【作者】 杨君；

【导师】 姚斌；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 嗜酸真菌Bispora sp. MEY-1分离自江西金属矿废水,具有极端嗜酸性,最适生长的pH为2.5-3.0,可分泌产生多种重要的工业用酶,包括β-葡聚糖酶、木聚糖酶、果胶酶、β-甘露聚糖酶、α-半乳糖苷酶、β-半乳糖苷酶、淀粉酶、CMCase以及β-葡萄糖苷酶等九种糖苷水解酶,是一株优良的工业产酶菌。本文从嗜酸真菌Bispora sp. MEY-1分离克隆到3个糖苷水解酶基因,即β-1,3-1,4-葡聚糖酶bgl7A、木聚糖酶xylD和多聚半乳糖醛酸酶pga1。对三个基因进行了序列分析,并进行了三维结构的预测。它们的氨基酸序列同已知序列一致性在50–68%之间,具有较高的新颖性。将这三个基因在毕赤酵母中进行表达,并对重组酶进行了纯化和性质测定。β-1,3-1,4-葡聚糖酶BGL7A与来源于Aspergillus terreus的假定第七家族内切葡聚糖酶EG-1氨基酸序列具有最高一致性(59.5%)。BGL7A的最适pH值有三个峰值分别是pH 1.5、3.5和5.0,其中pH 5.0为最高值,最适温度为60°C,在pH 1.0–8.0之间和60°C分别处理60 min酶的稳定性良好。以大麦葡聚糖为底物测得Km和Vmax分别是9.16 mg/ml和6,737μmol/min/mg。以大麦葡聚糖和地衣多糖为底物分析BGL7A的比活分别为4,040和2,740 U/mg,高于CMC-Na的比活395 U/mg,这一点不同于其他已报道的第七家族内切葡聚糖酶。木聚糖酶XYLD与来源于Talaromyces stipitatus的假定内切-1,6-β-D-葡聚糖酶氨基酸序列具有最高一致性(49.9%),与来源于Leptosphaeria maculans的假定木聚糖酶的序列一致性为38.8%,但经性质研究发现其只具有木聚糖酶活性,不具有葡聚糖酶活性,经分析确定其为第30家族的木聚糖酶。最适pH值为3.0,最适温度为60°C,pH 1.0–6.0之间和温度60°C下分别处理60 min稳定性良好。XYLD以山毛榉木聚糖、桦木木聚糖、4-O-甲基-D-葡糖醛酸木聚糖和燕麦木聚糖为底物的比活分别是2,463、2,144、2,020和1,429 U/mg。以山毛榉木聚糖为底物分析表观Km和Vmax分别是5.6 mg/ml和3,622μmol/min/mg。多聚半乳糖醛酸酶PGA1与来源于Colletotrichum lupini的内切多聚半乳糖醛酸酶氨基酸序列具有最高一致性(67.2%)。最适pH和最适温度分别为3.5和55°C。在pH 2.0–7.0之间和60°C下稳定性良好。PGA1以多聚半乳糖醛酸和果胶为底物的比活分别为1,520和725 U/mg。以多聚半乳糖醛酸为底物分析表观Km和Vmax分别为1.25 mg/ml和2,526μmol/min/mg。用10 U/mL的PGA1处理苹果汁,可使苹果汁的粘度降低7.7%,而透光率增加84%。结果表明,酸性条件下PGA1具有良好工业应用潜力。本实验中克隆到的三个酶都是嗜酸酶,在饲料、食品等领域有广阔的应用前景。更多还原

【Abstract】 Bispora sp. MEY-1 is an acidophilic fungal strain isolated from the acidic wastewater of Uranium Mine in Jiangxi, China. Strain MEY-1 shows optimal growth at pH 2.5–3.0 and can secrete several important industrial enzymes, such asβ-glucanase, xylanase, pectinase,β-mannanase, amylase, CMCase,α-galactosidase,β-galactosidase,β-glucosidase and so on.Based on sequence analysis, three glycoside hydrolase genes encoding an endo-β-1,3-1,4-glucanase (bgl7A), a xylanase (xylD) and an endo-polygalacturonase (pga1), were cloned from strain MEY-1 and expressed in Pichia pastoris GS115. The deduced protein sequences showed 50–68% identities to the known proteins, suggesting their novelty. All the proteins were subjected to tertiary structure prediction and catalytic sites were identified, respectively.bgl7A showed the highest amino acid sequence identity of 59.5% to a putative family 7 endoglucanase EG-1 from Aspergillus terreus. Purified BGL7A had three activity peaks at pH 1.5, 3.5, and 5.0 (maximum), respectively, and a temperature optimum at 60°C. The enzyme showed good pH stability at pH 1.0–8.0 and thermostability at 60°C. The Km and Vmax values for barleyβ-glucan were 9.16 mg/ml and 6,737μmol/min/mg, respectively. The specific activities of BGL7A towards barleyβ-glucan and lichenan (4,040 and 2,740 U/mg) were higher than that of carboxymethyl cellulose sodium (CMC-Na; 395 U/mg), which was different from other family 7 endo-β-glucanases.The xylanase gene xylD shared the highest amino acid sequence identity of 49.9% to a putative endo-1,6-β-D-glucanase from Talaromyces stipitatu. Purified recombinant XYLD was more homologous to members of GH 30 based on phylogenetic analysis. The maximal activity of XYLD occurred at pH 3.0 and 60°C. The pH stability and thermostability were good at pH 1.0–6.0 and 60°C, respectively. The specific activity of XYLD towards beechwood xylan, birchwood xylan, 4-O-methyl-D-glucuronoxylan, and oat spelt xylan was 2,463, 2,144, 2,020 and 1,429 U/mg, respectively. The apparent Km and Vmax values for beechwood xylan were 5.6 mg/ml and 3,622μmol/min/mg, respectively.The deduced catalytic domain sequence of PGA1 was 67.2% identical with the polygalacturonase from Colletotrichum Lupini. The optimum pH and temperature were pH 3.5 and 55°C, respectively. The enzyme showed good pH stability at pH 2.0–7.0 and thermostability at 55°C. The specific activity of PGA1 towards polygalacturonic acid and pectin was 1,520 and 725 U/mg. The apparent Km and Vmax values for polygalacturonic acid were 1.25 mg/ml and 2,526μmol/min/mg, respectively. The capacity of PGA1 to reduce the viscosity of apple juice was also determined. The intrinsic viscosity reduction was equal to 7.7% of the initial viscosity value, and the light transmittance was increased more than 84% after treatment with 10 U/ml PGA1.These three enzymes are all acidophilic, having a broad prospects for feed and food industrial applications.更多还原