一株嗜酸真菌的分离鉴定及其两种苷酶的酶学性质
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摘要
嗜酸微生物是极端环境微生物的重要类群,在酸性环境中发挥着重要的生态作用,对人们的生活生产具有潜在的生物技术价值。
本研究从新疆某矿区铀矿酸性废水分离出一株真菌。依据ITS序列并结合形态特征分析,该菌株初步鉴定为Penidiella sp.,暂命名为Penidiella sp.HEY-1。该菌株的最适生长pH值为3.0～4.0,在pH值2.0也能较好生长,具有嗜酸性。以麸皮和豆渣为基质进行发酵产酶研究,该菌株能分泌产生木聚糖酶、淀粉酶、果胶酶、α-半乳糖苷酶、β-半乳糖苷酶以及β-葡萄糖苷酶等糖苷水解酶。是一株具有应用潜力的工业用酶产生菌。
初步建立了能诱导菌株Penidiella sp.HEY-1同时产生上述六种酶的发酵方法。通过优化培养基中麸皮和豆渣的组成比例、培养的pH值和温度,建立了4种培养方法。方法1：只添加麸皮,pH值2.0,培养温度20℃,高产木聚糖酶。方法2：麸皮与豆渣比例为1：1,pH值2.5,培养温度25℃,高产淀粉酶、果胶酶。方法3：麸皮与豆渣比例为1：1,pH值4.0,培养温度30℃,高产α-半乳糖苷酶。方法4：只添加豆渣,pH值3.0,培养温度30℃,高产β-半乳糖苷酶和β-葡萄糖苷酶。
采用乙醇沉淀、DEAE-Sepharose Fast Flow离子交换层析与Sephadex G-100凝胶过滤层析等步骤后获得了凝胶电泳均一的α-半乳糖苷酶和β-葡萄糖苷酶,它们的相对分子质量分别为52.5 kDa、65.5 kDa,比活分别为15.83 U/mmg和3.30U/mg、纯化倍数分别为15.05和4.52、回收率分别为14.6%和2.2%。
α-半乳糖苷酶反应的最适温度为60℃,最适pH值为3.0,在60℃以下及pH值2.0～6.0范围内能保持稳定。Ba2+、Fe2+、Mg2+、Zn2+、Ca2+、Cu2+和K+对酶活有明显的激活作用,Mn2+和Fe3+对酶活性有极强的抑制作用,Hg+和Na+几乎完全抑制了酶活性。该酶可作用对-硝基苯-α-D-半乳糖苷(pNPGal)、棉籽糖和水苏糖,该酶作用于pNPGal的米氏常数(Km)值为0.768 mmol/L,酶促反应的最大速度(Vmax)为1.55×10-3mol/(L·min)。
β-葡萄糖苷酶反应的最适温度为60～70℃,最适pH值为3.0,在70℃以下及pH值2.0～8.0范围内均能保持稳定。Mn2+对酶有激活作用,K+、Fe3+微弱的激活作用,而Na+对酶有明显的抑制作用,其他金属离子对酶的活性影响不大。该酶作用于对硝基苯-β-D-葡萄糖苷(pNPGlu)、邻硝基苯-p-D-半乳糖苷(oNPG)的米氏常数(Km)值分别为0.434 mmol/L和0.411mmol/L,酶促反应的最大速度(Vmax)分别为1.0×1O-3和3.3×10-4mol/(L·min)。
研究表明α-半乳糖苷酶和β-葡萄糖苷酶均具有嗜酸性,可以应用于饲料、食品和医学等领域。
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.
本研究从新疆某矿区铀矿酸性废水分离出一株真菌。依据ITS序列并结合形态特征分析,该菌株初步鉴定为Penidiella sp.,暂命名为Penidiella sp.HEY-1。该菌株的最适生长pH值为3.0～4.0,在pH值2.0也能较好生长,具有嗜酸性。以麸皮和豆渣为基质进行发酵产酶研究,该菌株能分泌产生木聚糖酶、淀粉酶、果胶酶、α-半乳糖苷酶、β-半乳糖苷酶以及β-葡萄糖苷酶等糖苷水解酶。是一株具有应用潜力的工业用酶产生菌。
初步建立了能诱导菌株Penidiella sp.HEY-1同时产生上述六种酶的发酵方法。通过优化培养基中麸皮和豆渣的组成比例、培养的pH值和温度,建立了4种培养方法。方法1：只添加麸皮,pH值2.0,培养温度20℃,高产木聚糖酶。方法2：麸皮与豆渣比例为1：1,pH值2.5,培养温度25℃,高产淀粉酶、果胶酶。方法3：麸皮与豆渣比例为1：1,pH值4.0,培养温度30℃,高产α-半乳糖苷酶。方法4：只添加豆渣,pH值3.0,培养温度30℃,高产β-半乳糖苷酶和β-葡萄糖苷酶。
采用乙醇沉淀、DEAE-Sepharose Fast Flow离子交换层析与Sephadex G-100凝胶过滤层析等步骤后获得了凝胶电泳均一的α-半乳糖苷酶和β-葡萄糖苷酶,它们的相对分子质量分别为52.5 kDa、65.5 kDa,比活分别为15.83 U/mmg和3.30U/mg、纯化倍数分别为15.05和4.52、回收率分别为14.6%和2.2%。
α-半乳糖苷酶反应的最适温度为60℃,最适pH值为3.0,在60℃以下及pH值2.0～6.0范围内能保持稳定。Ba2+、Fe2+、Mg2+、Zn2+、Ca2+、Cu2+和K+对酶活有明显的激活作用,Mn2+和Fe3+对酶活性有极强的抑制作用,Hg+和Na+几乎完全抑制了酶活性。该酶可作用对-硝基苯-α-D-半乳糖苷(pNPGal)、棉籽糖和水苏糖,该酶作用于pNPGal的米氏常数(Km)值为0.768 mmol/L,酶促反应的最大速度(Vmax)为1.55×10-3mol/(L·min)。
β-葡萄糖苷酶反应的最适温度为60～70℃,最适pH值为3.0,在70℃以下及pH值2.0～8.0范围内均能保持稳定。Mn2+对酶有激活作用,K+、Fe3+微弱的激活作用,而Na+对酶有明显的抑制作用,其他金属离子对酶的活性影响不大。该酶作用于对硝基苯-β-D-葡萄糖苷(pNPGlu)、邻硝基苯-p-D-半乳糖苷(oNPG)的米氏常数(Km)值分别为0.434 mmol/L和0.411mmol/L,酶促反应的最大速度(Vmax)分别为1.0×1O-3和3.3×10-4mol/(L·min)。
研究表明α-半乳糖苷酶和β-葡萄糖苷酶均具有嗜酸性,可以应用于饲料、食品和医学等领域。
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.
引文
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