中国农业科学院农业信息研究所 中国农学会
Agricultural Information Institute of CAAS China Association of Agricultural Science Societies
En
Ch
中国农业科学院农业信息研究所 中国农学会 主办

AI检索

订阅

返回顶部
当前位置: 首页 > 文章 > 易错PCR技术定向进化褐藻胶裂解酶Alg-2的分析 食品科学 2019 (4) 146-151
Position: Home > Articles > Directed Evolution of Alginate Lyase Alg-2 Based on Error Prone PCR FOOD SCIENCE 2019 (4) 146-151

易错PCR技术定向进化褐藻胶裂解酶Alg-2的分析

作  者：
李树;张伟;赵春梅
单  位：
山东大学(威海)海洋学院;江南大学生物工程学院工业生物技术教育部重点实验室
关键词：
易错PCR;定向进化;褐藻胶裂解酶;氨基酸序列
摘  要：
为提高褐藻胶裂解酶活力,采用易错聚合酶链式反应(polymerase chain reaction,PCR)技术对海洋细菌Tamlanasp.S12中具有代表性的酶组分Alg-2进行体外定向进化。经过2轮易错PCR及96孔板发酵筛选,分别获得2株正突变和2株负突变菌株,其酶比活力分别是亲本(2 675.2 U/g)的162%、241%、53%、11%。酶的动力学分析显示,正突变株P2-81的Km值比亲本降低50%,而负突变株N2-47的Km值比亲本提高106%,表明突变株对底物的亲和力有极大的上升和下降。基因测序及氨基酸序列分析结果表明,Glu、Thr、Ser、Asp和Tyr对褐藻胶裂解酶活力提高起到正面的积极作用,而Lys的突变起到负面的消极作用,后续人工合成的定点突变则进一步证实Asp和Lys的正/负作用。本研究有助于深入理解褐藻胶裂解酶的催化机制,为后续利用理性设计手段改造褐藻胶裂解酶提供理论参考。
译  名：
Directed Evolution of Alginate Lyase Alg-2 Based on Error Prone PCR
作  者：
LI Shu;ZHANG Wei;ZHAO Chunmei;Marine College, Shandong University (Weihai);Key Laboratory of Industry Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University;
单  位：
Weihai
关键词：
error prone PCR;;directed evolution;;alginate lyases;;amino acid sequence
摘  要：
In order to improve the enzymatic activity of alginate lyase Alg-2, directed evolution based on error prone PCR was carried out on the marine bacterium Tamlana sp. S12. After two rounds of error prone PCR and 96-well plate fermentation, two positive and two negative mutants were obtained with 162%, 241%, 53% and 11% higher enzymatic activity as compared with the control, respectively. According to kinetic analysis, Km value of the positive strain P2-81 was reduced by 50% while that of the negative strain N2-47 was increased by 106% as compared with the parental one, implying that the substrate affinity of the enzyme in the mutant strains is greatly increased or reduced. The results of gene sequencing and amino acid sequence analysis showed that Glu, Thr, Ser, Asp and Tyr played a positive role in the increased activity of alginate lyase whereas mutation of Lys in the conserved region had a negative effect. The role of Asp and Lys was further confirmed by directed mutations. The results of this study can be helpful to gain a deep understanding of the catalytic mechanism of alginate lyases and to provide a theoretical basis for alginate lyases reconstruction using rational design methods.

相似文章

导出引用
计量
文章访问数: 14
HTML全文浏览量: 0
PDF下载量: 0

所属期刊

推荐期刊

友情链接Friendly link
农业信息研究所Agricultural information institute 农业智能知识服务平台Agricultural intelligent knowledge service platform 农业学术服务平台Agricultural Knowledge Service System 中国知网CNKI 维普网VIP 万方WANFANG DATA
联系我们Contact us

电话Tell：010-82109652-808

邮箱E-mail：agrijournal@caas.cn

服务Service
关于我们About us 意见建议Opinions and suggest
版权说明Copyright
单位：中国农业科学院农业信息研究所 中国农学会 © 2017中国农业科学院农业信息研究所 ICP备案号：京ICP备09089781号-46