Heterologous Expression And Catalytic Properties Of Glycoside Hydrolase From Cellulosimicrobium Sp. TH-20

Glycoside hydrolases,a widely distributed group of enzymes,can cleave glycosidic bonds in glycosides,glycans,and glycoconjugates,and play key roles in biofuel development and disease research.A strain of Cellulosimicrobium sp.TH-20was screened in our laboratory in the early stage.This strain has degradation and transformation activities on various polymers,such as cellulose,xylan,and ginsenoside.The genome sequencing of the strain was performed in the laboratory.Genomic annotation information was used to conduct related research on six glycoside hydrolases of Cellulosimicrobium sp.TH-20.The main experimental results are as follows.Six glycoside hydrolase genes were selected from Cellulosimicrobium sp.TH-20,cloned,and constructed into recombinant plasmids.These genes were heterologously expressed and purified in Escherichia coli.The recombinant enzymes were purified using affinity chromatography.The pure enzyme components were CAgl13,CXyl3,CAbf51,CMal65,CAmy13,and CGH43.Sequence analysis of CAgl13 revealed that the enzyme was an alpha-amylase of the GH13 family.CAgl13 displayed an optimum temperature of 40? and optimum pH of 8.0.This enzyme exhibited thermal stability below 30? and pH stability between pH 5.0–12.0.The CAgl13 substrate had a wide range of specificities,hydrolyzed pNP?Glu,pNP?Glu,pNP?Gal,pNP?Arap,malto-oligosaccharides,and starch,and had transglycoside activity on maltose.Glucose inhibited enzyme activity.In particular,2.5 M glucose led to approximately 40%residual activity of the enzyme.The calculated K_m and K_cat/K_m values of the enzyme were 6.82 mM and 0.01 s^-1 mM^-1for pNP?Glu,respectively.The enzyme activity was inhibited by Fe³⁺,Co²⁺,Zn²⁺,and Cu²⁺as well as by low concentrations of Mn²⁺,Ca²⁺,and Zn²⁺.The enzyme activity was promoted by high concentrations of Mg²⁺,Mn²⁺,and Na⁺as well as by propanol and glycerol.Butanol,EDTA,SDS,N-acetylimidazole,iodoacetic acid,TritonX-100,and Tween80 had inhibitory effects on enzyme activity.Sequence analysis of CXyl3 showed that the enzyme was a?-glucosidase from the GH3 family.Substrate specific results showed that the enzyme was capable of hydrolyzing pNP?Xyl,pNP?Araf,pNP?Arap,and pNP?Gal and had the highest activity against pNP?Xyl.CXyl3 displayed an optimum temperature of 40? and optimum pH of 7.0.The enzyme had good thermal stability below 40? and pH stability between pH 6.0–11.0.Xylose inhibited the enzyme activity.Treatment with2.5 M xylose led to approximately 30% residual activity of the enzyme.The calculated K_m and K_cat/K_m values of the enzyme were 1.47 mM and 0.07 s^-1 mM^-1 for pNP?Xyl,respectively.The enzyme activity was inhibited by Fe³⁺,Co²⁺,Zn²⁺,and Cu²⁺as well as by low concentrations of Mn²⁺,Mg²⁺,and Ca²⁺.The enzyme activity was promoted by high concentrations of Na⁺,Mn²⁺,and Mg²⁺.Ethanol,propanol,butanol,TritonX-100,Tween80,SDS,?-mercaptoethanol,N-acetylimidazole,and iodoacetic acid had inhibitory effects on the enzyme activity.Sequence analysis of CAbf51 showed that the enzyme was?-L-arabinofuranosidase from the GH51 family.CAbf51 displayed an optimum temperature of 60? and optimum pH of 7.0.The enzyme had good thermal stability below 40? and pH stability between pH 6.0–9.0.The CAbf51 substrate had a wide range of specificities and hydrolyzed pNP?Araf,pNP?Xyl,and pNP?Gal as well as arabinooligosaccharide to release arabinose.Arabinose promoted the enzyme activity.Treatment with 2.5 M arabinose increased the enzyme activity to 130% of the original activity.The calculated K_m and K_cat/K_m values of the enzyme were 3.07 mM and 0.01s^-1 mM^-1 for pNP?Araf,respectively.The enzyme activity was inhibited by Co²⁺,Zn²⁺,and Cu²⁺as well as by high concentrations of K⁺,Ca²⁺,Fe²⁺,Mn²⁺,and Fe³⁺.The activity was promoted by low concentrations of Fe²⁺,Mn²⁺,Ca²⁺,and Fe³⁺and by high concentrations of Na⁺and Mg²⁺.The enzyme activity was also promoted by ethanol,propanol,and glycerol.Butanol,?-mercaptoethanol,SDS,N-acetylimidazole,and iodoacetic acid had inhibitory effects on the enzyme activity.The sequence analysis results showed that CMal65,CAmy13,and CGH43 were maltose phosphorylase of the GH65 family,?-amylase of the GH13 family,and glycoside hydrolase of the GH43 family,respectively.Enzyme activity was measured with pNP substrate,and the results showed that CMal65 only had weak activity on pNP?Gal.CAmy13 and CGH43 had no hydrolytic activity on currently detected pNP substrates,and further research is needed.In this study,the recombinantly expressed glycoside hydrolase had new enzyme characteristics in the sequence.In-depth research is required to elucidate the hydrolysis mechanism of Cellulosimicrobium sp.TH-20 and provide a new potential enzyme source for industrial applications of glycoside hydrolases.