Gene Cloning Of Rhizopus Oryzae Lipase And Its High Level Expression In Pichia Pastoris

The lipase from Rhizopus oryzae with highly specificity of 1,3-sn and stereoselectivity, is widely used in the field of oil processing, production of specialty esters, chiral substances split, bio-diesel, etc. To achieve an efficient industrial production, genetic engineering is generally necessary. In this study, the Rhizopus oryzae lipase gene ProROL was cloned from Rhizopus oryzae. And through rational design and co-expression of Ero1p&PDI chaperone, the expression of the lipase was improved greatly. Enzymatic properties were determined after preliminary separation and purification and its application was studied prelimimarily. The main content is as follows:(1) The gene ProROL was cloned from the genomic DNA of R. oryzae by PCR. According to the gene sequencing, three nucleotides were different from the sequence in NCBI, but both encode the same amino acid sequence. The gene ProROL was cloned into the expression vector pPIC9K. The linearized expression plasmid was transformed into Pichia pastoris GS115. The recombinant strain (Mut+His+) was obtained by preliminary screening and molecular validation. The lipase activity of the supernatant was 10.6 U/mL towards p-NPP in shake flask, while in 7-liter fermenter it reached 126.8 U/mL which was 11 times than that in shake flask.(2)The prosequsence of lipase was very important for secretion and folding of the enzyme. By SOE PCR, the prosequence between the lipase from R. oryzae (ProROL) and the lipase from R. chinensis (ProRCL) was replaced and the new recombined gene was named as ProAROL. The expression vector pPIC9K-ProAROL was constructed and linearized and then transformed into Pichia pastoris GS115 and GS115/PDI/Ero1p (co-expressing Ero1p&PDI chaperone). We obtained a list of recombinant strains with different gene dosage determined by qPCR. The highest activity of the recombinant strain H248 and BH128 (co-expressing Ero1p&PDI chaperone) were 120.0U/mL and 152.3 U/mL respectively, which were about 12 and 15 times higher compared with the expression level of ProRCL. In 7-liter fermenter the highest activity of H248 was 4059.6 U/mL, which was 33.8 times than that in shake flask. We also found that with co-expression of Ero1p&PDI chaperone not only the enzyme activity was improved, but also the fermentation time was shortened.(3) The recombinant enzymes ProROL and ProAROL were further purified by a two-step of purification protocol, including cation exchange chromatography and hydrophobic chromatography. The purification factors of ProROL and ProAROL were 3.90 and 5.61, respectively.(4) The enzymatic properties of ProAROL and ProROL were similar. The optimum temperatures were both 40℃. Under 40℃, both enzymes showed high activity. The enzyme activity declined sharply when the temperature is higher than 45℃. The optimum pH were both 8.0. Between the pH 6.0-10.0, more than 50% of the enzyme activities were both retained. The Km value of ProAROL and ProROL was 0.440 mM and 0.442 mM, respectively, while the kcat value was 1.30×104 and 9.75×103, respectively. Both enzymes showed a higher selectivity towards C12-C16 long-chain fatty acid of p-nitrophenyl esters. The selectivity was more than 40% for C18, while had a lower selectivity for the short chain fatty acids. Both lipases had specificity of 1,3-sn by enzyme hydrolysis. In the study of esterification of 1-caprylic acid and 1-octyl alcohol catalyzed by ProAROL and ProROL, the conversion rates of 1-caprylic acid after 24 h were more than 95%.