Chymotrypsin from the hepatopancreas of cuttlefish (Sepia officinalis) with high activity in the hydrolysis of long chain peptide substrates: Purification and biochemical characterisation

Chymotrypsin from the hepatopancreas of cuttlefish (Sepia officinalis) was purified to homogeneity, with a 120-fold increase in specific activity and 23% recovery. The molecular weight of the purified chymotrypsin was estimated to be 28 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis. The optimum pH and temperature for the chymotrypsin activity were pH 8.5 and 55 °C, respectively, using succinyl-l-ala-ala-pro-l-phenylalanine-p-nitroanilide (SAAPFpNA) as a substrate. The enzyme was extremely stable in the pH range of 7.0–10.0 and highly stable up to 50 °C after 1 h incubation. This proteinase was strongly inhibited by chymostatin, soybean trypsin inhibitor, diisopropylfluorophosphate and phenylmethylsulfonyl fluoride, but was not inhibited by tosyl-l-phenylalanine chloromethyl ketone, N-carbobenzoxy-phenylalanine chloromethyl ketone or N_α-tosyl-l-lysine chloromethyl ketone. The enzyme hydrolysed long chymotrypsin peptide substrates SAAPFpNA, SAAPLpNA and ZAALpNA and did not hydrolyse short chymotrypsin substrates. Kinetic parameters of the enzymatic reaction demonstrated that the best substrate was SAAPFpNA, with k_cat 18 s^?1 and K_m 22 μM. However, the enzyme had a lower K_m for SAAPLpNA, 54 μM.The N-terminal amino acid sequence of the first 20 amino acids of the purified chymotrypsin was IVGGQEATIGEYPWQAALQV.     

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.     

Biochemical characterisation of an aminopeptidase with highest preference for lysine from Japanese flounder skeletal muscle

An aminopeptidase was purified from Japanese flounder skeletal muscle to homogeneity by ammonium sulphate fractionation and three chromatographies. The enzyme was approximately 100 kDa with isoelectric point of 5.7 as estimated by two-dimensional polyacrylamide gel electrophoresis. Its optimum temperature and pH were 45 °C and 7.5, respectively. According to peptide mass fingerprinting study, the enzyme revealed high identity to a puromycin-sensitive aminopeptidase. It had a broad specificity toward aminopeptidase substrates and preferred to hydrolyse Lys-MCA with k_cat/K_m of 8.1 × 10⁶ M^?1 s^?1, and the activation energy (E_a) of 72.5 kJ M^?1. Metal-chelating agents effectively inhibited the enzyme activity, and Zn²⁺, Mn²⁺ and Co²⁺ significantly restored the apoenzymatic activity dialysed by EDTA, whilst inhibitors to other proteinases did not show much effect. Furthermore, bestatin strongly inhibited its activity. These results indicate that the purified enzyme is a metalloaminopeptidase which would possibly contribute to free amino acids increase in fish muscle.     

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.     

Biochemical changes throughout the ripening of a traditional Spanish goat cheese variety (Babia-Laciana)

The physico-chemical characteristics, proteolysis (classical nitrogen fractions, caseins and their degradation products and free amino acids), and lipolysis (fat acidity and free fatty acids) were studied throughout the ripening of three batches of Babia-Laciana cheese, a Spanish traditional variety made from raw goats’ milk. The main compositional characteristics of this cheese at the end of the ripening are its high content of total solids (TS) (78.0±2.4 g 100 g⁻¹ of cheese) and fat (61.1±1.2 g 100 g⁻¹ of TS), the presence of residual lactose (1.6±0.8 g 100 g⁻¹ of TS) and its low content of sodium chloride (1.1±0.7 g 100 g⁻¹ of TS) and ash (2.8±0.5 g 100 g⁻¹ of TS). Its pH values (4.44±0.72) are extraordinarily low. The evolution and final values of the different nitrogen fractions show that this cheese undergoes a very slight proteolysis, a fact which was corroborated when the caseins and their degradation products were quantified: β-casein did not undergo any modification throughout ripening, while only 21% of the α_s-caseins were degraded. Free amino acids content increased by a factor of about 7 throughout ripening, resulting in a high content of γ-amino butyric acid and a low content of glutamic acid at the end of the process. Fat acidity increased very slightly, approximately 4.5 times, during ripening, reaching final values of 3.5±2.2 mg KOH g⁻¹ of fat. The total free fatty acids content showed a similar evolution to fat acidity. At the end of the ripening process, the main free fatty acid was C_18:1, followed by C₁₆ and C₁₀.     

Structure–mechanism relationship of antioxidant and ACE I inhibitory peptides from wheat gluten hydrolysate fractionated by pH

The aims of this study were to assess bioactive properties (ACE inhibition and antioxidant capacity) from wheat gluten hydrolysate peptides fractionated by pH (4.0, 6.0 and 9.0), to determine peptide action mechanism, and to relate it to the secondary structure and functional groups of peptides. Gluten hydrolysate extracts (GHE) were enriched in peptides with medium hydrophobicity and molecular weight (≈ 60% MH and 5.5 kDa, respectively). Gluten peptides inhibited ACE I by uncompetitive mechanism and a direct relationship between α-helix structure and IC50% value was obtained (r = 0.9127). TEAC and cooper chelating activity from GHE 6.5 were the highest and directly correlated with MH peptides. GHE 9.0 had high carotene bleaching inhibition (47.5 ± 0.3%) and reducing power activity (163.1 ± 2.9 mg S₂O₃^2 − equivalent g^− 1 protein), which were directly related to disulfide bonds content of peptides (r = 0.9982 and 0.9216, respectively). pH was a good alternative to select bioactive peptides from wheat gluten hydrolysate.     

Isolation and characterisation of a novel antibacterial peptide from bovine αS1-casein

Bovine casein was hydrolysed with a range of proteolytic enzymes including pepsin, trypsin, α-chymotrypsin and β-chymotrypsin, and assessed for antibacterial activity. The pepsin digest of bovine casein, which showed antibacterial activity, was fractionated using reverse phase high performance liquid chromatography and the antibacterial peptides isolated were characterised using electrospray ionisation mass spectrometry. Two antibacterial peptides were identified, a novel peptide (Cp1) which corresponded to residues 99–109 of bovine α_S1-casein and a previously reported peptide (Cp2) which corresponded to residues 183–207 of bovine α_S2-casein. The minimum inhibitory concentration (MIC) of Cp1 and Cp2 were determined against a range of bacterial cultures. Cp1 exhibited an MIC of 125 μg mL⁻¹ against all Gram-positive bacteria tested, and MIC ranging between 125 and >1000 μg mL⁻¹ against the Gram-negative bacteria tested. Cp2 was generally far more potent against the Gram-positive bacteria, exhibiting an MIC of 21 μg mL⁻¹, compared to MICs ranging from 332 to >664 μg mL⁻¹ against most of the Gram-negative bacteria tested.     

Purification and hypotensive activity of rapeseed protein-derived renin and angiotensin converting enzyme inhibitory peptides

Rapeseed protein isolate (RPI) was hydrolyzed with Alcalase followed by reverse-phase high performance liquid chromatography (RP-HPLC) purification of bioactive peptides. The rapeseed protein hydrolysate (RPH) obtained after 4 h digestion with Alcalase had a degree of hydrolysis (DH) of ～11%. Gel permeation chromatography separation showed high contents of low molecular weight peptides in the RPH when compared to the RPI. After preparative and analytical RP-HPLC separations, three peptides (LY, TF and RALP) were purified and amino acid sequence determined by tandem mass spectrometry. LY (IC₅₀, 0.11 mM) was the most potent (p < 0.05) against ACE activity when compared to TF (IC₅₀, 0.81 mM) and RALP (IC₅₀, 0.65 mM). However, RALP (IC₅₀, 0.97 mM) was the most potent (p < 0.05) against renin activity when compared to LY (IC₅₀, 1.87 mM) and TF (IC₅₀, 3.1 mM). Single oral administration (30 mg/kg body weight) to spontaneously hypertensive rats showed LY and RALP to be the more effective hypotensive agents with maximum blood pressure reduction of ?26 and 16 mmHg, respectively when compared to TF (?12 mmHg). The results suggest that the higher number of hydrophobic amino acid residues LY and RALP contributed to their higher in vitro and in vivo activities when compared to TF.     

Antibiotic resistance and susceptibility to some food preservative measures of spoilage and pathogenic micro-organisms from spices

Antibiogram of 84 strains of Bacillus cereus, 26 strains of Clostridium perfringens, four strains of Staphylococcus aureus, 51 strains of Enterobacteriaceae, two strains of each of Salmonella and Shigella; isolated from spices, were studied against 20 different antibiotics that are commonly used against foodborne diseases, mainly gastroenteritis. All the tested strains of B. cereus, Cl. perfringens, Staph. aureus, Escherichia coli, Salmonella and Shigella were found resistant to at least 3, 4, 7, 6, 10 and 9 antibiotics, respectively. In brain–heart infusion broth supplemented with glucose, the D_100°C-values for B. cereus were 3.5–5.9 min, and the z-values were 17–18°C. The D_100°C-values for Cl. perfringens in fluid thioglycolate medium were higher (10.0–19.8 min) than those of B. cereus. The minimum inhibitory concentrations (MICs) of sodium chloride were 45–80 mg ml⁻¹. While the MIC of benzoic acid for Cl. perfringens, tested on perfringens agar (pH 7.3) plates by incubating anaerobially at 35°C for 24 h, was 1.9–2.2 mg ml⁻¹, for others, tested on nutrient agar (pH 6.8) plates by incubating at 35°C for 18 h in static aerobic condition, it was much less. Similarly, the MIC of sorbic acid for all the tested isolates, excepting Cl. perfringens, was 0.6–1.1 mg ml⁻¹. Of the eight isolates of Cl. perfringens, only three were inhibited at 2.0 mg sorbic acid ml⁻¹, while others were resistant. Sixty percent and 75% of the respective strains of B. cereus and Cl. perfringens were resistant to 5000 IU Nisaplin ml⁻¹, whereas the MIC values of Staph. aureus were between 3000 and 5000 IU ml⁻¹. While studying combined effect of selected hurdles on the growth of enterotoxigenic Cl. perfringens 16-C2, the judicious combination considered was low acid (pH 6.0), 30 mg sodium chloride ml⁻¹ and 1.25 mg benzoic acid ml⁻¹.     

Controlled permeabilization of Lactococcus lactis cells as a means to study and influence cheese ripening processes

Cells of Lactococcus lactis were permeabilized by perturbing the membrane structure through the delipidating action of n-butanol to an extent that allowed normally excluded peptidase substrates to enter the cells and be accessible to intracellular enzymes. The degree of permeabilization of cells depended on the concentration of the solvent used, the duration of the treatment, the density of the cell suspension, pH and temperature. This was indicated by the optimum or maximum activities of the intracellular peptidases aminopeptidase N and aminopeptidase X. The possible usefulness of permeabilized cells for cheese-ripening studies was demonstrated with cells that were treated with 5 ml L⁻¹ n-butanol at pH 6.5 and 25 °C using a mixture of chymosin-generated primary cheese peptides as substrate. Production of amino acids could be correlated with conversion of peptides that had entered the permeabilized cells and reflected the course of amino acid production in cheese.     

Purification and identification of three novel antioxidant peptides from protein hydrolysate of bluefin leatherjacket (Navodon septentrionalis) skin

Skin protein from a bluefin leatherjacket (Navodon septentrionalis) processing by-product was hydrolyzed by trypsin, flavourzyme, neutrase, papain, alcalase, and pepsin, and protein hydrolysate (BSH) prepared using alcalase showed the highest DPPH, HO, and O₂⁻ · scavenging activities among all hydrolysates. Using ultrafiltration and consecutive chromatography, three novel peptides with strong antioxidant properties were purified from BSH, and their sequences were determined as Gly-Ser-Gly-Gly-Leu (GSGGL, BSP-A), Gly-Pro-Gly-Gly-Phe-Ile (GPGGFI, BSP-B), and Phe-Ile-Gly-Pro (FIGP, BSP-C) with molecular weights of 389.41, 546.63, and 432.52 Da, respectively. BSP-C exhibited the highest scavenging activities on DPPH (EC₅₀ 0.118 mg/ml), HO (EC₅₀ 0.073 mg/ml), and O₂⁻ · (EC₅₀ 0.311 mg/ml) among the three peptides. In addition, BSP-C could effectively inhibit autooxidation in a linoleic acid model system. The antioxidant activities of BSP-A, BSP-B, and BSP-C might be due to the small molecular sizes and the hydrophobic and/or aromatic amino acid residues in their amino acid sequences. The present results suggested that peptides purified from the skin protein hydrolysate of bluefin leatherjacket were excellent antioxidants and could be effectively used as food ingredients and additives, and pharmaceuticals.     

Angiotensin-converting enzyme inhibitory activity of milk protein hydrolysates: Effect of substrate,enzyme and time of hydrolysis

Nine milk protein substrates were hydrolysed in vitro with five proteases for various times (0, 3, 6, and 24 h), and the angiotensin-converting enzyme (ACE)-inhibitory activity of hydrolysates was assessed. Overall, the casein substrates gave rise to hydrolysates with significantly higher ACE-inhibitory activity than the whey protein (WP) substrates (85% vs. 79%). No significant difference between 3 and 24 h of hydrolysis was found. A reasonable correlation was found between the ACE inhibition of the 6 h hydrolysates determined in vitro and estimated by in silico modelling. The highest ACE-inhibitory activity was found in hydrolysates made with thermolysin followed by proteinase K, trypsin, pepsin and Bacillus licheniformis protease. The IC₅₀ values for thermolysin hydrolysates of caseins and WPs were 45–83 and 90–400 μg mL⁻¹, respectively, with α-lactalbumin giving the highest inhibitory activity. Thermolysin, proteinase K and trypsin were useful for the release of highly potent ACE-inhibitory peptides from both WPs and caseins.     

Digestive acid protease from zebra blenny (Salaria basilisca): Characteristics and application in gelatin extraction

The present study reports on the characterization and evaluation of a crude acidic protease from the viscera of zebra blenny (Salaria basilisca) for use in gelatin extraction. Using hemoglobin, zymogram analysis revealed the presence of at least one clear band. The crude acid protease was noted to be optimally active at pH 3.0 and 50 °C and highly stable over a pH range of 2.0 to 7.0. The enzymatic extract lost about 87% of its activity after incubation with pepstatin A for 30 min at 4 °C. The acidic protease from the viscera of zebra blenny was noted to be effective in the extraction of gelatin from the skin of zebra blenny, with an extraction yield of 14.65% based on the wet weight of zebra blenny skin. The extracted zebra blenny skin gelatin (ZBSG) was characterized based on its chemical composition, polypeptide pattern, gel strength, textural parameters, and functional properties. ZBSG had high protein (90.6%) and low ash (3.1%) and fat (0.6%) contents. It contained α₁ and α₂-chains as the major constituents and determined as belonging to type I. The bloom strength of solidified gelatin was 151.3 g. The findings from Fourier Transformed Infrared (FT-IR) spectroscopy suggested the presence of helical arrangements of ZBSG. The latter showed excellent concentration-dependent functional properties. While emulsion activity index (EAI) and emulsion stability index (ESI) decreased, foam expansion (FE) and foam stability (FS) increased as the concentration of gelatin increased. Overall, zebra blenny endogenous acid protease could open new promising opportunities for the extraction of gelatin.     

Purification and characterization of the 3-phosphoglycerate kinase from the thermophile Lactobacillus delbrueckii subsp. lactis

The 3-phosphoglycerate kinase (PGK) of the thermophile Lactobacillus delbrueckii subsp. lactis was purified to homogeneity and found to be a monomeric enzyme with a molecular weight of 45 kDa. PGK is a Michaelis–Menten type enzyme with a K_m=0.7 mm for ATP and a K_m=2.6 mm for 3-phosphoglycerate. pH tolerance of PGK was found to be oriented towards acidic conditions with an optimum at pH 6.8 and a plateau of 90% of the total activity between pH 6.0 and 7.4. The optimum temperature for PGK activity is 45°C, which is consistent with the value expected for a thermophilic bacterium with an optimal growth temperature of 45°C.     

Beta-lactoglobulin–polysaccharide complexes as nanovehicles for hydrophobic nutraceuticals in non-fat foods and clear beverages

For enrichment of non-fat clear drinks with hydrophobic nutraceuticals (HN), vitamin D₂ (VD₂), a model HN, was successfully entrapped within electrostatically stable nanoparticles ?50–70 nm in size. The particles, having electrophoretic mobility more negative than ?3 × 10^?8 m² V^?1 s^?1, comprised β-lactoglobulin (β-Lg) to which the HN was bound, and excess of a polyanionic polysaccharide (low methoxyl pectin), which provided electrostatic stability. As pectin concentration increased in 0.05% β-Lg solutions and pH 3.5–4.5, coacervation and turbidity first increased, but dropped beyond 0.01% pectin. The lowest turbidity, of about 0.035, was obtained at pH 4.25 and 0.05% pectin. As pH was increased from 3.5 to 4.25 the minimal particle size decreased and solutions became clearer. Optimal systems obtained were transparent, and may be suitable for enrichment of clear acid beverages. The nanocomplexes provided better protection to the vitamin against degradation than β-Lg alone, and stability was significantly better than that of unprotected vitamin dispersed in water.     

Controlling moisture transport in a cereal porous product by modification of structural or formulation parameters

Equilibrium and dynamic water sorption properties of sponge cakes with varying porosity (86–52%) and fat content (0–0.30 g/g d.b.) were determined using a water vapour sorption microbalance. Contrary to porosity, addition of fat decreased equilibrium moisture contents. The effective moisture diffusivity (D_eff) was identified from a numerical solution of Fick’s second law, taking into account an external mass transfer coefficient and the swelling of the solid matrix. D_eff increased from 1.61 to 8.67 × 10⁻¹⁰ m²/s with moisture content, reached a threshold at moisture content 0.15 g/g d.b. and then decreased until water saturation. D_eff decreased from 8.67 to 2.97 × 10⁻¹⁰ m²/s with decreasing porosity. This effect was attributed to a change of water diffusion mechanism, from predominant vapour to liquid. D_eff decreased from 8.67 to 2.12 × 10⁻¹⁰ m²/s with increasing fat content. Addition of fat had an effect on the water diffusion in two ways, decreasing porosity (sagging of the foam) and increasing tortuosity.     

Predicting cutting and clotting time of coagulating goat's milk using diffuse reflectance: effect of pH,temperature and enzyme concentration

A diffuse reflectance sensor, which used optical fibres and near-infrared radiation (NIR) at 880 nm, was used to monitor goat's milk coagulation. A randomised block design replicated three times was utilized to test the effects of pH, temperature and enzyme concentration on diffuse reflectance parameters. Milk pH was adjusted to three levels (5.5, 6.0 and 6.5) and coagulated at three different temperatures (28, 32 and 36°C), using three enzyme concentrations (0.020, 0.035 and 0.050 mL kg⁻¹ of milk). A linear cutting time prediction equation, T_cut=βT_max, was found to predict visual cutting time with a standard error of prediction of 84.5 s and an R² of 0.9785. β was affected by pH, temperature and enzyme concentration. The diffuse reflectance parameter (T_max) was strongly correlated to the Berridge clotting time (R²=0.9913). Parameters generated from the diffuse reflectance profiles, with the exception of response-based parameters, were found to be a function of coagulation rate.     

Isolation and characterization of three antioxidant pentapeptides from protein hydrolysate of monkfish (Lophius litulon) muscle

In the current study, an efficient method had been developed to acquire the antioxidant hydrolysate of monkfish muscle protein (MPH) using trypsin by an orthogonal (L₉(3)⁴) test. Under the optimum conditions of enzymolysis time 4 h, enzyme-to-substrate ratio (E/S) 2%, enzymolysis temperature 40 °C and pH 8.0, the DH (Degree of hydrolysis) and hydroxyl radical scavenging activity of MPH reached 19.83 ± 0.82% and 58.05 ± 3.01%, respectively. By using ultrafiltration, gel filtration chromatography and reversed phase high performance liquid chromatography (RP-HPLC), three antioxidant pentapeptides were isolated from MPH, and their amino acid sequences were identified as Glu-Trp-Pro-Ala-Gln (MPH-P1), Phe-Leu-His-Arg-Pro (MPH-P2), and Leu-Met-Gly-Gln-Trp (MPH-P3) with molecular weights of 629.68 Da, 668.80 Da, and 633.77 Da, respectively. MPH-P1, MPH-P2, and MPH-P3 exhibited good scavenging activities on hydroxyl radical (EC₅₀ 0.269, 0.114 and 0.040 mg/ml), DPPH radical (EC₅₀ 2.408, 3.751, and 1.399 mg/ml), and superoxide anion radical (EC₅₀ 0.624, 0.101, and 0.042 mg/ml) in a dose-dependent manner. MPH-P3 was also effective against lipid peroxidation in the model system. The antioxidant activities of MPH-P1, MPH-P2, and MPH-P3 were due to their small sizes and the presence of antioxidant and hydrophobic amino acid residues within their sequences. The results of this study suggested that the protein hydrolysate and/or its isolated peptides might be effectively used as food additives for retarding lipid peroxidation occurring in foodstuffs.     

Purification and partial characterisation of X-prolyl dipeptidyl aminopeptidase of Lactobacillus helveticus ITG LH1

A X-prolyl dipeptidyl aminopeptidase (EC 3.4.14.5, XPDAP) from Lactobacillus helveticus ITG LH1, a strain used for Swiss-type cheese, was purified by ion exchange and affinity chromatographies. The enzyme appeared to be a 140 kDa monomer. Optimal activity occurred at pH 7 and 40°C, but it was rapidly inactivated above 50°C. The enzyme was activated by NaCl and KCl up to 50–200 mm but its activity levelled off at higher salt concentrations. Its complete inhibition was caused by 0.1 mm HgCl₂, 1 mm SnCl₂ and 2.5 mm CuCl₂. It was inactivated by reagents specific for serine proteases, such as phenylmethylsulfonyl fluoride and sulfhydryl group-blocking reagents. The enzyme hydrolysed p-nitroanilide-substituted X-Pro and X-Ala dipeptides, as well as β-casomorphin 1-4.     

Purification and characterization of an antioxidant protein from Ginkgo biloba seeds

A new antioxidation protein named G4b was purified from Ginkgo biloba seed albumin. The results of chemical colorimetry and chemiluminescence methods showed that the hemi-inhibitory concentration (IC₅₀) of G4b on the hydroxyl radical were 78.1 μg/mL and 115.1 μg/mL, and the IC₅₀ on DPPH was 100.7 μg/mL, while the IC₅₀ on the superoxide anion and DNA injury were 23 μg/mL and 27 μg/mL, respectively, and the abundance of sulfur amino acids and aromatic amino acids contained in G4b might contribute to its strong antioxidation properties. G4b is a kind of homogeneous and novel protein with a molecular mass of 29,247 Da, consisting of two peptides with similar molecular weights linked by a disulfide bond. Result of β-elimination reaction showed that G4b is connected with a small quantity of polysaccharide by an oxygen-glucosidic bond.