ISSN:
0006-3592
Keywords:
preparative separation
;
continuous
;
free-flow zone electrophoresis
;
electrophoretic mobility
;
net charge
;
enzymes
;
proteins
;
crude extract
;
cell debris
;
Candida boidinii
;
Escherichia coli
;
formate dehydrogenase
;
formaldehyde dehydrogenase
;
methanol oxidase
;
β-galactosidase
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Process Engineering, Biotechnology, Nutrition Technology
Notes:
Continuous, single-step, state-of-the-art preparative separations of enzymes from microorganism crude extracts by free-flow zone electrophoresis are presented. In the first example, the enzymes formate dehydrogenase, formaldehyde dehydrogenase, and methanol oxidase were continuously separated from Candida boidinii crude extract. Yields of 85% to 95% and purification factors between 3 and 7 were obtained along with a simultaneous separation of the finer cell debris from the enzymes. Using multiple injections of sample, a throughput of 46.2 mg protein/h was recorded. In the second example, a fivefold purification of β-galactosidase from Escherichia coli was achieved along with complete, simultaneous cell debris separation from the enzyme. The yield of the enzyme was greater than 90%. The preparative free-flow zone electrophoresis experiments were run continuously for a period of 12 h and the separations were found to be stable; i.e., the enzymes and the cell debris eluted at their respective fraction numbers during the entire period. In both examples, choice of the type of buffer played a critical role and had to be investigated and optimized experimentally. Scale-up aspects of the separations are also discussed. Recently, by comparison of free-flow zone electrophoresis with ion-exchange chromatography, we have presented evidence that free-flow electrophoresis separations are governed by net surface charge (S. Nath et al., Biotechnol. Bioeng. 1993, 42: 829-835). Here, we offer further confirmation of this evidence by comparison of preparative free-flow zone electrophoresis experiments at various pHs on a mixture of two model proteins with analytical electrophoretic titration curves of the proteins. We are thus in a position to predict separations in free-flow zone electrophoresis. © 1996 John Wiley & Sons, Inc.
Additional Material:
8 Ill.
Type of Medium:
Electronic Resource
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