Purification and Partial Characterization of Trypsin from the Viscera of Tropical Sierra (Scomberomorus Sierra) from the Gulf of California

Trypsin from the viscera of sierra (Scomberomorus sierra) was purified by affinity chromatography on Sepharose‐4B coupled to soybean trypsin inhibitor and characterized with respect to its purity, sensitivity to temperature, pH and inhibition. Trypsin was purified from sierra viscera with 11.9‐fold and 29.7% yield. The enzyme had a molecular weight of 25.4 kDa estimated by SDS‐PAGE and two possible trypsin isoforms were observed in activity gels. Trypsin activity was strongly inhibited by soybean trypsin inhibitor and porcine trypsin inhibitor, showing a partial inhibition by a serine protease inhibitor. The optimal activity of the enzyme was observed at pH 9 and 60C with n‐α‐benzoyl‐dl‐arginine‐p‐nitroanilide as a substrate. The enzyme maintained more than 50% of its activity in temperatures up to 50C and within the pH range of 8–10 for a period of up to 2 h.     

PURIFICATION AND PROPERTIES OF TRYPSIN-LIKE ENZYMES AND A CARBOXYPEPTIDASE A FROM EUPHAUSIA SUPERBA

ABSTRACT Five proteases designated as A_1, A_2, B, C and D were isolated from Euphausia superba by the succesive steps of ammonium sulfate fractionation, acetone precipitation, gel filtration and DEAE-Sephadex A-50 chromatography, A_1, B, C and D were purified to homogeneity in disc gel electrophoresis by means of rechromatography on the DEAE-Sephadex A-50 column. Studies on substrate specificity of these enzymes revealed that A_2, B, C and D were trypsin-like enzymes and A₁ a carboxypeptidase A. A_1, B, C and D had molecular weights of about 24,000, 24,000 28,000 and 27,000; optimal pH at 9.0, 8.0, 7.5 and 7.5–8. 0; and optimal temperature at 48, 50, 55–60 and 55°C, respectively. The activity of B, C and D were not inhibited by sulfhydryl reagents and N-α-tosyl-L-phenylalanylchloro-methyl ketone, but inhibited by reducing agents, N-α-tosyl-L-lysylchloromethyl ketone and soybean trypsin inhibitor. The activity of protease A₁ was stimulated 3.5 fold by cobalt, but inhibited by 3-indolepropionate and D-phenylalanine.     

ENZYMATIC CHARACTERISTICS OF TRYPSIN FROM PYLORIC CECA OF SPOTTED MACKEREL (SCOMBER AUSTRALASICUS)

Trypsin was purified from the pyloric ceca of spotted mackerel (Scomber australasicus) by gel filtration on Sephacryl S‐200 and Sephadex G‐50. The purification and yield were 20‐fold and 81%, respectively, as compared to those in the starting crude extract. Final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and the molecular weight of the enzyme was estimated to be 24,000 Da by SDS–PAGE. The trypsin was stable at pH 5–11 for 30 min at 30C, and its maximal activity against N^α‐p‐tosyl‐L‐arginine methyl ester was pH 8.0. Trypsin was heat‐stable up to about 50C for 15 min at pH 8.0. Optimum temperature of the trypsin enzyme was 60C. The enzyme was stabilized by calcium ion. The purified trypsin was strongly inhibited by serine protease inhibitors such as N‐p‐tosyl‐L‐lysine chloromethyl ketone and soybean trypsin inhibitor, suggesting that it is a trypsin‐like serine protease. N‐Terminal amino acid sequence of spotted mackerel trypsin was IVGGYECTAHSQPHQVSLNS.     

PURIFICATION AND CHARACTERIZATION OF AN ALKALINE PROTEASE FROM THE VISCERA OF BOLTI FISH (TILAPIA NILOTICA)

An alkaline protease was extracted from acetone powder of the viscera of bolti fish (Tilapia nilotica) with distilled water, precipitated from the extract by 40–60% (NH₄)₂SO₄and then dialyzed. Enzyme homogeneity studies by polyacrylamide gel electrophoresis (PAGE) illustrated that the enzyme was homogenous. Sodium dodecyl sulfate–PAGE showed a molecular weight of 23.0 kDa. The optimal pH and optimal temperature were 8.0 and 45C, respectively, with casein as a substrate. A remarkable stability was observed under alkaline conditions (pH 6.0–10.0), where more than 90% of the enzyme activity was maintained. In the acidic region (pH 2.0–6.0), the enzyme retained more than 50% of its activity. Furthermore, the enzyme retained 85.4 and 41.8% of its activity after heating at 50 and 60C for 120 min, respectively, while the relative activity after heating at 70C for 120 min was 5.25%. The alkaline enzyme lost most of its activity when incubated at 80C for 30 min. A high percentage of total enzyme activity was inhibited when the enzyme was incubated with 50 mM of either soybean trypsin inhibitor or ethylenediaminetetraacetic acid. The presence of NaCl and CaCl₂at 10 mM concentration increased the enzyme activity by 6.9 and 10.6%, respectively.     

FURTHER CHARACTERIZATION OF THE 12 kDa PROTEASE/ALPHA AMYLASE INHIBITOR PRESENT IN MAIZE SEEDS

A 12 kDa protease/α-amylase inhibitor was purified from maize seeds and studied. Its trypsin- and amylase-inhibitor activities against enzymes from different origins were determined, as well as its optimal pH for inhibition. Eight different proteases, extracted from insects and fungi which attack grains during storage, were tested with the inhibitor. Bovine trypsin and trypsin-like proteases from the insect P. truncatus, and the fungi A. niger and A. fumigatus, were recognized by this inhibitor. Out of 11 α-amylases tested, only those from T. castaneum and C. maculatus were recognized by this inhibitor. The optimal pH's for the inhibition of trypsin and the trypsin-like protease from P. truncatus were 8.0 and 7.5, respectively. The optimal pH activity was 5.0 for the inhibition of amylases from T. castaneum and C. maculatus.     

CHARACTERIZATION OF TRYPSIN PURIFIED FROM THE PYLORIC CAECA OF THE SOUTHWEST ATLANTIC WHITE CROAKER Micropogonias furnieri (Sciaenidae)

A southwest Atlantic croaker protease was purified from the pyloric caeca by ammonium sulfate fractionation, acetone precipitation and affinity chromatography. The enzyme was classified as a trypsin on the basis of molecular weight, its ability to hydrolyze synthetic substrates N-α-benzoyl-arginine-p-nitroanilide (BAPA) and tosylarginine methyl ester (TAME), and its inhibition by known trypsin inhibitors. The isolated enzyme has a single band on SDS-PAGE with an estimated molecular mass of 24 kDa. Croaker trypsin activity was stable between pH 5 and pH 11 for 30 min at 0C, 10C and 25C and its maximal activity against BAPA was at pH 9.5. Thermostability was observed to about 55C for 30 min at pH 7.8 and its temperature optimum was 60C. Substrate turnover number was 850 BAPA units per μmol trypsin, and the K_mwas 0.081 mM at 25C. For the hydrolysis of TAME, V_maxwas 9273 units per μmol trypsin and the K_m was 0.155 mM at 25C. The catalytic efficiency V_max/K_mwas higher than that of trypsin from fish living in colder waters.     

Purification and characterisation of an acid protease from the Aspergillus hennebergii HX08 and its potential in traditional fermentation

A protease AP3 from Aspergillus hennebergii HX08 was purified by ammonium‐sulphate precipitation, followed by anion‐exchange chromatography and gel filtration. The molecular weight of acid protease AP3 was 33 kDa (SDS‐PAGE and MALDI‐TOF‐MS). The protease AP3 was identified as an acid protease with MALDI‐TOF/TOF tandem MS. Its optimal temperature and pH were 60°C and 4.0, respectively. Its K _max and V _max were 57.92 mg/mL and 32.57 U/mL, respectively. The enzyme was active over a broad pH and temperature range (pH 3.0–5.0 and 30–60°C), and exhibited high activity and stability in 2–12% (v /v) ethanol solvent. Subsequent studies suggest that the enzyme presents a relatively high substrate affinity to wheat protein (98% of total activity). Its application to solid‐state fermentation of wheat flour with Saccharomyces cerevisiae could increase the hydrolysis degree of wheat protein (28.26%) and amino acid nitrogen concentration of fermented grains (34.21%). Additionally, enhanced S. cerevisiae biomass (37.09%) and alcohol concentration (38.29%) were also observed during the process. Volatile compounds analysis of fermented grains by headspace solid‐phase micro‐extraction and GC‐MS revealed more flavour compounds. These results suggest its potential in food and alcohol industries. Copyright © 2017 The Institute of Brewing & Distilling     

CHARACTERIZATION OF THE PROTEASES INVOLVED IN HYDROLYZING PADDLEFISH (POLYODON SPATHULA) MYOSIN

An extract from paddlefish surimi possessed activities of B, L, and H‐like cathepsins. The optimal pH was around 5.0 for cathepsins B and L, and was between 6.0–6.5 for the H‐like cathepsin. The enzyme activities were not impaired by heating at 40Cfor 20 min. However, the protease extract lost about 20% of its cathepsin B, 50% B+L, and 90% H‐like cathepsin activities after heating at 50C for 20 min. The activity of H‐like cathepsin was not inhibited by E‐64, suggesting that it did not belong to ike known cysteme protease group. The protease extract was capable ofhydrolyzing myosin heavy chain, producing a major fragments) around 140 kDa. Degradation of myosin by the protease extract was substantially reduced by protease inhibitors including E‐64, a protease inhibitor mixture, and bovine plasma powder.     

Purification and Some Properties of a Protease from Sorghum Malt Variety KSV8–11

A protease from sorghum malt variety KSV8–11 was purified by a combination of dialysis against 4 M sucrose, ion‐exchange chromatography on Q‐Sepharose (Fast flow), gel filtration chromatography on Sephadex G‐100 and hydrophobic interaction chromatography on Phenyl Sepharose CL‐4B. The enzyme was purified 5‐fold to give a 14.1% yield relative to the total activity in the crude extract and a final specific activity of 1348.9 U mg^?1 protein. SDS‐PAGE revealed a single migrating protein band corresponding to a relative molecular mass of 16 KDa. Using casein as substrate, the purified protease had optimal activity at 50°C and maximal temperature stability between 30°C and 40°C but retained over 64% of its original activity after incubation at 60°C for 30 min. The pH optimum was 5.0 with maximum stability at pH 6.0 but 60% of the activity remained after 24 h between pH 5.0 and 8.0. The protease was inhibited by Ag⁺, Ca²⁺, Co²⁺, Fe²⁺, Mg²⁺, iodoacetic acid (IAA) and p‐chloromercuribenzoate (p‐CMB), stimulated by Cu²⁺, Sr²⁺, phenylmethylsulfonyl‐fluoride (PMSF) and 2‐mercaptoethanol (2‐ME) while Mn²⁺ and ethylenediaminetetraacetic acid (EDTA) had no effect. The purified enzyme had a K_m of 18 mg·mL^?1 and a V_max of 11.1 μmol · mL^?1 · min^?1 with casein as substrate.     

Isolation and properties of trypsin and chymotrypsin inhibitors from barley grits after storage

The occurrence of antiproteolytic activity in albumins of barley grits has been shown. As a result of storage lasting 6 months at a temperature of ?10 °C the freeze-dried albumin of barley grits lost its antiproteolytic activity and a proteolytic activity appeared. The antiproteolytic activity was partially regained after dissociation of the enzyme–inhibitor complex which had formed. From the reactivated freeze-dried albumins of stored barley grits two thermolabile protease inhibitors were isolated and partially purified. The first trypsin–chymotrypsin inhibitor B_7.1 inhibits, at a concentration of 2.5 μg, 1 μg of crystalline trypsin and 1.9 μg of this inhibitor inhibits 1 μg of crystalline alpha-chymotrypsin. Lower activity was demonstrated for the second inhibitor of chymotrypsin B_7.4; 4.8 μg of this inhibitor is needed to inhibit the activity of 1 μg of crystalline alpha-chymotrypsin. The occurrence of both inhibitors has been confirmed by means of starchgel electrophoresis. Their specific activity was small in comparison with that of some commercial crystalline preparations of inhibitors. The capacity to inhibit native proteases shows their important physiological function, based mainly on the control of proteolytic activity of endogenous enzymes. However it seems unlikely that they could have a negative influence on the nutritive value of proteins in barley grains or grits.     

PURIFICATION AND CHARACTERIZATION OF TRYPSIN FROM PYLORIC CAECA OF BIGEYE SNAPPER (PRICANTHUS MACRACANTHUS)

Trypsin from the pyloric caeca of bigeye snapper was purified and characterized. Trypsin had an apparent molecular weight of 23.8 kDa when analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and substrate‐gel electrophoresis. The trypsin fraction consisted of three isoforms as evidenced by the appearance of three different bands on native‐PAGE. Optimal activity was observed at 55C and pH range of 8–11. The activity of trypsin fraction was completely inhibited by soybean trypsin inhibitor and was partially inhibited by E‐64 and ethylenediaminetetraacetic acid. CaCl₂ partially protected the trypsin fraction from activity loss at 40C, while NaCl (0–20%) decreased the activity in a concentration‐dependent manner. The apparent Michaelis–Menten constant (K_m) and catalytic constant (k_cat) were 0.312 mM and 1.06 s, respectively when Nα‐Benzoyl‐dl ‐arginine ρ‐nitroanilide was used as a substrate. Trypsin from the pyloric caeca of bigeye snapper generally showed similar characteristics to other fish trypsins.     

Comparative study on proteolysis of two species of bigeye snapper,Priacanthus macracanthus and Priacanthus tayenus

Proteolytic activity in muscle from two species of bigeye snapper (Priacanthus macracanthus and Priacanthus tayenus) was studied. Autolysis of mince and washed mince at 50 and 60 °C was compared. Higher degradation of myosin heavy chain was observed in both mince and washed mince from P macracanthus than in those from P tayenus, especially when the incubation time was increased. Autolysis of washed mince from both species was inhibited by soybean trypsin inhibitor, suggesting that myofibril‐associated proteases were serine proteases. When sarcoplasmic proteolytic activity in P macracanthus muscle was studied, two activity peaks with an optimum temperature of 60 °C were observed at pH 6.5 and 8.5. The activities of both peaks were mostly inhibited by soybean trypsin inhibitor, suggesting that the major protease was a serine protease. Major sarcoplasmic proteolytic activity in P macracanthus muscle was found at M_r 62 000 on sodium dodecyl sulphate substrate gel. For P tayenus sarcoplasmic proteolytic activity, two activity peaks with an optimum temperature of 60 °C were found at pH 5.0 and 8.5. The pH 5.0 peak activity was effectively inhibited by pepstatin A, while the pH 8.5 peak activity was inhibited by several inhibitors. The results indicated that various sarcoplasmic proteases were present in P tayenus muscle. The two species contained different sarcoplasmic proteases in terms of composition and activity level. P macracanthus muscle generally had higher sarcoplasmic proteolytic activities than P tayenus muscle. Copyright © 2003 Society of Chemical Industry     

COMPARATIVE STUDY OF ENZYMATIC CHARACTERISTICS OF TRYPSINS FROM THE PYLORIC CECA OF YELLOW TAIL (SERIOLA QUINQUERADIATA) AND BROWN HAKELING (PHYSICULUS JAPONICUS)

Trypsins from the pyloric ceca of two fish species, yellow tail (Seriola quinqueradiata) and brown hakeling (Physiculus japonicus) were purified by a series of chromatographic separations. Purity increased 62‐ and 106‐fold with approximately 55 and 10% yield for yellow tail trypsin and brown hakeling trypsin, respectively. Final enzyme preparations were homogeneous in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and the molecular weights of both enzymes were estimated to be 24 kDa by SDS‐PAGE. Yellow tail and brown hakeling trypsins had maximal activity at pH 8.0 for hydrolysis of N_α‐p‐tosyl‐L‐arginine methyl ester hydrochloride and was unstable at acidic pH. The optimum temperatures for yellow tail and brown hakeling trypsins were 60 and 50C, respectively. Yellow tail trypsin was stable up to 50C, whereas brown hakeling was stable up to 40C. Both trypsins were stabilized by calcium ions. The activities of both trypsins were strongly inhibited by soybean trypsin inhibitor and N_α‐p‐tosyl‐L‐lysine chloromethyl ketone hydrochloride, and were partially inhibited by ethylenediaminetetraacetic acid. The N‐terminal amino acid sequences of yellow tail trypsin and brown hakeling trypsin were determined as IVGGYECTPYSQPHQVSLNS and IVGGYECPKHSQPHQVSLNS, respectively.     

PURIFICATION AND CHARACTERIZATION OF A SERINE PROTEINASE FROM THE TUNA PYLORIC CAECA

A 15.0 kDa serine proteinase with collagenase activity from pyloric caeca of tuna, Thunnus thynnus, was purified in four steps; acetone precipitation, gel filtration chromatography on a Sephadex G‐100, ion‐exchange chromatography on a DEAE‐Sephadex α‐50 and gel filtration chromatography on a Sephadex G‐75 column. The purification and yield were 30.5‐fold and 0.023%, respectively, as compared with those in the starting crude extract. The optimum pH and temperature for the purified collagenolytic enzyme were around pH 7.5 and 55C, respectively. The purified proteinase was strongly inhibited by metal ions (Hg²⁺ and Zn²⁺) and serine proteinase inhibitors (PMSF, TLCK and soybean trypsin inhibitor) suggesting it is a serine protease. The K_m and V_max of the purified enzyme for collagen type I were approximately 3.82 mM and 851.5 U, respectively.     

COMPARATIVE STUDIES ON PROTEOLYTIC ACTIVITY OF SPLENIC EXTRACT FROM THREE TUNA SPECIES COMMONLY USED IN THAILAND

Proteolytic activities of splenic extract from three tuna species including skipjack tuna (Katsuwonus pelamis), yellowfin tuna (Thunnus albacores) and tongol tuna (Thunnus tonggol) were studied. Optimal activity of splenic extract from all tuna species was at pH 9.0 and 55C when casein was used as a substrate. Among all species tested, yellowfin tuna showed the highest activity, followed by skipjack tuna and tongol tuna. The proteolytic activity was strongly inhibited by soybean trypsin inhibitor, TLCK and partially inhibited by ethylenediaminetetraacetic acid. E‐64, N‐ethylmaleimide, iodoacetic acid, TPCK and pepstatin A showed no inhibition. The effect of NaCl and CaCl₂ on proteolytic activity was also investigated. Activities continuously decreased as NaCl concentration increased, and no activity remained in the presence of 30% NaCl. On the other hand, activities increased as CaCl₂ concentration increased. The highest activity was obtained in the presence of 1 mM CaCl₂. SDS‐substrate gel electrophoresis revealed that major proteinases in splenic extract from different tuna species were different in apparent molecular weights and sensitivity to TLCK. Although the major activity bands of all species were strongly inhibited by soybean trypsin inhibitor, varying sensitivity to TLCK probably implied the differences in binding characteristic of enzyme to substrate and/or inhibitors. The results suggest that major proteinases in spleen of all tuna species were trypsin‐like serine proteinases.     

Antimicrobial and Antioxidant Activities of Cichorium Intybus Root Extract Using Orthogonal Matrix Design

Solvent, impregnation time, sonication repetitions, and ultrasonic power were important factors in the process of ultrasound‐assisted extraction from chicory (Cichorium intybus) root, while there were no studies about optimizing these 4 factors for extract yield, total phenolic content (TPC), antioxidant, antibacterial, and antifungal activity of the extracts using orthogonal matrix design. The present research demonstrated that the solvent composition played a significant role in the improving extract yield, TPC, antioxidant, and antibacterial activities. The other 3 factors had inequable effect on different purposes, ultrasonic power could improve TPC and antioxidant activity, but long time of extraction lowered antioxidant activity. The TPC increased from 22.34 to 27.87 mg GAE (gallic acid equivalents)/100 g (dry extracts) with increasing solvent polarity. The half inhibition concentration (IC_50, μg/mL) of the radical scavenging activity of the chicory extracts ranged from 281.00 to 983.33 μg/mL. The content of caffeoylquinic acids of root extract, which was extracted by the optimal combination was 0.104%. Several extracts displayed antibacterial activities against Escherichia coli, Staphylococcus aureus, Bacillus thuringiensis, Bacillus subtilis, and Salmonella typhi, while Penicillium sp. and Aspergillus sp. resisted against all the extracts. Combination of 70% ethanol v/v, 24‐h impregnation time, 3 sonication rounds, and 300‐W ultrasonic input power was found to be the optimal combination for the chicory extract yield, TPC, antioxidant activity, and antibacterial activity.     

Particle Size of Milk Protein Concentrate Powder Affects the Texture of High‐Protein Nutrition Bars During Storage

Milk protein concentrate powder with 85% protein (MPC85) was jet‐milled to give 2 particle size distributions (that is, JM‐Coarse and JM‐Fine) or freeze‐dried (FD), in order to improve the functional properties of MPC85 for use in high‐protein nutrition (HPN) bars. Volume‐weighted mean diameter decreased from 86 μm to 49, 22, and 8 μm in FD, JM‐Coarse, and JM‐Fine, respectively (P < 0.05). The MPC85 powders modified by jet‐milling and freeze‐drying were significantly denser than the control MPC85 (P < 0.05). Volume of occluded air in the modified powders decreased (P < 0.05) by an order of magnitude, yet only FD possessed a lower volume of interstitial air (P < 0.05). Particle size reduction and freeze‐drying MPC85 decreased its water holding capacity and improved its dispersibility by at least 20%. Contact angle measurements showed that these modifications increased initial hydrophobicity and did not improve wettability. HPN bars made from JM‐Fine or FD were firmer by 40 or 17 N, respectively, than the control on day 0 (P < 0.05). HPN bar maximum compressive force increased by 38%, 33%, and 242% after 42 d at 32 °C when formulated with JM‐Fine, FD, or control MPC85, respectively. HPN bars prepared with JM‐Fine were less crumbly than those formulated with control or FD MPC85. Physically altering the particle structure of MPC85 improved its ability to plasticize within HPN bars and this improved their cohesiveness and textural stability.     

Effects of tea polyphenols on the activities of soybean trypsin inhibitors and trypsin

Tea polyphenols (TPs) and other materials were extracted from Chinese green tea, and their effects on trypsin inhibitors and trypsin were analysed. TPs were found to have a deactivation effect on both Kunitz trypsin inhibitor (KTI) and Bowman–Birk trypsin inhibitor (BBTI). KTI was more easily deactivated than BBTI by complexing with TPs. The deactivation effect of TPs on KTI and BBTI reached a maximum at a TP/KTI ratio of 25 and a TP/BBTI ratio of 16. However, the deactivation effect of TPs on KTI and BBTI was reduced dramatically when KTI and BBTI were already complexed with trypsin. TPs were also found to inhibit trypsin. The inhibitory activity of TPs, KTI and BBTI on trypsin was found to decrease in the order BBTI > gtTI > gtPs. Complete inhibition of trypsin by TPs could not be achieved. When the TP concentration was increased to about 17 µg ml^?1, the residual activity of trypsin was maintained at 400 TU mg^?1, equivalent to 32% of the initial trypsin activity. In TP inhibition the K_M value for trypsin remained unchanged at 5.88 × 10^?4 mol l^?1 and V_max decreased when benzoyl‐DL ‐arginine‐p‐nitroanilide (BAPNA) was used as substrate. The pattern of trypsin inhibition by TPs is non‐competitive. Copyright © 2004 Society of Chemical Industry     

EFFECTS OF HYDROSTATIC PRESSURE ON ALPHA-MACROGLOBULIN AND SELECTED PROTEASES

The enzymes, chymotrypsin, trypsin, collagenase and cathepsin C, as well as the protease inhibitor, α₂-macroglobulin, have been shown to be susceptible to hydrostatic pressure inactivation. a₂-Macroglobulin lost about 15% activity at a pressure of 2,000 atm and as much as 80% at 3,000 atm pressure applied for 1 h in both cases. Thermal stability studies also indicated the protein to be completely denatured at about 75C. The enzymes were also inactivated to various extents depending on the amount of pressure and the duration of application, The degree of susceptibility to pressures ranging between 2,000–4,000 atm applied for time periods ranging from 5–60 min was found to be as follows: chymotrypsin < cathepsin < trypsin < collagenase. The hydrostatic pressure effects were irreversible when pressure-treated enzymes were stored at room temperature (20–25C), but showed various levels of reactivation when stored at refrigerated temperatures (4–7C).     

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.