Biochemical properties of pepsinogen and pepsin from the stomach of albacore tuna (Thunnus alalunga)

Pepsinogen (PG) from the stomach of albacore tuna (Thunnus alalunga) was purified to homogeneity by using a series of chromatographies involving Sephacryl S-200HR, Sephadex G-50 and DEAE-cellulose with a 658-fold increase in purity. Based on the native-PAGE and zymography, PG showed a single band with pepsin activity. Molecular weights (MW) of PG and active pepsin were estimated to be 39.9 and 32.7 kDa as determined by SDS–PAGE, respectively. PG was converted to the corresponding pepsin through an intermediate form (MW ≈ 36.8 kDa) and the complete activation was observed after 30–60 min. The N-terminal amino acid sequence of the first 15 amino acids of activation segment of pepsinogen was FHKLPLIKGKTAREE. The optimal pH and temperature for pepsin activity were 2.0 and 50 °C, respectively. The activity was stable in the pH range of 2–5. Residual activity more than 85% was found after heating at temperatures up to 50 °C for 30 min. Pepsin activity was strongly inhibited by pepstatin A, whilst E-64, ethylenediaminetetraacetic acid (EDTA) and soybean trypsin inhibitor exhibited the negligible effect. SDS and cysteine also showed inhibitory effects, whilst ATP, molybdate, NaCl and CaCl₂ had no impact on pepsin activity.     

Multiple α-galactosidases from <Emphasis Type="Italic">Aspergillus foetidus</Emphasis> ZU-G1: purification,characterization and application in soybean milk hydrolysis

α-Galactosidase had applied in food and feed industries for hydrolyzing raffinose series oligosaccharides (RO) that are the factors primarily responsible for flatulence upon ingestion of soybean-derived products. The objective of the current work was to purify the α-galactosidase of Aspergillus foetidus ZU-G1 and compared the biochemical and hydrolytic properties of three major α-galactosidase forms (α-gal I, α-gal II and α-gal III). The molecular mass of the purified enzyme as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 106.3, 49.7 and 109.9 kDa, respectively. Its optimum reaction temperature was 60 °C and stable below 50 °C. The optimum pH of α-gal I and α-gal III was 5.0 and α-gal II was 4.0. Under 28 °C conditions for 24 h, α-gal I was stable at pH 4.0, α-gal II was stable at pH 6.0, and α-gal III was pH 5.0. α-Galactosidase was completely inhibited by Ag⁺. CuSO₄·5H₂O and SDS were powerful inhibitors of α-gal I and α-gal III but had little effect to α-gal II. EDTA did not strongly affect α-gal I and α-gal III, while strongly affect α-gal II. CaCl₂·2H₂O, MgSO₄·7H₂O and MnSO₄·7H₂O were activation for α-gal I, α-gal II and α-gal III. No significant inhibition of enzymes activity was observed in the presence of raffinose, lactose as well as other sugars tested. Synthetic substrate p-nitrophenyl-α-d-galactopyranoside was not preferentially hydrolyzed than natural substrates, such as melibiose, stachyose and raffinose. Under 40 and 50 °C incubation for 1–5 h, the stachyose of soybean milk was degraded by α-gal I, α-gal II and α-gal III and strongly hydrolyzed by α-gal II, and the raffinose of soybean milk was completely hydrolyzed by α-gal II and weakly hydrolyzed by α-gal I and α-gal III. The distinct hydrolytic and biochemical properties of α-gal I, α-gal II and α-gal III further signify the α-galactosidase of A. foetidus ZU-G1 was propitious to soybean milk and related food industry.     

Effect of angiotensin I-converting enzyme (ACE) inhibitory peptide purified from enzymatic hydrolysates of <Emphasis Type="Italic">Styela plicata</Emphasis>

Angiotensin I-converting enzyme (ACE) inhibitory peptide was isolated from Styela plicata. The S. plicata was hydrolyzed with various proteases including Protamex, Kojizyme, Neutrase, Flavourzyme, Alcalase, trypsin, α-chymotrypsin, pepsin, and papain. The hydrolysate prepared with Protamex had the highest ACE inhibitory activity compared to the other hydrolysates. We attempted to isolate ACE inhibitory peptides from hydrolysate prepared with Protamex using ultra-filtration, gel filtration on a Sephadex G-25 column and reversed-phase high-performance liquid chromatography (RP-HPLC) on an ODS column. IC₅₀ value of the purified ACE inhibitory peptide was 24.7 μM, and Lineweaver–Burk plots suggest that the purified peptide from S. plicata acts as mixed-type inhibitor against ACE. Amino acid sequence of the purified peptide was identified as Met-Leu-Leu-Cys-Ser, with a molecular weight 566.4 Da. The results of this study suggest that peptides derived from S. plicata may be beneficial as anti-hypertension compounds in functional foods resource.     

Evaluation of angiotensin I-converting enzyme (ACE) inhibitory activities of smooth hound (Mustelus mustelus) muscle protein hydrolysates generated by gastrointestinal proteases: identification of the most potent active peptide

In this study, smooth hound protein hydrolysates (SHPHs), obtained by treatment with various gastrointestinal proteases, were analyzed for their angiotensin I-converting enzyme (ACE) inhibitory activities. Protein hydrolysates were obtained by treatment with crude alkaline enzyme extract, low molecular weight (LMW) alkaline protease, trypsin-like protease and pepsin from Mustelus mustelus, and bovine trypsin. All hydrolysates exhibited inhibitory activity toward ACE. Hydrolysate generated with alkaline protease extract displayed the highest ACE inhibitory activity, and the higher inhibition activity (82.6% at 2 mg/mL) was obtained with a hydrolysis degree of 18.8%. This hydrolysate was then fractionated by size exclusion chromatography on a Sephadex G-25 into five major fractions (P₁–P₅). ACE inhibitory activities of all fractions were assayed, and P₃ was found to display a high ACE inhibitory activity (62.24% at 1 mg/mL). P₃ was then fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC) and ten fractions of ACE inhibitors were found (F₁–F₁₀). Sub-fraction F₃ showed the strongest ACE inhibitory activity, being able to suppress more than 60% of initial enzyme activity at a concentration of 100 μg/mL. The amino acid sequence of peptide F₃ was determined by ESI/MS and ESI–MS/MS as Ala-Gly-Ser, and the IC₅₀ value for ACE inhibitory activity was 0.13 ± 0.03 mg/mL. Further, purified peptide F₃ maintained inhibitory activity even after in vitro digestion with gastrointestinal proteases in order to demonstrate gastrointestinal stability digestion to enable oral application. These results indicate that smooth hound protein hydrolysate possesses potent antihypertensive activity.     

Characterization of a β-glucosidase isolated from an alpeorujo strain of Candida adriatica

A β-glucosidase-producing strain, Candida adriatica CECT13142, was isolated from olive oil wastes (alpeorujo) and identified by PCR/restriction fragment length polymorphism of the rDNA internal transcribed spacer and sequence analysis of the D1/D2 region of the 26S rRNA gene techniques. The enzyme was purified by sequential chromatography on DEAE-cellulose and Sephadex G-100. The relative molecular mass of the enzyme was estimated to be 50 kDa by SDS-PAGE. The hydrolytic activity of the β-glucosidase had an optimum pH of 8.2 and an optimum temperature of 40°C. The enzyme displayed high substrate specificity and high catalytic efficiency (K_m 0.85 mM, V_max 12.5 U/g of cells) for p-nitrophenyl-β-D-glucopyranoside. Although β-glucosidases have been purified and characterized from several other organisms, the C. adriatica β-glucosidase is able to have optimal activity at alkaline pH.     

Characterization of purified xylanase from finger millet (<Emphasis Type="Italic">Eleusine coracana-</Emphasis>Indaf 15) malt

Xylanase (E.C. 3.2.1.8) was purified to apparent homogeneity from 96 h finger millet (Eleusine coracana, Indaf-15) malt by a three step purification procedure via ammonium sulphate fractionation, DEAE-cellulose ion exchange and Sephadex G-75 gel permeation chromatographies with a recovery of 4.0% and fold purification of 60. Xylanase, having a molecular weight of 29 ± 2 kDa was found to be monomeric on SDS-PAGE. pH optimum of the enzyme was found to be in the range of 5.0–5.5. The activation energy was 25 kJmol⁻¹. Xylanase showed maximum stability at 35 °C in a pH range of 5.0–6.0. K _m and V _max of purified xylanase were found to be 0.2% and 4.5 μmol min⁻¹, respectively. Metal ions such as Ca²⁺, Mg²⁺, Mn²⁺, Cu²⁺, Fe²⁺, Ag²⁺ and Ni²⁺ enhanced xylanase activity at 5 mM concentration. p-chloromercuribenzoate, citric, oxalic and boric acids inhibited the enzyme in concentration dependent manner. The mode of action of xylanase was found to be “endo” as determined by the analysis of products liberated from larchwood xylan by ESI-MS and H¹NMR. In vitro studies using Bifidobacterium and Lactobacillus sp. confirmed the prebiotic activity of the xylo-oligosaccharides.     

Optimization of Conditions for Enzymatic Production of ACE Inhibitory Peptides from Collagen

Enzymatic condition for producing angiotensin I-converting enzyme (ACE) inhibitory peptides from collagen was optimized with the aid of response surface methodology, which also derived a statistical model for experimental validation. The results showed that the optimal condition for the hydrolysis by pepsin was at pH 2, temperature 37 °C, and in enzyme to substrate ratio (E/S) of 2 when 8.23% collagen (w/v) and 3.82 h of hydrolysis time were applied. Through the single-enzyme hydrolysis, the ACE inhibitory activity could reach an average of 78.06%. In contrast, when a combination of pepsin and trypsin was used for a multiple-proteases hydrolysis, the ACE inhibitory activity could be significantly improved to an average of 88.25%. Furthermore, the IC₅₀ (μg/mL) value of the enzyme combination by pepsin and trypsin (141.64 ± 22.11) was significantly lower than that of the combinations of pepsin and papain (438.59 ± 84.37) or pepsin and protease M (336.76 ± 87.88; p<0.05). Our results have shown that collagen can be used for enzyme-mediated production of ACE inhibitory peptides.     

Properties of thermostable extracellular FOS-producing fructofuranosidase from <Emphasis Type="Italic">Cryptococcus</Emphasis> sp.

The present work was devoted to investigations concerning the fructooligosaccharide producing activity of Cryptococcus sp. LEB-V2 (Laboratory of Bioprocess Engineering, Unicamp, Brazil) and its extracellular fructofuranosidase. After cell separation, the enzyme was purified by ethanol precipitation and anion exchange chromatography. The enzyme showed both fructofuranosidase (FA) and fructosyl transferase (FTA) activity. With sucrose as substrate, the data failed to fit the Michaelis–Menten behaviour, showing a substrate inhibitory model. The K _m, K _i and v _max values were shown to be 64 mM, 3 M and 159.6 μmol mL⁻¹ min⁻¹ for FA and 131 mM, 1.6 M and 377.8 μmol mL⁻¹ min⁻¹ for FTA, respectively. The optimum pH and temperature were found to be around 4.0 and 65 °C, while the best stability was achieved at pH 4.5 and temperatures below 60 °C, for both the FA and FTA. Despite the strong FA activity, the high transfructosylating activity allowed for good FOS production from sucrose (35% yield).     

Natural plant enzyme inhibitors. Isolation and characterisation of two α-amylase inhibitors from Colocasia antiquorum tubers

Two α-amylase inhibitors I-1 and I-2, were purified to homogeneity from Colocasia antiquorum tubers by extraction with 0.02m phosphate buffer pH 7.6, treatment with diethyl amino ethyl-cellulose, ammonium sulphate fractionation, chromatography on Sephadex G-50 and chromatography on phenyl sepharose CL-4B. Both the inhibitors were basic proteins. I-1 alone contained carbohydrates to the extent of 5.4%. The molecular weights of I-1 and I-2 were found to be about 14 300 and 12 500, respectively, by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The inhibitors inactivated human salivary, human pancreatic and hog pancreatic amylases but they had no action on Bacillus subtilis and Aspergillus oryzae amylases and resisted attack by pepsin, trypsin, chymotrypsin and pronase. Chemical modification of amino groups and guanidino groups of I-1 and I-2 resulted in loss of inhibitory activities. Formation of enzyme inhibitor complexes between hog pancreatic amylase, I-1 and I-2 were demonstrated by gel chromatography on Sephadex G-100. Total amylase inhibitory activity in Colocasia tubers decreased gradually during plant growth.     

Purification of a lipase from the hepatopancreas of oil sardine (Sardinella longiceps linnaeus) and its characteristics and properties

Lipase has been purified from the hepatopancreas of oil sardine (Sardinella longiceps) by defatting, water extraction, ammonium sulphate fractionation and chromatography on DEAE Sephadex and Sephadex G-100. The preparation was homogeneous on polyacrylamide disc gel electrophoresis and on gel filtration through Sephacryl S-200. The enzyme showed a molecular weight of 54000±57000 with 6.1% of carbohydrate. The pH and temperature optima of purified sardine lipase were 8 and 37°C respectively. Sardine lipase remained stable up to 45°C (15 min) and in the pH range 5 to 9.5. The K_m values obtained for the substrates tributyrin and triacetin were 4 × 10^?2 and 30 × 10^?2, respectively. The effect of halogens and various metal ions on sardine lipase activity, substrate specificity, amino acid and carbohydrate composition are also reported.     

Purification,characterization, and in vitro digestion of novel antioxidant peptides from chicken blood hemoglobin

The study aimed to purify and characterize antioxidant peptides from chicken blood hemoglobin hydrolysate. The fraction M₂ (< 3 KDa) with the strongest antioxidant activity was isolated by ultrafiltration, and its DPPH (1,1-diphenyl-2-picryl-hydrazyl radical) free radical scavenging rate, ABTS [2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate)] free radical scavenging rate, and iron ion chelation activity were 82.91%, 77.49%, and 80.99%, respectively. After in vitro digestion, the antioxidant capacity of chicken blood hydrolysate was significantly higher than that before digestion (p < 0.05). M₂ exhibited the strongest antioxidant activity after stomach digestion, with a DPPH radical scavenging rate and iron ion chelating power of 82.91% and 79.61%, respectively. Component A was purified from M₂ by Sephadex G-25 gel chromatography. The peptide sequences were identified by LC-MS/MS from fraction A, and four peptides, AEDKKLIQ (944.54 Da), APAPAAK (625.36 Da), LSDLHAHKL (1033.57 Da), and LSNLHAYNL (1044.54 Da) were synthesized using the solid-phase peptide method, among which APAPAAK was a novel antioxidant peptide. Molecular docking was used to simulate the binding of these four peptides to the key active site of Keap1 via hydrogen bonding. This study suggests that chicken blood may provide a new natural source of antioxidant peptides.     

Purification and characterization of hydroperoxide lyase from amaranth tricolor (Amaranthus mangostanus L.) leaves

Hydroperoxide lyase (HPL) was extracted from amaranth tricolor leaves using Triton X-100, and purified to electrophoretic homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic interaction chromatography and hydroxyapatite chromatography. The purified HPL preparation consisted of a single band and spot with a molecular mass of about 55 kDa as shown in SDS–PAGE and 2-D PAGE, respectively; the isoelectric point was found to be about 5.4. The maximum activity of the enzyme was observed at pH 6.0 and 25 °C, respectively. The HPL showed higher activity against 13-hydroperoxy-linolenic acid compared to 13-hydroperoxy-linoleic acid. K _m value for 13-hydroperoxy-linolenic acid was 62.7 μM, and the corresponding V _max was 178.5 μM min⁻¹. The activity of HPL was significantly inhibited by nordihydroguaiaretic acid, HgCl₂ and 2(E)-hexenal but not by EDTA and hexanal.     

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.     

Partial Purification and Characterization of Extracellular Fructofuranosidase with Transfructosylating Activity from <Emphasis Type="Italic">Candida</Emphasis> sp.

The present work was carried out with the aim to investigate some properties of an extracellular fructofuranosidase enzyme, with high transfructosylating activity, from Candida sp. LEB-I3 (Laboratory of Bioprocess Engineering, Unicamp, Brazil). The enzyme was produced through fermentation, and after cell separation from the fermented medium, the enzyme was concentrated by ethanol precipitation and than purified by anion exchange chromatography. The enzyme exhibited both fructofuranosidase (FA) and fructosyltransferase (FTA) activities on a low and high sucrose concentration. With sucrose as the substrate, the data fitted the Michaellis–Menten model for FA, showing rather a substrate inhibitory shape for fructosyltransferase activity. The K _m and v _max values were shown to be 13.4 g L⁻¹ and 21.0 μmol mL⁻¹ min⁻¹ and 25.5 g L⁻¹ and 52.5 μmol mL⁻¹ min⁻¹ for FA and FTA activities, respectively. FTA presented an inhibitory factor K _i of 729.8 g L⁻¹. The optimum conditions for FA activity were found to be pH 3.25–3.5 and temperatures around 69 °C, while for FTA, the optimum condition were 65 °C (±2 °C) and pH 4.00 (±0.25). Both activities were very stable at temperatures below 60 °C, while for FA, the best stability occurred at pH 5.0 and for FTA at pH  4.5–5.0. Despite the strong fructofuranosidase activity, causing hydrolysis of the fructooligosaccharides (FOS), the high transfructosilating activity allows a high FOS production from sucrose (44%).     

PROPERTIES OF PEPSIN AND TRYPSIN ISOLATED FROM THE DIGESTIVE TRACT OF PARONA SIGNATA "PALOMETA"

Two digestive proteases from Parona signata (Palometa) were isolated from gastric and pyloric caeca tissues and characterized. The stomach enzyme was inhibited by pepstatin and was classified as pepsin. Palometa pepsin purified from a Sephadex G-100 column appeared as two bands on an SDS-PAGE gel and exhibited optimum activity at pH 3.5, and 37C. Palometa pepsin was inhibited by pepstatin to a similar exient as porcine pepsin. An enzyme isolated from the pyloric caeca was shown to be trypsin based on its molecular weight, its ability to hydrolyze the synthetic substrates, N-α-benzoyl-arginine-p-nitroanilide (BAPA), and tosyl-arginine methyl ester (TAME) and inhibition by the known trypsin inhibitors, SBTI, TLCK, PMSF and benzamidine. The purified trypsin from palometa pyloric caeca yielded a single band with a molecular mass of 24 kDa. Optima trypsin activity was obtained at pH 8.5 and the enzyme was stable over a pH range of 3 to 11. Palometa trypsin activity was stable for 30 min at 50C, but lost 50% of its activity after 30 min at 60C. The optimum temperature for activity was 65C. The biochemical properties of Palometa trypsin and pepsin were discussed in relation to the varying environment of Palometa in the Rio de la Plata estuary.     

Purification and characterization of Aspergillus oryzae LK-101 salt-tolerant acid protease isolated from soybean paste

A novel salt-tolerant acid protease was produced from Aspergillus oryzae LK-101 (AOLK-101). The AOLK-101 protease was purified to homogeneity by ammonium sulfate precipitation, DEAE-Sephadex A-50 and Sephadex G-100 chromatographies in order. The specific activity and the purification ratio of the purified protease were 2,301 unit/mg and 11.6 fold, respectively, with 25 kDa of molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrpphoresis (SDS-PAGE). Its optimal pH and temperature were pH 6.5 and 50°C, respectively. This protease was relatively stable at pH 4.5–7.5, below 40°C, and up to 10% salt concentration. The protease was moderately inhibited by Ag²⁺ and Zn²⁺, and strongly by ethylenediamide tetraacetic acid (EDTA) and phenylmethysulfonyl fluoride (PMSF), but activated by Cu²⁺ and Mn²⁺. Therefore, the AOLK-101 protease was a serine protease based on the influence of metal ions and inhibitors. K _m , V _max , k _cat , and k _cat /K _m values of AOLK-101 protease for hammastein milk casein were 1.04 mg/mL, 124.84 unit/L, 163.5/sec, and 3.9×10⁶/m·sec, respectively.     

Heat inactivation and kinetics of polyphenoloxidase from palmito (Euterpe edulis)

Polyphenoloxidase (PPO, EC 1.14.18.1)was extracted from palmito (Euterpe edulis Mart) using 0.1 M phosphate buffer, pH 7.5. Partial purification of the enzyme was achieved by a combination of (NH₄)₂SO₄precipitation (35–90% saturation) and Sephadex G-25 and DEAE-cellulose chromatography. The purified preparation gave five protein bands on polyacrylamide gel electrophoresis, three of them with PPO activity. The K_mvalues for chlorogenic acid, caffeic acid, catechol, 4-methylcatechol and catechin were 0.57, 0.59, 1.1, 2.0 and 6.25 mM , respectively. PPO has a molecular weight of 51 000 Da, maximum activity at pH 5.6 with chlorogenic acid as substrate, and was stable between pH 5.0 and 8.0. The enzyme was heat stable at 50–60°C and inactivated at 75°C. The heat stability of palmito PPO was found to be pH dependent; at 50°C and pH 4.0 the enzyme was fully inactivated after 30 min. The pH/activity studies showed two groups with pK values c 4.6 and 6.7 involved in PPO catalysis.     

Properties of <Emphasis Type="Italic">Aspergillus subolivaceus</Emphasis> free and immobilized dextranase

Aspergillus subolivaceus dextranase is immobilized on several carriers by entrapment and covalent binding with cross-linking. Dextranase immobilized on BSA with a cross-linking agent shows the highest activity and considerable immobilization yield (66.7%). The optimum pH of the immobilized enzyme is shifted to pH 6.0 as compared with the free enzyme (pH 5.5). The optimum temperature of the reaction is resulted at 60 °C for both free and immobilized enzyme. Thermal and pH stability are significantly improved by the immobilization process. The calculated K _m of the immobilized dextranase (14.24 mg mL⁻¹) is higher than that of the free dextranase (11.47 mg mL⁻¹), while V _max of the immobilized enzyme (2.80 U μg protein⁻¹) is lower than that of the free dextranase (11.75 U μg protein⁻¹). The immobilized enzyme was able to retain 76% of the initial catalytic activity after 5.0 cycles.     

Biochemical characteristics and thermal inhibition kinetics of polyphenol oxidase extracted from Thompson seedless grape

Polyphenol oxidase (PPO) was isolated from Thompson seedless grape (Vitis vinifera ‘Thompson Seedless’), and its biochemical characteristics were studied. The PPO showed activity to catechol and D, L-DOPA, but not towards monophenol l-Tyrosine, diphenols guaiacol and caffeic acid, and triphenols pyrogallic acid and gallic acid. Apparent Michaelis–Menten constant (K _m) and maximum velocity of the reaction (V _max) values were 45.0 ± 0.05 mM and 500.0 ± 15.3 OD_400 nm/min for catechol, and 34.6 ± 0.03 mM and 384.6 ± 11.7 OD_478 nm/min for D, L-DOPA, respectively. The obtained similar specificity values of V _max/K _m ratio of catechol and D, L-DOPA indicated their similar affinity to Thompson seedless PPO. The most effective inhibitor was l-cysteine, followed in decreasing order by ascorbic acid, sodium metabisulfite, EDTA, NaCl, and citric acid. It was discovered that metal ions of Mg²⁺ and Cu²⁺ increased, while Zn²⁺ and K⁺ reduced the PPO activity. Sugars showed inhibition on the PPO activity, with higher effect by sucrose and lower effect by fructose and glucose. Optimum pH and temperature for grape PPO activity were 6.0 and 25 °C with 10 mM catechol as substrate. The enzyme was heat stable between 10 and 25 °C, but showed significant activity loss at temperatures higher than 40 °C and completely inactivation at 70 °C for 10 min. Thermal inactivation of PPO showed a first-order kinetic with an activation energy (E _a) of 146.1 ± 10.8 kJ/mol at pH 6.0.     

PURIFICATION AND CHARACTERIZATION OF ACIDIC PROTEASES FROM THE STOMACH OF THE DEEPWATER FINFISH ORANGE ROUGHY (HOPLOSTETHUS ATLANTICUS)

Acidic proteases were extracted and purified from the stomach of orange roughy (Hoplostethus atlanticus). Protease I and II were glycoproteins with molecular weights of 33.5 and 34.5 KDa, respectively. Protease I had an isoelectric point of 5.30. The two forms of protease II (a and b) had isoelectric points of 4.35 and 4.40, respectively, and N-terminal sequence identity for 12 amino acids. The proteases exhibited optimal temperature activity at 37C. They had high activity at low temperatures and low thermal stability compared to mammalian pepsins. They were stable in the pH range of 2–4.5 and unstable above pH 6.5. Protease I and II had pH optima of 2.5 and 3.5, respectively, and K _m’values for the hydrolysis of hemoglobin (pH 3.0, 37C) of 124 μM and 517 μM, respectively. Enzyme activities were inhibited by pepstatin A and high NaCl concentrations, and were slightly stimulated by Ca²⁺ and Cu²⁺.