Isolation and characterization of polymorphic microsatellite loci in large yellow croaker

合集下载

WT1基因在骨髓增生异常综合征患者外同血中的表达及临床意义

WT1基因在骨髓增生异常综合征患者外同血中的表达及临床意义聂泽强;李晓云;马爻芳;方敏;鲍杰【摘要】目的:探讨WT1基因在骨髓增生异常综合征(MDS)患者中的表达情况及WT1基因与MDS发病及病情进展的关系.方法:采用逆转录-聚合酶链反应检测48例MDS患者,4例MDS-AL患者和10例正常人外周血中WT1基因的表达,并进行分析.结果:①4例MDS-AL患者的WT1阳性率100％,10例正常人的WT1阳性率0％,48例MDS患者WT1阳性率为27.1％.MDS组、MDS-AL组、对照组两两相比有统计学差异(P<0.05).② 将MDS按IPSS分组,WT1mRNA的表达水平在高危与中危及低危组之间差异均有统计学意义(P<0.05),中危与低危组、中危Ⅰ组与中危Ⅱ组之间无统计学差异(P>0.05).WT1基因表达水平与IPSS评分值有相关性(r=0.74,P<0.05).③WT1基因表达水平与原始细胞百分数密切相关(r=0.66,P<0.05).④动态随访2例MDS患者,随着疾病进展,WT1的表达水平逐渐升高.结论:WT1基因在各类型MDS均高表达.WT1基因的表达与疾病的进展呈正相关,WT1基因表达情况与原始细胞百分比及IPSS评分均有明显相关性.其可作为临床上对M DS病情的高危评估,预测病情变化和动态监测治疗效果及判断预后的重要指标之一.【期刊名称】《陕西医学杂志》【年(卷),期】2019(048)008【总页数】4页(P977-980)【关键词】骨髓增生异常综合征;急性白血病;国际预后评分系统;WT1基因;逆转录-聚合酶链反应;相关性【作者】聂泽强;李晓云;马爻芳;方敏;鲍杰【作者单位】山西省运城市中心医院血液科运城044000;山西省运城市中心医院血液科运城044000;山西省运城市中心医院血液科运城044000;山西省运城市中心医院血液科运城044000;山西省运城市中心医院血液科运城044000【正文语种】中文【中图分类】R551.3骨髓增生异常综合征(Myelodysplastic syndrome, MDS)是一种造血干细胞恶性髓系克隆性疾病，且高风险向急性白血病转换[1]。

苯乙烯微生物降解机理的研究进展(1)

[4]J un Hiraki,Takafumi Ichika wa,Shin-ichi Ninomi ya,et al .Use of AD ME studi es to confirm the s afety of -poly-L-l ysine as a preservative in food [J].Regulatory T oxicology and Pharmacology ,2003,37(2):328-340.[5]Ki to M,Takimoto R,Yoshida T,et al .Purification and characteriz ationof -poly-L-lysine-degrading enzyme from an -pol y-L-lysine-produc ing s train Stre ptomyce s albulus [J].Arch Microbiol ,2002,178:325-330.[6]Nishi kawa M,Ogawa K.Distri bution of microbes producing anti microbi al -poly-L-Lysine polymers in s oil microflora determined by a novel method[J].Applied and Environmental Microbiology ,2002,68(7):3575-3581.[7]G Szokan,M Almas,K Krizsan,e t al .Structure determination and s ynthesis of l ysi ne isopeptides i nfluencing on cell proli ferati on[J].B iopolymers ,1997,42(3):305-318.[8]Kahar P,T Iwata,J Hiraki,et al .Enhance ment of -polylys ine production by Stre pto myce s albulus s train 410usi ng pH control[J ].J.Biosci.B ioeng ,2001,91(2):190-194.[9]Kahar P,K Kobayashi,T Iwata,et al .Production of -polylysine i n an air lift bioreactor (ABR)[J].J.B iosci.Bioeng ,2002,93(3):274-280.[10]Ki to M,Onji Y,Yoshida T,et al .Occurrence of -poly-L-lysine-de gradi ng enz yme in -poly-L-l ysine-tolerant Sphingobacte rium multivorum OJ10:purification and charac terization [J ].FEMS Microbiology Letters ,2002,207(2):147-151.[11]Kito M,Rika T,Onji Y,et al .Puri fication and characterization of an -poly-L-l ysi ne-Degrading Enz yme from the -poly-L-lysine-tolerant Chryseobacte rium sp .057[J].J.B iosci.Bioeng ,2003,96(1):92-94.[12]Hi raki J,Hatakeyama M ,M ori ta H,et al .Improved -poly-L-lysine production of an S-(2-aminoe thyl)-L-cysteine resis tant mutant of Stre pto myc es alblus [J].Seibu tsu Kogaku K ais hi ,1998,76:487-493.[13]Takagi H,Hoshino Y,Nakamori S,e t al .Isolation and sequence analysis of plasmid pNO33in the -pol y-L-lysineproduci ng antino myce te Stre ptomyces albulus IFO14147[J].J.Bios ci.Bioeng ,2000,89(1):94-96.[14]Kawai T,Kubota T,Hiraki J,et al .Bi os ynthesis of -poly-L-lysine i n a cell-free system of Stre ptomyces albulus [J ].Biochemical and B iophys ical Research Communications ,2003,311(3):635-640.[15]Yu-Ting H,Shoichi ro I,Munehiko T Improving emulsi fying activi ty of -polylysine by conjugation w i th dextran through the Maillard reac tion[J].Food Chemistry ,2000,68(4):449-455.[16]Keiji I,Kyos uke Y,Atsus hi H,et al .Polyi on comple x micelles from plas mid DNA and pol y (e thylene gl ycol)-poly(L -lysine)bl ock copol ymer as serum-tolerable pol yple x system:physicochemical properties of micelles rele vant to gene trans fection efficiency[J ].Biomaterials ,2003,24(24):4495-4506.[17]W Cui,G Barr,K M Faucher,et al .A me mbrane-mimetic barrier for islet encaps ulation[J].Transplantation Proceedings ,2004,36(4):1206-1208.[18]M aureen D B,Alexander I G,Laurence T,et al .In vi tro and in vi vo gene transfer with poly(amino aci d )vesicles [J].Journal of Controlled Release ,2003,93(2):193-211.[19]Tris tan M ,Pascal D,Thierry B,e t al .Efficient gene trans fer into human epithelial cell lines using gl ycosylated cationic carriers and neutral gl ycosylated co-lipids [J ].Blood Cells,Molecules,an d Diseases ,2004,32(2):271-282.[20]Hana H,David F,Petr K.Growth and elec trochemical ac tivity of the poly-L-lysine-poly-L-glutamic acid thin layer fi lms:an EQCM and electro chemical study[J].Journal of Electroanalytical Chemistry ,2004,562(1):261-265.[21]孙宇峰,王连超,吴仲城,等.石英晶体振荡器海洛因快速测定生物合成酶膜制备及反应机理研究[J].传感器世界,2003,9(12):2-5.[22]Kunioka M ,Choi H J.Properties of biodegradable hydrogels prepared by -irradiation of microbial poly( -l ysi ne)aqueous sol utions[J].J Appl Polym Sci ,1995,58:801-806.苯乙烯微生物降解机理的研究进展吴献花1,2,孙石2,邵丹1,李海涛1,章新1,林洪1(1.玉溪师范学院化学与环境科学系,云南玉溪653100;2.昆明理工大学环境科学与工程学院,云南昆明650093)摘要:综述苯乙烯微生物降解的机理。

一株螺旋状铁氧化细菌的分离及特性研究

收稿日期:2003-10-17基金项目:国家自然科学基金资助项目(50170034)(30170026)作者简介:刘缨(1976-),女,助理工程师,主要从事自养微生物的分子生物学研究.文章编号:1671-9352(2004)02-0116-04一株螺旋状铁氧化细菌的分离及特性研究刘缨,刘相梅,郑力真,林建群,颜望明(山东大学微生物技术国家重点实验室,山东济南 250100)摘要:利用双层平板培养技术,从云南腾冲地区高温温泉边酸性水中分离出1株螺旋状铁氧化细菌ML -04,对该菌的理化特性研究结果显示,该菌专性化能自养,可利用亚铁和黄铁矿为能源,不能氧化硫磺、四硫酸盐、硫代硫酸盐,最适生长温度40e ,最适生长p H2.5.对砷黄铁矿的浸矿实验表明,ML -04菌株可以有效浸出矿粉中的铁和硫元素.关键词:螺旋状;铁氧化细菌;分离;理化特性中图分类号:Q93 文献标识码:AIsolation and characterization of a vibrioid -shaped iron -oxidizing bacteriumLIU Ying,LIU Xiang -mei,Z HENG L-i zheng,LIN Jian -qun &YAN Wang -ming(State Key Laboratory of Microbial Technol ogy,Shandong Univ.,Ji nan 250100,Shandong,China)Abstract :A vibrioid -shaped i ron -oxidizing bacteriu m,named strain ML -04,was isolated from Tenchong area,Yunnan province in China with the double layer culture technique.And the characterization resul ts showed that ML -04s train is obli gately autotrophic and could use ferrous iron and p yrite as sole energy sources,but not element sulfur,thiosulfate and tetrathionate.The opti mal tem -perature of ML -04strai n is 40e and the opti mal p H is 2.5for growth.The result of leaching test showed that iron and sulfur ele -ment could be effectively extracted from arsenopyrite by bio -oxidation process of the ML -04strain.Key words :Vibrioid -shaped;Iron -oxidizing bacterium;Isolati on;Physi ological characters生物浸矿技术具有悠久的历史,古代人们就用微生物浸出铜.和化学方法相比,生物浸矿具有多方面的优点,不仅耗能低,对环境污染小,还可以处理常规化学方法难处理的低品位矿石,因而在环境问题日益严重,金属富矿匮乏的今天,越来越受到人们的关注[1].生物浸矿技术在一些国家已成功应用于工业生产,如南非、巴西、澳大利亚、美国、加纳、秘鲁、乌兹别克斯坦、希腊等国都实现了生物浸矿的工业化应用.我国在这方面的工作也开始起步,2000年,山东莱州天承生物金业股份有限公司引进澳大利亚生物提金技术处理含砷难冶金精矿,日处理矿石能力可达120吨[2].浸矿微生物主要是一些在酸性环境中生长的铁或硫氧化细菌.多为化能自养,可利用低价态铁或还原态无机硫化物作为电子供体,具有嗜酸性,生长pH 在1.5~2.0左右[3].目前国外已广泛开展对浸矿细菌的研究,以更好的应用于生产实践.但研究过程中存在很多技术难点,尤其是浸矿细菌绝大多数都是专性化能自养,有机质的存在对其生长有抑制作用,因而在琼脂糖或琼脂固体培养基上难以生长,也就难以对其进行分离纯化.目前分离纯化这类细菌多采用双层平板培养技术,该技术是在底层培养基中加入1种嗜酸性异养细菌,通过底层异养细菌的生长,消耗了琼脂第39卷第2期Vol.39 No.2山东大学学报 (理学版)JOURNAL OF SHANDONG UNI VERSITY2004年4月 Apr.2004糖固体平板中的微量有机成分,如寡糖类物质,从而有利于上层平板中化能自养细菌的生长[4,5].本研究利用双层平板培养基技术,从采集自云南腾冲地区高温温泉边的酸性水样中分离纯化出1株螺旋状铁氧化细菌ML-04,对该菌的理化特性和浸矿能力进行了研究.1材料1.1样品采自云南腾冲地区高温温泉边酸性水样.1.2培养基ML-04菌株的分离用FeTSB双层平板培养基[5]、生长曲线的测定用9K矿粉培养基[6],即9K培养基中加入砷黄铁矿(该矿粉含S20.45%,Fe 24.9%,As5.16%),用1mol P L的硫酸调pH至2.5.ML-04菌株能源利用特性的测定用基础培养基[7].该培养基用1mol P L的硫酸调pH至2.5.2方法2.1ML-04菌株的分离将采集的样本在9K培养基中,37e富集培养10d左右,待培养基颜色变成红棕色,采用梯度稀释法,涂布Fe TSB双层平板培养基,37e培养7d左右,长出单菌落,挑取单菌落在Fe TSB双层平板培养基上连续分离纯化,镜检观察,直至菌体形态一致.记作ML-04.收集ML-04菌体,经革兰氏染色后在光学显微镜下观察并摄影.收集ML-04菌体,涂布在小块盖玻片上,自然晾干后,用导电胶贴于圆形贴片上,喷金后在扫描电镜下观察及摄影.2.2ML-04菌株最适生长温度的测定以相同接种量接种ML-04菌悬液到含8%砷黄铁矿的9K培养基中,分别置于25e、35e、40e、45e、55e五个温度梯度,摇床培养6d,用血球计数板计菌数,以温度为横坐标,以菌数的对数值为纵坐标,绘制ML-04的生长温度)))菌数曲线图.2.3初始pH对ML-04菌株生长的影响以相同接种量接种ML-04菌悬液到含8%砷黄铁矿的9K培养基中,培养基pH值分别用奥力龙828型pH计标定至0.5,1.5,2.5,3.5,4.5,40e摇床培养6d,用血球计数板计菌数,以初始pH为横坐标,以菌数的对数值为纵坐标,绘制ML-04的pH)))菌数曲线图.2.4ML-04菌株生长曲线的绘制接种ML-04菌悬液到含8%砷黄铁矿的9K培养基中,pH2.5,40e条件下摇床培养,转速120r P min,每隔2d用血球计数板计菌数,以培养天数为横坐标,以菌数的对数值为纵坐标,绘制ML-04的生长曲线图.2.5ML-04菌株能源利用特性的研究向基础培养基中分别加入蛋白胨(0.1%)、酵母粉(0.1%)、葡萄糖(0.1%)、硫磺(5%)、硫代硫酸钠(1%)、四硫酸钾(0.3%)、硫酸亚铁(0.1mol P L)和黄铁矿(5%).其中硫代硫酸钠、四硫酸钾、硫酸亚铁过滤除菌后加入培养基,硫磺隔水蒸煮1h灭菌,再加入培养基.以ML-04菌悬液接种,40e摇床培养6d,连续3代移种,在显微镜下涂片观察菌的生长情况,生长者为阳性.2.6ML-04氧化砷黄铁矿的研究接种ML-04菌悬液到含8%砷黄铁矿的9K培养基中,每隔2d测1次培养基中可溶性总铁的量及硫酸根离子的量.总铁的测定用邻二氮菲分光光度法,硫酸根离子的测定用比浊法[8].以溶液中可溶性总铁的量与培养基中矿粉总铁含量的比例为纵坐标,以培养天数为横坐标,绘制铁的氧化率曲线.以溶液中硫酸根离子含量与培养基中矿粉含硫总量的比例为纵坐标,以培养天数为横坐标,绘制硫的氧化率曲线.3结果3.1菌株的分离及形态学观察菌株ML-04的菌落形态为琥珀色同心圆状,直径1~3mm,表面湿润,凸起,光滑(见图1).菌体形态为螺旋状,大小(0.25~0.3)@(1~3L m),革兰氏染色阴性(见图2和图3).3.2ML-04菌株最适生长温度的测定由图4可以看出,ML-04最适生长温度在40e 左右.当培养温度达到55e时,ML-04的菌数迅速下降.3.3初始pH对ML-04菌株生长的影响从图5可见,ML-04菌株生长的最适初始pH为2.5左右.在此pH条件下生长最好.第2期刘缨,等:一株螺旋状铁氧化细菌的分离及特性研究117图1 ML -04菌株的菌落形态照片Fig.1 The Colony of ML -04图2 ML -04菌株的光学显微镜照片(@1000)Fig.2 M icrograph of ML -04图3 ML -04菌株的扫描电镜照片(@15000)Fig.3 Scanning electron micrograp h of ML -04图4 培养温度对ML -04生长的影响Fig.4 Effect of temperature on the growth of ML -04strain图5 初始pH 对ML -04生长的影响Fig.5 Effect of culture media p H on the growth of ML -04strain3.4 ML -04菌株的生长曲线从图6可见,ML -04经过4d 左右的生长延滞期,4~6d 为对数生长期,从第8天以后,菌数增长非常缓慢.图6 ML -04菌株的生长曲线Fig.6 The growth curve of ML -04strain3.5 ML -04能源利用特性的研究由表1可见,ML -04菌株可以利用硫酸亚铁和黄铁矿为能源生长,不能利用硫磺、硫代硫酸盐和四硫酸盐,也不能利用葡萄糖、蛋白胨等有机物.表1 ML -04能源利用特性的研究T ab.1 Energy sources utilization characters of ML -04strai n能源生长情况蛋白胨-酵母粉-葡萄糖-硫磺-硫代硫酸钠-四硫酸钾-硫酸亚铁+黄铁矿+3.6 ML -04菌株对砷黄铁矿中铁和硫的氧化浸出结果ML -04菌株对铁的氧化浸出率见图7.ML -04菌株可以有效浸出砷黄铁矿中的铁,随着培养天数的增加,溶液中总铁的含量不断提高,第18天时,铁的浸出率在90%以上.图7 ML -04菌株对砷黄铁矿中铁的氧化浸出曲线Fig.7 The curve of iron concentration in the extraction ofarsenopyrite by ML -04strain118山东大学学报 (理学版)第39卷ML -04菌株对砷黄铁矿中硫的氧化浸出率见图8.随着培养天数的增加,溶液中硫酸根的含量不断提高,第18天时,硫的浸出率达80%以上.图8 ML -04菌株对砷黄铁矿中硫的氧化浸出曲线Fig.8 The curve of sul fur concentration in the extraction ofarsenopyrite by ML -04s train4 讨论长期以来,人们一直认为氧化亚铁硫杆菌(Thiabacillus ferrooxidans )在浸矿过程中起主要作用,近些年的研究却发现,钩端螺旋菌属(Leptos pirillum )在浸矿过程中往往起关键作用.和T .ferroo xidans 相比,Leptospirillum 属菌株如氧化亚铁钩端螺旋菌(L .ferroo xidans )的氧化还原电势更高,对铁离子的耐受能力更强,生长不受高浓度三价铁离子的抑制.因而,在工业生产中,尤其是在连续反应浸矿系统中,Leptos pirillum 属菌株占微生物种群的主要组分,在浸矿过程中起主导作用[9,10].因此,有关钩端螺旋菌属资源的发掘及其浸矿特性的研究,对微生物浸矿技术的发展和应用具有十分重要的意义.本实验的研究结果表明,ML -04菌株是螺旋状铁氧化细菌.它的最适生长温度40e ,最适生长pH2.5,和Leptos pirillum 属的L .ferrooxidans 一样,既能氧化硫酸亚铁,又能氧化硫化矿物,不能氧化硫磺、硫代硫酸盐及四硫酸盐.不能利用有机物为能源进行生长,属于化能自养型微生物[3,11].ML -04菌株与T .ferroo xidans 和L .ferroo xidans 的比较见表2.铁氧化细菌中的氧化亚铁钩端螺旋菌(L .fer -roo xidans )虽然不能直接利用无机硫化物作为能源,但它们在矿物的生物氧化过程中,通过浸出矿物中的铁元素,产生的硫酸铁中间代谢物是一种有效的金属矿物氧化剂,可作用于矿物中的无机硫,将硫元素也浸提出来[12].对ML -04的浸矿实验研究表明,ML -04菌株可有效浸出砷黄铁矿中的铁和硫.因而,该菌株在实际生产中具有潜在的应用价值,有关该菌株更为广泛的浸矿特性研究,本室正在进一步进行中.表2 ML -04菌株与T .f e rrooxidans 和L .f e rrooxidans的比较T ab.2 Characteristics of Strain ML -04,T .ferrooxidans andL .ferrooxidans生理生化特性ML -04L .ferrooxidan T .ferrooxidan s 革兰氏染色G -G -G -菌体形态螺旋状螺旋状杆状菌体大小(L m)0.25~0.3@1~30.2~0.4@1~30.5~1@1~2最适生长温度(e )4037~4030最适生长pH 2.5 1.5~1.8 2.5~3.0生长类型化能自养化能自养化能自养能量来源Fe 2+Fe 2+Fe 2+,S 0参考文献:[1]童雄.微生物浸矿的理论与实践[M].北京:冶金工业出版社,1997.[2]杨显万,郭玉霞.生物湿法冶金的回顾与展望[J].云南冶金,2002,31(3):85~88.[3]Rawlings D E.Heavy metal mining using microbes[J].AnnuRev Microbi ol,2002,56:65~91.[4]Johnson D B,Macvicar J H M,Rolfe S.A new solid mediumfor the i solatation and enumeration of Thiabacillus ferrooxidan s and acidophilic heterotrophic bacteria [J].J Microbial M eth -ods,1987,7:9~18.[5]Johnson D B,M cGinness S.A highly efficient and universalsolid medium for growing mesophilic and moderately thermo -philic,iron -oxidizi ng ,acidophilic bacteria [J ].J M icrobial Methods,1991,13:113~122.[6]Silverman M P,Lundgren D G.Studies on the chemoautotro -phic iron rium Ferr obacterium f e rrooxidans I:An improved me -dium and harvesting procedure for securing high cell yields[J].J Bacterial,1959,77:642~647.[7]东秀珠,蔡妙英等.常见细菌系统鉴定手册[M].北京:科学出版社,2001.[8]南京大学5无机及分析化学实验6编写组.无机及分析化学实验(第三版)[M ].北京:高等教育出版社,1998.[9]Rawlings D E,Tributsch H,Hansford G S.Reasons why-Le ptospirillum .-like species rather than Thiobacillus f err ooxi -dans are the dominant iron -oxidizing bacteria in many commer -cial processes for the biooxidati on of pyri te and related ores[J].Microbi ology,1999,145:5~13.(下转第124页)第2期刘缨,等:一株螺旋状铁氧化细菌的分离及特性研究119点:1、ZAP Express载体具有包括EcoRÑ、NotÑ在内的12个单一酶切位点,可插入0-12kb的DNA片段;2、在克隆位点两侧,含有T3、T7、Lac、C MV等双向启动子满足在原核和真核中表达的条件;3、具有T3、T7等多条测序引物;4、含有Ne o r-Kan r抗性标记便于重组子的筛选;5、构建于该载体上的克隆可用DNA探针或抗体探针筛选;7、由于ZAP E xpress中引入了f1噬菌体的复制其始和终止信号,在得到阳性克隆噬菌斑后,利用辅助噬菌体E xAssist进行超感染,使插入片段连同pB K-C MV从噬菌体DNA上剪切下来,形成噬菌粒,省掉了插入片段从噬菌体DNA到质粒载体上的酶切、连接和转化这一过程,极大方便了在体外对插入DNA片段的亚克隆操作.关于ZAP E xpress更为详尽的讨论见文献[5].利用分离纯化的火菇素的免疫血清对金针菇表达型cDNA文库进行免疫筛选,再对阳性克隆用PC R、限制性内切酶进行酶切、大肠杆菌初步诱导分析,获得了目的基因相关的cDNA片段,进一步的序列分析、鉴定和克隆火菇素基因的工作正在进行之中.参考文献:[1]Komatsu N,T erakawa H,Nakanishi K.Flammulin,a basicprotein of Flammulina velutipes wi th ant-i tumor activities[J].Antibiotics,Ser A,1963,16(3):139~143.[2]Watanabe Y,Nakanishi K,Komaisu N.Flammulin,an ant-itumor substance[J].Bull Chem Soc,Japan,1964,37(5): 747~750.[3]周凯松,彭俊峰,张长铠,等.火菇素提取新工艺及其生物活性研究[J].中国生物化学与分子生物学报, 2003,(2),In press.[4]张龙翔.生化实验方法和技术[M].北京:高等教育出版社,1997.[5]Joseph Sambrook,David W.Russel.分子克隆(第三版)[M].黄培堂,等译.北京:科学出版社,2002.[6]Laemmli UK.Cleavage of structural proteins during the assem-bly of the head of bacteriophage T4[J].Nature,1970,227: 680~685.(编辑:于善清)(上接第119页)[10]Sand W,Rohde K,Sobotke B,et al.Evaluation of Leptospi-rillu m f err ooxidans for leaching[J].Appl Environ Microbiol, 1992,58:85~92.[11]Arthur P,Harrison J.Genomic and physiological diversi tyamongst s trains of Thiobacillus ferrooxidand,and genomic comparison wi th Thiobacillus thioox idans[J].Arch Microbial,1982,131:68~79.[12]Battaglia-Brunet F,d,Hugues P,Cabral T,et al.T he mutu-al effect of mixed Thiobacilli and Leptospirilli populations on pyrite bioleachina[J].Minerals Engineering,1998,11(2): 195~205.(编辑:于善清)124山东大学学报(理学版)第39卷。

真菌多聚半乳糖醛酸酶的研究进展

真菌多聚半乳糖醛酸酶的研究进展董章勇1，2，王振中2（1.仲恺农业工程学院园艺园林学院，广东广州510225；2.华南农业大学植物病理生理学研究室，广东广州510642）摘要：真菌多聚半乳糖醛酸酶是植物细胞壁果胶的主要降解酶之一，是植物病原真菌的重要致病因子。

综述了多聚半乳糖醛酸酶的特性、多聚半乳糖醛酸酶基因及其特征、多聚半乳糖醛酸酶的分子进化、多聚半乳糖醛酸酶的表达调控及其在真菌致病过程中的作用。

关键词：真菌；细胞壁降解酶；果胶酶；多聚半乳糖醛酸酶中图分类号：S432.4+4文献标识码：A文章编号：1004-874X(2011)18-0125-03果胶酶是参与果胶降解的一组复合细胞壁降解酶，能将高等植物的初生细胞壁和中胶层的果胶多聚物降解为片段，最初在产生软腐症状的植物组织中发现。

主要包括多聚半乳糖醛酸酶（polygalacturonase,PG）、果胶甲基半乳糖醛酸酶（Pectin methyl galacturonic acid enzymes,PM G）、多聚半乳糖醛酸反式消除酶（Poly-galacturonic acid trans-elimination enzyme,PGTE）、果胶甲基反式消除酶（Elimination methyl trans-eliminase,PMTE）和纤维素酶（Cellulase,Cx）等，其在病菌致病过程中起摄取营养和降解寄主组织的重要作用。

PG是首次从离体细胞壁中获得的由致病真菌产生的果胶酶，被认为是通过降解植物细胞壁中同源的多聚半乳糖醛酸区域起作用，从而引起组织浸解和原生质体死亡，与真菌的致病性和毒性相关[1-5]。

多聚半乳糖醛酸酶的研究发展迅速，本文对多聚半乳糖醛酸酶及其序列特征、多聚半乳糖醛酸酶基因及其序列特征、多聚半乳糖醛酸酶的分子进化、多聚半乳糖醛酸酶的表达调控以及其与病原真菌的致病关系等进行综述。

1多聚半乳糖醛酸酶的特征与分类多聚半乳糖醛酸酶是一种细胞壁结合蛋白，广泛存在于细菌、真菌和植物中，具有水解酶的性质，是降解植物果胶骨架结构的主要酶之一[6]。

聚赖氨酸应用

Biochemical Engineering Journal 65 (2012) 70–81Contents lists available at SciVerse ScienceDirectBiochemical EngineeringJournalj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /b ejReviewReview on production and medical applications of ␧-polylysineSwet Chand Shukla a ,Amit Singh b ,Anand Kumar Pandey c ,Abha Mishra a ,∗aSchool of Biochemical Engineering,Institute of Technology,Banaras Hindu University,Varanasi 221005,India bDepartment of Pharmacology,Institute of Medical Sciences,Banaras Hindu University,Varanasi 221005,India cSchool of Biomedical Engineering,Institute of Technology,Banaras Hindu University,Varanasi 221005,Indiaa r t i c l ei n f oArticle history:Received 3May 2011Received in revised form 28March 2012Accepted 2April 2012Available online 11 April 2012Keywords:␧-PolylysineHomopolyamideS.albulus Lysinopolymerus Conjugate Drug carrier Targetinga b s t r a c t␧-Polylysine (␧-PL)is a homopolyamide linked by the peptide bond between the carboxylic and epsilon amino group of adjacent lysine molecules.It is naturally occurring biodegradable and nontoxic towards human.This review article gives an insight about the various ␧-PL producing strains,their screening procedures,mechanism of synthesis,characterization,and its application in the medical ﬁeld.The poly cationic nature of ␧-PL at physiological pH makes it as one of the potential candidates in the ﬁeld of drug delivery.Most of the biomedical applications till date use synthetic ␣-PLL as a raw material.However,it is believed that naturally occurring ␧-PL would be an ideal substitute.© 2012 Elsevier B.V. All rights reserved.Contents 1.Introduction ..........................................................................................................................................712.Origin and distribution of ␧-PL ......................................................................................................................713.Mechanism of synthesis .............................................................................................................................714.Biosynthesis and molecular genetics ................................................................................................................715.Microbial production of ␧-polylysine ................................................................................................................726.Screening and detection of ␧-PL production in microbial system...................................................................................737.Puriﬁcation and characterization of ␧-PL ............................................................................................................738.Conformation of ␧-PL ................................................................................................................................749.Application of polylysine in medicine ...............................................................................................................749.1.Polylysine as a drug carrier ...................................................................................................................749.2.Polylysine as nanoparticles...................................................................................................................759.3.Polylysine as a gene carrier...................................................................................................................759.4.Polylysine as liposomes ......................................................................................................................769.5.Polylysine as interferon inducer .............................................................................................................769.6.Polylysine as lipase inhibitor .................................................................................................................779.7.Polylysine as hydrogel ........................................................................................................................779.8.Polylysine as coating material................................................................................................................779.9.Other applications ............................................................................................................................7810.Conclusion ..........................................................................................................................................78References ...........................................................................................................................................78Abbreviations:Pls,polylysine synthetase;NaSCN,sodium thiocynate;FTIR,Fourier transform infrared spectroscopy;NMR,nuclear magnetic resonance spectroscopy;MION,monocrystalline iron oxide nanoparticle;NPs,nanoparticles;IgM,immunoglobulin M.∗Corresponding author.Tel.:+919451887940.E-mail address:abham.bce@itbhu.ac.in (A.Mishra).1369-703X/$–see front matter © 2012 Elsevier B.V. All rights reserved./10.1016/j.bej.2012.04.001S.C.Shukla et al./Biochemical Engineering Journal 65 (2012) 70–81711.Introduction␧-Polylysine (␧-PL)is a basic polyamide that consists of 25–30residues of l -lysine with an ␧-amino group-␣-carboxyl group link-age (Fig.1).Polyamide can be grouped into two categories,one in which the polyamide consists of only one type of amino acid linked by amide bonds called homopolyamide and the other which consists of different amino acids in their chain called proteins [1].Furthermore,proteins are biosynthesized under the direction of DNA,while the biosynthesis of homopolyamides is catalyzed by peptide synthetases.Therefore,the antibiotics that are inhibitors of translation such as chloramphenicol,do not affect the biosyn-thesis of polyamides.Proteins in general exhibit exact length,whereas homopolyamides show a remarkable variation in molec-ular weight.Amide linkages in proteins are only formed between ␣-amino and ␣-carboxylic groups (␣-amide linkages),whereas amide bonds in homopolyamide involve other side chain functions such as ␤-and ␥-carboxylic with ␧-amino groups [1].Particularly,chemically synthesized polylysine were found to have linkages between ␣-carboxyl and ␣-amino group.Many workers investi-gated various applications of ␣-PL in the drug delivery system.However,␣-PL was reported to be toxic to human beings,and there-fore,research has now been diverted towards ﬁnding naturally occurring polymers [2,3].␧-PL is an unusual naturally occurring homopolyamide having linkages between the ␧-amino group and ␣-carboxylic group,and it shows high water solubility and sta-bility.No degradation is observed even when the ␧-PL solution is boiled at 100◦C for 30min or autoclaved at 120◦C for 20min [4].␧-PL was discovered as an extracellular material of Streptomyces albulus ssp.Lysinopolymerus strain 346during screening for Dra-gendorff’s positive substances [5–7].Mutation studies were made by nitrosoguanidine treatment on wild type Lysinopolymerus strain 346to enhance the ␧-PL production.As a result of mutation,S-(2-aminoethyl)-l -cysteine and glycine resistant mutant were isolated,with four times higher amounts of ␧-PL than the wild type [8].␧-PL is a cationic surface active agent due to its positively charged amino group in water,and hence they were shown to have a wide antimi-crobial activity against yeast,fungi,Gram positive,Gram negative bacterial species [4,9].The excreted polymer is absorbed to the cell surfaces by its cationic property,leading to the striping of outer membrane and by this mechanism the growth of microbes sensi-tive to ␧-PL is inhibited.␧-PL degrading enzyme plays an important role in self-protection of ␧-PL producing microbes [9].Due to its excellent antimicrobial activity,heat stability and lack of toxicity,it is being used as a food preservative [10,11].Naturally occurring ␧-PL is water soluble,biodegradable,edible and nontoxic toward humans and the environment.Therefore,␧-PL and its derivatives have been of interest in the recent few years in food,medicine and electronics industries.Derivatives of ␧-PL are also available which offers a wide range of unique applications such as emul-sifying agent,dietary agent,biodegradable ﬁbers,highly water absorbable hydrogels,drug carriers,anticancer agent enhancer,biochip coatings,etc.Polylysine exhibits variety of secondary struc-tures such as random coil,␣-helix,or ␤-sheet conformations in aqueous solution.Moreover,transitions between conformations can be easily achieved using,salt concentration,alcohol con-tent,pH or temperature as an environmental stimulus.There is aH NH*CH 2CH 2CH 2CH 2CH NH 2CO*OHnFig.1.Chemical structure of epsilon polylysine.growing interest in using ␧-PL and its derivatives as biomaterials and extensive research has been done leading to a large number of publications [4,12–15].The present review focuses on various pro-cess parameters for maximal yield of polymer by microbial system more speciﬁcally by actinomycetes,probable biosynthetic route and its application,especially in pharmaceutical industries.2.Origin and distribution of ␧-PLNot much is known about the ␧-PL producing microbial species existing in the environment.It is observed that ␧-PL producers mainly belong to two groups of bacteria’s:Streptomycetaceae and Ergot fungi .Besides Streptomyces albulus ,a number of other ␧-PL producing species belonging to Streptomyces,Kitasatospora and an Ergot fungi,Epichole species have been isolated [16].Recently,two Streptomyces species (USE-11and USE-51)have been isolated using two stage culture method [17].3.Mechanism of synthesis␧-Polylysine (␧-PL)is a homopolymer characterized by a pep-tide bond between ␣-carboxyl and ␧-amino groups of l -lysine molecules.Biosynthetic study of ␧-PL was carried out in a cell-free system by using a sensitive radioisotopic ␧-PL assay method,suggested that the biosynthesis of ␧-PL is a non ribosomal peptide synthesis and is catalyzed by membrane bound enzymes.In vitro ,␧-PL synthesis was found to be dependent on ATP and was not affected by ribonuclease,kanamycin or chloramphenicol [18].In a peptide biosynthesis,amino acids are activated either by adeny-lation or phosphorylation of carboxyl group.Adenylation occurs in translation and in the nonribosomal synthesis of a variety of unusual peptides [19,20];Phosphorylation has been suggested for the biosynthesis of glutathione [21].In the former,ATP is con-verted to AMP and pyrophosphate by adenylation,and in the latter,phosphorylation leads to ADP and phosphate as the ﬁnal prod-ucts.The synthesis of ␧-PL,a homopolypeptide of the basic amino acid l -lysine,is similar to that of poly-(␥-d -glutamate)in terms of adenylation of the substrate amino acid [18].Through the exper-imental observations,the probable mechanism of synthesis was suggested by Kawai et al.showed that in the ﬁrst step of ␧-PL biosynthesis l -lysine is adenylated at its own carboxyl groups with an ATP-PPi exchange reaction.The active site of a sulfhydryl group of an enzyme forms active aminoacyl thioester intermediates,lead-ing to condensation of activated l -lysine monomer.This is the characteristic feature of nonribosomal peptide synthetase enzyme [22–24].␧-PL producing strain of Streptomyces albulus was found to pro-duce ␧-PL synthetase (Pls).A gene isolated from the strain was identiﬁed as a membrane protein with adenylation and thiolation domains which are characteristic features of the nonribosomal pep-tide synthetases (NRPSs).␧-PL synthetase has six transmembrane domains surrounding three tandem soluble domains without any thioesterase and condensation domain.This tandem domain itera-tively catalyzes l -lysine polymerization using free l -lysine polymer as an acceptor and Pls-bound l -lysine as a donor,thereby yielding chains of diverse length (Fig.2).Thus,␧-PL synthetase acts as a ligase for peptide bond formation [25].Yamanaka et al.suggested that ␧-PL synthetase function is regulated by intracellular ATP and found that acidic pH conditions are necessary for the accumulation of intracellular ATP,rather than the inhibition of the ␧-PL degrading enzyme [26].4.Biosynthesis and molecular geneticsThe precursor of ␧-PL biosynthesis was identiﬁed to be l -lysine by radiolabeling studies using [14C]-l -lysine in Streptomyces72S.C.Shukla et al./Biochemical Engineering Journal 65 (2012) 70–81Fig.2.Mechanism for synthesis of ␧-polylysine.albulus 346[18].However,a high-molecular-weight plasmid (pNO33;37kbp)was detected in ␧-PL-producing S.albulus ,and the replicon of pNO33was used to construct a cloning vector for S.albu-lus strain [27].The order and number of NRPSs modules determine the chain length of the ␧-PL [24,28].However,the chain length of ␧-PL was shortened by the use of aliphatic hydroxy-compound and ␤-cyclodextrin derivative [29,30].␧-PL with more than nine l -lysine residues severely inhib-ited the microbial growth while the ␧-PL with less than nine l -lysine residues showed negligible antimicrobial activity.All the strains producing ␧-PL from glycerol showed lower number aver-age molecular weight (M n )than those obtained from glucose [31].The ␧-PL-degrading activity was detected in both ␧-PL tolerant and ␧-PL producing bacteria.The presence of ␧-PL-degrading activity in Streptomyces strains is closely related with ␧-PL-producing activ-ity,which indicates that tolerance against ␧-PL is probably required for ␧-PL producers.The presence of ␧-PL degrading enzyme is detri-mental to industrial production of ␧-PL.Therefore,␧-PL degrading enzyme of S.albulus was puriﬁed,characterized and the gene encoding an ␧-PL degrading enzyme of S.albulus was cloned,and analyzed [32].The ␧-PL-degrading enzyme of S.albulus is tightly bound to the cell membrane.The enzyme was solubilized by NaSCN in the presence of Zn 2+and was puriﬁed to homogeneity by phenyl-Sepharose CL-4B column chromatography,with a molecular mass of 54kDa.The enzymatic mode of degradation was exotype mode and released N-terminal l -lysine’s one by one.Streptomyces vir-giniae NBRC 12827and Streptomyces noursei NBRC 15452showed high ␧-PL-degrading aminopeptidase activity and both strains have the ability to produce ␧-PL,indicating a strong correlation between the existence of ␧-PL degrading enzyme and ␧-PL produc-ing activity [33].␧-PL degrading enzymes were also found in ␧-PL tolerant microorganisms,Sphingobacterium multivorum OJ10and Chryseobacterium sp.OJ7,which were isolated through enrichmentof the culture media with various concentrations of ␧-PL.S.mul-tivorum OJ10could grow well,even in the presence of 10mg/ml ␧-PL,without a prolonged lag phase.The ␧-PL-degrading enzyme activity was also detected in the cell-free extract of ␧-PL tolerant S.multivorum OJ10.The enzyme catalyzed an exotype degradation of ␧-PL and was Co 2+or Ca 2+ion activated aminopeptidase.This indicates the contribution of ␧-PL-degrading enzymes to the toler-ance against ␧-PL [34].An ␧-PL degrading enzyme of ␧-PL tolerant Chryseobacterium sp.OJ7,was also characterized and the puriﬁed enzyme catalyzed the endotype degradation of ␧-PL,in contrast to those of Streptomyces albulus and Sphingobacterium multivorum OJ10.Probably,their possession of proteases enables their growth in the presence of a high ␧-PL concentration.␧-PL degradation was also observed by commercially available proteases,such as Pro-tease A,Protease P and Peptidase R [34,35].5.Microbial production of ␧-polylysinePolylysine can be synthesized by chemical polymerization start-ing from l -lysine or its derivatives.Researchers described two different routes to polymerize lysine residues without the use of protection groups.However,linear ␧-PLL can be obtained by applying 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as an activating agent for the polycondensation of l -lysine in an aqueous medium.In contrast to this,␣-poly(l -lysine)can be obtained by using dicyclohexyl carbodiimide and 18-crown-6ether in chloro-form [36].Dendrimeric ␣,␧-polylysine were synthesized by using solid phase peptide synthesis method and used dendritic ␣,␧-polylysine as a delivery agent for oligonucleotides [37,38].Moccia et al.for the ﬁrst time reported ␣,␧-polylysine by assembling Fmoc and Boc protected l -lysine monomers by solid phase synthesis [39].Guo et al.synthesized ␧-PL-analogous polypeptides with not only similar ␣-amino side groups but also similar main chain throughS.C.Shukla et al./Biochemical Engineering Journal65 (2012) 70–8173microwave assisted click polymerization technique[40].Recently, Roviello et al.synthesized a cationic peptide based on l-lysine and l-diaminobutyric acid for theﬁrst time by solid phase synthesis [41].␧-PL was discovered as an extracellular material produced by ﬁlamentous actinomycetes group of micro-organism Streptomyces albulus ssp.Lysinopolymerus strain346more than35years ago [5].It is synthesized by a nonribosomal peptide synthetase and released extracellularly.In actinomycetes group of organisms l-lysine is synthesized through the diaminopimelic acid pathway. Diaminopimelate is formed via l-aspartate(Asp)produced by com-bining oxaloacetate in the tricarboxylic acid cycle with ammonium as a nitrogen source.Citrate was found to be facilitator for the production much more than other organic acids of TCA cycle[24].Studies revealed that decline in pH during the fermentation pro-cess is an essential condition for the accumulation of␧-PL.Shima et al.carried out two-step cultivation method for S.albulus.Strain wasﬁrst grown for24h in a culture medium containing glycerol as carbon source with yeast extract,then in second step medium was replaced by glucose,citric acid with(NH4)2SO4[42].It was found that the mutant of strain346decreases the culture pH from its initial value of6.8–4.2by36h,and slowly decreased thereafter to 3.2at96h.The accumulation of␧-PL in the broth increased signiﬁ-cantly when the culture pH was about4.0.The fed batch cultivation was adopted to enhance the␧-PL production with two distinct phases.In phase I,cell was grown at pH(6.8)optimum for cul-ture growth then in phase II,the pH was kept around4.0by the addition of glucose.Depletion of glucose causes an increase in pH of the culture broth leading to the degradation of the produced ␧-PL.Thus the pH control strategy in fed batch culture success-fully enhanced the yield of␧-PL to almost9fold[43].The airlift bioreactor(ABR)was also evaluated and compared with jar fer-mentor for␧-PL production.The results showed that the production level of␧-PL in a ABR with a power consumption of0.3kW/m3was similar to that in a5-l jar fermentor with power consumption of 8.0kW/m3.The leakage of intracellular nucleic acid(INA)-related substance into the culture broth in the ABR was70%less than that in the jar fermentor.Thus,ABR system with low intracel-lular nucleic acid-related substances minimize the difﬁculties of downstream processing for recovery and puriﬁcation of the poly-mer products.Furthermore,the use of ABR is promising tool for the low-cost production of␧-PL of high purity[44].In some␧-PL producing strains,the production of␧-PL is unstable and depen-dent on cell density which can cause problem such as high viscosity and low oxygen transfer efﬁciency.Furthermore,increase of agita-tion speeds leads to the rise of shear stresses which might cause undesired effects on mycelial morphology,product formation,and product yields.Bioprocesses using immobilized cells on various inert supports can increase overall productivity and minimize pro-duction costs[45].Bankar et al.reported that aeration and agitation of the fermentation broth markedly affect␧-PL production,cell mass formation,and glycerol utilization.Fermentation kinetics per-formed revealed that␧-PL production is growth-associated,and agitation speed of300rpm and aeration rate at2.0vvm supports higher yields of␧-PL[46].Many efforts have been made to opti-mize the media in order to enhance the productivity of␧-PL.Shih and Shen applied response surface methodology for optimization of␧-PL production by Streptomyces albulus IFO14147[47].It was found that␧-PL production started on agar plated with iron two or three days earlier than that on plates without iron.Manganese and cobalt were also found to have stimulating effect on␧-PL produc-tion.Kitasatospora kifunense strain produces␧-PL of shorter chain length about8–17lysine residues[48].Metabolic precursors such as amino acids,tricarboxylic acid cycle intermediates and cofactors have been investigated for improved production of␧-PL.Addition of citric acid after24h and l-aspartate after36h of fermentation medium had a signiﬁcant effect on␧-PL production[49].Zhang et al.investigated the production of␧-PL on immobilized cells of Kitasatospora sp.MY5-36on bagasse,macroporous silica gel,syn-thetic sponge,loofah sponge and found that loofah sponge gave highest production of␧-PL in shakeﬂask culture[50].6.Screening and detection of␧-PL production in microbial systemNishikawa and Ogawa developed a simple screening method to detect␧-PL producing microbes.Screenings were carried out on agar plates containing either basic or acidic dyes.The dyes used were,Poly R-478,Remazol Brilliant Blue-R(RBBR)and Methylene blue.The screening method was based on the rationale interac-tion that occurs between charged groups of the secreted␧-PL and charged group of the basic or acidic dyes.A synthetic glycerol(SG) medium containing either0.02%of acidic dye Poly R-478/RBBR or0.002%of Methylene blue was used for the primary screen-ing.The SG medium was composed of glycerol10g,ammonium sulfate0.66g,sodium dihydrogen phosphate0.68g,magnesium phosphate heptahydrate0.25g,yeast extract0.1g,and1.0ml of Kirk’s mineral solution in1l of distilled water.The pH was adjusted to7.0with1M NaOH solution,and the medium was solidiﬁed by adding1.5%agar.The plates were incubated at28◦C for about one week;microbes forming speciﬁc colonies interacting with dyes were picked up and puriﬁed after several culture transfers.The acidic dye condensed around the organism’s colonies while basic dye was excluded from the surrounding zone.A zone of at least ﬁve mm in diameter for each colony was needed to visualize the interaction between secreted substances and dyes[16].The concentrations of␧-PL in the culture broth can be deter-mined by using either the spectrophotometric method or HPLC method.The colorimetric method is based on the interaction between␧-PL and methyl orange,which is an anionic dye,and thus the interaction of cationic␧-PL with anionic methyl orange in the reaction mixture led to form a water insoluble complex[51].The HPLC method for␧-PL detection was reported by Kahar et al.in which HPLC column(Tsk gel ODS-120T,4.6mm×250mm)with a mobile phase comprising of0.1%H3PO4was used[43].7.Puriﬁcation and characterization of␧-PL␧-PL a cationic polymer,can be isolated at neutral pH,and puri-ﬁed from the culture broth by ion exchange chromatography using an Amberlite IRC-50(H+form)column[5,52].The culture super-natant can be passed through an Amberlite IRC-50column at pH 8.5with successive washing by0.2N acetic acid and water.The elution can be made with0.1N hydrochloric acid,and the eluate can be neutralized with0.1N sodium hydroxide to pH6.5.Sub-sequent puriﬁcation can be done by using CM-cellulose column chromatography to get␧-PL in homogeneity.The puriﬁcation of the product can be monitored by UV absorption at220nm and fur-ther characterized by amino acid analysis.The molecular weight of␧-PL can be estimated by gelﬁltration on a Sephadex column [16,53].Kobayashi et al.extracted the␧-PL from Kitasatospora kifu-nense.The pH of the cultureﬁltrate wasﬁrst adjusted to7.0,and the aliquot was mixed with Gly-His-Lys acetate salt as an inter-nal peptide standard.The resulting mixture was then applied to Sep-Pak Light CM cartridge.The cartridge was washed with water and␧-PL was eluted with0.1M HCl.The eluate was lyophilized and the residue was dissolved in0.1%pentaﬂuoropropionic acid [46].Recently,ultra-ﬁltration technique for fractionation of␧-PL of different molecular weight has been applied.The␧-PL with molec-ular weight higher than2kDa form a␤-turn conformation whereas molecular weight smaller than2kDa possesses a random coil74S.C.Shukla et al./Biochemical Engineering Journal65 (2012) 70–81conformation.The fraction of␧-PL with molecular weight higher than2kDa was found to have signiﬁcant antibacterial activity, while the fraction with molecular weight smaller than2kDa shows nominal antibacterial activity[54].8.Conformation of␧-PLStructure and conformation studies are prerequisite to under-stand the functional behavior of␧-PL.Numerous workers have investigated the conformation and the molecular structure of microbially produced␧-PL by NMR,IR and CD spectroscopy[55,56]. The thermal property of crystalline␧-PL was determined by Lee et al.[52].The glass transition temperature(T g)and the melting point(T m)was observed to be88◦C and172.8◦C respectively.The results from pH dependent IR and CD spectra,1H and13C NMR chemical shifts together with that of13C spin-lattice relaxation times T1indicated that␧-PL assumes a␤-sheet conformation in aqueous alkaline solution.␧-PL at acidic pH might be in an electro-statically expanded conformation due to repulsion of protonated ␣-amino group,whereas at elevated pH(above p K a of the␣-amino group)the conformation was found to be similar to the antiparallel ␤-sheet.The molecular structure and conformation of microbial␧-PL was studied by FT-IR and Raman spectroscopy.␧-PL was found to assumed a␤-sheet conformation in the solid state and solid state 13C NMR also revealed that␧-PL existed as a mixture of two crys-talline forms.Spin-lattice relaxation times yield two kinds of T1s corresponding to the crystalline and amorphous components,with the degree of crystallinity as63%[57].Solid-state high-resolution13C and15N NMR spectra of micro-bial␧-PL derivatives with azo dyes have been measured.These chemically modiﬁed␧-PL’s Exhibit15N NMR signals characteristic of the binding mode at the␣-amino groups.The spectral analy-sis reveals that the␧-PL/DC sample contains a small amount of ion complexes with methyl orange(MO).It has been shown that side chain␣-amino group of␧-PL does not make a covalent bond with methyl orange(MO)but forms a poly-ion complex,(␧-PL)-NH3+SO3−-(MO).On the other hand,dabsyl chloride(DC)makes covalent bond with␧-PL to form sulfonamide,(␧-PL)-NH-SO2-(DC). However,a few tens percent of DC change to MO by hydrolysis to form a poly-ion complex,(␧-PL)-NH3+SO3−-(MO)[58].Rosenberg and Shoham characterized the secondary structure of polylysine with a new parameter namely,the intensity ratio of the bands of charged side chain amine NH3+and amide NH bands.The enthalpy of the secondary structure transition,which is observed in PLL at the change of pH from11to1amounts to4.7kJ mol−1[59].9.Application of polylysine in medicinePolylysine is available in a large variety of molecular weights. As a polypeptide,polylysine can be degraded by cells effortlessly. Therefore,it has been used as a delivery vehicle for small drugs[60]. The epsilon amino group of lysine is positively charged at phys-iological pH.Thus,the polycationic polylysine ionically interacts with polyanion,such as DNA.This interaction of polylysine with DNA has been compacted it in a different structure that has been characterized in detail by several workers[61–66].In addition,the epsilon amino group is a good nucleophile above pH8.0and there-fore,easily reacts with a variety of reagents to form a stable bond and covalently attached ligands to the molecule.Several coupling methods have been reported for preparation of conjugated of␧-PL [67–70].(a)Modiﬁcation of epsilon amino groups of polylysine with bifunctional linkers containing a reactive esters,usually add a reac-tive thiol group to the polylysine molecule and consequent reaction with a thiol leads to a disulﬁde or thioether bond,respectively.This has been used to couple large molecules,such as proteins to polylysine.(b)Compounds containing a carboxyl group can be acti-vated by carbodiimide,leading to the formation of an amide bond with an epsilon amino group of polylysine.(c)Aldehydes,such as reducing sugars or oxidized glycoprotein,form hydrolysable schiff bases with amino groups of␧-PL,which can be selectively reduced with sodium cyanoborohydride to form a stable secondary amine.(d)Isothiocyanate reacts with epsilon amino groups by forming a thiourea derivative.(e)Antibody coupling can also be done specif-ically to the N-terminal amino group of polylysine[71,72].A variety of molecules such as proteins,sugar molecules and other small molecules have been coupled to polylysine by using these methods.Puriﬁcation of the conjugates are usually being achieved by dialysis or gelﬁltration in conjunction with ion-exchange chromatography or preparative gel electrophoresis. Fractionation of the ligand–polylysine ratio and conjugate size can be done by using acid urea gel electrophoresis in combination with cation-exchange HPLC,ninhydrin assay and ligand analysis (sugar,transferrin,etc.)[73].Galactose terminated saccharides such as galactose,lactose and N-acetylgalactosamine were found to be accumulated exclusively in the liver,probably by their hepatic receptor.These conjugates could therefore be excellent carriers for a drug delivery system to the liver.The other saccharides such as the mannosyl and fucosyl conjugates are preferentially delivered to the reticuloendothelial systems such as those in the liver,spleen and bone marrow.In particular,fucosyl conjugates accumulated more in the bone marrow than in the spleen whereas xylosyl con-jugates accumulated mostly in the liver and lung.Generally,the accumulated amount in the target tissue increased with increasing molecular weight and an increased number of saccharide units on each monomer residues of polymer[74].One of the disadvantages of polylysine from the pharmaceu-tical point of view is its heterogeneity with respect to molecular size.The size distribution of polylysine with degrees of polymer-ization(dp)can be reduced by gel permeation chromatography. Al-Jamal et al.studied sixth generation(G6)dendrimer molecules of␣-poly-l-lysine(␣-PLL)to exhibit systemic antiangiogenic activ-ity that could lead to solid tumor growth arrest.Their work showed that G6PLL dendrimer have an ability to accumulate and persist in solid tumor sites after systemic administration and exhibit antian-giogenic activity[75].Sugao et al.reported6th generation dendritic ␣-PLL as a carrier for NF␬B decoy oligonucleotide to treat hepatitis [76].Han et al.synthesized a new anti-HIV dendrimer which con-sisted of sulfated oligosaccharide cluster consisting with polylysine core scaffold.The anti-HIV activity of polylysine-dendritic sulfated cellobiose was found to have EC50-3.2␮g/ml for viral replication which is as high as that of the currently clinically used AIDs drugs. The results also indicated that biological activities were improved because of dendritic structure in comparison to oligosaccharide cluster which were reported to have low anti-HIV activity[77].9.1.Polylysine as a drug carrierPolylysine can be used as a carrier in the membrane transport of proteins and drugs.Shen and Ryser reported that␣-PLL was found to be easily taken up by cultured cells.In fact,the conju-gation of drug to polylysine markedly increased its cellular uptake and offers a new way to overcome drug resistance related to deﬁ-cient transport[60,78,79].Resistance toward methotrexate has been encountered in the treatment of cancer patients.The poly lysine conjugates of methotrexate(MTX)were taken up by cells at a higher rate than free drugs form.This increased uptake can overcome drug resistance due to deﬁcient MTX transport.Addi-tion of heparin at a high concentration restores growth inhibitory effect of MTX-poly lysine[11,60].Shen and Ryser worked conjuga-tion of␣-PLL to human serum albumin and horseradish-peroxidase。

ZnO纳米粒子的合成与表征

ZnO纳米粒子的合成与表征陈延明;贾宏伟【摘要】以乙醇为溶剂,乙酸锌为前驱物,聚乙烯吡咯烷酮为表面修饰剂,采用溶液化学法,制备了氧化锌纳米粒子。

考察反应时间、聚乙烯吡咯烷酮加入量及含水量的影响。

通过UV-Vis、FL和TEM等对ZnO纳米粒子进行表征。

结果表明,在PVP-乙醇反应体系中加入3 mL浓度33.4 mmol/L乙酸锌水溶液,3 mL水,0.5 g PVP,在80℃反应120 min时,制得氧化锌纳米粒子的效果较好,氧化锌纳米粒子呈规则的球形,具有较好的分散性,粒径约为200 nm,且具有较窄的尺寸分布,证明PVP 对ZnO纳米粒子表面具有较好的修饰效果。

%ZnO nanoparticles have been synthesized by using ethanol as solvent,zinc acetate as precursor and poly( vinyl pyrrolidone) as polymer stabilizer through wet-chemical route. The influence of reaction time,PVP and water additions on preparation of ZnO nanoparticles were studied. The synthesized ZnO nanoparticles were characterized by UV-Vis,FL and TEM methods. The results show that the synthesized ZnO nanoparticles could give better properties by adding 3 mL zinc acetate aqueous with concentration 33. 4 mmol/L,0. 3 mL water,0.5 g PVP under reaction temperature 80 ℃ and reaction time 120 min re-spectively in PVP-ethanol reaction system. The size of the synthesized ZnO nanoparticles is about 200 nm with a good dispersion and narrow size distribution. This work demonstrated that poly( vinyl pyrrolidone) could play a better role in the modification of nano-ZnO surface as polymer stabilizer.【期刊名称】《应用化工》【年(卷),期】2015(000)006【总页数】4页(P1064-1067)【关键词】纳米氧化锌;乙醇;聚乙烯吡咯烷酮;溶液化学法【作者】陈延明;贾宏伟【作者单位】沈阳工业大学石油化工学院，辽宁辽阳 111003;沈阳工业大学石油化工学院，辽宁辽阳 111003【正文语种】中文【中图分类】TQ13;TB383纳米ZnO 带隙约为3.37 eV，激子结合能高达60 meV，广泛应用于紫外激光发射器［1］、场效应晶体管［2］、催化剂［3］、光电探测器［4］和细胞标记材料［5］等领域。

漆酶的研究进展及其应用

漆酶的研究进展及其应用刘岩;刘锐;苏新国;赵冠里;杨昭【摘要】Laccase is a kind of polyphenol oxidase. Because of its wide distribution in nature,laccase has broad ap⁃plication prospects in environmental protection,textile,printing and dyeing,food and chemical synthesis,etc. It has received extensive attention and research in recent years.This paper introduces the research progress and outstand⁃ing application of laccase,providing the new prospects and direction for bacterial laccase.%漆酶是一种多酚氧化酶，由于其在自然界分布广泛，并且在环保、纺织、印染、食品、化学合成等方面都具有广泛的应用前景，近年来得到了广泛的关注和研究。

该文主要综述了国内外漆酶的研究进展及其应用，为细菌漆酶提供新的应用前景和方向。

【期刊名称】《安徽农学通报》【年(卷),期】2016(022)013【总页数】4页(P25-27,159)【关键词】漆酶;研究进展;应用【作者】刘岩;刘锐;苏新国;赵冠里;杨昭【作者单位】广东食品药品职业学院，广东广州 510520;仲恺农业工程学院，广东广州 510225;广东食品药品职业学院，广东广州 510520;广东食品药品职业学院，广东广州 510520;广东食品药品职业学院，广东广州 510520【正文语种】中文【中图分类】Q814漆酶（EC 1.10.3.2）又名蓝色多铜氧化酶，可以氧化包括酚类物质、多酚类物质、苯胺、木质素、多环芳香烃甚至无机物等一系列物质，以分子氧气为电子受体，生成反应过程中唯一的副产物水。

【最新word论文】苦瓜的现代研究概况【药学专业论文】

苦瓜的现代研究概况作者：陆建锋宋新波马百平【摘要】依据国内外近30年的文献报道，对苦瓜中化学成分的来源、结构类型以及药理活性等进行了综述，为对其进一步的开发利用提供参考。

【关键词】苦瓜化学成分药理作用苦瓜（MomordicacharantiaL.）为葫芦科（Curcubitaceae）苦瓜属植物苦瓜的未成熟果实，因具有特殊苦味而得名。

苦瓜不仅有良好的食用价值，而且入药历史十分悠久。

现代大量研究表明，苦瓜的根、茎、叶、花，尤其是果实和种子具有显著的生理活性，其显著的降血糖、抗肿瘤、抗生育作用[1、2]，越来越受到人们的重视。

苦瓜广泛分布于热带、亚热带地区，在亚洲许多国家和地区均有入药记载。

1化学成分长期以来苦瓜一直作为一种蔬菜被人们食用，对苦瓜的化学成分研究始于20世纪60年代，在近20年的时间里国内外学者对其作了大量研究。

研究表明，苦瓜中含有三萜[3、4]、甾类、生物碱、蛋白、有机酸及多糖等多种化学成分。

1.1三萜类化合物苦瓜中的三萜类成分[5、6]，主要为葫芦烷型四环三萜及少量齐墩果烷型五环三萜类皂苷及其衍生物，近年更是从中分离得到乌苏烷型三萜类化合物，现已从苦瓜中分离鉴定了40余种该类化合物，见表1。

表1苦瓜中三萜类化合物1.2甾体类化合物自1965年Sucrow[2]从苦瓜果实中分离得到甾体皂苷和豆甾醇皂苷以来，国内外学者从苦瓜中分离得到多种甾体类化合物，其中包括胡萝卜甾醇[15]、β-谷甾醇[16]等，见表2。

表2苦瓜中甾体类化合物1.3生物碱类及其它成分除了以上两大类化合物之外，人们还从苦瓜中分离得到其他类别的化合物，其中包括有生物碱类、蛋白多肽类、酰胺类以及脂肪酸类化合物等，见表3。

表3苦瓜中其他类化合物2药理作用研究概况2.1降血糖作用研究证明，苦瓜皂苷对糖尿病模型兔有明显的降血糖作用，与优降糖相比其降糖作用缓慢而持久，可能有类似胰岛素的作用，已被确定为苦瓜降血糖的有效成分[18]；1974年Khanna[19]发现苦瓜中存在类胰岛素样肽(insulin-likepeptide,polypetide-P)，给正常沙鼠、猴及糖尿病患者注射有降低血糖作用，口服同样有效；ShubhasishSarkar等发现苦瓜果肉的乙醇提取物，剂量为500mg/kg，在给药1h后抑制动物血糖达10%～15%；苦瓜醇提物能显著增加正常小鼠肝糖元的含量，并改善正常小鼠的糖耐量，能显著降低链脲佐菌霉素致糖尿病模型小鼠的血糖含量，明显降低四氧嘧啶糖尿病大鼠的血糖含量；对于葡萄糖喂养的正常大鼠，苦瓜的活性低于磺胺苯比唑类药物甲磺丁脲，但实验证实了苦瓜提取物对公认的糖尿病模型具有降血糖作用[20]。

玉竹多糖降血糖作用的研究进展

Advances in Clinical Medicine 临床医学进展, 2016, 6(4), 187-190 Published Online December 2016 in Hans. /journal/acm /10.12677/acm.2016.64035文章引用: 郭珊珊, 张雪, 刘宁宁, 修鹏程. 玉竹多糖降血糖作用的研究进展[J]. 临床医学进展, 2016, 6(4): 187-190.The Research Progress of Blood Decreasing Function about Polygonatum PolysaccharidesShanshan Guo, Xue Zhang, Ningning Liu, Pengcheng XiuTaishan Medical University, Taian ShandongReceived: Nov. 6th , 2016; accepted: Dec. 13th , 2016; published: Dec. 16th, 2016Copyright © 2016 by authors and Hans Publishers Inc. This work is licensed under the Creative Commons Attribution International License (CC BY)./licenses/by/4.0/AbstractPolygonatum polysaccharide is the highest ingredient in jade bamboo. The study shows that it has obvious function of regulating the blood sugar level. In this thesis, the active ingredients of polygonatum polysaccharides, the basic composition and extraction of polygonatum polysac-charides and hypoglycemic mechanism of polygonatum polysaccharides will be elaborated as follows. KeywordsPolygonatum Polysaccharides, Blood Decreasing, Basic Composition, Mechanism玉竹多糖降血糖作用的研究进展郭珊珊，张雪，刘宁宁，修鹏程泰山医学院，山东泰安收稿日期：2016年11月6日；录用日期：2016年12月13日；发布日期：2016年12月16日摘要玉竹多糖是玉竹中含量最高的成分，经研究表明，玉竹多糖具有明显的降血糖作用。

艾塞那肽长效缓释微球的制备及体内药效学研究

艾塞那肽长效缓释微球的制备及体内药效学研究刘斌1,2，董庆光1，王梦舒1，李纯1，孔维1,2，陈妍1*(1.吉林大学生命科学学院，长春130012；2.吉林大学超分子结构与材料国家重点实验室，长春130012.)摘要：目的制备艾塞那肽长效缓释微球，并考察其理化性质、体外释放特性及体内药效学。

方法采用复乳－溶剂挥发法（W/O/W）制备包裹艾塞那肽的聚乳酸－羟基乙酸嵌段共聚物（PLGA）微球；考察微球的粒径分布、表面形态、载药量和包封率；用micro－BCA试剂盒测定微球的体外释放速率；考察了微球在糖尿病模型小鼠体内的降血糖作用。

结果微球外观光滑圆整，分散性好，艾塞那肽载药量为3％，包封率为75％；微球40d之内体外累积释放达到88％，其体外释放曲线近似零级释放模式；体内药效学实验结果显示，40d内微球组小鼠与模型对照组小鼠血糖相比具有显著性差异（P<0.05）。

结论采用复乳－溶剂挥发法成功制备了可以缓释40d的艾塞那肽长效微球，且40d之内在糖尿病小鼠体内具有明显的降血糖作用。

关键词：艾塞那肽；聚乳酸－羟基乙酸嵌段共聚物；微球；血糖中图文类号：文献标识码：文章编号：Preparation of Long-acting Release Microspheres of Exenatide and Studies on Their in vivo PharmacodynamicsLIU Bin1,2，DONG Qing-Guang1，WANG Meng-Shu1，LI Chun1，KONG Wei1,2，CHEN Yan1*（1. College of Life Science, Jilin University, Changchun 130012, China； 2. State Key Laboratory of Supramolecular Structure & Materials, Jilin University, Changchun 130012.,China.）ABSTRACT: OBJECTIVE To prapere Exenatide loaded long-acting release microspheres, and to evaluate their physical and chemical characteristics、in vitro release behavior as well as in vivo pharmacodynamics.METHOD Exenatide loaded PLGA microspheres were prepared by double emulsion(W/O/W)method. The mean diameter、morphology、drug loading and encapsulation efficiency were evaluated. The micro-BCA protein assay was used for the in vitro release behavior. The effect of reducing plasma glucose were evaluated on the diabetes mice. RESULTS The microspheres possessed smooth and round appearance as well as good dispersive quality. The drug loading and encapsulation efficiency was 3% and 75%. The in vitro accumulated release within 40d reached up to 88%, and the release profile is consistent with zero-class release model. The results of in vivo pharmacodynamics indicated that the plasma glucose of mice injected with Exenatide microspheres was different significantly from the plasma glucose of the diabetes model mice within 40d (P<0.05). CONCLUSION The Exenatide loaded long-acting release microspheres which could yield 40d continuous release were preparedsuccessfully by double emulsion method. The microspheres displayed significant appearance of decreasing plasma glucose in the diabetes mice within 40d.KEY WORDS:Exenatide；poly ( lactide-co-glycolic acid)；microspheres；plasma glucose 艾塞那肽（Exenatide）是Exendin-4的人工合成物，Exendin-4是美国西南部的大毒蜥在进食时所分泌唾液中的一种含有39 个氨基酸的多肽，分子式C184H282N50O60S，分子量4186.57[1]。

2007_Isolation and characterization of antibacterial substances

Chiaki Imada1Ã, Naoko Koseki1, Masazumi Kamata2, Takeshi Kobayashi1, and Naoko Hamada-Sato1
1Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7, Konan, Minato-ku, Tokyo 108-8477, Japan
Table 1. Location of sampling stations in Otsuchi Bay
6
Station Latitude (N) Longitude (E) Depth (m)
1
39 22.0780
142 00.6640
100.0
5
2
39 21.9000
141 59.6260
INTRODUCTION
Natural organic compounds produced by microorganisms are an important screening target for a variety of bioactive substances. Compounds of actinomycetal origin, in particular, have been valuable in the ﬁeld of bioactives. However, the rate of discovery of novel substances from microorganisms, especially from actinomycetes of terrestrial origin, has decreased recently. Most actinomycetes were believed to terrestrial; however, some strains have also been found in marine environments, though no marine actinomycetes with special characteristics have been isolated. In 2002, Mincer et al.1) isolated actinomycetes from a marine environment, requiring seawater for growth, and these strains were designated marine actinomycetes.

BN-PAGE实验操作步骤

Supplemental MaterialAppendix S1. Experimental proceduresPigment analysis5Prior to pigment extraction, each sample was weighed. Seedlings were frozen in liquid nitrogen and ground to powder with a high-speed mixer mill (model MM301, Retsch,). Total chlorophyll and carotenoid were extracted from 30 seedlings with 100% acetone. The debris was removed by centrifugation at 10,000 rpm for 10 min.Absorbances of the supernatant were measured with a Beckman DU®650 spectrophotometer10(), and the contents of the pigments were determinedaccording to Lichtenthaler and Wellburn (1983).Anthocyanin extraction and quantification was performed as described previously (Kim et al., 2003). In brief, anthocyanin was extracted from 40 seedlings with 300 µL of acidified (1% HCl) methanol overnight at 4°C in dark conditions. The extract was separated by the15addition of 200 µL of water and 200 µL of chloroform, followed by centrifugation for 5 min at 3,000 rpm. The absorbance of the upper phase was determined spectrophotometrically at 530 nm and 657 nm, and the anthocyanin content was calculated as A530− 0.33A657.For Pchlide analysis, wild-type and heterozygous atterC-1 seeds were sown onto MS medium and grown in the dark for 4 d at 22°C. At the end of the dark treatment, plates were 20exposed to light for 20 h to select the pigment-deficient homozygous atterC-1 seedlings. The selected mutant seedlings were then transferred to fresh MS medium on a clean bench. The wild-type and mutant seedlings were kept in darkness again for an additional two days.Seedlings were harvested under a dim-green light, and Pchlide extraction was performed asdescribed previously (Terry and Kendrick, 1999). Seedlings were homogenized as described above. Extracts were obtained from 80 seedlings. After extraction with 0.5 mL of coldacetone (0.1 M NH4OH (90/10, v/v)), samples were centrifuged at 30,000 g for 10 min. The pellet was then re-extracted in 150 µL solvent and centrifuged again. The supernatants werecombined and washed twice with an equal volume and a one-third volume of hexane prior to5spectrophotometric analysis. Fluorescence spectroscopy was performed using a Hitachi F-2000 (). Fluorescence was measured using an excitation/emissionwavelength of 440/637 nm for Pchlide. All pigment measurements were repeated in threeindependent experiments.10Protein extraction and immunoblot analysisProtein extraction was performed as described by Frick et al. (2003), except that the concentration of SDS was changed to 0.5%. Protein extracts were obtained from frozen seedlings ground in liquid nitrogen and thawed in homogenization buffer (0.0625 M Tris-HCl, pH 6.8; 0.5% [w/v] SDS; 10% [v/v] glycerol; 0.5% [v/v] β-mercaptoethanol) at 95°C for 10 min.15After centrifugation at 16,000 g for 10 min, supernatant protein concentrations were determined with Bradford reagent (USB, ) using BSA as a standard, then separated by 10% SDS-PAGE. The proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, ) by electroblotting for 1 h at 90 V in 25 mM Tris, 0.2 M Glycine, and 20% (w/v) methanol. Blocking and immunodecorations were 20performed in Tris-buffered saline containing 5% nonfat dry milk. Protein gel blots were developed using a WEST-ZOL® plus western blot detection system (iNtRON). Antisera used in the immunoblot assay were as follows: Anti-D1 (Cat. no. AS06124A, diluted 1:2000), anti-PasA (Cat. no. AS03025, diluted 1:5000), anti-Cyt f (Cat. no. AS06119, diluted 1:5000), anti-AtpB (Cat. no. AS05085, diluted 1:2000), anti-rbcL (Cat. no. AS03037, diluted 1:10000), anti-Tic40 (Cat. no. AS06149, diluted 1:5000) antibodies were purchased from AgriSera(). Antibodies used for the immunological detection of CAB (Cat. no.sc-12691, diluted 1:2000) and GFP (Cat. no. sc-9996, diluted 1:1000) were purchased fromSanta Cruz Biotechnology (). The antiserum against Arabidopsis POR 5was kindly provided by G. Armstrong (Ohio State University, Columbus, OH). Anti-actinantibody (Cat. no. MA1-744, Affinity BioReagents, ) was used toconfirm equal loading of proteins.Polysome analysis10Polysomes were isolated as described previously by Barkan (1998). Frozen seedlings(0.2 g) were ground in liquid nitrogen,and 1 mL of polysome extraction buffer (0.2 M Tris-HCl,pH 9.0,0.2 M KCl, 35 mM MgCl2, 25 mM EGTA, 0.2 M sucrose, 1% [w/v] Triton X-100, 2%[w/v] polyoxyethylene-10-tridecy ether, 100 mMß-mercaptoethanol, 0.5 mg/mL heparin, 100µg/mL chloramphenicol, and 25 µg/mL cycloheximide) was added. Sample was ground 15further until thawed and placed on ice for 10 min. Nuclei and insoluble materials were pelletedby centrifugation at 13,000 rpm for 5 min at 4°C. Sodium deoxycholate was added to thesupernatant to a final concentration of 0.5% (w/v), and the sample was placed on ice for 5 min.Remaining insoluble material was subsequently pelleted by centrifugation at 13,000 rpm for 15min at 4°C. The supernatant (0.5 mL) was layered onto 4-ml sucrose gradients and centrifuged 20with a Beckman SW 55 swing rotor at 45,000 rpm for 65 min at 4°C. Four hundred and fiftymicroliters of each fraction was collected by gentle pipetting from the top of the gradient andadded to 50 µL of a solution consisting of 5% SDS and 0.2 M EDTA, pH 8.0. RNA waspurified from each fraction by phenol:chloroform:isoamyl alcohol (25:24:1) extraction andsubsequent ethanol precipitation. Finally, RNA samples were resuspended in 30 µL of TE buffer (10 mM Tris-HCl, pH8.0, and 1 mM EDTA), and 5 µL of each fraction was examined by RNA gel blot analysis.Chloroplast isolation and subfraction5Intact chloroplasts were isolated as previously described (Barneche et al., 2006) with little modification. Leaves were homogenized in ice-cold CIB buffer (0.45 M sorbitol, 50 mM Hepes-KOH pH 7.8, 2 mM EDTA, 0.1% BSA, 2.5 mM MgCl2, Protease Inhibitor Cocktail) using a mortar and pestle. The extract was filtered through two layers of Miracloth, debris were eliminated by centrifugation for 5 s at 1000 g, and the material was concentrated by 10centrifugation for 7 min at 1000 g. The pellet was resuspended in 1 mL of CIB and then layered on a discontinuous 40% (6 mL) / 80% (10 mL) Percoll gradient. After centrifugation for 15 min at 7000 g intact chloroplasts were isolated at the interface of the two layers.Chloroplasts isolated after two Percoll gradients were washed twice in HMS buffer (0.33 M Sorbitol, 50 mM Hepes-KOH pH 7.8, 2.5 mM MgCl2and protease inhibitor cocktail) with 15centrifugation for 5 min at 1000 g. The chloroplasts were broken by osmotic lysis in 1 mL of lysis buffer (10 mM Hepes-KOH pH 7.8, 4 mM MgCl2, 1 mM PMSF, Protease Inhibitor Cocktail) with vortex, and incubated on ice for 10 minPreparation of chloroplast subfractions was carried out as previously described (Robinson and Barnett, 1988). After addition of 150 µL of 80% (w/v) sucrose to the 1-mL 20lysed chloroplast, the mixture was layered on a discontinuous sucrose gradient of 0.98 M and0.6 M sucrose, buffered in each case with 10 mM Hepes-KOH pH 7.8, 4 mM MgCl2, and thencentrifuged with a Beckman SW 41 swing rotor at 90,000 g for 2 h. The stroma was obtained from the top fraction over 0.6 M sucrose. The envelope was collected at the interface betweenthe layers of 0.6 M and 0.98 M sucrose and thylakoids as a pellet. The stroma fraction wasprecipitated with 10% trichloroacetic acid. In order to remove most of the soluble stromalproteins sequestrated in the envelope vesicles, envelope membrane preparations were furthertreated by sonication as previously described (Miras et al., 2002). The collected envelopefractions were diluted with 3 vols of lysis buffer containing 0.5 M NaCl and incubated for 30 5min at 4°C. Then, the mixture was sonicated for 10 s and stored for 15 min on ice beforecentrifugation with a Beckman SW 55 swing rotor at 72,000 g for 35 min at 4°C, and envelopeproteins recovered in the pellet.Oligonucleotide sequences and cDNA fragments10Mutant identification: P1 (5′-CTTGGTGGA TTAAG GAGACTTTTCA-3′), P2 (5′-ACCGCAGTGCCAACCTATTGA-3′), LBa1 (5′-TGGTTCACGTAGTGGGCCATCG-3′), L1(5′-GTCTACA TTCACGTCCAAATGGGGGC-3′), and R1 (5′-GTTCCAACCACGTCTTCAAAGCAAGTG-3′). RNA gel blot and polysome analysis: NB-P1 (357-bp 5′ codingregion of AtTerC, NM_121251), 5′-ATGAGCTTAGCTTCAGTTA TCCACCACGGA-3′, 5′-15AGTCTTGAAGGAAGTTTTGTAAGTTTCTTCTTG-3′; NB-P2 (1155-bp full-length cDNAregion of AtTerC), 5′-ATGAGCTTAGCTTCAGTTA TCCACCACGGA-3′,5′-TTAGCTGTCGCT GGA TTTGTTTGTTAGGCTC-3′; NB-P3 (329-bp 3′ UTR region ofAtTerC), 5′-CTAAGAACTGAGAGCTGACTACTGAGAAAC-3′, 5′-CCAAAGGAATAA TCTTATAAGA TGAGC-3′; psbA(X79898), 5′-20ATGACTGCAA TTTTAGAGAGACGCGAAAG-3′, 5′-TTA TCCA TTTGTAGA TGGAGCCTCAACAG-3′; rbcL(U91966), 5′-ATGTCACCACAAACAGAGACTAAAGCAAG-3′, 5′-CTACTCTTGGCCATCTAATTTA TCGA TGG-3′; rbcS(NM_202369), 5′-CACCGGTTAA TTTCCCTTTGCTTTTGTG-3′, 5′-GTGAAACTAA TTTTCTTAATGA TTTCTTTAGCGAC-3′;psaA(A TCG00350), 5′-ATGA TTA TTCGTTCGCCGGAACCAG-3′, 5′-GCTGCTTTGTGA TAA TGGAACCAACCA-3′;petA(A TCG00540), 5′-ACCTTTTCTTGGATAAGGGAAGAGATTAC-3′, 5′-ACATCTTTA TTAGTAGCAGGGTCTGGAGC-3′; atpB(A TCG00480), 5′-TCCTACTAC 5TTCAAATCCA GAGGTTTCGA-3′, 5′-CACCAAATACGGATACACCACCATGAGCT-3′DFR (AJ251982), 5′-TTGAAGGTGTTGATGAGAATCTAAAGAGCA-3′, 5′-ACCGTTACAATCACACGCGATACAAAATC-3′. For CHS(M20308), a 1.3-kb Hind IIIfragment of pCHS3.9 (Feinbaum and Ausubel, 1988) was used. For CAB (AY045673), a 0.5-kb Bam HI/Bgl II fragment of the RAFL 08-09-P03 clone obtained from RIKEN BRC was used.10At18S ribosomal RNA (X16077) 5′-GGTTGA TCCTGCCAGTAGTCA TA TGC-3′ and 5′-GCCCTCCAATGGATCCTCGTTAAGG-3′. Complementation analysis: (5′-GCAGTCGACGATGAGGAACGAGAGGA-3′and 5′-GCACTGCAGGAAAACATCAGATGTTCACCT-3′). DNA gel blot analysis: 5′-GAGACCTGATTTCATAGTTATCTCAATAGG-3′ and 5′-15GTTACCGCAAATGCTATGTCACTGAG-3′. For AtTerC coding region of 35S::AtTerC:GFP:5′-ACTAGTATGAGCTTAGCTTCAGTTATCCACCAC-3′ and 5′-ACTAGTGCTGTCGCTGGATTTGTTTGTTAGG-3′Barkan, A.(1998) Approaches to investigating nuclear genes that function in chloroplast 20biogenesis in land plants. Methods Enzymol.297, 38-57.Barneche, F., Winter, V., Crèvecœur, M. and Rochaix, J.D. (2006) ATAB2 is a novel factor inthe signalling pathway of light-controlled synthesis of photosystem proteins. EMBO J.25,5907-5918.Feinbaum, R.L. and Ausubel, F.M. (1988) Transcriptional regulation of the Arabidopsisthaliana chalcone synthase gene. Mol. Cell. Biol.8, 1985-1992.Frick, G., Su, Q., Apel, K. and Armstrong, G.A. (2003) An Arabidopsis porB porC double mutant lacking light-dependent NADPH:protochlorophyllide oxidoreductases B and C is highly chlorophyll-deficient and developmentally arrested. Plant J.35, 141-153.5Kim, J., Yi, H., Choi, G., Shin, B., Song, P.S. and Choi, G. (2003) Functionalcharacterization of phytochrome interacting factor 3 in phytochrome-mediated light signaltransduction. Plant Cell, 15, 2399-2407.Lichtenthaler, H.K. and Wellburn, A.R. (1983) Determination of total carotenoids andchlorophylls a and b of leaf extracts in different solvents. Biochem. Soc. Trans.603, 591-592. 10Miras, S., Salvi, D., Ferro, M., Grunwald, D., Garin, J., Joyard, J. and Rolland, N. (2002) Non-canonical transit peptide for import into the chloroplast. J. Biol. Chem.277, 47770-47778.Robinson, C. and Barnett, L.K. (1988) Isolation and characterization of chloroplasts. In CH Shaw, ed, Plant Molecular Biology: A Practical Approach. IRI Press, Oxford, pp 67-78.Terry, M.J. and Kendrick, R.E. (1999) Feedback inhibition of chlorophyll synthesis in the15phytochrome chromophore-deficient aurea and yellow-green-2 mutants of tomato. PlantPhysiol.119, 143-152.。

骨髓间充质干细胞的主要表面标志

骨髓间充质干细胞的主要表面标志1 骨髓间充质干细胞的发现和来源骨髓组织中有多种细胞成分，除基质细胞等已经分化的细胞外，还含有两类多潜能干细胞：造血干细胞和间充质干细胞。

1987 年Friedenstein 等发现在塑料培养皿中培养的贴壁的骨髓单个细胞在一定条件下可分化为多种类型的细胞，而且经过20－30个培养周期仍能保持其多向分化潜能。

由于骨髓中的这种多能细胞能够分化为多种中胚层来源的间质细胞, 故称之为间充质干细胞(Mesenchymal stem cells，MSCs),或间质祖细胞(MPCs)，是成人多能干细胞的一类。

早期分离培养时，发现其形状呈成纤维细胞样而称其为成纤维细胞集落形成单位(Colony-forming unit-fibroblast，CFU-F)，或骨髓基质成纤维细胞(Marrow stromal fibroblast，MSF)。

Friedenstein AJ , Chailakhyan RK, Gerasimov UV. Bone marrow o steogenic stem cells: in vit ro cult ivat ion and t ransp lantat ion in diffusion chambers. Cell T issue Kinet, 1987, 20 (3) : 263-267]2 鉴于其强大的增殖能力及多向分化潜能，可在体外长期培养和遗传背景较稳定，而且用自体干细胞诱导构建的组织不涉及伦理问题，也不存在MHC限制，所以骨髓间充质干细胞日益受到重视。

但是与造血干细胞等其他细胞相比，骨髓中MSCs的数量非常少，约占整个骨髓有核细胞的十万分之一，并随年龄的增加，细胞数量逐渐减少。

因此，如何简便有效地从骨髓中获取高纯度的MSCs显得尤为重要，寻找高度特异性的MSCs的表面抗原也就成为MSCs研究中的一项重要任务和目标。

不仅如此，一种同样来源于骨髓、贴壁生长、被认为更原始（可以分化为MSCs）也具有更强增殖能力的干细胞也被鉴定，它就是多能成体祖细胞（multipotent adult progenitor cell (MAPC) or mesodermal progenitor cell(MPC)）[Reyes, M., Lund, T., Leuvik, T., Aguiar, D., Koodie, L., Verfaillie,C.M. (2001) Purification and in vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 98, 2615-2625]，因能和MSCs一起被纯化而统称BM stromal stem cell。

2016年度广东省科学技术奖项目公示

在多糖制备关键技术产业化应用、多糖质量控制、复合多糖配伍研究方面均做出了重要贡献。其对创新点4、5作出了创造性贡献，是代表性论文2的主要作者之一，是知识产权明细5、9、10的主要发明人之一。
14、黄生权，工程师，无限极（中国）有限公司
在中草药活性多糖制备关键技术的产业化应用方面做出了重要贡献，主要体现在利用真空带式干燥技术解决了传统中草药多糖产品干燥过程中存在的问题。其对创新点3做出了创造性贡献，其代表性论文见附件“其他代表性论文目录”，其专利情况见附件“其它知识产权明细目录”
Ultrasound-assited extraction and structural identification of polysaccharides from Isodon lophanthoides var. gerardianus (Bentham) H. Hara/ Carbohydrate Polymers/温玲蓉、林恋竹、游丽君、杨宝、蒋跃明、赵谋明
1、主要技术内容
（1）联合运用HPLC、GPC、GC、IR、MS等分析技术明确了单糖组成、单糖链接方式、分子量范围及空间构象等多糖化学结构信息，鉴定出香菇多糖MPSSS、灰树花多糖GFPBW1、枸杞多糖LBPF4-OL、黑果枸杞多糖LbGp1等100多种中草药多糖结构，建立了中草药多糖库。
（2）建立了基于石英晶体微天平技术（QCM）的中草药多糖高通量快速筛选平台，获得了结构明确、作用机制清晰、功效显著的系列中草药活性多糖；首次揭示香菇、灰树花、天麻多糖可以借助Clathrin和Rab5蛋白实现跨膜转运从而被小肠上皮细胞吸收，并证明多糖可以通过影响肠道粘膜免疫系统发挥作用；并在细胞及动物整体水平验证了活性多糖的免疫调节、抗肿瘤等活性，为中草药活性多糖应用于保健食品提供了科学依据。

Isolation and Structural Characterization of a Novel Polysaccharide

J OURNAL OF B I O SCI E NCE AND B I O ENGI N E E RING © 2007, The Society for Biotechnology, Japan V ol. 104, No. 2, 111–116. 2007DOI: 10.1263/jbb.104.111Isolation and Structural Characterization of a Novel PolysaccharidePrepared from Arca subcrenata LischkeYunmian He,1 Chunhui Liu, 2 Yuxing Chen, 3 Ancheng Ji,3Zilong Shen,1 Tao Xi,1 and Quansheng Yao3*School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, P.R. China, 1 Institute of Biochemical and Biotechnological Drugs, School of Pharmaceutical Sciences, Shandong University,Jinan 250012, P.R. China,2 and the Drug Safety Evaluation Center of Jiangsu Province,Nanjing 210009, P.R. China 3Received 7 March 2007/Accepted 12 May 2007A water-soluble polysaccharide was isolated from Arca subcrenata Lischke (named ASLP) byhot-water extraction, anion-exchange, and gel-permeation chromatography. The average molecu-lar weight of ASLP was estimated to be 3500 Da. The structural characterization of ASLP wasperformed by sugar composition analysis, methylation analysis, and partial acidic hydrolysis.Further analysis of ASLP was carried out by UV, FT-IR and NMR spectroscopies (1D, COSY, andHSQC, respectively). Our data suggests that ASLP is an α-(1→4)-D-glucan, with an α-(1 →6)-D-glucan at the C-6 position every fourth residue along the main chain. The branch chain has threeglucose residues. The possible structure, determined on the basis of structural analyses results,was also determined. Preliminary in vitro tests revealed that ASLP can stimulate mouse spleenlymphocyte proliferation and its branches are extremely important for its immunological activity.[Key words: Arca subcrenata Lischke, polysaccharide, gas liquid chromatograph/mass spectrometry, nuclearmagnetic resonance, structural characterization, spleen lymphocyte proliferation]In recent years, various polysaccharides and polysaccha-ride complexes have been isolated from marine life organ-isms and characterized. Some polysaccharides, such as those from marine green and brown algae were evaluated for in potential anticoagulant activities (1, 2). Some sulfated poly-saccharides extracted from marine sponges may function in species-specific aggregation of sponge cells or in structural integrity (3). Various of polysaccharides from edible sea-weeds were identified as important bioactive natural prod-ucts, possessing many important properties of pharmaco-logical relevance (4). Moreover, many polysaccharides from marine microorganisms have been reported, such as the marine thermotolerant Bacillus licheniformis, marine fila-mentous fungus Keissleriella sp., and marine bacteria Pseu-doalteromonas carrageenovora and Shewanella (5–8). Arca subcrenata Lischke, an Arcidae, is a marine inverte-brate. It is a popular seafood in China. A Chinese traditional medicine, wa leng zi (Concha Arcae), is extracted from A. subcrenata Lischke. In this paper, we report the character-ization of A. subcrenata Lischke polysaccharide (ASLP) by a series of chemical and instrumental analyses. In vitro ASLP showed significant immunological activity in spleen lymphocyte proliferation assay.MATERIALS AND METHODSSample collection and general methods A. subcrenata Lischke was collected from Huanghai Sea, China and transported to our laboratory packed in ice. A specimen of this marine animal is deposited in the Drug Safety Evaluation Center of Jiangsu Province, P.R. China.The specific rotation was determined at 20 ±1°C using an auto-matic polarimeter (model WZZ-2B; Shanghai Shengguang Instru-ment, P.R. China). UV-Vis absorption spectra were recorded using a Perkin-Elmer Lambda 2 spectrometer. The FT-IR spectra (KBr pellets) were recorded on a Nicolet 360 FTIR spectrophotometer. Elemental analysis (C, H and N) was conducted on an Elementar Vario EL III instrument. Total carbohydrate content was deter-mined by the phenol-sulfuric acid method as D-glucose equivalents (9). Uronic acid content was determined by an m-hydroxydiphenyl colorimetric method in which neutral sugars do not interfere (10).Extraction and fractionation of ASLP A. subcrenata Lischke (30 kg) was shelled, homogenized, and treated with acetone to re-move fats and pigments (1:1, 10 l ×6). After centrifugation (6000 rpm, 20 min) and overnight drying, the resulting pellets were kept in distilled water at 80°C for 8 h with constant stirring. The process was repeated three times. The supernatant was concentrated and precipitated in 4 volumes of ethanol. The precipitate collected by centrifugation was suspended in distilled water and protein was re-moved by the Sevag method (11). Then the crude polysaccharide fraction was obtained by precipitation in 4 volumes of ethanol and washed with acetone and ethyl ether several times. Ion exchange chromatography was performed on a column (3.5 ×30 cm) of DEAE52 (Whatman, Brentford, Middlesex, UK). Crude polysac-charide (200mg) was loaded onto the column each time. Then the column was eluted with distilled water, followed by 0 to 2mol/l* Corresponding author. e-mail: yaoqs2007@phone/fax: +86-25-83285226111HE ET AL. J. B I OS C I. B IOENG., 112linear gradient of sodium chloride at a flow rate of 1 ml/min. The yielded fractions were combined according to the total carbohy-drate content quantified by the phenol-sulfuric acid method at 490 nm (9). The fraction corresponding to major sugar peak was fur-ther chromatographed on a column (1.6 ×80 cm) of Sephadex-G50 (Pharmacia, Peapack, NJ, USA) with water at a flow rate of 0.2 ml/min. The main fraction was collected, dialyzed, and lyophilized for further investigation.Homogeneity and molecular weight Homogeneity and aver-age molecular weight were measured by high-performance gel-permeation chromatography (HPGPC) on a Waters instrument, us-ing a Waters 2414 refractive index detector (RID). The standard dextrans (the molecular weights were 270,000 Da; 133,800 Da; 84,000 Da; 50,000 Da; 21,400 Da; 4600 Da; and 2500 Da) were passed through a TSK-GEL G4000PWXL column (7.8 ×300 mm; Tosoh, Tokyo), and then the retention times were plotted against the logarithms of their corresponding average molecular weights.A sample solution (20 µl, 5 mg/ml) was injected in each run and eluted with 0.1% NaN3 water solution at a flow rate of 0.5 ml/min. The retention time of ASLP was then plotted in the same graph, and the average molecular weight of ASLP was determined.Composition analysis ASLP (10 mg) was dissolved in 2 ml of 2 M trifluoroacetic acid (TFA) and hydrolyzed at 120 °C for 2 h. After reduction with 20 mg of sodium borohydride, monosaccha-ride alditol acetate was prepared using a method described previ-ously (12). The prepared alditol acetates were analyzed by gas-liquid chromatography using a Hewlett-Packard model 6890 in-strument equipped with an HP-5 5% phenylmethyl siloxane column (30 m ×0.25 mm) and a flame-ionization detector. The temperature program was set to increase from 150 °C to 220°C at 2°C/min and from 220 °C to 280°C at 30°C/min with nitrogen as carrier gas. Peaks were identified and estimated using myo-inositol as the in-ternal standard. The quantity of fractions was determined from the peak area, using response factors.Methylation analysis ASLP (10 mg) in 10 ml of dimethyl sulfoxide was methylated using sodium hydroxide and iodo-methane three times in accordance with the method of Needs and Selvendran (13). The methylated polysaccharide was examined by IR spectroscopy. The absence of the absorption peak correspond-ing to hydroxyl indicated the complete methylation. The fully me-thylated product was depolymerized with 90% methanoic acid at 100°C for 6 h and converted into partially methylated alditol ace-tates by hydrolysis with TFA, reduction with sodium borohydride, and acetylation with acetic anhydride. The resulting product was subjected to linkage analysis by GLC-MS. Agilent 5975 inert-GLC/MSD was used for mass-spectral identification of GLC com-ponents. GLC-MS was performed using a DB-5 capillary column (30 m ×0.32 mm i.d.) with a film thickness of 0.25 µm. The GLC temperature program was isothermal at 140 °C for 2 min, followed by a 4 °C/min gradient up to 250 °C. The components were identi-fied by a combination of the main fragments in MS and relative re-tention times in GLC, and the molar ratios were estimated from peak areas and response factors (14, 15).Partial acidic hydrolysis ASLP (40.0 mg) was dissolved in 3 ml of 0.3 M TFA for 18 h at 100 °C. The solution was neutralized with sodium hydroxide and applied to a Sephadex-G10 desalting column (1 ×25 cm). The main chain was obtained (named H-ASLP) and a freeze-dried sample analyzed by 1D NMR (16).Nuclear magnetic resonance spectroscopy ASLP (30 mg) was kept over dry phosphorus oxide in vacuum for 2 d and then dissolved in 0.5 ml of 99% D2O. Spectra were recorded at 300 K on a Bruker AV-500 spectrometer operating at 500 MHz for 1H and 125 MHz for 13C. Chemical shift references are given in ppm, with DSS as an internal chemical shift reference. In addition to 1D spectra, H-H COSY and HSQC spectra were obtained. A H-H COSY experiment was performed using the Bruker standard pro-gram with 2 s acquisition time and 2 K data points in the F2dimen-sion. The data matrix was zero-filled in the F1 dimension to obtain a matrix of 1 K ×1 K points and resolution was enhanced in both dimensions by a shifted sine-bell function before Fourier transfor-mation. A nuclear Overhauser experiment was performed using the Bruker standard program, with a mixing time of 1 s. A hetero-nuclear experiment was performed using the pulse field gradient program HSQC.Spleen lymphocyte proliferation assay in vitro Male K un-ming mice (8 weeks old) were purchased from the Experimental Animal Center of China Pharmaceutical University. All mice were kept at the animal facilities under pathogen-free conditions until use. Sterile food and water were supplied.The mouse spleen cells were obtained by gently teasing the organ in RPMI-1640 medium supplemented with penicillin (100 IU/ml), streptomycin (50 µg/ml), and 10% newborn-bovine serum. Cells (6 ×106 cells/ml) were seeded on a 96-well plate (100 µl/well). ASLP (100 µl) in RPMI-1640 medium (100 µg/ml, 200 µg/ml, and 400 µg/ml) and 100 µl of H-ASLP (100 µg/ml, 200 µg/ml, and 400µg/ml) were added to the test groups. Concanavalin A (ConA, 100µl, 5 µg/ml) was added to the positive control group and 100 µl of RPMI-1640 medium was added to the negative control.The cells were cultured at 37 °C, in 5% CO2for 44 h, and further incubated for 4 h with 20 µl of 3-(4,5-dimethylthiazolyl-2)-2,5-di-phenyl tetrazolium bromide per well (MTT: 5 mg/ml, Sigma). Acidified isopropylalcohol (100 µl) was added to the culture and homogenized for at least 10 min to fully dissolve the stained mate-rial. The absorbance at 570 nm was measured using an ELISA reader (Thermo Multiskan Spectrum) (17).Statistical analysis GraphPad Instat ver. 3.05 (GraphPad Software, San Diego, CA, USA) was used to analyze the results. Data are presented as mean ±SD. Data were subjected to one-way analysis of variance (ANOV A), followed by a Bonferroni t test. A value of P<0.05 was considered to indicate a statistically signifi-cant difference.RESULTS AND DISCUSSION Preparation and physicochemical characteristics of the polysaccharide The water extract of A. subcrenata Lischke was precipitated in 80% ethanol, deproteinized by the Sevag method, washed with ethyl ether and acetone several times, and then dried in vacuum to obtain 9.32 g of crude polysaccharide. The obtained polysaccharide was suc-cessively loaded on the DEAE52 column (ion exchange chromatography). The major sugar fraction (P-1), corre-sponding to the main peak, eluted by water was collected and concentrated (Fig. 1). It was further chromato graphed on the Sephadex-G50 column (gel-permeation chro matog-raphy). Two completely separate fractions were obtained (Fig. 2). The main fraction was collected, dialyzed and lyo-philized to obtain 837 mg of white Arca subcrenata Lischke polysaccharide (name ASLP), which resulted in the com-plete isolation of the polysaccharide ASLP with a recovery of 9.0% relative to the crude A. subcrenata Lischke poly-saccharide (Fig. 3). The minor fractions (ASLP-1) obtained were not investigated further.The HPGPC profiles (Fig. 4) showed a single and sym-metrical peak, indicating that ASLP was a homogeneous polysaccharide with an average molecular weight of 3500 Da. After freeze-drying, ASLP appeared as a white powder and its total sugar content was found to be 99.6% as deter-mined by the phenol-sulfuric acid method. It had a negativePOLYSACCHARIDE PREPARED FROM A. SUBCRENATA LISCHKE V OL . 104, 2007 113response in the Bradford test and showed no absorption at 280 nm or 260 nm in the UV spectrum, indicating the ab-sence of protein and nucleic acid. Elemental analysis re-vealed the following: C, 41.21; H, 6.53; and N, 0.00; indi-cating it was a neutral polysaccharide. The relatively high positive value of optical rotation, +250° (c 0.2, H 2O),suggests the predominance of α-form glycosidic linkages and the D-configuration of the glucosyl residues in ASLP (18). A negative result of m -hydroxydiphenyl colorimetric determination suggests that no uronic acid was present in ASLP, which was in agreement with the conclusion that ASLP was not adsorbed onto DEAE52 column and could be eluted by water. The quantitative determination of the neu-tral monosaccharide component pattern by GC indicated that ASLP only comprises glucoses, suggesting that ASLP is a glucan. The physicochemical characteristics of ASLP are shown in Table 1.Structural features of the polysaccharide In the FT-IR spectrum of ASLP (Fig. 5), the band in the region of3413.74 cm –1corresponds to the hydroxyl stretching vibra-tion of the polysaccharide and that at 2931.41 cm –1 cor-responds to a weak C-H stretching vibration. The band inthe region of 1636.04 cm –1corresponds to associated water.A characteristic absorption at 843.24 cm –1 and 922.52 cm –1was also observed, indicating the α-configuration of thesugar units. There was no absorption at 890 cm –1for theβ-configuration. The absorption at 1022.87 cm –1, 1078.10cm –1 and 1152.22cm –1indicated α-pyranose of the glucose FIG. 1. DEAE52 elution profile of polysaccharides from Arca subcrenata Lischke . The column was eluted with distilled water, fol-lowed by 0 to 2 mol/l linear gradient of sodium chloride at a flow rate of 1 ml/min.FIG. 2. Carbohydrate profile on Sephadex-G50. The column waseluted with distilled water at a flow rate of 0.2ml/min.α[]D20FIG. 3. Summarized extraction scheme of ASLP from Arca sub-crenata Lischke .FIG. 4. Purity identification of ASLP by H PGPC with a TSK-GEL G4000PWXL column.TABLE 1. Physicochemical characteristics of ASLP ItemTotal sugar (wt%) 99.6Glc (mol%) 100Uronic acid a(wt%)0+250Carbon (wt%) 41.21Hydrogen (wt%) 6.53Nitrogen (wt%) 0Protein b(wt%) 0Mr c(Da)3500aDetermined by m -hydroxydiphenyl colorimetric method.bIndicated by the absence of absorbance at 280nm.cMolecular weight determined by HPGPC.α[]D20HE ET AL. J. B I OS C I . B IOENG .,114residue (18–20).Methylation analysis of ASLP by GLC-MS revealed four types of glucose derivative at a relative molar ratio of 1.0:2.8:2.1:1.1 corresponding to the peak areas (Table 2).The identification of 2,3-di-O -methylglucitol acetate indi-cated that ASLP is a branched glucan. The 2,3,4,6-tetra-O -methylglucitol acetate has terminal nonreducing glucose,whereas 2,3,4-tri-O -methylglucitol acetate has (1 →6)-glu-cosyl residues and 2,3,6-tri-O -methylglucitol acetate has (1→4)-glucosyl residues. The 13C NMR spectrum (Fig. 6A )of H-ASLP showed signals at δ78.7 ppm and δ63.5 ppm assigned to the substitution of C-4; therefore the backbone chain of ASLP is the 1→4 linkage. On the basis of these results, a 1→4 glucan with branches at the C-6 position is indicated.The structure of ASLP was analyzed by 1D NMR spec-troscopy, then further analyzed by NMR spectroscopy using two-dimensional spectra (H-H COSY and HSQC).In the 500-MHz 1H NMR spectrum of ASLP (Fig. 6B ),the signals appeared in the anomeric region at δ5.39 ppm (residue A) and δ4.95 ppm (residue B); accordingly in the anomeric region of the 13C NM R spectrum (Fig. 6C ), the carbon resonances appeared at δ102.7 ppm and δ100.7 ppm.Both results confirmed the presence of two types of gluco-pyranse residue. It also confirmed that the sugar residues are linked α-glycosidically, which is consistent with presenceof an IR band at 843.24 cm –1(21). In the 13C NM R spec-trum, the signal at δ63.2 ppm was reasonably assigned to the unsubstituted C-6 of residue A. The C-4 signal of resi-due A appeared at δ80.3 ppm. The downfield carbon chemi-cal shift at δ68.6 ppm, caused by the glycosylation effect,was assigned to substitute C-6 of residue B (22).In the 1H-13C HSQC spectrum, the signals at δ3.64/80.3ppm, δ3.84/63.2 ppm, δ3.77/63.2 ppm, δ3.96/68.6 ppm, and δ3.74/68.6 ppm were assigned to AH4/C4, H6a/C6, H6b/C6and residue B H6a/C6 (H6b/C6), respectively. These spe-cific signals confirmed the presence of O-4 and O-6 substi-FIG. 5.FT-IR spectrum of ASLP.FIG. 6. (A) 13C NMR spectrum of the partial-acidic-hydrolysis product. Signals at δ78.7 ppm and δ63.5 ppm assigned to the substitu-tion of C-4. (B) 1H NMR spectrum of ASLP. (C) 13C NMR spectrum of ASLP. The signals at δ5.39ppm and δ4.95ppm in panel B and the sig-nals at δ102.7ppm and δ100.7ppm in panel C suggest the presence of two types of glucopyrance residue. Spectra were recorded at 300K on a Bruker A V-500 spectrometer operating at 500MH z for 1H and 125MHz for 13C.TABLE 2.GLC-MS of alditol acetate derivatives from the methylated product of ASLPMethylated sugar (as alditol acetate)aType of linkage Retentiontime bMolar%Mass fragment (m /z )2,3,4,6-Me 4-GlcTerminal Glc p 1.00 1.043, 45, 71, 87, 101, 117, 129, 145, 161, 2052,3,4-Me 3-Glc1,6-linked Glc p 1.29 2.143, 45, 71, 87, 101, 117, 129, 161, 173, 189, 2332,3,6-Me 3-Glc1,4-linked Glc p 1.25 2.843, 45, 71, 87, 99, 101, 117, 129, 161, 173, 2332,3-Me 2-Glc1,4,6-linked Glc p 1.51 1.143, 85, 99, 101, 117, 127, 142, 159, 201, 261a2,3,4,6-Me 4-Glc =1,5-di-O -acetyl-2,3,4,6-tetra-O -methyl-glucose.bRelative retention times of corresponding alditol derivatives relative to 2,3,4,6-tetra-O -methyl-D -glucose.POLYSACCHARIDE PREPARED FROM A. SUBCRENATA LISCHKE V OL . 104, 2007 115tutions. The chemical shifts from 3.5 to 4.0 ppm were as-signed to protons of carbons C-2 to C-6 of a glycocidic ring.The 1H and 13C NMR spectra of ASLP were assigned on thebasis of the results of 2D 1H-1H COSY and 1H- 13C HSQC experiments (Table 3). The NMR spectral analysis confirmed that, as supported by the conclusions drawn from methyla-tion data and the 13C NMR spectrum of H-ASLP, ASLP has a backbone chain of (1→4)-glucosyl residues, with three (1→6)-glucosyl residues out of four (1→4)-glucosyl resi-dues being substituted at the C-6 position.Taken all together, it can be concluded the ASLP is com-posed of repeating units having a possible structure shown in Fig. 7.Splenocyte proliferation To clarify the immunomod-ulatory activity of ASLP, we investigated the effects of ASLP on the proliferation of mouse splenocytes in vitro .As observed in Fig. 8, ASLP significantly increased spleen lymphocyte proliferation in a dose-dependent manner (P <0.01); however, after the partial acid hydrolysis of ASLP (H-ASLP), it did not elicit such an immune response.This observation suggests that the branches are extremelyimportant for the biological activities of ASLP.A. subcrenata Lischke is a popular seafood in China. In this paper, we report on a novel glucan isolated and purified from the water extract of A. subcrenata Lischke . It is an α-(1→4)-D -glucan with α-(1→6)-D -glucan at the C-6 posi-tion. Preliminary immunopharmacological tests suggest that ASLP enhances spleen lymphocyte proliferation in vitro and the branches of ASLP are extremely important for its biological activities. Further investigations on the detailed pharmacological effects of ASLP are currently underway in our laboratory.ACKNOWLEDGMENTSThe financial support from the Drug Safety Evaluation Center of Jiangsu Province is gratefully acknowledged. The authors also wish to thank Mr. Dongjun Chen for help in NMR spectroscopy,the Center for Instrumental Analysis of China Pharmaceutical Uni-versity, Mr. Fengguo Zhang and Dr. Jianmin Liao, School of Life Science and Technology of China Pharmaceutical University. Mrs.Shuijuan Wang, Nanjing University, is acknowledged for conduct-ing GLC.REFERENCES1. Athukorala, Y., Lee, K. W., Kim, S. K., and Jeon, Y. J.:Anticoagulant activity of marine green and brown algae col-lected from Jeju Island in K orea. Bioresour. Technol., 98,1711–1716 (2006).2. Matsubara, K., Matsuura, Y., Bacic, A., Liao, M. L., Hori,K., and Miyazawa, K.: Anticoagulant properties of a sul-fated galactan preparation from a marine green alga, Codium cylindricum . Int. J. Biol. Macromol., 28, 395–399 (2001).3. Zierer, M. S. and Mourão, P. A. S.: A wide diversity of sul-fated polysaccharides are synthesized by different species ofmarine sponges. Carbohydr. Res., 328, 209–216 (2000).4. Shanmugam, M. and Mody, K. H.: Heparinoid-active sul-phated polysaccharides from marine algae as potential bloodanticoagulant agents. Curr. Sci., 79, 1672–1682 (2000).5. Arena, A., Maugeri, T. L., Pavone, B., Iannello, B.,Gugliandolo, C., and Bisignano, G.: Antiviral and immuno-regulatory effect of a novel exopolysaccharide from a marine thermotolerant Bacillus licheniformis . Int. Immunopharmacol.,6, 8–13 (2006).6. Han, F., Yao, W. B., Yang, X. B., Lin, X. N., and Gao,X. D.: Experimental study on anticoagulant and antiplatelet aggregation activity of a chemically sulfated marine polysac-charide YCP. Int. J. Biol. Macromol., 36, 201–207 (2005).7. Silipo, A., Molinaro, A., Nazarenko, E. L., Gorshkova,R. P., Ivanova, E. P., Lanzetta, R., and Parrilli, M.: The O-chain structure from the LPS of marine halophilic bac-terium Pseudoalteromonas carrageenovora -type strain IAM 12662T . Carbohydr. Res., 340, 2693–2697 (2005).8. Nazarenko, E. L., Komandrova, N. A., Gorshkova, R. P.,Tomshich, S. V ., Zubkov, V . A., Kilcoyne, M., and Savage,TABLE 3.1H and 13C NMR chemical shifts of polysaccharide ASLP in D 2OResidue13C/1H （ppm ）123456a 6b A (1→4)- α-D -Glc p102.7 73.8 75.7 80.3 75.9 63.2 –5.39 3.85 3.73 3.64 3.98 3.84 3.77B (1→6)- α-D -Glc p100.7 73.9 75.7 73.9 75.9 68.6 –4.95 3.91 3.73 3.60 3.98 3.96 3.74FIG. 7. Possible structure of polysaccharide ASLP isolated fromArca subcrenata Lischke.FIG. 8. Effects of ASLP and H-ASLP on spleen lymphocyte prolif-eration in vitro . Proliferation activity was expressed as the absorption at 570 nm. The activity response of ASLP was dose-dependent. ConA (5µg/ml) was used as the positive control. Values are means ±SD of 10 wells; ** P <0.01 vs. negative control; ## P <0.01 vs. positive con-trol.HE ET AL.J. B IOSCI. B IOENG., 116A.V.: Structures of polysaccharides and oligosaccharides ofsome gramnegative marine Proteobacteria. Carbohydr. Res., 338, 2449–2457 (2003).9.Dubois, M., Gilles, K.A., Hamilton, J.K., R ebers, P.A.,and Smith, F.: Colorimetric method for determination of sugars and related substances. Anal. Chem., 28, 350–356 (1956).10.Filisetti-Cozzi, T.M.C.C. and Carpita, N.C.: Measurementof uronic acids without interference from neutral sugars.Anal. Biochem., 197, 157–162 (1991).11.Staub, A.M.: Removeal of protein-Sevag method. MethodsCarbohydr. Chem., 5, 5–6 (1965).12.Blakeney, A.B., Harris, P.J., Henry, R.J., and Stone, B.A.:A simple and rapid preparation of alditol acetates for mono-saccharides analysis. Carbohydr. Res., 113, 291–299 (1983).13.Needs, P.W. and Selvendran, R.R.: Avoiding oxidativedegradation during sodium hydroxide/methyl iodine-mediated carbohydrate methylation in dimethyl sulfoxide. Carbohydr.Res., 245, 1–10 (1993).14.Sweet, D.P., Shapiro, R.H., and Albersherm, P.: Structuraland immunological studies of a major polysaccharide form spores of Ganoderma luci dum (Fr.) K arst. Carbohydr. Res., 40, 217–225 (1975).15.Tsumuraya, Y., Misaki, A., Takaya, S., and Torii, M.: A newfungal α-glucan, elaborated by Elsinoe leucospila. Carbohydr.Res., 66, 53–65 (1978).16.Omarsdottir, S., Petersen, B.O., Paulsen, B.S., Togola, A.,Duus, J.Ø., and Olafsdottir, E.S.: Structural characterisation of novel lichen heteroglycans by NMR spectroscopy and me-thylation analysis. Carbohydr. Res., 341, 2449–2455 (2006).17.Mossmann, T.: Rapid colorimetric assay for cellular growthand survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods, 65, 55–63 (1983).18.Liu, Ch.H., Lin, Q.X., Gao, Y., Ye, L., Xing, Y.Y., and Xi,T.: Characterization and antitumor activity of a polysaccha-ride from strongylocentrotus nudus eggs. Carbohydr. Polym., 67, 313–318 (2007).19.Zhao, G.H., Kan, J.Q., Li, Zh.X., and Chen, Z.D.:Characterization and immunostimulatory activity of an (1→6)-α-D-glucan from the root of Ipomoea batatas. Int. Immuno-pharmacol., 5, 1436–1445 (2005).20.Zhang, W.J.: Biochemical study technology in polysaccha-ride compounds [M]. Zhejiang University Press, Hangzhou, P.R. China (1999).21.Wu, Y.L., Sun, C.R., and Pan, Y.J.: Studies on isolationand structure features of a polysaccharide from the mycelium of an Chinese edible fungus (Cordyceps sinensis). Carbohydr.Polym., 63, 251–256 (2006).22.Zhao, Ch., Li, M., Luo, Y.F., and Wu, W.K.: Isolation andstructural characterization of an immunostimulating polysac-charide from fuzi, Aconi tum carmi chaeli. Carbohydr. Res., 341, 485–491 (2006).。

polysome profiling 原理

polysome profiling 原理Polysome profiling is a technique used to study gene expression at the translational level. It involves the isolation and characterization of polysomes, which are groups of ribosomes simultaneously translating the same mRNA molecule.The principle behind polysome profiling is based on the fact that actively translating mRNA molecules are associated with multiple ribosomes, forming polysomes. In contrast, non-translating or poorly translated mRNA molecules are usually associated with fewer or no ribosomes.To perform polysome profiling, cells or tissues are treated with a chemical called cycloheximide, which prevents ribosomes from dissociating from mRNA and allows the stabilization of polysomes. The cells are then lysed and the cytoplasmic fraction is separated by centrifugation.The cytoplasmic fraction is subjected to sucrose density gradient centrifugation. The density gradient separates polysomes based on their size and number of associated ribosomes. The gradient is usually composed of increasing sucrose concentrations, with the lighter fractions at the top and the heavier fractions at the bottom. After centrifugation, the gradient is fractionated, and each fraction is collected. RNA is isolated from each fraction, and the distribution of mRNA molecules across the gradient is analyzed using techniques such as reverse transcription and quantitative polymerase chain reaction (RT-qPCR) or RNA sequencing.The results of polysome profiling provide information about the translational status of mRNA molecules. mRNA molecules associated with multiple ribosomes (polysomes) are considered actively translated, while those associated with few or no ribosomes are considered poorly translated or non-translated. Polysome profiling can reveal changes in translational efficiency or regulation under different biological conditions or stimuli. It can also help identify specific mRNA molecules or gene networks that are preferentially translated or regulated at the translational level, providing insights into protein synthesis control and regulation.。

类球红细菌应用进展

类球红细菌应用进展陈琳;李祖明【摘要】类球红细菌(Rhodobacter Sphaeroides)属光合细菌,是目前研究最深入的光合微生物之一,具有多种代谢方式.不仅能够产生类胡萝卜素、辅酶Q10、超氧化物歧化酶、5-氨基乙酰丙酸和氢气,而且能够降解农药残留、有机废水和多环芳烃等有毒有害物质,应用领域十分广泛.本文综述了类球红细菌在食品、医药、农业、环境、氢气生产等领域中的应用进展,并对类球红细菌的应用前景进行展望.【期刊名称】《微生物学杂志》【年(卷),期】2016(036)003【总页数】4页(P105-108)【关键词】类球红细菌;应用;进展【作者】陈琳;李祖明【作者单位】北京联合大学应用文理学院,北京100191;北京联合大学应用文理学院,北京100191【正文语种】中文【中图分类】Q939.9类球红细菌属于紫色非硫细菌，是目前研究最深入的光合微生物之一[1] ，能在多种条件下生长，如有氧、无氧、黑暗等[2]。

类球红细菌不仅可以产生辅酶Q10[3]、类胡萝卜素[4]、超氧化物歧化酶(Superoxide dismutase, SOD)[5]、5-氨基乙酰丙酸(5-aminolevulinic, ALA)[6]、氢气[7]和D-阿洛酮糖[8]等，还能降解农药残留[9]、有机废水[10]和多环芳烃(Polyaromates aromatic hydrocarbons, PAHs)[11]等，还可以固定二氧化碳和氮气以及补救放射性污染[1]。

可见，类球红细菌应用领域十分广泛，已经成为一种具有巨大的工业化开发潜力的微生物[12]。

1.1 功能食品辅酶Q10是一个内生酶辅助因子，在人类所有活细胞中都能产生，分布在细胞膜上，是一种强力的抗氧化膳食补充剂[13]。

Kien等[3]筛选出一种生产辅酶Q10的类球红细菌突变株KACC 91339P，其辅酶Q10的产量达到8.12 mg/g DCW。

SOD属于金属酶类，可以通过催化超氧阴离子自由基的歧化反应，有效地清除体内的超氧阴离子自由基，实现抗氧化的功能[14]，从而应用于功能食品中，如生产SOD功能苹果[15]。

白术多糖的提取、纯化和活性研究

白术多糖的提取、纯化和活性研究郭志欣;梁中焕;娄丽秋;张丽萍【摘要】[目的]提取和纯化白术多糖,并探讨多糖中蛋白对小鼠脾淋巴细胞转化的影响.[方法]采用醇沉分级、脱蛋白和Sephadex G-75制备柱从中药白术中提取分离得到cPAM、zPAM和PAM 3种多糖,并对这3种多糖进行体外淋巴细胞转化试验.[结果]3种白术多糖对ConA诱导的T淋巴细胞增殖均有促进作用;浓度为5μg/ml的zPAM对小鼠脾淋巴细胞增殖有明显的促进作用;浓度为5和10μg/ml 的PAM对小鼠脾淋巴细胞增殖有明显的促进作用.[结论]白术多糖的纯化程度与脾淋巴细胞增殖有一定关系;纯化程度高的多糖对小鼠脾淋巴细胞的增殖有促进作用;白术粗多糖中的蛋白对小鼠脾淋巴细胞的增殖没有影响.%[ Objective] To extract and purify the polysaccharides from RHIZOMA ATRACTYLODIS MACROCEPHALAE ; and to discuss the effects of polysaccharides on lymphocyte transformation of murinespleen. [Method] Polysaccharides (cPAM, zPAM, PAM) were extracted and isolated from KHIZOMA ATRACTYLODIS MACROCEPHALAE by alcohol precipitation method, deproteinization, and Sephadex G-7S preparative column, l.ymgihocyle transformation in vitro of the three, kinds of polysaccharides was researrhed. [ Result ] The three kinds of pol-ysaccharides all had significant promotion effects on the T lymphocyte proliferation of murinespleen induced by ConA. S μg/ ml zPAM showed significant promotion effects on the proliferation of murinespleen lymphocyte; and 5 and 10 μg/ml PAM also had significant promotion effects on the proliferation of murinespleen lymphocyte. [Conclusion] The purification degree of RHIZOMAATRACTYIJODIS MACROCEPHAI.AF. polysaccharides had certain relationship with the proliferation of murinespleen lymphocyte. Polysaccharides with high purification degree could promote the proliferation of murinespleen lymphocyte. Crude polysaccharides from RHIZOMA ATRACTYIODIS MACROCEPHALAE had no effects on the proliferation of murinespleen lymphocyte.【期刊名称】《安徽农业科学》【年(卷),期】2012(040)024【总页数】3页(P12011-12013)【关键词】白术(RHIZOMA ATRACTYLODIS MACROCEPHALAE);多糖;纯化;淋巴细胞转化【作者】郭志欣;梁中焕;娄丽秋;张丽萍【作者单位】通化师范学院,吉林通化134002;东北师范大学生命科学学院,吉林长春134000;东北师范大学生命科学学院,吉林长春134000;东北师范大学生命科学学院,吉林长春134000【正文语种】中文【中图分类】Q539;S567.23+3白术(RHIZOMA ATRACTYLODIS MACROCEPHALAE)是我国传统的常用中药材，有悠久的生产栽培和应用历史。

甘蔗皮纤维的提取工艺初探

甘蔗皮纤维的提取工艺初探宗闪闪;杨旭红【摘要】甘蔗皮中含有可提取的纤维物质,但其木质素和半纤维的含量较高,因此选取的纤维提取工艺应能有效去除木质素和半纤维又不损伤纤维.本文选用正交试验的方法对甘蔗皮纤维的提取工艺进行研究,结果表明:经不同工艺处理后,甘蔗皮中的半纤维都基本得到有效去除,而木质素尚不能完全去除;不同工艺下甘蔗皮纤维的平均长度为0.73～1.07mm,平均直径为16.5～19.8μm.结合纤维的长度和红外光谱图分析,得出甘蔗皮在40 mol/L的硫酸溶液中100℃酸煮1 h、在10%的烧碱溶液中100℃碱煮2.5 h.再搅拌处理1 h为较优的提取工艺.【期刊名称】《南通大学学报（自然科学版）》【年(卷),期】2010(009)002【总页数】5页(P54-58)【关键词】甘蔗皮纤维;提取工艺;纤维长度;纤维直径【作者】宗闪闪;杨旭红【作者单位】苏州大学,纺织与服装工程学院,江苏,苏州,215021;现代丝绸国家工程实验室,江苏,苏州,215123;苏州大学,纺织与服装工程学院,江苏,苏州,215021;现代丝绸国家工程实验室,江苏,苏州,215123【正文语种】中文【中图分类】TS102.6甘蔗被榨汁以后，甘蔗皮和甘蔗渣大部分被废弃，不仅产生了大量的固体废弃物，还造成了资源浪费.为了合理利用这种廉价的资源，越来越多的人把甘蔗渣用于制备复合材料[1-2].有研究表明，和蔗渣相比用甘蔗皮纤维增强复合材料的性能更优[3].本文研究了甘蔗皮纤维的提取方法，为甘蔗皮纤维制备复合材料做准备.1 实验材料和方法1.1 实验原材料原料：甘蔗外皮试剂：浓硫酸、固体NaOH、无水乙醇、苯、草酸铵1.2 试样准备取适量甘蔗皮，放于烘箱中在105℃的条件下烘至恒重，迅速取出放入干燥器中冷却30 min，分别称取4个约5 g的甘蔗皮样品，用于甘蔗皮的成分分析.另取适量甘蔗皮，加入蒸馏水，浴比为50∶1，置于100℃的水浴中沸煮1 h，更换新的蒸馏水再沸煮2 h，再换水沸煮1 h，以去除甘蔗渣中的糖分.将甘蔗皮取出洗净，放在60℃烘箱中烘至恒重后，放入干燥器中干燥30 min后称量.每5 g为一份，共称量18份，用于做纤维提取实验.1.3 实验方法1.3.1 甘蔗皮成分分析甘蔗皮的成分分析参照《苎麻化学成分定量分析方法》（GB/T 5889—86）进行测试.首先用苯乙醇提取脂蜡质，再用蒸馏水沸煮去除水溶物，然后用质量浓度为5 g/L的铵溶液沸煮3 h以去除果胶物质，接着用质量浓度为20 g/L的氢氧化钠溶液沸煮3.5 h以去除半纤维素.在去除水溶物时，由于甘蔗皮所含的水溶物较多，经过多次实验分析后，采用水煮4 h，换水3次的方法，以便将水溶物充分去除.木质素含量的测定，先将提取脂蜡质后风干的试样剪碎，用质量分数为72%的硫酸溶液浸渍24 h后用蒸馏水稀释，再反复抽滤、洗涤.称取每个步骤处理前后的样品干重，根据重量变化计算各成分含量.1.3.2 甘蔗皮纤维的提取甘蔗皮纤维的提取工艺流程如下：酸煮→水洗→碱煮→水洗→搅拌→水洗→烘干在大量实验的基础上，结合纤维的长度和纤维的去杂率确定了酸浓度（40mol/L）、碱煮质量分数（10%）和搅拌时间（1 h）3个因素.固定以上3个因素，选取酸煮温度、酸煮时间、碱煮温度、碱煮时间4个因素，分别取3个水平进行正交试验.1.3.3 傅立叶红外光谱分析将样品剪成粉末，用溴化钾压片法制样后，在傅立叶Nicolet 5700(美国Nicolet公司)测试仪上进行红外光谱测定.1.3.4 纤维的形态结构分析利用Y172型纤维切片器，采用哈氏切片法制纤维切片，用扫描电镜Hitachi S-4700（日本日立公司）观察纤维的横截面形状.将待测的纤维用双面胶固定于测试台上，用于观察纤维的纵向结构.1.3.5 纤维的长度和直径测试采用Leica偏光显微镜拍摄纤维图像，用图像分析法直接测量纤维的直径，每个样品测100根.纤维的长度则采用AutoCAD 2004软件测量，每个样品测120根.2 实验结果与讨论2.1 甘蔗皮的化学成分经测试，甘蔗皮的化学成分组成如表1所示.由表1可知，甘蔗皮中的脂蜡质和果胶含量较低.与亚麻和黄麻相比，甘蔗皮的半纤维素和木质素含量较高，纤维素含量较低；与慈竹相比，甘蔗皮的半纤维素含量稍高，木质素和纤维素含量都和慈竹较为接近.表1 甘蔗皮及相近材料的化学成分材料各成分的质量分数/%脂蜡质果胶半纤维素木质素纤维素其它甘蔗皮 0.80 1.80 39.30 22.50 35.60慈竹[4] 3.20 2.50 27.50 21.70 39.10 6.00亚麻[5] 2.26 3.61 15.51 4.42 65.71 8.49黄麻[6] 0.34 13.371.19 16.01 9.93 59.162.2 纤维提取工艺的正交试验结果与分析本文以去杂率为主要考察指标，以纤维的长度为参照指标，对甘蔗皮纤维的提取工艺条件进行比较分析.正交试验设计及结果如表2所示.去杂率定义如下：表2 甘蔗皮纤维提取工艺的正交试验结果分析表实验号 A酸煮温度/℃B酸煮时间/h C碱煮温度/℃ D碱煮时间/h 去杂率/%平均长度/m m 160（1）1（1） 60（1） 2.5（1） 55.650.97260（1） 2（2） 80（2） 3.5（2） 62.850.79360（1） 3（3） 100（3） 4.5（3） 65.80 0.73456789去杂率/%I I I 80（2） 1（1） 80（2） 4.5（3） 63.80 0.7680（2） 2（2） 100（3）2.5（1） 66.75 0.8680（2） 3（3） 60（1）3.5（2） 61.95 0.80100（3）1（1） 100（3） 3.5（2） 69.20 0.91100（3） 2（2） 60（1）4.5（3）61.55 0.97100（3） 3（3） 80（2） 2.5（1） 64.45 1.0761.43 62.88 59.72 62.2864.17 63.72 63.70 64.67 I I I I平均长度/m m I I I I I 65.0764.07 67.25 63.720.8317 0.8800 0.9133 0.96500.8067 0.8733 0.87170.83330.98170.86670.8350 0.8217式中，W0为处理前样品干重，W1为处理后样品干重.根据表2中的去杂率可知，甘蔗皮脱胶的较优工艺为 A3、B3、C3、D2.由表3分析可知，碱煮温度（C）对去杂率的影响较为显著（Sig.＜0.05），其他因素（A、B、D）影响很小（Sig.＞0.1），因此除了 C 因素根据去杂率选择C3外，其他因素的水平再参考纤维长度进行调整.由长度分析可知，酸煮温度（A）和碱煮时间（D）对纤维长度影响显著.若以长度为参考，A因素仍以A3水平为佳，而D因素以D1水平为佳.B对去杂率和纤维长度的影响都甚微，因此选择时间较短的B1水平为好.综合考虑去杂率和纤维长度后得出较优工艺为 A3、B1、C3、D1.表3 正交试验方差分析表来源因变量F S i g.A 去杂率 1.559 0.262平均纤维长度 19.745 0.001 B 去杂率 0.161 0.854平均纤维长度 0.098 0.908 C 去杂率6.185 0.020平均纤维长度 3.386 0.080 D 去杂率 0.627 0.556平均纤维长度13.9650.0022.3 甘蔗皮的傅里叶红外光谱分析图1为经不同工艺处理后甘蔗皮纤维的红外光谱图.图1 甘蔗皮纤维红外光谱图由文献知，1733 cm－1处吸收峰是羰基的伸缩振动[7]，1508 cm－1附近的峰是芳香环的骨架振动[8].由图1可知，处理样品中除3号与0号未处理样品在1733 cm－1处存在吸收峰以外，其余样品在此处的吸收峰都已消失，说明除3号以外，其余处理工艺都基本去除了甘蔗皮中的半纤维素.1～9号样品在1508 cm －1处都有吸收峰存在，说明提取的甘蔗皮纤维中仍含有木质素[9].其中5号、7号样品在1508 cm-1处的峰相对较弱，说明5号、7号去除了较多的木质素. 2.4 甘蔗皮纤维的形态结构图2所示为按照1.3.2所述方法去除杂质前后甘蔗皮纤维的形态结构图.由图2a知处理前的甘蔗皮纤维表面存在明显的胶质，经处理后的甘蔗皮纤维表面的蜡质被去除，显露出明显的沟槽（图2b）.由图2c、d知：处理前甘蔗皮纤维是中空结构，空隙较大，呈椭圆型；处理后甘蔗皮纤维的中空结构消失，纤维截面形状不规则.图2 处理前后甘蔗皮纤维的扫描电镜图图3是按照1.3.1所述方法，甘蔗皮在化学成分定量分析过程中各个成分被逐步去除后的截面形状变化图.由图3a可知：当甘蔗皮中的脂蜡质去除后，和未处理的甘蔗皮2c相比，截面中腔变得扁平，说明纤维易被压扁；当水溶物去除后（图3b），甘蔗皮截面的空隙变小；去除果胶后（图3c），和3b相比，截面形态除了中腔更为扁平外没有明显变化；而当甘蔗皮纤维中的半纤维素去除后，纤维中的空隙消失，纤维粘结在一起（图3d），这可能是因为氢氧化钠溶液处理使得纤维沿纵向裂开，中腔消失，在制作切片时纤维受挤压而粘结在一起.2.5 甘蔗皮纤维的长度和直径图3 纤维截面变化图甘蔗皮纤维的长度和直径分布如图4所示.由图4可以看出，甘蔗皮纤维的长度分布集中在0.4～1.6 mm，直径分布集中在10～28 μm.由不同提取工艺得到的甘蔗皮单纤维的平均长度在0.73～1.07 mm，平均直径在16.5～19.8 μm.9个样品中最长的单纤维为2.51 mm，最短的仅有0.14 mm；直径最大值为41.36 μm，直径最小值为7.56 μm.9个样品的平均长度的差异较大，平均直径的差异不大.由此可知不同处理条件，对纤维的长度影响较大，而对直径影响较小.图4 甘蔗皮纤维长度和直径分布图3 结论1）经本文中不同工艺处理后，甘蔗皮中的半纤维素基本能去除，而木质素尚不能完全去除.2）未处理的甘蔗皮呈中空结构，且空隙较大，纵向表面有明显的包覆物.经去杂处理后，甘蔗皮的中空结构消失，表面包覆物减少，显露出明显的沟槽.3）通过以去杂率为指标的正交试验分析，结合纤维长度分析，本文得出甘蔗皮的较优提取工艺为 A3、B1、C3、D1，即甘蔗皮在浓度为 40 mol/L 的硫酸溶液中100℃下酸煮1 h、在质量分数为10%的烧碱溶液中100℃下碱煮2.5 h，再搅拌处理1 h.在此工艺下，可得到脱胶率较高、平均长度较长的纤维.参考文献：[1]曹勇，合田公一，吴义强.甘蔗渣纤维增强聚丙烯复合材料的制备和力学性能［J］.复合材料学报，2007，24（6）：1-6.[2]Corradini E，Ito E N，Marconcini J，et al.Interfacial behavior of composites of recycled poly(ethyelene terephthalate)and sugarcane bagasse fiber［J］.Polymer Testing， 2009， 28（2）：183-187.[3]Lee S C，Mariatti M.The effect of bagasse fibers obtained（from rind and pith component） on the properties of unsaturated polyester composites［J］.Materials Letters， 2008， 62（15）：2253-2256.[4]徐伟.天然竹纤维的提取及其结构和化学性能的研究［D］.苏州：苏州大学纺织与服装工程学院，2006.[5]赵小锋，张军，李素波，等.亚麻纤维化学成分定量分析方法研究［J］.上海纺织科技， 2008， 36（2）：49-51.[6]陈琼华，于伟东.黄麻纤维的形态结构及组分研究现状［J］.中国麻业， 2005，27（5）：254-258.[7]Mulinari D R，Voorwald H J，Cioffi M O，et al.Preparation and properties of HDPE/sugarcane bagasse cellulose composites obtained for thermokinetic mixer［J］.Carbohydrate Polymers.2009， 75（2）：317-321.[8]邱卫华，陈洪章.红外光谱分析木质素在漆酶酶法改性中的反应性［J］.光谱学和光谱学分析， 2008， 28（7）：1501-1505.[9]Sun J X，Sun X F，Zhao H，et al.Isolation and characterization ofcellulose from sugarcane bagasse［J］.Polymer Degradation and Stability，2004， 84（2）：331-339.。

1、下载文档前请自行甄别文档内容的完整性，平台不提供额外的编辑、内容补充、找答案等附加服务。
2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
3、如文档侵犯您的权益，请联系客服反馈,我们会尽快为您处理(人工客服工作时间：9:00-18:30)。