【作者】 李雪龙；

【导师】 包怡红；

【作者基本信息】 东北林业大学 ， 食品科学， 2008， 硕士

【摘要】 木聚糖酶是可分解世界上第二丰富的可再生有机碳源——半纤维素的主要酶制剂,在食品、造纸、饲料及能源转化等方面都有广阔的应用前景。本课题从木聚糖酶高产菌的筛选、鉴定、酶学性质研究到产酶培养条件的优化做了系统的研究,在国内首次筛选和鉴定了一株高产木聚糖酶的类芽孢杆菌,主要结果如下:本研究采用自制玉米芯木聚糖及桦木木聚糖纯品为唯一碳源进行初筛培养和复筛培养,以透明圈直径和透明度为指标,从来自不同的富含半纤维素的土壤样品中分离纯化得到30株木聚糖酶产生菌株；再通过摇瓶发酵及DNS法检测酶活,得到三株木聚糖酶的高产菌株:L0302、L0602、L0604,其木聚糖酶活力分别达到:110.79U、118.22U、117.94U。经形态学、培养特征、生理生化特性及菌种16SrRNA的鉴定,初步确定三株菌均为类芽抱杆菌属,并最终选定L0604为出发菌株做进一步的研究。基于对木聚糖酶研究的需要,探讨了木聚糖酶粗酶液的基本性质:粗酶液的最适反应pH为5.0、最适反应温度为50℃；酶液在pH5.0时稳定性最好,在pH4.0-10.0范围内相对酶活均保持在80%以上,说明该菌株所产木聚糖酶能在较大pH范围内保持稳定；酶液在40℃时稳定性最好,随保温时间的延长,酶活可得到相应的提升,在20℃-50℃之间均有较好的稳定性,酶活可保持在最适温度时的88%以上；Cu²⁺、Zn²⁺、Mg²⁺对酶活有较好的激活作用,且在一定范围内,浓度越大,作用越强,其中Cu²⁺的激活作用最强,使该酶酶活可提高到150%。经单因素试验和正交设计试验,得出该菌株的最佳产酶培养基为:玉米芯木聚糖3.0%、胰蛋白胨0.6%、K₂HPO₄ 0.5%、吐温80 0.4%。用此配方对菌株进行摇瓶培养,最佳培养条件为:培养基最适初始pH 7.0、培养温度32℃、摇床转数220r/min、在此条件培养96h,发酵液中木聚糖酶活力达到最大峰值194.67U,是未经优化的基础产酶培养条件产酶能力的1.58倍。更多还原

【Abstract】 Xylanases can hydrolyze hemicelluloses, which are the second abundance renewable resource. Xylanases have a broad prospect in food industry, paper industry, animal feed industry and energy bioconversion etc. This study carried out a series of experiments: screening, identification, properties of xylanase and the optimal culture conditions for high production. A high xylanase-producing Paenibacillus.sp was first isolated from the soilsamples and identified. The main results are follows:According to xylan-hydrolyzed halos method, the ingredients of the medium for screening of xylanase-producing strain in this study included self-made corncob xylan and birchwood xylan, respectively. With this method, the sizes and transparency of transparent zones surrounding colonies in Petri dishes were directly proportionate to their xylanase activities. 30 xylanase-producing strains were screened from the soil-samples which full of high hemicellulose. L0302, L0602 and L0604 strains presenting the higher activity were selected for further investigation by experiments in shake flasks. According to the morphology, cultural characters, physiological and biochemical characteristics and analysis of 16SrRNA sequence, three strains were all identified as Pacnibacillus.sp. the xylanase activities of them were 110.79U, 118.22U and 117.94U. respectively. L0604 was ultimately selected as a candidate due to its most prominent xylanase activity.Based on the study of xylanase. the enzymetic properties of crude enzyme were discussed. The crude xylanase of L0604 strain displayed an optimal pH at 5.0 and optimal temperature at 50℃. It was stable at pH5.0. And has a wide stable pH range from 4.0 to 10.0. At this situation. it still had 80% activity, and still had 80% activity. It kept stable at 40℃, The activity of xylanase at this temperature was observed increased with increasing the time of incubation. After incubated for 60 min at the temperature range from 20℃to 50℃, it still had 88% activity. In the metal ions investigated. Cu²⁺, Zn²⁺,Mg²⁺ promoted effects on xylanase, and the activity of xylanase was increased with increasing the concentration of these metal ions in certain does. The other ions all inhabited its activity more or less. Especially, Cu²⁺ promoted 150% activity in the enzyme reaction.After single factor tests and orthogonal array, the best condition for L0604 strain produce xylanase were corncob xylan used as carbon source and tryptone as nitrogen source. The optimal medium was consisted of corncob xylan 3.0%, tryptone 0.6%. K₂HPO₄ 0.5%, tween80 0.4%.the initial pH of medium was 7.0. Shaken at 220r/min and cultured in this optimal medium for about 96 hours at 32℃, the xylanase activity was up to 194.67U, which was 1.58 times of original enzyme activity.更多还原