期刊
JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE
卷 101, 期 12, 页码 5154-5162出版社
WILEY
DOI: 10.1002/jsfa.11161
关键词
alginate lyase; oligosaccharides; salt‐ activated; signal peptide; heterologous proteins; extracellular expression
资金
- National Natural Science Foundation of China [31871745]
- National First-Class Discipline Program of Food Science and Technology [JUFSTR20180203]
- Postgraduate Research and Practice Innovation Program of Jiangsu Province [KYCX18 1760]
The study identified and characterized a new salt-activated alginate lyase, Aly01, and successfully expressed it extracellularly in E. coli using its signal peptide. By optimizing the culture medium with glycine and calcium, the productivity of the enzyme was enhanced, demonstrating the potential of using the Aly01 signal peptide for producing other heterologous proteins in E. coli.
BACKGROUND Alginate lyases (EC 4.4.2.3/4.4.2.11) have been applied to produce alginate oligosaccharides, which have physiological advantages such as prebiotic and antidiabetic effects, and are of benefit in the food and pharmaceutical industries. Extracellular production of recombinant proteins in Escherichia coli presents advantages including simplified downstream processing and high productivity; however, the presence of certain signal peptides does not always ensure successful secretion, which make the extracellular production of alginate lyase in E. coli rarely reported but of great significance. RESULTS A PL7 family alginate lyase, Aly01, with its native signal peptide from Vibrio natriegens SK42.001, was identified, characterized, and extracellularly expressed in E. coli. The enzyme specifically released trisaccharide from alginate and was strictly NaCl activated. Green fluorescent protein (GFP) was fused with the Aly01 signal peptide and successfully secreted in E. coli to expand the feasibility of using this signal peptide to produce other heterologous proteins extracellularly. Through a synergistic strategy of utilizing Terrific Broth (TB) medium supplemented with 120 mmol L-1 glycine and 10 mmol L-1 calcium, the lag phase of protein secretion was reduced to 3 h from 12 h; meanwhile calcium remedied glycine-related cell growth impairment, leading to further enhancement of overall enzyme productivity, reaching a maximum of 4.55 U mL(-1). CONCLUSION A new salt-activated alginate lyase, Aly01, was identified and characterized. E. coli employed its signal peptide and extracellularly expressed both Aly01 and a GFP, which indicated the signal peptide of Aly01 could be a powerful tool for extracellular production of other heterologous proteins in E. coli. (c) 2021 Society of Chemical Industry
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