Production of lysosomal enzymes by continuous high-cell-density fermentation of the ciliated protozoon Tetrahymena thermophila in a perfused bioreactor

• Published in: Enzyme and microbial technology Vol. 18; no. 4; pp. 268 - 274
• Main Authors: Kiy, Thomas, Scheidgen-Kleyboldt, Gerda, Tiedtke, Arno
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Inc 01.03.1996; Elsevier Science
• Subjects: Biological and medical sciences; Biotechnology; ciliated protozoa; continuous high-cell-density fermentation; Enzyme engineering; Fermentation; Fundamental and applied biological sciences. Psychology; lysosomal enzyme production; Methods. Procedures. Technologies; perfused bioreactor; Production of selected enzymes; Tetrahymena thermophila; continuous high-cell-density fermentation; ciliated protozoa; Tetrahymena thermophila; perfused bioreactor; lysosomal enzyme production; High density; β-N-Acetylhexosaminidase; Phosphoric monoester hydrolases; Esterases; Phosphoric diester hydrolases; Cell density; Enzymatic activity; Production; Proteinases; Protozoa; Stability; Enzyme; Fermentation; Continuous process; Bioreactor; β-Glucosidase; α-Glucosidase; Perfusion; Glycosidases; α-Mannosidase; Hydrolases; Ciliata; O-Glycosidases; Acid phosphatase; Phosphodiesterase I
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/0141-0229(95)00076-3

The production of several hydrolytic enzymes of lysosomal origin, such as acid phosphatase, β-hexosaminidase, α- and β-glucosidase, α-mannosidase, protease, and phosphodiesterase I and II, by the ciliated protozoon Tetrahymena thermophila was studied in a perfused bioreactor. After an initial batch phase, the second phase—characterized by continuous exchange of the medium—was started. Because the cells were retained completely, cell densities of 2.2 × 10 7 cells ml −1 could be achieved. Compared to standard batch fermentations of Tetrahymena this value is about 20 times higher. The enzyme activities of the harvested culture broth were even 40 to 50 times higher. For example, 19,600 mU acid phosphatase ml −1, 10,500 mU β-hexosaminidase ml −1, and 370 U protease ml −1 were obtained in the cell-free harvested medium. The highest secretion rates were achieved with 1% glucose in the feed and a perfusion rate of 1.0 d −1. Under these conditions 10 6 cells secreted 790 ± 49 mU acid phosphatase d −1 and 226 ± 28 mU β-hexosaminidase d −1. Because the protein content of the exhausted culture medium mainly consisted of secreted hydrolases, the specific enzyme activities turned out to be high, e.g., 7635 ± 448 mU acid phosphatase mg −1 protein. The activities of β-hexosaminidase and acid phosphatase in the culture medium harvested via microfiltration remained stable for at least 500 h at room temperature even without the addition of protease inhibitors.