耐热β-甘露聚糖酶基因的克隆与表达及酶学性质  

Gene Cloning and Expression of a Thermostable β-Mannanase and Its Enzymatic Properties

作　　者：赵梅[1] 魏喜换[1] 王春娟[1] 董运海[1] 李剑芳[1] 邬敏辰[2] 

机构地区：[1]江南大学食品学院,江苏无锡214122 [2]江南大学无锡医学院,江苏无锡214122

出　　处：《食品与生物技术学报》2014年第6期590-596,共7页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金项目(31271811)

摘　　要：基于生物信息学和基因组学分析的结果,采用RT-PCR技术从宇佐美曲霉（AspergiLLus usamii）E001中克隆出一种糖苷水解酶（GH） 26家族β-甘露聚糖酶（AuMan26A）成熟肽编码基因（A uman26A）,成功实现其在毕赤酵母（Pichia pastoris）GS115中的异源表达.重组β-甘露聚糖酶（reAuMan26A）的发酵上清液对角豆胶的酶活性为281.9 U/mL,纯化后比酶活为2281.7 U/mg.其最造反应温度Topt为40℃;在80℃处理60 min后残留酶活性为60％;90℃时的半衰期T1/290为10 min,处理60 min后残留酶活性仍为33％;其最适pH为5.5,在pH 5.0～7.0的范围内较稳定;Fe^2＋和Fe^3＋对其有明显的抑制作用,其它所测金属离子和EDTA对其没有明显的影响;所测酶的最适底物为角豆胶,其次是魔芋粉和瓜尔豆胶,其对角豆胶的Km和Vmax值分别为15.25 mg/mL和7 841.9 U/mg.reAuMan26A良好的热稳定性使其在食品、饲料和纺织等工业具有广阔的应用前景.Based on the bioinformatics and genomics analysis,a glycoside hydrolase （GH） family 26 gene,Auman26A,which encodes β-mannanase mature peptide was cloned from Aspergillus usamii E001 by RT-PCR.Then,it was successfully expressed in Pichia pastoris GS115.The recombinant enzyme （reAuMan26A） activity of the fermentation supernatant towards locust bean gum was 281.9 U/mL.The specific activity of the purified enzyme was 2281.7 U/mg.The optimal temperature of reAuMan26A was 40 ℃.After incubating at 80 ℃ and 90 ℃ for 60 min,the residual activities were 60% and 33%,respectively.The half-life （t1/290） of reAuMan26A at 90 ℃ was 10 min.The optimal pH of reAuMan26A was 5.5,and it was stable over a range of pH 5.0～7.0.Its activity was not significantly affected by metal ions and EDTA,but inhibited by Fe^2＋ and Fe^3＋.Its most favorable substrate was locust bean gum,followed by konjac flour and guar gum.The Km and Vmax of reAuMan26A towards locust bean gum were 15.25 mg/mL and 7 841.9 U/mg,respectively.The superior properties of reAuMan26A,especially the excellent thermostability,make it have the broad application prospects in food,paper making and textile industry.

关 键 词：Β-甘露聚糖酶 耐热性 基因克隆和表达 酶学性质 

分 类 号：TS201.25[轻工技术与工程—食品科学]