Myrothecium verrucaria NF-08产漆酶条件的响应面法优化及粗酶染料脱色研究

Optimization of culture conditions for laccase production by Myrothecium verrucaria NF-08 via response surface methodology and performance of the crude laccase in dye decolorization

以半知菌属疣孢漆斑菌Myrothecium verrucaria NF-08为供试菌,采用响应面方法优化菌株产漆酶条件,并以其漆酶粗酶进行3种结构类型6种染料的脱色试验,考察了粗酶浓度、温度和p H值对脱色率的影响.两水平析因设计、中心组合设计和验证试验结果表明:葡萄糖浓度为15.4 g·L^(-1)、蛋白胨浓度为43.3 g·L^(-1)和培养基初始p H值为6.68时,漆酶活性达到最大值(25.58±1.60)U·m L^(-1),是单因素试验(16.82 U·m L^(-1))的1.52倍,提高了52.1%.以上述发酵条件获得的M.verrucaria NF-08漆酶粗酶对3种结构类型染料均有脱色效果,排序为偶氮类优于芳甲烷类优于蒽醌类.脱色率随粗酶浓度增大(2~8 U·m L^(-1))、反应温度升高(15~35℃)而上升,随p H值上升(4~10)而下降.粗酶浓度为5 U·m L^(-1),反应温度为25℃,p H值为4时,偶氮类染料铬黑T和芳甲烷类染料碱性品红即可几乎完全脱色,脱色率分别为99.2%和94.6%.

The deuteromycete fungus Myrothecium verrucaria NF-08 was under investigation. Response surface methodology was adopted to optimize the laccase-producing condition parameters by M.verrucaria NF-08. The effects of concentration of crude laccase,temperature and p H value on decolorization of six dyes belonging to three structural types were tested. The results of two level factorial design,central composite design and model validation showed that when the concentration of glucose and peptone were 15.4 g·L^（-1）and 43.3 g·L^（-1）,respectively and the initial p H value of fermentation medium was6.68,the laccase activity reached the maximum（ 25.58±1.60） U·m L^（-1）which was 1.52 folds of that after single factor experiment（ 16.82 U·m L^（-1）） and was enhanced by 52. 1%. Crude laccase of M. verrucaria NF-08 was collected under the above-mentioned fermentation parameters and applied in dye decolorization. The crude laccase exhibited remarkable decolorization effects on the three structural dyes,specifically the decolorization rate from higher to lower was azo,arylmethane and anthraquinone. Generally,the decolorization rate enhanced along with the increase of crude laccase concentration（ 2 to 8U·m L^（-1）） and temperature（ 15 ℃ to 35 ℃） while declined along with the increase of p H value（ acidic 4 to alkalic 10）. Under the condition of crude laccase 5 U·m L^（-1）,25 ℃ and p H 4,eriochrome black T and fuchsin basic could be almost totally decolorized with decolorization rate 99.2% and 94.6%,respectively.