【作者】 宋春丽；

【导师】 池振明；

【作者基本信息】 中国海洋大学 ， 生态学， 2010， 硕士

【摘要】 乳糖酶(β-D-半乳糖苷酶,β-D-galactoside-galactohydrolase, EC 3.2.1.23)为一种水解酶,可以将乳糖水解为葡萄糖和半乳糖,目前广泛应用于医药、食品、环保等领域。冷适应乳糖酶具有在低温下高活性的优点,应用在乳品加工和乳清引起的环境污染治理等方面,有着中温和高温乳糖酶无法达到的优越性。同时南极地区被认为是一个潜在的、重要的微生物资源库,可能是产生新型生物活性物质和先导化合物菌株的潜在种源地。我们实验室从采集自南极的海泥样品中筛选到33株酵母菌,本实验在15℃条件下,采用X-gal定性试验和ONPG定量试验从这33株酵母中筛选产乳糖酶的菌株,结果筛选到1株高产乳糖酶菌株17-1,经酵母菌常规方法和分子生物学方法鉴定,我们将其鉴定为一株普鲁兰久浩酵母(Guehomyces pullulans)。研究发现,G. pullulans 17-1菌株可同时产胞外和与细胞结合的(Cell-bound)乳糖酶,根据温度对菌体生长的影响,我们判定菌株17-1为一株耐冷型酵母。对耐冷酵母G. pullulans 17-1菌株产乳糖酶的培养基及培养条件进行了优化,确定了最佳培养基成分为(w/v)：乳糖3.0%,酵母粉0.7%,蛋白胨0.3%,紫苏油0.02%, KH2PO4 0.28%, MgSO4·7HO20.06%, MnSO4·3HO2 0.00085%,培养基初始pH为4.5,蒸馏水配制；最佳培养条件为：15℃、170 rpm振荡培养132 h,此时耐冷酵母G. pullulans 17-1菌株乳糖酶总产量可达13.9 U/mL。对发酵罐高密度发酵产酶条件进行了初探,实验结果表明可以将该乳糖酶的总产量提高到25.3U/mL。用粗酶样品水解乳糖溶液,水解产物为葡萄糖和半乳糖,水解牛奶可产生大量还原糖。G. pullulans 17-1菌株胞外乳糖酶经超滤法浓缩、SephadexTM G-200凝胶过滤、CM-Sepharose F.F.阳离子交换层析得到了纯化,比活力提高2.4倍。SDS-PAGE显示其单亚基分子量约为170.0 kDa,用凝胶过滤层析法测得该纯化酶的分子量约为335.0 kDa,因此推测G. pullulans 17-1菌株乳糖酶是一个同源二聚体。对该纯化酶的酶学性质进行了研究,结果表明：其最适作用温度和pH分别为50℃和4.0,受Ag+、Hg2+的强烈抑制(8.2%,8.4%),Li+离子对它有一定激活作用,对底物ONPG的Km值和Vmax分别为3.3 mM和9.2μmol/min。一级蛋白质谱分析表明,该乳糖酶的一个肽片ALEEYKK与其它已知酵母乳糖酶序列相匹配。将纯化的乳糖酶和4.6%乳糖溶液混合,50℃下孵育4 h后,测得乳糖溶液的水解率为43.5%,实验结果表明该乳糖酶在食品工业中有潜在的应用价值。更多还原

【Abstract】 β-galactosidase (β-D-galactoside-galactohydrolase, EC 3.2.1.23) hydrolyzes lactose to glucose and galactose. It has many applications in chemical, food industries, and entironment-protecting et al. In recent years, the enzymes from psychrophilic or psychrotolerant microorganisms have received increasing attention as they have many potential applications in food and chemical industries. A psychrotolerant yeast strain Guehomyces pullulans 17-1 was isolated from sea sediment in Antarctica. It was found that it could yield both extracellular and cell-boundβ-galactosidase.After optimization of the medium and cultivation conditions, it was found that the yeast strain 17-1 produced over 25.3 U/mL ofβ-galactosidase within 144 h. However, the optimal of pH and temperature for the crudeβ-galactosidase were 4.0 and 50℃, respectively. Lactose could be converted into glucose and galactose and a large amount of reducing sugar could be released from milk under catalysis of the yeast culture.The extracellularβ-galactosidase in the supernatant of the cell culture of the psychrotolerant yeast strain G pullulans 17-1 was purified to homogeneity with a 2.4-fold increase in specific activity as compared to the supernatant by ultrafiltration, concentration, gel filtration chromatography (SephadexTM G-200) and cation-exchange chromatography (CM-Sepharose Fast Flow cation-exchange). The molecular mass of the purified extracellularβ-galactosidase was estimated to be 335.0 kDa on the gel filtration chromatography (SephadexTM G-200). The optimal temperature and pH of the purifiedβ-galactosidase were 50℃and 4.0, respectively. Km and Vmax values of the purified P-galactosidase for o-nitrophenyl-β-D-galactopyranoside were 3.3 mM and 9.2μmol/min.43.5% lactose could be converted into glucose and galactose after 4 h at 50℃. The matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectroscopy identified a peptide ALEEYKK which was the conserved motif of the P-galactosidases from other yeasts. The results show that the enzyme may have potential applications in food industry.更多还原