title: Calcium leads to further increase in glycine-enhanced extracellular secretion of recombinant alpha-cyclodextrin glycosyltransferase in Escherichia coli.
abstract: 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 alpha-cyclodextrin glycosyltransferase (alpha-CGTase) from Paenibacillus macerans strain JFB05-01. It was found that glycine, as a medium supplement, could enhance the extracellular secretion of recombinant alpha-CGTase in E. coli. In the culture with glycine at the optimal concentration of 150 mM, the alpha-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 alpha-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, Ca(2+) could remedy cell growth inhibition induced by glycine, thereby leading to further increase in the glycine-enhanced extracellular secretion of recombinant alpha-CGTase. In the culture with 150 mM glycine and 20 mM Ca(2+), 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 Ca(2+) on the extracellular secretion of a recombinant protein in E. coli.
authors: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, People's Republic of China.
authors: Zhao-Feng
authors: ZF
authors: Li
authors: Bin
authors: B
authors: Li
authors: Zhi-Guo
authors: ZG
authors: Liu
authors: Miao
authors: M
authors: Wang
authors: Zheng-Biao
authors: ZB
authors: Gu
authors: Guo-Cheng
authors: GC
authors: Du
authors: Jing
authors: J
authors: Wu
authors: Jian
authors: J
authors: Chen
mesh_terms: D001419:Bacteria / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology*; D002463:Cell Membrane Permeability / Q000187:drug effects; D004357:Drug Synergism; D004926:Escherichia coli / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism*; D015870:Gene Expression; D005964:Glucosyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D005998:Glycine / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; D002118:Calcium; D005998:Glycine