Improvement Of Cholesterol Oxidase Thermotolerance By Site-Directed Mutagenesis

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.