Rhizopus Chinensis Lipase: Gene Cloning, Expression In Pichia Pastoris And Characterizations Of Recombinant Enzymes

In industry application, the most restrict factor was lipase catalyst. The amount of lipase yielded by wild-type strains was low naturaly. Industry requirement can not be satisfied using traditional breeding. So the lipase gene(RCL) was cloned from the genomic DNA isolated from Rhizopus chinensis CCTCCM201021, an important lipase producing fungus. The prosequence lipase and mature lipase was successfully expressed in Pichia pastoris GS115. The differences of characterizations between two lipases were studied after purification. Recombinant lipase might be used as a potent bioctalyst.The results were as follows:(1)Rhizopus chinensis 18S rDNA was cloned by PCR amplification using Rhizopus universal primers and Rhizopus chinensis genomic DNA as the template. the oligonucleotide primers used for cloning lipase gene were designed based on the alignment between Rhizopus stolonifer lipase and Rhizopus oryzae lipase gene. 3'end fragment of lipase and 5'end fragment of lipase were cloned from two PCR programs, then the whole-length sequence of lipase gene was gained by connecting with two lipase sequences. The sequence contained one complete open reading frame without intron, encoding a 389 amino acid protein including 26 amino acid signal sequence, 94 amino acid prosequence and 269 amino acid mature lipase sequence. The percentage of amino-acid identity compared with other Rhizopus sp. lipase sequences was 86%.( 2 )The recombinant plasmids pPIC9k-proRCL and pPIC9k-mRCL linearized with Bglâ…¡were transformed into a fungal host, P. pastoris GS115. Four phenotypes of recombinant GS115/pPIC9k-proRCL and GS115/pPIC9k-mRCL were His+muts and His+mut+, respectively. The result of SDS-PAGE showed that the molecular weight of the prolipase protein was nearly 37 KD, lipase in the supernatant has the highest level of expression of 0.85 mg/mL with the highest activity of 121.2 U/mL. And the the molecular weight of the mature lipase protein was nearly 30 KD, the specific lipase activity of mRCL was 26.9 U/mg after 96 h of inducing by methanol.(3)The recombinant enzymes proRCL and mRCL were further purified by a three-step of purification protocol, including ultrafiltration with 10 kD membrane, SP-Sepharose FF chromatography and Phenyl-Sepharose FF chromatography. The N-terminal amino acid sequence analysis of proRCL showed that the N-terminal 67 amino acid were degraded when recombinant protein was secreted, expressed recombinant protein was consist of 27 amino acid prosequence and 269 amino acid mature lipase sequence. No differences in the molecular weight of the recombinant protein before and after incubation with endo-β-N-acetylglycosamidase H could be observed, suggesting that no glycosylation had occurred.(4)The characterization of two lipases showed the pro27RCL and mRCL had similar pH and temperature optima. The optimal pH were 8.0 and 8.5, and the optimal temperature were 40℃and 35℃, respectively. Their substrate specificity and Km and Vmax for the hydrolysis of p-nitrophenyl palmitate were obviously different. pro27RCL was more specific to six-acyl-chains middle chain fatty acid of p-nitrophenyl esters while mRCL had a preference for the hydrolysis of two-acyl-chains short chain fatty acid of p-nitrophenyl esters, so it belong to esterase in classification. The Km and Vmax for the hydrolysis of p-nitrophenyl palmitate of pro27RCL and mRCL were 0.304 mmol/L,0.345 mmol/L and 3.38μmol/min/mL,0.074μmol/min/mL, respectively. Both lipases had nonspecific position for triacylglycerols hydrolysis. Both lipases were stimulated by 10 mmol/L Ca²⁺,Mg²⁺, while Hg²⁺,SDS and PMSF had strong inhibition on their activities. pro27RCL and mRCL had high level activities and good stabilities in hexane,heptane and isooctane(30% v/v).