Heterologous Expression And Characterization Of ?-Galactosidase From Fecal Microorganisms

Lactose is the mian carbohydrate found in mammalian and dariy products,When people drunk the milk,Lactose must be hydrolyzed by?-galactosidase to galactose and glucose during digestion to be absorbed by the body.It is reported that about one tenth people in the word suffer from lactose intolerance due to the lack of?-galactosidase in the body;after drinking milk,the body may have adverse reactions such as diarrhea and vomiting.Among them,?-galactosidase has a large commercial use situation,and the enzyme with excellent properties has important practical value in production and life.?-galactosidase is a kind of glycoside hydrolase and is widely existed in nature.Based on the similarity of amino acid sequences,?-galactosidase is classified into five Glycoside hydrolases?GH?families 1,2,35,42,59.Among them,part of?-galactosidase has a turbulent activity in addition to its hydrolysis activity.To date,there are many studies on?-galactosidase,but the enzyme from animal gastrointestinal tract and having transglycosylation activity have not been reported,and there are few reports of salt-tolerant?-galactosidase.Since?-galactosidase is widely used in the food industry,medical and biochemical analysis,salt tolerance is closely related to its application prospects.In this study,the?-galactosidase gene galRBM20₁?galRBM20₂?galRBM20₁3 and galRBM20₁5 were cloned by PCR from the fecal microbial metagenome genomic DNA of Rhinopithecus bieti.The four?-galactosidase genes were cloned into pEASY-E2 plasmid and transformed Escherichia coli BL21?DE3?to express the foreign protein successfully.The recombinant enzymes galRBM20₁?galRBM20₂?galRBM20₁3 and galRBM20₁5 were purified and characterized,The results show:?1?The optimum pH of?-galactosidase galRBM20₁ was 5.0,and can retain more than70%of activity between pH 4.0-7.0.The enzyme was stable at pH 5.0-8.0,The optimum temperature is 45°C,the enzyme was stable at 37°C and 45°C,retaining nearly 100%activity after pre-incubation for 1 h.Most metal ions and chemical agents have an activation effect on the activity of the enzyme.The enzyme has strong salt tolerance,the activity of the enzyme can be significantly improve for an appropriate concentration of NaCl in the reaction environment or treated with NaCl in a certain concentration range.?2?The optimum pH of?-galactosidase galRBM20₂ was 7.0,and can retain more than70%of activity between pH 7.0-9.0,the enzyme was stable at pH 5.0-7.0.The optimum temperature is 45°C,the residual activity was above 45%after incubated for 1 h at 55°C.Most metal ions and chemical agents have an activation effect on the activity of the enzyme.The enzyme has strong salt tolerance,the activity of the enzyme can be significantly improve for treated with NaCl in a certain concentration range.?3?The optimum pH of?-galactosidase galRBM20₁3 was 7.0,and can retain more than 70%of activity between pH 7.0-9.0,the optimum temperature is 45°C and stable at37°C?45°C?50°C and 55°C.Meta ions and organic reagents such as?-mercaptoethanol?Mg²⁺?Fe²⁺have positive effect on the galRBM20₁3.the activity of the enzyme can be significantly improve for treated with NaCl in a certain concentration range.?4?The optimum pH of?-galactosidase galRBM20₁5 was 7.0,and can retain more than 70%of activity between pH 7.0-9.0,galRBM20₁5 is strongly activated by pH.After 1h treatment in the range of pH 5.0-10.0,the enzyme activity is above 100%,and the relative enzyme activity reaches 180%after 1 hour of pH 10 treatment.The optimum temperature is45°C and stable at 50°C and 55°C.Meta ions and organic reagents such as?-mercaptoethanol?Mg²⁺?Mn²⁺have positive effect on the galRBM20₁5.The enzyme has a certain salt tolerance and still has 55%activity when 2 M NaCl is present in the reaction system.The results of TLC assay showed that the recombinant enzymes galRBM20₁,galRBM20₂,galRBM20₁3 and galRBM20₁5 had transglycosylation activity in addition to hydrolysis of lactose to galactose and glucose.Therefore,the?-galactosidase obtained in this study can not only hydrolyze whey,cheese and milk?pH 5.0-7.0?,but it can exert good reaction potential in the reaction with salt solution.Adapt to the wide range of uses for?-galactosidase.