Construction and Characterization of Novel Chimeric β -Glucosidases with Cellvibrio gilvus (CG) and Thermotoga maritima (TM) by Overlapping PCR

It is known that β-glucosidase is very important for obtaining clean energy from cellulolytic materials such as plants and wood. For most bioconversion processes with cellulose, endo(14)-β-D-glucan glucanhydrolases and exo(1-4)-β-D-glucan cellobiohydrolases are necessary for catalyzing the random hydrolysis of cellulose to produce cellobiose. On the basis of substrate specificity, β-glucosidases can be classified into aryl-β-glucosidases, cellobioses hydrolysing only oligosaccharides, and those hydrolysing both aryl-β-glucosides and oligosaccharides [1, 2]. β-glucosidases are important components of the cellulase enzyme complex required for the hydrolysis of cellulose into glucose by catalysing the final step, which is the conversion of cellobiose into glucose [3]. From the basis of sequence homology β-glucosidases have been divided into two sub-families, namely BGA (β-gucosidases and phospho-β-glucosidases from bacteria to mammals) and BGB (β-glucosidases from yeast, mold and rumen bacteria) [4]. The study of these enzymes has been facilitated by the use of recombinant DNA technology [5,6,7,8]. A number of cellulase genes including several forms of β-glucosidases, have been cloned and expressed in both E.coli and S.cereviciae [9,10]. In recent years, protein engineering has become an increasingly important tool in the development of novel hybrid enzymes with useful catalytic functions [11,12,13]. The catalytic activities and thermal stabilities of enzymes can be improved by the construction of chimeric enzymes with gene-shuffling from different species of genes [14]. For this purpose, chimeric genes are normally constructed by the application of one of two methods: using restriction enzymes or by overlapping polymerase chain reaction (PCR) [15]. Although the use of restriction enzymes is the easier method, a high level of identity is required between the parental DNA sequences to obtain common restriction enzyme sites in both genes. Often, the availability of common restriction enzyme sites limits the use of this strategy in the construction of chimeric genes. In contrast, there are no such limitations in defining shuffling sites for chimeric genes constructed via overlapping PCR technique.