| Acidophile playing important ecological roles in acid environment is an important member of the extremophilic family, which has potential biotechnology value in people's life and production.The strain HEY-1 isolated from the acidic waste water of some Uranium Mine in Xinjiang, China, was identified as(Penidiella sp.) according to its mycelia morphology and ITS sequence analysis, and was designated as Penidiella sp. HEY-1. The strain HEY-1 was able to grow at pH value 2.0, with the optimal growth at pH value 3.0～4.0. Wheat bran and soybean dregs were used as mediums to study the fermentation conditions for the strain HEY-1, experiments showed that the strain HEY-1 can secrete xylanase, amylase, pectinase, a-galactosidase, P-galactosidase andβ-glucosidase. being an application to potential enzyme producer.A fermentation system was initially established to induce HEY-1 to secrete the six enzymes of the above mentioned. By optimizing the proportion of wheat bran and soybean dregs in the medium, pH value and temperature, four methods were formulated to induce different enzymes. The first method for high-yield of xylanase: only wheat bran, while pH value 2.0 and 20℃. The second method for high-yield of amylase and pectinase:the proportion of wheat bran and soybean dregs was 1:1, while pH value 2.5 and 25℃. The third method for high-yield of a-galactosidase:the proportion of wheat bran and soybean dregs was 1:1, while pH value 4.0 and 30℃. And the fourth method for high-yield ofβ-galactosidase andβ-glucosidase:only soybean dregs, pH value 3.0 and 30℃.The purification of a-galactosidase and P-glucosidase from Penidiella sp.HEY-1 in fermentation medium, that has been studied to use ethanol precipitation, DEAE-Sepharose Fast Flow chromatography, followed by Sephadex G-100 chromatography. By running SDS-PAGE, their molecular weights were determined to be 52.5 kDa forα-galactosidase while 65.5 kDa forβ-glucosidase, their specific activities were 15.83 U/mg and 3.30 U/mg respectively, and the purification multiples were 15.05 and 4.52, their recovery rate were 14.6% and 2.2% respectively.The a-galactosidase characteristics research showed that its optimum pH value and temperature was 3.0 and 60℃respectively. It was stable in the pH value range of 4.0～6.0 and up to 60℃. And it was also clearly found that the enzyme can be significantly stimulated by Ba2+N,Fe2+,Mg2+,Zn2+,Ca2+,Cu2+ and K+, especially significantly inhibited by Mn2+ and Fe1+. But it is likely that Hg+ and Fe1+ inhibit the enzyme activity completely. The results showed that the enzyme can hydrolyze small galacto-oligosaccharides such as p-Nitrophenyl-a-D-Galactopyranoside (pNPGal). raffinose and stachyose. And the Km and Kmax were 0.768 mmol/L,1.55x10-3 mol/L·min when pNPGal as substrate.The (3-glucosidase characteristics research indicated that its optimum pH value and temperature was 3.0 and 60～70℃respectively. It was stable in the pH value range of 2.0～8.0 and up to 70℃. It was also found that theβ-glucosidase can be stimulated by Mn2+, little activated by K+ or Fe3+ and inhibited by Na+ obviously. But other metal ions had little effects on the activity of P-glucosidase. The results showed that the enzyme effects directly on (3-glucosidic compounds such as p-Nitrophenyl-β-D-glucopyranoside (pNPGlu) and o-Nitrophenyl-β-D-Galactopyranoside (oNPG). And the Km was 0.434 mmol/L and 1.0×10-3 mol/L·min respectively, when pNPGlu was as substrate. The parameters Vmax were 0.411 mmol/L and 3.3×10-4mol/L·min respectively, when oNPG was as substrate.The research showed that two enzymes are all acidophilic, can be used in feed,food and medicine industrial applications. |
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