Functional Analysis On Glycoside Hydrolases Of GH3 Family From Sphingomonas Sp.ATCC 31555 And The Preliminary Exploration Of The Function Of Fn3＿like Domain

Glycoside hydrolase（GH,EC 3.2.1）is a class of enzymes that hydrolyzes glycosidic bonds by either exo-or endo-activities,and turn the glycoside substrates into monosaccharides,oligosaccharides or sugar complexes.Glycoside hydrolase of GH3 family distribute widely in fungus,bacteria and plants.They exhibit wide substrate spectrum,some of them are multi-function enzymes,and showed great potential in producing active compounds such as resveratrol and Bohoroside I,converting lignocellulose,synthetizing compounds such as monoglycosides and polysaccharides,etc.However,glycoside hydrolase of GH3 family that are capable of hydrolyzing flavone glycosides and terpene glycosides has been rarely reported.To more efficiently hydrolyze these compounds,it is necessary to identify new enzymes with traits in request from the natural biological resources.The previous studies of our group demonstrated that Sphigomonas was potent in hydrolyzing flavone glycosides and terpene glycosides,suggesting it could be a good source for isolating glycoside hydrolase that are capable of hydrolyzing such compounds.In this study,a survey on the genome of Sphingomonas sp.strain ATCC31555 for genes encoding glycoside hydrolase was carried out.Five of the genes（Sp BGL1,2,3,4,5）were cloned and the proteins were purified successfully.Substrate spectrum analysis revealed that Sp BGL1 was a xylitosidase withβ-glucosidase activiy,Sp BGL2 was aβ-glucosidase and Sp BGL3 was a xylitosidase.The substrates include various sugar group-containing compounds,including monoglycosides,oligosaccharides,polysaccharides,saponins and glycoproteins,etc.β-glucosidase（EC 3.2.1.21）is a group of glycoside hydrolase that hydrolyze glycosidic bonds on glycosides and oligosaccharides to release non-reducing terminal glucose residues.β-glucosidases（EC 3.2.1.21）is one of glycoside hydrolases that hydrolyze glycosidic bonds on glycosids and oligosaccharides to release non-reducing terminal glucose residues.Through evolution or artificial design,β-glucosidase also catalyze synthesis glycosidine bonds by transglucosides.Through natural evolution or artificial design,β-glucosidase could be tuned into glycosynthases which are able to catalyze the synthesis of glycosidine bonds in the form of transglucosides.Glycoside hydrolases is abundant in diverse organisms,and an increasing amount of enzyme of this family are being cloned and studied.β-glucosidases are prominent in many biological applications such as producing biological active aglycone（resveratrol,Baohuoside I,etc.）,the conversion of lignocellulose,the synthesis of alkyl glucosides and glycoconjugates,etc.The biological production catalyzed byβ-glucosidases are usually performed in mild condition and are environment-friendly,such advantageous characteristics have madeβ-glucosidases the most studied glycoside hydrolases.Besides the identification of newβ-glucosidases with desired traits from microorganism,the optimization of the conversion process and the downstream product purification methods are also crucial for more effective and environment-friendly productions.Structural biology studies on enzymes can provide valuable information on fundamental questions such as the enzyme-substrate interactions and the catalyzations.However,despite the large amount ofβ-glucosidase being identified,the structure of only a small fraction of these enzymes have been resolved by crystallography.AI-powered protein structure prediction has been advanced dramatically in recent years.To date,among the algorithms published,Alpha Fold 2 is prominent in term of prediction efficiency and accuracy.It provides a fast and relatively low-cost way to dig into the enzyme-substrate interaction at atomic level with high confidence.The enzymatic properties of Sp BGL1,Sp BGL2 and Sp BGL3 were studied using p NPG and p NP-Xyl as substrates.The result showed that the optimum p H and optimum temperature for the enzymatic activity of Sp BGL1 on p NP-Xyl was 6.0 and 50℃,respectively.The values of K_m and V_max of Sp BGL1 were2.752±0.316 m M and 8.082±0.297μmol·min^-1mg^-1,respectively.Sp BGL1exhibited good stability under p H ranging from 5.5 to 6.5 and temperature between 25-35℃.The enzyme activity was maintained after 6 hours’incubation at 25°C.Sp BGL1 showed good tolerance to glucose with glucose inhibition constant K_i of 110.6±5.563m M,and xylose K_i of 175.264±6.105m M.Sp BGL1exhibited a broad substrate specificity towards aryl glycosides and showed the highest activity on p NP-Xyl,followed by p NPG.It can also hydrolyze p NP-β-D-mannopyranoside.Sp BGL1 showed good tolerance for various alcohol,and was able to remain 60.1%and 90.9%of activity under the presence of methanol or ethanol at 15%（v/v）.The optimum p H and temperature for the enzymatic activity of Sp BGL2 on p NPG was 6.0 and 45℃,respectively.The values of K_m and V_max of Sp BGL2were 4.06±0.53 m M and 38.35±1.92μmol·min^-1mg^-1,respectively.Sp BGL2exhibited good stability under the p H range 5.0-6.0 below 30℃.Sp BGL2 was sensitive to glucose and the inhibition constant K_i was 3.161 m M.Sp BGL2showed a broad substrate specificity towards aryl glycosides and showed the highest activity on p NPG,followed by p NP-β-D-cellobioside.Sp BGL2 was also capableofhydrolyzingweaklyo NP-β-D-galactopyranoside,p NP-β-D-galactopyranoside,4-Methylumbelliferyl-β-D-Glucuronide,Amygdalin,cellobiose and gentiobiose.The activity of Sp BGL2 could be stimulated by Triton X-100（1%,v/v）,β-mercaptoethanol（1%,v/v）and Tween 80（5%,v/v）up to 130%,121%and 141%,respectively.The optimum p H and temperature for the enzymatic activity of Sp BGL3 on p NP-Xyl was 5.5 and 40℃,respectively.The values of Km and Vmax of Sp BGL3 were 12.85±5.72 m M and 3.165±0.703μmol·min-1mg-1,respectively.Sp BGL3 showed good tolerance toward xylose,the 77%of the enzymatic activity was maintained under the presence of 1 M of xylose.However,Sp BGL3showed poor stability.Its enzymatic activity dropped to less than 30%after incubating at temperature above 30℃for 1 h.Among the substrate spectrum testes in this study,Sp BGL3 exhibited hydrolysis activity only on p NP-Xyl.Sp BGL3 was inhibited by SDS（10 m M）,while it was stimulated by Triton X-100（1%,v/v）,β-mercaptoethanol（1%,v/v）and Tween 80（5%,v/v）.Imidazole（10 m M）showed no effector on Sp BGL3.Studies on the transglycosylation activity of Sp BGL1 and Sp BGL2 were carried out.The results showed that Sp BGL1 exhibited transglycosylation activities onα-lactose,D-xylose,maltose and isomaltose.Surprisingly,Sp BGL1was able to catalyze transglycosidic reaction on rubusoside and resulted in a novel product.Sp BGL2 showed transglycosylation activities onα-lactose,D-galactose,D-xylose,D-fructose,sorbitol,ethylene glycol,1,2-propanediol,1,3-propanediol and glycerol.In this study,the potential of Sp BGL1 and Sp BGL2 in industrial conversions was investigated.Results showed that even though Sp BGL2 was capable of hydrolyzing dissolve soy isoflavones,epimedium flavonoids and polydatin,the reaction was time-consuming,thus made Sp BGL2 not a good choice for industrial applications.On the contrary,besides soy isoflavones,epimedium flavonoids and polydatin,Sp BGL1 was capable of hydrolyzing the ginsenosides such as Fc.Sp BGL1 showed high activity on polydatin convert to resveratrol.To optimized the reaction condition and product recovery in the polydatin-resvertrol conversion by Sp BGL1,a biphase conversion system was designed and optimized in this study.In a 3 m L system containing 180μg/m L of polydating,30 g/L of 20%crude polydatin was successfully converted into resveratrol of 3.34 g/L,demonstrating huge potential and advantages in the biphase conversion system.Alpha Fold 2 was deployed in this study to predict and compare the protein structure of Sp BGL1,Sp BGL2 and Sp BGL3.Amino acid sequence analysis revealed that all three proteins were three-domain proteins,which shared glyco＿hydro＿3 and Fn3＿like domains in common.Structural analysis revealed that despite the differences in biological function and domain composition,the three domains in all three proteins had similar spatial arrangement.The structure of Fn3＿like domains were well conserved,and this domain formed a potential pocket with the other two domains.Based on the 3D structure prediction by Alpha Fold 2,molecular docking simulations between Sp BGL1 and its substrates were carried out,and the pocket formed by Fn3＿like domains showed high potential in interacting with the substrates,indicating its importance in enzyme-substrate recognition.In this study,a high-throughput screening system was established for screening the enzymatic activities of the Sp BGL1 mutants.Site-directed random mutagenesis was performed on D765,Y657/N658,E292,Y136 and Y416,resulting in four single-site and one dual-site mutant libraries.The ability of mutants in hydrolyzing polydatin were screen using the high-throughput screening system.The results showed that D765 and Y657/N658 was highly correlated with the enzymatic activity on polydatin.Since D765 locates in the Fn3＿like domain and Y657/N658 locate in the glyco＿hydro＿3＿C domain,it was postulated that the pocket involving Fn3＿like domain was important for the enzyme-substrate interaction.It might facilitate the catalyzing pocket contact with the substrate by conformational changes,which might be mediated by the Fn3＿like domains-substrate interaction.