解淀粉芽孢杆菌淀粉酶催化活力改良及其在枯草芽孢杆菌中的高效表达

doi:10.13560/j.cnki.biotech.bull.1985.2019-0234

摘要/Abstract

摘要： α-淀粉酶（α-amylase）是一类作用于淀粉内部水解其α-1,4糖苷键从而将淀粉水解为糊精、低聚糖和单糖的酶。该类酶已被广泛应用于面包生产及洗涤等工艺中。从分子水平上改造淀粉酶,以提高水解活力及催化效率,已经成为多年的目标,也具有重要的研究意义。以目前广泛应用的来源于解淀粉芽孢杆菌的中温中性淀粉酶BAMYA出发,通过理性设计在该淀粉酶的C端结构域设计突变位点,利用定点突变提高其水解活力,并在枯草芽孢杆菌中实现高效表达。野生型酶BAMYA的最适作用温度和pH分别为55℃和6.0,比活为6 835 U/mg。突变体BAMYA-L473K/K474H/N475K最适作用温度为60℃,较野生型提高了5℃,作用的最适pH没有发生变化。突变体比活为10 148 U/mg,比野生型提高48%。野生型BAMYA和突变体BAMYA-L473K/K474H/N475K的Km值、催化效率分别为3.58 mg/mL和2.21 mg/mL,以及1 577 mL/（s·mg）和4 760 mL/（s·mg）。催化效率较野生型相比提高了2倍以上。该突变体应用于工业生产可有效提高其水解效率,降低应用成本。

关键词: α-淀粉酶, 定点突变, 枯草芽孢杆菌, 催化效率

Abstract: α-Amylase is a kind of enzymes that catalyzes the random internal hydrolysis of α-1,4 glycosidic linkages in starch chain into dextrin,oligosaccharides and monosaccharides. The enzyme is widely used in bread making and washing detergent etc. Modifying amylase at the molecular level to improve the hydrolysis activity and catalytic efficiency has been the goal for many years and has important research significance. In this study,based on BAMYA,a widely used neutral amylase from Bacillus amyloliquefaciens,the mutation sites（L473K/K474H/N475K）at the C-terminal similar substrate binding region（CBM）were rationally designed as the site-directed mutation was aimed to increase its hydrolysis ability,and its efficient expression in Bacillus subtilis was achieved. The optimal temperature and pH of wild BAMYA were 55℃and 6.0,respectively and the specific activity was 6 835 U/mg. The optimal temperature of mutant BAMYA-L473K/K474H/N475K was 60℃,5℃ higher than that of wild one,while no changes in pH. The specific activity of the mutant was 10 148 U/mg,and it increased by 48% compared to the wild one. The Km and catalytic efficiency of wild BAMYA and mutant BAMYA-L473K/K474H/N475K were 3.58 mg/mL and 2.21 mg/mL,1 577 mL/（s·mg）and 4 760 mL/（s·mg）,respectively. The catalytic efficiency increased over two times than that of wild one. In sum,the application of the mutant in industry will effectively improve the efficiency of hydrolyzing starch and reduce the application cost.

Key words: α-amylase, site-directed mutation, Bacillus subtilis, catalytic efficiency

邱锦, 黄火清, 姚斌, 罗会颖. 解淀粉芽孢杆菌淀粉酶催化活力改良及其在枯草芽孢杆菌中的高效表达[J]. 生物技术通报, 2019, 35(9): 134-143.

QIU Jin, HUANG Huo-qing, YAO Bin, LUO Hui-ying. Improvement of Catalytic Activity of Amylase from Bacillus amyloliquefaciens and Its High Expression in Bacillus subtilis[J]. Biotechnology Bulletin, 2019, 35(9): 134-143.

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