Expression And Directed Evolution Of Rhizopus Arrhizus Lipase Gene

Many kinds of lipases from Rhizopus species have been proved useful to produce structured lipids due to their strong 1,3-regiospecificity. Rhizopus arrhizus L-03-R-1,which is a mutant train by many generation mutagenesis and breeding from the original strain, produced lipase at a high level. We cloned and expression the lipase genes in E. coli and P. pastoris. The optimum temperature and thermostability of Rhizopus arrhizus lipase(RAL) were improved by directed evolution. The research is listed as follows:1．The genes of ProRAL and RAL were amplified by PCR using genome DNA from the Rhizopus arrhizus L-03-R-1 as a template. The amplified products were ligated to plasmid pMD-18T and the sequences were analyzed. The genomic DNA sequence of ProRAL was obtained and submitted to Genebank and the accession number was DQ489719．The genomic DNA of ProRAL and RAL are composed of 1101bp and 810bp respectively. The homology is about 96% between the genomic DNA sequence of ProRAL and that of Rhizopus oryzae lipase (ROL, Genebank: AF229435) . The deduced amino acid sequences of ProRAL are 97% identical to ROL. RAL gene was cloned to construct the expression vectors pET-28a-RAL and pET-22b-RAL, and was expressed in E. coli BL21(DE3) using the T7 promoter. The recombinant protein was expressed when the strain BL21( pET-28a-RAL) induced by IPTG, but the activity of the products was not detected.2．The expression vectors pPIC9K-ProRAL and pPIC9K-RAL were constructed and linearized by Sacâ… and Bglâ…¡, and then the linearized vectors were transformed into methylotrophic yeast Pichia pastoris (GS115) by electroporation. Fifty-six transformants, containing the multicopy of ProRAL and RAL genes, were selected from 2000 transformants by YPD-Geneticin plates (2mg/ml G418) . The lipase activity was detected by MM-rodamine plates. The transformant BQ1(pPIC9K-ProRAL) and SC77(pPIC9K-RAL) expressed and secreted 18 U/mL and 26U/mL lipase, respectively, when 0．5% methanol was fed during the cultivation.3．ProRAL and RAL were purified by ultrafiltration, SP-Sepharose Fast Flow chromatography, and Butyl-Sepharose Fast Flow chromatography. Molecular weight of ProRAL and RAL was 32 kDa and 30 kDa, respectively. The yield of ProRAL and RAL was 37% and 37．7% , respectively. The specific lipase activities of ProRAL and RAL were 1543U/mg and 2437U/mg, respectively. The molecular weight of ProRAL and RAL did not change by PNGase F treatment, which suggested that no glycosylation had occurred. The optimum temperature of ProRAL and RAL was 30℃and 35℃, respectively. The optimum pH of ProRAL and RAL was 8．0 and 8．5, respectively. ProRAL was more stable than RAL at pH 4-7, while RAL was more stable at pH 7-10．ProRAL had the highest lipase activity towards tributyrin (C₄) , whereas rRAL had the highest lipase activity towards tricaprylin ( C₈) . The distinct properties of ProRAL and RAL may be caused by the lipase prosequence.4．To improve the yield of the recombinant lipase, a 5L fermentor was used. Methanol feeding strategy under constant dissolved oxygen (DO) was applied. The supernatant contained 72U/ml ProRAL and 125U/ml RAL after 80h and 92h cultivation, respectively. ProRAL was a processed protein by KEX2 proteinase. RAL was possibly glycosylated under the fermentation condition.5．To expand the functionality of lipase from Rhizopus arrhizus (RAL) we have used error-prone PCR and DNA shuffling methods to create RAL mutants with improved thermostability and the optimum temperature. About 5500 yeast recombinants were screened on the olive oil plates at 50℃. One desirable mutant with three amino acids substitution(A9T, E190V, M225I) was obtained. The mutated lipase(Mut-RAL) was characterized. The optimum temperature of the mutant lipase was higher by10℃than that of the wild-type RAL(WT-RAL). In addition, the thermostability of the mutant was also improved as the result of directed evolution. The half-life (T_1/2 ) at 50℃of the mutant exceeded those of WT-RAL by 12-fold. To confirm which substitution contributed to enhance thermostability and the optimum temperature for lipase activity, the site-directed mutation lipase (E190V) from the WT-RAL gene was constructed. Amino acid substitution at position 190 was determined to be critical for lipase thermostability and the optimum temperature. The mutational effect is interpreted according to a simulated three-dimensional structure for the mutant lipase.6．The genes of P43 promoter and nprB signal peptide were amplified by PCR methodology using genome DNA from the B. subtilis1．1390 as a template. The expression cassette, P43+SP+RAL, was constructed by the ligation of P43, SP and RAL. The B. subtilis expression vector, pGJ103-RAL, was constructed by the ligation P43+SP+RAL and pGJ103．...