Screening,Expression And Characterization Of Novel Fungal Lipases And Cutinases For Baijiu Production

Ester aroma substances are the most important flavor substances in beverage wine,especially in Chinese Baijiu,ester substances account for 40～70%of the total flavor substances,and are an important factor determining the quality and flavor of liquor.Enzyme preparations such as esterase are added in the brewing process,which can increase ester and flavor,not only improve the rate of high-quality wine,but also shorten the fermentation cycle,which is of great significance for the realization of high-efficiency,low-carbon and green development of the liquor industry.At present,most of the researches on esterases used in winemaking at home and abroad focus on Candida antarctica lipase B,and there are also reports on lipases such as Rhizopus chinensis and Cladosporium.This topic took Baijiu Daqu as the research object,screened and isolated strains with excellent esterase-producing performance,and used transcriptome technology to mine high-expressed esterase genes.Its hydrolase activity was analyzed by the p-nitrophenol method,and its ability to catalyze the synthesis of flavor esters was evaluated in an aqueous system.In addition,this topic also discussed the synthesis of flavor esters catalyzed by fungal cutinase in water.This study provides good materials for the development of special enzyme preparations for Baijiu brewing,and also lays a foundation for scientific analysis of the catalytic mechanism of esterases under aqueous conditions.The specific research results are as follows:（1）Firstly,two strains of filamentous fungi with strong ester hydrolysis activity were isolated and screened from Daqu of Baijiu by the tributyrin plate method.They were identified by molecular biology as Lichtheimia corymbifera and Aspergillus flavus,named L.corymbifera B08 and A.flavus B09,were prepared as koji and their esterification ability was analyzed.The results showed that A.flavus B09 had better ethyl acetate and ethyl lactate synthesis ability,so this strain was selected as the follow-up research object.（2）In order to obtain novel fungal esterase genes,A.flavus B09 obtained in the previous screening and Aspergillus niger H7 preserved in the laboratory were selected as the research objects,and 4 genes in bran culture were screened by using transcriptome sequencing technology.The highly expressed lipase genes under the conditions were AFLA₀66160,AFLA₀25190,ANI₁₂66144,and ANI₁₂36084,respectively.（3）The target protein was expressed in P.pastoris GS115 or E.coli BL21（DE3）using p PIC9K and multiple expression vectors of p ET28（a）,p ET22（b）,p ET32（a）,and p COLD-TF,and finally successfully realized the heterologous expression of ANI₁₂36084,ANI₁₂66144 and AFLA₀66140,and the crude enzyme liquid was purified by affinity chromatography.In addition,the substrate specificity and optimum temperature of different lipases were analyzed by the p-nitrophenol hydrolysis method.The results showed that:the optimum substrate of lipase ANI₁₂66144 is p NPA（C₂）,AFLA₀66140 is p NPP(C₁₂),and ANI₁₂36084 The catalytic specificity expressed in different expression hosts is different,one is p NPA and the other is p NPB（C₄）,which means that the late modification of the protein by the expression host affects the catalytic properties;the optimum temperature of lipase ANI₁₂66144 is 20°C,AFLA₀66140 was 30°C,and ANI₁₂36084 was expressed at the optimum temperature of 40°C in different expression hosts.（4）The ability of lipases to catalyze the synthesis of flavor esters was tested in the aqueous system.When the substrate preference of lipases was studied,it was found that the ability of these esterases to catalyze the synthesis of long-chain acids and ethanol into esters is significantly higher than that of short-chain acids,whether under single acid or mixed acid conditions（octanoic acid>heptanoic acid>caproic acid>valeric acid>butyric acid）;in addition,ANI₁₂66144 showed higher ester synthesis ability than other lipases.（5）Through bioinformatics analysis,9 cutinase genes with potential ester synthesis ability were screened from different fungi,and were heterologously expressed and purified in E.coli,and 7 recombinant proteins were successfully obtained,namely:M.p₄69027,M.p₂60141,AFLA₀72700,AFLA₀23390,AFUA₄G14120,AFUA₂G09380,AFUA₄G03210.The substrate specificity and optimum temperature of different cutinases were analyzed by p-nitrophenol hydrolysis method.The results showed that the optimum substrates of cutinases M.p₄69027,AFLA₀72700,AFUA₄G14120,AFUA₂G09380and AFUA₄G03210 were all p NPB,M.p₂60141 is p NPA,AFLA₀23390 is p NPC（C₈）;cutinase M.p₄69027,AFUA₄G03210 has an optimum temperature of 20°C,AFUA₂G09380 is 50°C,M.p₂60141,AFLA₀72700,AFLA₀23390,AFUA₄G14120is 40°C.Similar to lipases,under aqueous conditions,these cutinases are more inclined to long-chain acids,except M.p₂60141,other cutinases have higher ester synthesis capacity,especially M.p₄69027 has a very high catalytic efficiency of ethyl octanoate.