Screening And Molecular Modification Of High-efficiency Esterification Enzymes Of Fusel Oil In Spirit

High content of fusel oil is one of the main factors affecting the quality of Chinese Spirit.Reducing the content of fusel oil in liquor is the technical bottleneck of production.Screening the strains with ability to produce fuser oil esterase,and expressing its esterase genes.Using molecular modification to construct an esterification enzyme with highly activities about fusel oil.Esterase esterification of fusel oil to ester is one of the effective ways to reduce the content of fusel oil in wine,and has great application potential.The strain producing the fusel oil esterification enzyme from the deep sea sediment was obtained,which was identified as Candida parapsilosis by ITS rDNA sequence.The crude enzyme solution was esterified at 30 ℃ for 1 d with the fusel oil content was 6%(n-propanol,isobutanol,isoamyl alcohol was 2%,respectively)and acetic acid content was 2%.The content of n-propyl acetate was 15.4661 mg/100mL;the content of isobutyl acetate was 8.7562 mg/100mL;the content of isoamyl acetate was 3.0515 mg/100 mL.The synthetic Candida parapsilosis esterase gene Lipase1 was expressed in Pichia pastoris GS115,and the synthesized esterase gene Lipase2 was expressed in Escherichia coli BL21.The activity of enzyme,expressed by Pichia pastoris GS115,was about: the content of n-propyl acetate was 8.6824 mg/100mL;the content of isobutyl acetate was 0.4371 mg/100mL;the content of isoamyl acetate was 0.2021 mg/100 mL.The activity of enzyme,expressed by Escherichia coli BL21,was about: the content of n-propyl acetate was 0.2684 mg/100 mL.The esterase expressed by E.Coil BL21 was molecular modification by error-prone PCR,then the PCR products was digested and ligated to transform.7 strains with significantly improved enzymatic activity were obtained.Amino acids sequence alignment analysis showed that the esterification enzyme with higher n-propanol esterification activity was mutated from SGW to VVG in 201-203 amino acids,and the synthesis of n-propyl acetate was increased from 0.2684 mg/100 mL to 37.0065 mg/100 mL,which was 137.89 times.The esterification enzyme with enhanced isobutanol esterification activity has a large number of mutations in the 170-200 amino acid range,and the maximum enzyme activity was increased to 0.2025 mg/100 mL.The esterase with higher isoamyl alcohol esterification activity has a large number of mutations in the 200-220 amino acid range,and the maximum enzyme activity was increased to 0.5683 mg/100 mL.The results of molecular modification showed that the amino acid mutation in the region of amino acid 190-220 significantly affected the performance of the esterification enzyme esterified fusel oil.The molecular model of fusel oil esterification enzymes was analysis,reduction of branch resistance of amino acid residues at active sites was significant for the activity of esterified n-propanol.A molecular model for the analysis of fusel oil esterification enzymes was constructed,and the decrease in the branch resistance of the amino acid residues at the active site was responsible for the significant increase in esterification enzyme esterification of n-propanol activity.The construction of esterification enzyme with high-efficiency esterified fusel oil,which can transport fusel oil and organic acid to ester and it is a novel way to reduce the content of fusel oil in Chinese spirit.The fusel oil esterification enzyme has a specific amino acid sequence,which can efficiently esterify the fusel oil,and has important practical application significance for reducing the content of liquor alcohol oil.The fusel oil esterification enzyme has a specific amino acid sequence,which can efficiently esterify the fusel oil,and has important practical application significance for reducing the content of fusel oil.