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

摘要

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.