Cloning and Expression of Bacillus pumilis Bg57 β-Glucanase Gene in Pichia pastoris: Purification and Characteristics of Recombinant Enzyme


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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.

Sobre autores

L. Borshchevskaya

State Research Institute for Genetics and Selection
of 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 Selection
of 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 Selection
of 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 Selection
of 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 Selection
of Industrial Microorganisms, Kurchatov Institute National Research Center (GOSNIIGENETIKA, Kurchatov Institute NRC)

Email: larisa.larbor3@yandex.ru
Rússia, Moscow, 117545

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