DETERMINANTS FOR THE ENHANCED THERMOSTABILITY OF HYBRID (1-3,1-4)-BETA-GLUCANASES

Hybrid (1-3,1-4)-beta-glucanases which contain an N-terminal region de rived from the Bacillus amyloliquefaciens enzyme and a C-terminal regi on of the closely related B. macerans enzyme may exhibit a thermostabi lity 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 fo ur of the N-terminal residues for the mature B. amyloliquefaciens (1-3 ,1-4)-beta-glucanase exhibit no significant changes in biochemical cha racteristics as compared with the parental B. macerans enzyme. However , significantly enhanced thermostability was observed in the hybrid en zyme containing an N-terminal segment of eight amino acid residues der ived from the B. amyloliquefaciens enzyme. Site-directed mutagenesis r evealed that the combined effect of Gln1, Thr2, Ser5 and Phe7 confer e nhanced stability on hybrid enzymes, probably by improving the hydroge n 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 Tyr13 in the hybrid enzyme containing the 12 N-terminal amino acids from the B. amyloliqu efaciens (1-3,1-4)-beta-glucanase results in a dramatic increase in st ability at 70-degrees-C with the half-life of 6 min increased to aroun d 4 h. This is twofold higher than the hitherto most stable hybrid enz yme in which the N-terminal domain consisted of 16 residues of the B. amyloliquefaciens enzyme.