Xuewu Liu,Xingfei Li,Zhengjun Xie,Xing Zhou,Long Chen,Chao Qiu,Cheng Lu,Zhengyu Jin,Jie Long
Agarose from biomass can be used to synthesize the rare sugar 3,6-anhydro- L -galactose ( L -AHG), and the new synthesis route and functional properties of L -AHG have always been the focus of research. Here we developed a novel method to co-immobilize Aga50D and Bp GH117 onto streptavidin-coated magnetic nanoparticles and achieved the conversion of agarose to bioactive L -AHG in one pot. Results showed that enzymes were successfully immobilized on the carrier. The activity of co-immobilized enzymes was 2.5-fold higher than that of single immobilized enzymes. Compared with free enzymes, co-immobilized enzymes exhibited enhanced thermal stability. The co-immobilized enzymes retained 79.45 % relative activity at 40 °C for 3 h, while the free enzymes only possessed 21.40 % residual activity. After eight cycles, the co-immobilized enzymes still retained 73.47 % of the initial activity. After silica gel chromatography, the purity of L -AHG obtained by co-immobilized enzymes hydrolysis reached 83.02 %. Furthermore, bioactivity experiments demonstrated that L -AHG displayed better antioxidant and antibacterial effects than neoagarobiose. L -AHG had broad-spectrum antibacterial activity, while neoagarobiose and D -galactose did not show an obvious antibacterial effect. This study provides a feasible method for the production of L -AHG by a co-immobilized multi-enzyme system and confirms that L -AHG plays a key role in the bioactivity of neoagarobiose.
Reversed micelles are helpful to solubilize otherwise insoluble membranotropic or membrane-bound enzymes in their functional form, thus enabling activity assay and inhibition analysis. However, in the case of redox enzymes, this task is further complicated by the necessity to select an appropriate electron-acceptor (EA) which, ideally, should be compatible with spectrophotometric measurements in reversed micelles. Here, we have identified such an EA and successfully used it in a reversed micellar environment to assay the activity of two homologous enzymes from mitochondria:l-galactone-1,4-lactone dehydrogenase (EC 1.3.2.3) fromArabidopsis thaliana(AtGALDH) and galactonolactone oxidase (EC 1.3.3.12) fromTrypanosoma cruzi(TcGAL), differing in their membranotropic properties, with TcGAL being almost insoluble in water and particularly difficult to assay. Furthermore, we have demonstrated the possibility to use this assay for inhibition analysis, with an elucidation of the mechanism and inhibition parameters, which otherwise could not be possible. In order to perform inhibition analysis, we improved the approach for the determination of activity of such membrane enzymes based on a reversed micellar system as membrane matrix, necessary for the functioning of membrane enzymes. A number of electron acceptors (EA) were tested for AtGALDH and optimal conditions of activity determination for AtGALDH were found. The suggested method was successfully applied to the study of the inhibition of AtGALDH by lycorine, and the mixed competitive mechanism of inhibition of AtGALDH by lycorine was determined. The developed approach to inhibitor analysis was applied for TcGAL, insoluble in water membrane, and the method provides new opportunities for searching effective inhibitors that may be potential drugs. Indeed, galactonolactone oxidase fromTrypanosoma cruzi(TcGAL) and AtGALDH are homologues, and the inhibition of TcGAL stops the vital biosynthesis of vitamin C in parasiteTrypanosoma cruzifrom causing Chagas disease. The approach proposed can be applied for the screening of inhibitors of AtGALDH and TcGAL, as well as to study properties of other membrane enzymes including determination of the mechanisms of inhibition, structure and catalytic properties, the impact of membrane components (for example lipids), and so on.Keywords:determination of membrane enzyme activity;spectrophotometric method;membrane enzymes;l-galactone-1,4-lactone dehydrogenase;Arabidopsis thaliana;galactonolactone oxidase;Trypanosoma cruzi;lycorine;drugs;Chagas disease
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