Molecular Mechanism Of Key Enzymes LIP05 And Bur01 Catalyze Formation Of Fatty Acid Ethyl Ester Bonds Of Baijiu

Short chain fatty acid ethyl esters are important flavor chemicals in strong-flavor Baijiu.Esterification of acid and alcohol catalyzed by esterase produced by microorganism is the main source of ester flavor chemicals of strong-flavor.However,the mechanism of enzymatic catalytic is scarce so far,resulting in the instability of ester synthesis in fermentation process of strong-flavor baijiu.In addition,the classical esterification reaction of acid and alcohol is organic phase system,while the moisture content of fermented grains is 53%-58%during fermentation,which belongs to aqueous phase system.Therefore,analyzing the mechanism of ester synthesis in aqueous system can provide a scientific theoretical basis for the quality improvement of strong-flavor baijiu.Therefore,in this study,the possible enzymatic catalytic mechanism of the enzyme LIP05 derived from Aspergillus purpureus YJX-8 and the enzyme Bur01 derived from Burkholderia BJQ0010 was proposed.The main content and conclusions of this study are as follows:(1)The protein structure of LIP05 was analyzed by homologous simulation,and the site-directed mutation of LIP05 were carried out with molecular modification technology.The results showed that the protein was a classicalα/β-hydrolase fold enzymes.The catalytic triad was Ser150-His215-Asp202.The residues Ile30 and Leu37 are located in the cap domain of the protein structure,where steric hindrance and changes in the polarity of amino acids play an important role in the catalytic function of the enzyme.Hydrogen bonding between enzyme and substrate at residues Gly73 and Thr74 is the key factor to maintain enzyme activity,and residues Gly73 and Thr74 together composed the oxygen anion hole of LIP05.Residues Leu83 is a hydrophobic amino acid at the substrate binding pocket.Steric hindrance and hydrophobic interaction play an important role in enzyme catalysis.The residues Tyr116 and Tyr149 need to maintain certain spatial structure,and must be specific aromatic amino acids,to maintainπbond stacking between the two sites,and thus stabilize the active site.Residues Ile204,Ile211 and Leu216 maintain enzyme catalytic function mainly through hydrophobic interaction.(2)Sixteen bacterial strains were screened for functional strains in the aqueous phase system,and the culture conditions of the strains were optimized.The genome of the strains was extracted and the whole genome was sequenced.The results showed that strain 1.3055had the ability to synthesize short-chain fatty acid esters under aqueous phase,named BJQ0010.The optimal carbon source was soluble starch with a concentration of 10 g/L.The optimal nitrogen source was peptone with a concentration of 10 g/L.The whole genome sequencing showed that the strain was identified as Burkholderia ambifaria,the genome contains two chromosome sequences and five plasmids.Genome composition analysis showed that strain BJQ0010 contained 6,089 genes,including 64 t RNA,15 r RNA and 9s RNA.The total gene length was 5,489,409 bp,the average gene length was 901.53 bp,and the GC content was 67.45%.(3)According to the annotation information of whole genome sequencing,60 enzymes related to ester synthesis were cloned and expressed,and the ester synthesis ability of aqueous system was determined.Six enzymes encoded by genes were found to have the ester synthesis ability in aqueous system,among which JG536＿RS05480 showed an obvious preference for decanoic acid.JG536＿RS25355 showed outstanding catalytic ability to synthesize small molecule fatty acid ethyl esters,named Bur01.Bioinformatics analysis was performed on the two enzymes,including basic enzymatic properties,signal peptide,transmembrane structure,hydrophilic/hydrophobic,secondary structure,tertiary structure and molecular bonding.(4)The protein structure of Bur01 was analyzed by crystal diffraction,and the site-directed mutation and structural modification of Bur01 were carried out combined with molecular modification technology.The crystal structure of Bur01 shows that there are three molecules stacked side by side in an asymmetric unit,where the interaction area between chain A and chain B reaches 896?~2,forming a stable interaction.Each monomer exhibits a classicalα/β-hydrolase fold enzymes structure,and the catalytic triad was Ser111-His274-Asp241.Hydrogen bonding between enzyme and substrate at residues Ala22 and Met112 is the critical force to maintain enzyme activity,residues Gly21,Ala22 and Met112 together composed the oxygen anion hole of Bur01.Theπbond stacking between Trp23 and His24residues is very important for stabilizing the catalytic active center of Bur01,and both sites form hydrogen bond interaction with substrate,which is the key force of enzyme catalysis.Residues Phe137,Val149,Leu159,Leu192,Phe217,Val243 and Ile244 together form the substrate channel of Bur01.Residues Phe137,Val149,Leu159,Phe217,and Val243 were located at the entrance of the channel,providing a hydrophobic entrance for substrate transport,while residues Leu192 and Ile244 were located in the interior of the channel,providing a hydrophobic environment for substrate binding to the active center with other hydrophobic residues.Theα-helicesα2,α3,α9,andα10 in the protein structure may prevents the substrate from entering the active center,but remove and replacement of these parts of the protein structure may cause the instability of the protein structure,suggesting that theseα-helices play an important role in the stability of the enzyme and can protect the catalytic active center from the destroy of the surrounding environment.In this thesis,the protein structures of key enzymes LIP05 and Bur01 were comprehensively analyzed,including the core catalytic domain and cap domain,as well as the catalytic active center,oxygen anion hole and key amino acid residues in the substrate binding pocket of the enzyme.The results showed that in addition to the amino acid residues in the active center have an effect on the catalytic function of the enzyme,the rest amino acid residues in other domain have an important influence on the substrate moving in and out of active center and the stability of the active center.The study is conducive to a comprehensive and systematic exploration of the enzymatic catalytic mechanism of ester synthetase and will provide important research ideas and theoretical guidance for improving the catalytic mechanism of ester synthase.