High Efficient Dioxyacetone Production By Synergistic Catalysis With Combined Biocatalyst

In this paper,we chose lipases from Psychrotrophic Bacterium sp. 7195（LIP7195）,lipase B from Candida antarctic（CALB） and glycerol dehydrogenase from Escherichia coli（EcogldA） to build a synergistic catalytic system.The LIP7195 and EcogldA were cloned and expressed in E.coli. Optimized soluble expression of Lip7195, and the purified recombinant enzymes were characterized.The synergistic catalysis system with combined LIP7195,CALB and EcogldA was performed to prepare 1,3-dihydroxyacetone（DHA） using plant oil as raw materials. The main results are as follows:（1）The GC content of modified Lip7195 was adjusted from 46% to 48%,the codon adaption index（CAI） of that was improved from 0.25（natural sequence） to 0.37（optimized synthetic gene）, which seems to be higher efficient for expression of LIP7195 in E. coli. Result showed the recombinant LIP7195 in E. coli mainly existed in inclusion body. Polycationic amino acid tag（polyarginine tag） enhanced the solubility of LIP7195 and decreased inclusion body formation in E. coli. The purified recombinant LIP7195 was estimated to possess a molecular mass of 35 kDa on SDS-PAGE. The maximum activity of LIP7195 was at 40 °C and pH 9.0. The recombinant LIP7195 was fairly stable under 30°C and at pH 8.0-10.0. It was activated in the presence of Co2.,while the different level inhibition of LIP7195 was observed with Zn²⁺, EDTA,Triton X-100, Tween-20 and SDS, respectively inhibited its enzymatic activity in different degrees.The recombinant LIP7195 had a Km of 0.18 mM,and Vmax of 28.95 mM·s^-1.The specific activity of recombinant LIP7195 was 10.9 U/mg for p-Nitrophenyl dodecanoate.（2） The EcogldA was over-expressed by pET-20 b in E. coli and purified by Ni-NTA chromatography. The recombinant EcogldA had an apparent molecular weight of 40.0 kDa on SDS-PAGE. The maximum activity of EcogldA was at 70°C and pH 11.0 with glycerol and NAD+ as substrates. The purified enzyme was stable from pH 6.0⁹.0 under 70°C.The recombinant EcogldA had a Km of 0.22 M and Vmax of 14.69 M·s^-1.The specific activity of EcogldA was 9.4 U/mg.（3）The optimum conditions of DHA yields catalyzed with the synergistic catalysis system with combined CALB,LIP7195 and EcogldA were as follows 28: 7: 15, totally 81 U/g Tributyrin at40℃ and pH 9.0 for 6 h, the highest conversion ratio of DHA was 48.7%（w/v）,while the corn oil and soybean oil as substrates, DHA yields were 32.3% and 35.6%.