Future enzyme modification

The structural varieties of hemicelluloses offer a number of possibilities for specific chemical, physical, and enzymic modifications. Future advancements will be based on the synthesis of hemicellulose-based polymers with new functionalities and with a well-defined and preset primary structure both on the level of the repeating imit and the polymer chain. Hemicelluloses have also started to be attractive to synthetic polymer chemists as... 

The core-enzymes, prepared in our laboratory, and containing the active centers, were successfully crystallized (Dr. Jones, Uppsala, communicated) and tertiary structures will be described in the near future. Chemical modification studies on these enzymes are currently being undertaken in our laboratory identification of important catalytic residues and location of the active centers will lead to more functional information on these enzymes. Other cellulases such as some endoglucanases from Clostridium thermocel-lum (EG A, EG B, EG D) (10) and EngA and Exg from Cellulomonas fimi (19) also contain sequences of conserved, terminally located and sometimes reiterated, amino acids. Some of these sequences are preceded by proline-serine rich domains. Thus, a bistructural-bifunctional organization seems to be a rather common feature among cellulases, at least for EngA and Exg from C. fimi and the enzymes from Trichoderma reesei. 

Another completely different type of future enzymes are the oxidases, which are able to oxidize selectively the hydroxyl groups in polymers. An example of this type of enzyme is galactose oxidase, which transforms the hydroxyl group in C-6 to the aldehyde group (44), This enzyme has been known for over 30 years, but its use in the modification of galactomannas has been restricted due to the lack of a suitable industrial enzyme preparation. The possible applications of galactose oxidases are discussed in more detail in another chapter in this book. Hitherto this is the only known oxidase to act on hemicelluloses, but hopefully more enzymes of this type will be discovered in the future. 

However, certain limitations do exist that need to be considered. Although enzymes necessary for post-translational modifications can be added, in principle there is currently no productive system available for the preparation of glycosylated proteins, although some interesting results have already been obtained [161]. Also, the expression of functional membrane proteins in quantities necessary for structural analysis will be a challenging task for the future. 

Aside from the Maillard reaction, other covalent modifications of amino acids and proteins are possible within the caries lesion, which merit future investigation. For example, certain oral microorganisms excrete y-glutamyl transferases. These enzymes catalyse the formation of cross-links between glutamic acid and lysine residues of proteins. In addition, N-acyl amino acids are present in plaque, which adsorb to mineral surfaces. 

Chemical modification of surface residues of HRP is one method which may offer some improvement in thermal or long-term stability of the enzyme. The -amino groups of the six surface Lys residues can be modified by reaction with carboxylic anhydrides and picryl sulfonic acid (296). In this example the number of sites modified was found to be more significant than the chemical nature of the modification, at least as a criterion for improved stability. Other methods explored include the use of bifunctional crosslinking reagents to couple surface sites on the enzyme (297). Future developments are likely to be concerned with the selection of site-directed mutants of HRP C that show enhanced thermal stability. Dramatic increases in thermal stability of up to 190-fold have been reported recently for mutants of Coprinus cinereus peroxidase (CIP) generated using a directed evolution approach (298). 

As biochemical tools, suitable and sensible imino sugar modifications will certainly become helpful intelligent probes and reporters addressing some of the future needs of research in carbohydrate-active enzymes. 

Thus, a brief puff of chemical neurotransmission from a presynaptic neuron can trigger a profound postsynaptic reaction, which takes hours to days to develop and can last days to weeks or even longer. Every conceivable component of this entire process of chemical neurotransmission is a candidate for modification by drugs. Most psychotropic drugs act on the processes that control chemical neurotransmission at the level of the neurotransmitters themselves or of their enzymes and especially their receptors. Future psychotropic drugs will undoubtedly act directly on the biochemical cascades, particularly on those elements that control the expression of pre- and postsynaptic genes. Also, mental and neurological illnesses are known or suspected to affect these same aspects of chemical neurotransmission. 

As mentioned earlier, the immunoassay can be done with a number of procedural modifications and in this instance one must substitute the isotope with another molecule (fluorescent dye, magnetic particle, enzyme) which can be measured and therefore serve as the source of tracer. For our initial studies we have chosen to use the enzyme immunoassay (EIA) system. At the present time the EIA is still in its infancy and although a number of successful EIA s have been developed the method cannot be considered a panacea (34). The future of this assay appears to be very bright and exciting, and there is considerable interest in the application of the EIA to problems in both microbiology and clinical medicine (34). Many of the procedures and protocols are derived from RIA procedures and the EIA, like the RIA, has the potential to be performed in a multitude of procedural variations but, for the purpose of this manuscript we will describe only the system we have chosen for our use. 

Plants represent a precious renewable resource and an important treasure chest for the rich variety and enormous diversity of flavonoid compounds. It is evident that flavonoids exhibit a broad range of functions, most of which are dependent on the presence of various modifications to the basic carbon skeleton. Several classes of enzymes are responsible for the wide array of metabolites produced. Their activities are not necessarily mutually exclusive, although they may act in a sequential manner, with one modification determining future substitution events. Nevertheless, there are numerous flavonoid compounds that have been isolated whose synthesis remains unclear. Efforts directed towards the elucidation of the biosynthesis and physiology of these novel compounds should yield valuable information that can be applied in various systems. 

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