Cloning and expression in E. coli of an organic solvent-tolerant and alkali-resistant glucose 1-dehydrogenase from Lysinibacillus sphaericus G10.
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.
Ding
Hai-Tao
HT
Institute of Microbiology, College of Life Science, Zhejiang University, Hangzhou 310058, China.
Du
Yi-Qing
YQ
Liu
Dan-Feng
DF
Li
Ze-Li
ZL
Chen
Xue-Jiao
XJ
Zhao
Yu-Hua
YH
D000468:Alkalies / Q000494:pharmacology*; D000595:Amino Acid Sequence; D001407:Bacillus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D005798:Genes, Bacterial / Q000235:genetics; D042843:Glucose 1-Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007477:Ions; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D009930:Organic Chemicals / Q000494:pharmacology*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012997:Solvents / Q000494:pharmacology*; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature
D000468:Alkalies; D007477:Ions; D008670:Metals; D009930:Organic Chemicals; D012997:Solvents; D004492:Edetic Acid; D042843:Glucose 1-Dehydrogenase