Gluconobacter oxydans is widely used in industrial application for its dehydrogenase system locating on cell membrane. These dehydrogenases have a character to oxidize sugars and sugar alcohols incompletely. There are two potential pathways known for glucose oxidization in G. oxydans: More than 90% of glucose is transformed into gluconate in the periplasmic space; only a minority of glucose (about 5%) is phosphorylated and taken into functional central metabolic pathways such as Entner-Doudoroff pathway (EDP) and pentose phosphate pathway (PPP) in the cytoplasmic compartment. In previous study, the Embden-Meyerhof-Parnas pathway (EMP) was found inactive in G. oxydans due to its lack of phosphofructokinase.In this study, a G. oxydans strain named DHA3-9 was screened which produced dihydroxyacetone (DHA) during glucose degradation. But DHA was not a product in EDP or PPP. A mutant strain of G. oxydans DHA3- 9 lacking of glucose dehydrogenase in cell membrane was constructed to study the possibility of other pathway of glucose metabolism in G. oxydans.A mgdh gene-disrupted mutant of G. oxydans DHA3-9 was constructed by the way of homologous recombination and its characteristic changes of the cells growth on glucose, glucose degradation, gluconate transformation, intermediate products and growth inhibition on acetate were studied.The results indicated that the growth of mutant strain on glucose showed an obvious delay and pH dropped much slower than that of wild type. The mutant lost most of its ability of glucose degradation and produced little gluconate. Instead, DHA formation of the mutant was recorded four times as that of wild type. Pyruvate and acetate were detected in the products of mutant whereas none of such products were found in wild type culture. Under the condition with glucose as the sole carbon source, 50 mmol/L acetate completely inhibited the growth of mutant, whereas this effect was remarkably low on wild type.These results prove that in G. oxydans DHA3-9 mutant strain, glucose is utilized in cytoplasmic compartment primarily through EMP and acetate can be produced by activities of pyruvate decarboxylase and acetaldehyde dehydrogenase.

OBJECTIVE:Gamma-butyrobetaine hydroxylase is an enzyme that catalyzes the last step in the biosynthesis of L-carnitine. We cloned, expressed and characterized a gamma-butyrobetaine hydroxylase gene bbh from Pseudomonas sp. L-1, to facilitate the production of L-carnitine using microorganisms.METHODS:We cloned bbh gene by PCR, and then cloned the open reading frame of bbh into pET-15b vector and expressed by Isopropyl beta-D-1-thiogalactopyranoside (IPTG) induction. After His-Bind Resin purification, the characteristics of BBH were studied. The three-dimensional structure of BBH monomer was modeled by SWISS-MODEL Workspace and resting cells were used for L-carnitine transformation.RESULTS:We cloned a gamma-butyrobetaine hydroxylase gene bbh (GenBank: JQ250036) from Pseudomonas sp. L-1 and expressed the gene in Escherichia coli BL21(DE3). BBH fusion protein was a homodimer, and the molecular weight of subunit was about 46.5kDa. The optimal temperature and pH was 30 degrees C and pH 7.5. The enzyme was stable below 45 degrees C. The enzyme was most stable at pH 6.0. We used resting cells of recombinant E. coli for L-carnitine biotransformation, after incubated at 30 degrees C and pH 7.0 for 31 h, the concentration of L-carnitine reached 12.7 mmol/L.CONCLUSION:The bbh gene from Pseudomonas sp. L-1 strain is remarkably different from that of reported one. The gamma-butyrobetaine hydroxylase expressed by this gene could effectively transform gamma-butyrobetaine for L-carnitine production. Beside by reporting of a bbh gene from bacteria, this research also provided a new process for biotransformation of L-carnitine.

In order to characterize a thermostable urate oxidase (Uox) from Microbacterium sp. strain ZZJ4-1, we cloned its gene (uox). The open reading frame of uox contained 894 base pairs and encoded a protein with 297 amino acids. Alignment of gene sequences indicated there was no obvious identity with the most reported uox and that 72% identity was found with uox from Arthrobacter globiformis. We inserted the gene into the plasmid pET-15b to construct an expression vector pET-15b-uox and got it induced expression in Escherichia coli BL21 (DE3). After the purification of the recombinant Uox by the HisBind column, we studied some properties of it. It was composed of subunits with a molecular mass of about 35 kDa. The optimal temperature and pH was 30 degrees C and pH 7.5. It was stable below 65 degrees C and from pH 8.5 to 11.0. The Km value was 0.22 mmol/L with the uric acid as the substrate. Ag+, Zn2+, CU2+ and SDS could totally inhibit its activity while Tween 20, Tween 80 and Triton X-100 had a slight promotion effect. The thermal stability of this enzyme was the most excellent among the reported recombinant Uox. Based on this property, it would be very useful in the application.

Response surface methodology was applied to optimise the enzymatic transformation process for enhancement of the dihydroxyacetone(DHA) fermentation concentration.It was demonstrated that with the parameters of a pH of 4.61,a temperature of 33.3 ℃ and the concentration of glycerol at 80.0 g·L-1,the glycerol dehydrogenase(GDH) had an optimum activity.GDH was stable in a narrow pH range,from 4.0 to 5.0,and at temperatures below 25 ℃;further,100 g·L-1 glycerol or 50 g·L-1 DHA were best to stabilise GDH.As the parameters for optimal microbial fermentation and GDH catalysis differed markedly,a two-stage process of enzymatic transformation was developed in a 5-L bioreactor that separated the growth of G.oxydans and the biotransformation process.This methodology showed high stability of GDH in a 136-hour transformation process and elevated DHA production to as high as 286.2 g·L-1.

We purified a sarcosine oxidase from Bacillus sp. strain BSD-8 isolated from soil. We purified the enzyme by ammonium sulfate precipitation, DEAE-cellulose, Toyopearl hydrophobic and Sephadex G-75 molecular sieve chromatography and characterized the purified sarcosine oxidase. This sarcosine oxidase was a flavin enzyme containing a noncovalently bound flavin with the subunit molecular mass of 51 kDa. The optimal temperature for this enzyme was 60 degrees C and it showed its highest activity at pH 8.5. It was stable in the pH range of 8.0-10.0 and at the temperature of 60 degrees C. Estimated by Lineveaver-Burk plots, the K(m) of the enzyme was 3.1 mmol/L. Ag+, Hg2+, SDS and Tween 80 dramatically inhibted the enzyme activity, whereas Tween 20 and Triton X-100 had no effect on enzyme activity. The thermostability of this enzyme was better than reported sarcosine oxidases, and it could be applied in enzymatic measuring of creatinine.