Chymotrypsin, applications

The successful use of these X-ray crysallographic techniques in studying the enzyme-substrate interactions of lysozyme (21) and chymotrypsin (22) has recently been reviewed by Blow and Steitz (16) and Blow (23). To date, however, these methods have had only limited application, since the detailed structures of only about ten enzymes have been elucidated by X-ray diffraction... 

Silica particles surface-imprinted with a TSA of a-chymotrypsin were applied for the enantio-selective hydrolyzation of amides. Surprisingly, the particles showed reverse enantio-selectivity, i. e., the sol-gel imprinted with the L-isomer of the enzyme s TSA showed a higher selectivity for the D-isomer of the substrate [125]. Also Ti02 gels have been imprinted, e.g., with 4-(4-propyloxypheny-lazo)benzoic acid. QCM coated with ultrathin films of this gel were prepared by an immersion process and showed selective binding of the template [ 126]. These examples demonstrate once more the broad applicability of the concept of molecular imprinting. 

Whilst chymotrypsin and trypsin are especially useful in peptide sequence analysis, they also have medicinal applications. Their ability to hydrolyse proteins makes them valuable for wound and ulcer cleansing (trypsin) or during cataract removal (chymotrypsin). 

Plunkett KN, Berkowski KL, Moore JS (2005) Chymotrypsin responsive hydrogel application of a disulfide exchange protocol for the preparation of methacrylamide containing peptides Biomacromolecules 6 632-637... 

RME shows particular promise in the recovery of proteins/enzymes [12-14]. In the past two decades, the potential of RME in the separation of biological macromolecules has been demonstrated [15-20]. RMs have also been used as media for hosting enzymatic reactions [21-23]. Martinek et al. [24] were the first to demonstrate the catalytic activity of a-chymotrypsin in RMs of bis (2-ethyl-hexyl) sodium sulfosuccinate (Aerosol-OT or AOT) in octane. Since then, many enzymes have been solubilized and studied for their activity in RMs. Other important applications of RME include tertiary oil recovery [25], extraction of metals from raw ores [26], and in drug delivery [27]. Application of RMs/mi-croemulsions/surfactant emulsions were recognized as a simple and highly effective method for enzyme immobilization for carrying out several enzymatic transformations [28-31]. Recently, Scheper and coworkers have provided a detailed account on the emulsion immobiUzed enzymes in an exhaustive review [32]. 

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. 

FIGURE 17.10 Effect of bovine pancreatic chymotrypsin on visual scaling after a 3 h occluded application. Vehicle (square), 0.5% chymotrypsin (diamonds), heat inactivated chymotrypsin (triangles). P < 0.05. From El-Kadi, K., Rawlings, A.V., Feinberg, C., Nunn, C., Battaglia, A., Chandar, R, Richardson, N., Sabin, R., and Pocalyko, D. Arch. Dermatol. Res., 293, 500-507, 2001. 

Application of ALPH to serine proteinases as an axiom thus results in a requirement for a separate kinetic event, nitrogen inversion. In the absence of additional evidence for such an event, therefore, serine proteinase action would be accounted an area in which ALPH probably fails, but two negative results provide some evidence that the tetrahedral intermediate is indeed first formed in conformation [102b]. Bizzozero and Zweifel (1975) found that amides [104] and [105] were not detectably hydrolysed by chymotrypsin,... 

The seeds of squash plants are rich in a family of trypsin and chymotrypsin inhibitors that are approximately 35 amino acids in size and have been extensively investigated not only for their enzyme inhibitory activity, but also because they are very stable mini-protein scaffolds with applications in protein engineering. The best studied examples are Ecballium elaterium trypsin inhibitor (EETI-II) and Cucurbita maxima trypsin inhibitor (CMTI). Both X-ray and NMR have been used to characterise their structures, which incorporate a cystine-knot motif formed by three conserved disulphide bonds.93 We will describe this motif in more detail in a later section describing the plant cyclotides. 

The facile reaction of CAA and BAA with nucleosides and nucleotides is one example of many of the applications of the bifunctional reactivity of halogenated aldehydes and ketones in modification of biomolecules. In an early example of the extensive use of halogenated ketones as protease substrate analogues, l-V-tosylamido-2-phenylethyl chloro-methyl ketone (TPCK) 30 was synthesized as a chymotrypsin substrate analogue. Stoichiometric inhibition was accompanied by loss of one histidine residue as a result of alkylation by the chloromethyl moiety68. A host of similar analogues were subsequently prepared and used as selective enzyme inhibitors, in particular for the identification of amino acid residues located at enzyme active sites69. 

In this figure the specific enzymatic activity (defined as activity per unit mass of protein) is plotted as a function of temperature. The data show that the enzyme loses activity due to adsorption. On the hydrophobic Teflon and PS surfaces the activity is completely vanished. The application of the (EO)x-oligomers on the PS surface leads to retention of some of the enzymatic activity of adsorbed a-chymotrypsin. The short (EO)g chains trapped between the adsorbed protein molecules and the PS surface, frustrate intimate contact. As a... 

Application and Principle This procedure is used to determine chymotrypsin activity in chymotrypsin preparations derived from purified extracts of porcine or bovine pancreas. 

Chymotrypsin Obtained from purified extracts of bovine or porcine pancreatic tissue. White to tan, amorphous powders soluble in water but practically insoluble in alcohol, in chloroform, and in ether. Major active principle chymotrypsin. Typical application hydrolysis of protein. 

Initial attempts to achieve an enzyme-catalyzed deprotection of the carboxy group of peptides centred around the use of the endopeptidases chymotrypsin, trypsin,and thermolysin.P l Thermolysin is a protease obtained from Bacillus thermoproteolyticus that hydrolyzes peptide bonds on the annino side of the hydrophobic amino acid residues (e.g., leucine, isoleucine, valine, phenylalanine). It cleaved the supporting tripeptide ester H-Leu-Gly-Gly-OEt from a protected undecapeptide (pH 7, rt). The octapeptide, thus obtained, is composed exclusively of hydrophilic annino acids. Due to the broad substrate specificity of thermolysin and the resulting possibility of unspecific peptide hydrolysis, this method is of limited application. 

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