新型Ⅰ型普鲁兰酶基因的克隆表达及酶学性质  

Cloning,Expression and Enzymatic Characterization of a New TypeⅠ Pullulanase Gene

作　　者：王青艳[1] 申乃坤[1] 朱婧[1] 秦艳[1] 朱绮霞[1] 谢能中[1] 李亿[1] 黄日波[1] 

机构地区：[1]广西科学院、国家非粮生物质能源工程技术研究中心、非粮生物质酶解国家重点实验室、广西生物炼制重点实验室,广西南宁530007

出　　处：《广西科学院学报》2016年第2期136-145,共10页Journal of Guangxi Academy of Sciences

基　　金：国家自然科学基金项目(31160023;31400079);广西科学研究与技术开发计划项目(桂科合14123001-19;桂科合15104001-1);广西自然科学基金项目(2015GXNSFBA139044);广西“八桂学者”、南宁市特聘专家和留学人员科技活动项目择优资助项目;广西科学院基本科研业务费项目(15YJ22SW01)资助

摘　　要：【目的】筛选并克隆表达高酶活且具有-定热稳定性的新型普鲁兰酶.【方法】克隆 Turne bacillus/ Zaelu GST4 的普鲁兰酶基因构建重组质粒后转化宿主菌大肠杆菌进行诱导表达,再运用亲和层析进行纯化并分析其酶学性质和结构.【结果】在大肠杆菌中实现可溶性表达,发酵液上清酶活力达到78 U / m L,粗酶液经纯化后比活力为258 U /mg.重组酶P u l B 最适反应温度和p H 值分别为55曟和5 . 0 ,在较窄的酸性范围内（ 日值4 . 5 -5 . 5 ） 酶活力比较稳定;对普鲁兰糖的Km= （ 1 6 . 2 8 ± 0 . 0 3 ） mg/mL , Vmax =（22. 0 5 ± 0 . 0 2 ） μmol . m i n^-1 . mg^-1.P u l B 的D N A 序列与GenBank数据库里的任何序列都没有同源性,在蛋白质序列上,由基因pulB 编码的氨基酸序列与T.aegyptius的环麦芽糖糊精酶相似性最高,B l a s t X 比对的Identities为54 % Positives 为69 % ,SMART 结构预测分析发现,pulB 具有淀粉酶的结构域.底物特异性分析表明,它可水解普鲁兰糖和支链淀粉生成线性的低聚糖或麦芽三糖.【结论】重组酶pilB是尚未报道的新型普鲁兰酶,它可水解普鲁兰糖和支链淀粉,属栺型普鲁兰酶.【Objective】Screening,cloning,heterologous expression of the pullulanase encoding gene fromTumebacillus flagellatus GST4 in order to obtain efficient expression of a new pullulanase and enhance its activity and thermosatbility.【Methods】Cloning,construction of recombinant and heterologous expression of pullulanase gene in Escherichia coli,purification by Ni-chelating affinity chromatography from cell free culture supernatant and characterization of pullulanase were carried out.【Results】The pullulanase gene pulB was cloned and expressed successfully in E.coli,and the activity of cultural supernatant can reach 78 U/mL.And PulB was purified to homogeneity and the specific activity was 258 U/mg.The optimal temperatureof purified PulB is 50℃,its optimal pH value is 5.0and activity remains stable within the acidic range of pH 4.5~5.5.PulB displayed typical Michaelis-Menten kinetics,where its Km is(16.28±0.03)mg/mL and Vmaxis(22.05±0.02)μmol·min-1·mg-1,respectively,when used pullulan as substrate.GenBank blast results show that there is no homologous DNA sequences with pulB,and the encoding protein of PulB had the highest identity(54%)with cyclomaltodextrinase fromThermicanus aegyptius.We find that it has amylase structure domain by online SMART searching.The substrate specificity analysis shows that it typically hydrolyze pullulan and amylopectin to produce liner oligosaccharides or maltotriose.【Conclusion】PulB is a new starch/pullulan hydrolase,which has not yet been reported.It can hydrolyze pullulan or amylopectin and belong to type I pullulanase.

关 键 词：Ⅰ型普鲁兰酶 膨胀芽孢杆菌 克隆表达 酶学性质 

分 类 号：Q93[生物学—微生物学]