Mutagenesis of N-terminal residues confer thermostability on a Penicillium janthinellum MA21601 xylanase

Abstract

Background: A mesophilic xylanase PjxA from Penicillium janthinellum MA21601 has high specific activity under acidic condition and holds great potential for applications in the animal feed industry. To enhance the thermostability of xylanase PjxA, two mutation strategies in the N-terminal region were examined and then integrated into the xylanase to further improvement. The recombinant xylanase PTxA-DB (The meaning of DB is disulfide-bridge.) was constructed by replacement of five residues in the mutated region in TfxA (T10Y, N11H, N12D, Y15F, N30 L), combined with an additional disulfide bridge in the N-terminal region. Results: The T m value of mutant PTxA-DB was improved from 21.3°C to 76.6°C, and its half-life was found to be 53.6 min at 60°C, 107-fold higher than the wild type strain. The location of the disulfide bridge (T2C-T29C) was between the irregular loop and the β-strand A2, accounting for most of the improvement in thermostability of PjxA. Further analysis indicated T2C, T29C, N30 L and Y15F lead to increase N-terminal hydrophobicity. Moreover, the specific activity and substrate affinity of PTxA-DB were also enhanced under the acidic pH values. Conclusions: These results indicated PTxA-DB could be a prospective additive to industrial animal feeds.

Production and biochemical characterization of xylanases synthesized by the thermophilic fungus Rasamsonia emersonii S10 by solid-state cultivation

“…Site directed mutagenesis can improve the stability significantly. Alterations of Nterminal residues of a xylanase from Penicillium janthinellum MA21601 improved the half-life 107-fold higher than the wild-type strain, increasing from 30 s to 53.6 min at 60 °C 58 .…”

Advances in Rational Protein Engineering toward Functional Architectures and Their Applications in Food Science

“…For example, there is evidence to suggest that the improvement of protein stability can be achieved through the introduction of a S−S bond, increase of hydrogen bonds, and alteration of hydrophobic properties, while substitution of residues such as Cys and Met to Ala, Ser, Gln, and Val can significantly enhance their oxidative stability. 24 The assembly behaviors of multimeric proteins are determined by the interfacial contacts of subunits. For instance, weakening the subunit−subunit interactions around C 4 interfaces yields ferritin nanocages with mild disassembly and reassembly properties, which can achieve bioactive compound encapsulation under moderate conditions.…”

“…The T m value of the designed mutant was improved from 21.3 to 76.6 °C, and its half-life was found to be 53.6 min at 60 °C. 24 Recently, the homologous modeling analysis of XynAF0, a GH10 family xylanase from Aspergillus fumigatus Z5, found that an α-helix composed of polythreonine in the linker region between the carbohydrate-binding module domain and the catalytic domain is of great importance for its thermostability. The introduction of the polythreonine fragment to other xylanase families including wild-type xylanase XynAS9 and XynAS9-CD significantly improved the overall thermostability.…”

Significantly Enhanced Thermostability of Aspergillus niger Xylanase by Modifying Its Highly Flexible Regions

“…22 In addition, the interaction between α-helix and β-strand B9 was enhanced by constructing a disulfide bond, 23−25 while the flexibility of loops was reduced by increasing its interaction with the adjacent α-helix or β-strand. 26 However, there were few reports regarding the modifications within the cord and thumb region, which are flexible and close to the catalytic site. 27,28 It has been demonstrated that the rigidity within the active site plays a critical role in the kinetic stability of the enzyme.…”