【作者】 杨俐；

【导师】 余蓉；

【作者基本信息】 四川大学 ， 微生物与生化药学， 2007， 硕士

【摘要】 甲壳素是地球上数量最大的含氮有机化合物，据估计自然界每年生物合成的甲壳素近100亿吨。壳聚糖是甲壳素的脱乙酰基的产物。近年来发现壳聚糖的低聚糖（壳寡糖）有重要的生物活性，尤其是六糖和七糖在抑制肿瘤方面的作用令人鼓舞。用专一性的壳聚糖酶（Chitosanase）水解胶体壳聚糖，控制反应条件，可获得含五糖和六糖较高的低聚糖。目前国内外真菌源壳聚糖酶的报道主要集中在曲霉属、青霉属和球孢白僵菌等，其纯化方法也多采用盐析、有机溶剂沉淀、离子交换层析等方法。寻找壳聚糖酶新酶源、简化壳聚糖酶的纯化工艺、利用理化因子诱变选育高产壳聚糖酶菌株极具研究价值。本研究在本实验室前期筛选到产壳聚糖酶活性较高的贵州绿僵菌AS 3.4608，并对其进行低成本的固态发酵的基础上，对贵州绿僵菌AS 3.4608固态发酵物提取液采用两步层析进行分离纯化，即采用自制交联壳聚糖树脂亲和吸附壳聚糖酶后用Cu²⁺洗脱，再以Superdex 75层析分离，经SDS-PAGE证实获得了电泳纯的壳聚糖酶，比活达106.39U／mg，每克干培养基可获壳聚糖酶328ug，活性回收率为43.25％，酶蛋白分子量为50.3KD。对样品理化性质研究表明，该酶在45℃以下有较好的稳定性，在弱酸性（pH 5.0-7.0）的溶液中也相当稳定。该壳聚糖酶降解壳聚糖反应最适温度为50℃，最适pH为4.0。其米氏常数K_m值为2.718g／L，V_max为1.152×10^-2g·L^-1·min^-1。Hg²⁺对该酶有强烈抑制作用，Mn²⁺、Mg²⁺、Zn²⁺、Fe³⁺、Ag⁺对其有较强抑制作用，Cu²⁺对其有较强激活作用。该酶是一种糖蛋白，含糖量约为26.33％。该酶最适底物为脱乙酰度为90％的胶体壳聚糖。本研究采用紫外线和氯化锂对贵州绿僵菌AS 3.4608进行复合诱变，筛选到了一株遗传稳定、产酶活力高的菌株Y0320。并对其发酵培养基进行了优化研究，经L9（3³）正交实验，最终确定其最佳发酵条件为：以黄豆粉∶麸皮=2.5∶1为培养基，添加22.5％的稻壳增加通气，添加3.25％的壳聚糖作为诱导物，经27℃～28℃培养120小时，每克干培养基产壳聚糖酶活力达600U，比野生菌株贵州绿僵菌AS3.4608（每克干培养基产壳聚糖酶活力可达83U）产酶活力增加6倍。本研究探索的简便的纯化壳聚糖酶方法和诱变选育到的稳定高产壳聚糖酶菌株有潜在工业化应用价值。更多还原

【Abstract】 Chitosan and its N-acetylated analogue, chitin, are among the most abundant glycans in nature. Conservative estimates of the amount of crustacean shells produced worldwide fall in the range of 100×10³ metric tons per annum. Chitosanases are hydrolytic enzymes acting on chitosan, a polymer composed ofβ-（1→4）-linked glucosamine residues. Chitosanases find application in the generation of size-specific chitosan oligomers. Such oligomers and their derivatives possess biological properties, such as induction of callose, immunopotentiation and inhibition of growth of plant pathogens. Chitosanases are found in bacteria, fungi and plants. For furthermore use, It is necessary to find new source of chitosanases, simplify its purification, and breeding for the high-yield strains.We found a new source of chitosanase from Metarhizium guizhouense. We studied the chitosanase production under solid state fermentation, and separated it from crude enzyme with affinity chromatography and molecular sieving. Cross-linked chitosan resins adsorbed chitosanase from crude enzyme, then eluted with 5‰Cu²⁺. The eluting was dialyzed、concentrated and then separated by Superdex 75 chromatography. Under the optimal conditions, maximum chitosanase activity reached 83.09±2.9U/g dry medium when cultured for 120h. The chitosanase was purified, and the enzyme activity was 43.52% of original activity. The specific activity of purified enzyme reached 106.39U/mg. The chitosanase had a molecular mass of 50.3kD. The enzyme showed a maximum activity at 50℃in pH 4.0. It was stable below 50℃and pH 5.0-7.0. Kinetic parameter Km was 2.718g/L. The chitosanase activity was strongly inhibited by Hg²⁺; also markedly inhibited by Mn²⁺、Mg²⁺、Zn²⁺、Fe³⁺、Ag⁺; but it was activiated by Cu²⁺. The chitosanase is a kind of glycoprotein, it contains about 26.33% of carbohydrate. The most susceptible substrate of the enzyme was 90% deacetylated colloid chitosan.We carried out UV & LiCl as mutagensis treatment to study the effectiveness of increasing the productivity of chitosanase producing strain Metarhizium guizhouense. By this mean, mutant Y0320 was obtained, and its productivity of chitosanase was increased by 600% over the parent strain.Furthermore, the ingredients of culture media were optimized. Many influencing factors and fermentation conditions were studied, such as carbon/nitrogen sources, chitosan, and so on. And then by designed orthogonal experiments, an optimum culture medium was established.更多还原