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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

Paper ID Volume ID Publish Year Pages File Format Full-Text
18291 42717 2007 7 PDF Available
Title
Enhanced expression of active recombinant alginate lyase AlyPEEC cloned from a marine bacterium Pseudoalteromonas elyakovii in Escherichia coli by calcium compounds
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.

Keywords
LB, Luria-Bertani medium; BCIP/NBT, 5-bromo-4-chloroindolyl phosphate/nitroblue tetrazorium; CBB, Coomassie Brilliant BlueAlginate lyase; Pseudoalteromonas elyakovii; AlyPEEC; Calcium; Expression
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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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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 40, Issue 2, 4 January 2007, Pages 285–291
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us