Study On The Excavation Of New Lipase And Its Regional Selective Diversity

Lipase can catalyze the hydrolysis of triacylglycerol to free fatty acids and glycerol.Antarctic candida lipase A(Candida antarctiac Lipase A(CALA)is a typical microbial lipase with good temperature stability and interfacial activity and has been commercialized.Because of its fatty acid selectivity,especially short-chain fatty acids and saturated fatty acids.Therefore,short chain saturated fatty acids can be removed by hydrolysis,therefore,it which is widely used in the preparation of functional oils and oils,and the enrichment of polyunsaturated fatty acids in microbial oils and oils.However,the selectivity of CALA for long chain fatty acids is so poor that it can not be used to hydrolyze polyunsaturated fatty acids directly.Therefore,it is necessary to develop new lipase with good temperature stability and long chain fatty acid selection activity.It has been reported that CALA has TG sn-2 activity,but current studies have shown that CALA does not have regioselectivity.In view of the good temperature tolerance of CALA and its representative of new types of lipase family,CALA was used as bait to mine and express new lipase,and its basic enzyme properties,regioselectivity and fatty acid selectivity in hydrolysis of microbial oils were investigated.The main contents and results are as follows:(1)The amino acid sequence of CALA was used as the query sequence to search for the related lipase sequence in the database,and the phylogenetic evolution tree was established.The results showed that CALA and its branches had low homology with other lipases.Finally,the lipases with homology of 97%,55% and 35% with CALA were selected,respectively.The three lipases were Man Lip(GAC73196.1),Ph Lip,(XP?012187489.1)and Us Lip(PWN48207.1),derived from Moesziomyces antarcticus T-34,Pseudozyma hubeiensis SY62 and Ustilaginomycotina sp.SA807,respectively.The recombinant plasmid p PIC9K-manlip,p PIC9K-phlip and p PIC9K-uslip were constructed by gene synthesis company.(2)Three recombinant plasmids were transferred into Pichia pastoris GS115 and the positive transformants were obtained.Lipase expression was induced by methanol,and lipase with theoretical molecular weight(about 50 k Da)was shown by SDS-PAGE electrophoresis,and it was found that the expression of lipase was the highest after 5 days of induction.The recombinant lipase was purified by ammonium sulfate precipitation method.The concentrations of three recombinant lipase Man Lip,Ph Lip and Us Lip proteins were 0.561 mg /m L and 0.486 mg/m L,1.17 mg/m L,respectively.(3)The basic enzyme properties of recombinant lipase Man Lip,Ph Lip and Us Lip were studied.the results showed that the optimum catalytic temperatures of Man Lip,Ph Lip and Us Lip were 70?,60? and 60?,respectively,and the optimum catalytic p H values were 9.0,9.0 and 8.0,respectively.Among the three recombinant lipases,Ph Lip has good temperature and p H stability.The activities of the three recombinant lipases against p-nitrophenol fatty acid ester with different carbon chain length were different,among which Ph Lip p-nitrophenol laurate(C12)and palmitate(C16)were the highest,and the other two lipases were also higher than CALA,while CALA had the highest activity against p-nitrophenol octanate(C8).(4)CALA and recombinant lipase were applied to hydrolysis of Mucor circinelloides oil,and the hydrolysate was analyzed by nuclear magnetic resonance(NMR)to explore the regioselectivity of recombinant lipase.The results showed that compared with the control group,the NMR spectra of the four experimental groups showed no difference in breaking the ester bond of TG,so CALA and three recombinant lipases had no regional selectivity in the hydrolysis of Mucor circinelloides oil.Ph Lip enzymes with better catalytic properties in three lipases were selected to study the preference of polyunsaturated fatty acids,and the free fatty acid bands in the hydrolyzed products were separated by TLC and analyzed by gas chromatograph.The results showed that when the lipase Ph Lip hydrolyzed the oil of Mucor circinelloides,it showed a certain preference for GLA(C18:3),and the selectivity of fatty acids was as follows: polyunsaturated fatty acids > monounsaturated fatty acids > saturated fatty acids,when the oil was hydrolyzed by recombinant lipase and the catalytic property is better than that of CALA.