青霉ZH1产纤维素酶条件的优化及其酶学性质研究

发布时间：2018-05-07 07:24

  本文选题：纤维素酶 + 筛选鉴定　； 参考：《沈阳农业大学》2017年硕士论文

【摘要】：纤维素是世界上最丰富的可再生资源,利用微生物生产的纤维素酶将纤维素降解为单糖,再转化为能源、食物和化工原料等。对人类解决环境污染和资源短缺等问题具有重大的现实意义。现有纤维素酶生产菌存在酶活力低、生产成本高等问题。所以筛选高效降解纤维素的菌株和一些相关内容的研究已成为国内外关注的热点。1.本研究从建平县农家柴垛附近7个采样点进行取样,通过分离纯化得到13株菌株,对其进行综纤维素、滤纸、微晶纤维素和刚果红四种筛选培养基初筛,并结合纤维素酶活力测定,得到高产纤维素酶的菌株E2.经形态鉴定及系统发育学分析,确定该菌株为赭绿青霉(Penicilliumochrochloron)。2.通过试验得到青霉液体发酵最佳产酶培养条件为:碳源为稻草,碳源浓度为3%,氮源为花生饼粉和KN03,氮源总浓度为0.4%,CaCl2浓度为0.05%;接种量为8%,初始pH为3,发酵温度为30℃,培养时间为144h.在此条件下,最终FPase为28.58U/mL,CMCase 为 54.88U/mL,β-Gase 为 194.16U/mL.3.通过试验得到青霉固体发酵最佳产酶培养条件为:氮源为花生饼粉,含水量(固体:液体)为1:3,接种量为5%(孢子悬浮液107个/mL),稻草粒径为40-60目;初始pH为4,发酵温度为30℃,培养时间为72h.在此条件下,最终FPase为148.29U/g,CMCase 为 256.68U/g,β-Gase 为 230.20U/g。4.将菌株液体发酵的粗酶液经硫酸铵沉淀、SephadexG-75层析得到一组内切葡聚糖酶,分子量为29.7kDa.对内切葡聚糖酶的酶学特性研究表明:酶的最适反应温度为40℃,在40℃时有良好的热稳定性;最适pH为5.0,在pH为3时稳定;金属离子K+和Ca2+对内切葡聚糖酶具有促进作用,Na+、Mrn2+、ZZn2+、Co2+和Fe2+对内切葡聚糖酶具有不同程度的抑制作用,其中Mn2+、Co2+和Fe2+抑制明显;对于内切葡聚糖酶的底物专一性,其对MCC和滤纸有作用,对水杨素、几丁质和pNPC没有作用。内切葡聚糖酶以羧甲基纤维素钠为底物的 Km 为 18.45mg/mL,Vmax 为 18.18mg/(mL · min).
[Abstract]:Cellulose is the most abundant renewable resource in the world. Cellulose is degraded into monosaccharide by cellulase produced by microorganisms and then converted into energy, food and chemical raw materials. It is of great practical significance for mankind to solve the problems of environmental pollution and shortage of resources. The existing cellulase producing bacteria have low enzyme activity and high production cost. Therefore, the screening of high efficient cellulose degradation strains and some related research has become a hot spot at home and abroad. 1. In this study, 13 strains were isolated and purified from 7 sampling sites near Nongjia Chaijong, Jianping County, and screened on four screening media: holocellulose, filter paper, microcrystalline cellulose and Congo red. Combined with the activity of cellulase, the high cellulase producing strain E _ 2 was obtained. After morphological identification and phylogenetic analysis, the strain was identified as Penicillium ochrochloron.2. The results showed that the optimum conditions for enzyme production by liquid fermentation of Penicillium were as follows: carbon source was rice straw, carbon source concentration was 3%, nitrogen source was peanut cake powder and KN03, total nitrogen source concentration was 0.410% and CaCl2 concentration was 0.05%, inoculation amount was 8%, initial pH was 3 鈩，

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