1. Cloning and expression in E. coli of an organic solvent-tolerant and alkali-resistant glucose 1-dehydrogenase from Lysinibacillus sphaericus G10.
- Author
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Ding HT, Du YQ, Liu DF, Li ZL, Chen XJ, and Zhao YH
- Subjects
- Amino Acid Sequence, Bacillus drug effects, Cloning, Molecular, Edetic Acid pharmacology, Electrophoresis, Polyacrylamide Gel, Enzyme Stability drug effects, Escherichia coli drug effects, Genes, Bacterial genetics, Glucose 1-Dehydrogenase chemistry, Glucose 1-Dehydrogenase isolation & purification, Hydrogen-Ion Concentration drug effects, Ions, Kinetics, Metals pharmacology, Molecular Sequence Data, Sequence Alignment, Sequence Analysis, DNA, Substrate Specificity drug effects, Temperature, Alkalies pharmacology, Bacillus enzymology, Bacillus genetics, Escherichia coli metabolism, Glucose 1-Dehydrogenase genetics, Organic Chemicals pharmacology, Solvents pharmacology
- Abstract
The gene gdh encoding an organic solvent-tolerant and alkaline-resistant NAD(P)-dependent glucose 1-dehydrogenase (LsGDH) was cloned from Lysinibacillus sphaericus G10 and expressed in Escherichia coli. The recombinant LsGDH exhibited maximum activity at pH 9.5 and 50 °C. LsGDH displayed high stability at a wide pH ranging from 6.5 to 10.0 and was stable after incubation at 30 °C for 1 week in 25 mM sodium phosphate buffer (pH 6.5) in the absence or presence of NaCl. The activity of LsGDH was enhanced by Li+, Na+, K+, NH4+, Mg2+, and EDTA at pH 8.0. LsGDH exhibited high tolerance to 60% DMSO, 30% acetone, 30% methanol, 30% ethanol, 10% n-propanol, 30% isopropanol, 60% n-hexanol and 30% n-hexane. The relationship between stability and chain length of the alcohols fit a Gaussian distribution model (R2≥0.94), and demonstrated lowest enzyme stability in C4-alcohol. The results suggested that LsGDH was potentially useful for coenzyme regeneration in organic solvents or under alkaline conditions., (Copyright © 2010 Elsevier Ltd. All rights reserved.)
- Published
- 2011
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