Purification and characterisation of trypsins from the spleen of skipjack tuna (Katsuwonus pelamis)

Three trypsin isoforms, trypsins A, B and C, from the spleen of skipjack tuna (Katsuwonus pelamis) were purified by a series of chromatographies including Sephacryl S-200, Sephadex G-50 and diethylaminoethyl-cellulose to obtain a single band on native-PAGE and SDS–PAGE. The molecular mass of all the trypsin isoforms was estimated to be 24 kDa by size exclusion chromatography and SDS–PAGE. The optimum pH and temperature of the three isoforms for the hydrolysis of N-p-tosyl-l-arginine methyl ester hydrochloride were 8.5 and 60 °C, respectively. Trypsins were stable to heat treatment up to 50 °C, and over a pH range of 6.0–11.0. All isoforms were stabilised by calcium ions. The trypsin activities were effectively inhibited by soybean trypsin inhibitor, TLCK and partially inhibited by ethylenediaminetetraacetic acid, while E-64, N-ethylmaleimide, iodoacetic acid, TPCK and pepstatin A showed no inhibitory effect. Activities decreased continuously as NaCl concentration (0–30%) increased. Trypsins A, B and C showed K_m of 0.11–0.29 mM and K_cat of 57.1–114 s⁻¹. The N-terminal amino acid sequence of 20 residues of three trypsin isoforms was IVGGYECQAHSQPHQVSLNS and had high homology to those of other fish trypsins.     

Purification and properties of an acid phosphatase from Lactobacillus curvatus DPC2024

An acid phosphatase was purified to homogeneity from a cell-free extract of Lactobacillus curvatus DPC2024 by chromatography on diethylaminoethyl-Sephacel, Phenyl Sepharose, chelating Sepharose Fast Flow and MonoQ. The purified enzyme was a tetramer with a subunit molecular mass of 26 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and gel filtration chromatography. Its optimum activity was at pH 4.5 and 70°C, with more than 65% of its activity retained after pre-heating for 30 min at 70°C. The enzyme was strongly inhibited by NaF (0.1 m ) and ZnCl₂ (1.0 m ); slightly inhibited by hexametaphosphate, tripolyphosphate or pyrophosphate at 1.0 m concentrations; but unaffected by 10 m ascorbic acid. The acid phosphatase hydrolysed a number of phosphate esters but not bis(p-nitrophenyl)phosphate nor uridine-5′-monophosphate. The N-terminal amino acid sequence of the first 20 residues of this enzyme showed 65% homology with an acid phosphatase from Lactobacillus plantarum DPC2739 and some homology with other phosphatases from mammals, yeasts and Escherichia coli.     

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.     

Simulation studies on the effects of solar heat on egg-laying, development and survival of Callosobruchus maculatus (F.) and Callosobruchus subinnotatus (Pic) in stored bambara groundnut Vigna subterranea (L.) Verdcourt

The effects of simulated solar heat on oviposition, development and survival of Callosobruchus maculatus (F.) and Callosobruchus subinnotatus (Pic) in stored bambara groundnut, Vigna subterranea (L.) Verdcourt, were evaluated at three high temperatures (40°C, 45°C and 50°C) at a constant, low humidity (30% relative humidity). Exposure to these temperatures for 6 h significantly reduced oviposition in C. maculatus and C. subinnotatus females. Females of both species that were exposed to 50°C laid significantly fewer eggs than those exposed to 40°C; and in the case of C. maculatus, females exposed to 45°C also laid significantly fewer eggs than those exposed to 40°C. The percentage of eggs laid by females of both species that reached adulthood after exposure to 50°C for 2–6 h was significantly lower than the percentage that developed from eggs laid by females that were exposed to 40°C. No adult developed from eggs of C. maculatus exposed for 6 h at 50°C or from eggs of C. subinnotatus exposed for 2 h at this temperature. For both species, no adult progeny subsequently emerged from seeds harbouring first instar larvae when exposed at 50°C for 2, 4 or 6 h. Older larvae of C. maculatus were more tolerant of exposure at 50°C: 26.8, 10.2 and 0.9% of late instar larvae exposed for 2, 4 and 6 h, respectively, developed to the adult stage. In contrast, no adults of C. subinnotatus emerged from seeds harbouring late instar larvae when exposed at 45°C for 6 h nor in seeds exposed to the temperature of 50°C for 4 or 6 h. On average, immature stages of C. subinnotatus were more susceptible to heat treatment than those of C. maculatus.     

Purification and characterization of a dipeptidase from Lactobacillus delbrueckii subsp. bulgaricus

A metal-dependent dipeptidase has been purified from a cell-free extract of Lactobacillus delbrueckii subsp. bulgaricus B14 by ammonium sulphate precipitation, anion exchange chromatography, metal chelating affinity chromatography with immobilized Cu²⁺, and repeated FPLC anion exchange chromatography. The molecular mass of the purified enzyme was estimated to be 51 kD by SDS-polyacrylamide gel electrophoresis as well as by gel filtration, which indicates that it does not consist of subunits. The enzyme was most active at pH 7 and 50°C. Reducing agents, like dithiothreitol and β-mercaptoethanol, increased enzyme activity while metal chelating agents had an inhibitory effect. Enzyme activity, inhibited by EDTA and EGTA, could be partially restored by Co²⁺ and Mn²⁺. The enzyme was most active on dipeptides containing an aminoterminal hydrophobic amino acid such as Leu-Leu and Leu-Gly. Kinetic studies indicated that the dipeptidase had a higher affinity for the first substrate mentioned. The K_m-values for both substrates were about 0·56 and 1·23 mM, with turnover numbers of 870 and 480 s⁻¹, respectively.     

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⁺.     

Effect of temperature on reproductive parameters and the longevity of Acarus farris (Acari: Acaridae)

Reproductive parameters and the longevity of Acarus farris were examined at six constant temperatures, ranging from 7 to 29 °C, and a relative humidity of 90±5%. As temperature increased, the preoviposition period rapidly decreased to a minimum, above 25 °C. Fecundity was adversely affected by extreme temperatures while the oviposition period increased as temperature was reduced. The relationship between reproductive parameters and temperature was fitted using different polynomial and non-linear models. Male and female longevity increased as temperature decreased, except for the males at 7 °C, which showed significantly lower longevity than at 10 °C. Moreover, males showed significantly greater longevity than females. The effect of temperature on the capacity for increase of A. farris populations was also established. The non-linear Lactin model provided a reliable fit of the relationship between intrinsic rate of increase and temperature (R²>0.99). The optimum temperature for development was calculated to be 25 °C. At this temperature, the population doubling time was 2.8 days. The lower and upper thermal thresholds for A. farris populations were established at 6.4 and 29.9 °C, respectively.     

Pepsinogen and pepsin from the stomach of smooth hound (Mustelus mustelus): Purification,characterization and amino acid terminal sequences

Pepsinogen from the stomach of smooth hound (Mustelus mustelus) was purified to homogeneity by 20–70% ammonium sulphate precipitation, Sephadex G-100 gel filtration and DEAE-cellulose anion exchange chromatography with a 9.4-fold increase in specific activity and 38.36% recovery. Upon activation at pH 2.0, M. mustelus pepsinogen was converted to active form in one-step pathway. Molecular weights of the purified pepsinogen and the active pepsin were estimated to be 40,000 and 35,000 Da using SDS-PAGE and gel filtration, respectively. The optimum pH and temperature for the pepsin activity were pH 2.0 and 40 °C, respectively, using haemoglobin as a substrate. Activity was completely inhibited by Pepstatin A but not by phenylmethylsulphonyl fluoride, a serine-protease inhibitor and ethylenediaminetetraacetic acid, a metalloenzyme inhibitor. The N-terminal amino acid sequences of the first 15 amino acids of the activation segment of the pepsinogen and the first 20 amino acids of the active pepsin were LLRVPLRKGKSTLDV and ATEPLSNYLDSSYFGDISIG, respectively. M. mustelus pepsinogen, which showed high homology to rat C pepsinogen, had Thr-Leu-Asp sequence at amino acid positions 12–14 not found in all pepsinogen sequences. A remarkable substitution was found in the activation segment of M. mustelus pepsinogen: the Arg-13 conserved in all gastric proteinases, whose sequences are known, is replaced by Leu-13.     

Thermal-death kinetics of fifth-instar Amyelois transitella (Walker) (Lepidoptera: Pyralidae)

Information on kinetics for thermal mortality of navel orangeworm, Amyelois transitella (Walker) (Lepidoptera: Pyralidae), is needed for developing post-harvest phytosanitation thermal treatments of walnuts. Thermal-death kinetics for fifth-instar navel orangeworms were determined at temperatures between 46°C and 54°C at a heating rate of 18°C min⁻¹ using a heating block system. Thermal-death curves for fifth-instar navel orangeworms followed a 0.5th-order of kinetic reaction. The time required to achieve 100% mortality (N₀=600) decreased with increasing temperature in a logarithmic manner. Complete kill of 600 insects required a minimum exposure time of 140, 50, 15, 6, and 1 min at 46°C, 48°C, 50°C, 52°C, and 54°C, respectively. The reaction rate (k) was affected by treatment temperatures following an Arrhenius relationship. The activation energy for thermal kill of fifth-instar navel orangeworms was estimated to be between 510 and 520 kJ mol⁻¹.     

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.     

Some properties of the polygalacturonase from four Zimbabwean wild fruits (Uapaca kirkiana, Zizphus mauritiana, Tamarindus indica and Berchemia discolor fruits)

Polygalacturonase was extracted from ripe Uapaca kirkiana, Zizphus mauritiana, Tamarindus indica and Berchemia discolor fruits of Zimbabwe. Protein concentrations and activities of the enzymes in the extracts were determined in the four fruit extracts. The protein concentrations in the enzyme extracts ranged from 0.82 ± 0.17 to 1.97 ± 0.13 mg/ml and enzyme activities from 1.99 ± 0.13 to 6.64 ± 0.38 mmol min⁻¹ mg⁻¹ in the four fruit extracts. Optimum pH of the enzyme ranged from 4.5 to 5 and optimum temperature from 25 to 37 °C. The enzyme extracts reduced the viscosity of 1% pectin solutions in an experiment which was done together with assay for reducing sugars to prove the activities of the enzyme extracts in the four wild fruits. The K_m and V_max ranged from 0.115 to 0.252 mg/ml and 0.0057 to 0.0119 mmol reducing groups/min/mg protein, respectively, in the four polygalacturonase extracts. Calcium chloride and sodium chloride activated the PG from all sources to a greater extend than magnesium chloride and barium chloride. PG from the other three fruits had very little effect on the polysaccharide from U. kirkiana.     

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.     

Life history studies of internally feeding pests of stored sorghum: Sitotroga cerealella (Ol.) and Sitophilus oryzae (L.)

A laboratory study of the ecology of single species populations of Sitotroga cerealella (Olivier) and Sitophilus oryzae (L.) was made at 3 temperatures (25, 30 and 35°C) and 3 relative humidities (60, 70 and 80% r.h.). The effects of 2 lower humidities (40 and 50% r.h.) on both species were also studied at 30°C. The optimum conditions for multiplication of S. cerealella were found to be 25–30°C and 60–80% r.h. while for S. oryzae the optimum was more clearly defined (30°C and 80% r.h.). However S. cerealella could tolerate a higher temperature (35°C) and lower humidities (40 and 50% r.h. at 30°C) than S. oryzae. The results are compared with those of other workers on sorghum and other cereals, and discussed in relation to the distribution of the two species in Sudan.     

Purification, partial characterisation and mode of action of enterococcin EFS2, an antilisterial bacteriocin produced by a strain of Enterococcus faecalis isolated from a cheese

Enterococcus faecalis strain EFS2, isolated from the surface of a traditional cheese, produced a bacteriocin active against Gram-positive bacteria including Listeria spp. and some Staphylococcus aureus strains. The bacteriocin, named enterococcin EFS2, has been purified to homogeneity by ammonium sulphate precipitation and reversed-phase high performance liquid chromatography (RP-HPLC). The molecular weight was determined by mass spectrometry to be 7149.6. The amino acid composition of enterococcin EFS2 revealed that it contained 67 amino acid residues and had a blocked amino-terminal end. Enterococcin EFS2 induced viability loss, efflux of K⁺ ions and ATP, and cell lysis. Kinetic study of bactericidal activity of enterococcin EFS2 on Listeria innocua strain LIN 11 indicated slower cell destruction than by nisin. At pH 7.0, the activity of enterococcin EFS2 was the highest at 35 °C and was lost at 15 °C. The bacteriocin was more active against L. innocua strain LIN11 in broth adjusted to pH 6.0, 7.0 and 8.0 than to pH 4.5 at 30 °C.     

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.     

Effect of feed texture, meal frequency and pre-slaughter fasting on carcass and meat quality, and urinary cortisol in pigs

The gelation characteristics of myofibrillar proteins are indicative of meat product texture. Defining the performance of myofibrillar proteins during gelation is beneficial in maintaining quality and developing processed meat products and processes. This study investigates the impact of pH on viscoelastic properties of porcine myofibrillar proteins prepared from different muscles (semimembranosus (SM), longissimus dorsi (LD) and psoas major (PM)) during heat-induced gelation. Dynamic rheological properties were measured while heating at 1 °C/min from 20 to 85 °C, followed by a holding phase at 85 °C for 3 min and a cooling phase from 85 to 5 °C at a rate of 5 °C/min. Storage modulus (G′, the elastic response of the gelling material) increased as gel formation occurred, but decreased after reaching the temperature of myosin denaturation (52 °C) until approximately 60 °C when the gel strength increased again. This resulted in a peak and depression in the thermogram. Following 60 °C, the treatments maintained observed trends in gel strength, showing SM myofibrils produced the strongest gels. Myofibrillar protein from SM and PM formed stronger gels at pH 6.0 than at pH 6.5. Differences may be attributed to subtle variations in their protein profile related to muscle type or postmortem metabolism. Significant correlations were determined between G′ at 57, 72, 85 and 5 °C, indicating that changes affecting gel strength took effect prior to 57 °C. Muscle type was found to influence water-holding capacity to a greater degree than pH.     

Peptides in rainbow trout (Oncorhynchus mykiss) muscle subjected to ice storage and cooking

Although proteolysis during post mortem storage is an important factor affecting fish texture, little is known about degradation end-products. This study was performed to investigate the occurrence of low molecular weight peptides (<5 kDa) in post mortem rainbow trout muscle, during ice storage, and to evaluate their stability during cooking. It combined quantitative (amino acid analysis) and qualitative approaches (mass spectrometry). The results showed that muscle of trout was poor in peptides. These were mainly anserine and glutathione. Their concentration was almost unaffected by the seven days of ice storage and vacuum cooking for 5 min at 70 °C. MS analysis revealed a limited but highly reproducible appearance of small peptides in trout muscle during the ice storage and after cooking. The compounds detected by MS analysis remain to be characterised.     

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%).     

Purification and characterization of a glucoamylase from Rhizopus oryzae

Glucoamylase (EC 3.2.1.3) of Rhizopus oryzae NRRL 395 was purified approximately sevenfold by sequential ammonium sulfate fractionation, Biogel P-100 gel filtration, Q-Sepharose anion exchange and S-Sepharose cation exchange. The pH and temperature optima were 4·8 and 60°C, respectively. Enzyme was stable at temperatures up to 40°C and pH values between 3 and 8. The molecular weight was 67 000 daltons as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and the pI was 8·7 as determined by chromatofocusing. The K_m for amylopectin and soluble starch were 0·98 and 1·34 mg/ml, respectively. The V_max for amylopectin and soluble starch were 782 and 136 μmoles of glucose produced per mg of protein per min, respectively. The enzyme activity was inhibited by Hg²⁺, Pb²⁺ and Cd²⁺, but not by EDTA.     

Thermal death kinetics and heating rate effects for fifth-instar Cydia pomonella (L.) (Lepidoptera: Tortricidae)

Thermal death kinetic parameters of fifth-instar codling moths (Cydia pomonella (L.)) and the effect of three heating rates (1°C min⁻¹, 10°C min⁻¹, and 18°C min⁻¹) on larval mortality were determined by a heating block system. The insects were heated to four temperatures (46°C, 48°C, 50°C, and 52°C) held for predetermined periods followed by 24 h storage at 4°C before mortality evaluation. Thermal death kinetics for fifth-instar codling moths followed a 0.5th order of kinetic reaction. Minimum time required to achieve 100% mortality of a given population decreased with temperature in a semi-logarithmic manner. No larval survival was observed in samples of 600 insects after exposure to 46°C, 48°C, 50°C, and 52°C for 50, 15, 5, and 2 min, respectively. Activation energy for thermal kill of fifth-instar codling moths at the heating rate of 18°C min⁻¹ was estimated to be about 472 kJ mol⁻¹. The lethal time accumulated during the ramp period was about 1.8, 0.2, and 0.1 min for the heating rates of 1°C min⁻¹, 10°C min⁻¹, and 18°C min⁻¹, respectively.