杏鲍菇菌渣固态发酵产漆酶及其在染料脱色中的应用
发布时间：2018-11-25 09:29
【摘要】：食用菌营养美味、种植逐年增加,但其菌渣处理量大,已成为食用菌行业亟需解决的难题。漆酶是食用菌中重要的木质纤维素酶,研究食用菌渣漆酶的合成,不仅能降低漆酶生产成本,还可高值化利用食用菌菌渣,符合绿色化学要求。论文旨在探究利用杏鲍菇菌渣固态发酵产漆酶的可行性及其产酶条件的优化、漆酶的酶学性质及其在染料脱色上的应用,其主要研究结果如下:1)通过传统的单因素方法优化了杏鲍菇菌渣固态发酵产漆酶培养基,以杏鲍菇菌渣为菌种和基料,优化后培养基组成为:碳源为6%甘蔗渣(w/w),氮源为1.2%硝酸铵(w/v),诱导物为1%碱木素(w/w)和150%(v/w)的无机盐液,无机盐液组分(g·L~(-1))如下:KH_2PO_4 0.8,Na_2HPO_4·7H_2O 0.75,ZnSO_4·7H_2O 0.002,MgSO_4·7H_2O 0.5,FeSO_4·7H_2O0.005,CuSO_4·7H_2O 0.02,CaCl_2·2H_2O 0.06,MnSO_4·H_2O 0.05。优化后培养条件为:培养液pH值为7,培养温度为28℃,培养时间培6 d。最优条件下菌渣固态发酵漆酶活力达到434.9 U·gds~(-1),是初始酶活12.3 U·gds~(-1)的35.3倍。2)通过文库构建、高通量测序以及生物信息学分析研究菌渣本身含有的微生物菌群对杏鲍菇菌丝产酶的影响,发现杏鲍菇菌渣固态发酵过程中细菌群落对漆酶合成无影响,真菌菌群主要为杏鲍菇,并含有子囊菌门的毛壳菌属(Chaetomium)、青霉菌属(Penicillium)、弯孢霉属(Curvularia)、曲霉属(Aspergillus),其丰度与原始样和加水培养的样品相比有所提高,因此部分真菌菌属可能会对杏鲍菇合成漆酶有促进作用;通过电镜扫描观察到杏鲍菇菌丝及甘蔗渣在发酵过程有较大的变化。3)应用硫酸铵沉淀、离子交换层析和分子筛层析三步纯化后得到的纯酶进行SDS-PAGE,进行质谱分析后确定漆酶由杏鲍菇产生,通过计算得到漆酶大小为65.18kDa,并初步研究了其酶学性质。在pH值2~6下漆酶具有较高的活力,在pH值5~8下稳定性较好;在20~70℃下反应相对酶活均高于70%,温度高于60℃时酶容易失活;对DMP的最适pH和温度分别为4和60℃,对ABTS最适pH和温度分别为4和40℃。0.1和1 mmol·L~(-1)金属离子对漆酶酶活抑制作用不明显,10 mmol·L~(-1)金属离子及各浓度的NaN3对漆酶活性有较大的抑制。4)利用粗酶液对四种常见合成染料进行了脱色研究,发现在无介体存在的条件下,发酵粗酶液作用3 h,对甲基红、活性艳蓝K3R、酸性蓝209、活性艳蓝KNR的脱色率均超过65%;在添加介体的条件下粗酶液作用3 h,对活性艳蓝K3R、酸性蓝209、活性艳蓝KNR的脱色率均超过80%。
[Abstract]:The edible fungi are nutritious and delicious, and the cultivation is increasing year by year, but it has become a difficult problem that needs to be solved in the edible fungus industry because of the large amount of the bacteria dregs. Laccase is an important lignocellulase in edible fungi. To study the synthesis of laccase from edible fungus residue can not only reduce the production cost of laccase, but also make use of edible fungus residue in high value, which meets the requirements of green chemistry. The purpose of this paper is to explore the feasibility and conditions of laccase production by solid state fermentation of Pleurotus eryngii residue, the enzymatic properties of laccase and its application in dye decolorization. The main results are as follows: 1) the laccase culture medium for laccase production from Pleurotus eryngii residue was optimized by traditional single factor method. The optimized medium consisted of 6% bagasse (w / w), 1.2% ammonium nitrate (w / v), 1% alkali-lignin (w / w) and 150% (v / w) inorganic salt solution. The composition of inorganic salt solution (g L ~ (-1) is as follows: KH_2PO_4 0.8 / Na2HPO4 / 7H_2O 0.75/ ZnSO4 / 7H_2O 0.002 / MgSO4 / 7H_2O / 0.5-FeSO-47H _ 2O _ 0.005, CuSO_4 7H_2O 0.02,CaCl_2 2H_2O 0.06,MnSO_4 H_2O 0.05. The optimized culture conditions were as follows: the pH value of the culture medium was 7, the culture temperature was 28 鈩，
本文编号：2355621
[Abstract]:The edible fungi are nutritious and delicious, and the cultivation is increasing year by year, but it has become a difficult problem that needs to be solved in the edible fungus industry because of the large amount of the bacteria dregs. Laccase is an important lignocellulase in edible fungi. To study the synthesis of laccase from edible fungus residue can not only reduce the production cost of laccase, but also make use of edible fungus residue in high value, which meets the requirements of green chemistry. The purpose of this paper is to explore the feasibility and conditions of laccase production by solid state fermentation of Pleurotus eryngii residue, the enzymatic properties of laccase and its application in dye decolorization. The main results are as follows: 1) the laccase culture medium for laccase production from Pleurotus eryngii residue was optimized by traditional single factor method. The optimized medium consisted of 6% bagasse (w / w), 1.2% ammonium nitrate (w / v), 1% alkali-lignin (w / w) and 150% (v / w) inorganic salt solution. The composition of inorganic salt solution (g L ~ (-1) is as follows: KH_2PO_4 0.8 / Na2HPO4 / 7H_2O 0.75/ ZnSO4 / 7H_2O 0.002 / MgSO4 / 7H_2O / 0.5-FeSO-47H _ 2O _ 0.005, CuSO_4 7H_2O 0.02,CaCl_2 2H_2O 0.06,MnSO_4 H_2O 0.05. The optimized culture conditions were as follows: the pH value of the culture medium was 7, the culture temperature was 28 鈩，
本文编号：2355621
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