Purification of Proteins and the Disruption of Microbial Cells

Abstract

Intracellular proteins with catalytic or biological activity are of growing importance for developments in enzyme technology, as well as for the production of mammalian proteins by recombinant‐DNA technology. The release of these proteins from microorganisms is an important unit operation, as it is the first step in their isolation. Gram‐scale disruption of microorganisms can be performed by a variety of established methods based on chemical, enzymatic, physical, or mechanical principles. For the large scale disruption of microorganisms, mechanical disintegrators, such as high‐speed agitator bead mills or high‐pressure industrial homogenizers, are commonly employed. Both types of equipment were designed originally for other tasks; in the paint industry or in the milk industry, respectively. Therefore, it appeared necessary and possible to improve design and performance for the application in cell disintegration. The goal is a uniform exposure of the microbial cells and a minimal exposure of solubilized protein to high shear forces, in order to obtain high yields and to avoid the generation of too small cell wall fragments, which are difficult to separate. Both types of machines have been investigated for the disintegration of different microbial cells and the influence of the operating parameters analyzed on protein solubilization and enzyme yield. We will summarize the state of the art and discuss new data to illustrate trends in process development.

C-phycocyanin extraction from Spirulina platensis wet biomass

“…Thus, the use of variations in the osmotic pressure, abrasive conditions, chemical treatment, freezing-thawing and sonication, amongst other disruption methods, are necessary. Mechanical cell disintegration methods are currently preferred for large-scale operations (Gacesa and Hubble, 1990;Kula and Schütte, 1987) since a complete disintegration of the biomass is desired, with high product and activity yields.…”

Ondas ultrassônicas e pérolas de vidro: um novo método de extração de beta-galactosidase para uso em laboratório

“…Para leveduras e bactérias, diâ-metros maiores ou menores que 0,5 mm, respectivamente, são considerados ótimos. Pérolas de diâmetro entre 0,10-0,15 mm são indicadas para a ruptura de células bacterianas; enquanto que pérolas entre 0,50-0,75 mm de diâmetro favorecem a ruptura de leveduras 10 . A mudança no diâmetro e na carga, das pérolas de vidro foi de suma importância para a eficiência da ruptura.…”

“…As características do processo de ruptura podem variar com base na estabilidade mecânica do microrganismo, a qual depende da espécie, idade da cultura, taxa de crescimento específico, temperatura de cultivo e meio de cultura 6,9 . Diferentes métodos podem ser empregados para extração de proteínas intracelulares, os quais dependem da força física da parede celular dos microrganismos, localização dentro da célula, estabilidade e do uso desejado para o composto de interesse 10,11 . Métodos mecânicos, físicos, químicos, enzimáticos e a combinação destes podem ser aplicados.…”

Significance of location of enzymes on their release during microbial cell disruption

“…Of the methods available, physical methods are often favored due to the economic and operational limitations of the chemical and enzymatic methods. The celldisruption process is dependent on cell properties such as the physical strength of the cell wall of the microorganism (Sauer et al, 1988) and on the intracellular location of enzyme in the microbial cell : Hettwer and Wang, 1989: Kuboi et al, 1995: Kula and Schutte, 1987: Melendres et al, 1993Schutte et al, 1983). Thus, location of the enzyme can in¯uence its release rate during the process of disruption.…”