Cloning,Expression,and Characterization Of Galactosidase Genes From Special Environmental Microorganisms

Galactosidase is a glycoside hydrolase,which can catalyzes the hydrolysis of heteropolysaccharide such as melibiose,lactose and raffinose.It also has important applications in food processing,medical treatment,animal feed and other industries.Galactosidase-producing strains have adapted to the special environments they are living and obtaining enzymes with special properties.In this study,4 galactosidase genes were cloned from microorganisms that were isolated from soda lake,mangrove forest and flaming mountains,and 3 of them have been heterologously expressed in E.coli or yeast.Then the recombinant enzymes were purified and characterized in detail.The results are as follow:1.Using soyabean and lactose as the sole carbon source of primary screening medium,7 alactosidase-producing strains were obtained.Four galactosidase gene fragments were amplified from 3 strains.These genes showed the highest identities from 74%to 100%with known galactosidases.2.Four full-length galactosidase genes were obtained by using TAIL-PCR or homologous cloning.Then the full-length genes were cloned and were analyzed:agalSL3-GH27,bgalSL3-GH42,agalMF5-GH27 and bgalFM3-GH2 were 1167,2058,1311 and 3045 bp long,encoding the polypeptide of 388,685,436 and 1014 amino acid residues,respectively.These four galactosidases had similarities of 99%,97%,99%and 71%with known galactosidases respectively.3.The enzymological of purified recombinant a-galactosidase rAgalSL3-GH27 are as follows:the optimum pH and temperature was 5.5 and 55?,respectively.The enzyme has good pH stability in pH 5.0?10.0 and had good thermal stability at 50?.The enzyme activity is had a good tolerance at a concentration of NaCl within 1.5 mol·L-1 and has good stability in the presence of 3 mol·L-1 NaCl.With PNPG as substrate,The specific activity,Km,Vmas and kcat of rAgalSL3-GH27 were 379.78±5.80 U·mg-1,2.61 ±0.02?mol·mL-1,452.30 ± 0.57 ?mol·(mg·min)-1 and 345.53 ± 1.27 s-1,respectively.The recombinant enzyme hydrolyzed melibiose and raffinose,but did not have detected activity to ONPG and guar gum.4.The enzymological of purified recombinant ?-galactosidase rBgalSL3-GH42 are as follows:the optimum pH and temperature was 6.5 and 40?,respectively.The enzyme has good pH stability in pH 6.0?9.0 and had good thermal stability within 40?.The enzyme activity were affected little by NaCl and has better stability within 1.5 mol·L-1 NaCl.With ONPG as substrate,The specific activity,Km,Vmax and kcat of rBgalSL3-GH42 were 354.48± 7.68 U·mg-1,10.76±0.38 ?mol·mL-1 833.33 ± 1.21 ?mol.(mg·min)-1 and 1110.42 ±1.36 s-1,respectively.The recombinant enzyme hydrolyzed lactose,but did not have any activity to PNPG.5.The enzymological of purified recombinant a-galactosidase rAgalMF5-GH27 are as follows:the optimum pH and temperature was 4.5 and 50?,respectively.The enzyme has good stability in pH 5.0?10.0 and good thermal stability within 50?.The enzyme activity is greatly influenced by NaCl and unstable in the presence of 2.0 mol·L-1 NaCl and above.With PNPG as substrate,the specific activity,K.,Vmax and kcat of rGalSL3-GH27 were 87.45±0.10 U·mg-1,0.15±0.01 ?mol.mL-1,80.13±0.02?mol·(mg·min)-1 and 80.51±0.05 s-1,respectively.The relative enzyme activity of recombinant enzyme hydrolysis of melibiose,raffinose and guar gum were 1.04%,3.75%and 2.3 1%,respectively.