【作者】 董桂清；

【导师】 谢庆武；

【作者基本信息】 广西大学 ， 微生物学， 2007， 硕士

【摘要】 经过富集培养、分离纯化，从土壤初步筛选出50株产葡甘聚糖酶的菌株；通过观察平板培养基中水解圈的大小和透明程度，得到10株酶活较高的菌株；经过摇瓶复筛（用DNS法测定酶活力），得到一株酶活力高、产酶较快的菌株（编号为DK3）。对DK3进行16S rDNA序列测定，并在互联网上与其它菌株的相关序列进行比对，结果显示DK3是一株成团泛菌（Pantoeaagglomerans）。对DK3菌株所产葡甘聚糖酶的产酶条件进行了初步研究，得到的最佳培养基配方为：葡甘聚糖0.6％，硝酸钠0.3％，酵母膏0.5％，蛋白胨0.3％，MgSO₄·7H₂O 0.5％，KCl 0.5％，K₂HPO₄ 0.1％，FeSO₄·7H₂O 0.001％，pH为6.0；最佳产酶条件为：接种量1％，发酵温度37℃，摇床转速160rpm。在此优化条件下培养24h，菌液中的葡甘聚糖酶活力达58.54U／mL，是优化前的1.27倍。对葡甘聚糖酶的分离纯化方法进行研究：经超滤浓缩、阴离子交换柱层析和凝胶过滤柱层析，所得酶液在SDS—PAGE电泳图谱中呈单一蛋白质区带，分子量为54kDa。对DK3菌株所产葡甘聚糖酶的酶学性质进行了初步研究，结果表明，酶促反应的最适温度为37℃，最适pH为6.0；该酶在40℃以下较为稳定，在pH5.0～9.0相对稳定。Fe²⁺、Cu²⁺对该酶有强烈的抑制作用，EDTA有较强的抑制作用，NH₄⁺、Mg²⁺有较弱的抑制作用，Na⁺则有较小的激活作用。对固定化细胞产葡甘聚糖酶的条件进行了初步的研究，结果表明，在培养基中加入2％CaCl₂，固定化细胞能反复使用4次左右，后期产酶效率降低。对葡甘聚糖酶酶法制备葡甘低聚糖进行了初步的研究，采用反复加入固体葡甘聚糖，反复液化葡甘聚糖溶胶的方法，使葡甘低聚糖溶液的最终浓度达到24％。硅胶G薄层层析结果表明，葡甘聚糖被酶解后所得到的低聚糖的成分比较复杂。更多还原

【Abstract】 50 glucomannanase producing strains were preliminarily screened from soilby enriching culture, isolation and purification. 10 strains with high enzymeactivity were obtained by flat screening. Then the strain DK3 with the highestenzyme activity was obtained after the second screening by shaking culture. 16srDNA sequence blast analysis in Genbank revealed that DK3 was most relatedto the strain Pantoea agglomerans （Identities=99%）.The glucomannanase production conditions for DK3 strain were studiedpreliminarily. The optimal medium composition is as follows: 0.6%glucomannan, 0.3% NaNO3, 0.5% yeast extract paste, 0.3% peptone, 0.5%MgSO₄·7H₂O, 0.5% KCl, 0.1% K₂HPO₄, 0.001% FeSO₄·7H₂O, pH6.0. Theoptimal cultivation conditions: volume of inoculum 1%, 37℃, 160rpm. Underthis optimal condition for 24h, the glucomannanase activity reach 58.54 U/mL,1.27 fold as before.The glucomannanase was then separated and purified to homogeneity by ultrafiltration, ion-exchange and gel flitration. The purified mannanase appearedas a single protein band on SDS-PAGE gel with a molecular mass of approx. 54kDa.The characteristics of glucomannanase produced by DK3 strain werestudied preliminarily. The optimal temperature and pH for the glucomannanaseactivity was 37℃and pH6.0 respectively. The glucomannanase was stablewithin pH 5.0-9.0 and up to 40℃at pH6.0. Fe²⁺ and Cu²⁺ strongly inhibit theglucomannanase; EDTA slightly inhibit the glucomannanase; NH₄⁺, Mg²⁺weekly inhibit this enzyme; the glucomannanase was gently activated by Na⁺.The conditions of immobilized cells producing konjac glucomannanasewere preliminarily studied. When 2% CaCl₂ was added to the culture mediumthe immobilized cells could be repeatly used for approx. 4 times after which theefficiency of enzyme production decreased.Enzymolysis of glucomannan by glucomannanase was preliminarilystudied. By repeatly supplying solid glucomannan, the final concentration ofglucomannan oligosaccharides in the solution reached 24%. The results ofsilica-gel G thin-layer chromatography showed that the composition of theoligosaccharides obtained by enzymolysis of glucomannan was verycomplicated.更多还原