木聚糖酶基因XynB在毕赤酵母中的表达及酶学性质研究  

Expression and enzymological properties of XylB gene xylanase in Pichia pastoris

作　　者：马腾飞[1] 杨江科[2] 韩正刚 肖璐梅 晁群芳[1] MA Teng-fei1, YANG Jiang-ke2, HAN Zheng-gang2, XIAO Lu-mei1, CHAO Qun-fang1(1 .College of Life Sciences and Technology, Xinjiang University, Urumqi 830046; 2.School of Biology and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan 43002)

机构地区：[1]新疆大学生命科学与技术学院,乌鲁木齐830046 [2]武汉轻工大学生物工程与制药学院,武汉430023

出　　处：《食品科技》2018年第5期1-9,共9页Food Science and Technology

基　　金：国家"863"项目(2007AA05Z417;2009AA03Z232)

摘　　要：目的:将来源于Thermotoga maritima的酸性耐高温木聚糖酶基因Xyn B通过密码子优化整合到毕赤酵母GS115基因组上,构建高效表达木聚糖酶Xyn B的毕赤酵母工程菌并研究其酶学性质。方法:通过分子克隆手段构建目的基因重组质粒以及二拷贝重组质粒,并将重组质粒导入毕赤酵母进行发酵表达,用DNS法测定发酵液酶活,并对酵母工程菌分泌的木聚糖酶进行一系列的酶学性质研究。结果:木聚糖酶的最适p H和温度分别为6.0和60℃;在p H4~6之间,稳定性较好,相对比酶活均在80%以上;在60℃和70℃保温2 h残留酶活分别为80%和70%左右;Co^(2+)对木聚糖酶活性有较明显的促进作用,Fe^(2+)、K^+和Zn^(2+)对酶的活性有微弱的促进作用,而Cu^(2+)、Ca^(2+)、Mg^(2+)、Mn^(2+)、Na^+对木聚糖酶活性有不同程度的抑制作用。TLC检验该木聚糖酶可以将木聚糖水解为二糖和少量单糖。通过10 L发酵罐发酵,最大酶活达到了8000 U/m L。结论:木聚糖酶在毕赤酵母中实现了高效表达,并获得了木聚糖酶的最适反应条件等一系列酶学性质,该酶性质优良适合用于饲料添加剂。Objective：To integrate the acid hyperthermophilic xylanase gene Xyn B from Thermotoga maritima on the Pichia pastoris GS115 genome through codon optimization.The Pichia pastoris engineering bacteria of the highly expressed xylanase Xyn B was constructed and its enzymatic property was studied.Methods：The target gene recombination plasmid and dual-copy recombination plasmid was constructed via molecular clone.The recombination plasmid was imported into the pichia pastoris to make fermentation expression.Use DNS method to measure the enzyme activities of fermentation broth,and make a series research on the enzymatic properties of xylanase excreted by the yeast engineering bacteria.Results：The most appropriate p H and temperature of xylanase were 6.0 and 60℃,respectively.It had a better stability at p H 46,and the most relative specific enzyme activities were above 80%.The residual enzyme activities at 60℃and 70℃after keeping the temperatures for 2 h were about 80%and70%.Co（2＋）had an obvious effect in accelerating the enzyme activity of xlynase,Fe（2＋）,K＋and Zn（2＋）had a weak effect in accelerating the enzyme activity,while Cu（2＋）,Ca（2＋）,Mg（2＋）,Mn（2＋）and Na＋can inhibit the enzyme activity of xylanase at different levels.TLC inspection showed that the xylanase could hydrolyze the xylan into disaccharide and a bit of monosaccharide.After the xylan was fermented in a 10 L fermentation tank,the maximum enzyme activity reached 8000 U/m L.Conclusion：Xylanase realized the high expression in Pichia pastoris,and a series of enzymatic properties such as the appropriate reaction condition of xylanase was obtained.The properties of this enzyme could be excellent and suitable for feed additives. Key words：

关 键 词：酸性 耐高温 木聚糖酶 酵母表达 酶学性质 

分 类 号：TS201.2[轻工技术与工程—食品科学]