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KMID : 0545120180280081293
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Journal of Microbiology and Biotechnology
2018 Volume.28 No. 8 p.1293 ~ p.1298
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Expression of manB Gene from Escherichia coli in Lactococcus lactis and Characterization of Its Bifunctional Enzyme, Phosphomannomutase
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Li Ling
Kim Seul-Ah
Fang Ruosi
Han Nam-Soo
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Abstract
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Phosphomannomutase (ManB) converts mannose-6-phosphate (M-6-P) to mannose-1- phosphate (M-1-P), which is a key metabolic precursor for the production of GDP-D-mannose used for production of glycoconjugates and post-translational modification of proteins. The aim of this study was to express the manB gene from Escherichia coli in Lactococcus lactis subsp. cremoris NZ9000 and to characterize the encoded enzyme. The manB gene from E. coli K12, of 1,371 bp and encoding 457 amino acids (52 kDa), was cloned and overexpressed in L. lactis NZ9000 using the nisin-controlled expression system. The enzyme was purified by Ni-NTA column chromatography and exhibited a specific activity of 5.34 units/mg, significantly higher than that of other previously reported ManB enzymes. The pH and temperature optima were 8.0 and 50°C, respectively. Interestingly, the ManB used in this study had two substrate specificity for both mannose-1-phosphate and glucose-1-phosphate, and the specific activity for glucose-1-phosphate was 3.76 units/mg showing 70% relative activity to that of mannose- 1-phosphate. This is the first study on heterologous expression and characterization of ManB in lactic acid bacteria. The ManB expression system constructed in this study canbe used to synthesize rare sugars or glycoconjugates.
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KEYWORD
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Lactococcus lactis, phosphomannomutase, mannose-1-phosphate, mannose-6-phosphate
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