Heterologous Expression And Characterization Of Alkaline Lipase In Trichoderma Reesei

Penicillium expansum is one of the main strains producing alkaline lipase.Its alkaline lipase(PEL)has the characteristics of high efficiency,specificity and mild conditions.It is widely used in detergent,food,oil processing and other fields.However,the alkaline lipase produced by the fermentation of P.expansum is mainly the spray dried product mixed with crude enzyme solution of the fermentation broth,which has been unable to meet the purity requirements of modern industry for enzyme products.The alkaline lipase produced by P.expansum is extremely unstable in the liquid environment,which may be degraded by the protease in the fermentation broth.This disadvantage limits the application of the lipase product in the field of liquid detergent.In addition,P.expansum is a necrotizing plant pathogen with the ability to infect a wide range of fruit hosts,which has a certain potential safety hazard in the production of industrial enzyme preparations.The industrial production capacity of Trichoderma reesei has been proved to be an important strain for the production of cellulase.In order to solve many adverse factors in the production of alkaline lipase by P.expansum as an industrial strain,in this paper,the alkaline lipase gene pel from P.expansum was heterologously expressed in T.reesei.Through codon optimization and changing different promoters,the alkaline lipase was succefully expressed and secreted in in T.reesei.The main research results are as follows:1.Effects of different promoters on heterologous expression of P.expansum lipase gene in T.reeseiFirstly,the basic lipase gene pel of P.expansum was expressed by using the promoter and signal peptide of cellobiohydrolase I(CBHI)in T.reesei.Using plasmid p Bluescript II SK(+)as backbone,The protein expression vector pCbh1-pel was constructed by inserting the DNA fragments of uridine deficient screening marker gene pyr4、Pcbh1 promoter and signaling peptide,pel c DNA sequence,and Tcbh1 sequence.pCbh1-pel was transformed into T.reesei Tu6Δku70Δpyr4.The pel gene was integrated into the cbh1 gene locus of T.reesei.The transformants harboring the pel gene expression cassette were cultivated in the medium with cellulose as induced carbon source for 120 hours.The result showed that the recombinant lipase was not successfully expressed in T.reesei.Subsequently,the inducible promoter Pcbh1 was replaced with the constitutive promoter Pcdna1 to obtain the alkaline lipase heterologous expression vector pCdna1-pel.After transforming T.reesei to obtain the transformant,the recombinant lipase was successfully expressed on the medium with glucose as the sole carbon source.The activity of olive oil was determined by Na OH titration,and the lipase activity in the supernatant was 359.4 U/m L.2.Effect of codon optimization on the expression of lipase gene pel in P.expansumIt is well known that codon bias could affect the expression levels of foreign genes in certain host organisms.In order to make the efficient expression of P.expansum alkaline lipase in T.reesei,the codon of lipase gene pel was optimized.The coding regions of 9352 genes of T.reesei were analyzed to elucidate its codon bias reference.The results showed that the GC content of 97% genes of T.reesei was 50% ～ 68%,and the average content of GC3 was 70.4%.21 highly expressed superior codons and 4 highly expressed optimal codons(CUC,GCC,CGC and GGC)were determined.The optimized alkaline lipase gene was defined as pelc,and the pCdna1-pelc expression vector was constructed.The lipase activity of the strain Tcdna1p-pelc was determined.The activity of olive oil was determined by Na OH titration,and the results showed that the lipase activity was 459.4 U/m L,which was nearly 1.5 times increased compared to that in the strain containing the original pel gene.3.Analysis of enzymatic properties of recombinant lipaseThe Tcdna1p-pelc strains obtained in the above experiment was used to study the enzymatic properties of recombinant lipase,and the optimum temperature,thermal stability,pH value and pH stability of recombinant lipase were analyzed.By measuring the activity of recombinant lipase at different temperatures,it was found that the optimum reaction temperature of the enzyme was 35℃,the enzyme activity decreased rapidly over50℃,while the enzyme activity was relatively stable at 30℃～50℃,and the remaining enzyme activity remained at about 90%.The lipase activity was measured in 50 mmol/L buffer with pH 5.0～10.5,and the optimum pH was 9.5.In the range of pH 7.0～10.0,the remaining enzyme activity is more than 90%,which shows that the enzyme can maintain relatively high enzyme activity in the alkaline region.The results were similar to the optimum temperature and pH of lipase in the supernatant of P.expansum fermentation broth.The effect of surfactants on enzyme activity was determined by adding 1% SDS,Triton X-100 and Tween 20.The results showed that all three surfactants could affect the activity of recombinant lipase,and the enzyme activity after surfactant treatment was only about 20%.However,the lipase activity of P.expansum fermentation supernatant will increase to a certain extent in 1% non-ionic surfactant,which is the biggest difference from recombinant enzyme.It is likely that some components in the fermentation supernatant of P.expansum will interact with surfactants,resulting in the single expression of alkaline lipase in T.reesei,which will reduce the tolerance of lipase to non-ionic surfactants.Through the further analysis of the components in the fermentation supernatant of P.expansum,the tolerance related proteins can be expressed in T.reesei,and the alkaline lipase and tolerance related proteins expressed by T.reesei can be added and applied as a composite enzyme preparation.In this study,the codon of P.expansum alkaline lipase gene pel was optimized to realize its high-efficiency heterologous expression in T.reesei.The preliminary analysis of the enzymatic properties of recombinant lipase not only lays a foundation for its industrial application,especially in detergent,but also provides gene resources for the study of its structure and function.