Abstract
The recovery of cutinase of Fusarium solani pisi produced by the yeast Saccharomyces cerevisiae was studied in a fluidised bed adsorption system directly integrated with a productive fermenter (so-called direct product sequestration; DPS). The relative efficiency of this system was compared with the one of a conventional purification process by discrete sequences of fermentation, broth clarification, ultrafiltration and fixed bed anion exchange chromatography. By direct product sequestration of the extracellular heterologous cutinase it was possible, through only one unit operation: (i) to perform broth clarification, (ii) to obtain a high cutinase concentration factor, and (iii) to recover cutinase with a specific activity that equalled that obtained with the conventional purification process. It was also possible (iv) to substantially reduce the total process time, (v) to improve the overall yield, and (vi) to increase cutinase productivity. Furthermore, the procedure outlined is suitable for large scale bioprocess exploitation.
Similar content being viewed by others
References
Bradford M (1976) A rapid and sensitive method for the quantification of micrograms quantities of protein utilising the principle of protein-dye binding. Anal. Biochem. 72: 248–254.
Blum H, Hildburg B and Gross H (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8: 93–99.
Carvalho CML, Aires-Barros MR and Cabral JMS (1999) Cutinase: from molecular level to bioprocess development. Biotechnol. Bioeng. 66: 17–34.
Chase HA and Draeger NM (1992) Affinity purification of proteins using expanded beds. J. Chromatogr. 597: 129–145.
Chase HA and Draeger NM(1992) Expanded bed adsorption of proteins using ion-exchangers. Sep. Sci. Technol. 27: 2021–2039.
Chase HA (1994) Purification of proteins by adsorption chromatography in expanded beds. Trends Biotechnol. 12: 296–303.
Curran BPG and Buceja VC (1993) Yeast cloning and biotechnology. In: Walker J M and Gingold E B (eds). Molecular biology and biotechnology. 3rd edn. Royal Society of Chemistry, Glasgow, UK.
Datar RV, Cartwright T and Rosen C-G (1993) Process economics of animal cell and bacterial fermentations: A case study analysis of tissues plasminogen activator. Bio/Technol. 11: 349–357.
Egmond MR, van der Hijden HWTM, Musters W, Peters H and Verrips CT (1994) Patent WO 94/14963.
Egmond MR, van der Hijden HWTM, Musters W, Peters H and Verrips CT (1994) Patent WO 94/14964.
Hamilton GE, Morton PH, Young TW and Lyddiatt A (1999) Process intensification by direct product sequestration from batch fermentations: Application of a fluidised bed, multi-bed external loop contactor. Biotechnol. Bioeng. 64: 311–333.
Hamilton GE, Luechau F, Burton SC and Lyddiatt A (2000) Development of a mixed mode adsorption process for the direct product sequestration of an extracellular protease from microbial batch cultures. J. Biotechnol. 79: 103–115.
Hjorth R, Kampe S and Carlsson M (1995) Analysis of some operating parameters of novel adsorbents for recovered of protein in expanded beds. Bioseperation 5: 217–223
Jelsch C, Longhi S and Cambillau C (1998) Packing forces in nine crystal forms of cutinase. Prot. Struct. Funct. Genet. 31: 320–333.
Kolattukudy PE (1984) Cutinases from fungi and pollen. In: Borgstrom B and Brockman H, (eds) Lipases. Elsevier, Amsterdam.
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
Lan JC-W, Hamilton GE and Lyddiatt A (1999) Physical and biochemical characterisation of a simple intermediate between fluidised and expanded bed contactors. Bioseparation 8: 43–51.
Lauwereys M, De Geus P, De Meutter J, Stranssens P and Matthyssens G (1990) Cloning, expression and characterization of cutinase, a fungal lipolytic enzyme. In: Schmid RD, Alberghina L and Verger R (eds) Lipases: Structure, Mechanism and Genetic Engineering. GBF Monographs, Vol. 16, Weiheim, Germany.
Longhi S, Nicolas A, Creveld L, Egmond M, Verrips CT, de Vlieg J, Martinez C and Cambillau C (1996) Dynamics of Fusarium solani cutinase investigated through structural comparison among different crystal forms of its variants. Prot. Struct. Funct. Genet. 26: 442–458.
Martinez C, De Geus P, Lauwereys M, Matthyssens G and Cambillau C (1992) Fusarium solani cutinase is a lipolytic enzyme with a catalytic serine accessible to the solvent. Nature 356: 615–618.
Morton PH and Lyddiatt A (1992) Direct recovery of protein products from whole fermentation's broth: A role for ion exchange adsorption in fluidised bed. In: Ion Exchange Advances. Slater M J Eds. Elsevier Applied Sciences.
Novick P, Ferro S and Scheckman R (1981) Order Events in the Yeast Secretory Pathway. Cell 25: 461–469.
Ollis DL, Cheah E, Cygler M, Dijkstra B, Frolow F, Franken SM, Harel M, Remington S J, Silman I M, Shrag J, Sussman JL, Verschueren KHG and Goldman A (1992) The α/β hydrolase fold. Protein Eng. 5: 197–211.
Romanos MA, Scorer CA and Clare JJ (1992) Foreign gene expression in yeast: a review. Yeast 185: 423–488.
Smith RA, Duncan MJ and Moir DT (1985) Heterologous protein secretion from yeast. Science 229: 1219–1224.
Thommes J (1997) Fluidised bed adsorption as a primary recovery step in protein purification. Adv. Biochem. Eng. 58: 185–230.
van Gemeren IA, Musters W, van den Hondel CAMJJ and Verrips CT (1995) Construction and heterologous expression of a synthetic copy of the cutinase cDNA from Fusarium solani pisi. J. Biotechnol. 40: 155–162.
Verger R and de Haas GH (1979) Interfacial enzyme kinetics of lipolysis. Ann. Rev. Biophys. Bioeng. 5: 77–117.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Calado, C.R.C., Hamilton, G.E., Cabral, J.M.S. et al. Direct product sequestration of a recombinant cutinase from batch fermentations of Saccharomyces cerevisiae. Bioseparation 10, 87–97 (2001). https://doi.org/10.1023/A:1012464218516
Issue Date:
DOI: https://doi.org/10.1023/A:1012464218516