Expression in Lactococcus lactis of a ß-1,3-1,4-glucanase gene from Bacillus sp. SJ-10 isolated from fermented fish.
Protein Expr Purif
; 162: 18-23, 2019 10.
Article
em En
| MEDLINE
| ID: mdl-31112758
ABSTRACT
Bacterial ß-1,3-1,4-glucanase (BG) is an endoglucanase that hydrolyzes linear ß-glucans containing ß-1,3 and ß-1,4 linkages, such as barley ß-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⯵molâ¯min-1, 1.97â¯mgâ¯ml-1, 588.54 s-1, and 298.26â¯mlâ¯s-1âmg-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 ß-glucan-containing liquid medium confirmed ß-glucan degradation by BG. The transformed strain degraded ß-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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Assunto principal:
Bacillus
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Proteínas de Bactérias
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Expressão Gênica
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Lactococcus lactis
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Endo-1,3(4)-beta-Glucanase
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Peixes
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Alimentos Fermentados
Limite:
Animals
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article