Enzyme source

The large majority of enzymes used for biotransformations in organic chemistry are employed in a crude form and are relatively inexpensive. The preparations typically contain only about 1-30% of actual enzyme, the remainder being inactive proteins, stabilizers, buffer salts, or carbohydrates from the fermentatirai broth from which they have been isolated. It should be kept in mind, that crude preparations are often more stable than purified enzymes. 

Roberts SM, Turner NJ, Willetts AJ, Turner MK (1995) Introduction to Biocatalysis Using Enzymes and Micro-organisms. Cambridge University Press, Cambridge 

Hough DW, Danson MJ (1999) Curr. Opinion Chem. Biol. 3 39 

Bell G, Hailing PJ, Moore BD, Partridge J, Rees DG (1995) Trends Biotechnoi. 13 468 

Koskinen AMP, Klibanov AM (eds) (1996) Enzymatic Reactions in Organic Media. Blackie 

The first question that should be addressed is on the source of the enzymes that are to be screened. There are advantages and disadvantages to each source, and these have been compiled in Table 1. 

In view of the high rate of hydrolysis by cholinesterases, it is usually possible to work with tissue homogenates, whole cells (erythrocytes), or partly purified enzymes. Only in a few cases have methods for the preparation of highly purified or crystalline ChE s been developed. 

The enzyme is present in the stroma of red blood cells and can be isolated, after hemolysis by distilled water, according to the method of Cohen and Warringa (2). The procedure gives a 250-400-fold purification. 

True Cholinesterase from the Electric Organs of Eleclrophorus electricus or Torpedo marmorata 

The electric organs of various fish are the richest source of true cholinesterase and yield very pure enzyme preparations by the method of Rothen- 

This cholinesterase has been brought to the highest stage of purification by Edsall (4) and co-workers during their work on plasma fractionation. Pseudo-cholinesterase is contained in fraction IV-6-3 and has been obtained in crystalline form. 

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Enzymatic Process. Chemically synthesized substrates can be converted to the corresponding amino acids by the catalytic action of an enzyme or the microbial cells as an enzyme source, t - Alanine production from L-aspartic acid, L-aspartic acid production from fumaric acid, L-cysteine production from DL-2-aminothiazoline-4-catboxyhc acid, D-phenylglycine (and D-/> -hydtoxyphenylglycine) production from DL-phenyUiydantoin (and DL-/)-hydroxyphenylhydantoin), and L-tryptophan production from indole and DL-serine have been in operation as commercial processes. Some of the other processes shown in Table 10 are at a technical level high enough to be useful for commercial production (24). Representative chemical reactions used ia the enzymatic process are shown ia Figure 6. 

A variety of enzyme sources have been used. Some examples are given in Table 63. 

Adenylyl Cyclases. Table 4 Nucleotide inhibitors of adenylyl cyclase. Enzyme source and assay conditions were as for Table 3. Values obtained for 3 -ATP are overestimations due to the formation of 2 3 -cAMP from 3 -ATP that occurs nonenzymatically in the presence of divalent cation... 

Enzyme (Source) K (Lactone) (mAf) K, (Hexose) (mAf) K, (Hexose) Kf (Lactone) References... 

The peroxidase-catalyzed polymerization of m-alkyl substituted phenols in aqueous methanol produced soluble phenolic polymers. The mixed ratio of buffer and methanol greatly affected the yields and the molecular weight of the polymer. The enzyme source greatly affected the polymerization pattern of m-substituted monomers. Using SBP catalyst, the polymer yield increased as a function of the bulkiness of the substituent, whereas the opposite tendency was observed when HRP was the catalyst. 

Rapidase C-80 (Gist -brocades) was used as enzyme source. The fractionation procedure of the crude preparation included chromatography on Bio-Gel PIO (100-200 mesh), DEAE-Bio-Gel A, and Bio-Gel HTP (Bio-Rad, Richmond, CA, USA). Other column materials used were cross-linked alginate (degree of cross-linking 2.34, prepared in our laboratory). Phenyl Superose HR 5/5 and Mono Q HR 5/5 (Pharmacia Biotech, Uppsala, Sweden). 

The A. niger PG preparation was suitably diluted with distilled water and was used as the enzyme source. Polygalacturonic acid was prepared as a one percent solution of in distilled water. 

Nutrient molecule Enzyme Enzyme action Enzyme source Action site... 

Vieille, C. and Zeikus, G.J. (2001) Hyperthermophilic enzymes sources, uses, and molecular mechanisms for thermostability. Microbiology and Molecular Biology Reviews, 65 (1), 1 43. 

It was recently reported that. >97% of BaP 4,5-epoxide metabolically formed from the metabolism of BaP in a reconstituted enzyme system containing purified cytochrome P-450c (P-448) is the 4S,5R enantiomer (24). The epoxide was determined by formation, separation and quantification of the diastereomeric trans-addition products of glutathione. Recently a BaP 4,5-epoxide was isolated from a metabolite mixture obtained from the metabolism of BaP by liver microsomes from 3-methylcholanthrene-treated Sprague-Dawley rats in the presence of the epoxide hydrolase inhibitor 3,3,3-trichloropropylene oxide, and was found to contain a 4S,5R/4R,5S enantiomer ratio of 94 6 (Chiu et. al., unpublished results). However, the content of the 4S,5R enantiomer was <60% when liver microsomes from untreated and phenobarbital-treated rats were used as the enzyme sources. Because BaP 4R,5S-epoxide is also hydrated predominantly to 4R,5R-dihydro-... 

Monophenolase activity shows a characteristic lag period before the maximum velocity of the hydroxylation step is reached. The time required to reach the steady-state rate depends on several factors enzyme source concentration of monophenol ... 

Restriction enzyme Source DNA recognition sequence and cleavage sitea... 

In clinical chemistry the activity of an enzyme should be expressed as units and must be related to the amount of material used as enzyme source. Many authors define the unit of enzyme activity as that amount of enzyme in a given volume or weight unit of material which causes a certain change of absorbance [A log (I0//)] at 340 or 366 mp per time unit under defined, but varying conditions, e.g., A A, = 1.000 or 0.001 per minute at 25°C. In European literature activities are often given as Biicher-Einheiten (B5) where the unit is defined as the amount in 1.00 ml medium causing the change of A, of 0.1 per 100 seconds at 366 mp and 25°C. 

The assay methods reported in the literature vary with respect to the use of methylene blue or another dye, and to the coenzyme used, depending on enzyme source, etc. 

Kumar, V., Rock, D.A., Warren, C.J., Tracy, T.S. and Wahlstrom, J.L. (2006) Enzyme source effects on CYP2C9 kinetics and inhibition. Drug... 

During a 30 minute incubation period, low levels of aldrin epoxidation (30-150 picomoles dieldrin/mg protein) were measured compared to those observed using enzyme sources such as aquatic Trichoptera Limnephilus sp. gut homogenates (1 pmole/mg protein 28) or rat liver homogenates (3000 pmoles/mg protein unpublished) under similar incubation conditions. Anisole metabolism based upon substrate disappearance was detectable but less than 5 picomoles/mg protein were transformed during the incubation period. Characteristics of the enzyme system are incompletely described owing to the low and variable levels of activity which have been obtained. 

Several modifications of incubation conditions have neither stabilized the system nor enhanced activity. Acetone and methanol have been used as substrate carriers without affecting activity. Similarly, addition of NADH to the incubation media did not effect epoxidation. The enzymatic nature of the system has been confirmed by use of heat treated homogenates (100 C, 1 min). Incubation temperatures of 8, 20, and 30 resulted in progressively greater epoxidation rates and provided no evidence of heat lability. Thus, at this time it is not possible to identify a superior enzyme source for comparative studies in spite of the fact that in vivo measurements indicate oxidative metabolic activity in living mussels. 

All fish were sacrificed between 5 00 and 9 00 a.m. Washed micro-somes were prepared from liver homogenates as described previously (3). When whole liver homogenates were used as the enzyme source, 10% w/v homogenates were prepared in 0.15 M KC1, 0.02 M HEPES (N-2-hydroxypiperazine-N -2-ethane sulfonic acid Sigma) buffer (pH... 

Enzyme source (100 mg) Total activity (U) Total soluble Specific activity... 

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