| ?-glucosidases are enzymes that calalyze the transfer of glycosyl group between oxygen nucleophiles.They can catalyze the simultaneously breakage and synthesis of ?-glcosidic bond,and have a wide range of sources and veriety functions.?-glucosidases have important applications in the fields of food,pharmacy and chemical engineering.In the study,two ?-glucosidase genes bgl2 and bgl4 of GH1,and one?-glucosidase gene bgl5 of GH3 from Streptomyces sp.GXT6 were cloned into expression vector pSE380.The recombinant plasmids were constructed and transformed into E.coli XL 1-blue for expression.The active proteins BGL2,BGL4 and BGL5 were successfully purified by nickel-nitrilortiacetic chromatography.And the substrates hydrolysis and transglycosylation of the recombinant proteins were studied in detail.In addition,based on the work of the research group,a supplementary research was carried out on the other two ?-glucosidases BGL1 and BGL3 from Streptomyces sp.GXT6.The properties of BGL1 and BGL3 for substrate hydrolysis and transglycosylation were complemented.And the study on the transglycoside function of the five mutants with enzymatic activity at W81 of BGL3 was carried out.The enzymatic properties of BGL2,BGL4 and BGL5 were studied using pNPG as substrate.The results showed that the optimum pH and temperature of BGL2 were 7.0 and 45 ?,the values of Km and Vmax were 0.2871±0.01586 mmol/L and 10.24±0.1577 ?mol·min-1mg-1,respectively.The value of glucose inhibition constant Ki was 75.63±5.499 mmol/L.BGL1 exhibited a broad substrate specificity towards aryl glycosides and showed the highest activity towards pNPG,only weak hydrolytic activity on trehalose and pNPC.It was capable of hydrolyzing soy isoflavones,epimedium flavanoids and polydatin,and the enzyme activity of BGL2 was obviously activied at a final concentration of 5%ethylene glycol and 1,3-propanediol.The optimum pH and temperature of BGL4 were 6.5 and 55 ?,the values of Km and Vmax were 0.4414±0.02575 mmol/L and 9.181±0.1673 ?mol·min-1mg-1,respectively.The value of glucose inhibition constant Ki was 192.3± 13.47 mmol/L.BGL4 was capable of hydrolyzing soy isoflavones,epimedium flavanoids and polydatin,with only weak hydrolysis of pNPC,oNPG,gentiobiose and lactose.The optimum pH and temperature of BGL5 were 6.5 and 40 ?,the values of Km and Vmax were 0.8562±0.03653 mmol/L and 0.09314±0.001156?mol·min-1mg-1,respectively.The value of glucose inhibition constant Ki was 50.7±2.585 mmol/L.BGL5 exhibited a broad substrate specificity towards aryl glycosides,and it was capable of hydrolyzing soy isoflavones,epimedium flavanoids and polydatin.Transglycosylation activity of BGL2,BGL4 and BGL5 were studied.The results showed that both BGL2 and BGL4 exhibited transglycosylation activity on a few alcohols,such as ethanol,ethylene glycol,1,2-propanediol,1,3-propanediol.At the same time,BGL2 has transglycosidic activity on hexyl-?-D-glucopyranoside,n-octyl-?-D-glucopyranoside and octanol,and BGL4 has transglycosidic activity on heptyl-?-D-glucopyranoside.However,BGL5 found no transglycosylation function in the investigated substrates.The hydrolysis and transglycosylation function of BGL1 and BGL3 were supplemented.The results showed that both BGL1 and BGL4 could hydrolyze epimedium flavonoids and polydatin.BGL 1 found no transglycosylation function in the investigated substrates.BGL3 exhibited transglycosylation activity on hexyl-?-D-glucopyranoside,heptyl-?-D-glucopyranoside,n-octyl-?-D-glucopyr-anoside,nonyl-?-D-glucopyranoside,decyl-?-D-glucopyranoside,hexanol,octanol,nonanol and decanol,pNPG as glycosyl donors.The results showed transglycosylation activities of five mutants of the W81 site of BGL3 were studied and found that the transglycosylation capacity of the five mutants did not increase compared to BGL3. |
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