Determinants for the enhanced thermostability of hybrid (1‐3,1‐4)‐β‐glucanases

Hybrid (1 -33 -4)-P-glucanases which contain an N-terminal region derived from the Bacillus amyloliquefaciens enzyme and a C-terminal region of the closely related B. macerans enzyme may exhibit a thermostability superior to both parental enzymes. A systematic series of hybrid enzymes were constructed in order to delineate the amino acid residues that affect protein stability. Hybrid enzymes with between one and four of the N-terminal residues for the mature B. amyloliquefaciens (1 -3,1-4)-P-glucanase exhibit no significant changes in biochemical characteristics as compared with the parental B. macerans enzyme. However, significantly enhanced thermostability was observed in the hybrid enzyme containing an N-terminal segment of eight amino acid residues derived from the B. amyloliquefaciens enzyme. Site-directed mutagenesis revealed that the combined effect of Glnl , Thr2, Ser5 and Phe7 confer enhanced stability on hybrid enzymes, probably by improving the hydrogen bonding that stabilizes the interactions between the N-terminal and the centre of the folded molecule, as well as between the two termini of the polypeptide chain. Furthermore, deletion of Tyrl3 in the hybrid enzyme containing the 12 N-terminal amino acids from the B. amyloliquefaciens (1-3,1-4)-P-glucanase results in a dramatic increase in stability at 70°C with the half-life of 6 min increased to around 4 h. This is twofold higher than the hitherto most stable hybrid enzyme in which the N-terminal domain consisted of 16 residues of the B. amyloliquefaciens enzyme.