Comparison of primary, secondary and tertiary structure of xylanase of Bacillus pumilus and cellulase of Aspergillus acleatus.

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The tertiary structure of F1-CM-cellulase of Aspergillus acleatus was found to be very similar to that of xylanase of Bacillus pumilus by X-ray crystallographic analysis, though no significant homology was observed in the amino acid sequence. It was considered that such a topology of both enzymes might be widely distributed among hydrolases of small molecular mass. We obtained many mutant xylanases by random mutagenesis to investigate the relationship between the function of xylanase and the substitution in the amino acid sequence.Four heat-resistant mutants of xylanase (N56, N102, N104 and F1) were obtained. The mutant xylanases had the following amino acid changes : N56, S26 to W, G38 to D, and Y126 to S ; N102, G38 to D ; N104, G38 to S and R48 to K ; F1, S12 to C.Kinetic studies showed that N104 is stabilized by an increase in the activation enthalpy, while the other mutants are stabilized by a decrease in the activation entropy.p-Nitrophenyl-beta-D-xylopyranobioside (pNPX2) was found to be a good model substrate of the xylanase to investigate the interaction between enzyme molecule and substrate. One mutant of xylanase having higher activity to pNPX2 was obtained. The amino acid change was S76 to G in the mutant. Kinetic studies showed that the higher activity was caused by an increase of Vmax value, not by a decrease of Km value.