Production and Isolation of Enzymes

Hirose, Y, Production and isolation of enzymes. Enzyme Catcdysis in Organic Synthesis, Vol. 1, pp. 41-66, Drauz, K. and Waldmann, H. (Eds.), Wiley-VCH, Weinheim, 2002. 

One of the most important criteria in the evaluation of a new process is the availability of an enzyme114 (see Chapter 20 Tabular Survey of Commercially Available Enzymes). If the enzymes are not commercially available, their isolation and purification can be expensive and time consuming (see Chapter 2 Production and Isolation of Enzymes). The importance of the product to be synthesized may sometimes justify the additional effort. 

Enzyme Production and Isolation. The production and isolation of veratryl alcohol oxidase (VAO) was described earlier (25). Laccase produced from the same 12-day culture (8 litres) was isolated from the supernatant by precipitation at 0°C with ammonium sulfate (80% saturation). The precipitate was suspended in 0.05 M Na acetate buffer, pH 5.0 and dialysed overnight against 4 litres of buffer. The soluble material was concentrated by ultrafiltration (Amicon PM10) to about 60 mL and applied to a DEAE-Bio-gel A column (2.5 cm x 35 cm). The column was washed with 20 mL of the same buffer, then eluted with a linear gradient from 0 to 0.6 M NaCl (total volume 550 mL). Fractions were monitored for VAO and laccase activity as described below. 

Several methods for the production and isolation of extra- and intracellular enzymes from wild-type strains are established during the last years. Highly efficient methods for the scale-up of dehydrogenases have been developed for the enzyme formate dehydrogenase [40, 41]. Alternately, gene cloning can be used successfully to obtain an efficient yield of the desired catalyst. 

Recombinant Proteins from Piants Production and Isolation of Clinically Useful Compounds, edited by Charles Cunningham and Andrew J. R Porter, 1998 2. Protocois in Bioremediation, edited by David Sheehan, 1997 1. Immobilization of Enzymes and Cells, edited by Gordon F. Bickerstajf, 1997... 

The genes that code for the two enzymes necessary for the conversion of EPA into DHA have been identified and cloned from the genomes of the marine microalgae Pavlova and Isochrysis (Pereira et al., 2004). These genes were expressed in yeast and conversion of EPA to DHA was successfully carried out. This study marks the final step in the elucidation of the biosynthetic pathway and isolation of enzymes necessary for production of DHA. Future work in this area may lead to commercial production of DHA using genetically enhanced microorganisms. 

Bio-Research Products Inc., was founded in 1975, and specialized in the isolation, purification and characterization of enzymes and proteins. The company is well known for its production of wheat germ phosphoenolpyruvate carboxylase (PEPC). Currently, it is involved in finished goods and raw material production, through a biomedical contract. Bio-Research Products runs custom services on enzymes, proteins production, diagnostic assays, and other goods for industry, governments, or academia. Bio-Research Products, Inc. also markets a number of enzymes and associated products, and carries out custom synthesis projects. 

Greater purification could be achieved, usually on a smaller scale, if the desired protein had easily measurable properties (e.g. was an enzyme), so that the specific activity of the product and extent of purification could be estimated. Well into the 1950s any enzyme required in a laboratory had first to be isolated by those needing it. With increased knowledge of enzyme properties two further methods of purification were commonly tried heat denaturation of contaminating proteins (with the incidental discovery of some remarkably heat-resistant enzymes), and protein precipitation at the iso-electric point. 

A product of an enzyme-catalyzed reaction should be stable enough over the time course of the experiment, including isolation, identification, and measurement of that product. If not, it may be necessary to alter the reaction conditions to increase the stability of a product. A number of enzymes have been studied at lower temperatures for this reason. The comments made in the substrate stabiUty entry are also pertinent here. See Substrate Stability... 

NORTHRUP, JOHN H. (1891-1987). An American chemist who won a Nobel prize in chemistry in 1946 along with James B. Sumner and Wendell M. Stanley. His work was primarily concerned with isolation and crystallization of enzymes. Many first included the production of the enzyme trypsin in the laboratory and isolation of the first bacterial virus. He was also responsible for producing diphtheria antitoxin in crystalline form. His education was at eastern schools including Harvard. Yale, and Princeton. 

Adapting techniques based on in vitro protein synthesis to the isolation of enzymes requires establishing a link between a nucleic acid-protein complex and product formation. Methods based on binding, analogous to those developed for phage displayed libraries, may be used to enrich catalysts from noncatalysts. In addition, Tawfik and Griffiths (1998) exploited the aqueous core of reverse micelles as artificial compartments... 

G. N. Zaitseva and A. N. Belozersky (1960). The production and utilization of polyphosphates by an enzyme isolated from Azotobacter vinelandii (in Russian). Dokl.Akad. NaukSSSR, 132, 950-954. G. N. Zaitseva and L. Yu. Frolova (1961). The effect of chloramphenicol on phosphorus and nucleic acid metabolism in Azotohacter vinelandii (in Russian). Biokhimiya (Moscow), 26, 200-208. 

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