A novel aspartic protease from the viscera of Sardinelle (Sardinella aurita): Purification and characterisation

A novel aspartic protease was extracted from the defatted viscera of sardinelle (Sardinella aurita) and purified, with a 9.5-fold increase in specific activity and 23.3% recovery. The molecular weight of the purified enzyme was estimated to be 17 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The purified enzyme appeared as a single band on native-PAGE. The optimum pH and temperature for protease activity were around 3.0 and 40 °C, respectively. The enzyme showed pH stability between 2.0 and 5.0 and retained more than 50% of its activity after heating for 30 min at 50 °C. The enzyme lost 90% of its activity after incubation with pepstatin A at room temperature, but was not inhibited by soybean trypsin inhibitor or phenylmethylsulfonyl fluoride. Its K_m value was determined to be 0.73 × 10⁻⁴ M using haemoglobin as a substrate. The N-terminal 12 amino acid sequence of the purified acidic protease was R V I I E D X D Q F C T. This sequence showed low homology with aspartic peptidases of several other species of fish, suggesting that the enzyme is a new aspartic protease.     

Low molecular weight serine protease from the viscera of sardinelle (Sardinella aurita) with collagenolytic activity: Purification and characterisation

A new low molecular weight (LMW) serine-protease from sardinelle (Sardinella aurita) viscera was purified using ammonium sulphate precipitation and Sephadex G-100 gel filtration, with a 3.82-fold increase in specific activity. The molecular weight of the enzyme was estimated to be 14.2 kDa by SDS-PAGE. The optimum pH and temperature for the enzyme activity were around pH 8.0 and 60 °C, respectively. The purified protease was strongly inhibited by phenylmethylsulphonyl fluoride, a serine-protease inhibitor, and soybean trypsin inhibitor. The N-terminal amino acid sequence of the first 10 amino acids of the purified protease was APVQPCVVVI. This sequence showed low homology with several peptidases, suggesting that the enzyme is a new protease. Interestingly, the protease was found to cleave collagen type I and hydrolyze succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPFpna), an amide substrate of chymotrypsin. Our findings indicate that the S. aurita protease is a new LMW enzyme with collagenolytic activity.     

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 27 kDa. 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, K_m and k_cat for BAPNA, were 0.6 mM and 1.38 s^?1, respectively.     

Bowman-Birk inhibitors in lentil: Heterologous expression,functional characterisation and anti-proliferative properties in human colon cancer cells

A full-length cDNA, encoding a Bowman-Birk protease inhibitor (BBI), was isolated from lentil immature seeds. The deduced amino acid sequence was longer than that of the BBI extracted from lentil seeds and contained two binding sites; the first inhibitory site inhibits trypsin whereas the second one inhibits chymotrypsin. In order to characterize this lentil BBI, a longer (complete) and its C-terminally processed (mature) form were heterologously expressed in the yeast Pichia pastoris. The recombinant BBI proteins proved to be active against trypsin and chymotrypsin, showing K_i values at nanomolar levels. Mass spectrometry analysis revealed that complete BBI was composed of an array of molecular masses, whereas mature BBI showed the presence of a major peak of the expected size. The effects of mature BBI on the growth of human colon adenocarcinoma HT29 and colonic fibroblast CCD-18Co cells were evaluated. Lentil BBI was able to inhibit the growth of such cells at concentrations higher than 19 μM, in a concentration-dependent manner; by contrast, the CCD18-Co cells were unaffected. These data broaden our knowledge of the beneficial biological activities of naturally-occurring BBI proteins and address the need for systematic evaluation of natural variants in order to design novel strategies in preventive medicine.     

Characterisation of trypsin purified from the viscera of Tunisian barbel (Barbus callensis) and its application for recovery of carotenoproteins from shrimp wastes

Trypsin was purified from the viscera of barbel by precipitation using ammonium sulphate (0-80%), Sephadex G-100, and Mono Q-Sepharose ion exchange chromatography. The trypsin was purified 27-fold, with 79 U/mg specific activity and 31% recovery. The enzyme had a molecular weight of 24 kDa; purified trypsin appeared as a single band on native-PAGE. The optimum pH and temperature for enzyme activity were pH 10.0 and 55 °C with BAPNA used as a substrate. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGYECTPYSQ. The Michaelis-Menten constant (K_m) and catalytic constant (k_cat) values of the enzyme were 0.018 mM and 1.21 s⁻¹, respectively. The study also investigated the effects of purified trypsin on the recovery of carotenoproteins from shrimp (Parapenaeus longirostris) shells through hydrolysis using 1.0 U barbel trypsin/g shrimp shells for 1 h at 30 °C. The freeze-dried carotenoproteins recovered contained 71.09% protein, 16.47% lipid, 7.78% ash, and 1.79% chitin.     

Characteristics of trypsin from the pyloric ceca of walleye pollock (Theragra chalcogramma)

Trypsin was purified from the pyloric ceca of walleye pollock (Theragra chalcogramma) by gel filtration on Sephacryl S-200 and Sephadex G-50. The final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and the molecular mass of the enzyme was estimated to be 24 kDa by SDS–PAGE. Trypsin activity was effectively inhibited by serine protease inhibitors, such as soybean trypsin inhibitor and TLCK. Trypsin had maximal activities at around pH 8.0 and 50 °C for the hydrolysis of N^α-p-tosyl-l-arginine methyl ester hydrochloride. Trypsin was unstable above 30 °C and below pH 5.0, and was stabilized by calcium ions. Walleye pollock trypsin was more thermally unstable than trypsin from the Temperate Zone fish and Tropical Zone fish. The N-terminal amino acid sequence of the trypsin, IVGGYECTKHSQAHQVSLNS, was found, and the sequential identity between the walleye pollock trypsin and Frigid Zone fish trypsin was higher (85–100%) than with Temperate Zone fish trypsin (75–90%), Tropical Zone fish trypsin (75–85%), or mammalian trypsin (60–65%).     

Antioxidant and free radical-scavenging activities of smooth hound (Mustelus mustelus) muscle protein hydrolysates obtained by gastrointestinal proteases

We have investigated the antioxidative activity of five hydrolysates from smooth hound (Mustelus mustelus) meat obtained by various gastrointestinal proteases: crude enzyme extract, low molecular weight (LMW) alkaline protease and trypsin-like protease from M. mustelus intestine, pepsin from M. mustelus stomach, and bovine trypsin.     

Biochemical properties of two isoforms of trypsin purified from the Intestine of skipjack tuna (Katsuwonus pelamis)

Two trypsins (A and B) from the intestine of skipjack tuna (Katsuwonus pelamis) were purified by Sephacryl S-200, Sephadex G-50 and DEAE-cellulose with a 177- and 257-fold increase in specific activity and 23% and 21% recovery for trypsin A and B, respectively. Purified trypsins revealed a single band on native-PAGE. The molecular weights of both trypsins were 24 kDa as estimated by size exclusion chromatography and SDS–PAGE. Trypsin A and B exhibited the maximal activity at 55 °C and 60 °C, respectively, and had the same optimal pH at 9.0. Both trypsins were stable up to 50 °C and in the pH range from 6.0 to 11.0. Both trypsin A and B were stabilised by calcium ion. Activity of both trypsins continuously decreased with increasing NaCl concentration (0–30%) and were inhibited by the specific trypsin inhibitors – soybean trypsin inhibitor and N-p-tosyl-l-lysine chloromethyl ketone. Apparent K_m and K_cat of trypsin A and B were 0.22–0.31 mM and 69.5–82.5 S⁻¹, respectively. The N-terminal amino acid sequences of the first 20 amino acids of trypsin A and B were IVGGYECQAHSQPPQVSLNA and IVGGYECQAHSQPPQVSLNS, respectively.     

Giant Amazonian fish pirarucu (Arapaima gigas): Its viscera as a source of thermostable trypsin

A trypsin was purified from pyloric caeca of pirarucu (Arapaima gigas). The effect of metal ions and protease inhibitors on its activity and its physicochemical and kinetic properties, as well its N-terminal sequence, were determined. A single band (28.0 kDa) was observed by SDS–PAGE. Optimum pH and temperature were 9.0 and 65 °C, respectively. The enzyme was stable after incubation for 30 min in a wide pH range (6.0–11.5) and at 55 °C. The kinetic parameters K_m, k_cat and k_cat/K_m were 0.47 ± 0.042 mM, 1.33 s⁻¹ and 2.82 s⁻¹ mM⁻¹, respectively, using BApNA as substrate. This activity was shown to be very sensitive to some metal ions, such as Fe²⁺, Hg²⁺, Zn²⁺, Al³⁺, Pb²⁺, and was highly inhibited by trypsin inhibitors. The trypsin N-terminal sequence IVGGYECPRNSVPYQ was found. The features of this alkaline peptidase suggest that it may have potential for industrial applications (e.g. food and detergent industries).     

Purification and characterisation of trypsins from the pyloric caeca of mandarin fish (Siniperca chuatsi)

Two trypsins of anionic form (trypsin A) and cationic form (trypsin B) from the pyloric caeca of mandarin fish (Siniperca chuatsi) were highly purified by a series of chromatographies, including DEAE-Sephacel, Sephacryl S-200 HR, Q-Sepharose or SP-Sepharose. Purified trypsins revealed a single band on native-PAGE. The molecular weights of trypsin A and B were 21 kDa and 21.5 kDa, respectively, as estimated by SDS–PAGE, both under reducing and non-reducing conditions. Zymography analysis showed that both trypsins were active in degrading casein. Trypsin A and B exhibited maximal activity at 35 °C and 40 °C, respectively, and shared the same optimal pH of 8.5, using Boc-Phe-Ser-Arg-MCA as substrate. The two trypsins were stable up to 45 °C and in the pH range from 4.5 to 11.0. Trypsin inhibitors are effective on these two enzymes and their susceptibilities were similar. Both trypsins were activated by metal ions such as Ca²⁺ and Mg²⁺ and inactivated by Fe²⁺, Zn²⁺, Mn²⁺, Cu²⁺, Al³⁺, Ba²⁺ and Co²⁺ to different degrees. Apparent K_m values of trypsin A and B were 2.18 μM and 1.88 μM, and K_cat values were 81.6 S⁻¹ and 111.3 S⁻¹ for Boc-Phe-Ser-Arg-MCA, respectively. Immunoblotting analysis using anti-common carp trypsin A positively cross-reacted with the two enzymes, suggesting their similarity. The N-terminal amino acid sequence of trypsin B was determined as IVGGYECEAH, which is highly homologous with trypsins from other species of fish.     

Glycosylation,amino acid analysis and kinetic properties of a major Kunitz-type trypsin inhibitor from Acacia victoriae Bentham seeds

An Acacia victoriae trypsin inhibitor (AvTI) was purified from the seeds of prickly wattle (A. victoriae Bentham) by salt precipitation, ion-exchange and gel filtration chromatography, and its degree of glycosylation, amino acid composition, and kinetic properties were determined. Gel electrophoresis revealed at least four glycoprotein bands in the crude extract, salt-precipitated and ion-exchange protein fractions, while the purified AvTI showed only one band and a degree of glycosylation of 2.06%. Glutamate (13.3%), aspartate (10.3%), leucine (7.62%) and lysine (7.01%) were the major amino acids in AvTI while the contents of sulphur-containing amino acids, cysteine (1.38%) and methionine (0.75%), as well as of tryptophan (1.17%) were low. Its dissociation constant (K_i) for the inhibition of bovine trypsin was found to be 1.06 × 10⁻⁸ M, indicating a high affinity between AvTI and this enzyme, and its role as a competitive inhibitor was confirmed by a double reciprocal plot. These results complement our earlier studies which indicated the presence of three isoforms of this Kunitz-type trypsin inhibitor in prickly wattle seed.     

Purification and characterization of trypsin from the viscera of sardine (Sardina pilchardus)

Trypsin from the viscera of Sardina pilchardus was purified by fractionation with ammonium sulphate, heat treatment and Sephadex G-100 gel filtration with a ninefold increase in specific activity and 9% recovery. The molecular weight of the enzyme was estimated to be 25,000 Da on SDS–PAGE. This enzyme showed esterase-specific activity on Nα-benzoyl-l-arginine ethyl ester (BAEE). The purified enzyme was inhibited by benzamidine, a synthetic trypsin inhibitor, and phenylmethylsulphonyl fluoride (PMSF) a serine-protease inhibitor, but was not inhibited by the β-mercaptoethanol. The optimum pH and temperature for the enzyme activity were pH 8.0 and 60 °C, respectively. The relative activity at pH 9.0 was 95.5% and the enzyme showed pH stability between 6.0 and 9.0. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGYECQKYSQ. S. pilchardus trypsin, which showed high homology to other fish trypsins, had a charged Lys residue at position 9, where Pro or Ala are common in fish trypsins. The enzyme was strongly inhibited by Zn²⁺ and Cu²⁺.     

Cloning, characterization, expression and antifungal activity of an alkaline serine protease of Aureobasidium pullulans PL5 involved in the biological control of postharvest pathogens

An alkaline protease gene was amplified from genomic DNA and cDNA of the antagonistic yeast-like fungus Aureobasidium pullulans PL5, a biocontrol agent effective against Monilinia laxa on stone fruit and Botrytis cinerea and Penicillium expansum on pome fruits. An open reading frame of 1248 bp encoding a 415-amino acid (aa) protein with a calculated molecular weight (M_r) of 42.9 kDa and an isoelectric point (pI) of 4.5 was characterized. The cDNAALP5 gene had an 18-amino acid signal peptide, one N-gylcosylation, one histidine active site, and one serine active site. The ALP5 gene with a M_r of 1351 bp contained two introns. One intron was of 54 bp, while the other was of 50 bp. Protein BLAST and phylogenetic tree analysis of the deduced amino sequences from the cDNAALP5 gene showed that the encoded protein had 100% homology to a protease enzyme (ALP2) of a sea strain of A. pullulans, suggesting that the protein ALP5 was an alkaline serine protease. Expression of ALP5 in Escherichia coli BL21 (DE3), followed by identification with Western-blotting, purification with Ni-NTA and analysis of enzymatic activity, yielded an homogeneous recombinant ALP5 which hydrolysed the substrate casein and inhibited the mycelial growth of the pathogens. At its optimal pH of 10.0 and reaction temperature of 50 °C, the recombinant protease exhibited the highest activity towards the substrate casein, though the highest stability was at lower temperatures and pH between 7.0 and 9.0. This study provided the direct evidence that extracellular proteases secreted by the antagonist A. pullulans PL5 played a role in the biocontrol activities against some postharvest pathogens of apple and peach.     

Angiotensin I-converting enzyme (ACE) inhibitory activities of sardinelle (Sardinella aurita) by-products protein hydrolysates obtained by treatment with microbial and visceral fish serine proteases

The angiotensin I-converting enzyme (ACE) inhibitory activities of protein hydrolysates prepared from heads and viscera of sardinelle (Sardinella aurita) by treatment with various proteases were investigated. Protein hydrolysates were obtained by treatment with Alcalase^®, chymotrypsin, crude enzyme preparations from Bacillus licheniformis NH1 and Aspergillus clavatus ES1, and crude enzyme extract from sardine (Sardina pilchardus) viscera. All hydrolysates exhibited inhibitory activity towards ACE. The alkaline protease extract from the viscera of sardine produced hydrolysate with the highest ACE inhibitory activity (63.2 ± 1.5% at 2 mg/ml). Further, the degrees of hydrolysis and the inhibitory activities of ACE increased with increasing proteolysis time. The protein hydrolysate generated with alkaline proteases from the viscera of sardine was then fractionated by size exclusion chromatography on a Sephadex G-25 into eight major fractions (P₁–P₈). Biological functions of all fractions were assayed, and P₄ was found to display a high ACE inhibitory activity. The IC₅₀ values for ACE inhibitory activities of sardinelle by-products protein hydrolysates and fraction P₄ were 1.2 ± 0.09 and 0.81 ± 0.013 mg/ml, respectively. Further, P₄ showed resistance to in vitro digestion by gastrointestinal proteases. The amino acid analysis by GC/MS showed that P₄ was rich in phenylalanine, arginine, glycine, leucine, methionine, histidine and tyrosine. The added-value of sardinelle by-products may be improved by enzymatic treatment with visceral serine proteases from sardine.     

A heat-stable trypsin from the hepatopancreas of the cuttlefish (Sepia officinalis): Purification and characterisation

Thermostable trypsin from the hepatopancreas of Sepia officinalis was purified by fractionation with ammonium sulphate, Sephadex G-100 gel filtration, DEAE-cellulose an ion-exchange chromatography, Sephadex G-75 gel filtration and Q-Sepharose anion-exchange chromatography, with a 26.7-fold increase in specific activity and 21.8% recovery. The molecular weight of the purified enzyme was estimated to be 24,000 Da by SDS-PAGE and size exclusion chromatography. The purified enzyme showed esterase specific activity on Nα -benzoyl-L-arginine ethyl ester (BAEE) and amidase activity on Nα -benzoyl-DL-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for the enzyme activity were pH 8.0 and 70 °C, respectively, using BAPNA as a substrate. The enzyme was extremely stable in the pH range 6.0–10.0 and highly stable up to 50 °C after 1 h of incubation. The purified enzyme was inhibited by soybean trypsin inhibitor (SBTI) and phenylmethylsulphonyl fluoride (PMSF), a serine-protease inhibitor. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGKESSPYNQ. S. officinalis trypsin, which showed high homology with trypsins from marine vertebrates and invertebrates, had a charged Lys residue at position 5 and a Ser residue at position 7, where Tyr and Cys are common in all marine vertebrates and mammalian trypsins. Further, the enzyme had an Asn at position 11, not found in any other trypsins.     

An alkaline protease from Kocuria kristinae F7: properties and characterization of its hydrolysates from soy protein

An alkaline serine protease (SF1) from Kocuria kristinae F7 was purified. The molecular mass of the SF1 protease was estimated to be 57 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The N-terminal amino acid sequence of the first 14 amino acids of the SF1 protease showed low homology with bacterial proteases, suggesting that the enzyme had not been described previously. The SF1 protease exhibited maximal activity at pH 9.0 and 60 °C. The activity of the SF1 protease was enhanced by the presence of Ca²⁺ and Mg²⁺ ions. It showed high stability in the presence of NaCl and ethanol. Reverse-phase high-performance liquid chromatography (RP-HPLC) analyses indicated that soybean protein isolates treated with the SF1 protease generated four principal new hydrophilic peptides. Mass spectrometry analyses indicate that the distribution of molecular weight of these peptides was from 0.705 to 1.305 kDa. Hydrolysis of soybean protein isolates with the SF1 protease increased the level of total free amino acids, essential amino acids and flavor amino acids. The SF1 protease may decrease the bitterness of soy protein hydrolysates. The results showed that the SF1 protease of Kocuria kristinae F7 appears to be good candidate enzyme for potential application in acceleration of fermented soybean food ripening.     

五种蛋白酶对牡蛎酶解产物滋味特性的影响

以牡蛎为研究对象,在水解度一致的情况下,探究胰蛋白酶、动物蛋白酶、碱性蛋白酶、中性蛋白酶和风味蛋白酶对牡蛎酶解产物感官、肽分子量和游离氨基酸的影响。结果表明:五种酶解产物均具有较强的鲜味。碱性蛋白酶酶解产物在五种酶解产物中整体感官评价最佳。分子量<5 kDa肽比例从大到小是胰蛋白酶(74.99%)、动物蛋白酶(73.44%)、中性蛋白酶(71.53%)、碱性蛋白酶(68.46%)和风味蛋白酶(58.77%)。五种酶解产物中游离氨基酸百分比均呈现出谷氨酸百分比最高、组氨酸百分比最低的特点。游离氨基酸总量从高到低分别为胰蛋白酶(22.816 mg/g)、中性蛋白酶(20.775 mg/g)、风味蛋白酶(20.530 mg/g)、动物蛋白酶(16.287 mg/g)、碱性蛋白酶(16.232 mg/g)。胰蛋白酶、动物蛋白酶、碱性蛋白酶、中性蛋白酶和风味蛋白酶酶解产物中TAV大于1的游离氨基酸数量分别为10、8、8、9和11种。5种蛋白酶酶解产物中,TVA值最大的氨基酸均是谷氨酸。

     

Blue-back fish: Fatty acid profile in selected seasons and retention upon baking

The effects of the catching season (either Autumn/Winter or Spring) on lipid content, fatty acid profile and true retention values after oven baking were determined in anchovy (Engraulis encrasicholus), sardine (Sardina pilchardus), sprat (Sprattus sprattus) and horse mackerel (Trachurus trachurus) from the North Adriatic sea. In the raw state, the catching season induced significant changes in the flesh lipid contents of sardine and sprat. Anchovy was the species whose fatty acid composition of flesh lipids was most clearly affected by the season of catch. Oven baking had a significant, though rather modest, effect on some fatty acids and indices of sardine and anchovy lipids. When compared to the other species within the same season, spring sardine and winter sprat gave significantly higher true retention values for several individual fatty acids and some sums. Between 1 and 2.5 servings/week (depending on season) of either cooked anchovy, sardine, sprat, or 3 servings of cooked spring horse mackerel would suffice to satisfy human weekly requirements of EPA + DHA.     

24 kDa Trypsin: A predominant protease purified from the viscera of hybrid catfish (Clarias macrocephalus × Clarias gariepinus)

Trypsin was purified to homogeneity from the viscera of hybrid catfish (Clarias macrocephalus × Clarias gariepinus) through ammonium sulphate fractionation and a series of chromatographies including Sephacryl S-200, Sephadex G-50 and DEAE-cellulose. It was purified to 47.6-fold with a yield of 12.7%. Based on native-PAGE, the purified trypsin showed a single band. The molecular weight of purified trypsin was estimated as 24 kDa by size exclusion chromatography and SDS–PAGE. The optimum pH and temperature for N^α-p-tosyl-l-arginine methyl ester hydrochloride (TAME) hydrolysis were 8.0 and 60 °C, respectively. Trypsin was stable to heat treatment up to 50 °C, and over a pH range of 6.0–11.0. Trypsin was stabilized by calcium ion. The trypsin activity was strongly inhibited by soybean trypsin inhibitor and N-p-tosyl-l-lysine chloromethyl ketone and partially inhibited by ethylenediaminetetraacetic acid. Activity decreased continuously as NaCl concentration (0–30%) increased. Apparent K_m value of trypsin was 0.3 mM and K_cat value was 92.1 S⁻¹ for TAME. The N-terminal amino acid sequence of 20 residues of trypsin was IVGGYECQAHSQPPTVSLNA, which is highly homologous with trypsins from other species of fish.     

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.