Enzymatic Characteristics,catalytic Mechanism And Application Of β-Glucosidase From Oenococcus Oeni

Oenococcus oeni is the most commonly used lactic acid bacteria in the process of wine fermentation.O.oeni can convert malic acid into lactic acid to reduce the acidity of wine and theβ-glucosidase produced by O.oeni can also convert the glycosidic-bonded aroma compounds into free aroma substances to significantly increase wine aroma.As a kind of cellulase,β-glucosidase was widely used in the fields of biomass energy conversion,food and medical.At present,researchers have done a lot of research onβ-glucosidase derived from microorganisms,but theseβ-glucosidases were mainly derived from Aspergillus niger or yeast.There was little research onβ-glucosidase derived from O.oeni and the necessary elucidations on the enzymatic properties and catalytic mechanism ofβ-glucosidase were also lacking.Therefore,in order to deeply understand and explain the enzymatic properties and catalytic mechanism ofβ-glucosidase from O.oeni,in this paper,firstly,the gene family analysis ofβ-glucosidase from O.oeni was carried out.Secondly,the recombinantβ-glucosidase BGL0224 from O.oeni SD-2a was isolated by gene cloning and heterologous expression.The enzymatic properties,structure and catalytic mechanism of BGL0224 were further studied.In addition,with commercialβ-glucosidase as a comparison,the effect of BGL0224 on the quality characteristics of Cabernet Sauvignon wine was analyzed.The main results were as follows:（1）Gene family analysis ofβ-glucosidase from O.oeni.A total of 35β-glucosidases was screened from O.oeni.The length of amino acid sequence of these 35β-glucosidases were between 129 and 752,the isoelectric point were between 4.84 and 9.30,the molecular weight were between 15 k Da and 84 k Da.A total of 6 different conserved domains was searched from the 35β-glucosidases,and the conserved domain Bgl B was the most common conserved domain among them.The 35β-glucosidases were divided into three evolutionary branches.In different branches,the length of amino acid sequences and motif compositions were also different.Theβ-glucosidases in branches I and II belonged to GH1 family,while theβ-glucosidase in branch III belonged to GH3 family.（2）Cloning,heterologous expression and purification ofβ-glucosidase from O.oeni SD-2a.Three genes encodingβ-glucosidase in the genome of O.oeni SD-2a were successfully cloned,namely OEOE-1569,OEOE-1210 and OEOE-0224.Compared with p ET-28a,the expression plasmid Pcold I was more suitable for expressing theβ-glucosidase gene of O.oeni SD-2a.The Pcold I plasmid could not only successfully express all threeβ-glucosidase genes,but its expression of the two genes OEOE-0224 and OEOE-1210 also avoid the formation of inclusion bodies.The activity of BGL0224 was significantly higher than that of BGL1569 and BGL1210.After a series of purifications of BGL0224,the specific activity reached 5.92μkat/mg,the purity was increased by 147.98 times and the recovery rate was 14.58%.LC-MS/MS results showed that the molecular weight of BGL0224 was55151.84 Da and the isoelectric point was 6.14.（3）Enzymatic characteristics of BGL0224.BGL0224 was consisted of 480 amino acid residues and belonged to GH1 family.It was a hydrophilic protein in cytoplasm and did not contain signal peptides.The optimal reaction temperature of BGL0224 was 50℃,the optimal reaction p H was 5.0 and the thermal stability and p H stability were all good.The ethanol concentration of 0～20%had a significant promotion effect on enzyme activity,especially when the ethanol concentration was 12%.K⁺,Na⁺and Hg⁺had basically no effect on the activity of BGL0224,Ba²⁺and Li⁺had a slight promotion effect on its activity.The other metal ions and additives had an inhibitory effect on the activity of BGL0224,especially Triton-X100 and Tween-80,the inhibition rates were all exceeded 80%.BGL0224had a standard ultraviolet absorption peak at 260～280 nm.When the excitation wavelength was 339 nm,the enzyme had a maximum fluorescence emission wavelength at 440 nm.The four types of chemical bonds（O-H、N-H、C=C and C-O）in chemical structure of BGL0224were relatively active.The chemical shifts of C in chemical structure of BGL0224 were between 30 ppm and 160 ppm and the chemical shifts of H were between 0.03 ppm and10.25 ppm.The V_max,K_m,K_cat and K_cat/K_m of BGL0224 acting on p-NPG were 382.81±7.76μM/min/mg,0.34±0.04 m M,351.88 S^-1 and 1034.94 S^-1?μM^-1,respectively.（4）Catalytic mechanism of BGL0224.BGL0224 had catalytic effects on all seven kinds of substrates.The optimum reaction temperature of BGL0224 acting on p-Nitrophenylβ-D-glucuronide and p-Nitrophenylβ-D-xylopyranoside were 45℃and 40℃respectively.The optimal reaction temperature for catalyzing the other five substrates was 50℃and the optimal reaction p H for catalyzing the seven substrates was 5.0.When catalyzing the seven substrates（p-Nitrophenylβ-D-glucopyranoside,p-Nitrophenylβ-D-galactopyranoside,p-Nitrophenylβ-D-xylopyranoside,p-Nitrophenylβ-D-cellobioside,p-Nitrophenylβ-D-glucuronide,p-Nitrophenylα-D-glucopyranosideandp-Nitrophenylα-D-galactopyranoside）,the K_m were 0.34、0.95、1.10、0.43、1.87、1.42、1.70 m M,V_maxwere 382.81、270.88、256.43、359.54、24.17、39.27、39.64μM/min/mg,K_cat/K_m were 1034.94、262.09、214.28、768.58、11.88、25.42、21.43 S^-1?μM^-1,and E_a were 23.70、28.72、34.26、25.83、76.96、67.09、73.89 k J/mol,respectively.The fluorescence quenching mechanism ofthe seven substrates on the protein group of BGL0224 were all static quenching.The quenching constants K_q were 8.07、5.25、4.93、7.51、0.93、1.10、0.78×10¹² L?M^-1 and the binding constants K_b were 8.09、4.53、3.94、7.62、0.73、0.83、0.73×10⁴ L?M^-1,respectively.Using the crystal structure ofβ-glucosidase Bgl A as a template,the three-dimensional structure model of BGL0224 was constructed,which was proved to conform to the energy law of stereochemistry.The results of molecular dynamics simulation showed that the composite system“BGL0224-p NPG”was stable after 40 ns,and the binding energy of“BGL0224-p NPG”was-202.00±20.72 k J/mol.Hydrogen bonding andπ-πinteraction were main driving forces in the binding process of BGL0224 and p-NPG.The catalytic mechanism of BGL0224 acting on p-NPG followed the double-shift reaction mechanism.The glutamate residues Glu178 and Glu377 played a vital role in the whole catalytic process.（5）The effects of BGL0224 on quality characteristics of Cabernet Sauvignon wines.The results of PCA showed that the sum of the variances of the two principal components accounted for 80.20%of the total variance,indicating that the E-nose could distinguish Cabernet Sauvignon wines with different treatments.Adding BGL0224 before alcohol fermentation significantly increased the“aromatic index”of Cabernet Sauvignon wines.A total of 78 aroma compounds was detected in all wine samples,of which 47,62,63,63 and49 aroma compounds were detected in CK,A1,A2,B1 and B2 group,respectively.Adding BGL0224 before alcohol fermentation significantly increased the concentrations of medium-chain fatty acid ethyl esters,long-chain fatty acid ethyl esters and terpenes,and can also enhance the“tropical fruity”,“sweet fruity”and“floral notes”of Cabernet Sauvignon wines.Meanwhile,the sensory characteristic of“citrus flavor”was inhibited.The“tropical fruity”of Cabernet Sauvignon wines were positively correlated with medium-chain fatty acid ethyl esters and long-chain fatty acid ethyl esters,were negatively correlated with short-chain fatty acid ethyl esters and other esters.The“floral notes”of Cabernet Sauvignon wines were positively correlated with terpenes and were negatively correlated with short-chain fatty acid ethyl esters and alcohols.