title: Effective approach to greatly enhancing selective secretion and expression of three cytoplasmic enzymes in Escherichia coli through synergistic effect of EDTA and lysozyme.
abstract: An effective approach to greatly enhancing the selective secretion and expression of recombinant cytoplasmic enzymes in Escherichia coli was successfully developed through the synergistic effect of ethylenediaminetetraacetate (EDTA) and lysozyme. The method was applied to two endoglucanases (EGs) and an amylase. The optimal culture conditions of temperature and concentration of isopropyl-β-D: -1-thiogalactopyranoside (IPTG) were 23-30 °C and 0.2 mM, respectively, under which the three enzymes could be expressed in active form. Among all the chemicals tested, EDTA was found to be most suitable for enhancing the secretion of EG-I-1A into the medium. Addition of lysozyme alone had little influence on the secretion and expression. In contrast, on the basis of the addition of 5 g EDTA/L at the induction time of 12 h, the simultaneous addition of 0.15 g lysozyme/L further significantly increased the secretion and expression of the three enzymes, demonstrating the synergistic effect of EDTA and lysozyme. The production of EG-I-1A in the culture medium by adding 5 g EDTA/L and 0.15 g lysozyme/L under the optimal culture conditions of 23 °C and 0.2 mM IPTG was over 260-fold higher than that without EDTA and lysozyme under the standard conditions of 37 °C and 1 mM IPTG. In summary, the advantage of this novel cultivation approach for secretion was that not only did it selectively enhance the secretion of the proteins of interest, but also greatly increased the expression of the three enzymes by over 80 %.
authors.affiliation: Shenzhen Key Laboratory for Microbial Gene Engineering, College of Life Science, Shenzhen University, Shenzhen 518060, People's Republic of China. liulsl@szu.edu.cn
authors.forename: Sen-Lin
authors.initials: SL
authors.lastname: Liu
authors.forename: Kun
authors.initials: K
authors.lastname: Du
authors.forename: Wei-Zhao
authors.initials: WZ
authors.lastname: Chen
authors.forename: Gang
authors.initials: G
authors.lastname: Liu
authors.forename: Miao
authors.initials: M
authors.lastname: Xing
mesh_terms: D000681:Amylases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D002480:Cellulase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology*; D004926:Escherichia coli / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D009113:Muramidase / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D003470:Culture Media; D004492:Edetic Acid; D000681:Amylases; D009113:Muramidase; D002480:Cellulase