Benzoyl Arginine P-nitroanilide Research Articles

Solid phase chemical modification of agarose glyoxyl-ficin: Improving activity and stability properties by amination and modification with glutaraldehyde

Abstract Ficin extract immobilized on glyoxyl-agarose was chemically modified using carbodiimide and ethylenediamine (full or partial amination of the carboxylic groups of the enzyme) or glutaraldehyde (modifying primary amino groups) under different conditions. Aminated enzyme altered its activity versus casein and benzoyl-arginine-p-nitroanilide (BANA), e.g., the activity versus casein even slightly increased while versus BANA it decreased, being this more significant at pH 9 (the fully aminated biocatalyst increased the activity versus casein by 10% but it decreased more than 5 folds versus BANA), greatly altering the enzyme specificity. The amination improved the enzyme stability at pH 5 while stability was impoverished at pH 9. Glutaraldehyde treatment usually decreased the activity versus BANA while it improved the activity versus casein (by around a factor of 2 when using 1% glutaraldehyde). Enzyme thermostability was enhanced, mainly at pH 7. This permitted to have more linear casein hydrolysis courses at pH 7 and 66 °C compared to the unmodified enzyme. Amination followed by glutaraldehyde treatment drove to the almost full enzyme inactivation. Thus, these treatments may be considered interesting for preparing an improved biocatalyst of ficin, mainly in proteolytic applications, and may be considered (mainly the amination) a possibility to further improve enzyme immobilization.

넙치(Paralichthys olivaceus) 알로부터 Serine Protease Inhibitors의 분획 특성

Protease inhibitors (PI) of trypsin and papain as target proteases from the roe of bastard halibut Paralichthys olivaceus were fractionated out using ammonium sulfate precipitation (A), DEAE 650M anion exchange chromatography (D), and Sephacryl S-300 gel filtration (S). The recovery percentages of the fractions with the strongest inhibitory activity for each fractionation method were 13% for the A4 fraction, 21.2% for the D3 fraction, and 21.3% for the S2 fraction, with specific inhibitory activities of the fractions toward trypsin and casein of 168, 139, and 218 U/mg, respectively, while no inhibition of papain was observed. The for the trypsin-specific substrate -benzoyl--arginine-p-nitroanilide (BAPNA) was 0.65, 1.55, 2.26, and 2.85 mg/mL for the A4, S2, A3, and D3 fractions, respectively. These results suggest that chromatographic fractionation methods (D and S) based on the molecular mass and charge of the protein were more effective at fractionating PI than was ammonium sulfate precipitation based on protein solubility, and that the bastard halibut roe extract acts as a serine protease inhibitor. Therefore, the PI fraction from fish roe might be useful for inhibiting proteases in foodstuffs, and could constitute an alternative food-grade inhibitor for the surimi industry.

Protein hydrolysis by immobilized and stabilized trypsin

The preparation of novel immobilized and stabilized derivatives of trypsin is reported here. The new derivatives preserved 80% of the initial catalytic activity toward synthetic substrates [benzoyl-arginine p-nitroanilide (BAPNA)] and were 50,000-fold more thermally stable than the diluted soluble enzyme in the absence of autolysis. Trypsin was immobilized on highly activated glyoxyl-Sepharose following a two-step immobilization strategy: (a) first, a multipoint covalent immobilization at pH 8.5 that only involves low pK(a) amino groups (e.g., those derived from the activation of trypsin from trypsinogen) is performed and (b) next, an additional alkaline incubation at pH 10 is performed to favor an intense, additional multipoint immobilization between the high concentration of proximate aldehyde groups on the support surface and the high pK(a) amino groups at the enzyme surface region that participated in the first immobilization step. Interestingly, the new, highly stable trypsin derivatives were also much more active in the proteolysis of high molecular weight proteins when compared with a nonstabilized derivative prepared on CNBr-activated Sepharose. In fact, all the proteins contained a cheese whey extract had been completely proteolyzed after 6 h at pH 9 and 50°C, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Under these experimental conditions, the immobilized biocatalysts preserve more than 90% of their initial activity after 20 days. Analysis of the three-dimensional (3D) structure of the best immobilized trypsin derivative showed a surface region containing two amino terminal groups and five lysine (Lys) residues that may be responsible for this novel and interesting immobilization and stabilization. Moreover, this region is relatively far from the active site of the enzyme, which could explain the good results obtained for the hydrolysis of high-molecular weight proteins.

Purification and properties of trypsin‐like enzyme from the midgut of Morimus funereus (coleoptera, cerambycidae) Larvae

Trypsin-like enzyme (TLE) from the anterior midgut of Morimus funereus larvae was purified by anion exchange chromatography and gel filtration chromatography and characterized. Specific TLE activity was increased 322-fold by purification of the crude midgut extract. The purified enzyme had a pH optimum of 9.0 (optimum pH range 8.5-9.5) and temperature optimum of 45 degrees C with the K(M) ratio of 0.065 mM for benzoyl-arginine-p-nitroanilide (BApNA). Among a number of inhibitors tested, the most efficient was benzamidine (K(I) value of 0.012 mM, Ic(50) value of 0.204 mM) while inhibition of TLE activity by SBTI, TLCK, and PMSF was partial. Almost all divalent cations tested enhanced the enzyme activity, amongst them Co2+ and Mn2+ stimulated TLE activity for 2.5 times. The purified TLE (after gel-filtration on Superose 12 column) had a molecular mass of 37.5 kDa with an isoelectric point over 9.3. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed one band of 38 kDa, suggesting that the enzyme is a monomer.

Trypsin from the Processing Waste of the Lane Snapper (Lutjanus synagris) and Its Compatibility with Oxidants, Surfactants and Commercial Detergents

A trypsin from the viscera of the lane snapper (Lutjanus synagris) was purified by heat treatment, fractionation with ammonium sulfate and affinity chromatography. The molecular weight of the enzyme was estimated to be 28.4 kDa (SDS-PAGE). The purified enzyme was capable of hydrolyzing the specific substrate for trypsin benzoyl-arginine-p-nitroanilide (BApNA) and was inhibited by benzamidine and tosyl lysine chloromethyl ketone (TLCK), synthetic trypsin inhibitors and phenylmethylsulfonyl fluoride (PMSF), which is a serine-protease inhibitor. The enzyme exhibited maximal activity at pH 9.0 and 45 degrees C and retained 100% of the activity after incubation at the optimal temperature for 30 min. At a concentration of 10 mM, activity was slightly activated by Ca(2+) and inhibited by the following ions in decreasing order: Cd(2+) > Hg(2+) > Cu(2+) > Zn(2+) > Al(3+). The effects of Ba(2+), K(1+) and Li(1+) proved to be less intensive. Using 1% (w/v) azocasein as substrate, the enzyme revealed high resistance (60% residual activity) when incubated with 10% H(2)O(2) for 75 min. The enzyme retained more than 80% activity after 60 min in the presence of different surfactants (Tween 20, Tween 80 and sodium choleate). The alkaline protease demonstrated compatibility with commercial detergents (7 mg/mL), such as Bem-te-vi, Surf and Ala, retaining more than 50% of initial activity after 60 min at 25 degrees C and 30 min at 40 degrees C. The thermostability and compatibility of this enzyme with commercial detergents suggest a good potentiality for application in the detergent industry.

The Effect of Organic Solvents and Other Parameters on Trypsin-Catalyzed Hydrolysis of Nα[alpha]-Benzoyl-arginine-p-nitroanilide. A Project-Oriented Biochemical Experiment

The study of enzymatic catalysis is a classical biochemistry experiment for undergraduate classes. We propose the utilization of the serine protease trypsin to discuss several parameters affecting enzyme catalysis. Hydrolysis of the chromogenic substrate Nα -benzoyl-arginine-p-nitroanilide (BApNA) was followed by spectrophotometric monitoring. The optimal pH and temperature values were found to be 8.0 and 40 °C, respectively. Km and Vmax values were obtained by adjustment to Michaelis-Menten, Lineweaver-Burke, and Hanes equations. We then investigated the effect of organic solvents (a series of alcohols) on the hydrolysis of the chromogenic substrate. The reaction rate was reduced in the presence of methanol and further reduced by ethanol, 1-propanol, and 2-propanol, when compared to the data obtained with buffer. Finally the students were asked to measure the molar absorptivity of p-nitrophenol in the presence of the alcohols employed for the kinetic experiments. Thus they could learn that the value of t...

Does Catalytic Activity of Bence-Jones Proteins Contribute to the Pathogenesis of Multiple Myeloma?

Some Bence-Jones proteins have been found to be capable of hydrolyzing DNA, chromogenic amide substrates, such as benzoylarginine p-nitroanilide, and natural oligopeptides, such as arginine vasopressin. Patients who excrete Bence-Jones protein with the DNA-nicking activity have shown moderately severe symptoms. When incubated with LLC-PK1 (porcine kidney proximal tubule) cells, some Bence Jones proteins penetrated the cytoplasm, and entered the nucleus with little or no degradation of epitopes. Intranuclear Bence Jones proteins ultimately induced DNA fragmentation in situ and cell death. This cytocidal activity was not directly associated with the DNA-nicking activity, since Bence Jones proteins with no detectable DNase activity also produced cell death. These results, however, suggest that the biological activities of Bence Jones proteins described here makes a significant contribution to the development and/or deterioration of multiple myeloma.

Identification and Expression of the cDNA-encoding Human Mesotrypsin(ogen), an Isoform of Trypsin with Inhibitor Resistance

The cDNA encoding a novel isoform of human trypsinogen was identified. The isoelectric points of the proenzyme and active forms calculated from the deduced amino acid sequence are consistent with those of mesotrypsin(ogen), known to be an inhibitor-resistant trypsin isoform. The cDNA attached with a bacterial signal peptide sequence was expressed in Escherichia coli. The recombinant proenzyme purified from periplasm showed enterokinase-dependent activation similar to a major isoform of human trypsinogen. The enzyme was far less inhibited by trypsin inhibitors such as soybean trypsin inhibitor, aprotinin, or pancreatic secretory trypsin inhibitor than the control trypsin. A gel filtration assay showed that the enzyme and aprotinin did not form a stable complex. It is noteworthy that the amino acid at position 198, which is in close vicinity to the active Ser, is Arg while those of other major trypsins are all Gly. It is concluded that the cloned cDNA encodes human mesotrypsinogen, a unique isoform of trypsinogen with inhibitor resistance.

Biofunctional membranes: an EPR study of active site structure and stability of papain non-covalently immobilized on the surface of modified poly(ether) sulfone membranes through the avidin-biotin linkage

Over the past few years the use of membrane-based bioreactors has become more widespread in various industrial processes. Membrane-based bioreactors or immobilized enzymes are one example of biofunctional membranes, defined as entities composed of biological molecules attached to micropores of polymeric membranes. Immobilized enzymes provide several advantages over their free soluble forms, including their relative ease of recoverability and reusability, their higher storage and thermal stability, and consequent lower costs. A detailed understanding of structure and function of enzymes upon immobilization is essential to develop a membrane bioreactor with optimal properties in order to get a best mix of enhanced stability/recoverability with minimal conformational changes at the active site of the enzyme. In this study, the high binding affinity between avidin and biotin has been utilized to create a non-covalent spacer for enzyme immobilization. NHS-LC-biotin, a derivative of biotin, was selected to enhance complex formation with the biotin-binding cleft of avidin. A sulfhydryl protease, papain (EC 3.4.22.2), was non-covalently immobilized onto the poly(ether)sulfone membrane via the avidin-biotin complex. Kinetic parameters for the amidase activity of non-covalently bound papain, using the substrate benzoyl arginine p-nitroanilide hydrochloride (BAPNA), were determined and the results were compared with those obtained from studies of papain in solution and papain directly immobilized onto the modified poly(ether)sulfone membrane. As expected, there was a decrease in the enzymatic activity upon direct immobilization. However, insertion of the avidin-biotin complex as a non-covalent spacer increased the apparent maximum enzymatic rate [ V max(app)], and decreased the apparent Michaelis-Menten constant [ K m(app)], relative to directly-immobilized papain. This non-covalently attached enzyme bioreactor also showed significat increase in stability and reusability as compared to the free enzyme. Electron paramagnetic resonance (EPR) spin labeling techniques were used for the first time to characterize the active site conformational changes of an enzyme immobilized on poly(ether)sulfone membranes through the avidin-biotin complex. Two EPR spectral subpopulations were seen corresponding to the active and denatured forms of papain. This paper reports the studies of pH dependence, reusability and storage stability of biofunctional membranes using this non-covalent spacer. There was a good correlation between the active site conformational changes and the amidase activity of papain upon non-covalent immobilization onto the poly(ether)sulfone membrane. All these findings indicate that the EPR spin labeling technique shows great promise as a powerful method for studying enzyme systems immobilized on polymeric membranes.

Hydrolysis of N'-Benzoyl-Arginine- p-Nitroanilide Stereoisomers as a Phenotypic Test: a Study of Gram-Positive Halotolerant Bacteria

We tested a variety of halotolerant Gram-positive coccoid bacteria and other halotolerant bacteria for hydrolytic activity towards the L- and the D-isomer of N '-benzoyl-arginine- p -nitroanilide (BAPA). D-BAPA cleaving activity was earlier shown to be associated with endospore-forming bacteria and related organisms. Four groups were distinguished on the basis of their behavior towards the BAPA isomers: 1. D-BAPA cleaving bacteria: several Bacillus strains, two Planococcus species, and Marinococcus hispanicus . 2. Strain M96/12b, an endospore-forming bacterium, that cleaves both isomers. 3. L-BAPA cleaving bacteria: Gram-negative halotolerant bacteria ( Halomonas elongata , Vibrio costicola ), the Gram-positive coccoid bacterium Micrococcus luteus , and Bacillus pasteurii . 4. A number of Gram-positive cocci that cleave neither L-BAPA nor D-BAPA. Also a few ectoine-producing Bacillus species ( B. halophilus , B. pantothenticus ) fall in this category. We recommended to include the tests for L-BAPA and D-BAPA hydrolysis in phenotypic tests and numerical taxonomy studies of Gram-positive bacteria.

Raw Soybeans Stimulate Human Pancreatic Proteinase Secretion

Intraduodenal instillation of raw soybeans stimulated pancreatic proteinase secretion in humans. Raw soybeans almost abolished the activity of chymotrypsin and severely reduced (50%) the tryptic activity. Immunoreactive tryptic and chymotryptic material simultaneously appeared in amounts 2 to 4 times basal concentrations. This increase, demonstrated with rocket immunoelectrophoresis, was begun within the first 10 min of soybean instillation. The enhanced secretion also persisted throughout the succeeding saline instillation, and it is suggested that the presence of Kunitz trypsin inhibitor contributed to this postprandial stimulation. An amidase that hydrolyzes low-molecular-weight substrates (i.e., benzoyl-arginine p-nitroanilide) was found in raw soybeans. Its low activity was not assumed to substantially bias standard trypsin assays. The increased proteinase secretion was, as previously published, not preceded by an elevated plasma cholecystokinin concentration. The raw soybeans also caused a nonparallel secretion of amylase and proteinases. Nervous, perhaps cholinergic, regulation mediates the inhibitor-stimulated proteinase secretion in humans. This stimulation yields both a general increase of proteinases and also a specific inhibitor-resistant trypsin. This is consistent with the physiologic need for proenzyme-activation in the presence of inhibitors and for restoration of the proteolytic capacity of the duodenal juice.

Partial purification and properties of proteases from fig (Ficus carica) callus cultures

Cell culture of fig (Ficuscarica) was evaluated as a source of thermostable cysteine proteases. Extracts of 28 day old callus contained a benzoyl-arginine-p-nitroanilide (BAPNA) hydrolase activity of 2200 U/g dry wt (49.5 U/mg protein) at 25°C. Thermal stability and thermal denaturation (at 70°C) properties showed some similarities to those of commercial ficin (Sigma). High performance gel filtration revealed the presence of a peak with BAPNA hydrolase and caseinolytic activity which matched commercial ficin. Covalent chromatography using Thiopropyl Sepharose 6B (Pharmacia) demonstrated that approx 50% of the total BAPNA hydrolase activity could be attributed to thiol-proteins.

Partial characterization of proteinase activity in the larval midgut of the European corn borer, Ostrinia nubilalis Hübner (Lepidoptera: Pyralidae)

Protease activity in the alimentary tract of the European corn borer, Ostrinia nubilalis, can be attributed to at least three endoproteinases. A high alkaline trypsin with maximal hydrolysis of benzoyl arginine p-nitroanilide at pH values higher than 10.0, a low alkaline trypsin with maximal activity against benzoyl arginine ethyl ester at pH 9.0, and chymotrypsin, which hydrolyzed benzoyl tryosine ethyl ester at pH 7.5–8.0, were detected in gut homogenates. Total proteolysis, measured using azocasein, had maximal activity at pH 10.0 or higher. Corn borer chymotryptic activity had characteristics similar to the vertebrate enzyme. Both tryptic activities differed from vertebrate trypsin by being insensitive to ovomucoid trypsin inhibitor. High and low alkaline trypsin differed from each other by their pH optima. High alkaline trypsin was activated by magnesium and calcium, and low alkaline trypsin was not affected by inclusion of either chemical in the assay mixture.

Properties of a new alkaline proteinase from Aspergillus niger.

A. niger LCF 9 synthesizes a new aspergillopeptidase of potential interest in therapeutics. The properties and operating range of the enzyme were determined. It is a semi-alkaline aspergillopeptidase (EC 3.4.23.4) with one endopeptidase activity. Its pI is 4.10, its molecular weight is 21000 Da and its A1%(1 cm) at 280 nm is 9.75. It rapidly hydrolyzes casein and hemoglobin. Its optimal pH is 7.8 and optimal temperature is 45 degrees C. It is thermally labile above 40 degrees C but can be stabilized by adding calcium ions. It is inhibited by phenylmethylsulfonyl fluoride (PMSF), by ethylenediaminetetraacetic acid (EDTA) and by certain metals ions, e.g. copper, manganese and cobalt ions. It has no dipeptidase or tripeptidase activity and its esterase activity is weak. It has a high collagenase activity and is to our knowledge the only aspergillopeptidase that is active towards benzoyl-arginine p-nitroanilide (BAPNA).

Isolation and purification of a new protease from Aspergillus niger LCF no 9

Aspergillus niger LCF No9 synthesizes large amounts of a semi-alkaline protease highly active on Benzoyl arginine p-nitroanilide (BAPNA) that is likely to find industrial use. An industrial-grade enzyme of around 80% purity was prepared and a reference enzyme was obtained in high yield from the industrial product by ultrafiltration and HPIEC on a mono Q column. The industrial-grade enzyme was produced on a pilot scale with a yield of 0.52% of medium solid starting material by adsorption on CM-Sephadex A50, precipitation with acetone at −30°C, ion-exchange chromatography on DEAE-Sepharose and diafiltration.

Neutral proteinases from two mucoraceous fungi: a comparative study

Neutral proteinases from the mycelial extracts of two mucoraceous fungi, Choanephora cucurbitarum and Phascolomyces articulosus, were purified by salting out, heat treatment, and Sephadex G75 and DEAE-Sephadex chromatography. The proteinase from P. articulosus possessed higher molecular weight (Mr 25 000) as compared with that from C. cucurbitarum (Mr 15 000). The enzyme from both fungi hydrolyzed casein rapidly, but the proteinase of P. articulosus registered higher carboxy-peptidase and leucine aminopeptidase activities than that of C. cucurbitarum. Benzoyl-arginine p-nitroanilide was not hydrolyzed by either of them. Both enzymes had a pH optimum of 7.0, were stable up to 40 °C, and were inhibited by EDTA and N-α-p-tosyl-L-lysine chloromethyl ketone, more so by the latter. The proteinase of C. cucurbitarum was also sensitive to diisopropyl fluorophosphate, benzamidine, L-1-tosylamide 2-phenylethyl chloromethyl ketone, leupeptin, and soybean trypsin inhibitor. Cysteine and dithiothreitol did not have any effect on the enzyme activity. The P. articulosus proteinase was more sensitive to almost all the heavy metal ions tested than the neutral proteinase of C. cucurbitarum.

Are the proteinase inhibitory activities in lenticular tissues real?

The possibility of proteinase inhibitory activities in lenses measured with synthetic substrates being spurious, due to the effective competition of lens proteins as substrates for the target enzymes, was investigated. Goat, sheep and human cataractous lens proteins were found to be poor substrates for trypsin, elastase and papain compared to casein or bovine serum albumin. Further, the inhibition of elastase catalyzed hydrolysis of succinyl trialanyl p-nitroanilide by casein (500 μg, 53%) and albumin (500 μg, 49%) and of trypsin-catalyzed hydrolysis of benzoyl argininep-nitroanilide by albumin (1 mg, 24%) were significant only at high protein concentrations. These data indicated that the relatively high antielastase and antitryptic activities observed in human cataractous lenses were real. On the other hand, coincident lens protein hydrolysis elevating the true antitryptic and antielastase activities in goat and sheep lenses (that have low activities) could not be ruled out The lesser papain inhibitory activities observed in lenses when albumin was used as substrate compared to activities with benzoyl arginine p-nitroanilide as substrate, appeared to be partly due to lens protein hydrolysis masking the actual inhibition in the former method. Preincubation of goat, sheep and human lens extracts with trypsin for 1 h resulted in complete loss of antitryptic and antielastase activity except in the case of human lens antielastase activity which underwent 50% loss. Papain inhibitory activity was fully stable. Similar papain treatment caused loss of 80–100% of antielastase activity and 45–55% loss of antitryptic activity.

Improved purification and some properties of bovine lysosomal carboxypeptidase B

Lysosomal carboxypeptidase B (peptidyl- L-amino-acid hydrolase, EC 3.4.18.1) from bovine spleen purified to apparent homogeneity was found to have a molecular weight of 52 000 in the absence and of 25 000 in the presence of urea, determined by gel filtration, indicating the existence of two subunits of identical size. The amount of approx. 15% carbohydrate after cleavage by endoglycosidase H was shown to be insignificant for enzymatic activity. The isoelectric focusing separated lysosomal carboxypeptidase B into several protein bands - each enzymatically active - with a range of isoelectric points between 4.6 and 5.2 The tiltration of the sulphydryl group in the active site of the enzyme with the proteinase inhibitor E-64 yielded one thiol group per molecule. A maximum of activation was achieved by the addition of selenocystamine together with dithioerythritol and EDTA in the incubation solution. Under these conditions the carboxypeptidase hydrolyzed benzoylglycylarginine (80 kat/mol enzyme), benzolyarginine amide (38 kat/ mol enzyme) and carbobenzoxyglutaryltyrosine (110 kat/mol enzyme). Slight enzymatic activities towards benzoylarginine 2-naphthylamide and benzoylarginine p-nitroanilide could be measured. With the oxidized insulin B chain, lysosomal carboxypeptidase B exhibit only carboxypeptidase activity.

Enzymatic subunit splitting of lupine storage proteins

AbstractThe purified vicilin‐like and legumin‐like proteins, major storage globulins of lupine seeds, undergo partial proteolytic breakdown when self‐incubated at 37 °C under moderately alkaline pH. The splitting preferentially involves the high molecular weight subunits of the globulins. The whole globulin extract and the purified globulin fractions split benzoylarginine‐p‐nitroanilide (BAPA), a known substrate for endopeptidases. Further purification of globulins 4 and 8 by gel filtration at high ionic strength or by adsorption chromatography on Concanavalin‐A Sepharose removes only partially this activity and does not abolish self‐digestion. This activity is attributed to an endogenous endopeptidase associated to each globulin. BAPAase is inactive in the conditions of protein extraction and isolation and no significant modifications of the subunits of the storage proteins occurred during these steps.

Proteolytic activity of a rumen anaerobic fungus

A strain of the anaerobic phycomycetous fungus Neocallimastix frontalis isolated from the rumen of a sheep had a high proteolytic activity which became predominantly extracellular during growth. Proteolytic activity appeared to be due to a metalloprotease, as it was inhibited by 1,10-phenanthroline, EDTA and other chelators but not by phenylmethylsulphonyl fluoride (PMSF). Inhibition by EDTA was fully reversed by the addition of Zn2+, Ca2+ or Co2+, whereas addition of metal ions in the presence of 1,10-phenanthroline restored only a little activity. p-Chloromercuribenzoate (PCMB) was also inhibitory in dialysed supernatant fluid. N-α-p-Tosyl-l-lysine chloromethylketone (TLCK) inhibited proteolysis, suggesting that the protease(s) has a trypsin-like specificity, but benzoylarginine p-nitroanilide was not hydrolysed. Protease activity has a broad pH profile with a maximum at pH 7.5. Gel fractionation indicated that most of the activity was in a high-Mr form.