【作者】 蔡红英；

【导师】 杨培龙；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 青霉能分泌多种重要的工业用酶,可以产生各种具有优良性状的半纤维素糖苷水解酶,包括木聚糖酶、β-甘露聚糖酶、β-葡聚糖酶等。本实验以从自云南锡矿的酸性废水（pH 3.0）中分离的三株青霉（C1,C6和C7）为实验菌株,以基因克隆与异源表达的方法对其半纤维素酶进行了初步的探索。通过设计简并引物,结合TAIL-PCR和RT-PCR技术,从青霉C1,C6和C7菌株中分别克隆到3种5个糖苷水解酶的全长基因,包括2个木聚糖酶基因（xyn10C1和xyn11C1）,2个β-甘露聚糖酶基因（man5C1和man5C6）与1个葡聚糖酶基因（bgl7C7）。通过同源比对和序列一致性分析,其推导氨基酸序列与已知序列的最高一致性在49–83%之间。结合三维结构的模拟和催化位点的分析,表明这些基因均具有一定的新颖性。将Penicillium sp. C1来源的xyn10C1在毕赤酵母中进行了重组表达,纯化和酶学性质分析。在摇床水平上木聚糖酶XYN10C1的表达量为477 U/mL。XYN10C1的最适pH为4.0–5.5,最适作用温度为75℃,在pH 2.5–6.5能维持69%以上的酶活性,在70–80℃温度范围内能保持91%以上的活性,甚至在90℃还能保持22%的活性,并在65℃条件下保持稳定。表明其在较宽的酸性pH和中高温范围内具有极好的适应性。以可溶性阿拉伯小麦木聚糖为底物,XYN10C1的比活,K_m及V_max值分别为137 U/mg,6.9 mg/mL,209μmol/min/mg。以桦木木聚糖为底物,比活,K_m及V_max值分别为101 U/mg,4.3 mg/mL,893μmol/min/mg。多数金属离子对XYN10C1酶活力没有明显的影响或是对酶有激活作用,该酶能抗胃蛋白酶与胰蛋白酶的水解。在模拟降解麦芽汁的实验中,80 U的XYN10C1能明显地提高酿酒麦芽的过滤速率（27%）并降低粘度（9.8%）。因此,XYN10C1具有潜在优势应用于食品与动物饲料工业。2个β-甘露聚糖酶基因man5C1,man5C6在毕赤酵母中进行了重组表达,其重组蛋白表达量分别为161.5 U/mL,575.4 U/mL。经高密度细胞发酵,Man5C6发酵5天后表达量达到2.5 g/L,明显高于目前所报道的大多数β-甘露聚糖酶。Man5C6纯化步骤简单,表达水平可通过分子生物学或蛋白组学的手段进一步提高,从而在工业应用中有助于节约成本。2个重组甘露聚糖酶Man5C1和Man5C6的最适pH分别为4.0和4.5,最适温度较高,都为70℃。Man5C1具有极好的抗胃蛋白酶和胰蛋白酶的能力,37℃条件下处理60 min后的酶活力是处理前的93–116%。Man5C6在胰蛋白酶处理后,剩余酶活为87%。大部分金属离子对这三种酶都有激活作用或不影响酶活力。以角豆胶为底物,Man5C1和Man5C6的比活分别为1035 U/mg和226 U/mg。K_m分别为5.6 mg/mL和12.3 mg/mL。V_max分别为2785μmol/ min/mg和2400μmol/ min/mg。此外,在模拟人工胃液的条件下,Man5C1在胃蛋白酶存在的条件下,能有效的水解角豆胶,释放出更多的还原糖。综合酶学性质,Man5C1具有很好的工业应用前景;Man5C6具有较好的热稳定性,pH适应性以及在酸性条件下较好的稳定性,且蛋白表达量高,因此在饲料行业应用中更有价值。综上所述,本实验从三株青霉菌株中克隆得到5个半纤维素酶基因,并对其中的3个基因进行了异源表达与酶学性质分析。三个重组酶都偏酸性,并耐受高温,具有良好的金属离子和化学试剂抗性,以及蛋白酶抗性,在应用试验中都具有明显的作用效果。这些优良的酶学性质特性表明它们在食品与饲料行业都有很好的应用前景。更多还原

【Abstract】 Penicillium can secrete a variety of important industrial enzymes, and produces kinds of excellent hemicellulases, including xylanase,β-mannanase andβ-glucanase etc. In this study, three Penicillium strains （C1, C6 and C7） were isolated from the acidic wastewater （pH 3.0） of a tin mine in Yunnan province, China, and used as donors for gene cloning and heterologous expression of hemicellulases.By combining TAIL-PCR and RT-PCR techniques with designing of degenerate primers, five full-length glycoside hydrolase genes encoding for three kinds of enzymes were cloned from Penicillium strains C1, C6 and C7. They are two xylanase genes （xyn10C1 and xyn11C1）, twoβ-mannanse genes （man5C1 and man5C6） and oneβ-glucanase gene （bgl7C7）, respectively. Homology searches in Genbank and sequence alignments indicated that their deduced amino acid sequences shared 49–83% identities with known proteins. Further homology modeling and catalytic residue analysis revealed that these six genes have certain sequence and structure novelty.xyn10C1 from Penicillium sp. C1 was expressed in Pichia pastoris, and the recombinant enzyme was purified and characterized. In shaker flask XYN10C1 was produced with the activity of 477 U/mL. XYN10C1 showed the optimal activity at pH 4.0–5.5 and 75°C, and exhibited good adaptability to broad ranges of acidic pHs and temperatures. It retained >69.0% of the maximal activity at pH 2.5–6.5 and >91.0% of the activity at 70–80°C, and 22.2% even at 90°C. The enzyme was stable at 65°C. The specific activity, K_m and V_max values towards soluble wheat arabinoxylan were 137 U/mg, 6.9 mg/mL and 209μmol/min/mg, respectively. Using birchwood xylan as the substrate, the specific activity, K_m and V_max values were 101 U/mg, 4.3 mg/mL and 195μmol/min/mg, respectively. XYN10C1 was strongly resistant to most metal ions and proteases （pepsin and trypsin）. Under simulated mashing conditions, addition of XYN10C1 （80 U） to the brewery mash significantly increased the filtration rate by 27% and reduced the viscosity by 9.8%, respectively. All these favorable properties make XYN10C1 attractive for potential applications in food and animal feed industries.Twoβ-mannanases Man5C1 and Man5C6 were produced in P. pastoris with the activity of 162 U/mL and 575 U/mL, respectively. The yield of Man5C6 was up to 2.5 g/L after 5-day fermentation in a 3.7 L fermentor, which was higher than that of most knownβ-mannanases. Man5C6 was easily purified, and its yield might be further improved by molecular and protein engineering methods, thus Man5C6 was potential in low-cost industrial applications.The optimum pHs of Man5C1 and Man5C6 were 4.0 and 4.5, respectively. Their temperature optima were 70°C. MAN5C1 had strong resistance to pepsin and trypsin, retaining 93–116% activities at 37°C for 60 min. Man5C6 only retained 86.6% activity after trypsin treatment under the same conditions. Most metal ions and chemical reagents enhanced or had no effects on their enzyme activities. Using locust bean gum as the substrate, the specific activities, K_m and V_max values for Man5C1 and Man5C6 were 1035 U/mg, 5.6 mg/mL, 2785μmol/min/mg; 226.5 U/mg, 12.3 mg/mL and 2400μmol/min/mg, respectively. Under simulated gastric conditions, Man5C1 released more reducing sugars from locust bean gum in the presence of pepsin. Based on the enzymatic properties, Man5C1 have good prospects in industrial applications. Man5C6 was produced with high yield, and showed good thermal stability, pH adaptability and pH stability under acidic conditions. These properties make it more valuable in the animal feed industry.In conclusion, five hemicellulase genes were cloned from three Penicillium strains, and three of them were expressed and characterized. The three recombinant enzymes were all acidic, tolerant to high temperatures, and had excellent resistance to metal ions, chemical reagents, and proteases. Moreover, these enzymes had significant effects in application tests. All these properties indicate that they have good prospects for application in food and feed industries.更多还原