Psychrophilic and acidophilic β-1,4-xylanase and its Ca2+-dependent carbohydrate-binding module

Abstract:

[Objective] β-1,4-xylanase is one of the key enzymes in the biodegradation of xylan. Psychrophilic and acidophilic xylanases play an important role in preparing functional xylooligosaccharides, whereas little is known about these enzymes. [Methods] We discovered a novel xylanase gene by functional annotation of the genome of the deep-sea bacterium Flammeovirga pacifica strain WPAGA1. The sequence alignment suggested 60% identity of this sequence with the verified β-1,4-xylanase from Clostridium saccharobutylicum (ID: P17137). Then, we constructed the recombinant plasmid and transformed it into the host cells for expression. After purifying the enzyme by nickel column, we examined the enzyme properties. [Results] The full-length β-1,4-xylanase (Xyl4513) had two conserved domains: a catalytic module belonging to the glycoside hydrolase family 11 (Xy14513-T) and a carbohydrate-binding module (CBM) belonging to family 60 (CBM4513). This was a rare phenomenon that the GH11 xylanase contained CBM. The purified Xyl4513 showed the highest activity at 30 °C and pH 3.0, being a psychrophilic and acidophilic β-1,4-xylanase. The truncated β-1,4-xylanase (Xy14513-T) demonstrated the highest activity at 20 °C and pH 4.0 and the catalytic efficiency (k_cat/K_m) 20% lower than that of Xyl4513, indicating the positive effects of CBM on the stability and catalytic performance of β-1,4-xylanase. In addition, Ca²⁺, Mg²⁺, and Ni²⁺ improved the catalytic activities, and Ca²⁺ showed the best performance. Only in the presence of Ca²⁺, CBM4513 had the specific binding ability to β-1,4-xylan, demonstrating a Ca²⁺-dependent CBM, and the maximum binding amount was 9.13 μmol/g. [Conclusion] We obtained a novel psychrophilic and acidophilic β-1,4-xylanase with a Ca²⁺-dependent CBM, which enriched the related gene and protein resources. The findings of this study will provide valuable information for exploring the stability, catalytic mechanism, and engineering of xylanases and CBMs.