Abstract
Alginate lyase digestion is an efficient way to degrade alginate into oligosaccharides, which are useful in various areas including chemistry, pharmacy and food industry. Here we determined the sequence of Vibrio sp. QY102 sourced alginate lyase, and set up its heterologous expression in E. coli. We improved its secretion efficiency by replacing the original secretive sequence by E. coli specific signal peptide ompA. We successfully purified the full-length protein in shake flask culture, however, degradation happened during fed batch cultivation. By domain and disorder examination, we found that the protein was completely functional by expressing the C terminal fragment alone. For the final strain we constructed (HMS-ompA-CF), the extracellular enzyme activity reached 375 U/ml in shake flask and 1789 U/ml in fed batch cultivation (5 L bioreactor). And the final protein yield reached 0.58 g/L in fed batch cultivation. We determined that the optimal pH and temperature for the shortened alginate lyase were 7.0 and 39 °C, respectively.
Keywords:
Alginate lyase; Bioreactor; Ni(2+) affinity chromatography; ompA signal peptide.
Copyright © 2018 Elsevier Inc. All rights reserved.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Alginates / chemistry*
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Alginates / metabolism
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Amino Acid Sequence
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Aquatic Organisms
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Bacterial Outer Membrane Proteins / genetics*
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Bacterial Outer Membrane Proteins / metabolism
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Bacterial Proteins / chemistry
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Bacterial Proteins / genetics*
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Bacterial Proteins / isolation & purification
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Bacterial Proteins / metabolism
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Batch Cell Culture Techniques
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Chromatography, Affinity
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Cloning, Molecular
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Escherichia coli / genetics
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Escherichia coli / metabolism
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Gene Expression
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Genetic Vectors / chemistry
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Genetic Vectors / metabolism
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Hydrogen-Ion Concentration
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Hydrolysis
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Models, Molecular
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Molecular Weight
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Oligosaccharides / chemistry
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Oligosaccharides / metabolism
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Polysaccharide-Lyases / chemistry
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Polysaccharide-Lyases / genetics*
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Polysaccharide-Lyases / isolation & purification
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Polysaccharide-Lyases / metabolism
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Protein Structure, Secondary
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Sequence Alignment
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Sequence Homology, Amino Acid
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Temperature
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Vibrio / enzymology*
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Vibrio / genetics
Substances
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Alginates
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Bacterial Outer Membrane Proteins
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Bacterial Proteins
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Oligosaccharides
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Recombinant Proteins
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OMPA outer membrane proteins
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Polysaccharide-Lyases
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poly(beta-D-mannuronate) lyase