| Termite is a kind of insect which can efficiently degrate lignocellulose in nature. It plays a very important role in carbon recycling and energy flowing. There are a plenty of diverse symbiotic microorganisms in termite gut, such as bacteria, archaea and eukaryotic protozoa. Many microorganisms have the ability to produce cellulases which might make contributions to lignocellulose degradation. Meanwhile, in order to adapt to lignocellulose degradation, termites developed a very efficient and complicated cellulase system in the long-term evolution. In this study, we cloned and expressed two ?-glucosidase genes (Tmbg1 and Rcbg3) derived from the intestinal bacteria of Reticulitermes chinensis Snyder, and characterized the enzyme properties, which lays the foundation for further elucidating the importance of symbiotic gut microorganisms in cellulose digestion of termite hosts. The main results of this study are as follows:1. A ?-glucosidase gene Tmbgl was obtained from symbiotic bacteria Opitutacrae sp.TSB-47 in the gut of Reticulitermes chinensis Snyder. The genes were 2070 bp length, encoding 690 amino acids. Tmbgl was heterogeneously expressed in E. coli BL21 and the soluble recombinant protein TmBG1 was used for enzyme characterization. The optimal pH for the activity of the purified TmBG1 was 6.0 and the optimum reaction temperature was 45?. TmBG1 had a wide pH reaction range and good pH stability between 5.2 and 9.2. The specific activity of TmBG1 was 0.348 U/mg. The Km and Vmax values were 8.47 mg/ml and 0.145 U/mg, respectively, with pNPG as substrate.1 mM and 5 mM various metal ions had different influence on the activity of TmBG1. Zn2+ and Ag+ displayed strong inhibition to the enzymatic activity of TmBG1. Unexpectedly, Ca2+, Ni2+, Al3+, K+, Fe2+ and Co2+ could neither stimulate the enzymatic activity of TmBG1 nor inhibit its activity. In the other hand, at the concentration of 5 mM, Mg2+ and Mn2+ increased its activity to more than 45% and 65% of the highest activity, respectively. pNPG and pNPC can be hydrolyzed by TmBG1. The hydrolysis pattern analysis by TLC indicated that the main degradation products of cellobiose (G2), cellotriose (G3) and cellotetraose (G4) by TmBG1 were glucose (G1), cellobiose (G2) and cellotriose (G3). As a hydrolysis product of ?-glucosidase, glucose has a primary influence on the activity of ?-glucosidase. The results showed that the activity of TmBG1 was gradually decreased with the increasing of glucose concentration. When glucose concentration reached up to 1.0 M,60% of the original activity was retained, which indicated a high tolerance on glucose.2. A P-glucosidase gene Rcbg3 was derived from the intestinal free-living bacteria of Reticulitermes chinensis Snyder. The genes were 1995 bp length, encoding 664 amino acids. Rcbg3 was heterogeneously expressed in E. coli BL21 and the soluble recombinant protein RcBG3 was used for enzyme characterization. The optimal pH for the activity of the purified RcBG3 was 5.6 and the optimum reaction temperature was 45?. The specific activity of RcBG3 was 0.601 U/mg. The Km and Vmax values were 0.193 mg/ml and 0.345 U/mg, respectively, with pNPG as substrate. The influence of metal ions on the activity of RcBG3 was studied at 1 mM and 5 mM. Zn2+ and Al3+ displayed strong inhibition to the enzymatic activity of RcBG3. Otherwise, Cu2+ and Ag+ almost completely inhibited the enzymatic activity of RcBG3. Unexpectedly, Ca2+, Mg2+, Al3+, K+, Fe2+ and Co2+ could neither stimulate the enzymatic activity of RcBG3 nor inhibite its activity. In the other hand, Mn2+ increased its activity to more than 60% of the highest activity, respectively at the concentrations of 1 mM and 5 mM. When glucose concentration reached up to 1.0 M,40% of the original enzyme activity was retained. The Ki value of recombinant RcBG3 was 0.46 M, which indicated a high tolerance on glucose. |
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