来源于嗜碱芽孢杆菌N16-5甘露聚糖利用基因簇的乙酰酯酶AesA的克隆及性质分析  

Cloning and Characterization of Acetylesterase AesA Derived from Mannan Utilization Gene Cluster of Bacillus sp.N16-5

作　　者：马翠萍 刘朵朵 潘炳菊 申会涛 宋亚囝 MA Cui-ping;LIU Duo-duo;PAN Bing-ju;SHEN Hui-tao;SONG Ya-jian(Key Lab of Industrial Fermentation Microbiology of the Ministry of Education,Tianjin Key Lab of Industrial Microbiology,College of Biotechnology,Tianjin University of Science&Technology,Tianjin 300457,China)

机构地区：[1]天津科技大学生物工程学院,工业发酵微生物教育部重点实验室暨天津市工业微生物重点实验室,天津300457

出　　处：《中国生物工程杂志》2020年第3期65-71,共7页China Biotechnology

基　　金：国家重点研发计划(2017YFD0400304)资助项目。

摘　　要：天然来源的多糖底物上常存在乙酰基取代,特异性的乙酰酯酶能够切割这些底物上的乙酰基,从而有利于聚糖底物的进一步降解。对Bacillus sp.N16-5甘露聚糖利用基因簇上编码的乙酰酯酶AesA进行了基因克隆和异源表达,并对其酶学性质进行了研究。aesA基因长957bp,编码318个氨基酸,属于碳水化合物酯酶第7家族。AesA对4-甲基伞形酮乙酸酯(4-methylumbelliferyl-acetate)表现出较好的催化活性,金属离子Fe^3+、Fe^2+、Mn^2+及Cu^2+对AesA活性均有不同程度的促进作用。AesA与甘露聚糖酶ManA对乙酰化的甘露聚糖底物具有显著的协同作用。此项研究有助于理解嗜碱芽孢杆菌Bacillus sp.N16-5对甘露聚糖的水解机制,并且在甘露聚糖降解中具有潜在的应用前景。The residues of natural polysaccharide substrates were often substituted by acetyl groups,and acetyl esterase can cut acetyl groups on these substrates,which is conducive to the further degradation.The gene aesA which encoding an acetyl esterase was cloned from mannan utilization gene cluster of Bacillus sp.N16-5 and heterologously expressed in prokaryotic host.The gene aesA is 957 bp long and encodes 318 amino acids,belonging to the carbohydrate esterase family 7(CE7).AesA showed good catalytic activity for 4-methylumbelliferyl-acetate and p NP-acetate,however,there was no active effect on alpha-naphthyl acetate.The enzyme activity for 4-methylumbelliferyl-acetate is 1.68 U/mg,and the kinetic parameters Km,Vmaxand kcat/Km,were measured by 3.27 mmol/L,0.044 mmol/min and 289.71 ms^-1,respectively.Metal ions Fe^3+,Fe^2+,Mn^2+and Cu^2+all promoted the activity of AesA,and Cu2+exhibited the most significant promoting effect.AesA has a significant synergistic effect withβ-mannanase ManA on degrading acetylated mannan substrates,and the synergy degree reached 1.47 when using acetylated locust bean gum as substrate.It is helpful to understand the mannan utilization mechanism of Bacillus sp.N16-5,and has potential application prospects in mannan degradation.

关 键 词：嗜碱芽孢杆菌N16-5 乙酰酯酶 酶学性质 甘露聚糖 

分 类 号：Q946.5[生物学—植物学]