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Calcium Leads to Further Increase in Glycine-Enhanced Extracellular Secretion of Recombinant α-Cyclodextrin Glycosyltransferase in Escherichia coli

Li, Zhao-Feng, Li, Bin, Liu, Zhi-Guo, Wang, Miao, Gu, Zheng-Biao, Du, Guo-Cheng, Wu, Jing, Chen, Jian
Journal of agricultural and food chemistry 2009 v.57 no.14 pp. 6231-6237
calcium, glycine (amino acid), extracellular fluids, protein secretion, recombinant DNA, glycosyltransferases, Escherichia coli, gene expression, cell culture, synergists
Overexpression of recombinant genes in Escherichia coli and targeting recombinant proteins to the culture medium are highly desirable for the production of industrial enzymes. However, a major barrier is inadequate secretion of recombinant protein across the two membranes of E. coli cells. In the present study, we have attempted to circumvent this secretion problem of the recombinant α-cyclodextrin glycosyltransferase (α-CGTase) from Paenibacillus macerans strain JFB05-01. It was found that glycine, as a medium supplement, could enhance the extracellular secretion of recombinant α-CGTase in E. coli. In the culture with glycine at the optimal concentration of 150 mM, the α-CGTase activity in the culture medium reached 23.5 U/mL at 40 h of culture, which was 11-fold higher than that of the culture in regular TB medium. A 2.3-fold increase in the maximum extracellular productivity of recombinant α-CGTase was also observed. However, further analysis indicated that glycine supplementation exerted impaired cell growth as demonstrated by reduced cell number and viability, increased cell lysis, and damaged cell morphology, which prevented further improvement in overall enzyme productivity. Significantly, Ca2+ could remedy cell growth inhibition induced by glycine, thereby leading to further increase in the glycine-enhanced extracellular secretion of recombinant α-CGTase. In the culture with 150 mM glycine and 20 mM Ca2+, both extracellular activity and maximum productivity of recombinant enzyme were 1.5-fold higher than those in the culture with glycine alone. To the best of our knowledge, this is the first article about the synergistic promoting effects of glycine and Ca2+ on the extracellular secretion of a recombinant protein in E. coli.