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王青艳,申乃坤,朱婧,秦艳,朱绮霞,谢能中,李亿,黄日波.新型Ⅰ型普鲁兰酶基因的克隆表达及酶学性质[J].广西科学院学报,2016,32(2):136-145. [点击复制]
- WANG Qingyan,SHEN Naikun,ZHU Jing,QIN Yan,ZHU Qixia,XIE Neng-zhong,LI Yi,HUANG Ribo.Cloning, Expression and Enzymatic Characterization of a New TypeⅠ Pullulanase Gene[J].Journal of Guangxi Academy of Sciences,2016,32(2):136-145. [点击复制]
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| 新型Ⅰ型普鲁兰酶基因的克隆表达及酶学性质 |
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王青艳, 申乃坤, 朱婧, 秦艳, 朱绮霞, 谢能中, 李亿, 黄日波
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| (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) |
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| 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 flagellatuss GST4的普鲁兰酶基因pul B,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]pul B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pul B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,pul B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 |
| DOI： |
| 投稿时间：2016-03-23 |
| 基金项目: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和广西科学院基本科研业务费项目(15YJ22SW01)资助。 |
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| Cloning, Expression and Enzymatic Characterization of a New TypeⅠ Pullulanase Gene |
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WANG Qingyan, SHEN Naikun, ZHU Jing, QIN Yan, ZHU Qixia, XIE Neng-zhong, LI Yi, HUANG Ribo
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| (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) |
| Abstract: |
| [Objective] Screening, cloning, heterologous expression of the pullulanase encoding gene from Tumebacillus flagellatuss 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 Nichelating affinity chromatography from cell free culture supernatant and characterization of pullulanase were carried out.[Results] The pullulanase gene pul B 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 MichaelisMenten kinetics, where its Km is(16.28±0.03) mg/mL andVmax 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 pul B, 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 flagellatuss cloning and expression enzymatic characterization |
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