Site-saturation mutagenesis of mutant l-asparaginase II signal peptide hydrophobic region for improved excretion of cyclodextrin glucanotransferase

The excretion of cyclodextrin glucanotransferase (CGTase) into the culture medium offers significant advantages over cytoplasmic expression. However, the limitation of Escherichia coli is its inability to excrete high amount of CGTase outside the cells. In this study, modification of the hydrophobic...

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Bibliographic Details
Published in:Journal of industrial microbiology & biotechnology 2017-12, Vol.44 (12), p.1627-1641
Main Authors: Ismail, Abbas, Illias, Rosli Md
Format: Article
Language:English
Subjects:
Amino acids
Asparaginase
Asparaginase - chemistry
Asparaginase - genetics
Asparaginase - metabolism
Biochemistry
Bioinformatics
Biomedical and Life Sciences
Biotechnology
Cell culture
Cell death
Cyclodextrin
Cytoplasm
E coli
Escherichia coli - genetics
Escherichia coli - metabolism
Excretion
Genetic Engineering
Genetic recombination
Genetics and Molecular Biology of Industrial Organisms - Original Paper
Glucosyltransferases - chemistry
Glucosyltransferases - genetics
Glucosyltransferases - secretion
Hydrophobic and Hydrophilic Interactions
Hydrophobicity
Inorganic Chemistry
L-asparaginase
Life Sciences
Microbiology
Mutagenesis
Mutagenesis, Site-Directed
Mutants
Mutation
Peptides
Protein folding
Protein Sorting Signals - genetics
Protein Sorting Signals - physiology
Recombinant Proteins - biosynthesis
Recombinant Proteins - genetics
Recombinant Proteins - secretion
Saturation
Saturation mutagenesis
Secretion
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Summary:The excretion of cyclodextrin glucanotransferase (CGTase) into the culture medium offers significant advantages over cytoplasmic expression. However, the limitation of Escherichia coli is its inability to excrete high amount of CGTase outside the cells. In this study, modification of the hydrophobic region of the N1R3 signal peptide using site-saturation mutagenesis improved the excretion of CGTase. Signal peptide mutants designated M9F, V10L and A15Y enhanced the excretion of CGTase three-fold and demonstrated two-fold higher secretion rate than the wild type. However, high secretion rate of these mutants was non-productive for recombinant protein production because it caused up to a seven-fold increase in cell death compared to the wild type. Our results indicated that the excretion of CGTase is highly dependent on hydrophobicity, secondary conformation and the type and position of amino acids at the region boundary and core segment of the h-region.
ISSN:1367-5435
1476-5535
DOI:10.1007/s10295-017-1980-6