Characteristic of a GH10 Xylanase From The Anaerobic Rumen Fungus Anaeromyces Robustus and Application in Bread Making

The rumen of ruminants contains a variety of fungi capable of producing xylanases to break down plant cell walls. In this study, a new GH10 xylanase gene ArXyn10c20 from anaerobic rumen microorganism Anaeromyces robustus was successfully expressed in Pichia Pastoris GS115, with a protein molecular weight of approximately 42 kDa and showed the similarity by 64.08% with the β-Xylanase form Neocallimastix Californiae. The optimal pH and temperature for ArXyn10c20 was 5.5 at 40℃. ArXyn10c20 was stable in the pH range 5.0 – 9.0 for 1h which the residual enzyme activity was all above 75%. The activity of recombinant xylanase was significantly enhanced by 1 mM Cu 2+ . The products of ArXyn10c20 hydrolysis of beechwood xylan were xylobiose, xylotriose and xylotetraose by TLC analysis. In food applications, ArXyn10c20 can significantly improve the quality of dough and bread. With the addition of 7.5 mg ArXyn10c20, the hardness, gumminess and chewiness of the bread decreased by 42.24%, 45.33% and 55.36% respectively and the reducing sugar increased by 18.67%. The new discovered xylanase ArXyn10c20 has great potential in food industry.

The mutation of Thr315 to Asn of GH10 xylanase XynR increases the alkaliphily but decreases the alkaline resistance

XynR is a thermophilic and alkaline GH10 xylanase, identified in the culture broth of alkaliphilic and thermophilic Bacillus sp. strain TAR-1. We previously selected S92E as a thermostable variant from a site saturation mutagenesis library. Here, we attempted to select the alkaliphilic XynR variant from the library and isolated T315N. In the hydrolysis of beechwood xylan, T315N and S92E/T315N exhibited a broader bell-shaped pH-dependent activity than the wild-type XynR (WT) and S92E. The optimal pH values of T315N and S92E/T315N were 6.5–9.5 while those of WT and S92E were 6.5–8.5. On the other hand, T315N and S92E/T315N exhibited a narrower bell-shaped pH-dependence of stability: the pHs at which the activity was stable after the incubation at 37 °C for 24 h were 6.0–8.5 for T315N and S92E/T315N, but 6.0–10.0 for WT and S92E. These results indicated that the mutation of Thr315 to Asn increased the alkaliphily but decreased the alkaline resistance.