【作者】 张莉；

【导师】 贾志宽；

【作者基本信息】 西北农林科技大学 ， 作物栽培学与耕作学， 2009， 博士

【摘要】 漆酶是一种含铜的多酚氧化酶,可催化酚类化合物和芳香胺的氧化。担子菌纲中的白腐菌是生产漆酶的一类重要的真菌。随着漆酶研究的深入,漆酶在含酚废水的处理、环境中酚类毒物的降解、秸秆生物降解、土壤生物修复和饲料工业等方面得到了越来越广泛的关注和应用。本论文以白腐菌T. pubescens MB89为漆酶生产菌,研究了发酵产漆酶以及酶的分离纯化的条件,游离漆酶和固定化漆酶的酶学性质,同时研究了漆酶对秸秆木素、氯酚化合物和染料的降解作用,得到如下结论:⑴T. pubescens MB89漆酶的发酵生产T. pubescens MB89发酵生产漆酶的最佳培养条件为:接种量6.0 mL;碳源为20 g/L葡萄糖,氮源为5.0 g/L蛋白胨;pH5,25℃,110 r/min振荡培养。发酵中期（10 d）添加1.0μmol/L的Cu²⁺能促进漆酶的分泌,黎芦醇、愈创木酚和苯甲醇对漆酶的分泌都具有诱导作用,其中苯甲醇的诱导效果最强。⑵T. pubescens MB89漆酶的分离和纯化80%硫酸铵沉淀可将90%左右的漆酶从发酵液中分离出来。漆酶粗酶液经过DEAE-Sepharose处理后,在280～610nm之间有两个漆酶主吸收峰,特征峰以峰1的构型为主。用Sephadex G-100分离纯化峰1处得到的漆酶液后,所得的样品的比活力为20.5,活力回收率达到了6%,纯化倍数为16.7。⑶T. pubescens MB89漆酶基本酶学性质①是一种含糖量为13.3%的分泌型胞外糖蛋白酶,分子量约为60000Da,pI2.8。②最适催化温度为50℃,热稳定性较差。属于耐酸性酶,在偏酸性环境下的酶稳定性优于偏碱性环境中的稳定性。③作用底物广泛,底物为酚类化合物或者是含I-的非酚类化合物时,最适合pH范围为4.0～4.5。④NaN₃是酶促反应的强抑制剂,浓度为0.1 mmol/L的EDTA和1.0 mmol/L的H₂O₂对漆酶有轻微的抑制作用。卤族离子对漆酶活性的抑制效果为F^->Cl^->Br^-,Cl^-和Br^-对漆酶催化活性的抑制具有瞬时的特点。Cu²⁺、Co²⁺、K⁺、Ag⁺对漆酶酶活性有促进作用,Fe²⁺、Fe3+、Ca²⁺使漆酶的相对酶活下降,具有抑制作用,Na⁺、Mg²⁺、Zn²⁺、Mn²⁺等离子对漆酶酶活性无显著影响。⑤以ABTS作为底物时,酶促反应的催化效率最高,达到48×10⁶ M^-1·s^-1,其次是丁香醛连氮,催化效率可达47×10⁶,对具有羟基或甲氧基取代苯酚以及无机亚铁离子的底物,也具有较高的催化活性,对碘离子基本上没有催化活性。⑷T. pubescens MB89漆酶固定化固定化方法和最佳固定条件:①壳聚糖固定化漆酶（IE1）:戊二醛的最佳浓度为4%,最适交联时间为8 h,最佳给酶量为1.5 mg/g;②壳聚糖铜固定化（IE2）:CuSO₄·5H₂O的最佳添加量为0.6 mg,最适络合时间为10 h,最佳给酶量为0.25 mg/g;③海藻酸钠/壳聚糖固定化（IE3）:2%海藻酸钠,2% CaCl₂,1%戊二醛,1.5%壳聚糖。最佳给酶量1.5 mg/g;④TEOB/PEG固定化（IE4）:最适PEG分子量为600～800,最佳使用浓度为1.5%,最佳给酶量为1.5mg/g。IE1和IE4的最适温度均为60℃,IE2和IE3最适合温度为30℃。IE1、IE3和IE4的最适pH值均为4.5,IE2的最适pH为4。与Lac的最适pH值相比,IE的最适pH值均向碱性方向偏移。IE的酸碱稳定性、热稳定性和贮存稳定性都优于Lac。⑸T. pubescens MB89漆酶对秸秆木素的降解①玉米秸秆可以T. pubescens MB89发酵生产漆酶的碳源。漆酶对木素的降解作用明显优于对纤维素和半纤维素的降解。②秸秆分离木素经过漆酶处理后其聚合度和多分散性都降低。漆酶降解木素主要发生在高分子量木素部分。③漆酶处理使木素酚羟基含量增加,漆酶/ABTS体系使木素酚羟基含量降低。⑹T. pubescens MB89漆酶对环境的生物修复对氯酚化合物的降解:①Lac与DCP、TCP和PCP三种氯酚化合物反应4h,就可以使氯酚降解率达到50%左右;IE降解反应2h,降解率就已经超过50%。②在相同pH值条件下,IE和Lac对TCP的降解效率最高,其次是DCP,PCP的降解效果最差。Lac和IE降解DCP的最佳pH均为5.5, IE1的降解率最大为89.6%,Lac的降解率最小,为82.9%。TCP降解的最佳pH为5.5,IE1、IE3和IE4最大降解率均为92.4%,Lac与IE2的最大降解率接近89%左右。PCP降解结果表明Lac、IE2、IE3和IE4的最佳降解pH为5,IE1的最大降解率出现在pH5.5。③Lac对DCP、TCP和PCP降解的最适温度分别为35℃、45℃和45℃,PCP降解处理中,IE1和IE4的最适作用温度为45℃,IE2和IE3的最适温度为35℃。④Lac降解TCP、DCP和PCP的最佳活力为30 U/mL。IE1降解DCP的添加量为40U/mL,降解TCP的添加量为20U/mL。IE2降解DCP、TCP和PCP的最适添加量为20U/mL,20U/mL和40U/mL。IE3和IE4降解三种氯酚化合物的最佳添加量分别为30U/mL,20U/mL和40U/mL。⑤随着DCP初始浓度的升高,其降解率逐渐降低,但变化趋势缓慢; DCP初始浓度为10 mg/L时,降解率为100%;初始浓度低于10 mg/L,降解率维持在100%的水平,初始浓度高于10 mg/L时,降解率呈缓慢下降趋势。PCP的降解率随初始浓度升高而降低,5 mg/L的降解率最高,达到37.8%,高浓度的PCP （20 mg/L）降解率仅为16.9%。IE在降解氯酚类化合物的时候,底物浓度对降解率的影响要小于对游离漆酶降解效果的影响。⑥在漆酶中添加5 mmol/L ABTS,可在反应30 min内去除69%的PCP,而不加ABTS时,反应17 h后只有24%的PCP被去除。固定化酶在有ABTS存在的环境下,对氯酚的降解作用于游离漆酶差异不大。对染料的降解:①Lac和IE4对DB作用的最佳pH为4,IE1、IE2和IE3在pH5时对DB的降解率达到最大,在RRG的降解过程中,IE4的最大降解率出现在pH4,Lac和其他几种固定化酶的最适pH为5。Lac和IE对PCP的催化作用都随着pH的升高呈下降趋势,其中IE4对PCP的降解效果随着浓度的变化幅度最大。②IE对DB、RRG和IB的降解效果比Lac的降解效果好。Lac和IE对三种染料的降解率都随着温度的升高呈先上升后下降的趋势。IE3和Lac在40℃时对DB的降解效率最高,IE2在50℃时对RRG的降解率最大,IB最佳降解率出现在IE2,40℃的降解处理中。③随着酶活力的提高,DB、RRG和IB三种染料降解速率的变化趋势相同,即开始时增加显著,随后缓慢上升到逐渐趋于平稳,当酶活力为10U/mL时,三种染料的降解率均达60%。在DB降解过程中,同浓度的IE降解效果要好于Lac,IE1、IE4在浓度为5 U/mL时,对DB脱色率就超过了50%。在RRG降解过程中,Lac和IE的作用无明显差异。IB的降解体系中,IE1和IE3在5 U/mL浓度时,降解率就超过了50%。④染料降解速率随着染料浓度的增加而增加,Lac和IE反应体系中,染料浓度在0～10 mg/mL范围时,反应速率快速增长,继续增加染料浓度,反应速率变化的幅度减小。IE1对三种染料的降解速率要大于Lac和其他三种IE的作用效果。⑤漆酶与染料在反应初期的10min,降解速率较快,Lac体系在反应20min后,降解率趋于稳定,IE体系则在反应40min后,降解率趋于稳定。因此,在Lac反应体系中,反应时间以不超过20min为宜。IE体系中,反应时间以40min为宜。⑥ABTS的添加对蒽醌类染料DB的降解率没有显著影响,在漆酶和偶氮类染料RRG以及靛青类染料IB反应体系中,ABTS的加入明显的提高了降解率,表明漆酶在降解RRG和IB两种染料的过程中,对于体系中的一些小分子物质具有依赖作用。更多还原

【Abstract】 Laccase, a copper-containing polyphenol oxidase, had been widely researched in the treatment of phenolic wastewater, environmental degradation of toxic phenolic, straw biodegradation, bioremediation of soil and feed industries. T. pubescens MB89, a kind of wihte-rot fungi,has a high laccase productivity. In present study, the culture and purification conditions suitable for laccase in T. pubescens MB89 were established first; and then enzymatic properties were examined. In addition, methods and characteristics of immobilized laccase, straw lignin decomposing, chlorophenol and dyes degradation were studied.Results of the above-mentioned studies indicate that:⑴The optimal culture conditions for laccase production were glucose 20 g / L, peptone 5.0 g / L, inoculation volume 6.0 mL, pH5, 25℃, 110 r / min.The to Adding 1.0μmol / L of Cu2 + in mid-fermentation （10 d） can promote laccase activity. All of veratryl alcohol, guaiacol and benzyl alcohol can induce laccase produce.⑵The results of laccase separation and purification showed that about 90% of laccase could be separated from fermentation broth by using 80% ammonium sulfate. After DEAE-Sepharose treatment, two characteristic peaks were detected between 280 to 610 nm. With Sephadex G-100 peak separation and purification of the paint to be an enzyme, the samples from the specific activity of 20.5, dynamic recovery rate reached 6 percent, for the purification of a multiple of 16.7.⑶Laccase characteristics were that:①It is a sugar content of 13.3% of the secreted glycoprotein extracellular protease, the molecular weight was about 60000Da and pI2.8.②The optimum catalytic temperature was 50℃, thermal stability was poor. It was an acid-resistant enzyme which stability was superior in alkaline conditions.③For phenolic substrate with I-or non-phenolic compounds, the most suitable pH range were between 4.0 to 4.5.④NaN3 was a strong inhibitor to laccase while 0.1 mmol / L EDTA and 1.0 mmol / L H₂O₂ were slight inhibition. The inhibitory of Chatham halogen ions were F-> Cl-> Br-, Cl-and Br- had transient suppression characteristics. Ions of Cu2 +, Co2 +, K +, Ag + could speed laccase activity, but ions of Fe2 +, Fe3 +, Ca2 + slowly enzyme reaction, and other ions such as Mg2 +, Zn2 +, Mn2 + had no significant effect.⑤with ABTS as substrate, the catalytic efficiency was the highest, reaching 48×106 M-1 ? s-1, followed by lilac aldehyde azine, catalytic efficiency up to 47×106, with hydroxy or methoxy - substituted phenols as well as the inorganic substrate of ferrous ions, but also have a high catalytic activity of the iodine ions are basically no catalytic activity.⑷The best methods and fixed conditions for immobilization①chitosan immobilized laccase （IE1）: the best concentration of glutaraldehyde 4%, the optimum crosslinking time for 8 h, the best to the enzyme was 1.5 mg / g;②chitosan immobilized copper （IE2）: CuSO4 ? 5H2O its adding the best of 0.6 mg, the optimal time for complex 10 h, the best to the enzyme was 0.25 mg / g;③alginate / immobilized chitosan （IE3）: 2% sodium alginate, 2% CaCl2, 1% glutaraldehyde, 1.5% chitosan. The volume of the best enzyme to 1.5 mg / g;④TEOB / PEG immobilized （IE4）: The optimum PEG molecular weight of 600 800, the best use of the concentration of 1.5%, the best to the enzyme was 1.5mg / g.The optimum temperature of IE1 and IE4 were 60℃, IE2 and IE3 were 30℃. IE1, IE3 and IE4 are the optimum pH value of 4.5, IE2 optimum pH of 4. IE pH stability, thermal stability and storage stability are superior to Lac.⑸The results of lignin degradation were①corn stalk can be used as carbon source for T. pubescens MB89 fermentation. Laccase had good degradation effect on lignin than on the cellulose and hemicellulose degradation.②The degree of polymerization and dispersion of separated straw lignin were reduced after laccase treatment. The degradation was mainly occured in parts of high molecular weight of lignin.③The phenolic hydroxyl contents of lignin increased with laccase treatment, but was decreased while ABTS existed.⑹Degradation of chlorophenol compounds:①The degradation time for DCP, TCP and PCP wer 4h by Lac, and degradation rate can reach about 50%; IE degradation 2h, the degradation rate has exceeded 50%.②With same pH value, IE and Lac efficiency on the degradation of TCP, followed by DCP, PCP degradation effects were the worst. DCP degradation by Lac and IE the best pH of 5.5, IE1 maximum degradation rate of 89.6%, Lac minimize the degradation rate, was 82.9%. TCP best pH for the degradation of 5.5, IE1, IE3 and IE4 are the largest degradation rate of 92.4%, Lac and IE2 close to the maximum degradation rate about 89%. The results show that the PCP degradation Lac, IE2, IE3 and IE4 best pH for the degradation of 5, IE1 degradation rate of the largest in the pH5.5.③Lac on the DCP, TCP and PCP degradation of the optimum temperature of 35℃, 45℃and 45℃, PCP degradation process, IE1 and IE4 the optimal temperature of 45℃, IE2 and IE3 the optimum temperature was 35℃.④Lac degradation of TCP, DCP and PCP the best dynamic for 30 U / mL. IE1 of its adding DCP degradation 40U/mL, its adding the TCP degradation 20U/mL. IE2 degradation of DCP, TCP and PCP its adding the optimum 20U/mL, 20U/mL and 40U/mL. IE3 and IE4 chlorophenol degradation of three compounds were the best addition 30U/mL, 20U/mL and 40U/mL.⑤With the increase of initial concentration of DCP, the degradation rate decreased, but the slow change in the trend; DCP initial concentration of 10 mg / L, the degradation rate was 100%; the initial concentration of less than 10 mg / L, the degradation rate of 100% of the level, the initial concentration is higher than 10 mg / L, the degradation rate was slow downward trend. PCP degradation rates increased with lower initial concentration, 5 mg / L the degradation rate of the highest, reaching 37.8%, high concentrations of PCP （20 mg / L） degradation rate was only 16.9%. IE in the degradation of chlorophenol compounds, the substrate concentration on the degradation rate of less than free laccase on the effect of degradation.⑥laccase added in 5 mmol / L ABTS, the reaction can be within 30 min to remove 69% of the PCP, without ABTS, the reaction after 17 h only 24% of PCP was removed.更多还原