Molecular engineering of a cold-active xylanase TsTA1 from a straw-decomposing fungus Talaromyces stipitatus.

Straw return is widely used to enhance soil organic matter in black soil regions; however, low temperatures severely constrain the microbial degradation of straw. In this study, a wild fungal strain Talaromyces stipitatus with robust hydrolytic activity under cold conditions was isolated from black soil. A xylanase, TsTA1, secreted by this fungus, was shown to be active at low temperatures. Through molecular engineering, a series of TsTA1 mutants were generated, and three variants, M142Y, I161V, and F215Y, were demonstrated have substantially (1.5-2-fold) increased catalytic efficiency compared to the wild-type enzyme. Structural analyses indicated that the M142Y and F215Y substitutions enhance activity by introducing additional hydrogen bonding with the substrate. Interestingly, the I161V mutation markedly altered the pH range in which the enzyme is active, conferring alkaline pH tolerance. These findings enhance the understanding of straw decomposition by cold-adapted microbes and demonstrate a successful strategy for improving enzyme performance under challenging environmental conditions.