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  • Title: Expression in Lactococcus lactis of a β-1,3-1,4-glucanase gene from Bacillus sp. SJ-10 isolated from fermented fish.
    Author: Tak JY, Jang WJ, Lee JM, Suraiya S, Kong IS.
    Journal: Protein Expr Purif; 2019 Oct; 162():18-23. PubMed ID: 31112758.
    Abstract:
    Bacterial β-1,3-1,4-glucanase (BG) is an endoglucanase that hydrolyzes linear β-glucans containing β-1,3 and β-1,4 linkages, such as barley β-glucans. In this study, a BG gene was transformed into the food-grade plasmid pNZ8149 and successfully expressed in Lactococcus lactis NZ3900 using the nisin-controlled gene expression system. To facilitate extracellular secretion, the signal peptide Usp45 was added during vector construction. A histidine tag was also added for affinity purification. BG was extracellularly secreted and was also present in the cells in soluble form. N-terminal amino acid residue analysis of secreted BG revealed that the Usp45 peptide was removed. The optimum temperature and pH for both intracellular and extracellular BG were 40 °C and 6, respectively. The enzyme kinetic parameters, Vmax, Km, kcat, and kcat/Km, of extracellular BG were 1317.51 μmol min-1, 1.97 mg ml-1, 588.54 s-1, and 298.26 ml s-1∙mg-1, respectively. There was no significant difference in the enzyme kinetic parameters of intracellular and extracellular BG. The growth pattern of transformed L. lactis NZ3900 in β-glucan-containing liquid medium confirmed β-glucan degradation by BG. The transformed strain degraded β-glucans, produced gluco-oligosaccharide, and produced lactic acid. The strain and expression system constructed in this study could be applied to industrial fields requiring BG produced in food-grade lactococcal secretory expression system.
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