Casein hydrolysis by Bifidobacterium longum KACC91563 and antioxidant activities of peptides derived therefrom

Milk protein is a well-known precursor protein for the generation of bioactive peptides using lactic acid bacteria. This study investigated the antioxidant activity of bovine casein hydrolysate after fermentation with Bifidobacterium longum KACC91563 using the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay and total phenolic content (TPC). The antioxidant activities of the 24-h and 48-h hydrolysates were higher than that of the 4-h hydrolysate (2,045.5 and 1,629.3 μM gallic acid equivalents, respectively, vs. 40.3 μM) in the ABTS assay. In contrast, TPC values showed activities of 43.2 and 52.4 μM gallic acid equivalents for the 4-h and 24-h hydrolysates, respectively. Three fractions (≥10 kDa, ≥3 but <10 kDa, and <3 kDa) were separated from the 24-h hydrolysate by ultrafiltration. Among these fractions, the <3 kDa fraction exhibited the highest antioxidant activity (936.7 μM) compared with the other fractions (42.1 and 34.2 μM for >10 kDa and 3–10 kDa fractions, respectively). Through liquid chromatography-electrospray ionization-tandem mass spectrometry analysis, 2 peptides, VLSLSQSKVLPVPQK and VLSLSQSKVLPVPQKAVPYPQRDMPIQA, containing the fragment VLPVPQ that has antioxidant properties, were identified in the <3 kDa fraction after 24 h of hydrolysis. The present study demonstrates the possibility of antioxidant peptide production from bovine casein using Bifidobacterium longum.     

Angiotensin I-converting enzyme-inhibitory peptide fractions from albumin 1 and globulin as obtained of amaranth grain

A number of biopeptides promoting health benefits have been isolated from food-protein hydrolysates and can be released during enzymatic digestion. Antihypertensive peptides can be part of protein fractions from amaranth grain. The objective of this work was to obtain ACE-inhibitory peptide fractions from albumin 1 and the globulin of amaranth (Amaranthus hypochondriacus) grain. Albumin 1 and globulin were hydrolysed with alcalase; hydrolysis was monitored by proteolytic degradation and by ACE-inhibitory activity. The highest ACE-inhibitory activity was 40% and 35% as obtained after 18 and 15 h hydrolysis for albumin 1 and globulin, respectively. Further separation and purification of the ACE-inhibitory peptide fractions were carried out by gel filtration and C₁₈ RP–HPLC. The IC₅₀ was 0.35 ± 0.02 mg/ml for albumin 1 peptide fraction and 0.15 ± 0.03 mg/ml for globulin peptide fraction. Albumin 1 peptide fraction showed an competitive mode of ACE inhibition, whereas the globulin peptide fraction was competitive. The globulin peptide fraction may have one of the most active naturally-occurring ACE-inhibitory peptides.     

ACE-inhibitory and antioxidant properties of potato (Solanum tuberosum)

Proteins were isolated from potato tubers (Solanum tuberosum) at different physiological states, and by-products from the potato industry were used to evaluate their ACE-inhibitory and radical-scavenging potencies. Protein isolates and by-products were autolysed or hydrolysed by alcalase, neutrase and esperase. Hydrolysis increased the inhibition of the angiotensin-converting enzyme (ACE) and the radical-scavenging activity. The ACE-inhibitory potencies of the hydrolysates were high (IC₅₀ = 0.018 − 0.086) and the by-product fractions showed ACE-inhibition also before hydrolysis. All samples exhibited low radical-scavenging activity, and hydrolysis for 2 h with proteases was needed to produce an increase in the activity. Ultrafiltration through 10–3 kDa membranes efficiently separated the ACE-inhibitory compounds into permeate fractions. The results of this study suggest that potato is a promising source for the production of bioactive compounds as ingredients for developing functional foods with a beneficial impact on cardiovascular health.     

ACE-inhibitory activity of tilapia protein hydrolysates

Fish processing wastes can be used for preparing bioactive peptides with various functionalities. Our objective was to evaluate the in vitro angiotensin converting enzyme (ACE) inhibitory activity of tilapia protein hydrolysates and its corresponding fractionates. Tilapia protein was alkali-solubilised at pH 11.0 and recovered at pH 5.5 to obtain a stable substrate. This substrate was hydrolysed using two enzymes, Cryotin-F or Flavourzyme, to 7.5% and 25% degree of hydrolysis (DH). The hydrolysates were ultra-filtered into three fractions: >30 kDa fraction, 10–30 kDa fraction, and <10 kDa fractions. Both hydrolysates and fractionates were tested for ACE inhibition. Results showed that both Cryotin and Flavourzyme hydrolysates with 25% DH gave maximum ACE inhibitory activity. Low MW peptides showed higher ACE inhibition than high MW peptides. The inhibitory activity of fractionates was lower than that of the corresponding hydrolysates, possibly due to separation and removal of synergistic peptides by ultrafiltration. Amongst fractionates, all the 7.5% DH Cryotin fractions and 25% DH Flavourzyme fractions exhibited optimum % ACE inhibition. The results of this research could be used for optimising enzyme parameters to obtain peptides from tilapia with optimum in vitro ACE inhibitory activity.     

Chemical properties and antioxidant activity of exopolysaccharides from mycelial culture of Cordyceps sinensis fungus Cs-HK1

Exopolysaccharide (EPS) was produced in a mycelial liquid culture of the Cordyceps sinensis fungus, Cs-HK1, at a rate proportional to mycelial growth. The crude EPS isolated from the culture medium by ethanol precipitation contained 65–70% sugar and about 25% protein, suggesting that the EPS was composed of polysaccharide–protein complexes. It exhibited a wide molecular weight (MW) range from about 5 kDa to more than 200 kDa. The intrinsic viscosity as an index of average MW of EPS, increased during the period of rapid mycelial growth to 11.0 dL/g on day 5, and remained relatively stable between day 5 to day 8 (stationary growth period). Both infrared spectrometry of EPS and gas chromatography of acetylated EPS suggested that the EPS had a β-d-glucan backbone. The EPS showed moderate antioxidant activities with a Trolox equivalent antioxidant capacity of 35–40 μmol Trolox/g and a ferric reducing ability of plasma of 50–52 μmol Fe(II)/g.     

A novel angiotensin I converting enzyme inhibitory peptide from tuna frame protein hydrolysate and its antihypertensive effect in spontaneously hypertensive rats

Tuna frame protein was hydrolysed using Alcalase, Neutrase, pepsin, papain, α-chymotrypsin and trypsin. Peptic hydrolysate exhibited the highest ACE I inhibitory activity among them and was fractionated into three ranges of molecular weight (below 1, 1–5 and 5–10 kDa) using an ultrafiltration membrane bioreactor system. The 1–5 kDa fraction showed the highest ACE inhibitory activity and was used for subsequent purification steps. During consecutive purification, a potent ACE inhibitory peptide from tuna frame protein (PTFP), which was composed of 21 amino acids, Gly-Asp-Leu-Gly-Lys-Thr-Thr-Thr-Val-Ser-Asn-Trp-Ser-Pro-Pro-Lys-Try-Lys-Asp-Thr-Pro (MW: 2,482 Da, IC₅₀: 11.28 μm), was isolated. Lineweaver–Burk plots suggest that PTFP plays as a non-competitive inhibitor against ACE. Furthermore, antihypertensive effect in spontaneously hypertensive rats (SHR) also revealed that oral administration of PTFP can decrease systolic blood pressure significantly (P < 0.01). These results suggest that the PTFP would be a beneficial ingredient for nutraceuticals and pharmaceuticals against hypertension and its related diseases.     

Value-added utilization of yak milk casein for the production of angiotensin-I-converting enzyme inhibitory peptides

Yak (Bos grunniens) milk casein derived from Qula, a kind of acid curd cheese from northwestern China, was hydrolysed with alcalase. The hydrolysates collected at different hydrolysis times (0 min, 60 min, 120 min, 180 min, 240 min, 300 min, 360 min) were assayed for the inhibitory activity of angiotensin-I-converting enzyme (ACE), and the one obtained at 240 min hydrolysis showed the highest ACE inhibitory activity. The active hydrolysate was further consecutively separated by ultrafiltration with 10 kDa and then with 6 kDa molecular weight cut-off membranes into different parts, and the 6 kDa permeate showed the highest ACE-inhibiting activity. This active fraction was further purified to yield two novel ACE-inhibiting peptides, whose amino acid sequences were Pro–Pro–Glu–Ile–Asn (PPEIN)(κ-CN; f156–160) and Pro–Leu–Pro–Leu–Leu (PLPLL) (β-CN; f136–140), respectively. The molecular weight and IC₅₀ value of the peptides were 550 Da and 566.4 Da, and 0.29 ± 0.01 mg/ml and 0.25 ± 0.01 mg/ml, respectively.     

Molecular characteristics of partially hydrolyzed fucoidans from sporophyll of Undaria Pinnatifida and their in vitro anticancer activity

The effects of molecular characteristics on the anticancer activity of fucoidans were investigated after hydrolysis by copper acetate and then fractionation with 30 and 5 kDa membranes, which produced three fucoidan fractions: F_>30 K, F_5–30 K and F_<5 K. The F_>30 K and F_5–30 K consisted of mostly carbohydrate (58.2–61.3%) and sulphate (31.7–35.5%) with small amounts of proteins (1.2–6.4%). However, the major constituents of F_<5 K were sulphate (31.8%) and ash (37.5%) with smaller amounts of carbohydrate (15.5%) and protein (1.2%). The molecular weights (M_w) of F_>30 K, F_5–30 K and F_<5 K, obtained by a light scattering technique, were 262, 5.6 and 1.6 kDa, respectively. The observed anticancer activities were 18.0–28.5% for F_>30 K, 19.2–57.5% for F_5–30 K and 26.5–36.5% for F_<5 K, respectively, in the concentration range of 0.2–0.8 mg/mL. The results suggest that the anticancer activity of fucoidans could be considerably improved by lowering their M_w and by improving the binding properties of sulphate groups possibly through changing the molecular conformation.     

Partial purification and characterization of trypsin-like proteinases in Indian anchovy (Stolephorus spp.)

Four fractions (P111, P21, P31, and P4) of proteinases were obtained from various purification steps including heat treatment (60 °C, 10 min), 30–60% ammonium sulfate precipitation, anion exchange, hydrophobic interaction, and gel filtration chromatography. Optimal temperature and pH of all fractions were 50–60 °C and 8.5, respectively. All partially purified proteinases preferably hydrolyzed substrates containing Arg at the P₁ position. All proteinases were inhibited by soybean trypsin inhibitor, leupeptin, and N-tosyl-l-lysine chloromethyl ketone. Partially purified proteinases were stable at 35 °C up to 12 h. However, their activity decreased about 40% when incubated at the optimal temperature (50–55 °C) for 2 h. Only P111 was stable at its optimal temperature (60 °C) up to 12 h. Molecular weight (MW) of P111, P21, and P31 was estimated to be 27, 33, 37, 43, 48, 55, 60, and 65 kDa, while MW of P4 was 39 kDa based on activity staining. All partially purified proteinases hydrolyzed washed anchovy mince at 4.0 M NaCl, pH 8.5, at 35 °C and at their optimal temperatures (50–60 °C).     

A NaCl-stable serine proteinase from Virgibacillus sp. SK33 isolated from Thai fish sauce

An extracellular proteinase from Virgibacillus sp. SK33, isolated from 1 month-old fish sauce, was purified to electrophoretic homogeneity, using hydrophobic interaction chromatography and hydroxyapatite with purification fold of 2.5 and 7% yield. The anomalous molecular weight (MW) of 19 kDa was obtained from SDS–PAGE, whereas a MW of 33.7 kDa was determined by MALDI-TOF. Optimum conditions for catalytic activity were 55 °C and pH 7.5. The proteinase was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferentially hydrolysed Suc-Ala-Ala-Pro-Phe-AMC, indicating a serine proteinase with subtilisin-like characteristics. K_m and k_cat of the purified proteinase were 27 μM and 12 s⁻¹, respectively. Proteinase activity, toward both synthetic and anchovy substrates, increased with NaCl up to 25%. The proteinase exhibited high stability in both the absence and presence of NaCl up to 25%. Approximately 2.5-fold increase in activity was observed in the presence of divalent cations, including Ca²⁺, Mg²⁺ and Sr²⁺ at 100 mM. MALDI-TOF MS and LC–ESI-MS/MS analyses, as well as N-terminal sequences, revealed that the purified enzyme did not match microbial proteinases in the database, indicating it to be a novel proteinase.     

Angiotensin I-converting enzyme inhibitor derived from soy protein hydrolysate and produced by using membrane reactor

Five different proteolytic enzymes, including Alcalase, Flavourzyme, trypsin, chymotrypsin and pepsin were employed to hydrolyze isolated soy protein (ISP) to produce the hydrolysates, respectively. The result indicated that hydrolysis of ISP for 0.5–6 h with Alcalase produced the highest ACE inhibitory activity. Therefore, Alcalase was selected for further study on optimization of hydrolysis conditions. The optimum conditions for Alcalase to hydrolyze ISP to produce the lowest IC₅₀ value were: E/S = 0.01, hydrolysis temperature = 50 °C, pH 9.0 and hydrolysis time = 6 h. Under these conditions, the IC₅₀ value of ISP was significantly reduced from 66.4 to 0.67 mg protein/ml. The lower IC₅₀ value represented the higher the ACE inhibitory activity. Moreover, several membranes with molecular weight cut-offs (MWCFs) of 1000–30,000Da were used to filter the hydrolysate. The 10 kDa permeate obtained from the treatment of the hydrolysate by 10,000 Da MWCF membrane could further reduce its IC₅₀ value from 0.668 to 0.078 mg protein/ml with a peptide recovery of 67.5%. An operation stability study showed that the membrane reactor system could maintain a steady production of ISP hydrolysate for over 8 h. The in vitro effect of gastrointestinal protease on ACE inhibitory activity of 10 kDa permeate was also investigated. The results suggested that gastrointestinal proteases have very little effect on the ACE inhibitory activity of 10 kDa permeate.     

Oxidative stability of mahi mahi red muscle dipped in tilapia protein hydrolysates

The oxidative stability of mahi mahi red muscle dipped in tilapia protein hydrolysates was evaluated. Alkali solubilised tilapia protein isolate was hydrolysed using Flavourzyme to 13% degree of hydrolysis. Whole tilapia protein hydrolysate and ultrafiltered fraction <10 kDa were used as dip solutions. Mahi mahi red muscle was dip treated either for 2 or 4 min and stored at 4 °C. Lipid hydroperoxides (PV), thiobarbituric acid reactive substances (TBARS) and a∗ value were measured at regular intervals. Results showed that dip treatments significantly decreased (p < 0.05) the formation of PV and TBARS over 90 h storage time. There was no significant (p > 0.05) difference between WH and <10 kDa fractions, and between 2 and 4 min treatments. Red colour of treated samples measured as a∗ value decreased with storage time, but was not significantly different from the control. It could be concluded that dip treatment for 2 min in whole tilapia protein hydrolysate may be used as a potential antioxidant treatment for improving oxidative stability of fish fillets.     

Interaction of milk α- and β-caseins with tea polyphenols

The interaction of α- and β-caseins with tea polyphenols (+)-catechin (C), (−)-epicatechin (EC), (−)-epigallocatechin (EGC) and (−)-epigallocatechin gallate (EGCG) was examined at a molecular level, using FTIR, UV–visible, CD and fluorescence spectroscopic methods as well as molecular modelling. The polyphenol binding mode, the binding constant and the effects of polyphenol complexation on casein stability and conformation were determined. Structural analysis showed that polyphenols bind casein via both hydrophilic and hydrophobic interactions with overall binding constants of K_C–α-cas = 1.8 (±0.8) × 10³ M⁻¹, K_EC–α-cas = 1.8 (±0.6) × 10³ M⁻¹, K_EGC–α-cas = 2.4 (±1.1) × 10³ M⁻¹ and K_{EGCG–α-cas} = 7.4 (±0.4) × 10³ M⁻¹, K_C–β-cas = 2.9 (±0.3) × 10³ M⁻¹, K_EC–β-cas = 2.5 (±0.6) × 10³ M⁻¹, K_EGC–β-cas = 3.5 (±0.7) × 10³ M⁻¹ and K_{EGCG–β-cas} = 1.59 (±0.2) × 10⁴ M⁻¹. The number of polyphenol bound per protein molecule (n) was 1.1 (C), 0.9 (EC), 1.1 (EGC), 1.5 (EGCG) for α-casien and 1.0 (C), 1.0 (EC), 1.1 (EGC) and 1.5 (EGCG) for β-casein. Structural modelling showed the participation of several amino acid residues in polyphenol–protein complexation with extended H-bonding network. Casein conformation was altered by polyphenol with a major reduction of α-helix and β-sheet and increase of random coil and turn structure suggesting further protein unfolding. These data can be used to explain the mechanism by which the antioxidant activity of tea compounds is affected by the addition of milk.     

Antihypertensive and free radical scavenging properties of enzymatic rapeseed protein hydrolysates

In this study, rapeseed protein isolate (RPI) was digested with various proteases to produce rapeseed protein hydrolysates (RPHs), which were then separated into different peptide fractions (<1, 1–3, 3–5, and 5–10 kDa) by membrane ultrafiltration. Membrane fractionation showed that peptides with sizes <3 kDa had significantly (p < 0.05) reduced surface hydrophobicity when compared to the RPHs and peptide fractions with sizes >3 kDa. In contrast, the <3 kDa peptides showed significantly (p < 0.05) higher oxygen radical scavenging ability when compared to the >3 kDa peptides and RPHs. In vitro inhibition of angiotensin I-converting enzyme (ACE) was significantly (p < 0.05) higher for the Thermolysin, Proteinase K and Alcalase RPHs when compared to the pepsin + pancreatin (PP) and Flavourzyme RPHs. The Alcalase RPH had significantly (p < 0.05) higher renin inhibition among the RPHs, while with the exception of Thermolysin, the 5–10 kDa peptide fraction had the least renin-inhibitory ability when compared to the <5 kDa peptide fractions. Oral administration (100 mg/kg body weight) of the RPHs and RPI to spontaneously hypertensive rats (SHR) showed the Alcalase RPH to be the most effective in blood pressure (BP) reduction (∼24 mm Hg) while Proteinase K RPH was the least effective (∼5 mm Hg) after 8 h. However, the PP RPH had the most prolonged effect with BP reduction of ∼20 mm Hg after 24 h of oral administration. We conclude that the strong BP-lowering ability of Alcalase and PP RPHs could be due to high resistance of the peptides to structural degradation coupled with high absorption rate within the gastrointestinal tract.     

Purification and identification of novel antioxidant peptides from enzymatic hydrolysates of sardinelle (Sardinellaaurita) by-products proteins

In order to utilise sardinelle (Sardinellaaurita) protein by-products, which is normally discarded as industrial waste in the process of fish manufacturing, heads and viscera proteins were hydrolysed by different proteases to obtain antioxidative peptides. All hydrolysates showed different degrees of hydrolysis and varying degrees of antioxidant activities. Hydrolysate generated with crude enzyme extract from sardine (Sardinapilchardus) displayed high antioxidant activity, and the higher DPPH radical-scavenging activity (87 ± 2.1% at 2 mg/ml) was obtained with a degree of hydrolysis of 6%. This hydrolysate was fractionated by size exclusion chromatography on a Sephadex G-25 into eight major fractions (P₁–P₈). Fraction P₄, which exhibited the highest DPPH scavenging activity, was then fractionated by reversed-phase high performance liquid chromatography (RP-HPLC). Seven antioxidant peptides were isolated. The molecular masses and amino acids sequences of the purified peptides were determined using ESI-MS and ESI-MS/MS, respectively. Their structures were identified as Leu-His-Tyr, Leu-Ala-Arg-Leu, Gly-Gly-Glu, Gly-Ala-His, Gly-Ala-Trp-Ala, Pro-His-Tyr-Leu and Gly-Ala-Leu-Ala-Ala-His. The first peptide displayed the highest DPPH radical-scavenging activity (63 ± 1.57%; at 150 μg/ml) among these peptides.     

Determination of physicochemical properties of sulphated fucans from sporophyll of Undaria pinnatifida using light scattering technique

The weight average molecular weight (M_w) of the intact sulphated fucans extracted with water from the sporophyll of Undaria pinnatifida without acid treatment was determined using the high performance size exclusion chromatography coupled to multiangle laser light scattering and refractive index detection (HPSEC-MALLS-RI) system. The effects of different heating conditions on the determination of M_w were investigated. The extracted intact fucoidans mostly consisted of carbohydrates (54.9%) and sulphate (41.5%) with monosaccharide composition of 78.8% fucose and 21.2% galactose. The M_w of the intact fucoidans was reduced from 23,600 to 5200 kDa when heated in boiling water for 1–15 min. Microwave heating for 30 s decreased the M_w of fucoidans to 2400 kDa despite no significant polymer degradation. The results indicate that the 30 s-microwave heating yielded a more accurate M_w value of the intact fucoidans than any other heat treatments used in this study.     

Purification and identification of polysaccharide derived from Chlorella pyrenoidosa

The conditions for extracting and purifying polysaccharides from Chlorella pyrenoidosa, including intensity and duration of ultrasound, the temperature and incubation time, and ethanol concentration, were investigated through an orthogonal design of L₁₆(4⁵) in this work. High performance liquid chromatography (HPLC) and gas chromatography (GC) were used to characterize the compounds in C. pyrenoidosa. The highest yield of 44.8 g kg⁻¹ was achieved at 400 W of ultrasound for 800 s and then followed by incubation in water bath at 100 °C for 4 h in 80% ethanol. Two polysaccharide fractions (S1 and S2) were separated from the extracts of C. pyrenoidosa using Sepharose 4B column chromatography. The average molecular weights (M_w) of S1 and S2 were 81,877 Da and 1749 Da, respectively. Gas chromatographic (GC) traces of the hydrolyzed polysaccharides showed that most of the majority of monosaccharide in both fractions was mannose (78.0% and 76.5% of relative mass from S1 and S2, respectively) with low levels of glucose (13.2% and 8.4% of relative mass from S1 and S2, respectively). The Fourier-transform infrared spectra (FT-IR) of S1 and S2 revealed typical characteristics of polysaccharides. Both samples had the characteristics of hydroxyl groups, weak C–H band and α-pyranoses; however, only S2 had a carboxyl group.     

Antioxidant properties of papain hydrolysates of wheat gluten in different oxidation systems

Enzymatic hydrolysis was used for preparing hydrolysates from wheat gluten which is by-product during production of wheat starch. The enzyme used for the hydrolysis was papain. The hydrolysate was separated based on the molecular weight of the peptides by membrane ultrafiltration (UF) with a molecular weight cut-off of 5 kDa into permeate (P) and retentate (5-K) fractions. The antioxidative activities of the hydrolysate and its UF fractions were investigated by using the TBA method and scavenging effect of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. The three fractions showed strong antioxidative activities in the linoleic acid oxidation system, and exhibited DPPH radical scavenging activity. The antioxidative activity of the P fraction was almost the same as that of vitamin E at pH 7.0. The molecular weight distribution of the P fraction was concentrated in 4.2 kDa (86.5%) after gel permeation chromatography fractionation using an HPLC system.The P and 5-K fractions had higher surface hydrophobicities (H₀) at pH7.0 compared with the hydrolysate. The resulting UF fractions were superior to the hydrolysate in terms of antioxidative activities.     

Characterisation and tissue distribution of polyphenol oxidase of deepwater pink shrimp (Parapenaeus longirostris)

Characterisation and tissue distribution of polyphenol oxidase (PPO) was studied in deepwater pink shrimp (Parapenaeus longirostris) post mortem. PPO activity was the highest in the carapace, followed by that in the abdomen exoskeleton, cephalotorax, pleopods and telson. No PPO activity was found in the abdomen muscle and in the pereopods and maxillipeds using the enzymatic assay. Storage of whole shrimps and of the different organs showed that melanosis (blackening) required the presence of the cephalotorax to be initiated, indicating that its development depends on other factors in addition to the PPO levels. Further characterisation was carried out in extracts partly purified using 40–70% ammonium sulfate fractionation. The enzyme had the highest activity at pH 4.5 and was most stable at pH 4.5 and 9.0. No clear maximum was observed in the 15–60 °C range but the higher stability was achieved at 30–35 °C. Apparent kinetic constants in the partly purified PPO from carapace were K_M = 1.85 mM and V_max = 38.5 U/mg of protein, pointing to a high affinity and reactivity of the enzyme when assayed with DOPA. Electrophoretic mobility was studied in native PAGE and non-reducing SDS–PAGE followed by staining with DOPA. Approximate MW of 500 kDa and 200 kDa were observed, respectively. These two forms could correspond to aggregates of minor PPO subunits that could not be resolved in these electrophoretic systems. The peptide mass fingerprinting obtained by MALDI-TOF analysis showed some peptides whose homology with hemocyanins and different PPO subunit precursors has already been demonstrated in the same species.     

A β-galactosidase from chick pea (Cicer arietinum) seeds: Its purification,biochemical properties and industrial applications

A β-galactosidase from Cicer arietinum seeds has been purified to apparent electrophoretic homogeneity using a combination of various fractionation and chromatographic techniques, giving a final specific activity of 220 units mg⁻¹, with approximately 1840 fold purification. Analysis of the protein by SDS–PAGE revealed two subunits with molecular masses of 48 and 38 kDa, respectively. These bands were further confirmed with LC–MS/MS, indicating that Chick pea β-galactosidase (CpGAL) is a heterodimer. Molecular mass was determined to be 85 kDa by Superose-12 FPLC column, which is in agreement with the molecular mass suggested by mass spectroscopy to be 83 kDa. The optimum pH of the enzyme was 2.8 and it hydrolysed o-nitrophenyl β-d galactopyranoside (ONPG) with a K_m value of 1.73 mM at 37 °C. The energy of activation (E_a) calculated in the range of 35 to 60 °C, using Arrhenius equation, was determined to be 11.32 kcal mol⁻¹. The enzyme could also hydrolyse lactose, with an optimum pH of 4.0 at 40 °C. K_m and E_a for lactose hydrolysis was found to be 10 mM and 10.57 kcal mol⁻¹, respectively. The enzyme was found to be comparatively thermostable showing maximum activity at 60 °C for both ONPG and lactose. Galactose was found to be the competitive inhibitor. β-Galactosidase also exhibited glycoproteineous properties when applied on Con-A Sepharose column. The enzyme was localised in germinated seeds with X-gal activity staining and shown to be expressed prominently at grown radical tip and seed coat. Sequence alignment of CpGAL with other known plant β-galactosidase showed high amino acid sequence homology.