Commonly Used Enzyme Preparations

Amino acid acylase Porcine kidney, Aspergillus melleus 

Alcaligenes sp. Amano, Biocatalysts, Meito Sangyo (QL), Boehringer (Chirazyme L-10) 

Aspergillus niger Aldrich, Amano (A, AP6), Biocatalysts, Fluka, Novo (Palatase), Evonik 

Candida antarctica A Boehringer (Chirazyme L-5), Ruka, Novo (SP526) 

Candida antarctica B Ruka, Novo (SP525 or SP435 ), Boehringer (Chirazyme L-2) 

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The majority of commonly used enzyme preparations are available through chemical suppliers. Nevertheless, for economic reasons, it may be worth contacting an enzyme producer directly, in particular if bulk quantities are required. For a list of enzyme suppliers see the appendix (Chap. 5). After all, the exact structure of a Grignard-reagent is still unknown. 

There has been a steady growth ia the economic importance of therapeutic ensymes, with sales reaching hundreds of millions of dollars per year as of 1992. Table 2 Hsts the trade names and costs of some of the more commonly prescribed enzyme preparations. Despite the magnitude of use of these products, the manufacture and sale of therapeutic enzymes represents a comparatively small fraction of the production and profits of the pharmaceutical houses that market them. 

Although acid hydrolysis is most commonly used to prepare the starting sirup, the liquefying amylase from B. subtilis can be used to thin the initial starch paste without the need for add resistant tanks. The process then becomes an all-enzyme process. The all-enzyme process produces a smaller amount of reversion products than the acid-enzyme process. 

For preparative purposes fermenting baker s yeast (Saccharomyces cerevisiae) is commonly used instead of a purified enzyme preparation. However, isolated pyruvate decarboxylates can also be used30. In this context, the most important substrate is benzaldehyde31 which is converted by n-glucosc fermenting yeast to (7 )-l-hydroxy-l-phenyl-2-propanone. This conversion has gained considerable industrial importance because ( )-l-hydroxy-1-phenyl-2-propanonc is an important precursor for the synthesis of (-)-cphedrin. 

Currently, a common form of activated mPEG used for preparation of therapeutic enzymes is mPEG-succinate-N-hydroxysuccinimide ester (SS-PEG) (11). It reacts with proteins in short periods of time under mild conditions, producing extensively modified conjugates with well preserved biological activity. However, the ester linkage between the polymer and the succinic acid residue has limited stability in aqueous media (5,12). 

A variation of the above method can be used, wherein the enzyme is first activated with SMCC and conjugated to a thiolated (strept)avidin molecule. This approach probably is the most common way of preparing (strept)avidin-enzyme conjugates, and since the preactivated enzymes are readily available (Thermo Fisher), it also may be the easiest. 

In view of the conductive and electrocatalytic features of carbon nanotubes (CNTs), AChE and choline oxidases (COx) have been covalently coimmobilized on multiwall carbon nanotubes (MWNTs) for the preparation of an organophosphorus pesticide (OP) biosensor [40, 41], Another OP biosensor has also been constructed by adsorption of AChE on MWNTs modified thick film [8], More recently AChE has been covalently linked with MWNTs doped glutaraldehyde cross-linked chitosan composite film [11], in which biopolymer chitosan provides biocompatible nature to the enzyme and MWNTs improve the conductive nature of chitosan. Even though these enzyme immobilization techniques have been reported in the last three decades, no method can be commonly used for all the enzymes by retaining their complete activity. 

In AChE-based biosensors acetylthiocholine is commonly used as a substrate. The thiocholine produced during the catalytic reaction can be monitored using spectromet-ric, amperometric [44] (Fig. 2.2) or potentiometric methods. The enzyme activity is indirectly proportional to the pesticide concentration. La Rosa et al. [45] used 4-ami-nophenyl acetate as the enzyme substrate for a cholinesterase sensor for pesticide determination. This system allowed the determination of esterase activities via oxidation of the enzymatic product 4-aminophenol rather than the typical thiocholine. Sulfonylureas are reversible inhibitors of acetolactate synthase (ALS). By taking advantage of this inhibition mechanism ALS has been entrapped in photo cured polymer of polyvinyl alcohol bearing styrylpyridinium groups (PVA-SbQ) to prepare an amperometric biosensor for... 

Asymmetric synthesis refers to the conversion of an achiral starting material to a chiral product in a chiral environment. It is presently the most powerful and commonly used method for chiral molecule preparation. Thus far, most of the best asymmetric syntheses are catalyzed by enzymes, and the challenge before us today is to develop chemical systems as efficient as the enzymatic ones. 

A requirement of biocatalysis in neat organic solvent is the use of a dehydrated form of an enzyme that displays the desired activity. A number of techniques are available for the preparation of dehydrated enzymes, some of which are discussed in a recent review by Griebenow and Barietta.The techniques that have been most commonly used are ... 

Numerous testing systems and protocols have been used to study 5-LO inhibitors in different laboratories, complicating attempts to compare compounds and series. In vitro, a variety of both cell-free and cellular preparations have been employed as primary screens. The most commonly used cell-free system is the crude cytosolic fraction from broken RBL-1 cells [25] various broken neutrophil preparations are also used, and more recently purified enzymes have occasionally been employed. The formation of 5-LO products is generally determined by radioimmunoassay or (in older work) HPLC or bioassay methods. 

Industrial enzymes are commonly used at levels of 0.1-0.5 per cent of the substrate being processed, with rare exceptions above these levels. Therefore, when the actual amount of any constituent of the preparation is evaluated as a constituent of the final processed product, it is unlikely that it will make a significant contribution in relation to other similar materials, present or introduced, in the total process. 

Isozymes are also a common presence in enzyme preparations and they can often be detected via polyacrylamide gel electrophoresis. The detected presence of isozymes may result in the need for further purification steps and the kinetic characterization of each isozyme. It may be necessary to use nondenaturing electrophoretic procedures to separate the different isozymes. See Isozymes Enzyme Concentration... 

The pyrophosphorylase procedures have been applied widely for preparation of various naturally occurring a-D-glucopyranosyl esters of nucleoside pyrophosphates. For synthetic purposes, it is common to use crude or partially purified enzyme preparations, which may be a mixture of individual, specific enzymes. The synthesis of adenosine 5 -(a-D-glucopyranosyl pyrophosphate) was achieved with pyrophos-phorylases from Arthrobacter viscosus 216,217 Azotobacter vinelandii,52... 

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