A b-glucosidase cDNA from the termite, Neotermes koshunensis, was successfully overexpressed in Escherichia coli, and the product was purified to homogeneity by affinity purification against His-tags. The molecular weight of the recombinant enzyme was 60 kDa. The expressed b-glucosidase preferentially hydrolyzed laminaribiose and cellobiose rather than synthetic substrates such as p-nitrophenolic compounds. The K m value of cellobiose was 3.8 mM and V max was 220 U (mmol of glucose/min)/mg. The optimum pH and thermostability were 5.0 and 45°C, respectively. These enzymatic characters are mostly consistent with the partially-purified b-glucosidase from the salivary glands of N. koshunensis. However, the specific activity of the recombinant enzyme was 156.7 U/mg, which is almost 3-folds of that of the partially purified b-glucosidase of N. koshunensis. Owing to the successful expression of the termite b-glucosidase in E. coli, it may provide an opportunity of termite b-glucosidase for further improvement of the enzymatic properties for potential industrial applications with the aid of bioengineering.

Among cellulase genes, those of animals are known for their difficulty in overexpression. We constructed a chimeric library by family shuffling of endo-beta-1,4-glucanase genes from four different termite species (Reticulitermes speratus, Nasutitermes takasagoensis, Coptotermes formosanus, and Coptotermes acinaciformis) sharing 78.5-96% homology in amino acid sequence. The constructed library was screened by Congo red plate assay combined with 96-well micro-enzyme assay, and clones showing enhanced CMCase activities were obtained. The mutated genes were overexpressed in Escherichia coli intracellularly as an active form. The endo-beta-1,4-glucanase (CMCase) activity in soluble fractions of E. coli harboring the mutant genes was 20-30 fold higher than that of wild-type genes. The mutant enzyme showed high activity against CMC and properties similar to those of the native enzymes.

There have been two major problems preventing applications of termite cellulases; one was difficulty for their hetelologous overexpression, and another is their low thermostability. We previously achieved adaptation of termite cellulase genes to an overexpression system of Escherichia coli by family shuffling of four orthologous cDNAs (Biosci. Biotechnol. Biochem., 2005; 69: 1711-1720). Using the adapted mutant cDNAs as parental genes combined with native-form cDNAs, we performed further family shuffling and obtained mutant cDNAs, which gave enzymes with improved thermostability. The best-evolved clone (PA68) was improved by 10 degrees C in maximum stability (retaining 90% original activity for 30 min incubation) from the parental enzymes, and kept 54% of its original activity for 150 min at 50 degrees C, whereas the most thermostable enzyme amongst the parents (A18) retained 30% of its original activity. PA68 showed 889 (micromoles of reducing sugars/min/mg of protein) in V(max) and 560 (micromoles of reducing sugars/min/mg of protein) in the specific activity against carboxymethylcellulose, which corresponds to 9.8 and 13.1 times of those of one of the ancestral enzymes rRsEG. In summary, we improved thermostability of the termite cellulase and increased the V(max) value and specific activity by combining only cDNAs encoding enzymes adapted for normal temperatures.

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