Enzymes screening kits

Enzyme immunoassay kits are now available for qualitative field testing or for laboratory screening and semiquantitative analysis of pesticides, herbicides, polychlorinated biphenyls (PCBs), mononuclear and polynuclear aromatic hydrocarbons, pentachlorophenol, nitroorganics, and many other compounds in aqueous and soil samples. Certain analytes may be quantitatively determined as well, with a degree of accuracy comparable to gas chromatography or high performance liquid chromatography determination. The method is rapid and inexpensive. 

In current research, oxidoreductases are second in the number of applications of enzymes in organic synthesis. The number of commercially available biocatalysts of this class has increased tremendously during the last few years and various screening kits for oxidation and reduction are sold. Many oxidoreductases are rather easy to handle, though, in contrast to hydrolases, they are dependent on cofactors [22]. 

Fig. 2. Enzyme library organization. Enzymes discovered in organisms and gene banks are organized by activity type into enzyme libraries. These are characterized and can be subdivided into screening kits based on their substrate preferences and synthetic utility...

Screening kits/sets containing samples of the normal commercially available enzymes are also provided by other enzyme suppliers, such as Boehringer Mannheim/Roche (Chirazyme sets for lipases/esterases, aldol reaction kits), Altus Biologies (ChiroScreen Kits TE and EH (based on CLECs, see section 5) for the chiral resolution of alcohols, amines, and esters), Biocatalysts (kits with alcohol dehydrogenases), Enzymatix (lipase biotransformation research kit), and others. 

As more enzyme kits become commercially available, the screening for a suitable catalyst can now be performed in a matter of hours rather than days or weeks. Furthermore, both the screening and biotransformation can be performed by nonspecialists. This increases the likelihood of uptake of a biocatalytic process, as a proof of concept can be more readily obtained without the commitment of considerable resource. For these reasons, the use of ADHs by pharmaceutical companies has increased considerably in recent years. 

Levels of lactate in buttermilk and yoghurt (and blood) were estimated using disposable sensors formed from screen-printed graphite laminated between two polymer sheets [18]. Platinum (deposited by sputter-coating) was the transducing surface. Layers of Nation were added to reduce interference and were surmounted by lactate oxidase in a mixture of polyethyleneimine and poly (carbamoyl) sulphonate hydrogel. The samples were measured in stirred buffer. A good correlation between biosensor results and those obtained with an enzyme kit was claimed but the data had a considerable amount of scatter—if the enzyme kit is taken as the reference method then a more severe analysis of the biosensor results [33] would not have shown them in a... 

Polynuclear aromatic hydrocarbons (PAH) are aromatic compounds that contain two or more benzene rings fused together. These substances may be analyzed by HPLC, GC, GC/MS and enzyme immunoassay techniques. The latter is a rapid screening method that may be applied for a qualitative or semiquanti-tative determination Test kits are commercially available for such screening. U.S. EPA (1995) has specified a method (Draft Method 4035) that detects a range of PAHs to different degrees and measures the composite of individual responses to determine the total PAHs in the sample. 

PCBs in soils and wastewaters can be rapidly screened on site or in the laboratory by immunoassay technique (Chapter 1.13). Immunoassay test kits are now commercially available from many suppliers. The samples can be tested at the calibration levels of 1 to 50 ppm. The kit primarily contains antibody-coated test tubes or magnetic particles, assay diluent, PCB-enzyme conjugate, a color-forming substance, and a solution to quench the reaction. The method does not distinguish accurately one Aroclor from another. PCBs can be measured semiquantitatively by comparing the optical density of the color formed in the sample against a set of calibration standards using a spectrophotometer. 

Although many aldolases have been characterized for research purposes, these enzymes have not been developed commercially to any significant extent. This is likely due to the availability of the various biocatalysts and the need for dihydroxyacetone phosphate (DHAP) (44), the expensive donor substrate required in nearly all aldolase reactions. A number of chemical and enzymatic routes have been described for DHAP synthesis, which could alleviate these concerns [12], In terms of the enzyme supply issue, this may change with the introduction of products from Boehringer Mannheim and their Chirazyme Aldol reaction kit. They have three kits, each containing a different aldolase fructose-1,6-diphosphate FruA) (EC 4.1.2.13), L-rhamnulose-1-phosphate RhuA (EC 4.1.2.19), and L-fuculose-1-phosphate (FucA) (EC 4.1.2.17). As more screening... 

FDA approves the first enzyme linked immunosorbent assay (ELISA) test kit to screen for antibodies to HIV... 

Screening test kits - enzyme linked immuno-sorbent assay (ELISA)... 

The ELISA kits are based on the use of selective antibodies raised against specific PAHs, which are attached to a solid matrix support. These are combined with sensitive enzyme reactions (see Section 6.4.4). The kits provide high selectivity and can provide an immediate answer on site, but the disadvantages are that they can be difficult to use in the less than ideal conditions on site and they can only usually give a range of concentrations, e.g. >50, 20-50, 1-20 or <1 mg/kg of total PAHs and mainly rely on a manual rapid cold shake extraction technique which may only extract a small fraction of the PAHs in some samples. In addition, the test kits can prove quite expensive for a screening analysis. 

Every development of a biocatalytic reaction starts with a screening for the most appropriate enzyme. Some companies offer screening sets (or kits) containing the most commonly used enzymes (Table 20-2). Some Sets are single use (Altus, ThermoGen) while others contain enough material to perform depending on the scale 5-20 experiments (BioCatalytics, Fluka, Roche). These sets may include enzymes available on industrial scale or on research scale only. 

The following companies offer screening set/kits for quick enzyme selection (Table 20-2). While some companies include only industrial scale enzymes, others contain enzymes only available at lab quantities. Diversa Co. offers an enzyme subscription program for lipases, esterases, nitrilases, cellulases, glycosidases, phosphatases, and transaminases (aminotransferases). 

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