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| Trypsin-resistant Improvement of Bacillus subtilis β-1,4-endoxylanase by Rational Design Based on Molecular Structure Evaluation |
| YU Xiao-dan1,2, WU Xiu-xiu1,2, YAO Dong-sheng1,2, LIU Da-ling1,3, XIE Chun-fang1,3 |
1. National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou 510632, China;
2. Institute of Microbial Technology, Jinan University, Guangzhou 510632, China;
3. Department of Bio-engineering, Jinan University, Guangzhou 510632, China |
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Abstract In the feeding process, the decomposition of the digestive tract proteases is one of the important reasons affecting the efficiency of feed enzymes. Therefore, the feed enzymes having protease resistance is a very important property. The rational design of protein modification with resistance to protease based on the method of the biocatalysis and computational chemistry was used, which has been established to improve Bacillus subtilis 168 1,4-β-endoxylanase with resistance to protease and to get the excellent properties. Single site mutation to R121 and K39 and double sites mutations to R121/K98 were utilized to get the fine properties of mutant enzymes XynAR121C, XynAR121C/K98Q and XynAK39I. Fortunately, the excellent properties were gotten finally. The optimal temperature of XynA, XynAR121C, XynAR121C/K98Q was 60℃,while XynAK39I was 40℃,and its temperature tolerance was lower than the wild type. The optimal pH of the wild and mutant enzymes was 6.0. During the treatment with simulated intestinal fluid (pH 6.8, 10mg/ml trypsin solution), the remained enzyme activity of mutants was much higher than the wild type. As for XynAR121C, its half-life period was 193 min, 1.52 times higher than XynA. As for XynAR121C/K98Q, its half-life period was 257 min, 2.02 times higher than XynA. As for XynAK39I, after incubation with simulated intestinal fluid at 40℃ for different time, its half-life period was 90 min, only 37min shorter than XynA.
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Received: 04 March 2016
Published: 25 August 2016
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