漆酶/HOBT体系对苯胺蓝的脱色条件及机理研究

On the technical condition and the mechanism of the aniline blue decolorization under the impact of the laccase/HOBT mediator system

研究了Pleurotus sp.N3固态发酵所得漆酶对三芳基甲烷类染料苯胺蓝的脱色条件和机理。通过可见光光谱扫描和非变性凝胶电泳的分析发现漆酶能够对苯胺蓝进行脱色,介体可以显著增强脱色效果,提高脱色率。分别考察了介体、酶活和温度对苯胺蓝脱色的影响,结果表明,在酶活为200 U/L、1-羟基苯并三唑(HOBT)浓度为4mmol/L、温度为40℃的条件下,反应6 h后苯胺蓝(24 mg/L)的脱色率达到90.42%。对脱色0、4 h、11 h和23 h的产物进行了超高效液相色谱-质谱(UPLC-MS)分析,推断出漆酶/HOBT体系对苯胺蓝的脱色过程中有显色基团碳氮双键(C=N)的断裂。

The paper is intended to disclose the mechanism of the aniline blue decolorization under the impact of the laccase/hobt mediator system and the necessary technical conditions for the decolorization.We have first of all conducted the visible light wavelength scanning and native polyacrylamide gel electrophoresis（Native-PAGE）analysis.Then,we have managed to make the technical condition including mediator（1-hydroxybenzotriazole,HOBT） concentration,laccase activity and the temperature optimized through a single-factorat-a-lime experiments.Based on our experimental results,we have proposed the hypothetical mechanism of the decolorization on the basis of our repetitive analyses of the decolorizing products.The results of the visible light wavelength scanning and Native-PAGE indicate that it is possible to decolorize the aniline blue by using the laccase derived from Pleurotus sp.N3 and then to enhance the effects of the decolorization by using the mediator（HOBT） remarkably,that is to say,the decolorization effects of the aniline blue can be enhanced with the increase of HOBT concentration in a range from 0 to 4 mmol/L.Our research also proves that the decolorization effects can also be enhanced with the intensification of laccase activities,though no considerable difference can be found with the decolorization effects when the activity changes only from 200 U/L to 250 U/L.Our experiments also demonstrate that,when temperatures were kept between 30℃ and50℃,the decolorization effects can be found increased with the increase of temperature.Moreover,no considerable difference can be found with the effect of the decolorization in a temperature range from40℃ to 50℃.The optimized condition we have found is made of the temperature of 40℃ for the laccase activity of 200 U/L,and HOBT concentration of 4 mmol/L.Under such a technical condition,the decolorization rate for aniline blue（24 rng/L） is expected to increase by 90.42%in the incubation of 6 h.The above said results suggest that it is optimistic to use the laccase/HOBT system to decolorize the Aniline Blue effectively.Furthermore,we have also analyzed it in decolorizing the products in the decolorization process by way of the ultra-high performance liquid chromatography-mass spectrometry（UPLC-MS）.As a result,we have identified three compounds for their molecular weight,which are 566,545 and 522,respectively,suggesting that the decolorization behavior is by nature a function of the carbon-nitrogen double bond（—C=N—） rupture in the molecules of the aniline blue.