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Expression of the β-Glucanase Gene from Paenibacillus jamilae Bg1 in Pichia pastoris and Characteristics of the Recombinant Enzyme

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Abstract

The isolation, heterologous expression, and characterization of a new, thermostable β-glucanase from Paenibacillus jamilae are described. The bgl26 gene from the P. jamilae Bg1 VKPM B-13 093 strain, which consists of 714 nucleotides, encodes endo-1,3-1,4-β-glucanase (EC 3.2.1.73), which contains 213 amino acids and 24 residues of the putative signal peptide in the N-terminal region. The nucleotide sequence of the bgl26 gene and the amino acid sequence of the mature Bgl26 protein have the greatest homology with the sequences of the Paenibacillus macerans endo-1,3-1,4-β-glucanase (82 and 88%, respectively). A gene fragment encoding the mature protein was expressed in Pichia pastoris. The purified recombinant enzyme Bgl26 was active against barley β-glucan. The optimal pH for the enzyme activity was 7.0, and the optimum temperature range was 40–45°C. The specific β-glucanase activity was at the level of 6650 U/mg of protein; KM and Vmax were equal to 6.4 ± 0.3 mg/mL and 9450.1 ± 471.2 μmol/(min mg), respectively. The recombinant protein Bgl26 was characterized by a high pH and thermal stability, as well as resistance to digestive enzymes. It was also shown that Co2+ ions have a positive effect on enzyme activity.

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ACKNOWLEDGMENTS

The work was carried out on the equipment of the Multipurpose Scientific Installation of the All-Russia Collection of Industrial Microorganisms of the Kurchatov Institute National Resource Center (GOSNIIgenetika).

Funding

The work was financially supported by the Ministry of Science and Higher Education of the Russian Federation (Unique Project Identifier RFMEFI60717X0179).

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Authors and Affiliations

  1. State Research Institute for Genetics and Selection of Industrial Microorganisms, Kurchatov Institute National Research Center (Kurchatov Institute NRC, GOSNIIgenetika), 117545, Moscow, Russia

    L. N. Borshchevskaya, T. L. Gordeeva, A. N. Kalinina, A. V. Serkina, A. S. Fedorov & S. P. Sineoky

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  1. L. N. Borshchevskaya
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  2. T. L. Gordeeva
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  3. A. N. Kalinina
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  4. A. V. Serkina
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  5. A. S. Fedorov
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  6. S. P. Sineoky
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Correspondence to L. N. Borshchevskaya.

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Translated by I. Gordon

Abbreviations: aa—amino acid residue(s); CL—culture liquid; CMC—carboxymethylcellulose; DNS—3,5-dinitrosalicylic acid; DNS method—method for xylanase activity measurement with DNS; EDTA—ethylendiamine tetraacetic acid; GH—glycosyl hydrolase; GH16—glycosyl hydrolase family 16; LB medium—lysogeny-broth medium; PAGE—polyacrylamide gel electrophoresis; PCR—polymerase chain reaction; SDS—sodium dodecyl(lauryl)sulfate; YPD medium—medium containing yeast extract, peptone, and dextrose.

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Borshchevskaya, L.N., Gordeeva, T.L., Kalinina, A.N. et al. Expression of the β-Glucanase Gene from Paenibacillus jamilae Bg1 in Pichia pastoris and Characteristics of the Recombinant Enzyme. Appl Biochem Microbiol 56, 854–860 (2020). https://doi.org/10.1134/S0003683820080025

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