Expression of the Xylanase Gene from Paenibacillus brasilensis X1 in Pichia pastoris and Characteristics of the Recombinant Enzyme

• Published in: Applied biochemistry and microbiology 2019-12, Vol.55 (8), p.797-804
• Main Authors: Kalinina, A. N., Gordeeva, T. L., Sineoky, S. P.
• Format: Article
• Language: English
• Subjects: Amino acid sequence; Amino acids; Biochemistry; Biology; Biomedical and Life Sciences; Cereals; Digestive enzymes; endo-1,4-β-Xylanase; Enzymatic activity; Enzyme activity; Enzymes; Gene Engineering; Gene expression; Gene sequencing; Homology; Life Sciences; Manganese ions; Medical Microbiology; Microbiology; Nucleotide sequence; Nucleotides; Paenibacillus; pH effects; Pichia pastoris; Producers; Proteins; Selection; Substrates; Thermal resistance; Thermal stability; XYL1 gene; Xylan; Xylanase; Yeast
• ISSN: 0003-6838; 1608-3024
• DOI: 10.1134/S0003683819080064

The heterologous expression, isolation, and characterization of a novel xylanase from Paenibacillus brasilensis are described. The xyl 1 gene from the Paenibacillus brasilensis strain X1 VKPM B-13092, which consists of 639 nucleotides, encodes a secreted endo-1,4-β-xylanase (EC 3.2.1.8) containing 184 amino acids and 28 residues of the putative signal peptide in the N-terminal region. The nucleotide sequence of the xyl 1 gene and the amino acid sequence of the mature Xyll protein have the greatest homology with the Bacillus subtilis endo-1,4-β-xylanase sequences (78 and 83%, respectively). A gene fragment encoding the mature protein was expressed in Pichia pastoris . The purified recombinant Xyl1 enzyme was able to use birch xylan and arabinoxylan as substrates. With birch xylan, the optimal pH for the enzymatic reaction was 6.0, the optimal temperature was 40–50°C, and K m and V max , were equal to 1.1288 mg/mL and 5124.3 μmol/(min mg), respectively. The recombinant Xyl1 protein showed high pH and thermal stability, and the resistance to digestive enzymes and xylanase protein inhibitors from cereals. It was also shown that Mn 2+ and Со 2+ ions stimulate enzyme activity.