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Enhanced expression of active recombinant alginate lyase AlyPEEC cloned from a marine bacterium Pseudoalteromonas elyakovii in Escherichia coli by calcium compounds

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Title: Enhanced expression of active recombinant alginate lyase AlyPEEC cloned from a marine bacterium Pseudoalteromonas elyakovii in Escherichia coli by calcium compounds
Authors: Sawabe, T. Browse this author →KAKEN DB
Takahasi, H. Browse this author
Saeki, H. Browse this author →KAKEN DB
Niwa, K. Browse this author
Aono, H. Browse this author
Keywords: Alginate lyase
Pseudoalteromonas elyakovii
AlyPEEC
Calcium
Expression
Issue Date: 4-Jan-2007
Publisher: Elsevier Inc.
Journal Title: Enzyme and Microbial Technology
Volume: 40
Issue: 2
Start Page: 285
End Page: 291
Publisher DOI: 10.1016/j.enzmictec.2006.04.008
Abstract: Recombinant protein production in Escherichia coli is well suited to applications in the basic and applied sciences due to its associated simplicity, cost-effectiveness and the large number of genetic strategies available. However, the active form of the marine bacterial enzyme, alginate lyase, was difficult to express in E. coli cells under standard culture conditions. In this study, we found various calcium compounds that enhanced the expression of the active enzyme. The alyPEEC gene encoding extracellular alginate lyase of Pseudoalteromonas elyakovii IAM 14594 was comprised of a 1197 bp open reading frame encoding 398 amino acid residues with the domain G165 to N398 functioning as the mature enzyme. Three clones, pTPB24 with a 2.7 kb insert containing alyPEEC gene and paeX and their respective promoter regions, pTPB31 with a 1.6 kb insert containing only the alyPEEC gene its own promoter, and pCD11 containing the truncated domain encoding G165 to N398 of AlyPEEC inserted into the pTrcHisB expression vector were constructed and their expression was analyzed. Alginate lyase activity for the three clones was detected in cell-free extract cultured in LB broth containing 50% artificial seawater (ASW), but not in media with LB broth alone. Maximum activity was observed in the clones cultured in broth containing 50–100% ASW, respectively. Further expression analysis using one-by-one element deficient ASW showed that calcium sulfate affected active AlyPEEC expression. Furthermore, in contrast to inorganic calcium, calcium lactate, glyceric acid calcium and calcium propionate enhanced active AlyPEEC expression markedly.
Relation: http://www.sciencedirect.com/science/journal/01410229
Type: article (author version)
URI: http://hdl.handle.net/2115/18852
Appears in Collections:水産科学院・水産科学研究院 (Graduate School of Fisheries Sciences / Faculty of Fisheries Sciences) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 澤辺 智雄

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