摘要
为了通过定向进化的手段获得耐温性能提高的短杆菌胆固醇氧化酶突变子,首先建立具有通用性的高通量筛选耐温性酶的方法,并通过散在点突变和活性中心点突变的方法对短杆菌来源的胆固醇氧化酶基因进行分子改造,选取性能改善的突变株在7L发酵罐中引入延时诱导培养基发酵培养重组菌。本论文主要内容涉及:
(1)建立了一种以转印膜为基础的高通量筛选耐温性胆固醇氧化酶的方法。利用定位膜转印技术将原始平板保存,高温处理转印膜,将酶的热处理、酶与底物反应以及菌体生长分开。解决了重组菌筛选耐温性突变酶时菌体不耐温死亡的困难,只需要根据酶的不同显色机理调整筛选平板,即可适用于其他不耐温菌株中耐温突变酶的筛选。
(2)对胆固醇氧化酶活性中心附近的氨基酸进行定点突变改造,突变酶的耐温性均得到不同程度的提高。50℃下保温2 h,原酶完全失活,而突变酶残留酶活性分别为9.06%、10.21%和40.69%。其中Q153E突变酶耐热性提高较为显著,在60℃下保温2 h,残留酶活仍可达36.46%。
(3)对短杆菌胆固醇氧化酶基因进行点组合突变,以耐热性提高较为显著的Q153E突变酶为基础,突变获得M2(Q153E和F128L)和M3(Q153E、F128L和S143H)两个点组合突变酶。其中突变酶M2耐热性和酶催化能力均有所提高,50℃保温2 h残留酶活可达43.06%,其K_m值从原酶的820μmol/L减小至640μmol/L。
(4)考察了不同诱导方式对菌体生长和产酶的影响,实验结果显示延时诱导方式培养比IPTG诱导更适合菌体生产胆固醇氧化酶。对7L发酵罐中延时诱导重组菌产酶进行优化,采用分阶段控制发酵过程温度、依据恒pH-star法发酵前期流加葡萄糖,后期流加含甘油和乳糖的复合诱导液,发酵终点时菌体浓度和酶活力可分别高达34.28 g/L和25.88 U/mL。
In this paper, in order to obtain the cholesterol oxidase mutations with improved thermotolerance by directed evolution, a common high-throughput screening method for selecting cholesterol oxidase mutants with improved thermotolerance had been estabilished. Site-directed mutagenesis was explored to transform the gene form Brevibacterium. The mutant with improved enzymatic properties was cultured in 7L fermentor by auto delay-induction. The main results are as follows:
(1) A high-throughput screening method based on transfer film for selecting cholesterol oxidase mutants with improved thermotolerance had been estabilished. The original plate was saved by using the film transfer technology. The method takes advantage of the fact that the process of enzyme treatment and the reaction between enzymes with the substrate was separated effectively. Moreover, it solved a difficult problem that recombinant strain was easy to die when screening temperature was too high. It will also appling to other enzymes screening only by adjust the plate according to different mechanism.
(2) Site-directed mutagenesis was applied to enhance the cholesterol oxidase thermotolerance by change the amino acid residue around active site. The thermotolerance of three mutants, Q153E, F128L and S143H, increased to varying degrees comparing with original enzyme. The relative activity was 9.06%, 10.21% and 40.69% respectively when incubated 2 hours at 50℃. Substitution of Gln153 with Glu revealed that the relative activity increased 36.46% when incubated 2 hours at 60℃.
(3) The multiple mutagenesis based on Q153E with higher thermaltoletance improved was performed. Mutations of ChOAb -M2 (Q153E and F128L) and ChOAb -M3 (Q153E, F128L, and S143H) were constructed. The relative activity was 43.06% when incubated 2 hours at 50℃. The K_m values of ChOA_b -M2 was reduced from 820 to 640μmol / L.
(4) The effects of cell growth and enzyme production induced by different methods were investigated. The result showed auto delay-induced was more suitable than IPTG-induced for cholesterol oxidase expression. Different culture strategies of recombinant strain in 7L fermentor were studied. Phased controls of fermentation temperature and pH were operated by different pH controlling mode during batch culture and pH-stat fed-batch culture. Two step fermentation processes was conducted based on the pH contract strategy. Glucose feeding solution was added during the first phase of culture. And lactose and glycerol mixture solution was added during the second phase. Then the cell density and enzyme activity reached 34.28 g/L and 25.88 U/mL, respectively.
引文
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