兽疫链球菌的改良及其发酵生产透明质酸论文

学号：08109117

常州大学

硕士学位论文

兽疫链球菌的改良及其发酵生产透明质酸

研究生孙晓慧

指导教师王利群副教授

学科、专业名称有机化学

研究方向生物有机化学

2010年 3 月

Improved Streptococcus Zooepidemicu s For Producing Hyaluronic Acid By Fermentation

A Dissertation Submitted to

Changzhou University

By

Sun Xiao-Hui

Organic Chemistry

Dissertation Supervisor: Prof. Wang Li-Qun

March，2011

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中文摘要

透明质酸(hyaluronic acid, HA)是由(β-1,4)-D-葡糖醛酸-(β-1,3)-N-乙酰-D-氨基葡糖的双糖单位重复连接组成的高分子链状聚合物。HA因其专门的生物功能学、独特的流体力学特性和极强的持水保湿性，广泛应用于医药、化妆品、食品等领域。本研究旨在筛选HA产生菌株，通过多种诱变育种的方法提高透明质酸的产量，并建立发酵液透明质酸含量的快速检测方法，对改良后菌种的培养条件进行优化，最后对菌种的透明质酸合成酶基因进行克隆和序列分析，为通过基因工程技术手段提高菌种HA产量打下基础。

从160份牛鼻粘膜中成功筛选到一株野生型产透明质酸菌株。样品经梯度稀释后涂布血琼脂平板，观看菌落的形态特征和溶血特征，筛选到一株有荚膜的乙型溶血链球菌。将该菌株的发酵液多糖精提物和HA标准样的红外谱图进行对比，特征汲取峰的位置和形状差不多相同，峰的相对强度相似，讲明该菌株为产HA菌株。

对透明质酸含量检测方法CTAB比浊法和Bitter-Muir法进行优化和比较，并用于发酵液中透明质酸含量的检测，成功建立了一种更快速、更精确、更准确、更高效的透明质酸检测方法。线性回归方程为y=0.0071x-0.1018，R=0.99987。

以筛选到的菌株为动身菌株，通过紫外诱变得到溶血突变型菌株UV1。以突变菌株作为动身菌株进行NTG、微波复合诱变，筛选出一株HA产量明显提高的菌株W2，产量达到1.446 g/L，同时遗传稳定性优良。对突变菌株的摇瓶发酵条件进行了初步优化，确定了最佳发酵条件为：温度35 ℃，初始pH值8.0，装液量50 mL / 250 mL，

转速180 r/min，发酵时刻20 h时，HA产量达到1.86 g/L，比初始条件下提高了29%。在发酵过程中添加脯氨酸和葡萄糖酸钠能够提高HA产量，发酵10 h时添加1 g/L的脯氨酸，HA的产量提高至1.986 g/L；发酵12 h时加入12.5 g/L葡萄糖酸钠作为补充碳源，HA的产量提高至2.267 g/L。

从筛选到的产透明质酸原始菌株中获得其基因组DNA，以此为模板通过PCR扩增获得透明质酸合酶基因has A序列，用限制性内切酶EcoR I和BamH I对目的基因和质粒pET-22b进行双酶切，然后将目的基因与质粒pET-22b进行连接，转入E. coli DH5α，得到转化子pET-22b-hasA，对转化子插入片段进行核酸序列分析，并与基因库中has A基因序列进行比对，为进一步通过基因工程手段改良菌种打下了良好的基础。

关键词：透明质酸；筛选；诱变；发酵；基因工程

ABSTRACT

Hyaluronic acid is a linear macromolecular polymer which is comprised of alternated disaccharide units of D-glucuronic acid and N-acetyl glucosamine by β-1, 3 and β-1, 4 glycosidic bonds. Depending on its special physiological characters, unique hydromechanical properties and excellent ability of holding water, HA is widely used in the fields of pharmacy, cosmetics and food etc. The purpose of this study was to screen HA- producing strain, to enhance the yield of HA through mutation breeding, to establish a rapid method to analyze the concentration of hyaluronic acid in fermentation broth, to optimize the fermentation conditions of improved strain, and to clone and analyze the hyaluronidase A gene (has A) sequence which is helpful to improve HA yeild by genetic engineering.

One strain of hyaluronic acid-producing bacteria was islolated successfully from 160 collected cattle tunica mucosa samples. Samples were coated on blood agar flat plates after gradient dilution, a strain of β-hemolytic streptococcus with capsule was identified by observation of morphplogical and hemolytic properties of colonies on the plates.The infrared spectroscopy of hyaluronic acid standard and fermentation broth extract of the screened bacterium was compared, and the size, shape and relative intensity of their typical IR peaks are similar. These results demonstrated that the β-hemolytic streptococcus produces HA.

The Bitter-Muir method and CTAB turbidimetric method were optimized to measure the concentration of hyaluronic acid in fermentation broth. The results showed that CTAB method is superior to Bitter-Muir method in terms of accuracy, precission and efficiency. The regression equation is y = 0.0071 x - 0.1018, R = 0.99987.

Using the screened bacteria as original strain, a hemolysin deficient mutant named UV1 was obtained by ultraviolet irradiation. UV1 was mutated subsequently by the combination of NTG treatment and microwave irradiation. A mutant named W2 with higher production capacity of hyaluronic acid was selected from all mutants by analyzing the HA concentration in fermentation broth. The yield of HA was stable after transferring five generations and reached to 1.446 g/L. Its fermentation conditions in shaking flask were optimized primarily. Under the conditions of 35℃, pH 8.0, 50 mL fermentation medium in 250mL flask, stiring speed 180 rpm and the fermentation time 20 h, the yield of HA reached to 1.86 g/L. Compared to the original conditions, the yield was increased by 29%. The adding of proline and sodium gluconate during the fermentation process could ehance the HA yield. The HA yield could reach 1.986 g/L through the addition of 1 g/L proline by the time of 10 h fermentation, and could reach 2.267 g/L through adding 12.5 g/L sodium gluconate as a carbon source

complement by the time of 12 h fermentation.

The genome DNA was extracted from the original strain, and the gene has A was obtained by PCR. The restriction enzymes BamH I and EcoR I were selected to digest the aimed gene DNA sequence and the vector pET-22b. Then the hasA gene was inserted into the vector pET-22b, and the recombinant plasmid pET-22b-hasA was transformed into E. coli DH5α. The sequence of insert fragment was analized and compared with other hasA gene sequences in gene bank. The results provided a good basis for further improving strains by genetic engineering.

KEYWORDS:Hyaluronic acid; Screening; Mutagenesis; Fermentation ; Genetic engineering

目录

1 绪论 (1)

1.1 透明质酸的理化性质 (1)

1.2 透明质酸的分布及生理功能 (2)

1.2.1 透明质酸的分布 (2)

1.2.2 透明质酸生理功能 (2)

1.3 透明酸的应用 (3)

1.3.1 在化妆品中的应用 (4)

1.3.2 在医学方面的应用 (4)

1.4 透明质酸生产现状 (5)

1.4.1 获得高产菌株 (6)

1.4.2 培养基及发酵条件优化 (8)

1.4.3 发酵液中透明质酸含量检测方法 (9)

1.4.4 透明质酸的提取 (10)

1.5 课题研究内容和目的 (11)

1.5.1 本课题研究内容 (11)

1.5.2 本课题要紧目的 (11)

2 透明质酸产生菌的分离、筛选与初步鉴定 (12)

2.1 实验材料 (12)

2.1.1 采样 (12)

2.1.2 要紧培养基及溶液组成 (12)

2.2 实验方法 (14)

2.2.1 透明质酸的分离纯化 (14)

2.2.2 菌种的筛选 (15)

2.2.3 菌种的初步鉴定 (16)

2.3 实验结果与讨论 (17)

2.3.1 菌种的筛选 (17)

2.3.2 菌种的分类鉴定 (19)

2.4 本章小结 (20)

3 透明质酸含量检测方法建立 (21)

3.1 实验材料 (21)

3.1.1 要紧仪器 (21)

3.1.2 要紧试剂 (22)

3.2 实验方法 (23)

3.2.1 透明质酸的初步纯化 (23)

3.2.2 CTAB比浊法 (23)

3.2.3 改良Bitter-Muir法 (23)

3.3 结果与讨论 (24)

3.3.1 CTAB比浊法 (24)

3.3.2 Bitter-Muir法 (30)

3.3.3 两种方法准确性比较 (32)

3.3.4 两种方法精确性比较 (33)

3.3.5 两种方法直接测定发酵液中HA含量的比较 (34)

3.4 本章小结 (35)

4 透明质酸高产菌的诱变选育 (36)

4.1 实验材料 (37)

4.1.1 菌种 (37)

4.1.2 要紧培养基及溶液 (37)

4.1.3 要紧仪器 (37)

4.2 实验方法 (38)

4.2.1 LiCl处理 (38)

4.2.2 紫外诱变 (38)

4.2.3 NTG诱变 (39)

4.2.4 微波诱变 (39)

4.2.5 紫外+NTG诱变 (40)

4.2.6 紫外+NTG+微波诱变 (40)

4.2.7 中间培养 (40)

4.2.8 稀释涂板 (40)

4.2.9 突变菌株筛选 (41)

4.3.10 分析方法 (41)

4.3 结果与讨论 (42)

4.3.1 紫外诱变最佳时刻的选择 (42)

4.3.2 NTG诱变最佳条件 (43)

4.3.3 微波诱变最佳条件选择 (45)

4.3.4 诱变结果分析 (46)

4.4 本章小结 (50)

5 透明质酸发酵法生产工艺条件的初步研究 (51)

5.1 实验材料 (52)

5.1.1 菌种 (52)

5.1.2 培养基 (52)

5.2 实验方法 (52)

5.2.1 发酵条件的优化 (52)

5.2.2 添加剂对发酵的阻碍 (54)

5.3 结果与讨论 (54)

5.3.1 发酵条件优化结果 (54)

5.3.2 添加剂的阻碍 (59)

5.4 本章小结 (61)

6 透明质酸合成酶基因克隆与载体构建初步研究 (62)

6.1.1 菌株、质粒 (63)

6.1.2培养基 (63)

6.1.3 酶、化学试剂 (63)

6.1.4要紧实验试剂配方 (63)

6.1.5 PCR扩增用引物 (65)

6.2 实验方法 (65)

6.2.1 提取兽疫链球菌总DNA (65)

6.2.2 目的基因的PCR扩增 (66)

6.2.3 PCR扩增DNA片段的回收纯化 (67)

6.2.4感受态细胞的制备 (68)

6.2.5 质粒DNA（PET-22b）的提取与检测 (68)

6.2.6目的基因片段和质粒的双酶切 (69)

6.2.7 双酶切产物回收 (70)

6.2.8 双酶切后的目的基因和质粒连接 (70)

6.2.9 重组质粒PET-22b导入E.coli DH5а感受态细胞

(70)

6.2.10 重组质粒的提取 (71)

6.3 结果与讨论 (71)