摘要
本文通过对东北林业大学微生物及免疫学实验室保存的27株(种)大型真菌木素氧化酶系组成、活性及对染料脱色作用的研究,筛选出木蹄层孔菌作为产漆酶的菌株。并进一步对该菌产漆酶发酵条件、漆酶的纯化及酶学性质进行了研究。获得如下结果:
(1)采用丁香醛连氮、苯胺蓝平板脱色等4种方法,定性检测了株(种)大型真菌的木素氧化酶系组成。结果表明5个菌种同时具有Lac、Lip、Mnp 3种酶的活性,5个菌种同时具有Lac、LiP 2种酶的活性,8个菌种具有1种酶的活性;12个菌种具有漆酶活性。选择其中生长速度快、漆酶活性高的5个菌种进行漆酶产生的研究,发现木蹄层孔菌在诱导、静止培养条件下产生的漆酶酶活峰值高达9496 U/mL,远远高于其它已报道过的菌种,说明木蹄层孔菌静止培养可代替振荡培养进行漆酶产生的后续研究。木蹄层孔菌等5种真菌对刚果红等4类染料均具有高效广谱的脱色作用,其中木蹄层孔菌效果最好。
(2)利用正交实验对木蹄层孔菌产漆酶发酵条件进行研究,得到优化培养基组成为(/L):淀粉20 g;藜芦醇2.5 g;蛋白胨2.5 g;Tween80 0.15%;CuSO_4 0.1 mM(调节pH至4.0,温度为30℃)。
(3)通过DEAF-Sepharose FF离子交换层析和Sephadex G-75凝胶过滤等纯化技术对木蹄层孔菌胞外漆酶进行了纯化,最终纯化倍数达11.93,回收率为20.18%。
(4)以ABTS作为底物时木蹄层孔菌纯化后的漆酶的最适pH为2.8,在pH 2.0-5.0之间维持较高的酶活,超过pH 5.0漆酶活性急剧下降;纯化后的漆酶最适反应温度为60℃,在20-65℃范围内较稳定,超过65℃酶活力下降迅速。纯化后的漆酶对底物ABTS的催化效率较高,米氏常数为0.068 mmol/L。几种漆酶的抑制剂中的NaN_3、半胱氨酸和DTT对漆酶活力有强烈的抑制,而金属离子鳌合剂EDTA对漆酶活力的抑制程度较低。Zn~(2+)、Mg~(2+)、Ca~(2+)等金属对漆酶活性有明显的激活作用。染料降解实验发现木蹄层孔菌与染料的共培养体系、木蹄层孔菌纯化后的漆酶均对偶氮类染料刚果红、三苯甲烷类染料结晶紫、杂环类染料亚甲基兰、蒽醌类染料茜素红四类染料有很高的降解作用。显示了木蹄层孔菌及其所产生的漆酶在染料降解的应用上具有很强的工业应用潜力。
In this study, the lignin oxidative system, laccase activities and decolorization assay of the 27 fungi were analyzed, which were preserved in the Laboratory of Microbiology and Immunology, Northeast Forestry University. From the results, Fomes fomentariu had been chosen as the strain produced laccase with its excellent capability and researched detailedly in fermentation conditions, purification and enzymatic properties on extracellular laccase which it produced. Main results as followed:
(1) The lignin oxidative system of 27 fungi species were analyzed qualitatively by four methods besides using syringaldazine as the substrate and the decolorization of aniline blue in plate cultivation. The result showed that 5 species had three enzymes activity of Lacese, Lip and Mnp, and 5 species had two. Another 8 species only had one enzymes activity. There were also 12 species produced laccase activities. The 5 fungi, which grew quickly and produced high laccase activity, were selected to study how the cultivation mode and inducer could affect the production of laccase activity. The laccase activities was produced by Fomes fomentarius reaching a peak of 9496 U/ml on induction and static cultivation, which was obviously higher than other fungi. It proved that shaking cultivation could be taken place by static cultivation to produce and prepare laccase in the follow up work for Fomes fomentarius. By the decolorization assay, it was found that all of this 5 species have efficient and broad-spectrum discolored function to 4 head kinds of dye and Fomes fomentarius had most excellent capability.
(2) Utilizing the orthogonal design experiment method, the best response system of media (g/L): starch 20, veratryl alcohol 2.5, peptone 2.5, Tween80 0.15 %, CuSO_4 0.1 mM (pH4.0, 30℃).
(3) Laccase from Fomes fomentarius was purified using DEAE-Sepharose FF anion-exchange chromatography and Sephadex G-75 gel filtration. The purification fold was 11.93 and recovery of total laccase activity was 20.18%.
(4) The pH for laccase activity against ABTS showed a peak of maximum activity at pH 2.8. The enzyme was stable at pH range from 2.0 to 5.0. The optimum temperature of the purified enzyme was observed at 60℃and was stable at range from 20℃to 65℃. But the activity decreased rapidly beyond 65℃. The purified laccase showed activity against various substrates. The highest Km value (0.068 mmol/L) was found for ABTS. The purified enzyme was strongly inhibited by sodium azide, L-cysteine and DTT, whereas the metal ion chelator EDTA showed only a slight inhibitory effect. The metal ion Zn~(2+), Mg~(2+) and Ca~(2+) could activate the laccase activities.
(5) In the dye degradation assay, it was found that the co-culture system of Fomes fomentarius and dye had high degradation effect as the laccase purified on the congo red, crystal violet, methylene blue and alizarin red, which were belong azo compounds, triphenylmethane, heterocyclic and anthraquinones, respectively. This result showed that the laccase produced by Fomes fomentarius had industrial application potential on the dye degradation.
引文
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