【作者】 张哲；

【导师】 李正强；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2017， 硕士

【摘要】 透明质酸（hyaluronic acid）,简称HA做为一种重要的医学应用材料和化妆品添加原料,现如今已经被列为新兴食品资源。自从1953年从牛眼玻璃体中首次提取获得HA以来到从各动物组织中提取生产HA以达到市场需求,再到近年来微生物发酵逐步取代动物组织提取成为生产HA的主要手段,HA的生产来源逐渐发生了更替。而微生物发酵当中由于链球菌属特异性含有生产HA所必须的关键酶-透明质酸合酶（hyaluronan synthase,简称HAS）,因此链球菌成为企业生产HA首选微生物菌种。但链球菌属具有β-溶血性等缺点,科研人员常选用物理或化学诱变等方法降低链球菌毒性。于此同时此类诱变方法具有随机性和遗传不稳定性等风险因素,因此成为链球菌作为发酵菌种生产HA的主要限制因素。随着合成生物学的发展,使得利用遗传背景清楚、生物安全性高的底盘细胞生产HA成为可能,枯草芽孢杆菌作为具有除HAS外含所有HA合成代谢途径及关键酶并且被广泛应用于工业生产的菌种,被本课题选定为改造对象;本课题通过克隆HAS基因,利用合成生物学原理及分子生物学技术构建一个新的代谢通路使得枯草芽孢杆菌可以通过自身代谢生产HA。本文考察了十六烷基三甲基溴化铵（cetyltrimethylammonium bromide,简称CTAB）与HA反应产生浑浊的特性,优化了CTAB浊度法,确定了检测条件和检测范围:使用2.5 g/L CTAB-NaCl溶液;检测波长为400 nm;在室温条件下,检测样品与CTAB溶液反应5 min;测定时间误差控制在5 s以内,通过制作标准曲线计算待测样品中HA含量。该条件下测定结果显示,各杂质对CTAB浊度法测定影响较小,准确性高。且该法安全性高,操作简便,适用于大量发酵液中检测HA含量。结果显示摇瓶水平重组枯草芽孢杆菌生产HA产量为1.2 g/L,高于国内非链球菌属发酵生产HA平均产量。采用乌氏粘度计对枯草芽孢杆菌发酵生产HA相对分子质量测定,测得相对分子质量约为35万Da。并且经多次传代,HA产量和HA分子量保持稳定,为进一步研究枯草芽孢杆菌发酵生产HA建立了基础。对构建新代谢通路的枯草芽孢杆菌合成HA效率进行了初步测定,针对重组枯草芽孢杆菌,通过单因素试验和正交设计试验优化了培养基组成:酵母粉5 g/L;蛋白胨20 g/L;葡萄糖20 g/L;MgSO₄·7H₂O 100 mol/L;最佳培养条件:pH 7.5;37℃;200 rpm/min;培养36 h。将此培养基应用5 L发酵罐小试,测得发酵36 h时产量为5 g/L;建立了CTAB沉淀法对发酵液中HA进行分离纯化。优化后分离条件是:采用HA终浓度为0.5-1 g/L培养基浓缩液,加入质量分数为10%的CTAB溶液,使其达到混合液总体积的15%,室温下静置1 h,用2 mol/L的NaCl溶液在磁力搅拌器搅拌下解离2 h,最后加入无水乙醇至占总体积的70%沉淀蛋白,蛋白去除率可达94%。本文以合成生物学理论为基础,建立了枯草芽孢杆菌生产HA代谢通路,在实验室水平条件下,进行了HA产量初步测定,优化了制备条件及检测方法,产量高于非链球菌产HA平均水平,为后续高生物安全级别生产HA提供了可能性。更多还原

【Abstract】 Hyaluronic acid（HA）as an important medical application materials and cosmetics to add raw materials,now has been listed as emerging food resources.Since the first extraction of HA from the bovine vitreous in 1953 to the HA from the animal tissue to extract the production of HA to meet the market demand,and then in recent years to gradually replace the microbial fermentation of animal tissue extraction HA production of the main means,HA production sources gradually Has changed.Streptococcus is the preferred microbial strain for the production of HA,because Streptococcus is a specific enzyme known as hyaluronan synthase（HAS）which is necessary for the production of HA.But the streptococcus has β-hemolytic and other shortcomings,researchers often use physical or chemical mutagenesis and other methods to reduce the toxicity of streptococcus.At the same time,such mutagenesis methods are risk factors such as randomness and genetic instability,and thus become the main limiting factor for the production of HA as a fermentative strain.With the development of synthetic biology,making the use of genetic background clear,high biosafety chassis cells to produce HA as possible,Bacillus subtilis as with HAS contains all HA synthetic metabolic pathways and key enzymes and is widely used in industrial production Which was selected as the object of transformation by this study.By using the synthetic biology principle and molecular biology technology to construct a new metabolic pathway,Bacillus subtilis can produce HA by self-metabolism by cloning the HAS gene.In this paper,the properties of cetyltrimethylammonium bromide（CTAB）and HA reaction were observed,and the CTAB turbidity method was optimized to determine the detection conditions and detection range: 2.5 g/L CTAB-Na Cl solution;detection wavelength of 400 nm;at room temperature,the test sample and CTAB solution reaction 5 min;measurement time error control within 5 s,through the production of standard curve to calculate the sample HA content.The results showed that the impurity had little effect on the determination of CTAB turbidity method.And the method is safe and easy to operate,and it is suitable for detecting HA content in a large amount of fermentation broth.The results showed that the yield of HA was 1.2 g/L,which was higher than that of domestic non-Streptococcus production.The relative molecular mass of HA produced by fermentation of Bacillus subtilis was measured by Ubbelohde viscometer.The relative molecular mass was about 350,000 Da.And after several passages,HA production and HA molecular weight remained stable,in order to further study the fermentation of Bacillus subtilis production HA established the foundation.The results showed that the composition of the culture medium was as follows: Yeast powder 5 g/L;peptone 20 g/L（2 g/L）was used to determine the efficiency of the synthesis of Bacillus subtilis in the construction of new metabolic pathways,and the recombinant medium was optimized by single factor test and orthogonal design experiment;Glucose 20 g/L;MgSO₄·7H₂O 100 mol/L;the best culture conditions: p H 7.5;37 ℃;200 rpm/min;The medium was applied to 5 L fermentor and the yield was 5 g/L at 36 h.The separation and purification of HA in fermentation broth were established by CTAB precipitation method.The optimized separation conditions were as follows: the concentration of HA was 0.5-1 g/L,and the CTAB solution with the mass fraction of 10% was added to reach 15% of the total volume of the mixture.The mixture was dissociated with 2 mol/L Na Cl solution for 2 h with stirring in a magnetic stirrer.Finally,anhydrous ethanol was added to 70% of the total volume of precipitated protein.The protein removal rate was 94%.Based on the synthetic biology theory,the HA metabolic pathway was established by Bacillus subtilis.At the laboratory level,the HA production was preliminarily determined,and the preparation conditions and detection methods were optimized.The yield was higher than that of non-streptococcus Level,providing a possibility for the subsequent production of high biosafety levels.更多还原