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 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)-beta-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 Gln1, 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 Tyr13 in the hybrid enzyme containing the 12 N-terminal amino acids from the B. amyloliquefaciens (1-3,1-4)-beta-glucanase results in a dramatic increase in stability at 70 degrees 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.
Politz
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O
Institute of Genetics, Microbiology and Biochemistry, Humboldt University, Berlin, Germany.
Simon
O
O
Olsen
O
O
Borriss
R
R
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology; D001483:Base Sequence; D004277:DNA, Single-Stranded; D004795:Enzyme Stability; D004926:Escherichia coli; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D004277:DNA, Single-Stranded; D011993:Recombinant Fusion Proteins; D006026:Glycoside Hydrolases; C028020:licheninase