Objective: Construct the whole cell biocatalysis system for the reduction of prochiral carbonyal compounds. And valuate the function of the disordered C terminus in the catalysis of formate dehydrogenase from Candida boidinii. Methods: The recombinant strains for the expression of LbADH ,CbFDH and CbFDHΔ354-364 were constructed by the genetic engineering methods. The expression of recombinant proteins were analyzed by SDS-PAGE, and the activity of the whole soluble proteins was determined photometrically at 340 nm. Rossetta(DE3)-pETDuet-1-adh and Rossetta(DE3)-pETDuet-1-fdh were mixed and incubated with acetophenone, the products were analyzed by high performance liquid chromatography (HPLC). To compare the NADH regeneration rate of Rossetta(DE3)-pETDuet-1-fdhΔ354-364 and Rossetta(DE3)-pETDuet-1-fdh, the two strains were mixed with Rossetta(DE3)-pETDuet-1-adh separately, and incubated with acetophenone, the products were detected timely. Results: The recombinant plasmid pETDuet-1-adh, pETDuet-1-fdh and pETDuet-1-fdhΔ354-364 were constructed correctly. The recombinant proteins LbADH, CbFDH and CbFDHΔ354-364 could be expressed solublely. The two recombinant strains harboring LbADH and CbFDH separately can be used coupled for the asymmetric reduction of acetophenone, the conversion rate reached 24.4% and enantiomeric excess reached 96.79%; the soluble expression of CbFDHΔ354-364 has no significant difference compared with CbFDH, but when using the whole soluble proteins of Rossetta(DE3)-pETDuet-1-fdhΔ354-364 catalyzed the regeneration of NADH, the conversion rate was sharply decreased to 8% when compared with Rossetta(DE3)-pETDuet-1-fdh. Conclusions: The recombinant strains Rossetta(DE3)-pETDuet-1-fdhΔ354-364 and Rossetta(DE3)-pETDuet-1-adh could be used coupled for the reduction of acetophenone, resulted in a chemical yield of 24.4% and an enantiomeric excess of 96.79%. Rossetta(DE3)-pETDuet-1-adh and Rossetta(DE3)-pETDuet-1-fdh were mixed and incubated with acetophenone, the conversion rate was decreased 32.79% compared with Rossetta(DE3)-pETDuet-1-fdh mixed with Rossetta(DE3)-pETDuet-1-adh. The disordered residues in C terminus of CbFDH may play an important role in the catalysis process of CbFDH.
A Bacillus licheniformis strain named G2 with esterifying enzyme activity screened from high temperature Daqu was mutagenized by using low temperature plasma and three strains with high esterifying enzyme activity was screened as parents .The parent protoplast was inactivated for genome shuffling. After the fused strains were purified by selection, the enzyme activity of fusants was determinated by fermentation, the strains of higher enzyme activity were screened to passage and their stability of enzyme activity were detected.The highest and stable enzyme activity of 6# fusant can reach 10.7U/ml ,which has been improved by 102% compared with the 5.3U/ml of original strain G2.The fermentation broth of the 6# fusant was analyzed by GC ,the results show that the strain can produce a certain amount of acids ,alcohols and esters, and the acetoin of the fermentation broth can reach 2565mg/L.
S-adenosylmethionine (SAM) and glutathione (GSH) are both important small S-contained compounds in cells. The effects of sodium citrate on the fermentative co-production of SAM and GSH with Candida utilis CCTCC M 209298 were investigated in flasks. Sodium citrate was found to be beneficial for the high co-production of SAM and GSH. The response surface analysis was applied in the optimization of sodium citrate concentration and addition time, and a strategy of 10 g/L sodium citrate addition at 6 h was predicted by a statistical model and verified to be the best approach for increased co-production of SAM and GSH. Based on the results derived from the kinetic analysis on the batch fermentation processes, intracellular levels of NADH and ATP could be significantly improved by sodium citrate, and which in turn provided essential energy substance needed for the over-production of SAM and GSH. The results also provide a potential approach for efficient production of analogical useful chemicals biosynthesized with the consumption of energy.
The effect of the addition of corn oil, rapeseed oil, soybean oil, methyl oleate, the mixture of rapeseed oil and metyl oleate on the production of spinosad by Saccharopolyspora spinosa LU 104 was studied. The results indicated that the yield of spinosad was increased 2.11-fold up to 804.59 mg/L when adding 3% mixture oil (rapeseed oil:metyl oleate=1:2) at 96 h and 1% corn steep liquor at 96 h and 144 h.
In the present study, the trehalose synthase gene of Thermobifida fusca was cloned and expressed in Escherichia coli BL21(DE3). The optimized composition of fermentation medium and culture conditions for the recombinant E. coli were investigated in shake flasks. The optimal fermentation medium was as follows: 12 g/L glycerol, 24 g/L yeast extract, 12 g/L peptone, 60 mmol/L PO43-, 2 mmol/L Zn2+. And the optimal culture conditions were that: volume 20 ml in 250 ml shake flask, induction temperature 25℃, induced by 0.4 mmol/L IPTG at 2 h of culture. Under these conditions, the maximal enzyme activity reached 28.6 U/ml, which was 3.4 times as high as that not optimized.
