Enzyme source selection

Earlier work in this field [28] indicated that acetylcholinesterase enzymes would be suitable biomolecules for the purpose of pesticide detection, however, it was found that the sensitivity of the method varied with the type and source of cholinesterase used. Therefore the initial thrust of this work was the development of a range of enzymes via selective mutations of the Drosophila melanogaster acetylcholinesterase Dm. AChE. For example mutations of the (Dm. AChE) were made by site-directed mutagenesis expressed within baculovirus [29]. The acetylcholinesterases were then purified by affinity chromatography [30]. Different strategies were used to obtain these mutants, namely (i) substitution of amino acids at positions found mutated in AChE from insects resistant to insecticide, (ii) mutations of amino acids at positions suggested by 3-D structural analysis of the active site,... 

Irrespective of the type of biomass used for ethanol production, the biomass needs to be pretreated to make the carbohydrates available for fermentation. However, which enzymes can be used depends on the source of the biomass. In addition, the biomass needs pretreatment before the enzymes are used. The first step of the pretreatment can be of a physical nature. Once the biomass is physically pretreated, the cellulose structures are open for enzyme action. In biomass from forests, the substance is mainly in the form of cellulose. Targeted enzymes are selective for the reaction of cellulose to glucose, and therefore there are no degradation byproducts, as occurs in acid conversion technology. There are at least three ways this can be performed. Firstly, in separate hydrolysis and fermentation, the pretreated biomass is treated with cellulase, which hydrolyzes the cellulose to glucose at 50 °C and pH 4.8. Secondly, in simultaneous fermentation and saccharification (SSF) the hydrolysis and fermentation take place in the same bioreactor. Thirdly,... 

Tailoring Opportunities. There are many methods or approaches available to tailor enzyme products. Early in the history of enzyme companies, methods such as source selection, microbial strain selection, growth conditions, media, purification, and recovery systems, were primarily used to make each enzyme preparation unique. Later, immobilization, encapsulation, and chemical modification of the enzyme molecule itself were added as methods of tailoring enzymes to better fit industrial applications. Today, all of these methods are still being used, and now we have added genetic engineering to our tailoring expertises. 

Scheme 2.13 shows a few examples of resolutions and desymmetrization using esterases. Entry 1 shows the partial resolution of a chiral ester using a crude enzyme source. The enantioselectivity is only moderate. Entries 2 to 5 are examples of desymmetrization, in which prochiral ester groups are selectively hydrolyzed. Entries 6 and 7 are examples of selective hydrolysis of unsaturated esters that lead to isomeric monoesters. These cases are examples of diastereoselectivity. In Entry 8, the f ,f -enantiomer of a racemic diester is selectively hydrolyzed. In all these cases, the... 

One approach to the fabrication of such hosts is to utilize the many such receptors available from biological sources. Selective binding of species is demonstrated by enzymes for their substrates (and cofactors), by transport proteins for their transportees, and by antibodies for their antigens. In particular, because antibodies can be raised to many antigens, the biotic creation of hosts proves a powerful method for binding analytes with selectivity. Indeed, it is fair to say that... 

For synthesis of chiral c/5-diols, biotransformations using bacterial strains containing dioxygenase have advantages over purely chemical reactions, including enantiospecificity, high yields, low economic cost, and environmental friendliness (Reddy et al. 1999). And there are two key issues that have to be addressed one is the selection of an appropriate enzyme source and a bacterial strain the other is the development of appropriate process technology (Ballard et al. 1994). 

A variety of cell disruption methods are available. Physical, chemical, and enzymatic methods have all been used. The proper method should be carefully selected to ensure maximum cell disruption with minimum enzyme damage. This depends on the enzyme source, nature and stability. 

Almost 200 pesticides were analyzed in various systems using dichloromethane and ethyl acetate (129). These pesticides were visualized by the following selective detection methods (a) o-tolidine-potassium iodide, (b) p-nitro-benzene-diazonium-fluoborate, (c) silver nitrate with UV irradiation, (d) p-dimethylaminobenzaldehyde, (e) bioassays with either fungispores of Aspergillus nigeror an enzyme inhibition method. When horse blood serum was the enzyme source, acetylcholine iodide was its substrate in the presence of 2,6-dichloro-phenol-indophenol. Naphthyl acetate was applied as the substrate for the human blood plasma esterases. The Rf values and detection limits were also published (129). 

Microbial cell factories [194] have recently been developed for the production of terpenoids such as sclareol [195], and, as was shown in the previous section, valencene and nootkatone. The genes responsible for the biosynthesis of the desired compound, such as prenyltransferases, terpene synthases, and additional transformation enzymes, are selected from a natural source and transferred into a host, usually S. cerevisiae or . coli, suitably engineered to overproduce IPP and DMAPP. High productivity values for a commercially exploitable production are achieved by further metabolic and bioprocess engineering improvements of the microbial system. 

Using purified recombinant GGTase-I as an enzyme source and GGpp and Ras-CVLL as substrates, seven hit compounds were tested in vitro as a matter of the experimental validation. The selection was based on high predicted activity, availability, and structural imiqueness. All tested compounds showed inhibition of GGTase-1 with the pICso ranging from 3.63 to 5.44... 

In nature, enzymes play an important role in the survival and reproduction of their source organism. Therefore, many enzymes in their natural form are not suitable for application directly as biocatalysts in bioprocessing. For example, most enzymes are active at relatively mild conditions, thus may not be viable under the harsher conditions encountered in most indnstrial production systems. Various methods have been used to improve the properties of enzymes, including selectivity, activity and thermostability, in order to enable them to function as efficient industrial biocatalysts. Enzyme engineering, which encompasses rational design and directed evolution, is an efficient method to improve enzyme properties. Rational design seeks for beneficial mutations or protein sequences by applying empirically derived rules or theoretical models. Meanwhile, directed evolution uses a combinatorial approach to create libraries of enzymes from which enhanced variants can be identified... 

Properties of Ehirine Biosynthetic Enzymes from Selected Sources... 

A microbial source for a food enzyme must be nonpathogenic and nontoxicogenic. Manufacturers of microbial food enzymes have always selected their production microorganisms from the safe end of the spectmm of available sources. Consequendy, a few species have acquired a record of safe use as sources of a wide variety of food enzymes. 

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