Enzymes, large-scale purification

The biotransformation process has been improved by significant advances in biochemical engineering advances in genetic and protein engineering, microbiological manipulations for the production of enzymes, and the use of biocatalysts in immobilized form and large-scale purification methods. 

MD Scawen. Large scale purification of enzymes. In Enzymes in Organic Synthesis. London Pitman Publishing, 1985, pp 40-56. 

F. S. Furbish, H. R Blaii I. ShOoach, R G. Pentdiev, and R. O. Brody. Enzyme replacement therapy in Gaucher s disease large-scale purification of ghicocoe-bromdase suitable for human administration. Pmc. Nail Acad. ScL USA 74 3560... 

ATP extraction has been used for the separation and large-scale purification of enzymes.45 47 Proteins and nucleic acids partition between the two phases differently due to differences in their surface charges and hydrophobic-hydrophilic domains. Proteins generally prefer the aqueous phase rich in... 

R. K. Gerding and R. G. Wolfe, J. Biol. Chem., 244 1164 (1%9). Malic Dehydrogenase. VIII. Large Scale Purification and Properties of Supernatant Pig Heart Enzyme. 

Typical biological compounds that are separated by HIC include, cytochrome P-450, enzymes, DNA polymerase, epidermal growth factor, glycoprotein hormones, human immunoglobulins, human recombinant DNA and canine pancreatic juice proteins. Many HIC techniques have been used for large scale purification of proteins. 

Large scale purification of enzymes in PEG/dextran and PEG/salt have been reported and reviewed by Kula et. al.(18. 19. 20). Examples include pullulanase and 1,4-j-glucan phosphorylase <21 , a-amylase (22), b-galactosidase (jg3), and the general economics of extractive enzyme recovery (24). Process studies have covered the use of continuous centrifuges (18. 19) and countercurrent distribution trains (2j 25). 

Budtz P (1994) Microbial rennets for cheese making. Novo-Nordisk Technical Report, A-6045a Burgess RR (1969) A new method for the large scale purification of Escherichia coli deoxyribonucleicacid-dependent ribonucleic acid polymerase. J Biol Chem 244(22) 6160-6167 Cao L (2005) Carrier-bound immobilized enzymes. Principles, application and design. Wiley-VCH, Weinheim, 563 pp... 

Many reports on IMAC used for purifying pharmaceutically interesting proteins, such as interferons, vaccines and antibodies, have been published but relatively few data exist on actual large-scale purifications of pharmaceutical proteins. However, IMAC offers possibilities for large-scale purification of many industrial enzymes as well as proteins for research in genetics, molecular biology and biochemistryi" 1. 

In previous experiments, wild type and mutant MCAD enzymes were overexpressed on a snM scale, using the plasmid pWt and mutant derivatives, for studies on the cellular lysate. ° However, the yields for large scale purification purposes were very low. To obtain larger amounts of pure protein, the plasmid pTrc has been obtained, which in the case of the wild type increased the yield by about 5-fold (data not shown). 

Recombinant proteins with unique properties can potentially generate new markets and penetrate into existing markets if they can be supplied on a large scale. An ideal system would produce the safest biologically active material at the lowest cost, and would be used in combination with an inexpensive and simple purification process. So far, there have been several examples of the high-yield production of recombinant proteins in transgenic crop plants, mainly in the area of molecular medicines such as antibodies, enzymes and vaccines [45, 48-50]. Modern agricultural practices offer... 

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