【摘要】：食用菌營養(yǎng)美味、種植逐年增加,但其菌渣處理量大,已成為食用菌行業(yè)亟需解決的難題。漆酶是食用菌中重要的木質(zhì)纖維素酶,研究食用菌渣漆酶的合成,不僅能降低漆酶生產(chǎn)成本,還可高值化利用食用菌菌渣,符合綠色化學(xué)要求。論文旨在探究利用杏鮑菇菌渣固態(tài)發(fā)酵產(chǎn)漆酶的可行性及其產(chǎn)酶條件的優(yōu)化、漆酶的酶學(xué)性質(zhì)及其在染料脫色上的應(yīng)用,其主要研究結(jié)果如下:1)通過傳統(tǒng)的單因素方法優(yōu)化了杏鮑菇菌渣固態(tài)發(fā)酵產(chǎn)漆酶培養(yǎng)基,以杏鮑菇菌渣為菌種和基料,優(yōu)化后培養(yǎng)基組成為:碳源為6%甘蔗渣(w/w),氮源為1.2%硝酸銨(w/v),誘導(dǎo)物為1%堿木素(w/w)和150%(v/w)的無機(jī)鹽液,無機(jī)鹽液組分(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。優(yōu)化后培養(yǎng)條件為:培養(yǎng)液pH值為7,培養(yǎng)溫度為28℃,培養(yǎng)時(shí)間培6 d。最優(yōu)條件下菌渣固態(tài)發(fā)酵漆酶活力達(dá)到434.9 U·gds~(-1),是初始酶活12.3 U·gds~(-1)的35.3倍。2)通過文庫構(gòu)建、高通量測(cè)序以及生物信息學(xué)分析研究菌渣本身含有的微生物菌群對(duì)杏鮑菇菌絲產(chǎn)酶的影響,發(fā)現(xiàn)杏鮑菇菌渣固態(tài)發(fā)酵過程中細(xì)菌群落對(duì)漆酶合成無影響,真菌菌群主要為杏鮑菇,并含有子囊菌門的毛殼菌屬(Chaetomium)、青霉菌屬(Penicillium)、彎孢霉屬(Curvularia)、曲霉屬(Aspergillus),其豐度與原始樣和加水培養(yǎng)的樣品相比有所提高,因此部分真菌菌屬可能會(huì)對(duì)杏鮑菇合成漆酶有促進(jìn)作用;通過電鏡掃描觀察到杏鮑菇菌絲及甘蔗渣在發(fā)酵過程有較大的變化。3)應(yīng)用硫酸銨沉淀、離子交換層析和分子篩層析三步純化后得到的純酶進(jìn)行SDS-PAGE,進(jìn)行質(zhì)譜分析后確定漆酶由杏鮑菇產(chǎn)生,通過計(jì)算得到漆酶大小為65.18kDa,并初步研究了其酶學(xué)性質(zhì)。在pH值2~6下漆酶具有較高的活力,在pH值5~8下穩(wěn)定性較好;在20~70℃下反應(yīng)相對(duì)酶活均高于70%,溫度高于60℃時(shí)酶容易失活;對(duì)DMP的最適pH和溫度分別為4和60℃,對(duì)ABTS最適pH和溫度分別為4和40℃。0.1和1 mmol·L~(-1)金屬離子對(duì)漆酶酶活抑制作用不明顯,10 mmol·L~(-1)金屬離子及各濃度的NaN3對(duì)漆酶活性有較大的抑制。4)利用粗酶液對(duì)四種常見合成染料進(jìn)行了脫色研究,發(fā)現(xiàn)在無介體存在的條件下,發(fā)酵粗酶液作用3 h,對(duì)甲基紅、活性艷藍(lán)K3R、酸性藍(lán)209、活性艷藍(lán)KNR的脫色率均超過65%;在添加介體的條件下粗酶液作用3 h,對(duì)活性艷藍(lán)K3R、酸性藍(lán)209、活性艷藍(lán)KNR的脫色率均超過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，

本文編號(hào)：2355621