Improving the thermostability of a mesophilic family 10 xylanase, AuXyn10A, from Aspergillus usamii by in silico design.

Abstract

To improve the thermostability of a mesophilic GH family 10 xylanase, AuXyn10A, from Aspergillus usamii E001, its modification was performed by in silico design. Based on the comparison of B-factor values, a mutant xylanase ATXyn10 was predicted by substituting a segment YP from Tyr(25) to Pro(34) of AuXyn10A with the corresponding one from Asn(24) to Ala(32) of TaXyn10, a thermophilic GH family 10 xylanase from Thermoascus aurantiacus. Analysis of a TaXyn10 crystal structure indicated that there is a close interaction between segments YP and FP. For that reason, another mutant xylanase ATXyn10(M) was designed by mutating Ser(286) and His(288) of ATXyn10 into the corresponding Gly(285) and Phe(287) in the FP of TaXyn10. Then, two ATXyn10- and ATXyn10(M)-encoding genes, ATxyn10 and ATxyn10 (M), were expressed in Pichia pas toris GS115. The temperature optimum of recombinant (re) ATXyn10(M) was 60 °C, 10 °C higher than that of reAuXyn10A. Its thermal inactivation half-life (t(1/2)) at 55 °C was 10.4-fold longer than that of reAuXyn10A. As compared with reAuXyn10A, reATXyn10(M) displayed a slight decrease in K(m) value and a significant increase in V(max) value from 6,267 to 8,870 U/mg.