草浆用木聚糖酶生产菌株的筛选、分离纯化及应用效果研究

【作者】 陈建宏；

【导师】 曲音波；

【作者基本信息】 山东大学 ， 微生物学， 2005， 硕士

【摘要】 自从1986年首次提出木聚糖酶用于纸浆漂白能够减少化学漂白剂的用量以来,各国科学家对此都进行了大量的研究工作。在我国造纸业主要以麦草作为浆料来源,落后的制浆及漂白工艺对环境造成了极大的压力。国内科研工作者也为此做了很多研究工作,先后筛选到了一些高产木聚糖酶的菌株,我们实验室也筛选到了黑曲霉An76（Aspergillus niger An76）和短小芽孢杆菌A30（Bacillus pumilusA30）等优良菌株。目前主要存在的问题在于真菌的木聚糖 酶耐碱性较差,这大大阻碍了其在生产中的应用,而细菌木聚糖酶在麦草浆漂白中的效果却不够明显,鉴于上述原因,使得筛选能够耐碱性高产木聚糖酶的真菌成为一个新的研究方向。筛选到具有较好耐碱性的且对麦草浆漂白有明显效果的真菌,它在生产中的应用将会成为可能,也会大大促进我国造纸业的发展。 从来源于造纸厂的碱性土样中共分离得到数株能够产碱性或中性木聚糖酶的真菌和细菌。经摇瓶复筛后,筛选到4株相对酶活较高的菌株,经过对麦草浆漂白实验,最终筛选出一株漂白效果较好的真菌ZCF57。通过对ZCF57形态学观察,初步确定其属于青霉属（Penicillium）。对其酶的基本性质研究表明:ZCF57木聚糖酶作用的最适温度为55℃,最适作用pH为6.0,在45℃以内和pH5.0～10.0之间有较好的稳定性。金属离子Fe²⁺、Mn²⁺和Mg²⁺对酶有激活作用。 对ZCF57液体摇瓶产木聚糖酶的条件进行了优化,确定其最适产酶条件为:3%玉米芯粉为最佳碳源,1%豆饼粉为最佳氮源,pH为9.0,温度为32℃,转速为150rpm,300ml三角瓶装液量为50ml,在此条件下培养72小时,ZCF57木聚糖酶酶活可以达到550IU/ml以上。经优化后酶活比优化前提高了四倍。Mg²⁺、Ca²⁺和Fe²⁺能够促进其生长及产酶,而Co²⁺和Cu²⁺则抑制其生长。表面活性剂吐温-80的添加能够促进其产酶,而SDS的添加抑制产酶。 对ZCF57发酵液经过硫酸铵分级沉淀、超滤浓缩、凝胶过滤层析色谱和阴离子交换层析,由ZCF57发酵液中最终分离得到了两个电泳纯的木聚糖酶组分Ⅺ和Ⅻ。用SDS-PAGE测定其分子量Ⅺ为23kD,Ⅻ为16kD。其中Ⅺ的最适反应温度为55℃,在45℃以下比较稳定,最适反应pH为7.0,在pH6.0～更多还原

【Abstract】 Since the first report that the use of xylanase in pulp bleaching could dramatically reduce the amount of chemical bleaching reagents in 1986, scientists all over the world have done lots of related research. In China, wheat straw is the main resource of pulp industry. Conventional pulpmaking and bleaching techniques have brought severe environmental pollution. Researchers in our country have also done lots of work referencing to this area. A lot of microbes, which can produce xylanase, have been isolated. Our laboratory also isolated Aspergillus niger An76 and Bacillus pumilus A30 et ah from nature. At present, the main problem is that xylanse from fungi was not alkalophilic and xylanse from bacteria improved wheat straw pulp brightness unconspicuously. So it is most important of isolating alkalophilic fungi that produce xylanase and using it in the paper industry. Perhaps this can accelerate the development of paper industry in our country.In this paper, lots of alkalophilic and neutrophilic microorganism, Which produce xylanase, were isolated from alkaline soil. Four of them that exhibited high activities of xylanase were obtained. By comparing to enhanced effectiveness of bleaching in xylanase-boosted bleaching of wheat straw pulp with the xylanase, fungus ZCF57 was screened out and preliminarily identified as Pemcillium. The optimum pH and temperature for crude xylanase from ZCF57 was 7.0 and 55℃, respectively. It was stable in the range of pH5.0-10.0 and below 45℃. Some of transition metal ions, such as Fe2+, Mn~2+ and Mg2+ can increase xylanase activity of ZCF57 dramatically.The culture conditions for producing xylanase were optimized by shaking flask liquid fermentation, and the optimal conditions are as follows: initial pH9.0, 32℃ and shaken at 150 rpm for 72h. The 3% of corncob powder and 1% of soybean cake powder was used as optimum carbon source and nitrogen source, respecitively. The highest xylanase activity was above 550 IU/ml under the optimum conditions of cultivation. When ZCF57 cultured in the optimized conditions, the xylanase activity is increased by 4 times.The crude culture filtration of ZCF57 was purified by ammonium sulfate fractionation, Sephadex G-100 and DEAE-Sephadex A-50 chromatographies, and two pure enzyme, XIand XII were obtained. It was indicated that the molecular weights of XIand Xllare 23kD and 16kD by SDS-PAGE analysis. As far as the enzyme activity is concerned, the optimum temperature and pH was 55℃ and pH 7.0 for the enzyme XI, and 50℃ and pH 6.0 for the enzyme XII, respectively. It was stable in the conditions of below 45℃ and pH 6.0~9.0 for enzyme XIand of below 50℃ and pH5.0~8.0 for enzyme XIIXylanse ZCF57 from the crude culture filtration of fungus ZCF57 was used for pretreating wheat straw pulp prior to bleaching to investigate effectiveness of enhanced bleaching with the xylanase. Results of the experiments showed that the brightness of pulps could be increased about 3~4 % ISO with a dose of 10 IU xylanase per gram pulp of oven dry weight, indicating that it was feasible for improving bleaching effectiveness of wheat straw pulp using xylanase of ZCF57.更多还原