摘要
采用固体平板法分离纯化得到多株白腐菌菌株。通过分析不同白腐菌的产酶特性，选育得到了可高产纤维二糖脱氢酶(CDH)的菌株W14和高产漆酶的菌株LZ-8。初步研究表明菌株W14产CDH的最佳碳源和氮源分别为棉花和尿素。利用优化后的碳源和氮源，菌株W14发酵第17天时，酶活达到208.4 U／L，达到国内外报道的较高水平。菌株LZ-8具有较高的产漆酶能力，并具有生长迅速、产酶周期较短等特点，具有良好的开发价值。
对菌株LZ-8产漆酶的最适培养基组成和最佳培养条件进行了优化。表明菌株LZ-8产漆酶时，最佳的碳源和氮源分别是土豆和豆饼粉。培养基中加入表面活性剂PEG4000以及同时添加Cu~(2+)和Mn~(2+)时均有利于促进漆酶产生。菌株LZ-8较佳的产漆酶培养基为(单位：L~-)：土豆150g；豆饼粉10g；NaH_2PO_4 1.5g；K_2HPO_4 3g；MgSO_4.7H_2O 0.2g；PEG4000 3g；CuSO_4·5H_2O 0.0025g；MnSO_4.H_2O 0.05mg；NaCl0.1g；CaCl_2 0.01g；VA 1g；pH 6.0-7.0。较佳的培养条件为：300ml三角瓶装80ml培养基，30℃，150rpm条件下培养。采用优化后的培养基和培养条件，在第9天时漆酶酶活达到10.86 U／ml，是优化前酶活的9.74倍。
对LZ-8所产漆酶粗酶液的酶学性质进行了初步研究。结果表明，其较适宜的缓冲体系为柠檬酸-柠檬酸钠缓冲体系，最适pH值和最适温度分别为pH 4和40℃。在pH值3-9和30℃～40℃的范围内，该漆酶有较好的稳定性。缓冲液中加入Ag~+或Zn~(2+)可以显著提高LZ-8漆酶的酶活，但Fe~(2+)和Fe~(3+)对漆酶酶活有明显的抑制作用。
以不同浓度的造纸中段废水为底物，采用逐级驯化定向选育方法，从上述分离的菌株和实验室保藏的菌株中，筛选出了抗造纸中段废水毒性的优良菌株。利用其中的多株菌株处理造纸中段废水，均可以降低废水的COD含量，但对废水色度的影响有所不同。其中选育的菌株Z-1和白腐菌L02可直接应用于造纸废水处理，在有效降低废水COD含量的同时，也有效地降低了废水的色度和pH值。例如，采用菌株Z-1处理造纸中段废水，处理4天后，废水色度降低82.61％，处理6天，COD含量降低63.44％，废水pH值从中性下降到pH 2左右的水平；菌株L02处理可使中段废水COD含量降低84％以上，废水的色度降低93％以上。采用白腐菌L02处理造纸废水(包括中段废水和次氯酸盐漂白废水)时，处理工艺条件如处理时间和温度、废水的初始pH值和浓度、通氧量等对最终的处理效果将产生一定的影响，因此，在应用时需要在处理成本和处理效果之间进行综合考虑。
Some fungi were isolated , properties of enzymes produced by these fungi and the stock cultures in our laboratory were analyzed. In which, the fungus W-14 and fungus LZ-8 were selected because of their high abilities to produce cellobiose dehydrogenase (CDH) and laccase respectively. Preliminary experiments showed that the optimum carbon source and nitrogen source for CDH production by fungus W-14 were dewaxed cotton powder and urea respectively. The activity of CDH was 208.4 U/L after 17 days cultivation when fungus W-14 was grown in the medium with the suitable carbon source and nitrogen source.
The culture components and fermentation conditions for laccase production in flasks by fungus LZ-8 were investigated in the paper. The results showed that the optimum carbon source was potato and nitrogen source was bean cake powder. The addition of surfactant PEG4000, metal ion Cu~(2+) and Mn~(2+) to basal media enhanced the enzyme production. The suitable culture were as follows: potato 150 g/L, bean cake powder 10 g/L, NaH_2PO_4 1.5 g/L, K_2HPO_4 3 g/L, MgSO_47·H_2O 0.2 g/L, PEG4000 3 g/L, CuSO_45·H2O 0.0025 g/L, MnSO_4·H_2O 0.05 mg/L, NaCl 0.1 g/L, CaCl_2 0.01 g/L, veratryl alcohol 1 g/L, and pH 6.0-7.0. The activity of laccase reach 10.86U/ml when fungus LZ-8 was cultured at 30℃ for 9 days with shaking at 150 rpm in the suitable laccase production medium (50 ml in 300 ml conical flask).
The properties of laccase produced by fungus LZ-8 were studied. The results showed that the suitable buffer was citric acid-sodium citrate buffer system and the optimum pH and temperature of laccase were pH 4.0 and 40℃ . The enzyme was stable in the range of pH 3.0-9.0 and below 50℃. The activity of laccase was enhanced by Ag~+ and Zn~(2+), but was strongly inhibited by Fe~(2+) and Fe~(3+).
Strains suitable for pulp and paper mill waster water treatment were screened from more than 10 white-rot fungi from the isolated fungi and the stock cultures in our laboratory by cultivating continuously in a series waste water-contained mediums which had increased gradually amount of waster water. Several strains were obtained which were waste water toxicity-resistance and could decrease COD(Chemical oxygen demand) content of waste water effectively. In which, fungus Z-1 and fungus L02 could be used directly for treating middle-stage effluent from pulp and paper mill and led to COD
引文
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