采用(NH4)2SO4分步沉淀法对诺维信中国公司生产的漆酶制剂DeniLite IIS进行了纯化,并用丁二酸酐(SA)对纯化酶进行了化学修饰,运用三硝基苯磺酸法、紫外光谱法及荧光光谱法对修饰效果进行了初步表征,比较了天然酶和修饰酶的pH稳定性、热稳定性及除酚效率.结果表明,修饰酶的平均氨基修饰度为85％,其紫外吸收峰和荧光发射峰均出现蓝移,而且紫外吸收减小、荧光强度增加.尽管采用SA化学修饰未能改变漆酶的最适反应温度,但使其最适反应pH值由4.5提高到5.5,并且使酶活提高60％.与天然酶相比,修饰酶的pH稳定性和热稳定性更高,催化效率(kcat)和酶与底物的亲和力(kcat/Km)分别提高了53％和122％,对邻、间和对苯二酚的除酚效率分别提高了48％,57％和18％.这预示着这些修饰漆酶可望应用于工业生产和酚类污染废水的治理.
Chemical modification is a useful method to change the properties of enzymes.Laccase is a phenol oxidase belonging to a multicopper protein,which catalyzes the oxidation of many phenolics.DeniLite IIS,a commercial laccase preparation from the Novozymes China Company,was purified by ammonium sulfate fractional precipitation.Succinic anhydride (SA) was used as a modifier for the chemical modification of the purified laccase.The effects of modification were characterized using the 6-trinitrobenzene sulfonic acid method,ultraviolet spectroscopy,and fluorescence spectroscopy.The pH stability,thermal stability,and the phenolics removal efficiency for the native and modified laccases were compared.The results showed that the average amino modification yield of the modified laccase was 85％ and the modified laccase had a blue shifted ultraviolet peak and fluorescence emission peak as well as a decrease in the ultraviolet absorbance and an increase in the fluorescence intensity.Although chemical modification with SA did not change the optimum temperature for the catalysis of the laccase,it caused the optimum pH of the catalysts to shift fiom 4.5 to 5.5 and the enzymatic activity increased by 60％.Compared with the native laccase,the modified laccase exhibited remarkably higher pH stability and thermal stability and its catalysis efficiency (kcat) and substrate affinity (kcat/gm) increased by 53％ and 122％,respectively.The phenolics removal efficiency (o-,m-,p-dihydroxybenzene) of the modified laccase increased by 48％,57％,and 18％,respectively.These results indicate that the modified laccase with higher stability and higher efficiency is suitable for application in industrial production and for the treatment of phenolics-polluted water.
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