Characterization Of Chitinase And ?-galactosidase In Fecal Microbial Metagenome From Rhinopithecus Bieti

Chitinase?EC 3.2.1.14?has been further developed by degrading chitin into chitosan oligosaccharides with different degrees of polymerization by cleaving?-1,4-D-glycosidic bonds between N-acetyl-D-glucosamine.Widely used,it also alleviatesenvironmentalpollutionwhileavoidingwasteofresources.?-galactosidase?EC 3.2.1.23?hydrolyzes lactose to galactose and glucose by breaking the?-1,4-D-galactopyranoside linkage in lactose for better absorption and utilization by the human body.Widely used in food and other fields.At present,the development and research of novel chitinases and?-galactosidases from different sources and with better characteristics are of great significance for further expanding the application fields and scope of chitinase and?-galactosidase.Animals have a large number of unique microbial flora in the gastrointestinal tract,closely related to animal immunity,food digestion,etc.At the same time,animal gastrointestinal microorganisms are a relatively independent,hugely-to-be-developed enzyme gene library,so they are excavated from them.Various microbial enzyme resources are important for research and development and utilization.In this dissertation,starting from the microbial metagenomic library of the rhesus feces of rhesus monkeys,combined with bioinformatics analysis,the chitinRBM1 and?-galactosidase gene galRBM1 were amplified by PCR using Fosmid plasmid DNA as a template.After heterologous expression and purification in Escherichia coli BL21?DE3?,the recombinases were studied for their related properties and preliminary applications.The result is as follows:?1?The optimal substrate for the recombinant enzyme ChitiRBM1 is 1%colloidal chitosan,the optimum pH is 6.0,the optimum temperature is 50°C,and the corresponding K _m,V _maxax and K _catat They were 4.2419 mM,52.63?mol/h/mg,and0.7252 min^-1,respectively.After 20 h of tolerance at 30°C and 40°C,the relative enzyme activity was maintained above 70%.After 50°C tolerated for 20 h,the relative enzyme activity was still 47.29%.More than 70%of the relative enzymatic activity was tolerated for 20 h at pH 5-9.After 12 h of tolerance at 0-15%NaCl,galRBM1 maintained relative activity of more than 80%,and the final concentration of 30%NaCl retained more than 30%of relative enzyme activity after 12 h of tolerance.The 10 mM final concentrations of Mn²⁺and Fe²⁺had a strong activation effect on the activity of galRBM1.The final concentration of 10 mM Co²⁺and 1%?-mercaptoethanol significantly promoted its enzyme activity,and the final concentration was 10 mM.Cu²⁺,Fe³⁺,Ag⁺,Ni²⁺,1%Tween-80 had obvious inhibitory effects on their enzymatic activity,while other ions or reagents had no significant effect on the enzymatic activity of the recombinase.?2?The optimal substrate for the recombinant enzyme galRBM1 is ONPG?o-nitrophenyl?-D-galactopyranoside?,the optimum temperature is 50°C,the optimum pH is 7.0,and the corresponding K _m,V _maxax and K _catat were 0.9862 mmol/L,5.296 6 mmol/min/mg,and 3.0896 s^-1,respectively.Tolerated for 4 h at 30-50°C,remaining enzyme activityKeep 85%or more.In the pH3 and pH4 acidic environment,the relative enzyme activities were 17.46%and 37.87%,respectively,within 1 h of the acid environment.Within the range of pH 5-10,the enzyme activity was maintained above?80%?,and the pH 11 alkalinity environment remained.More than 70%of the enzyme activity.When the final concentration of 0-10%NaCl was tolerated for 1 h,the relative remaining enzyme activity was maintained at more than80%.Under the condition of high concentration of 30%NaCl,the relative enzyme activity remained 40.87%after 1 h of tolerance.The final concentration of 10 mM Fe³⁺and Fe²⁺had a strong activation effect on the enzyme activity of the recombinase galRBM1.Cu²⁺,Ag⁺and 1%Tween-80 had significant inhibitory effects on its enzyme activity.The remaining metal ions and chemical reagents have no significant effect.In summary,a chitinase and?-galactosidase were obtained in this study,which has a wide range of pH tolerance and good pH and temperature stability,can tolerate various metal ions and has good salt tolerance,etc.Excellent industrial properties have potential applications in the fields of medicine,food,agriculture and dairy products processing and development.