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
王青艳,申乃坤,朱婧,秦艳,朱绮霞,谢能中,李亿,黄日波
WANG Qingyan,SHEN Naikun,ZHU Jing,QIN Yan,ZHU Qixia,XIE Nengzhong,LI Yi,HUANG Ribo

(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)
(State Key Laboratory of Nonfood Biomass and Enzyme Technology,National Engineering Research Center for Nonfood Biorefinery,Guangxi Key Laboratory of Biorefinery,Guangxi Academy of Sciences,Nanning,Guangxi,530007,China)
摘要:【目的】筛选并克隆表达高酶活且具有一定热稳定性的新型普鲁兰酶。【方法】克隆Tumebacillus flagellatus GST4的普鲁兰酶基因pulB,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pulB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/mL,粗酶液经纯化后比活力为258 U/mg。重组酶PulB最适反应温度和pH值分别为55℃和5.0,在较窄的酸性范围内(pH值4.5~5.5)酶活力比较稳定;对普鲁兰糖的Km=(16.28±0.03)mg/mL,Vmax=(22.05±0.02)μmol·min-1·mg-1。PulB的DNA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,BlastX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PulB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Ⅰ型普鲁兰酶。
关键词:Ⅰ型普鲁兰酶   膨胀芽孢杆菌    克隆表达   酶学性质
中图分类号:Q93     文献标识码:A     文章编号:1002-7378(2016)02-0136-10
Abstract:【Objective】Screening,cloning,heterologous expression of the pullulanase encoding gene from Tumebacillus 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 temperature of purified PulB is 50℃,its optimal pH value is 5.0 and 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 Vmax  is (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 from Thermicanus 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.
Key words:typeⅠpullulanase,Tumebacillus flagellatus,cloning and expression,enzymatic characterization

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发布日期:2016/6/23