Enzymes Used

The key to producing Enzyme-modified cheeses (EMCs) is the proper choice of enzymes and their apphcation under optimal conditions. The majority of research in this area has been on the hpase enzymes. A wide variety of hpase enzymes are commercially available. These enzymes come primarily from animal or microbial sources. Lipase choice is critical because each hpase has a specificity for hydrolysis thereby determining the free fatty acid profile (a key determinant of flavor) in the EMC [54]. 

Kid PGE - sharp peppery often called piccante flavor, which quickly clears from the palate 

Lamb PGE - peccorino flavor described as dirty sock  

The use of any animal source enzyme brings concern for religious (Kosher or Halal) or dietary choices (e.g., vegetarian). These concerns as well as cost issues have promoted the development of miCTObial lipases and esterases for this purpose. If these enzymes are approved as GRAS (Generally Recognized as Safe), they can be used in this application. 

As one would expect, microbial enzymes range in specificity. For example, lipases from Candida cylindracea and Staph aureus have little specificity while those from Geotrichum candidum are specific for long chain acids [57], which are of little value in this application. Fortunately, most microbial hpases show specificity for the one and three positions of the triglyceride where the short chain fatty acids are found. While substantial information exists in the literature characterizing lipase enzyme activity to help in enzyme choice [58], enzymes must be evaluated for function in a model system. This often is done in a curd slurry system, which is produced by blending two parts of fresh cheese curd plus one part sterile dilute salt solution (5.2% 

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Bulk Enzymes. Enzymes such as proteases, amylases, glucose isomerases, and rennin are used in food processing. Similarly proteases and Hpases are used in detergents. CeUulases and xylanases are used in the paper pulp industry. The genes for most of the enzymes used in the various commercial processes have been cloned and overexpressed. Rennin (chymosin) produced from E. coli and A. nigerhas been approved by FDA for use in the dairy industry. 

Low Temperature Process. The low temperature process was developed when B. licheniformis and B. stearothermophilus a-amylases became commercially available in the 1970s. These enzymes ate more thermostable, more acidutic, and requite less calcium for stabiUty than the B. subtilis enzyme used in the EHE process. Consequendy, the high temperature EHE heat treatment step was no longer requited to attain efficient Hquefaction. 

Immobilization. Enzymes, as individual water-soluble molecules, are generally efficient catalysts. In biological systems they are predorninandy intracellular or associated with cell membranes, ie, in a type of immobilized state. This enables them to perform their activity in a specific environment, be stored and protected in stable form, take part in multi-enzyme reactions, acquire cofactors, etc. Unfortunately, this optimization of enzyme use and performance in nature may not be directiy transferable to the laboratory. 

Enzymes as Antidotes. Rhodanese [9026-04-4] given along with thiosulfate to counteract cyanide poisoning in mice (224) was the first enzyme used as an antidote. This combination raised the LD q of potassium cyanide in mice by eightfold (224). 

To decide between these alternatives, a fixed point of reference, such as one of the ends of the fragment, must be identified or labeled. The task increases in complexity as DNA size, number of restricdon sites, and/or number of restricdon enzymes used increases. 

Studies on the oxygen activation mechanisms by new heme enzymes using hemoprotein mutants and synthetic heme models 96YGK1046. 

Therapeutic Function Proteolytic enzyme used in chemical nucleolysis Chemical Name See "Structural Formula" below. 

Therapeutic Function Enzyme used to prevent wound adhesions Chemical Name Sae Structural Formula Common Name —... 

Auxotrophic mutants are mutants that miss one or more of the enzymes used in the biosynthetical pathway for one or more amino adds. In practice this means that the mutant needs one or more key metabolites which it cannot synthesise for growth in its growth medium. For example, consider Figure 8.4. 

The enzymes used for modification of oils and fats are extraoelluar microbial lipases. They are excreted by micro-organisms into the growth medium to catalyse the degradation of lipids, and can be produced on a large scale by fermentation. 

The preparations of luciferin (Ln, an electron acceptor) and soluble enzyme used were crude or only partially purified. The luciferase was an insoluble particulate material, possibly composed of many substances having various functions. Moreover, the luciferin-luciferase reaction was negative when both luciferin and luciferase were prepared from certain species of luminous fungus. It appears that the light production reported was the result of a complex mechanism involving unknown substances in the test mixture, and probably the crucial step of the light-emitting reaction is not represented by the above schemes. 

For production of an enzyme used for synthesis of a sun protection lotion, the required kinetic data and constants are ... 

P-Lactamases are enzymes that hydrolyze the P-lactam ring of P-lactamantibiotics (penicillins, cephalosporins, monobactams and carbapenems). They are the most common cause of P-lactam resistance. Most enzymes use a serine residue in the active site that attacks the P-lactam-amid carbonyl group. The covalently formed acylester is then hydrolyzed to reactivate the P-lacta-mase and liberates the inactivated antibiotic. Metallo P-lactamases use Zn(II) bound water for hydrolysis of the P-lactam bond. P-Lactamases constitute a heterogeneous group of enzymes with differences in molecular structures, in substrate preferences and in the genetic localizations of the encoding gene (Table 1). 

Similar reaction mechanisms, involving general base and metal ion catalysis, in conjunction with an OH nucleophilic attack, have been proposed for thermolysin (Ref. 12) and carboxypeptidase A (Refs. 12 and 13). Both these enzymes use Zn2+ as their catalytic metal and they also have additional positively charged active site residues (His 231 in thermolysin and... 

The permeability of the films to paracetamol as a model compound was dependent on film composition and was markedly increased after exposure to pectinolytic enzymes, used to mimic conditions in the colon. Similar formulations, apphed as a film coat to tablets, were used with colonic conditions for an increased release rate [242],... 

A number of enzymes are in common use and each of these cleaves the polypeptide backbone adjacent to a particular amino acid residue. The one used for a particular investigation is therefore chosen for the specificity with which it will cleave the polypeptide backbone of the protein being studied. A number of the enzymes used for this purpose are shown in Table 5.4. 

Table 5.4 Enzymes used for the cleavage of proteins for sequence investigations and the corresponding amino acid residues at which they break the peptide backbone...

The presence of three polypeptides in Table 5.8 tliat were not predicted from the relationship between the amino acid sequence and the enzyme used for digestion is worthy of note when interpretation of data of this sort is undertaken. The MALDI data showed six further unexpected polypeptides, none of which were detected in the LC-MS data ... 

Each of these compounds, 53-56, was shown to be a very effective competitive inhibitor of the enzyme with respect to the fructose 1,6-diphosphate, whereas several other analogs, including acyclic structures, had no effect. These and other results suggest that the furanose form of the sugar diphosphate is the active form in the enzymatic reaction (105). More recent studies using rapid quenching techniques and C-nmr measurements have confirmed this hypothesis and indicate that the enzyme uses the a anomer 52 much more rapidly than the 3 anomer 50 and probably uses the a anomer exclusively (106). 

Glyphosate kills plants by specifically inhibiting one critical plant enzyme used in the biosynthesis of aromatic amino acids. As such, glyphosate was one of the first commercially successful herbicides to have a primary identified enzyme site of action in plants (4,5). 

Plasma also contains numerous other enzymes that perform no known physiologic function in blood. These apparently nonfunctional plasma enzymes arise from the routine normal destruction of erythrocytes, leukocytes, and other cells. Tissue damage or necrosis resulting from injury or disease is generally accompanied by increases in the levels of several nonfunctional plasma enzymes. Table 7-2 lists several enzymes used in diagnostic enzymology. 

Table 7-2. Principal serum enzymes used in clinical diagnosis. Many of the enzymes are not specific for the disease listed.

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