The pro-peptide of Streptomyces mobaraensis transglutaminase functions in cis and in trans to mediate efficient secretion of active enzyme from methylotrophic yeasts
| Autor: | Yoshimi Kikuchi, Nobuo Kato, Hiroya Yurimoto, Hiroshi Matsui, Maiko Yamane, Yasuyoshi Sakai |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
Glycosylation
Tissue transglutaminase Peptide Protein Sorting Signals Applied Microbiology and Biotechnology Biochemistry Analytical Chemistry law.invention law Secretion Cloning Molecular Protein precursor Molecular Biology Candida chemistry.chemical_classification Enzyme Precursors Transglutaminases integumentary system biology Organic Chemistry Mutagenesis General Medicine Yeast Streptomyces Enzyme chemistry biology.protein Recombinant DNA Mutagenesis Site-Directed Biotechnology |
| Zdroj: | Bioscience, biotechnology, and biochemistry. 68(10) |
| ISSN: | 0916-8451 |
| Popis: | Transglutaminase (TGase) from the actinomycete Streptomyces mobaraensis is a useful enzyme in the food industry, and development of an efficient production system for it would be desirable. Herein we report secretion of TGase in an enzymatically active form by methylotrophic yeasts as expression hosts. Secretory production of active TGase required a pro-peptide from TGase. When an artificial Kex2-endopeptidase recognition site was placed between the pro-peptide and mature TGase, secretion and in vitro maturation of TGase depended on Kex2-dependent cleavage. Unexpectedly, coexpression of unlinked pro-peptide with mature TGase yielded efficient secretion of the active enzyme. These results indicate that the pro-peptide from TGase functions not only in an intramolecular but also in an intermolecular manner. Site-directed mutagenesis of putative N-glycosylation sites increased the productivity of the active TGase further. A recombinant Candida boidinii strain was found to secrete active TGase up to 1.83 U/ml (about 90 mg/l) after 119 h of cultivation. |
| Databáze: | OpenAIRE |
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