Chymotrypsin-catalyzed hydrolysis of N-acetyl- and N-benzoyl-l-tyrosine p-nitroanilides

Bundy, Hallie F.

doi:10.1016/0003-9861(63)90249-2

Cited by 72 publications

(24 citation statements)

References 21 publications

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“…For the assay, 26 2.8 mL of 12 mmol L -1 Bz-L-TyrpNA/70 mmol L -1 phosphate buffer, pH 7.6, were placed in a 4.5 mL quartz cuvette. The reaction at 25 °C was initiated by adding 0.2 mL of cPPL solution (0.5 mg per mL of 0.01 mol L -1 HCl/water).…”

Section: Amidase Activity Against Bz-l-tyr-pna In Cpplmentioning

confidence: 99%

“…Considering these results, we then assayed only cPPL against synthetic substrates of proteases, [25][26][27] an approach used in combination with SDS-PAGE 20,21,38 to examine enzyme preparations. The high activity of cPPL against Bz-L-Tyr-pNA, Bz-L,D-Arg-pNA and Z-Gly-Phe (usually employed to assay the amidase activity of the proteases chymotrypsin) trypsin and carboxypeptidase A, was consistent with the results reported by Maruyama et al 20 In fact, these authors tested 13 commercial lipases, found that only PPL was active against Bz-L-Tyr-pNA and suggested that the peptidase activity of the commercial PPL was due to contamination by pancreatic α-chymotrypsin.…”

Section: Analysis Of Cppl and Ppplmentioning

confidence: 99%

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Dipeptide synthesis in biphasic medium: evaluating the use of commercial porcine pancreatic lipase preparations and the involvement of contaminant proteases

Liria

Romagna

Rodovalho

et al. 2008

J. Braz. Chem. Soc.

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Sínteses bem sucedidas de dipeptídeos a partir de Ac-L-Tyr-OEt ou Z-L-X-OMe (X: Asp, Tyr, Phe, Arg, Lys ou Thr) e glicina amidada em meio reacional bifásico foram realizadas usando dois tipos de preparações comerciais de lipase pancreática suína (PPL) (bruta (cPPL) e purificada (pPPL)). Nas mesmas condições reacionais, a α-quimotripsina, protease pancreática que também apresenta atividade esterásica, catalisou a síntese de Ac-L-Tyr-Gly-NH 2 com alta produtividade. Na maioria das sínteses também ocorreu a hidrólise do produto formado. Eletroforese em gel de poliacrilamida, ensaios enzimáticos com substratos cromogênicos específicos e cromatografia de exclusão molecular demonstraram que cPPL e pPPL apresentam proteases contaminantes e, portanto, atividades esterásica e amidásica. Em conjunto, esses resultados indicam que tais proteases, e não a PPL, possam ser os catalisadores majoritários da síntese da ligação peptídica quando ésteres de N α -acil-L-aminoácidos e preparações comerciais de PPL são usados. Por outro lado, tais dados não contradizem a possibilidade de usar cPPL como fonte barata de catalisadores para a síntese de dipeptídeos em condições reacionais brandas.Dipeptide syntheses starting from Ac-L-Tyr-OEt or Z-L-X-OMe (X: Asp, Tyr, Phe, Arg, Lys or Thr) and glycine amide in biphasic reaction media were achieved using two commercially available porcine pancreatic lipase (PPL) preparations (crude (cPPL) and purified PPL (pPPL)). Under the mild conditions employed, α-chymotrypsin, a pancreatic protease that also presents esterase activity, catalyzed Ac-L-Tyr-Gly-NH 2 synthesis with high productivity. Product hydrolysis also occurred in most of the syntheses studied. Polyacrylamide gel electrophoresis, enzymatic assays employing specific chromogenic substrates and size-exclusion chromatography revealed that cPPL and pPPL contain contaminant proteases and, therefore, exhibit esterase and amidase activities. Overall, these data indicate that those contaminants may be the main catalysts of peptide bond synthesis when N α -blocked-L-amino acid esters and the commercial PPL preparations are used. On the other hand, such data do not contest the possibility of using such enzyme preparations as an inexpensive source of catalysts for dipeptide synthesis under soft conditions. Keywords: biocatalysis, enzymes, peptide bond synthesis, n-hexane IntroductionIn the last decade, interest in environment friendly technologies has increased drastically. As a result, the ability of enzymes to catalyze reactions required for the manufacture of fine chemicals has been explored extensively. 1 Proteases that also present esterase activity have been widely used to catalyze reactions related to peptide synthesis, such as ester hydrolysis, esterification, transesterification and peptide bond formation. 2,3 However, it is well known that the main concern in protease-catalyzed peptide bond synthesis is protease-catalyzed product consumption. [4][5][6] Therefore, the following approaches have been used to minimize such an undesirable r...

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Section: Amidase Activity Against Bz-l-tyr-pna In Cpplmentioning

confidence: 99%

Section: Analysis Of Cppl and Ppplmentioning

confidence: 99%

Dipeptide synthesis in biphasic medium: evaluating the use of commercial porcine pancreatic lipase preparations and the involvement of contaminant proteases

Liria

Romagna

Rodovalho

et al. 2008

J. Braz. Chem. Soc.

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“…The activation of zymogen was performed with enterokinase according to Gorril and Thomas (11). The activity of trypsin (EC 3.4.4.4) was assayed spectrophotometrically on the substrate a-N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) in dimethylformamide, at 410 nm, and that of chymotrypsin (EC 3.4.4.5) on the substrate N-acetyl-i-tyrosine-p-nitroanilide-hydrochloride (ATPNA) also at 410 nm (7,8).…”

Section: Methodsmentioning

confidence: 99%

The Comparative Effect of Prolonged Feeding with Raw and Heated Soybean Meal on the Growth Response, Pancreatic Enlargement and Pancreatic Enzymes of Chicks

Madar¹,

Tencer²,

Gertler³

et al. 1976

Ann Nutr Metab

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Prolonged feeding of chicks with raw soybean meal as the main protein source in the diet resulted in growth inhibition, pancreas enlargement, and increased content of pancreatic proteolytic enzymes. The effect of feeding with raw soybean meal as compared to heated soybean meal upon total intestinal enzymatic activities in chicks was tested. The tendency of chicks to overcome the effects of raw soybean meal is expressed mainly by the increase of biosynthesis and secretion of proteolytic enzymes in the pancreas.

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“…After a 10-fold dilution of the homogenates in a 50-m.V/ Tris-base buffer (pH 8.0) containing 20 mM CaCL. trypsinogen and chymotrypsinogen were activated separately at 4°C for 24 h and for 30 min with 1:100 and 1:10 trypsin (w/w, enzyme: protein substrate ratio), respectively [29], Activities of trypsin were assayed with N-benzoyl-DL-arginine /> nitroanilide [30] and chymotrypsin with N-benzoyl-Ltyrosinc /r-nitroanilide [31]. The resulting enzymatic units were expressed as pmol of/Miitroanilinc released per min at 25 and 35°C, respectively.…”

Section: Rna Dna Protein and Enzyme Assaysmentioning

confidence: 99%

Response of the Calf Pancreas to Differently Processed Soya Bean and Pea Diets

Dréan¹,

Huërou-Luron²,

Philouze-Romé³

et al. 1995

Ann Nutr Metab

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The purpose of this study was to determine the effect of replacing skim-milk powder by differently treated soya bean or pea products on growth, pancreas size and pancreatic enzyme activities in calves. Three separate experiments have been performed. In experiments 1 and 2, 28 and 21 male Holstein calves were divided into 4 or 3 groups, respectively, and fed either dairy products or milk substitutes in which protein was mainly provided by soya bean products differing in their protein concentration due to the technological processing applied. In experiment 3, 45 male Holstein calves were divided into 3 groups and were fed either dairy products, or raw or flaked pea flour as a protein source. After an experimental period of 99 ± 4 days in experiments 1 and 2, and of 88 days in experiment 3, animal growth rate was significantly lower with raw pea flour (16%) and with the soya bean diet, which was highly concentrated in carbohydrates and allergenic proteins (13-27%). Pancreas weight decreased significantly (16-18%) with pea diets and tended to be lower (NS) with the water extracted, concentrated and heated flour (soya bean). Amylase-specific activity increased significantly (43%) with pea diets but showed opposite tendencies with the most refined soya bean products. Proteolytic enzyme activities were slightly influenced by dietary protein source, but this was not as obvious as in the literature reviewed. Specific messenger RNAs corresponding to amylase, trypsin and chymotrypsin seemed to increase (NS) with the soya bean diets, particularly with the less elaborated one. However, further investigations are required before any conclusions may be drawn concerning regulation levels of pancreatic adaptation to dietary protein. According to this study and the literature, results concerning pancreatic response to diets were different suggesting that the origin of soya bean, pea seeds and technological treatments applied to them were of great importance. Also, the level of incorporation into milk substitute and the presence of more or less antinutritional factors could influence pancreatic enzyme variations by complex mechanisms.

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Chymotrypsin-catalyzed hydrolysis of N-acetyl- and N-benzoyl-l-tyrosine p-nitroanilides

Cited by 72 publications

References 21 publications

Dipeptide synthesis in biphasic medium: evaluating the use of commercial porcine pancreatic lipase preparations and the involvement of contaminant proteases

Dipeptide synthesis in biphasic medium: evaluating the use of commercial porcine pancreatic lipase preparations and the involvement of contaminant proteases

The Comparative Effect of Prolonged Feeding with Raw and Heated Soybean Meal on the Growth Response, Pancreatic Enlargement and Pancreatic Enzymes of Chicks

Response of the Calf Pancreas to Differently Processed Soya Bean and Pea Diets

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