ad木聚糖酶(XYNB)的分离纯化与性质研究

南京工业大学硕士学位论文耐热木聚糖酶（XYNB）的分离纯化与性质研究姓名：孙雷申请学位级别：硕士专业：生物化工指导教师：韦萍;李环20060601摘要木聚糖是植物半纤维素的重要组成部分，在自然界中是继纤维素之后含量第二丰富的再生物质资源。

木聚糖结构复杂，它的完全降解需要多种水解酶的共同作用。

内切-β-1,4-木聚糖酶（1,4-β-D-木聚糖水解酶，EC3.2.1.8）以内切的方式作用于木聚糖的主链，产生不同链长的寡糖及少量的木糖，是木聚糖降解酶系中关键的酶。

木聚糖酶的耐高温和热稳定性是工业化应用的理想特性，在生物转化、制浆造纸，食品饲料等工业中存在很大的应用潜力。

本文综述了木聚糖酶的分离纯化技术以及性质和结构研究进展，研究了重组大肠杆菌1020产生的耐热木聚糖酶（XYNB）的纯化方法与性质。

采用不同的破碎方法，对表达的木聚糖酶在细胞中的分布进行分析，确定了表达的耐热木聚糖酶XYNB主要分布中可溶性细胞质中。

纯化前对表达的酶进行细胞定位是本论文的一个特色。

XYNB是胞内酶，采用反复冻融和超声波联合的方法破碎，发现对湿菌泥反复冻融三次后，酶的释放量最大；50 mL 20%的菌悬液，采用500 W，间歇时间10 s，超声波破碎15 min后，酶的释放量最大。

利用热变性除去杂蛋白，选择变性温度70℃，时间30 min，回收率可达到69.4%，纯化倍数4.9。

结合Ni-NTA 亲和层析，采用梯度洗脱方法，一步得到电泳纯XYNB，回收率29.4%，纯化倍数13.4。

采用热变性和一步亲和层析分离得到电泳纯的耐热木聚糖酶XYNB，简化了分离纯化步骤，是本论文的一个特色。

酶学性质研究表明XYNB的最适pH在6.5左右，在pH 6.0-10.0能保持最高活力的60%以上，在pH值低于6.0和高于10.0时，活力显著下降。

在50-100℃范围内，酶催化活力随着温度的升高不断上升，酶在80-100℃范围内表现出50%以上的酶活力。

在pH8.0，70℃，保温6 h后，酶活力变化不大；100℃保温1.5 h 后，残余50%的酶活力。

1mmol/L Hg2+显著影响酶活力，其它金属阳离子和EDTA 对酶活的影响不大。

XYNB对Oat spelt xylan 酶促反应的K m为0.23 mg/mL，最大反应速度V max为0.36 μmol/(min﹒mL)。

采用生物信息学手段分析XYNB的序列和结构，发现XYNB属于F/10族，与Thermotoga sp. strain FjSS3-B.1的xyn A有85%一致性，与Thermotoganeapolitana的xylanase B有83%一致性。

XYNB序列中Glu647(E727)，Glu753(E862)，His598(H656)，Trp602(W660)是保守的，对酶表现活力有重要影响。

XYNB前19个氨基酸构成酶的信号肽。

整个肽链折叠成β/α桶形，这与大多数F/10族的结构相似。

分析XYNB的氨基酸组成与结构，并与其它木聚糖酶比较，发现XYNB中不稳定氨基酸（Ser, Thr, Cys, Asn, Gln）含量低，Pro的含量高于嗜温木聚糖酶，结构上含有较多的离子对，以及低的溶剂可及疏水表面积和高的带电残基溶剂可及表面积，这些因素稳定了酶的构象，增强了酶的热稳定性。

采用化学修饰的方法讨论酶的必需氨基酸，进一步证实了Trp, Glu是酶表现活力的必需氨基酸，Trp对底物和酶的结合起关键作用，Glu是酶表现活力的重要氨基酸。

对His, Cys, Ser的修饰后，酶活力变化不明显，这些氨基酸对酶的构象或活性影响不大。

WRK修饰的XYNB热稳定性下降，进一步证明了离子对对酶热稳定性的重要作用。

在序列和结构分析的基础上对保守氨基酸进行化学修饰，提高了化学修饰的针对性，同时从序列和结构的角度对化学修饰的结果进行合理的解释，这是本文的一个创新点。

关键词：耐热木聚糖酶分离纯化酶学性质序列和结构化学修饰ABSTRACTXylan is a major component of hemicellulose fraction of the plant walls, second to cellulose in natural abundance. The backbone may carry various substitutions and its depolymerization is accomplished by the action of endo-xylanases and other enzymes. Xylanase can hydrolyze β-1, 4-glycosidic linkages of the xylan backbone to produce short chain xylo-oligosaccharides of various length. Hence endo-β-xylanase is the crucial enzyme component of microbial xylanolytic systems. The character of high temperature resistance and thermostability is good for industry applications, such as in biotransformation, paper pulp, food and feedstuff.In the present dissertation, the separation and purification methods of xylanase were summarized, including the properties and structure investigation. Thermostable xylanase XYNB from bioengineering E.coli 1020 was investigated.Different methods of disruption were used to determine the location of expressed XYNB, and the main fraction of xylanase was in soluble cytoplasm. It is a character of locating the expressed enzyme in cell before separation.XYNB was an enzyme in the cell. Repeated freezing and thawing, as well as ultrasonic disruption were used in destroying the cell. Three times of repeated freezing and thawing were the best in XYNB releasing. 50 mL cell solution with the concentration of 20% was disrupted by 500 W, with the intermittent time of 10 s and total time was 15 min. Heat treatment was used to concentrate the crude enzyme solution, and the purified factor was 4.9 with enzyme recovery of 69.4%. XYNB was purified with Ni-NTA immobilized metal affinity chromatography using gradient eluting style, and the purified factor was 13.4 with the recovery of 29.4%. It is characteristic of purifying XNNB to electrophoresis purity by combining heat treatment and affinity chromatography.℃h pH of 6.0 to 10.0, the activity The optimum pH was 6.5 (at 70), and withold 60% of the maximum. The activity was inceasing with the elevating temperature between 50-100℃, and the activity was above 50% of the maximum between the℃2+of 1mmol/L concentration inhibited the activity heavily, temperature of 80-100. Hgand EDTA had no influence on the activity of XYNB. The K m and V max of the enzyme catalyzing oat spelt xylan were 0.23 mg/mL and 0.36 μmol/(min﹒mL) individually.Amino acid sequences and structure of XYNB were analyzed by bioinformatics methods. XYNB was belonged to F/10 glyco-hydrolyzing enzyme, and of 85% identity with xyn A of Thermotoga sp. strain FjSS3-B, 83% identity with xylanase B of Thermotoga neapolitana. Glu647(E727), Glu753(E862), His598(H656), Trp602(W660) were conserved in the evolution of the xylanases. The front 19 amino acids formed a single peptide. The structure of XYNB was a β/α barrel. The low unstable amino acids (Ser, Thr, Cys, Asn, Gln), and high proline of XYNB were contributed to the thermostability. Salt bridges were important in stabilizing the structure and low accessible solvent areas (ASA) and high exposed charged ASA contributed the themostability.Chemical modification was used to determine the important amino acids related to activity and thermostability. Tryptophan and glutamic acid were important in the enzyme activity. Tryptophan was the binding amino acid with the substrate, and glutamic acid was the active site. Modification of Histidine, Cysteine, Serine had little influence in activity. Glutamic acid is crucial in high temperature resistance. That chemical modification was based on the analysis of sequence and structure elevated the pertinence of certain amino acid modification. And explanations were given to the chmical modification results. These were characteristics of this dissertation.KEYWORDS: Themostable xylanase；Separation and purification；Enzymic property；Sequence and structure；Chemical modification南京工业大学学位论文独创性声明及使用授权的声明一、学位论文独创性声明本人声明所呈交的学位论文是我个人在导师指导下进行的研究工作及取得的研究成果。