Types of enzymes used

We set up a restriction digest using 10 pg of DNA in a final reaction volume of approximately 10 pL. The reaction is mixed and incubated in a 37°C water bath for 1 h (temperature and incubation time may vary with type of enzyme used check supplier s protocol). 

When students learn about enzymes, it is nearly always stated, very dogmatically, that enzymes will act on only one chemical—they have very narrow substrate specificity. Indeed, when introducing the concept of enzyme action at an elementary level, the analogy of the lock and key is often used to illustrate the concept of specificity. In other words, students are told that for every product found in a cell, there will be one enzyme that has made that product and that enzyme will make no other product. This idea was extended to the idea of one gene-one enzyme-one reaction. There is indeed a considerable body of evidence to support the view that many enzymes do have narrow substrate specificity. However, exceptions were known to this rule . But more importantly, the types of enzymes used as good examples of the lock and key concept were drawn... 

As well as alternative substrates, there have been a number of studies on inhibitors of flavocytochrome 62- Known inhibitors include D-lactate (16, 92-95), pyruvate (16, 58, 60, 96), propionate (96), DL-man-delate (90, 91), sulfite (60), and oxalate (16, 60, 97). Values of K, for these inhibitors and the conditions and types of enzyme used can be found in the papers referenced above. All of the above inhibitors show typical competitive inhibition except pyruvate and oxalate, for which mixed inhibition has been observed (60, 97). Inhibition has also been reported for excess substrate with the intact enzymes from both S. cerevisiae (16) and H. anomala (92), though not apparently with the cleaved enzyme from S. cerevisiae (16). It is possible that inhibition by excess substrate arises either from different binding modes at the active site or from a second lower affinity binding site elsewhere on the enzyme. 

The types of enzymes used by organic chemists vary widely and include such well-known biocataiysts as lipases, esterases, oxidoreductases, oxinitrilases, transferases and aldolases [4]. An example which illustrates the industrial application of a lipase concerns the kinetic resolution of a chiral epoxy ester used as the key intermediate in the synthesis of the calcium antagonist Diltiazem, a major therapeutic in the treatment of high blood pressure [6] (Fig. 1). In developing the industrial process for the production of this drug, many different lipases were screened, but only the bacterial lipase from Serratia marescens showed both a sufficiently high activity and enantioselectivity. The intermediate is produced industrially on a scale of 50 tons/year. 

All this may explain why many publications give only incomplete information on the exact type of enzyme used in the work described and why many references to enzymes are simply wrong. The author strongly recommends to provide at least the following information ... 

Fig.1. Overview of the types of enzymes used in around 100 commercialised biotransformations [3]...

So far, most peroxidase-catalyzed oxidative polymerizations have been carried out using the enzyme horseradish peroxidase (HRP). Another useful peroxidase that catalyzes the oxidative polymerization of phenols is soybean peroxidase (SBP). While the use of either HRP or SBP may often lead to similar products and results [77], the enzyme activity, yield, and molecular weight of the resulting polymers can also sometimes depend strongly on the type of enzyme used for the polymerization process. For example, SBP was foimd to be superior to HRP for the efficient polymerization of bisphenol A [140], but the polymerization of phenol with SBP afforded... 

One of the merits of an enzyme biosensor is its versatility. The selectivity of biosensors directly relates to the type of enzyme used for constructing biosensors. Up to now, many kinds of enzyme sensors have been reported, other lhan glucose biosensors, using a variety of enzymes. A miniaturization of the sensor body is another possible advantage of enzyme sensors. In fact, miniature sensors sized in microns have been prepared... 

In the next section, recent studies on the determination of OP nerve agents and pesticides based on electrochemical biosensors will be discussed. The electrochemical biosensors used for detecting OP compounds can be divided into three types, depending on the type of enzymes used for constructing biosensors (i) choline esterase (ChE)-ChOx bienzyme-modified biosensors, (ii) ChE-modified biosensors, and (iii) organophospho-rus hydrolase (OPH)-modified biosensors. Also, OP biosensors based on transducers other than electrochemical devices are discussed in this chapter. 

Undried starch, usually from wet milling, is pumped as a slurry to the conversion plant where it undergoes one or more hydrolytic processes to yield mixtures of various carbohydrates in the form of syrups or as crystalline dextrose. The kind and amount of the various carbohydrates obtained depend upon the type of hydrolysis system used (acid, acid-enzyme, or enzyme-enzyme), the extent to which the hydrolytic reaction is allowed to proceed, and the type of enzyme used. The fact that most starches consist of two different kinds of polymers (amylose and amylopectin) also has an effect on the nature of the products obtained. 

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. 

Because enzymes can be intraceUularly associated with cell membranes, whole microbial cells, viable or nonviable, can be used to exploit the activity of one or more types of enzyme and cofactor regeneration, eg, alcohol production from sugar with yeast cells. Viable cells may be further stabilized by entrapment in aqueous gel beads or attached to the surface of spherical particles. Otherwise cells are usually homogenized and cross-linked with glutaraldehyde [111-30-8] to form an insoluble yet penetrable matrix. This is the method upon which the principal industrial appHcations of immobilized enzymes is based. 

The reactant is referred to as a substrate. Alternatively it may be a nutrient for the growth of cells or its main function may require being transformed into some desirable chemical. The cells select reactants that will be combined and molecules that may be decomposed by using enzymes. These are produced only by living organisms, and commercial enzymes are produced by bacteria. Enzymes operate under mild conditions of temperature and pH. A database of the various types of enzymes and functions can be assessed from the following web site http //www.expasy.ch/enzyme/. This site also provides information about enzymatic reactions. 

At present, photosynthetic organisms are not generally used as biocatalysts for bioconversion of organic compounds except for bioremediation of pollutants in the environment, although they are environment-friendly catalysts, and they may contain unusual type of enzymes to establish new reactions. Development of bioreactors specially developed for photosynthefic organism-catalyzed reaction as well as finding effective photosynthetic organisms as a biocatalyst are required in the future. 

The availability of these novel enzymes, next to the known pectic enzymes, offer new opportunities to use them as analytical tools in revealing the structure of oligo- and polysaccharides [31,32]. In contrast with frequently used chemical degradation methods, which usually have a poor selectivity, these enzymes act in a deflned way. To be able to recognize different structural units within the polymer, endo-acting types of enzyme are preferred, although accessory enzymes might be essential as well [30]. 

Most suitable would be the use of a perfectly NH4+ ion-selective glass electrode however, a disadvantage of this type of enzyme electrode is the time required for the establishment of equilibrium (several minutes) moreover, the normal Nernst response of 59 mV per decade (at 25° C) is practically never reached. Nevertheless, in biochemical investigations these electrodes offer special possibilities, especially because they can also be used in the reverse way as an enzyme-sensing electrode, i.e., by testing an enzyme with a substrate layer around the bulb of the glass electrode. 

Molecular methods used to uncover mutations are subject to several variables. The anticoagulants used for blood collection can affect digestion with restriction enzymes and amplification reactions. The type of detergent used in cell lysis can affect amplification of DNA by inhibiting the DNA-amplifying enzyme such as the taq polymerase used in the polymerase chain reaction (116). The control of contamination is crucial in ensuring the quality of results obtained by molecular analysis (117). 

Liposome conjugates may be used in various immunoassay procedures. The lipid vesicle can provide a multivalent surface to accommodate numerous antigen-antibody interactions and thus increase the sensitivity of an assay. At the same time, it can function as a vessel to carry encapsulated detection components needed for the assay system. This type of enzyme-linked immunosorbent assay (ELISA) is called a liposome immunosorbent assay or LISA. One method of using liposomes in an immunoassay is to modify the surface so that it can interact to form biotin-avidin or biotin-streptavidin complexes. The avidin-biotin interaction can be used to increase detectability or sensitivity in immunoassay tests (Chapter 23) (Savage et al., 1992). 

The concept of microbial models of mammalian metabolism was elaborated by Smith and Rosazza for just such a purpose (27-32). In principle, this concept recognizes the fact that microorganisms catalyze the same types of metabolic reactions as do mammals (32), and they accomplish these by using essentially the same type of enzymes (29). Useful biotransformation reactions common to microbial and mammalian systems include all of the known Phase I and Phase II metabolic reactions implied, including aromatic hydroxylation (accompanied by the NIH shift), N- and O-dealkylations, and glucuronide and sulfate conjugations of phenol to name but a few (27-34). All of these reactions have value in studies with the alkaloids. 

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