Isolation And Identification Of Stachybotrys Sp. And Characterization Of Its β-glucosidase

In order to obtain the fungi strains which may hydrolyze cellulose with high efficiency, 45 soil samples were collected from different places of Xinjiang Autonomos Region. 40 fungi strains that utilized cellulose as the unique carbon course were gained. 9 strains secreted cellulase and p-glucosidase were determined. The cellulase and P-glucosidase activity in the strain X20-2 was the highest one.The rDNA fragment including 18S rDNA (1785bp), 5.8S rDNA (137bp) and two internal transcript spaces (ITSI and ITS2, 468bp) of strain X20-2 were sequeced. After comparing with related strains in GenBank, the sequence homology of 18S rDNA and 5.8S rDNA with strains in genus of Stachybotrys were 99 % and 100 % respectively. By observing the morphological characters such as filamentous form and conidial fructification, the strain X20-2 belongs to Stachybotrys sp. The toxic tests indicated that there was no toxin produced in the culture solution of X20-2.One-fact-at-a-time was used to optimize the fermentation conditions of P-glucosidase production. The results showed that the bran is the most suitable carbon course and the p-glucosidase activity in culture solution increased 90% after optimizing the producing enzyme conditions. It was found that the crude enzyme may survive in high concentration of glucose and its activity remained 28% in the presence of 10g glucose per liter.The β-glucosidase produced by X20-2 was purified with four steps: ammonium sulfate precipitation, DEAE-52 cellulose, HiTrapDEAE sepharose and Mono_Q ion-exchange chromatography. The purified enzyme showed that a single protein band on SDS-PAGE and its molecular mass was estimated to be 100 kDa. The enzyme was purified 143.5-fold to homogeneity and the specific activity reached to 29903.45 units /mg. The optimal temperature and pH for the enzyme activity were 60℃ and pH 5.6. The enzyme was stable in the range of pH 5.6-9.8 and within 60℃. The enzyme was inhibited by Ag+, Cu²⁺, Zn²⁺ and Fe³⁺ with different level. The Km and F_max of the enzyme for p-nitrophenyl-β-D-glucopyranoside (pNP-G) were 0.21mM, 178.6 umol(mlmin)^-1. and the glucose acted as competitive inhibitor of pNP-G hydrolysis with Ki value of 1.54mg/ml.Ten amino acid residues in the N-terminal of the enzyme was determined to be N-Ser-Trp-Pro-Leu-Asp-Phe-Ala-Asp-Trp-Pro.