Cloning，Expression and Characterization of Gluconate 5-dehydrogenase from Gluconobacter oxydans

Abstract

Abstract: Guconate 5-dehydrogenase gene from Gluconobacter oxydans H24 was amplified with primes designed based on NCBI database published sequence information. Ga5DH was cloned and expressed in Escherichia coli Rosetta strain. Results showed that the molecular mass of the enzyme was 26.5 kD by SDS-PAGE gel analysis. After purification, the enzyme activity could reach 7.83 U/mg. It was suggested that the recombinant protein had a maximum activity at 40℃, pH 11. The characterization of purified enzyme indicated that Ga5DH was highly resistant to organic solvents.

Key words: Gluconate 5-dehydrogenase, Gluconobacter oxydans, Expression, Enzymatic properties

Li Ling, Xu Lin,Wei Miao, Li Yan, Yan Ming. Cloning，Expression and Characterization of Gluconate 5-dehydrogenase from Gluconobacter oxydans[J]. Biotechnology Bulletin, 2014, 0(9): 157-163.

References

［1］Adachi O, Shinagawa E, Matsushita K, et al. Crystallization and properties of 5-keto-D-gluconate reductase from Gluconobacter sub-oxydans［J］. Agricultural and Biological Chemistry, 1979, 43（1）：75-83.
［2］Silberbach M, Maier B, Zimmermann M, et al. Glucose oxidation by Gluconobacter oxydans：characterization in shaking-flasks, scale-up and optimization of the pH profile［J］. Applied Microbiology and Biotechnology, 2003, 62（1）：92-98.
［3］Hanke T, N?h K, Noack S, et al. Combined fluxomics and transcriptomics analysis of glucose catabolism via a partially cyclic pentose phosphate pathway in Gluconobacter oxydans 621H［J］. Applied and Environmental Microbiology, 2013, 79（7）：2336-2348.
［4］Klasen R, Bringer-Meyer S, Sahm H. Biochemical characterization and sequence analysis of the gluconate：NADP 5-oxidoreductase gene from Gluconobacter oxydans［J］. Journal of Bacteriology, 1995, 177（10）：2637-2643.
［5］Elfari M, Ha SW, Bremus C, et al. A Gluconobacter oxydans mutant converting glucose almost quantitatively to 5-keto-D-gluconic acid［J］. Applied Microbiology and Biotechnology, 2005, 66（6）：668-674.
［6］Matzerath I, Kl?ui W, Klasen R, et al. Vanadate catalysed oxidation of 5-keto-D-gluconic acid to tartaric acid：the unexpected effect of phosphate and carbonate on rate and selectivity［J］. Inorganica Chimica Acta, 1995, 237（1）：203-205.
［7］袁建锋, 吴绵斌, 林建平, 等. 基于 5-酮基-D-葡萄糖酸生物制造 L-（+）-酒石酸的研究进展［J］. 现代化工, 2013, 33（9）：13-16.
［8］Merfort M, Herrmann U, Bringer-Meyer S, et al. High-yield 5-keto-d-gluconic acid formation is mediated by soluble and membrane-bound gluconate-5-dehydrogenases of Gluconobacter oxydans［J］. Applied Microbiology and Biotechnology, 2006, 73（2）：443-451.
［9］Ameyama M, Chiyonobu T, Adachi O. Purification and properties of 5-ketogluconate reductase from Gluconobacter liquefaciens［J］. Agricultural and Biological Chemistry, 1974, 38（7）：1377-1382.
［10］Galante E, Lanzani GA, Sequi P. Variations of 2-ketogluconate and 5-ketogluconate oxidoreductases during growth in Acetobacter suboxydans［J］. Enzymologia, 1966, 30（4）：257-264.
［11］Zhang Q, Peng H, Gao F, et al. Structural insight into the catalytic mechanism of gluconate 5-dehydrogenase from Streptococcus suis：Crystal structures of the substrate-free and quaternary complex enzymes［J］. Protein Science, 2009, 18（2）：294-303.
［12］Kubota K, Miyazono K, Nagata K, et al. Crystallization and preliminary X-ray analysis of 5-keto-D-gluconate reductase from Gluconobacter suboxydans IFO12528 complexed with 5-keto-D-gluconate and NADPH［J］. Acta Crystallographica Section F：Structural Biology and Crystallization Communications, 2010, 66（12）：1680-1682.
［13］Ge X, Zhao Y, Hou W, et al. Complete genome sequence of the industrial strain Gluconobacter oxydans H24［J］. Genome Announcements, 2013, 1（1）. doi：10.1128/genomeA.00003-13.
［14］王嘉乐, 魏东芝, 林金萍. 氧化葡萄糖酸杆菌胞内脱氢酶 Gox0525 的酶学性质研究［J］. 中国医药工业杂志, 2011, 42（10）：742-746.
［15］Green M R, Sambrook J. Molecular cloning：a laboratory manual［M］. New York：Cold Spring Harbor Laboratory Press, 2012.
［16］Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding［J］. Analytical Biochemistry, 1976, 72（1）：248-254.

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