Expression of the Xylanase Gene from Pyromyces finnis in Pichia pastoris and Characterization of the Recombinant Protein
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UCERS, BIOLOGY, SELECTION, AND GENE ENGINEERING
Expression of the Xylanase Gene from Pyromyces finnis in Pichia pastoris and Characterization of the Recombinant Protein A. N. Kalininaa, *, L. N. Borshchevskayaa, T. L. Gordeevaa, and S. P. Sineokya a
State Research Institute for Genetics and Selection of Industrial Microorganisms, Kurchatov Institute National Research Center (Kurchatov Institute NRC—GOSNIIGENETIKA), Moscow, 117545 Russia *e-mail: [email protected] Received June 7, 2019; revised June 17, 2019; accepted July 11, 2019
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. Keywords: xylanase, xylan, feed enzyme, Pichia pastoris, Pyromyces finnis DOI: 10.1134/S0003683820070054
INTRODUCTION Xylans are one of the main structural components of the plant cell wall; they account for about a third of the renewable plant biomass on Earth [1]. They are heteropolymers consisting of a linear chain of the D-xylose residues linked y β-1,4-glycosidic bonds that are partially acetylated and contain D-glucuronic acid, α-L-arabinose and ferulic and para-cumaric acid residues as substituents in the side chains [2, 3]. The xylan structure is highly dependent on plant species. Endo-1,4-β-xylanases (EC 3.2.1.8) are the basic components of the enzyme complex that decomposes xylans in nature. These enzymes are widely used in the processes of the lignocellulosic waste bioconversion, the pulp and paper industry, and the production of combined feed for farm animals and poultry [4, 5]. Bacteria, archaea, fungi, and yeasts are the main sources of xylanases [6, 7]. However, not all of the enzymes from these organisms have the necessary properties for their industrial use: sufficient thermal 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—pol
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