Calcium Leads to Further Increase in Glycine-Enhanced Extracellular Secretion of Recombinant α-Cyclodextrin Glycosyltransferase in Escherichia coli
Calcium Leads to Further Increase in Glycine-Enhanced Extracellular Secretion of Recombinant α-Cyclodextrin Glycosyltransferase in Escherichia coli
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DOI：
10.1021/jf901239k
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发表时间：
2009-07-22
影响因子：
6.1
通讯作者：
Chen, Jian
中科院分区：
文献类型：
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作者：
Li, Zhao-Feng;Li, Bin;Chen, Jian
在大肠杆菌中高效表达重组基因并将重组蛋白定向到培养基中是生产工业酶的非常理想的方法。然而，一个主要的障碍是重组蛋白在大肠杆菌细胞的两层膜上分泌不足。在本研究中，我们试图解决淡色拟杆菌JFB05-01重组α-环糊精糖基转移酶(α-CGTase)的分泌问题。研究发现，添加甘氨酸能促进重组α-CGTase在大肠杆菌中的分泌。在添加150 mM甘氨酸的条件下，培养40h时，α-CGTase活力可达23.5U/mL，是普通TB培养基活力的11倍。重组α-CGTase的最大胞外产量也提高了2.3倍。然而，进一步的分析表明，补充甘氨酸会损害细胞的生长，表现为细胞数量和活力的减少，细胞裂解的增加，以及细胞形态的破坏，这阻碍了整体酶生产率的进一步提高。值得注意的是，钙离子可以纠正甘氨酸对细胞生长的抑制，从而进一步增加甘氨酸促进的重组α-CGTase的胞外分泌。在含150 mM甘氨酸和20 mM Ca~(2+)的培养条件下，重组酶的胞外活力和最大产酶能力均比单独使用甘氨酸时提高1.5倍。据我们所知，这是第一篇关于甘氨酸和钙离子对重组蛋白在大肠杆菌细胞外分泌的协同促进作用的文章。
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.