Alkaline Trypsin Research Articles

Alkaline trypsin from the viscera and heads of Engraulis anchoita : partial purification and characterization

ENWEndNote BIBJabRef, Mendeley RISPapers, Reference Manager, RefWorks, Zotero AMA Lamas D, Yeannes M, Massa A. Alkaline trypsin from the viscera and heads of Engraulis anchoita: partial purification and characterization. BioTechnologia. 2017;98(2):103-112. doi:10.5114/bta.2017.68309. APA Lamas, D., Yeannes, M., & Massa, A. (2017). Alkaline trypsin from the viscera and heads of Engraulis anchoita: partial purification and characterization. BioTechnologia, 98(2), 103-112. https://doi.org/10.5114/bta.2017.68309 Chicago Lamas, Daniela L., Maria I. Yeannes, and Agueda E. Massa. 2017. "Alkaline trypsin from the viscera and heads of Engraulis anchoita: partial purification and characterization". BioTechnologia 98 (2): 103-112. doi:10.5114/bta.2017.68309. Harvard Lamas, D., Yeannes, M., and Massa, A. (2017). Alkaline trypsin from the viscera and heads of Engraulis anchoita: partial purification and characterization. BioTechnologia, 98(2), pp.103-112. https://doi.org/10.5114/bta.2017.68309 MLA Lamas, Daniela L. et al. "Alkaline trypsin from the viscera and heads of Engraulis anchoita: partial purification and characterization." BioTechnologia, vol. 98, no. 2, 2017, pp. 103-112. doi:10.5114/bta.2017.68309. Vancouver Lamas D, Yeannes M, Massa A. Alkaline trypsin from the viscera and heads of Engraulis anchoita: partial purification and characterization. BioTechnologia. 2017;98(2):103-112. doi:10.5114/bta.2017.68309.

Biochemical and molecular characterisation of a new alkaline trypsin from Liza aurata: Structural features explaining thermal stability

This study investigated the fine structure and biochemical characterization of trypsin from the viscera of Liza aurata. The purified enzyme displayed an apparent molecular weight of 23kDa as determined by sodium dodecyl sulphate–polyaccrylamide gel electrophoresis (SDS–PAGE). The optimum pH and temperature for the proteolytic activity were 10.0 and 50°C, respectively. Trypsin was strongly inhibited by serine protease inhibitor. The cDNA of the mature trypsin was cloned and sequenced. It encodes a protein of 222 amino acids, having only 86% of identity with its most homologous trypsin II of the Salmo salar. A phylogenic analysis showed that L. aurata trypsin (LAT) is close to fish enzymes. Given the high amino acid sequence homology between fish enzymes, a 3-D structure model was built using the structure of S. salar as a template. According to this model, structural features common with warm-active trypsins would explain why LAT acts at high temperatures, unlike cold adapted enzymes.

IN VITRO ANTIVIRAL ACTIVITY OF AN ALKALINE TRYPSIN FROM THE DIGESTIVE JUICE OF Bombyx mori LARVAE AGAINST NUCLEOPOLYHEDROVIRUS

Alkaline trypsin protein of molecular mass 25,436 Da purified from the digestive juice of Bombyx mori larvae indicated strong antiviral activity against Bombyx mori nucleopolyhedrovirus (BmNPV) under in vitro conditions. Partial N-terminal amino acid sequence of the protein was determined and the cDNA was cloned based on the amino acid sequence. A homology search of the deduced amino acid sequence of the cDNA showed 55% identity with Helicoverpa armigera trypsin and the active site of this protein was completely conserved. Hence, the protein was designated B. mori trypsin (Bmtryp). The results suggest that Bmtryp, an insect digestive enzyme, can be a potential antiviral factor against BmNPV at the initial site of viral infection.

Trypsin from zebra blenny (Salaria basilisca) viscera: Purification, characterisation and potential application as a detergent additive

An alkaline trypsin was purified from the viscera of zebra blenny (Salaria basilisca) by ammonium sulphate (40−80% saturation) precipitation, Sephadex G-100, Mono Q-Sepharose and ultrafiltration. A yield of 12% with a purification-fold of 4.2 was obtained. The trypsin had an apparent molecular weight of 27kDa. Soybean trypsin inhibitor and phenylmethylsulfonyl fluoride showed a strong inhibitory effect on the purified trypsin. Trypsin had maximal activity at pH 9.5 and 60°C for the hydrolysis of Nα-benzoyl-dl-arginine-p-nitroanilide (BAPNA). It was stable at low temperatures and in the pH range of 7.0−12.0. The N-terminal amino acid sequence of the first 12 amino acids of the purified protease was IVGGRECTEPSQ. S. basilisca trypsin, which showed high homology with other fish trypsins, had a charged Arg residue at position 5, where Tyr is common in marine vertebrates and mammalian trypsins. The trypsin kinetic constants, Km and kcat for BAPNA, were 0.6mM and 1.38s−1, respectively.

Earthworm Protease

The alimentary tract of earthworm secretes a group of proteases with a relative wide substrate specificity. In 1983, six isozymes were isolated from earthworm with fibrinolytic activities and called fibriniolytic enzymes. So far, more isozymes have been found from different earthworm species such asLumbricus rubellusandEisenia fetida. For convenience, the proteases are named on the basis of the earthworm species and the protein function, for instance,Eisenia fetidaprotease (EfP). The proteases have the abilities not only to hydrolyze fibrin and other protein, but also activate proenzymes such as plasminogen and prothrombin. In the light of recent studies, eight of theEfPs contain oligosaccharides chains which are thought to support the enzyme structure. Interestingly,EfP-II has a broader substrate specificity presenting alkaline trypsin, chymotrypsin and elastase activities, butEfP-III-1 has a stricter specificity. The protein crystal structures show the characteristics in their specificities. Earthworm proteases have been applied in several areas such as clinical treatment of clotting diseases, anti-tumor study, environmental protection and nutritional production. The current clinical utilizations and some potential new applications of the earthworm protease will be discussed in this paper.

A Laundry Detergent-Stable Alkaline Trypsin from Striped Seabream (Lithognathus mormyrus) Viscera: Purification and Characterization

An alkaline trypsin from the intestine of striped seabream (Lithognathus mormyrus) was purified and characterized. The enzyme was purified to homogeneity by precipitation with ammonium sulfate, Sephadex G-100 gel filtration and CM-Sephadex cation-exchange chromatography, with a 24.9-fold increase in specific activity and 13% recovery. The molecular weight of the purified alkaline trypsin was estimated to be 27.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography. The purified trypsin appeared as a single band on native PAGE. Interestingly, the enzyme was highly active over a wide range of pH from 8.0 to 11.0, with an optimum at pH 10.0 using Nalpha-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as a substrate. The relative activities at pH 8.0, 11.0, and 12.0 were 73%, 67% and 50.4%, respectively. The enzyme was extremely stable over a broad pH range (5.0-12.0). The optimum temperature for enzyme activity was 50 degrees C. The purified enzyme was strongly inhibited by soybean trypsin inhibitor (SBTI). In addition, the enzyme showed excellent stability toward various surfactants and bleache agents and compatibility with some commercial solid and liquid detergents. The trypsin kinetic constants, Km and kcat of the enzyme for BAPNA, were 0.29 mM and 1.36 s(-1), respectively, while the catalytic efficiency kcat/Km was 4.68 s(-1) mM(-1).

New alkaline trypsin from the intestine of Grey triggerfish (Balistes capriscus) with high activity at low temperature: Purification and characterisation

A highly alkaline trypsin from the intestine of Grey triggerfish (Balistes capriscus), with high activity at low temperature, was purified and characterised. The enzyme was purified to homogeneity using acetone precipitation, Sephadex G-100 gel filtration and Mono Q-Sepharose anion-exchange chromatography, with a 13.9-fold increase in specific activity and 41.3% recovery. The molecular weight of the purified alkaline trypsin was estimated to be 23.2 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and size exclusion chromatography. Purified trypsin appeared as a single band on native–PAGE. Interestingly, the enzyme was highly active over a wide range of pH, from 9.0 to 11.5, with an optimum at pH 10.5, using Nα-benzoyl-DL-arginine-p-nitroanilide (BAPNA) as a substrate. The relative activities at pH 9.0, 11.5 and 12.0 were 86.5%, 92.6% and 52.4%, respectively. The enzyme was extremely stable in the pH range 7.0–12.0. In addition, the enzyme had high activity at low and moderate temperatures with an optimum at around 40 °C and had more than 80% of its maximum activity at 20 °C. The purified enzyme was strongly inhibited by soybean trypsin inhibitor (SBTI) and phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor. The enzyme showed extreme stability towards oxidising agents, retaining about 87% and 80% of its initial activity after 1 h incubation at 40 °C in the presence of 1% sodium perborate and 1% H2O2, respectively. In addition, the enzyme showed excellent stability and compatibility with some commercial solid detergents. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGYECTPNST. B. capriscus trypsin, which showed high homology with trypsins from marine vertebrates, had a basic residue, Asn, at position 10, where His and Tyr are common in all marine vertebrates trypsins. The trypsin kinetic constants, Km and kcat for BAPNA, were 0.068 mM and 2.76 s−1, respectively, while the catalytic efficiency, kcat/Km, was 40.6 s−1 mM−1.

Synthesis of a Precursor Dipeptide of Thymopentin in Organic Solvents by an Enzymatic Method

The protease‐catalyzed, kinetically controlled synthesis of a precursor dipeptide of thymopentin(TP‐5), Z‐Arg‐Lys‐NH2 in organic solvents was studied. Z‐Arg‐OMe was used as the acyl donor and Lys‐NH2 was used as the nucleophile. An industrial alkaline protease alcalase and trypsin were used to catalyze the synthesis of the target dipeptide in water‐organic cosolvent systems. The conditions of the synthesis reaction were optimized by examining the effects of several factors, including organic solvents, water content, temperature, pH, and reaction time on the yield of Z‐Arg‐Lys‐NH2. The optimum conditions using alcalase as the catalyst are pH 10.0, 35°C, in acetonitrile/DMF/Na2CO3‐NaHCO3 buffer system (80∶10∶10, V/V), 6 h, with the dipeptide yield of 71.1%. Compared with alcalase, the optimum conditions for trypsin are pH 8.0, 35°C, in ethanol/Tris‐HCl buffer system (80∶20, V/V), 4 h, with the dipeptide yield of 76.1%.

Studies of adsorption of alkaline trypsin by poly(methacrylic acid) brushes on chitosan membranes

AbstractPoly(methacrylic acid)‐grafted chitosan membranes (chitosan‐g‐poly(MAA)) were prepared in two sequential steps: in the first step, chitosan membranes were prepared by phase‐inversion technique and then epichlorohydrin was used as crosslinking agent to increase its chemical stability in acidic media; in the second step, the graftcopolymerization of methacrylic acid onto the chitosan membranes was initiated by ammonium persulfate (APS) under nitrogen atmosphere. The chitosan‐g‐poly(MAA) membranes were first used as an ion‐exchange support for adsorption of trypsin from aqueous solution. The influence of pH, equilibrium time, ionic strength, and initial trypsin concentration on the adsorption capacity of the chitosan‐g‐poly(MAA) membranes have been investigated in a batch system. Maximum trypsin adsorption onto chitosan‐g‐poly(MAA) membrane was found to be 92.86 mg mL−1 at pH 7.0. The experimental equilibrium data obtained for trypsin adsorption onto chitosan‐g‐poly(MAA) membranes fitted well to the Langmuir isotherm model. The adsorption data was analyzed using the first‐ and second‐order kinetic models, and the experimental data was well described by the second‐order equation. More than 97% of the adsorbed trypsin was desorbed using glutamic acid solution (0.5M, pH 4.0). In addition, the chitosan‐g‐ poly(MAA) membrane prepared in this work showed promising potential for various biotechnological applications. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2008

The content of abscisic acid and the activities of proteinases and trypsin inhibitory proteins, in the germinating seed of common beans under water stress conditions

Specific features of changes in the contents of free and bound abscisic acid (ABA) and the activities of neutral and alkaline proteinases and trypsin inhibitory proteins were determined in the embryonic axis and cotyledons of the common bean (Phaseolus vulgaris L.) after drying. The changes in ABA content, observed following the loss of 5% seed weight, were regarded as an adaptive reaction to stress, whereas the corresponding changes after the loss of 10% of the seed’s weight, was regarded as a result of a pathological disturbance of ABA metabolism. Both drying modes had a negative effect on the state of the proteinase-inhibitory system, as was apparent from the disruption of the regular inverse correlation between the activities of proteinases and serine proteinase inhibitory proteins. A comparison of the dynamics of these characteristics with the buildup of water stress demonstrates an inverse correlation between the content of free ABA and the activity of the proteinases studied. This suggests a potential inhibitory effect of this hormone on the function of the hydrolases in question in the germinating seed.

Enzymic Modification of Feather Keratin Hydrolysates with Lysine Aimed at Increasing the Biological Value

The lysine content of feather protein hydrolysate was modified via activation with alkaline proteinase and trypsin at pH 6·2 and 8·3, respectively. Lysine was used as the hydrochloride and the diacetyl derivative in the first case and as the methyl ester in the trypsin modification. The results indicate a lysine content of 26–35 g kg-1 and 59–64 g kg-1 after alkaline proteinase activation and 52–65 g kg-1 after trypsin activation. Considerable changes in the molecular mass profile of peptides were observed. Thus, enzyme modification is a way to raise the nutritive value of feather protein hydrolysate by increasing its lysine content (the main limiting essential amino acid). © 1997 SCI.

Macroporous and hydrophilic polymer resins modified with isothiocyanate groups for immobilization of enzymes

Hydrophilic and macroporous polymer resins composed of glycerylmethacrylate, styrene, and divinylbenzene were quite easily modified with isothiocyanate groups using a Friedel-Crafts reaction with 3-chloropropionyl chloride and subsequent nucleophilic reaction with KSCN. Alkaline phosphatase and trypsin could be covalently bound to the isothiocyanate-carrying polymer resins, and the immobilized enzymes obtained were sufficiently active and stable due to a covalent bonding via a spacer group between the carrier resins and the enzymes and also due to a hydrophilic environment around the enzymes. A heterogeneity of the immobilized enzyme was taken into account to interpret the thermal denaturation process of the enzyme immobilized onto the resins.

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.

Alzheimer dementia and Pick's disease: neurofibrillary tangles and Pick bodies are associated with identical phosphorylated neurofilament epitopes

Sections of formaldehyde-fixed paraffin-embedded cortical and hippocampal brain tissue from five cases with senile dementia of Alzheimer type (SDAT) and five cases with Pick's disease (PD) were immunostained with the monoclonal antibodies (mabs) 147, RT 97, BF 10 and 8D8 with and without pretreatment with alkaline phosphatase (AP) or trypsin (Tr). The mabs 147, RT 97 and BF 10 had previously been demonstrated to bind exclusively to phosphorylated epitopes of neurofilament proteins, while mab 8D8 is shown in this report to bind mainly, but not exclusively, to phosphorylated neurofilament epitopes. The mabs RT 97, BF 10 and 8D8, but not 147 stain most, if not all, Pick bodies (PB) and Alzheimer neurofibrillary tangles (NFT). When sections are pretreated with AP or Tr the immunostaining with mab BF 10 is very resistent in both PB and NFT. This resistance of PB and NFT is in contrast to the reduced staining of axons and of swollen cells in PD by the same enzymatic pretreatment. Immunostaining with mab RT 97 of PB and NFT is reduced moderately by AP and considerably by Tr. Only when stained with mab 8D8 is there a discrepancy between PB and NFT in their reaction to the pretreatment with AP: NFT staining with mab 8D8 is not affected, while that of PB is abolished. Thus, in spite of their different ultrastructure, PB and NFT are very similar immunocytochemically and in the accessibility of their phosphorylated epitopes to enzymatic treatment.

Changes in the intestinal enzyme activity of lambs during chronic infection with Trichostrongylus vitrinus

The pathology and enzymology of the intestinal mucosae of lambs dosed daily with 2500 Trichostrongylus vitrinus larvae and killed at five, nine or 14 weeks were compared with worm-free animals. The proximal small intestines of the infected lambs exhibited extensive mucosal damage at five and nine weeks, but only isolated lesions were found at 14 weeks. Activities of the brush border enzymes alkaline phosphatase, leucine amino-peptidase, maltase and glycyl-L-leucine dipeptidase were all significantly depleted during infection, although the magnitude, time of onset and duration of the individual enzyme responses varied. Mucosal activities of the pancreatic enzymes, trypsin and to a lesser extent chymotrypsin were also markedly decreased particularly during the first nine weeks of infection. Specific acetylcholinesterase activity was significantly increased throughout the study, maximal levels being observed at five weeks. In contrast 'pseudo'-cholinesterase levels were consistently within the control range. During the early stages of infection (five weeks) glutamine-oxaloacetate transaminase activity was significantly decreased, while aldolase and creatine phosphokinase levels were significantly elevated. At nine weeks low glutamine-oxaloacetate transaminase activities were again detected and lactate dehydrogenase activity was also markedly reduced. At 14 weeks the mean activities of all four enzymes were within the normal range as were superoxide dismutase levels throughout. Significant correlations were found between alkaline phosphatase, trypsin, chymotrypsin, aldolase and glutamine-oxaloacetate transaminase activities and the degree of mucosal damage within the individual lambs.

Proteolytic capacity in mouse cardiac muscle following strenuous exercise.

Proteolytic capacity in mouse cardiac muscle was analyzed 1, 3, and 6 days after exhaustive intermittent or submaximal prolonged running. No significant changes were recorded in the activities of acid or alkaline proteases, β-glucuronidase or trypsin inhibitor. Similarly, no changes were found in the rates of acid or neutral autolysis.

Thein vivo effect of mercuric chloride on some digestive enzymes of a fresh water teleost fish,Channa punctatus

Alterations in the activities of alkaline phosphatase, acid phosphatase, glucose-6-phosphatase amylase, trypsin, pepsin, aminotripeptidase, glycylglycine dipeptidase and carnosinase due to exposure of Channa punctatus to a sublethal concentration (0.30 mg/L) of mercuric chloride by bath for 20 days have been studied in the different parts of the digestive system. Afall in the activities of the three phosphatases was recorded except for alkaline phosphatase which showed a slight elevation in activity in intestine and pyloric caeca. An increase in the activity of amylase and the two proteases was observed in all the portions of the digestive system. The three peptidases revealed a decrease in activity.

Chronic mercuric chloride intoxication in digestive system ofChanna punctatus

The effect of exposure to a sublethal concentration (0.30 mg/l) of mercuric chloride on the activities of alkaline phosphatase, acid phosphatase, amylase, trypsin, and pepsin has been examined at intervals of 7, 15, and 30 d in the digestive system of a teleost fish, Channa punctatus. Inhibition of the activities of all these enzymes was noted after the first week of treatment. Treatment of the fish for 15 d resulted in marked increases in the activities of all the enzymes. A slight fall in enzyme activity was recorded after 30 d, but the overall activity was higher than in control fish.

Fibrous and Cystic Lesions in the Ovaries of Aged Atlantic Cod (Gadus morhua): a Preliminary Report

Ovarian stromal fibrosis occurred in four Atlantic cod (Gadus morhua L.) from the northeastern Gulf of St. Lawrence (49°07′N, 59°18′W) and off Fox Harbour, Labrador (52°18′N, 55°52′W), with accompanying cystic lesions in two specimens.Externally the ovaries varied. Specimen 1 (115 cm fork length, age 18 years) possessed clear areas (0.5–1.5 mm) where the underlying stroma was relatively thin. Specimen 2 (107 cm, 19 years) possessed scattered cream or yellow nodules (< 1 mm) and coalesced nodules (1.5–2.0 mm). Specimen 3 (118 cm, 19 years) had many colourless fluid-filled cysts (1.5–2.0 mm) and a few larger cysts (5.0 × 7.5–7.0 × 12.0 mm), the latter confined to the tapering posterior regions of the ovary. Specimen 4 (99 cm, 16 years) possessed large (up to 8 mm) multilocular cysts anteriorly.Stromal fibrosis occurred to varying degrees in each specimen, containing predominantly fibrinoid as identified by Mallory's PAH, Weigert's fibrin test, and retention of a PAS-positive reaction after incubation in alkaline trypsin. Widespread degeneration of ovigerous lamellae occurred in specimens 3 and 4.The condition of specimens 3 and 4 suggests involutional ovarian stromal fibrosis, but the endocrine studies necessary to confirm this opinion were not performed.

EFFECT OF ALTERNATING ACID AND ALKALINE DIGESTIVE FLUIDS ON THE GASTROINTESTINAL TRACT OF THE RAT.

SummaryPrevious experiments have demonstrated that exposure of a frog's leg alternately to acid-pepsin and alkaline pancreatic secretion produced rapid digestion of the leg. It was suggested that in man the rapid interchange between acid pepsin and alkaline trypsin solution in the duodenal bulb might play a role in peptic ulcer. For this reason, 5 kinds of perfusion from esophagus to ileum were conducted in rats. Alternating perfusion with acid pepsin and alkaline pancreatin (Group 3) had more marked effects in duodenum, jejunum, and ileum, than perfusion with acid pepsin alone (Group 2), or other perfusions (Group 1, 4, 5), for 90 minutes. Similar results were observed on cats and rabbits. The esophagus of the rat seemed to be more resistant to such perfusions than that of the cat. Changes of blood pH due to alternating perfusion with acid-alkaline enzyme solutions did not seem to affect the deleterious effects. While these results are suggestive of a role of alternation between gastric and pancreatic ju...