Improvement of the enzymatic properties of kojibiose phosphorylase from Thermoanaerobacter brockii by random mutagenesis and chimerization

Random mutagenesis by error-prone PCR was introduced to kojibiose phosphorylase (KP; EC 2.4.1.230) from Thermoanaerobacter brockii ATCC35047. One thermostable mutant and two DP-mutants that were defined as the mutant producing kojioligosaccharides with a degree of polymerization (DP) different from that of wild-type KP were isolated. The half-lives of a thermostable mutant, D513N were estimated to be 135 h at 60 deg C, 110 min at 70 deg C and 6 min at 75 deg C, respectively. They were about 1.6-fold, 7-fold and 6-fold longer than those of the wild-type enzyme, respectively. Two DP-mutants, S676N and N687I showed higher productivity of kojioligosaccharides of DP=4 than the wild-type KP under the same condition of reaction. In the case of S676N, the amount of kojipentaose increased more than 3 fold that for the wild type. Furthermore, kojioligosaccharides with a DP of ten or greater was detected by using S676N, but not by the wild type. Chimeric phosphorylases were constructed from the KP gene and trehalose phosphorylase (TP; EC 2.4.1.64) gene from T. brockii. One unique enzyme, chimera V-III was isolated by chimerization from KP and TP. Although only 125 amino acid residues in 785 residues of chimera V-III were from that of wild-type KP, chimera V-III showed not TP-type but KP-type enzyme. Since both KP and TP catalyze transglucosylation using beta-G1P as the glucosyl donor, it is strongly suggested that 125 amino acid residues exchanged from KP play an important role in the binding of glucosyl acceptors. The Km value of chimera V-III for kojibiose was remarkable increased, and this chimera accepted little or no monosaccharide other than glucose; furthermore, the enzyme was able to act on laminaribiose in the absence of inorganic phosphate, and produced trisaccharides, 6-O-beta-D-glucosyl-laminaribiose and laminaritriose from laminaribiose. From these results, it is assumed that chimerization causes a conformational change of the catalytic site where the reducing-end of molecules of substrates would bind, and the marked decrease of the substrate-binding activity in this region.