Identification Of A Novel GH42 Family Bifunctional β-Galactosidase/α-arabinopyranidase And Its Preliminary Rational Design

P-galactosidase(EC3.2.1.23)is a kind of glycosidase that can catalyze the break of βD-galactosidic bond in β-galactosidic compounds.The enzyme can not only catalyze the hydrolysis of β-galactoside compounds,such as lactose,to release galactose,but also catalyze the transformation of galactose into galactosides to synthesize various galactoside compounds.Therefore,β-galactosidases are widely used in food,and medicinal industries,and chemical analysis.β-galactosidase is wide spread in microorganisms,plants,and animals,in which microorganisms are widely distributed in various physical and chemical environments,thus microbial β-galactosidases exhibit a variety of biochemical properties.Therefore,exploring β-galactosidases from microorganisms is a feasible way to obtain enzyme with excellent characteristics.In this study,a new GH42 family β-galactosidase gene ba3238,was identified from the genome of Bacillus sp.KW1.The full length of the gene is 2067 bp,encoding a protein composed of 688 amino acid residues.Amino acid sequence analysis showed that Ba3238 exhibits the highest similarity with the GH42 family β-galactosidase,Gan42B,derived from Geobacillus stearothermophilus(≈57%homology).The gene was clone,the recombinant plasmid of pET28a-ba3238 was constructed and transformed into Escherichia coli strain BL21(DE3)for protein expression.The soluble recombinant protein was obtained and purified,and the purified recombinant Ba3238 was used for further biochemical analysis.The results of the enzyme analysis showed that the optimum pH of Ba3238 was 6.5 when using lactose as the substrate,and it had good enzyme activity under the condition of pH of fresh milk(pH6.4-6.8).The optimum temperature of Ba3238 was 45℃,and the maximum enzyme activity remained above 27-48%in the low-temperature range(5-20℃).The stable pH range of Ba3238 was analyzed at 45℃.It was found that the activity of the enzyme did not decrease significantly after being treated in pH 5.5-11.0 buffer for 24 h,which indicated that the enzyme had good pH stability.After treatment at 40℃ and 45℃for 7 h,the residual enzymatic activity of the enzyme remained above 90%,indicating that Ba3238 possessed good stability around the optimum temperature.Ca2+,Mg2+,Co2+significantly stimulated the activity of Ba3238,while Ag+and Cu2+significantly inhibited the activity.Low temperature hydrolysis of milk showed that the lactose in milk was completely hydrolyzed when 2U Ba3238 was incubated with 1 mL milk at 10℃ for 12 h.The specific activity of lactose,pNPG,oNPG,pNPA,and pNPF were 15.43,158.72,86.99,31.5 and 20.03 U·mg-1,respectively.After incubating Ba3238 separately with galactan,arabinan,wheat arabinoxylan,paeonolide,ginsenoside Rb2,and fucoidan,the hydrolysates were analyzed by HPAEC-PAD.Ba3238 can hydrolyze galactan and arabinogalactans to release galactose,and can hydrolyze wheat arabinoxylan,paeonolide,and ginsenoside to release arabinose.However,Ba3238 can’t hydrolyze algae polysaccharides to release monosaccharides and/or oligosaccharides.These results proved that Ba3238 is a novel GH42 family bifunctional β-galactosidase/α-arabinopyranidase.The results also showed that the Km of Ba3238 to lactose,pNPG,oNPG,and pNPA were 29.81,2.798,7.858 and 14.21 mM,Vmax:22.51,251.69,179.46 and 16.62 μmol·min-1·mg-1,respectively.Furthermore,we found that Ba3238 exhibited good synergistic effect with its cognate xylanase Ba104 and cognate endo-galactanase Ba3239:the addition of Ba3238 to Ba104 could increase the release of reducing sugar to 3.4-fold as compared to that released by Ba104 in the hydrolysis of wheat arabinoxylan,while supplementing Ba3238 with Ba3239 enhanced the liberation of reducing sugars to 1.75-fold of that liberated by Ba3239 alone.β-galactosidases are widely used in dairy industry,and the activity of enzymatic hydrolysis of lactose is a key index to determine the application potential of β-galactosidase.In order to improve the activity of Ba3238 to lactose,the method of rational design based on structure simulation can be used to improve the activity of lactose.Since the tertiary structure of Ba3238 is unknown,the structure of Gan42B(PDB code:4oif),the highest homolog of Ba3238,was used as the template for structure simulation.On this basis of enzyme-substrate molecular docking and molecular dynamics simulation,we found that the binding energy(AG)of Ba3238 with α-lactose and β-lactose were-25.20 and-14.71 kcal·mol-1.respectively,which suggested that Ba3238 had higher affinity in binding αlactose,the conformation of Ba3238 binding to α-lactose was more compact.By studying the conformation and decomposition of binding energy between the enzyme and substrates,a group of key amino acid residues which are favorable for enzyme-substrate binding and catalysis were identified.Among them,the key amino acid residues contributing to the binding of Ba3238 with α-lactose are ARG119,HIE374,PHE361,GLN362,TRP331,GLU323,GLU371,SER370,and ASN22,while LYS338,LYS372,GLU158,T YR286,PRO326,ASP284,GLY375,and ASP20 have adverse effect for enzyme-substrate binding.The residues contributing the binding of Ba3238 with β-lactose are GLU158,TRP331,ARG119,GLU323,and HIE374,whereas LYS338,TYR286,PRO326,GLU371,and ASP284 are unfavorable for the binding of enzyme with β-lactose.The identification of the key amino acid residues establishes the foundation for further modification of Ba3238 with better enzymatic activity by site-directed mutagenesis.