A novel glycosyl hydrolase family 11 xylanase gene, xynMF13A, was cloned from Phoma sp. MF13, a xylanase-producing fungus isolated from mangrove sediment. xynMF13A was heterologously expressed in Pichia pastoris, and the recombinant XynMF13A (rXynMF13A) was purified by Ni-affinity chromatography. The temperature and pH optima of purified rXynMF13A were 45 °C and pH 5.0, respectively. rXynMF13A showed a high level of salt tolerance, with maximal enzyme activity being seen at 0.5 M NaCl and as much as 53% of maximal activity at 4 M NaCl. The major rXynMF13A hydrolysis products from corncob xylan were xylobiose, xylotriose, xylotetraose, and xylopentaose, but no xylose was found. These hydrolysis products suggest an important potential for XynMF13A in the production of xylooligosaccharides (XOs). Furthermore, rXynMF13A had beneficial effects on Chinese steamed bread production, by increasing specific volume and elasticity while decreasing hardness and chewiness. These results demonstrate XynMF13A to be a novel xylanase with potentially significant applications in baking, XOs production, and seafood processing.

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Proteinase K (PROK) from Parengyodontium album hydrolyzes keratin, a major protein component of poultry feathers, which are an inexpensive and renewable protein resource. Based on structural studies for analysis of amino acid flexibility near the catalytic center, identification of highly conserved residues, and experimental screening, we obtained a mutant R218S with residual activity 1.6-fold higher than that of PROK after incubation at 60 °C for 1 h. Molecular dynamics simulation indicated that substitution of Arg218 with Ser leads to three hydrogen bonds being introduced into the structure, stabilizing the β-sheet in which Ser218 is located, and thus improvement of thermostability. Additionally, the mutant R218S had a 15% increase in specific activity compared to PROK and improvement in the rate and thoroughness of feather degradation compared with PROK. We confirmed the positive effects of enhancing catalytic center rigidity on enzyme thermostability, a finding which may have broad applications.