摘要
表征酶与底物有效吸附是认识纤维素酶促水解反应过程、揭示预处理作用机制的重要手段之一.成功构建了纤维素外切酶(Cel7A)、纤维素内切酶(Cel5A)和葡萄糖苷酶(BglⅠ)荧光探针,优化吸附条件,并考察其对细胞壁的吸附.结果表明,Cel7A、Cel5A、BglⅠ基因N端分别连接至荧光蛋白基因mCherry、eGFP、eBFP C端,依次连入pPICZα载体,成功在毕赤酵母(X33)菌株表达;mCherry-Cel7A、eGFP-Cel5A和eBFP-BglⅠ荧光探针的最佳激发波长分别为573、469 nm和385 nm,最佳发射波长分别为613、511 nm和447 nm;以滤纸为底物,测定最佳吸附浓度分别为0.058、0.19 U/mL和1.93 U/mL,最佳吸附时间分别为 7、7 min和6 min,最佳观察时间分别为<2 min、<12 s和<12 s.纤维素酶荧光探针能够结合生物质样品,并反应出不同底物与纤维素酶亲和力的差异.上述工作为生物质抗降解屏障、生物质预处理、纤维素高效酶促水解等研究提供了技术支持.
Abstract
Characterization of the valid adsorption between enzyme and substrate is one of the most important strategies to understand the process of cellulase catalyzing hydrolysis reaction and reveal the mechanism of pretreatment.In this research,cellulose exonuclease(CeI7A),cellulose endonuclease(CeI5A)and glucosidase(BgIⅠ)fluorescent probes are successfully constructed.The adsorption conditions are optimized and their adsorption on cell wall are also observed.Results show that N-terminal of Cel7A,Cel5A and BglⅠ genes are connected to C-terminal of mCherry,eGFP and eBFP fluorescent protein genes,respectively,and successfully expressed in Pichia pastoris(X33).The optimal excitation wavelengths of mCherry-Cel7A,eGFP-Cel5A and eBFP-BglⅠfluorescent probes are 573 nm,469 nm and 385 nm,respectively and their optimal emission wavelengths are 613 nm,511 nm and 447 nm,respectively.Using filter paper as substrate,the optimal adsorption concentrations are 0.058 U·mL-1,0.19 U·mL-1 and 1.93 U·mL-1,respectively for these probes,the optimal adsorption time is 7 min,7 min and 6 min,and the optimal observation time is<2 min,<12 s and<12 s,respectively.Fluorescent probes can bind to biomass samples,reflecting the adsorption affinity between cellulase and substrates.This work provides technical support for biomass anti-degradation barrier,biomass pretreatment and cellulase catalyzing hydrolysis reaction.
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