The Construction Of Secreted And Soluble Expression System Of Chlorpropionic Acid Dehalogenases

S-2-chloropropionic acid dehalogenase (DehII-B2), R-2-chloropropionic acid dehalogenase (Deh DIV-E7) and3-chloropropionic acid dehalogenase (BHD) have great application in preparation of chemical midbodies. It has important significance in the consctuction of recombinant expression system of these dehalogenases. Until now, there were little reports about recombinant expression of2-chloropropionic acid dehalogenases. Scanty reports mainly choosed the prokaryotic system, especially Escherichia coli as the host. However, intracellular expression in E.coli has some disadvantages like low enzyme activity and easy to produce inclusion bodies. Extracellular recombinant expression or intracellular expression with proper vector and host can solve problems mentioned above well.This paper constructed and optimized secreted expression system of2-chloropropionic acid dehalogenases in Pichia pastoris. S-2-chloropropionic acid dehalogenase and R-2-chloropropionic acid dehalogenase genes were cloned into pPIC9K carrier. The recombinant plasmids were linearized and transformed into GS115yeast strain by electroporation. Screening of multiple copies of the transformants was performed on YPD plates containing geneticin G418. After a lot of screening, recombinants with12copies were chosen and extracellular expressed by methanol. We researched effections of copy numbers, methanol concentrations and induction temperature on recombinant DehII-B2and found that one with the highest activity containing12copy numbers for30℃induction at the concentration of ’methanol was1%. The expressed recombinant DehII-B2activity was up to236U/L The optimum temperature and pH of the recombinant DehII-B2was50℃and9.5, respectively. The expressed recombinant Deh DIV-E7activity was94U/L Additionally, recombinant DehII-B2and Deh DIV-E7both showed the quite good genetic stability.In this paper, We tried to choose a variety of carriers and host bacteriums to construct3-chloropropionic acid dehalogenase recombinants considering the promoter, molecular chaperone and rare codon. Seven E.coli recombinants were restructured. One recombinant expressing the highest activity and soluble protein was Rosetta(DE3) pLysS-Pcold TF-BHD. The amount of target protein expression was nearly doubled up to0.96g/L. The enzymatic activity was6.02U/L, comparing to the original increased by38%. Because the recombinase containing the molecular chaperone TF which could help BHD fold correctly. At the same time Rosetta (DE3) pLysS host contained a variety of rare codon supplement and was conducive to BHD soluble expression as BHD containing a large number of rare codon.