High-level Expression Of Lipases In Pichia Pastoris And Their Application In Conversion Of Lipid Compounds

A 1,3-specific lipase gene from the fungus Rhizomucor miehei was previously cloned and expressed in methylotrophic yeast Pichia pastoris strain GS115.The enzyme produced(termed RML)was able to catalyze methanolysis of soybean oil and showed strong position specificity.However,the enzyme activity and amount of enzyme produced were not adequate for industrial application.One of our goal in this study was to improve the enzyme properties of RML in order to apply it for conversion of microalgae oil to biofuel.Several new expression plasmids were constructed by changing the expression system,adding the propeptide of the target gene,optimizing the signal peptide,increasing the number of target gene copies.Each plasmid was transformed separately into P.pastoris strain X-33.The recombinant strain with 2-copies of the target gene was screened by flask culture showed maximal enzyme activity for RML(21.4-fold increased)to 1200 U/mL and enzymatic properties of the thermostability and methanol and ethanol tolerance were enhanced.The enzyme was termed Lipase GH2,and the expressed protein with the propeptide was a stable glycosylated protein,which can be stable for>6 months at 4?.Quantitative real-time RT-PCR analysis revealed two major reasons for the increase in enzyme activity:1)the modified recombinant expression system gave an increased transcription level of the target gene(rml),and 2)the enzyme was suitable for expression in host cells without causing endoplasmic reticulum(ER)stress.When overexpression PDI gene in the recombinant strain(containing 4-copies of target gene)relieved the protein folding pressure and increased enzyme activity 1.3 times to 1480 U/mL.The modified enzyme was tried to use as a free enzyme for catalytic conversion of microalgae oil mixed with methanol or ethanol for biodiesel production in an n-hexane solvent system.The lipid components of microalgae oil extracted from Chlorella vulgaris powder was evaluated.The major fatty acid components were myristic acid,palmitic acid,palmitoleic acid,stearic acid,oleic acid,linoleic acid,and linolenic acid.Optimal values were determined for five reaction conditions:reaction temperature,water content,alcohol/oil molar ratio,alcohol additional strategy,and enzyme content.Conversion rates of two major types of biodiesel,fatty acid methyl ester(FAME)and fatty acid ethyl ester(FAEE),reached maximal values(>90%)after 24 h.In the FAME reaction system,liquid enzyme could be reused 5 times or more.When using Lipase GH2 as catalyst,the process of FAME production is generally more simple and economical than that of FAEE production,even though the two processes show similar conversion rates.In spite of the damaging effect of ethanol on enzyme activity,we successfully obtained ethyl ester by the enzymatic method.Our findings indicate that Lipase GH2 is a useful catalyst for conversion of microalgae oil to FAME or FAEE,and that this system provides efficiency and reduced costs in biodiesel production.Next,a mono-and diacylglycerol lipase gene(mdl)was cloned from Penicillium cyclopium and expressed in Pichia pastoris strain GS115.The recombinant enzyme was named Lipase GH1.High cell density fermentation was performed by culture in a 7.5-L fermenter using BSM-Na2GP medium,in which the phosphate in basal salt medium was replaced by sodium glycerophosphate(Na2GP).The maximal lipase activity detected was 18,000 U/mL,and total protein content in the fermentation supernatant was 3.94 g/L.The liquid enzyme was stable under alkaline conditions at 4? for 6 months with no change and was 50%after one year.Lipase GH1 was used for the synthesis of mono-and diacylglycerols(MAGs and DAGs),which are commonly used emulsifiers for industrial applications.A conversion rate of 84%after 24 h of reaction was obtained using glycerol/oleic acid molar ratio 11:1,water content 1.5 wt%,enzyme dosage 80 U/g,and reaction temperature 35?.Lipase GH1 was more efficient for the synthesis of MAGs and DAGs than was Lipase G50(a similar,commercially available lipase derived from Penicillium camemberti)when oleic acid was used as an acyl donor.Lipase GH1 has potential for food emulsifier preparation.