期刊
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
卷 57, 期 14, 页码 6231-6237出版社
AMER CHEMICAL SOC
DOI: 10.1021/jf901239k
关键词
Cyclodextrin glycosyltransferase; Paenibacillus macerans; extracellular secretion; glycine; Escherichia coli; Ca2+
资金
- Natural Science Foundation of Jiangsu Province [BK2007019]
- Major State Basic Research Development Program of China (973 Program) [2007CB714036]
- National Outstanding Youth Foundation of China [20625619]
- National High-Tech Research and Development Program of China (863 Program) [2006A-A10Z335]
- Research Program of State Key Laboratory of Food Science and Technology [SKLF-MB-200802]
- Graduate Student Creative Research Program of Jiangsu Province [CX08B_127Z]
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 a-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, Ca2+ 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 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.
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