No abstract

Increasing the yields of short xylooligosaccharides by enzymatic production is efficient to improve prebiotic effects. Previously, C-terminal oligopeptide C60 was found to accelerate short xylooligosaccharides. Herein, in order to further understand the molecular mechanism of C60, the sequence analysis firstly showed that C60 displays typical properties of a linker (rich in proline/alanine/glycine/glutamine/arginine, 8.33–20.00%). C60 shared the highest identity with the N-terminal region of esterase (98.33%) and high identity with the linker between xylanase and esterase from Prevotella sp. (56.50%), it is speculated to originate from an early linker between XynA and another domain. Besides, structure simulation showed that C60 enhances the molecular interactions between substrate and active residues to improve catalytic efficiency. Moreover, three truncated variants with different lengths of C-terminal regions were successfully generated in Escherichia coli. The specific activities of variants were 6.44–10.24 fold of that of XynA-Tr, and their optimal temperature and pH were the same as XynA-Tr. Three truncated variants released more xylooligosaccharides, especially xylobiose (46.33, 43.41, and 49.60%), than XynA-Tr (32.43%). These results are helpful to understand the molecular mechanism of C60, and also provide new insight to improve the yields of short xylooligosaccharides by molecular modification at the terminal of xylanases.

Improving the specific activity and thermostability of psychrophilic xylosidase is important for improving its enzymatic performance and promoting its industrial application. Herein, a psychrophilic xylosidase AX543 exhibited activity in the temperature range between 0 and 35 °C, with optimum activity at 20 °C, which is lower than that of other reported psychrophilic xylosidases. The thermostability, specific activity, and catalytic efficiency of the site-directed variants G110S, Q201R, and L2 were significantly enhanced, without affecting the optimal reaction temperature. Comparative protein structural analysis and molecular dynamics simulation indicated that these improvements might be the result of the increased hydrogen bonds interaction and improved structural rigidity. Furthermore, homologous module substitution with four segments demonstrated that the psychrophilic characteristics of AX543 are the results of the whole protein structure, and the C-terminal segment A4 appears to be more essential in determining psychrophilic characteristics, exhibiting potentiality to produce more psychrophilic xylosidases. This study provides valuable structural information on psychrophilic xylosidases and also offers attractive modification strategies to modify catalytic activity, thermostability, and optimal reaction temperature.

Fumonisin (FB) is one of the most common mycotoxins contaminating feed and food, causing severe public health threat to human and animals worldwide. Until now, only several transaminases were found to reduce FB toxicity, thus, more fumonisin detoxification transaminases with excellent catalytic properties required urgent exploration for complex application conditions. Herein, through gene mining and enzymatic characterization, three novel fumonisin detoxification transaminases—FumTSTA, FumUPTA, FumPHTA—were identified, sharing only 61–74% sequence identity with reported fumonisin detoxification transaminases. Moreover, the recombinant proteins shared diverse pH reaction ranges, good pH stability and thermostability, and the recombinant protein yields were also improved by condition optimum. Furthermore, the final products were analyzed by liquid chromatography-mass spectrometry. This study provides ideal candidates for fumonisin detoxification and meets diverse required demands in food and feed industries.