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.

Aspartic proteases exhibit optimum enzyme activity under acidic condition and have been extensively used in food, fermentation and leather industries. In this study, a novel aspartic protease precursor (proTlAPA1) from Talaromyces leycettanus was identified and successfully expressed in Pichia pastoris. Subsequently, the auto-activation processing of the zymogen proTlAPA1 was studied by SDS-PAGE and N-terminal sequencing, under different processing conditions. TlAPA1 shared the highest identity of 70.3 % with the aspartic endopeptidase from Byssochlamys spectabilis (GAD91729) and was classified into a new subgroup of the aspartic protease A1 family, based on evolutionary analysis. Mature TlAPA1 protein displayed an optimal activity at 60 °C and remained stable at temperatures of 55 °C and below, indicating the thermostable nature of TlAPA1 aspartic protease. During the auto-activation processing of proTlAPA1, a 45 kDa intermediate was identified that divided the processing mechanism into two steps: formation of intermediates, and activation of the mature protein (TlAPA1). The former step was completely induced by pH of the buffer, while the latter process depended on protease activity. The discovery of the novel aspartic protease TlAPA1 and study of its activation process will contribute to a better understanding of the mechanism of aspartic proteases auto-activation. IMPORTANCEThe novel aspartic protease TlAPA1 was identified from T. leycettanus and expressed as a zymogen (proTlAPA1) in P. pastoris. Enzymatic characteristics of the

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Platform chemicals and polymer precursors can be produced via enzymatic pathways starting from lignocellulosic waste materials. The hemicellulose fraction of lignocellulose contains aldopentose sugars, such as d-xylose and l-arabinose, which can be enzymatically converted into various biobased products by microbial non-phosphorylated oxidative pathways. The Weimberg and Dahms pathways convert pentose sugars into α-ketoglutarate, or pyruvate and glycolaldehyde, respectively, which then serve as precursors for further conversion into a wide range of industrial products. In this review, we summarize the known three-dimensional structures of the enzymes involved in oxidative non-phosphorylative pathways of pentose catabolism. Key structural features and reaction mechanisms of a diverse set of enzymes responsible for the catalytic steps in the reactions are analysed and discussed.

ABSTRACTAspartic proteases exhibit optimum enzyme activity under acidic condition and have been extensively used in food, fermentation and leather industries. In this study, a novel aspartic protease precursor (proTlAPA1) from Talaromyces leycettanus was identified and successfully expressed in Pichia pastoris. Subsequently, the auto-activation processing of the zymogen proTlAPA1 was studied by SDS-PAGE and N-terminal sequencing, under different processing conditions. TlAPA1 shared the highest identity of 70.3 % with the aspartic endopeptidase from Byssochlamys spectabilis (GAD91729) and was classified into a new subgroup of the aspartic protease A1 family, based on evolutionary analysis. Mature TlAPA1 protein displayed an optimal activity at 60 °C and remained stable at temperatures of 55 °C and below, indicating the thermostable nature of TlAPA1 aspartic protease. During the auto-activation processing of proTlAPA1, a 45 kDa intermediate was identified that divided the processing mechanism into two steps: formation of intermediates, and activation of the mature protein (TlAPA1). The former step was completely induced by pH of the buffer, while the latter process depended on protease activity. The discovery of the novel aspartic protease TlAPA1 and study of its activation process will contribute to a better understanding of the mechanism of aspartic proteases auto-activation.IMPORTANCEThe novel aspartic protease TlAPA1 was identified from T. leycettanus and expressed as a zymogen (proTlAPA1) in P. pastoris. Enzymatic characteristics of the mature protein were studied and the specific pattern of zymogen conversion was described. The auto-activation processing of proTlAPA1 proceeded in two stages and an intermediate was identified in this process. These results describe a new subgroup of aspartic protease A1 family and provide insights into a novel mode of activation processing in aspartic proteases.