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
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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
·研究报告·
生物技术通报
BIOTECHNOLOGY BUL143
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
邱锦 黄火清 姚斌 罗会颖
（中国农业科学院饲料研究所，北京 100081）
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。
Key words: α-amylase ；site-directed mutation ；Bacillus subtilis ；catalytic efficiency
关键词 ： α- 淀粉酶，定点突变，枯草芽孢杆菌，催化效率 DOI ：10.13560/ki.biotech.bull.1985.2019-0234
Improvement of Catalytic Activity of Amylase from Bacillus amyloliquefaciens and Its High Expression in Bacillus subtilis
QIU Jin HUANG Huo-qing YAO Bin LUO Hui-ying
（Feed Research Institute，Chinese Academy of Agricultural Sciences，Beijing 100081）
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.
·研究报告·
生物技术通报
BIOTECHNOLOGY BUL143
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
邱锦 黄火清 姚斌 罗会颖
（中国农业科学院饲料研究所，北京 100081）
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。
Key words: α-amylase ；site-directed mutation ；Bacillus subtilis ；catalytic efficiency
关键词 ： α- 淀粉酶，定点突变，枯草芽孢杆菌，催化效率 DOI ：10.13560/ki.biotech.bull.1985.2019-0234
Improvement of Catalytic Activity of Amylase from Bacillus amyloliquefaciens and Its High Expression in Bacillus subtilis
QIU Jin HUANG Huo-qing YAO Bin LUO Hui-ying
（Feed Research Institute，Chinese Academy of Agricultural Sciences，Beijing 100081）
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.