Optimization of pullulanase production in Escherichia coli by regulation of process conditions and supplement with natural osmolytes.
In this study, the effects of temperature, IPTG (Isopropyl β-D-1-thiogalactopyranoside) concentration, and osmolytes (proline, K-glutamate, and betaine) on cell growth and soluble pullulanase productivity of recombinant Escherichia coli were investigated. The yield of soluble pullulanase was found to be enhanced with decrease in cultivation temperature, lower IPTG concentration, and betaine supplementation in a shake flask. In addition, a modified two-stage feeding strategy was proposed and applied in a 3-L fermentor supplied with 20mM betaine, which achieved a dry cell weight of 59.3 g L(-1). Through this cultivation approach at 25 °C, the total soluble activity of pullulanase reached 963.9 U mL(-1), which was 8.3-fold higher than that observed without addition of betaine at 30 °C (115.8 U mL(-1)). The higher expression of soluble pullulanase in a scalable semisynthetic medium showed the potential of the proposed process for the industrial production of soluble enzyme.
State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China.
Xuguo
X
Duan
State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China.
Jian
J
Chen
State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China. Electronic address: jingwu@jiangnan.edu.cn.
Jing
J
Wu
D001622:Betaine / Q000737:chemistry; D018533:Biomass; D019149:Bioreactors; D003470:Culture Media / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004798:Enzymes / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D018698:Glutamic Acid / Q000737:chemistry; D005990:Glycerol / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000096:biosynthesis*; D007218:Industrial Microbiology / Q000379:methods*; D007544:Isopropyl Thiogalactoside / Q000737:chemistry; D011392:Proline / Q000737:chemistry; D012995:Solubility; D013696:Temperature; D013997:Time Factors
D003470:Culture Media; D004798:Enzymes; D007544:Isopropyl Thiogalactoside; D018698:Glutamic Acid; D001622:Betaine; D011392:Proline; D006026:Glycoside Hydrolases; C100181:pullulanase; D005990:Glycerol
Betaine; Escherichia coli; Pullulanase; Soluble expression