【作者】 王慧；

【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】 α-半乳糖苷酶(EC 3.2.1.22)和β-半乳糖苷酶(EC 3.2.1.23)是自然界中广泛存在的两类糖苷水解酶,能从一系列化合物中释放以α-或β-键连接的D-半乳糖。利用α-半乳糖苷酶可以对半乳甘露聚糖进行酶法改性,使改性后的产品具有更多优良的性能,从而使其能够广泛的应用于食品,医药和造纸等行业。β-半乳糖苷酶则可以将乳制品中的乳糖水解为葡萄糖和半乳糖,利用微生物产的β-半乳糖苷酶生产口服剂,可以有效地缓解因不能吸收乳糖而引起的“乳糖不耐症”。本研究从嗜酸真菌Bispora sp. MEY-1中克隆到三个α-半乳糖苷酶基因和一个β-半乳糖苷酶基因,使其在毕赤酵母中进行表达并进行相关酶学性质的研究。三个α-半乳糖苷酶AgalA、AgalB和AgalC都属于27家族糖苷水解酶。AgalA和AgalC与Lachancea thermotolerans (耐热克鲁维酵母)来源的α-半乳糖苷酶相似性最高均为52%,而AgalA与AgalC两者间的氨基酸一致性仅为57.6%; AgalB则与Penicillium simplicissimum (青霉)来源的α-半乳糖苷酶有最高一致性为36%, AgalB与AgalA和AgalC的氨基酸一致性分别为24.9%和27.2%。三个重组α-半乳糖苷酶的最适pH分别为5.5、3.5和7.0,最适温度分别为55°C、55°C和45°C。AgalA、AgalB和AgalC对不同底物表现出不同的特异性: AgalA只对合成底物pNPG有降解能力,对蜜二糖、棉子糖和水苏糖等半乳寡糖均不表现其活性;而AgalB则不仅能催化pNPG、蜜二糖、水苏糖和棉子糖等小分子半乳寡糖,而且对瓜尔豆胶和角豆胶等半乳甘露聚糖也有较高的水解能力; AgalC对不同底物的特异性与AgalB类似。AgalA、AgalB和AgalC的比活分别为31.2 U/mg、581 U/mg和128.8 U/mg。AgalB与同来源的β-甘露聚糖酶Man5A协同降解瓜尔豆胶的实验结果表明: AgalB不仅能单独对瓜尔豆胶侧链的半乳糖残基进行降解,而且能与Man5A一起协同降解瓜尔豆胶。延长AgalB对侧链的作用时间,能显著地促进Man5A对主链的降解。而且随着作用温度的提高,反应速率加快,产物中的还原糖含量也随之增加。AgalA与AgalC则均不能与Man5A发生协同作用。β-半乳糖苷酶BgalA属于糖苷水解酶35家族,其最适pH为1.5,最适温度为60°C。BgalA在整个pH范围内均能保持稳定。有很好的抗胃蛋白酶和胰蛋白酶的能力。大多数金属离子对其酶活性没有显著地影响。重组酶的比活为99.5 U/mg。BgalA对ONPG和乳糖两种底物的Km值分别5.22 mM和0.31 mM, Vmax值分别为120.8μmol/(min·mg)和137.3μmol/(min·mg)。与商业化的Aspergillus oryzae来源的β-半乳糖苷酶相比, BgalA在整个模拟人工胃液条件下均能稳定的存在,且重组BgalA对牛奶中的乳糖的水解率可达85.8%,而作为对照的A. oryzae来源的β-半乳糖苷酶在动态模拟人工胃液条件下对乳糖的水解率只能达到3.5%。综合以上实验结果:α-半乳糖苷酶AgalB和β-半乳糖苷酶BgalA不仅耐酸,而且对胃蛋白酶和胰蛋白酶都有很好的抗性,具有很好的工业应用前景。本研究为其将来的工业应用打下了良好的基础。更多还原

【Abstract】 α-Galactosidase (EC3.2.1.22) andβ-galactosidase (EC 3.2.1.23) are two types of glycoside hydrolases widely existed in nature, which can cleaveα- orβ-linked galactose residues from a series of compounds. The modification of galactomannan byα-galactosidase extended its applications in food, pharmacy, and pulp industries.β-Galactosidase hydrolyze lactose in milk into galactose and gluctose. Supplementation of microbialβ-galactosidase could alleviate the symptoms caused by lactose intolerance. In this study, threeα-galactosidase genes and oneβ-galactosidase gene from the acidophilic fungus Bispore sp. MEY-1 were cloned, expressed and characterized.All the threeα-galactosidases AgalA、AgalB and AgalC from Bispore sp. MEY-1 belong to glycoside hydrolase family 27. Both of AgalA and AgalC exhibited the highest amino acid sequence identity (52%) with theα-galactosidase from Lachancea thermotolerans; however, the amino acid identity between AgalA and AgalC was only 57.6%. The deduced amino acid sequence of AgalB showed the highest identity (36%) with that of theα-galactosidase from Penicillium simplicissimum, and 24.9% and 27.2% to that of AgalA and AgalC, respectively. The optimal pH of AgalA、AgalB and AgalC were 5.5、3.5 and 7.0, respectively. The temperature optima of the threeα-galactosidases were 55°C、55°C and 45°C, respectively. AgalA、AgalB and AgalC exhibited different activity towards different substrates. AgalA only hydrolyzes synthetic substrate pNPG; AgalB and AgalC have ability to not only hydrolyze small galacto-oligosaccharides (melibiose, raffinose and stachyose), but also galactomannan polysaccharides (guar gum and locust bean gum). The specific activity of AgalA、AgalB and AgalC towards pNPG was 31.2 U/mg、581 U/mg and 128.8 U/mg, respectively. The synergistic action of AgalB and family 5β-mannanase Man5A from the same organism to degrade guar gum showed that AgalB not only degrade the side-chain of guar gum alone, but also acts synergisitically withβ-mannanase. When increased the incubation period of AgalB, the action of Man5A was enhanced significantly. Neither AgalA nor AgalC had ability to act synergisitically withβ-mannanase.Belonging to glycoside hydrolase family 35,β-galactosidase BgalA showed optimal activity at pH 1.5 and 60°C. BgalA was stable over the whole pH range and was strongly resistant to pepsin and trypsin digestion. Most of metal ions have no significant effects on its activity. The specific activity of r-BgalA towards ONPG was 99.5 U/mg. The Km and Vmax of BgalA for ONPG and lactose are 5.22 mM and 120.8μmol/ (min·mg), and 0.31 mM and 137.3μmol/ (min·mg), respectively. Compared with the low hydrolysis ratio (3.5%) of commercialβ-galactosidase from Aspergillus oryzae, BgalA was more stable and showed higher hydrolysis ability (85.8%) toward milk lactose under simulated gastric conditions.In summary,α-galactosidase AgalB andβ-galactosidase BgalA were resistant to acid and proteases, and have great potential in industrial applications. This study provides more information for development of future industrial enzymes.更多还原