Gene Cloning, Expression And Characterization Of The Thermostable Endoglucanases From Thermotoga Maritima MSB8

Biofuels are a new priority in efforts to reduce dependence on fossil fuels, and bioethanol is one of the most significative clean renewable energy using in industrial production. Biomass is the most abundant renewable resource on earth, and lignocellulosic biomass has long been recognized as a potential sustainable source of mixed sugars for fermentation to biofuels and other biomaterials. Cellulase is a kind of glycoside hydrolase which can degrade cellulose, and it plays an important role in the process of biomass conversion. Thermotoga maritima, is originally isolated from a geothermal environment,and has an optimum growth temperature of 80?, is a heterotrophic organism which encodes the largest number of glycoside hydrolases compared to any archaeal or bacterial genome sequence. In this study, the gene engineering technique was used to construct the expression system of Escherichia coli for thermophilic endoglucananses manufacture, and to explore the enzymatic activity of these thermophilic enzymes. The main contents of this study are as follows:(1) Construction of recombinant overexpression plasmids for thermophilic endoglucanases produce in E. coli. pHsh, a novel heat shock expression vector of E. coli,as template to get the plasmid fragment by inverse PCR and a small peptide was fused to N terminal for improve the level of expression. PCR amplified eight segments of thermophilic endoglucanase coding mature peptide sequence with the T. maritima genome DNA as template, join these genes and pHsh expression vector fragment to generate recombinant plasmid pHsh-EGs. Heterogeneous expression of recombinant thermophilic endoglucanase in E. coli and SDS-PAGE analysis of the recombinant enzyme showed that the eight enzymes were expressed clearly.(2) Purification and characterization of eight recombination endoglucanases. Two steps, heat treatment and nickel-affinity chromatography, the Tma-EGs were purified to homogeneity. Tma-EG12 A, Tma-EG12 B, Tma-EG5 A and Tma-EG74 have showed a strong ability to hydrolyze cellulose, and Tma-EG5 B, Tma-EGA, Tma-EGB and Tma-EGC only have very low activity using CMC(1.0 %) as substrate. Tma-EG12 A, Tma-EG12 B,Tma-EG5 A and Tma-EG74 showed the highest activity at pH 5.6, pH 5.6, pH 5.2, pH 5.2and 95?, 90?, 85?, 85?, and specific activity were 55.5, 213.5, 31, 4.5 U/mg,respectively. The all four enzymes showed high thermal stability and pH stability.(3) Discovery and characterization of a novel ?-mannase. Tma-EG5 B is labeled as a endoglucanase in T. maritima genome, but it is also defined as a lichenase of a research abroad. In this study, Tma-EG5 B has much higher ?-mannase activity(112 U/mg)than CMCase(0.96 U/mg) and lichenase(0.88 U/mg), and homology analysis of the Tma-EG5 B amino acid sequence reveal that it no mannase homology, suggests that the Tma-EG5 B is a new type of mannase. Using LBG as substrate, Tma-EG5 B showed the highest activity at pH 5.6 and 80?, and at the range of pH 5.0-8.0 enzyme activity could maintain at above 70%. The thermal stability of Tma-EG5 B showed that activity was maintained at 70% after incubation at 70? for 2 h. Co2+ is founded to be critical to increase the recombinant enzyme activity, but Cu2+ was significantly inhibited.