Secreted Enzyme Analysis, Gene Cloning And Heterologous Expression Of Glycoside Hydrolases Of Acidophilic Fungus Bispora Sp. MEY-1

Extremophiles play important roles in the ecosystem and provide rich microbial resources for human beings. Along with the isolation and identification of more and more extremophiles, various extremozymes have been isolated and purified, leading to the expansion of more applications of extremozymes in biocatalysis and biotransformation.In this study, an acidophilic fungus, MEY-1, was isolated from the acidic waste water of "721" Uranium Mine in Jiangxi, China. Isolate MEY-1 was able to grow at pH1.0～6.0, with the optimal growth at pH 2.5～3.0. Based on morphology and ITS sequence analysis, isolate MEY-1 was identified to belong to the genus Bispora. Being an excellent industrial enzyme producer, Bispora sp. MEY-1 can secrete nine important industrial enzymes, including xylanase,β-glucanase,β-mannanase, CMCase, amylase, pectinase,α-galactosidase,β-galactosidase andβ-glucosidaset.A fermentation system was initially established to induce MEY-1 to secrete the nine enzymes mentioned above. By optimizing the proportion of wheat bran, corncob powder, konjac flour and soybean meal in the medium, pH and temperature, four methods were used to induce different enzymes. Method 1 for high-yield ofβ-mannanase, CMCase, amylase and pectinase: the proportion of wheat bran, corncob powder and soybean meal was 2:5:2, pH 2.0 and 20℃. Method 2 for high-yield ofβ-glucanase: the proportion of wheat bran, corncob powder and soybean meal was the same as Method 1, pH 2.0 and 30℃. Method 3 for high-yield ofα-galactosidase,β-galactosidase andβ-glucosidase: the proportion of wheat bran, corncob powder and soybean meal was 1:1:7, pH4.0 and 30℃. Method 4 for high-yield of xylanase: the proportion of wheat bran, corncob powder and soybean meal was 5:2:2, pH 4.0 and 30℃.Based on sequence analysis, nine glucoside hydrolase genes belonging to seven kinds of enzymes were cloned from the acidophilic fungus Bispora sp. MEY-1. They are three xylanase genes, oneβ-Mannanase gene, oneα-amylase gene, oneα-galactosidase gene, oneβ- galactosidase gene, oneβ-glucosidase gene and oneα-L- rhamnosidase gene, respectively. Homology searches in GenBank using BLAST and multiple alignment using CLUSTALW were performed. The highest identity of nucleotide and amino acid sequences of these enzymes to known glucoside hydrolases are: xylanase gene xyl11A, 85.3% and 93.7%; xylanase gene xyl11B, 61.4% and 57.0%; xylanase gene xyl10A, 49.8% and 43.6%;β-mannanase gene man5A, 45.4% and 47.1%;α-amylase gene amyA, 60.2% and 62.0%;α-galactosidase gene agalA, 54.2% and 49.7%;β-galactosidase gene bgalA, 56.8% and 55.5%;β-glucosidase gene bglA, 49.5% and 34.8%;α-L- rhamnosidase gene rhaA, 55.0% and 44.9 %. Except for rhaA, all other enzymes were subject to three-dimensional structure and catalytic site analysis, indicating the novelty of eight enzymes besides xyl11A. Theβ-mannanase gene man5A, and three xylanase genes xyl11A, xyl11B and xyl10A were expressed in Pichia pastoris with the activity of 64, 83, 54 and 101 U/mL in flasks, verifying the function of these genes.The recombinantβ-mannanase MAN5A (MAN5A) and three xylanases (XYL11A, XYL11B and XYL10A) were purified and characterized. MAN5A had the optimum pH of 1.0~1.5, lower than all the knownβ-mannanases, and retained >70% activity over pH 1.0~5.5. The optimum pHs of XYL11A, XYL11B and XYL10A were 2.4, 2.6 and 3.0, respectively. The optimum temperatures of MAN5A, XYL11A, XYL11B and XYL10A were 65, 60, 65 and 80℃, respectively. All the recombinantβ-mannanase and three xylanases had strong resistance to pepsin and trypsin treatmen, retaining 85~150% activities at 37°C for 60 min. Metal ions and chemical reagents enhanced or had no effects on enzyme activities. The Km and Vmax values for MAN5A were 1.56 mg/mL and 6587.6μmol/min ? mg, respectively, with the specific activity of 3373 U/mg. The specific activity of XYL11A, XYL11B and XYL10A were 8334, 2049 and 5437 U/mg, respectivley. The Km and Vmax values for XYL11A, XYL11B and XYL10A were 19.907 and 7308.5, 29.96 and 1757.2, and 1.005 mg/mL and 2709μmol/min ? mg, respectively. All these four enzymes are acidicphilic with very high specific activities, suggesting their good prospects in industrial applications.