Thermal Stability Molecular Modification And Catalytic Mechanism Of Rice α-Galactosidase

α-Galactosidase(α-Gal,EC 3.2.1.22)can specifically catalyze the hydrolysis of α-1,6-galactosidase bond in galacto-oligosaccharides,polysaccharides,glycolipids and glycoproteins.It is widely used in food,feed,medicine and light industry.Therefore,it has been studied in recent years.However,α-Gal has some problems,such as poor thermal stability,low catalytic efficiency,and low production,which limit its application.It is great significance to construct α-Gal with good thermal stability,high specific enzyme activity and high production.In this study,α-Gal Ⅱ gene from rice(Oayza.sativa L.subsp.Japonica var.Nipponbare)was successfully anchored and expressed on the cell surface of Saccharomyces cerevisiae by a-agglutinin to obtain the Yeast Surface Display(YSD)whole cell catalytic unit YSD rice α-Gal Ⅱ.It simplified the steps of enzyme protein separation and purification,and optimized the components of culture medium and continuous fed fermentation to improve the enzyme activity per cell,dry cell weight and total production,and the addition of dominant cofactors further improved the activity and stability;Through the rational design of molecular dynamics simulation to analyze the key functional amino acids that affect the thermal stability,the mutants were screened by molecular dynamics secondary simulation to obtain good thermal stability mutants,and the actual mutation was carried out.Finally,the wild-type enzyme and the mutants with good stability were docking with different galacto-oligosaccharide and galactomannan small models substrates to study the mechanism of enzyme structure on the catalytic activity of different substrates.The main results are as follows:1.Rice α-Gal Ⅱ was expressed using food safety yeast EBY100 as the host to obtain the whole cell catalytic unit YSD rice α-Gal Ⅱ.The enzyme activity was 240.6 U/g,dry cell weight was 1.0 g,and total production was 243.6 U(in 1 L YNB-CAA medium).Adding complex nitrogen sources((NH4)2SO4/urea = 2/1,w/w)0.6 g/L,sucrose 5 g/L,continuous supplement galactose to the final concentration of 20 g/L(flow rate 1.5 m L/h),the optimal enzyme activity was 507.2 U/g,dry cell weight was 3.0 g and total production was 1548.5 U.In addition,it was found that the co-express with 5 m M Vc increased the enzyme protein content by 5.1 times.Combined with the above two methods,the enzyme activity,dry cell weight and total production were significantly increased to 1905.3 U/g,4.2 g and 8021.3 U,respectively,which were 7.9 times,4.2 times and 32.9 times of those before optimization.2.The optimal reaction temperature and p H value of YSD rice α-Gal Ⅱ were 45 oC and 5.0 respectively.The thermal stability decreased sharply with the increase of temperature,and the range of p H stability was 4.0-6.0.The hydrolytic activity of p-nitrophenyl-α-D-Galactopyranoside(p NPG)and galac-oligosaccharides was the highest,and hydrolysis of defatted soybean meal and guar gum was maintaining good stability in the long-term reaction system.Six dominant cofactors,VC,Fe3+,EDTA,urea,arginine and xylitol were found to increase the enzyme activity and stability.Arginine increased the enzyme activity by 178.7%.Its thermal stability was 10-20 oC and 4.5-6.5,and the stability of p H 7.0-10.0 was also improved.Xylitol extended the thermal and p H stability of YSD rice α-Gal Ⅱ to 10-25 oC and 3.5-7.0,respectively.After freeze-drying and storage at room temperature for 90 days,the residue activity was still 90.0%.3.The three-dimensional structure of rice α-Gal Ⅱ was simulated by SWISS-MODEL.GROMACS molecular dynamics simulation was used to predict the thermal instability amino acid residues and secondary simulation of mutants were screened positive mutants.Based on the simulation results,the thermostable mutants R167E、R167V、D285V、D285G and N9 were obtained by site-directed and N-terminal removed mutagenesis.The residual activity of wild enzyme was 19.5%,and that of mutants R167E、D285V、D285G and N9 was increased to 30.4%,65.9%,45.8% and 78.5%,respectively at 50 oC.The residual activity of wild enzyme was completely deactivated,and that of mutants R167E、D285V、D285G and N9 was increased to 30.0%,49.3%,35.3% and 39.7%,respectively at 60 oC.R167 E,R167V and N9 broaden the range of p H stability from the original 4.0-6.0 to 4.0-6.5,D285 V,D285G and N9 improved the stability of enzyme in alkaline p H range.The Kcat/Km value of R167 V and D285 G to p NPG was higher than that of wild type,and the result of enzymatic kinetics was better than that of wild type.R167 V was 108.0 % of the wild enzyme for hydrolysis of p NPG,but the hydrolysis of natural substrate was low.D285 V and D285 G were higher than the wild type in the hydrolysis of raffinose and stachyose,and the hydrolysis of melibiose and guar gum was decreased.N9 increased the hydrolysis of melibiose,and the hydrolysis of guar gum was only 56.6% of the wild type.4.Based on the hydrolytic differences of mutants to different substrates,molecular docking was used to melibiose,raffinose and Gal3Man3(G3M3)and Gal3Man4(G3M4)with rice α-Gal I,rice α-Gal Ⅱ and mutants D285 G,D285V and N9.The binding ability of rice α-Gal I with melibiose was stronger than rice α-Gal Ⅱ,but the binding ability to raffinose,G3M3 and G3M4 was much lower than rice α-Gal Ⅱ.The reason is that the structure of loop1,α 2-helix,α 5-helix,α 6-helix,α 7-helix and α 8-helix structure of enzyme catalytic center and the amino acid difference on β 9-strand which has recognition function between C-terminal and substrate,changing the direction of substrate entering the catalytic cavity of rice α-Gal I.The substrate was not completely embedded in the active cavity.The two key catalytic amino acids Asp130 and Asp185 in rice α-Gal I did not participate in the catalytic action,which resulted in the difference of the binding effect of the two enzymes on the substrate.The binding ability of three mutants to melibiose was improved.The binding ability of D285 V and rice α-Gal Ⅱ to raffinose was similar,but the binding ability of D285 G and N9 was significantly lower than the former two.Rice α-Gal Ⅱ has the highest hydrolysis on G3M3/G3M4,so it has the best hydrolysis on galactomannan.The other mutants in turn were D285 V,D285G and N9.The reason for the sharply decrease of the hydrolysis of N9 on the two galactomannan small molecules is that the amino acid Pro not belonged to catalytic center after removed N-terminal amino acids.The Pro formed a steric hindrance on the substrate entering the active cavity,which affected the substrate entering the active cavity internal binding with key amino acids.The results of molecular docking were consistent with the actual substrate specific,and the catalytic mechanism of rice α-Gal for different substrates was further explained at the molecular level.