Characteristics of a XIP‐resistant xylanase from Neocallimastix sp. GMLF1 and its advantage in barley malt saccharification

Abstract

The well-studied xylanase inhibitor protein (XIP-I) regularly inhibits fungi-derived xylanases, yet some fungal xylanases are not inhibited by XIP-I. Based on the sequence alignment with the known XIP-I resistant NpXyn11A from Neocallimastix patriciarum, a new annotated xylanase from ruminal fungus Neocallimastix sp. GMLF1 was found, noted as Xyn1B, which shared 77.8% of sequence identity with NpXyn11A and was proved to be resistant to XIP-I. To evaluate the performance of a XIP resistant xylanase used in barley malt saccharification, a XIP-I sensitive xylanase ThXyn1 from Trichoderma harzianum was chosen as a control. The barley malt saccharification experiment was carried out on the condition with or without extra XIP-I added. The results showed that Xyn1B displayed only slight difference in presence and absence of added XIP-I, with the difference of xylose released (DX) and the difference of mash clarity (DA) being 0.17 g L À1 (P > 0.05) and 0.007 (P > 0.05), respectively; while those for ThXyn1 group reached 0.96 g L À1 (P < 0.01) and 0.095 (P < 0.05), indicating that XIP-I did not adversely affect Xyn1B's function, but did affect ThXyn1's function. Our work for the first time suggested that a xylanase with resistance to xylanase inhibitor proteins might have an advantage in barley malt saccharification over an inhibitor sensitive xylanase.

Directed Modification of a GHF11 Thermostable Xylanase AusM for Enhancing Inhibitory Resistance towards SyXIP-I and Application of AusMPKK in Bread Making

“…The Km and Vmax values of the purified AusM and AusM PKK towards birchwood xylan were graphically determined by using Origin non-linear curve fi ing as shown in Figure 5B to be 21.31 mg/mL and 3360.87 U/mg for AusM and 32.96 mg/mL and 3419.45 U/mg, respectively. The Vmax values of AusM and AusM PKK were unchanged before and after the mutation, which was comparable to those previously reported in the range of 400-5000 U/mg while Km is relatively higher than those previously reported in the range of 4-5 mg/mL [21,40]. The Km value of AusM PKK was relatively higher than that of AusM, indicating that the affinity of AusM PKK for the xylan substrate was less than that of AusM.…”

“…The inhibitory activity of these inhibitors exhibits variation contingent upon the source and type of the validated xylanase. Both TAXI and TLXI inhibitors have been found to inhibit GHF11 xylanases from fungal and bacterial origins but not GHF10 xylanases, while XIP-type inhibitors mostly inhibit GHF11 and GHF10 xylanases from fungal sources [20,21]. Additionally, it has been demonstrated that XIP-I can effectively suppress the enzymatic function of both AMY1 and AMY2 (barley-amylase ioszymes) [22].…”