Preparation Of Cellulase Fluorescent Probe And Its Preliminary Application

Precise characterization of the effective adsorption of cellulase and substrates is an important prerequisite for optimizing pretreatment and exploring the mechanism of enzymatic hydrolysis of cellulose.In this paper,cellulase fluorescent probes were prepared by heterologous expression in Pichia pastoris;the adsorption conditions of cellulase fluorescent probes were clarified and the adsorption characteristics of different cell walls and cellulase were investigated.The main results were as follows:(1)To obtain recombinant strains,three recombinant plasmids were constructed and heterologous expression in Pichia pastoris.During fermentation,the enzyme activity of Cel7A-M(MW: 80 k D)was 0.21 U/m L,the enzyme activity of Cel5A-G(MW: 70 k D)was 0.41 U/m L,and the activity of BglⅠ-B(MW: 106 k D)was 0.21U/m L.The constructed cellulase fluorescent probe can be successfully adsorbed to the cellulose substrate.(2)The optimal excitation wavelengths of Cel7A-M,Cel5A-G and BglⅠ-B were573 nm,469 nm,and 385 nm,respectively,and the optimal fluorescence emission wavelengths were 613 nm,511 nm,and 447 nm,respectively.(3)When the filter paper was used as the substrate,the optimal adsorption concentration,adsorption time and observation time of Cel7A-M were 0.058 U/m L,7min and 2 min,respectively.The optimal adsorption concentration,adsorption time and observation time of Cel5A-G were 0.193 U/m L,7 min and 12 s,respectively.The optimum adsorption concentration,adsorption time and observation time of BglⅠ-B were 0.193 U/m L,6 min and 24 s,respectively.(4)Different tissues have different adsorption capacity to cellulase fluorescent probe,sclerenchyma(ST)> vascular tissue(VT)> parenchyma(PT);cellulase fluorescent probe can clearly reflect the pretreatment effect.