构巢曲霉转化子菌株产MnP条件优化及对3种染料的脱色

摘要

Objective]The initialized culture conditions for two Aspergillus nidulans tranformant stains TN02 A7-He-mnp1 and TN02A7-He-mnp2 to produce manganese preoxidase( MnP) were optimized so as to enhance MnP activity and yield significantly. The decolorizing capacity of optimized culture solutions of two stains to 3 kinds of dyes was determined.[Method]Single-factor test was conducted to screen the optimal temperature for two strains to produce MnP. An orthogonal test was conducted to determine the effects of four factors-hemin concentration,Mn2 + concentration,pH value,and rotation speed on MnP activity,and a confirmation test was conducted to measure MnP activity. The absorbance value was determined to evaluate the decolorizing capacity of optimized culture solutions to 3 kinds of dyes.[Result]Results showed that the most optimal culture parameters for TN02A7-He-mnp1 to produce MnP were Mn2 +267 μmol·L -1 ,hemin 0. 05 g· L -1 ,2 tablets of φ =10 mm inoculum,50 mL culture solution of pH =7. 5 in 100 mL flask at 180 r·min -1 shaking condition,and under culture temperature of 37 ℃,on the basis of MMPGRT liquid medium plus to 2 g wood chip of Populus ussuriensis as substrate. The most optimal culture parameters for TN02A7-He-mnp2 to produce MnP were similar to those of TN02A7-He-mnp1,but only hemin 0. 03 g·L -1 and at 220 r·min -1 shaking condition. Under above-mentioned optimal culture conditions,the highest MnP activity reached 133. 62 and 147. 09 U·L -1 after 120 h culture for TN02A7-He-mnp1 and TN02A7-He-mnp2,respectively,which was 2. 89 and 3. 46 times higher than that produced at initial conditions. Decolorization tests showed that decolorization percents of two strains to heterocyclic dye neutral red and azo dye congo red surpassed 58% at 4 h,as high as or nearly to 100% at 16 h to neutral red and 20 h to congo red, respectively.[Conclusion]The MnP yield produced by two A. nidulans tranformants could be enhanced under optimal medium composition and culture conditions,and optimized extracellular MnP solutions of two strains could effectively decolorize heterocyclic dye neutral red and azo dye congo red.%【目的】对转入猴头菌 MnP1和 MnP2基因的2个构巢曲霉转化子菌株 TN02A7-He-mnp1和 TN02A7-He-mnp2产 MnP的初始培养条件进行优化，然后用优化后的酶液对3种染料进行脱色研究，以期提高锰过氧化物酶( MnP)活性。【方法】采用单因素试验进行最适产酶温度的筛选；正交试验检测血红素浓度、Mn2+浓度、pH 值和转速4个因素在3个水平下对酶活性的影响，并通过优化培养验证试验检测酶活性；吸光度法检测优化后的酶液对3种染料的脱色率。【结果】菌株 TN02A7-He-mnp1产 TN02A7-He-MnP1最适培养条件为：在 MMPGRT 培养基的基础上，加入2 g青杨木屑作为产酶底物，Mn2+浓度为267μmol·L －1、氯化血红素浓度为0.05 g·L －1、培养液 pH值为7.5，100 mL三角瓶中装液量为50 mL、接入2个φ=10 mm的菌块，于37℃、180 r·min －1的摇床中培养120 h；菌株 TN02A7-He-mnp2产 TN02A7-He-MnP2最适培养条件与 TN02A7-He-mnp1相似，只是氯化血红素溶液浓度为0.03 g·L －1，摇床转速为220 r·min －1，其他条件相同。在上述最适培养条件下，2个构巢曲霉转化子菌株产TN02A7-He-MnP1和 TN02A7-He-MnP2的酶活性分别为133.62和147.09 U·L －1，是初始培养条件的2.89和3.46倍。染料脱色试验表明，2个菌株对杂环类染料中性红和偶氮类染料刚果红在4 h内达到58%以上的脱色率，16 h对中性红达到或接近100%，20 h对刚果红达到或接近100%。【结论】通过优化培养基成分与条件可以提高2个构巢曲霉转化子菌株 MnP的产酶量，2个菌株对中性红和刚果红都有高效的脱色作用。