Cloning and expression of theClostridium thermocellumL-lactate dehydrogenase gene inEscherichia coliand enzyme characterization

Author:

Özkan Melek,Yllmaz Ebru I,Lynd Lee R,Özcengiz Gülay

Abstract

The structural gene for L-lactate dehydrogenase (LDH) (EC.1.1.1.27) from Clostridium thermocellum 27405 was cloned in Escherichia coli by screening the Lambda Zap II phage library of C. thermocellum genomic DNA. In one positive clone, an open reading frame of 948 base pairs corresponded to C. thermocellum ldh gene encoding for the predicted 315-residue protein. The ldh gene was successfully expressed in E. coli FMJ39 (ldh mutant) under the lac promoter. The recombinant enzyme was partially purified from E. coli cell extracts and its kinetic properties were determined. Clostridium thermocellum LDH was shown to catalyze a highly reversible reaction and to be an allosteric enzyme that is activated by fructose-1,6-diphosphate (FDP). For pyruvate, partially purified LDH had Kmand Vmaxvalues of 7.3 mmol/L and 87 µmol/min, respectively, and in the presence of FDP, a 24-fold decrease in Kmand a 5.7-fold increase in Vmaxwere recorded. The enzyme exhibited no marked catalytic activity for lactate in the absence of FDP, whereas Kmand Vmaxvalues were 59.5 mmol/L and 52 µmol/min, respectively, in its presence. The enzyme did not lose activity when incubated at 65 °C for 5 min.Key words: L-lactate dehydrogenase purification, thermophilic bacteria.

Publisher

Canadian Science Publishing

Subject

Genetics,Molecular Biology,Applied Microbiology and Biotechnology,General Medicine,Immunology,Microbiology

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3