Purification and biochemical characterization of a novel ene- reductase from Kazachstania exigua HSC6 for dihydro-ß-ionone from ß-ionone.

PURPOSE: We purified and characterized a novel ene-reductase (KaDBR1) from Kazachstania exigua HSC6 for the synthesis of dihydro-ß-ionone from ß-ionone. METHODS: KaDBR1 was purified to homogeneity by ammonium sulfate precipitation and phenyl-Sepharose Fast Flow and Q-Sepharose chromatography. The purified enzyme was characterized by measuring the amount of dihydro-ß-ionone from ß-ionone with LC-MS analysis method. RESULTS: The molecular mass of KaDBR1 was estimated to be 45 kDa by SDS-PAGE. The purified KaDBR1 enzyme had optimal activity at 60 °C and pH 6.0. The addition of 5 mM Mg2+, Ca2+, Al3+, Na+, and dithiothreitol increased the activity of KaDBR1 by 25%, 18%, 34%, 20%, and 23%, respectively. KaDBR1 favored NADH over NADPH as a cofactor, and its catalytic efficiency (kcat/Km) toward ß-ionone using NADH was 8.1-fold greater than when using NADPH. CONCLUSION: Owing to its unique properties, KaDBR1 is a potential candidate for the enzymatic biotransformation of ß-ionone to dihydro-ß-ionone in biotechnology applications.

A novel thermophilic ß-carotene 15,15'-monooxygenase with broad substrate specificity from the marine bacterium Candidatus Pelagibacter sp. HTCC7211.

To characterize a novel thermophilic ß-carotene 15,15'-monooxygenase BCMO7211 isolated from the marine bacterium Candidatus Pelagibacter sp. HTCC7211. BCMO7211 was functionally overexpressed in Escherichia coli and purified to homogeneity by Ni-NTA affinity chromatography and Superdex-200 gel filtration chromatography. Labeling experiments with H218O demonstrated that the oxygen atom in the terminal aldehyde group of the produced retinal molecules was provided from both molecular oxygen and water, indicating that BCMO7211 is the first characterized bacterial ß-carotene 15,15'-monooxygenase. BCMO7211 exhibited broad carotenoid substrate specificity toward α-carotene, ß-cryptoxanthin, ß-carotene, zeaxanthin, and lutein. The optimum temperature, pH, and concentrations of the substrate and enzyme for retinal production were 60 °C, 9.0, 500 mg ß-carotene/L, and 2.5 U/ml, respectively. Under optimum conditions, 888.3 mg/L retinal was produced in 60 min with a conversion rate of 89.0% (w/w). BCMO7211 is a potential candidate for the enzymatic synthesis of retinal in biotechnological applications.

Production, characterization, and bioactivities of exopolysaccharides from the submerged culture of Ganoderma cantharelloideum M. H. Liu.

In this study, the submerged culture conditions for exopolysaccharide (EPS) production by Ganoderma cantharelloideum M. H. Liu were screened and optimized, and the physicochemical and biological properties of EPS were investigated. Results showed that the glucose and tryptone were the best C and N sources for the maximum EPS production, respectively. Under the optimal culture conditions, the EPS production achieved 1.60 g/L at day 6 in a 5 L stirred tank reactor. Two purified fractions (i.e., Fr-I and Fr-II) were obtained from the G. cantharelloideum EPS by gel permeation chromatography. Fr-II had a higher yield (87.54%), carbohydrate (95.86%), and protein (3.07%) contents and lower molecular weight (74.56 kDa) than that of Fr-I. Both Fr-I and Fr-II were polysaccharide-protein complexes with different monosaccharide compositions and chemical structures. Fr-II also exhibited stronger radical scavenging abilities, antioxidant capacities, and α-amylase and α-glycosidase inhibitory activities in vitro than that of Fr-I. Therefore, Fr-II obtained from G. cantharelloideum EPS might be explored as potential natural functional components or supplements for applications in food, medicine, and cosmetics.

Enhancing production of L-serine by increasing the glyA gene expression in Methylobacterium sp. MB200.

Microbial fermentation using methylotrophic bacteria is one of the most promising methods for L-serine production. Here we describe the metabolic engineering of a Methylobacterium strain to increase the production of L-serine. The glyA gene, encoding serine hydroxymethyltransferase (SHMT), was isolated from the genomic DNA of Methylobacterium sp. MB200, using a DNA fragment encoding Methylobacterium extorquens AM1 SHMT as a probe, and inserted into the vector pLAFR3. The resulting construct was transformed into Methylobacterium sp. MB200 using triparental mating. The genetic-engineered strain, designated as Methylobacterium sp. MB202, was shown to produce 11.4 + or - 0.6 mg/ml serine in resting cell reactions from 30 mg/ml wet cells, 20 mg/ml glycine, and 70 mg/ml methanol in 2 days, representing a 4.4-fold increase from that of the wild strain. The results demonstrated the potential for improving L-serine production by manipulating the glyA in bacteria and should facilitate the production of L-serine using Methylobacterium sp. strains.

Isolation and characterization of an operon involved in sulfate and sulfite metabolism in Sinorhizobium fredii.

A gene cluster ORFabcd from a Sinorhizobium fredii HN01 mutant strain HSMRalpha was isolated. We showed that it was an operon involved in sulfur metabolism. Functional studies revealed that, except for ORFb, the three genes ORFa, ORFc and ORFd were involved in sulfite reduction. ORFa and ORFc were similar to the cysG and cysI from Sinorhizobium meliloti 1021 and Rhizobium etli CFN 42, respectively. ORFd encodes a conserved hypothetical protein in other bacteria. We demonstrate here, for the first time, that it was a new locus involved in sulfate assimilation in S. fredii HN01 and we designated it as cysII.

Significant improvement of Serratia marcescens lipase fermentation, by optimizing medium, induction, and oxygen supply.

Production of an extracellular lipase from Serratia marcescens ECU1010, which is an industrially important biocatalyst for the stereospecific synthesis of Diltiazem precursor, was carefully optimized in both shake flasks and a fermenter, using Tween-80 as the enzyme inducer. Dextrin and beef extract combined with ammonium sulfate were indicated to be the best carbon and nitrogen sources, respectively. With the increase of Tween-80 from 0 to 10 g l-1, the lipase production was greatly enhanced from merely 250 U l-1 to a maximum of 3,340 U l-1, giving the highest lipase yield of ca 640 U g-1 dry cell mass (DCW), although the maximum biomass (6.0 g DCW l-1) was achieved at 15 g l-1 of Tween-80. When the medium loading in shake flasks was reduced from 20 to 10% (v / v), the lipase production was significantly enhanced. The increase in shaking speed also resulted in an improvement of the lipase production, although the cell growth was slightly repressed, suggesting that the increase of dissolved oxygen (DO) concentration contributed to the enhancements of lipase yield. When the lipase fermentation was carried out in a 5-l fermenter, the lipase production reached a new maximum of 11,060 U l-1 by simply raising the aeration rate from 0.5 to 1.0 vvm, while keeping the dissolved oxygen above 20% saturation via intermittent adjustment of the agitation speed (> or =400 rpm), in the presence of a relatively low concentration (2 g l-1) of Tween-80 to prevent a potential foaming problem, which is easy to occur in the intensively aerated fermenter.