【作者】 葛方兰；

【导师】 李维；

【作者基本信息】 四川师范大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 木聚糖（xylanase EC 3．2．1．8）是植物体内存在的半纤维素的主要成分，是自然界中仅次于纤维素的最为丰富的多糖。木聚糖酶是戊聚糖酶的一种，可以将木聚糖降解成低聚木糖和木糖的复合酶系，主要应用于造纸、饲料、医药、能源、纺织等行业，具有极大的商业价值和广阔的应用前景。本研究从四川青城山、九寨沟、拉萨、山东临祈等地采集林木长期自然覆盖下的土壤。用RBB-xylan选择培养基进行初筛，根据不同菌株所形成的透明圈的大小，确定需要复筛的菌株，经产酶基本培养基复筛后，筛选到4株产酶稳定且活力较高的菌株，选择其中产木聚糖酶较高的一株X-18，作为进一步发酵条件优化的实验菌株。该菌细胞形态为杆状，革兰氏染色为阳性，有芽孢，分类鉴定的结果表明，菌株X-18属于芽孢杆菌属（Bacillus sp．）细菌，因此，该菌命名为Bacillus sp．No X-18。进行单因素试验，结果显示甘露糖、蔗糖为最佳产酶的碳源，其木聚糖酶活力分别为17．24 IU／mL、16．68 IU／mL。最佳氮源为蛋白胨，其木聚糖酶活力为18．5 IU／mL：双因素试验，结果显示木聚糖和阿拉伯糖的碳源组合菌株酶活最高，为19．86 IU／mL,蛋白胨和草酸铵的组合最有利于产酶，酶活为19．71 IU／mL；培养基成分的综合优化试验，结果显示最佳培养基是：1．5％蔗糖、1％阿拉伯糖、0．37％NH₄Cl、0．37％酵母膏、0．5％K₂HPO₄、0．02％MgSO₄·7H₂O，pH 8．0，其木聚糖酶活力可达74．21IU／mL。最佳培养条件为：起始PH8．0，50ml装液量，32℃，发酵时间72h。对Bacillus sp．No X-18木聚糖酶的部分酶学特性进行了研究，结果表明，菌株X-18酶活最适温度为50℃，其热稳定性较好，在50℃保温1小时仍达到100％的酶活力。该酶pH值范围较广，在pH 5．0至pH8．0范围内均能保持较高的活性，最适pH值为5．0，与其相关报道的研究相比，该木聚糖酶酸碱性较好。更多还原

【Abstract】 Xylan is the main component of hemicellulose in the plant, and it is the second only to cellulose in natural amylose. Xylanase is a kind of pentosanase which can degrade the xylan to XyLo-oligosaccharides and xylose. It’s mainly applied in paper making, foodstuff, medicine energy sources, and textile industry and so on. Both commerce value and application foreground are considerable. All bacteria in our study were collected from soil covered by lots of wood and leaves for long time in different area,the districts contain Qing Cheng mountain, Jjiuzhaigou valley in Si chuan Province, Lhasa, Linqi city in Shandong Province and else. The bacteria were first screened by RBB-xylan culture medium according to whether appear hydrolyzed holecircle or not. And the bacteria with clarity circle were cultivated in ferment medium for the next screening. We obtained four excellent xylanase enzyme producing strains through the anterior process. Finally we choose the high enzyme activity strain No. X-18 to carry out ferment condition optimization experiment. The Gram stain and spore stain are both Positive Rod toward the bacteria. The result of classification and characterization indicate the No. X-18 belongs to Bacillus sp. The result of single factor experiment show the most appropriate carbon sources are mannose and cane sugar corresponding to the enzyme activity is 17.24 IU/mL 16.68 IU/mL respectively, the most appropriate nitrogen sources is peptone corresponding to the enzyme activity is 18.5 IU/mLon the other hand, the result of double factors reveal the most high activity 19.86 IU/mL is made up of the compound of xylan and arabinose refer to the carbon source, When use the compound of peptone and ammonium oxalate as nitrogen source the enzyme is considerable up to 19.71 IU/mL.The optimal medium composition as 1.5% sucrose, 1% arabinose,0.37% NH₄Cl, 0.37% yeast extract, 0.5% K₂HPO₄, 0.02%MgSO₄·7H₂O, pH 8.0 after a series of the single factor experiment, double factors experiment and medium component optimization experiment. Furthermore, the optimal culture condition described below: 50mL initial PH 8.0 ferment medium in 250ml flask at 32℃for 72h. The enzyme activity can reach 74.21IU/mL at the optimal condition.We investigate part zymological property about the Bacillus sp.No X-18 xylanase, find the optimum temperature is50℃,And its heat stability is fine, it keep 100% activity after incubating at 50℃for 1 hour. Its pH range also is wide.the activity can keep high from pH5.0to pH8.0,and the optimum pH is 5.0,tolerance of the enzyme is much beffer than it reported already.更多还原