Chymotrypsin enzyme efficiency

The first application of ReAL reagents to proteins showed that there are unanticipated difficulties. Compounds 3a and 3b (Fig. 5) were designed to react with chymotrypsin. The tyrosine derivative 3a did not alter the catalytic activity significantly after 2 hours of incubation although 80% of the activity was lost after 5 days. The acid 4b, however, inactivates chymotrypsin very efficiently 88% of the activity was lost in one minute. Attempts to restore the activity were not successful the control enzyme lost 28% of its activity when irradiated for 2 h with light of roughly 350 nm and the derivatized enzyme under the same conditions dropped from 12% to 7% of the original activity. 

Numerous suggestions have been made that enzymes might owe part of their catalytic efficiency to the opportunity they afford for stabilization of intermediates or transition states by hydrogen bonding to functional groups near the active site. For example, in the case of (x-chymotrypsin this might be represented as in [43] where... 

Cabral and coworkers [253] have investigated the batch mode synthesis of a dipeptide acetyl phenylalanine leucinamide (AcPhe-Leu-NH2) catalyzed by a-chymotrypsin in a ceramic ultrafiltration membrane reactor using a TTAB/oc-tanol/heptane reverse micellar system. Separation of the dipeptide was achieved by selective precipitation. Later on the same group successfully synthesized the same dipeptide in the same reactor system in a continuous mode [254] with high yields (70-80%) and recovery (75-90%). The volumetric production was as high as 4.3 mmol peptide/l/day with a purity of 92%. The reactor was operated for seven days continuously without any loss of enzyme activity. Hakoda et al. [255] proposed an electro-ultrafiltration bioreactor for separation of RMs containing enzyme from the product stream. A ceramic membrane module was used to separate AOT-RMs containing lipase from isooctane. Application of an electric field enhanced the ultrafiltration efficiency (flux) and it further improved when the anode and cathode were placed in the permeate and the reten-tate side respectively. 

In order to understand why enzymes are such efficient catalysts, it is necessary to understand first why uncatalyzed reactions in solution are so slow. As illustrations, we consider the reactions that may be catalyzed by chymotrypsin or lysozyme. 

The pH-stat method can be modified and applied to a particular enzyme or substrate to assay any enzyme/substrate combination. The substrate can come from any source of protein such as poultry, milk, soybean, or fish processing byproducts. The amount of protein in the reaction should not exceed 8% (as calculated in Basic Protocol 1). The enzyme can be any alkaline endopeptidase such as Alcalase, trypsin, or chymotrypsin, and should be used in the proportions indicated in Basic Protocol 1. The selection of the appropriate enzyme depends on its efficiency and cost. 

Protein aggregates [107, 109, 121] or dye crystals [122-126] can serve as templates for LbL polyelectrolyte adsorption. Chymotrypsin aggregates encapsulated by PSS and PAH deposition contain a high protein amount and the enzyme keeps its bioactivity [107], The aggregates prepared in this manner have high incorporation efficiency and a protein content of 50-70% [109]. An encapsulated catalase has been shown to be stable against protease degradation [121],... 

For many years a solution of crude acetone powder of bovine or porcine pancreas has been used to disaggregate tissues and to release cultured cells from their substratum. These preparations — referred to as trypsin 1 250 (based on an international standard) — contain not only trypsin but a range of enzymes including chymotrypsin and elastase which are equally important. Purified trypsin seldom is as efficient as the cruder preparations, especially on tissue disaggregation, when mucinous clumps result (Ronaldoni, 1959). 

Another immobilization method was described by Maeda and coworkers [344], They developed a facile and inexpensive preparation method for the formation of an enzyme-polymeric membrane on the inner wall of the microchannel (PTFE) through cross-linking polymerization in a laminar flow. With this approach, a-chymotrypsin was immobilized successfully. The activity of the immobilized enzyme was tested using N-glutaryl-L-phenylalanine p-nitroanilide as substrate, and the reaction products were analyzed offline by HPLC. There was no significant difference in the hydrolysis efficiency compared to solution-phase batchwise reactions using the same enzyme/substrate molar ratio (Scheme 4.87). 

Efficient modification steps through the proper orientation of the inhibitor reactive group to the enzyme nucleophile is realized by covalent bond formation. A classic example of this type is the modification of a methionine residue of chymotrypsin by /7-nitrophenyl bromoacetyl a-aminoisobutyrate (26)47). In this instance, the reactive group (bromoacetyl) is fixed at the locus near the active site through a covalent bond by means of acyl enzyme intermediates. 

Generally speaking, the role of the enzyme consists of the selective and specific attraction of substrate and the highly efficient catalysis. Every enzyme has its own characteristic feature for example, the specificity in the binding and a charge-relay action in the catalysis in a-chymotrypsin, the contribution of the imidazole moiety as an electron donor to the electrophilicity of zinc ion in carboxypeptidase, the change in the spin state and the reactivity of the transition metal ion by the coordination of the imidazole in the hemochrome. These typical characteristic features are the result of the cooperative actions of the constituents. 

I Cyclodextrins are excellent enzyme models Catalysis and induced fit. Due to their cavities, which are able to accommodate guest (substrate) molecules, and due to the many hydroxyl groups lining this cavity, cyclodextrins can act catalytically in a variety of chemical reactions and they therefore serve as good model enzymes. Thus, benzoic acid esters are hydrolyzed in I aqueous solution by factors up to 100 times faster if cyclodextrins are added. The reaction in- j volves an acylated cyclodextrin as intermediate which is hydrolyzed in a second step of the j reaction, a mechanism reminiscent of the enzyme chymotrypsin. The catalytic efficiency can. be further enhanced if the cyclodextrins are suitably modified chemically so that a whole range of artificial enzymes have been synthesized [551-555, 556, 563, 564]. 

Life as we know it would be impossible without the astonishing characteristics of enzymic catalysis. This catalysis is not only highly efficient, so that reactions may proceed at low temperature and at neutral pH with the speed required by living cells, but it exhibits also a remarkable specificity. Let us cite two typical examples First, the enzyme urease catalyzes the hydrolysis of urea but of no other compound (1). Second, the catalytic action is frequently restricted to one of the antipodes of optically active substrates. Thus, chymotrypsin will catalyze the hydrolysis of acylated L-tyrosinamides, but will not catalyze the reaction of the corresponding derivatives of D-tyrosine (2). 

We have noted previously that the catalytic effect of the hydrolytic enzyme chymotrypsin depends critically on the interaction of a hydroxyl and an imidazole group placed in juxtaposition in the enzyme molecule. It was, therefore, particularly tempting to see if an analogous interaction could enhance the catalytic efficiency of PVI, by using copolymers of 4(5)-vinylimidazole with p-vinylphenol (VI/VP) or with vinyl alcohol (VI/VA)... 

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