Expression of the Xylanase Gene from Pyromyces finnis in Pichia pastoris and Characterization of the Recombinant Protein

Abstract

The heterologous expression and characteristics of a new xylanase from Pyromyces finnis are described. The endo-1,4-β-xylanase XylP (EC 3.2.1.8) consists of 223 amino acids and 19 residues of a putative signal peptide in the N-terminal region. The amino-acid sequence of the mature protein has the greatest homology (84%) with the sequence of the native catalytic N-terminal domain of Neocallimastix patriciarum endo-1,4-β-xylanase. A synthetic nucleotide sequence encoding the mature XylP protein was expressed in Pichia pastoris. The purified recombinant enzyme was active with birch xylan and arabinoxylan as substrates. The optimal pH and temperature for enzyme activity were established as 5.0 and 50°C, respectively, with the use of birch xylan. The specific activity of xylanase was 4700 U/mg protein, and KM and Vmax were equal to 0.51 mg/mL and 7395.3 μmol/(min mg), respectively. The recombinant XylP protein showed moderate thermal and high pH stability, as well as resistance to digestive enzymes and protein xylanase inhibitors from cereals. It was also shown that Mg2+, Co2+ and Li+ ions have a positive effect on enzyme activity.

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Funding

The work was performed with the financial support of the Ministry of Education and Science of Russia (Unique Project Identifier RFMEFI60717X0180) with the Unique Scientific Installation, the Kurchatov Institute National Resource Center of the All-Russia Collection of Industrial Microorganisms (Kurchatov Institute NRC, GOSNIIGENETIKA).

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Correspondence to A. N. Kalinina.

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Translated by I. Gordon

Abbreviations: aa—amino-acid residue(s); CL—culture liquid; CMC—carboxymethylcellulose; DNS—3,5-dinitrosalicilic acid; DNS method—method to assess xylanase activity with dinitrosalicylic acid; EDTA—ethylendiamine tetraacetate; KM—Michaelis constant; PAGE—polyacrylamide gel electrophoresis; PCR—polymerase chain reaction; SDS—sodium dodecy(lauryl)sulfate; Vmax—xylanase maximum reaction rate.

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Kalinina, A.N., Borshchevskaya, L.N., Gordeeva, T.L. et al. Expression of the Xylanase Gene from Pyromyces finnis in Pichia pastoris and Characterization of the Recombinant Protein. Appl Biochem Microbiol 56, 787–793 (2020). https://doi.org/10.1134/S0003683820070054

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Keywords:

  • xylanase
  • xylan
  • feed enzyme
  • Pichia pastoris
  • Pyromyces finnis