Cloning and Expression of Bacillus pumilis Bg57 β-Glucanase Gene in Pichia pastoris: Purification and Characteristics of Recombinant Enzyme
- Autores: Borshchevskaya L.N.1, Gordeeva T.L.1, Kalinina A.N.1, Bulushova N.V.1, Sineoky S.P.1
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Afiliações:
- State Research Institute for Genetics and Selection of Industrial Microorganisms, Kurchatov Institute National Research Center (GOSNIIGENETIKA, Kurchatov Institute NRC)
- Edição: Volume 55, Nº 8 (2019)
- Páginas: 771-779
- Seção: Producers, Biology, Selection, and Gene Engineering
- URL: https://journals.rcsi.science/0003-6838/article/view/153103
- DOI: https://doi.org/10.1134/S0003683819080039
- ID: 153103
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Resumo
The isolation, heterologous gene expression, and characteristics of a new β-glucanase from Bacillus pumilus is described. The bgl1 gene from the Bacillus pumilus Bg57 VKPM В-13195 strain, which consists of 729 nucleotides, encodes a secreted endo-β-l,3(4)-D-glucanase (EC 3.2.1.6) containing 214 amino acids and 28 residues of the putative signal peptide in the N-terminal area. The nucleotide sequence of bgl1 and amino acid sequence of the mature Bgl protein have the highest homology (89 and 95%, respectively) with the sequences for β-l,3-l,4-glucanase from Bacillus licheniformis. A fragment of the gene encoding the mature protein was expressed in Pichia pastoris. The purified recombinant enzyme of Bgl was active towards barley β-glucan and lichenin. With barley β-glucan, the optimal pH and temperature were shown to be 6.0 and 50°C, respectively; Km and Vmax were 2.2 mg/mL and 3036.4 μmol/(min mg), respectively. The recombinant protein Bgl showed a high specific activity, thermal stability, and resistance to digestive enzymes. Other characteristics of the recombinant protein, including pH stability and sensitivity to metal ions and chemical reagents, were also determined.
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Sobre autores
L. Borshchevskaya
State Research Institute for Genetics and Selectionof Industrial Microorganisms, Kurchatov Institute National Research Center (GOSNIIGENETIKA, Kurchatov Institute NRC)
Autor responsável pela correspondência
Email: larisa.larbor3@yandex.ru
Rússia, Moscow, 117545
T. Gordeeva
State Research Institute for Genetics and Selectionof Industrial Microorganisms, Kurchatov Institute National Research Center (GOSNIIGENETIKA, Kurchatov Institute NRC)
Email: larisa.larbor3@yandex.ru
Rússia, Moscow, 117545
A. Kalinina
State Research Institute for Genetics and Selectionof Industrial Microorganisms, Kurchatov Institute National Research Center (GOSNIIGENETIKA, Kurchatov Institute NRC)
Email: larisa.larbor3@yandex.ru
Rússia, Moscow, 117545
N. Bulushova
State Research Institute for Genetics and Selectionof Industrial Microorganisms, Kurchatov Institute National Research Center (GOSNIIGENETIKA, Kurchatov Institute NRC)
Email: larisa.larbor3@yandex.ru
Rússia, Moscow, 117545
S. Sineoky
State Research Institute for Genetics and Selectionof Industrial Microorganisms, Kurchatov Institute National Research Center (GOSNIIGENETIKA, Kurchatov Institute NRC)
Email: larisa.larbor3@yandex.ru
Rússia, Moscow, 117545
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