兽疫链球菌的改良及其发酵生产透明质酸论文

1、学号： 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 Zooepidemicus For Producing Hyaluronic Acid By FermentationA Dissertation Submitted toChangzhou UniversityBySun Xiao-HuiOrganic ChemistryDissertation Supervisor: Prof. Wang Li-QunMarch，2011常州大学学位论文原创性声明本人郑重声明：所呈交的学位论文是本人在导师指导下独立进行的研究工作及取得的研究成果。除文中已经注明引用的内容外，本论文不含任何其他个人或集体已经发表或撰写过的作品成果。对本文的研究做出重要贡献的个人和集体，均已在论文中以明确方式标明。本人已完全意识到本声明的法律结果由本人承担。作者签名： 签字日期： 年 月 日学位论文版权使用授权的说明本学位论文作者完全了解 常州大

2、学 有关保留、使用学位论文的规定，即：研究生在校攻读学位期间论文工作的知识产权单位属常州大学。学校有权保留并向国家有关部门或机构送交论文的复印件和磁盘，允许论文被查阅和借阅。学校可以公布学位论文的全部或部分内容，可以采用影印、缩印或扫描等复制手段保存、汇编本学位论文。保密论文注释：本学位论文属于保密范围，在 年解密后适用本授权书。非保密论文注释：本学位论文不属于保密范围，适用本授权书。学位论文作者签名： 签字日期： 年 月 日 导师签名： 签字日期： 年 月 日 中 文 摘 要透明质酸(hyaluronic acid, HA)是由(-1,4)-D-葡糖醛酸-(-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因其特殊的生物功能学、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

3、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，观察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，特征吸收峰的位置和形状基本相同，峰的相对强度相似，说明该菌株为产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。以筛选到的菌株为出发菌株，经过紫外诱变得到溶血突变型菌株UV1。以突变菌株作为出发菌株进行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，并且遗传稳定性优良。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，发酵时间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可以提高HA产量，发酵10

4、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 DH5，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ABSTRACTHyaluronic acid is a linear macromolecular polymer which is comprised of alternated disaccharide units of D-glucuronic acid and N-acetyl glucosamine by -1, 3 and -1, 4 glycosidic

5、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

6、 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 -hemolytic streptococcus with capsule was identified by observation of morphplogical and hemolytic properties of colonies on the plates. The infrared spectroscopy of hyaluronic acid standar

7、d 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 -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

8、 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 t

9、o 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 tim

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