Ammonium Sulfate Fractionation Research Articles

Condition Optimization and Production of Extracellular l-Arginine Deiminase from Vibrio Alginolyticus 1374

Objective: This is a study on the condition optimization of production of l-arginine deiminase (ADI) by Vibrio alginolytics 1374. To increase the production quantity, it could be helpful in the production of ADI for the therapeutic use in the future. Method: Vibrio alginolyticus 1374; GU726873 is a gram negative rod shaped marine bacteria isolated from chirala beach of Andhra Pradesh. It was screened for the enzyme production in minimal arginine medium by dye based method. Further the activity was confirmed calorimetrically by estimating the levels of l-citrulline. The potential of isolated novel strain for ADI production was analyzed under submerged fermentation with different process parameters and medium constituents. The enzyme thus obtained was purified by ammonium sulphate fractionation, ion exchange and gel permeation chromatography. The purity of enzyme was detected by polyacrylamide gel electrophoresis. Result: The maximum yield of enzyme production was achieved in a seawater based medium at pH 8, 37°C, 2% inoculum concentration and 2% l-arginine concentration for 120 h. The medium when supplemented with carbon source, it improved the enzyme production from 172 to 192 IU/ml with 2% maltose. Addition of 2% soybean meal also improved the ADI production (183.56 IU/ml). The enzyme was purified to near homogeneity (1161.2 fold).The enzyme was found to have specific activity of about 280.6 IU/mg with molecular weight of about 48kDa. Conclusion: This novel strain has an immense potential as an industrial organism for the large scale production of ADI for the therapeutic use in the future. Keywords: L-Arginine deiminase (ADI), Vibrio alginolyticus 1374; GU726873, ion exchange chromatography, gel permeation chromatography.

Investigating on the Stability of Peroxidase Extracted from Soybean (Glycine max var. Williams) and Effects of Na+ and K+ Ions on its Activity

In the present study, some techniques were used for Soybean peroxidase (SBP) purification including: ammonium sulfate fractionation, DEAE Sephadex anion exchange chromatography and Concanavalin A Sepharose 4B affinity chromatography. Molecular weight of purified SBP was estimated about 44 kDa by SDS-PAGE as a single polypeptide band. The optimal pH and temperature for enzyme activity were found to be 4.5 and 70◦C, respectively. The enzyme was more stable in alkaline pH than acidic ones and could tolerate 10 minutes heating in 40-50◦C without any loss of its activity. Both NaCl and KCl were found to have significant effects on the enzyme stability, but presence of NaCl was more effective than KCl. Our results showed that after 24 hours incubation of the enzyme in the presence of 20 mM NaCl, more than 60٪ of the enzyme activity was remained while it would fall to 3٪ if incubation was not accompanied by NaCl. Purified peroxidase from seed hull of soybean relative to the other identified peroxidases was more stable, for this reason a lot of benefit will be considered by use of this enzyme in different industry.

Matrix Metalloproteinase 2 (MMP-2) Plays a Critical Role in the Softening of Common Carp Muscle during Chilled Storage by Degradation of Type I and V Collagens.

Matrix metalloproteinases (MMPs) are proposed to play important roles in the degradation of collagens, thus causing the post-mortem softening of fish muscle, although the specific mechanism remains largely unresolved. Previously, we reported the existence of gelatinase-like proteinases in common carp (Cyprinus carpio) muscle. The primary structures of these proteinases, however, have never been investigated. In the present study, two MMPs with molecular masses of 66 and 65 kDa were purified to homogeneity from common carp muscle by ammonium sulfate fractionation and a series of column chromatographies. Matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS/MS) analysis indicated that they are completely identical to MMP-2 from common carp. During chilled storage of common carp at 4 °C, the enzymatic activity of MMP-2 increased to 212% in 12 h while the texture profile increased over the first 2 h and gradually decreased. On the other hand, type V collagen was purified to homogeneity and a specific polyclonal antibody against this protein was prepared. Both type I and V collagens were effectively hydrolyzed by MMP-2 at 30 °C and even at 4 °C. Furthermore, injection of metalloproteinase proteinase inhibitor EDTA into the blood vessel of live common carp suppressed post-mortem tenderization significantly. All of these results confirmed that MMP-2 is a major proteinase responsible for the degradation of collagens, resulting in the softening of fish muscle during chilled storage.

A Novel Aldo-Keto Reductase, HdRed, from the Pacific Abalone Haliotis discus hannai, Which Reduces Alginate-derived 4-Deoxy-l-erythro-5-hexoseulose Uronic Acid to 2-Keto-3-deoxy-d-gluconate

Abalone feeds on brown seaweeds and digests seaweeds' alginate with alginate lyases (EC 4.2.2.3). However, it has been unclear whether the end product of alginate lyases (i.e. unsaturated monouronate-derived 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH)) is assimilated by abalone itself, because DEH cannot be metabolized via the Embden-Meyerhof pathway of animals. Under these circumstances, we recently noticed the occurrence of an NADPH-dependent reductase, which reduced DEH to 2-keto-3-deoxy-D-gluconate, in hepatopancreas extract of the pacific abalone Haliotis discus hannai. In the present study, we characterized this enzyme to some extent. The DEH reductase, named HdRed in the present study, could be purified from the acetone-dried powder of hepatopancreas by ammonium sulfate fractionation followed by conventional column chromatographies. HdRed showed a single band of ∼ 40 kDa on SDS-PAGE and reduced DEH to 2-keto-3-deoxy-D-gluconate with an optimal temperature and pH at around 50 °C and 7.0, respectively. HdRed exhibited no appreciable activity toward 28 authentic compounds, including aldehyde, aldose, ketose, α-keto-acid, uronic acid, deoxy sugar, sugar alcohol, carboxylic acid, ketone, and ester. The amino acid sequence of 371 residues of HdRed deduced from the cDNA showed 18-60% identities to those of aldo-keto reductase (AKR) superfamily enzymes, such as human aldose reductase, halophilic bacterium reductase, and sea hare norsolorinic acid (a polyketide derivative) reductase-like protein. Catalytic residues and cofactor binding residues known in AKR superfamily enzymes were fairly well conserved in HdRed. Phylogenetic analysis for HdRed and AKR superfamily enzymes indicated that HdRed is an AKR belonging to a novel family.

Identification of a new alpha-2-macroglobulin: Multi-spectroscopic and isothermal titration calorimetry study

A α2M homologue was isolated from sheep (Ovis aries) blood plasma, using a simple two-step procedure, ammonium sulphate fractionation and gel filtration chromatography. Sheep α2M was found to be a large tetrameric glycoprotein of 630kDa with monomeric subunit of 133kDa each. Each subunit of sheep α2M was found to be made up of two fragments of 102 and 31kDa respectively. The proteinase inhibitor from sheep was found to have Stokes radius of 79Ǻ, which makes it much more compact than its human homologue. It entraps only 1mol of trypsin per mole of inhibitor, like its caprine counterpart. The use of isothermal titration calorimetry has become gold standard for exploring thermodynamics of binding interactions. In this study, binding interaction of trypsin with alpha-2-macroglobulin is studied using ITC. The thermodynamic signatures – enthalpy change (ΔH), entropy change (ΔS) and Gibb's free energy change (ΔG), along with number of binding sites (N) and affinity constant (K) are explored for α2M-trypsin binding for the first time for any known α2M molecule. The thermodynamics of proteinase-antiproteinase association suggests that trypsin–α2M interaction is enthalpy driven event.

Purification and characterization of a novel and versatile α-amylase from thermophilicAnoxybacillussp. YIM 342

An extracellular thermophilic α-amylase (1, 4-α-d-glucanohydrolase, EC 3.2.1.l) from Anoxybacillus sp. YIM 342 was partially purified by ultrafiltration followed by ammonium sulfate fractionation and dialysis. This procedure was followed by a single purification step using gel filtration chromatography to give a 10.41% yield, 1912.2 U/mg specific activity, and 32-fold purification enrichment. The molecular mass of the purified α-amylase was determined to be 68 kDa using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The optimum temperature and pH of the α-amylase activity were 80°C and 9.0, respectively. Furthermore, the high hydrolysis rate toward amylose and amylopectin suggested the enzyme has significant potential for applications in starch degradation. The Km and Vmax of the amylase toward soluble starch was 4.18 mg/mL and 7.48 µmol/min/mg, respectively. This enzyme hydrolyzes soluble starch to glucose, maltose, and maltotriose, indicating that the amylase represents a promising candidate for applications in the biofuel industry.

Purification of a dimeric arginine deiminase from Enterococcus faecium GR7 and study of its anti-cancerous activity

The arginine deiminase (ADI, E.C 3.5.3.6) – a key enzyme of ADI pathway of Enterococcus faecium GR7 was purified to homogeneity. A sequential purification strategy involving ammonium sulfate fractionation, molecular sieve followed by Sephadex G-100 gel filtration was applied to the crude culture filtrate to obtain a pure enzyme preparation. The enzyme was purified with a fold of 16.92 and showed a final specific activity of 76.65IU/mg with a 49.17% yield. The dimeric ADI has a molecular mass of about 94,364.929Da, and comprises of hetrodimers of 49.1kDa and 46.5kDa as determined by MALDI-TOF and PAGE analysis. To assess anti-cancerous activity of ADI by MTT assay was carried out against cancer cell lines (MCF-7, Sp2/0-Ag14 and Hep-G2). Purified ADI exhibited the most profound antiproliferative activity against Hep-G2 cells; with half-maximal inhibitory concentration (IC50) of 1.95μg/ml. Purified ADI from E. faecium GR7 was observed to induce apoptosis in the Hep-G2 cells by DNA fragmentation assay. Our findings suggest the possibility of a future use of ADI from E. faecium GR7 as a potential anticancer drug.

Characterization of a Solvent-Tolerant Manganese Peroxidase (MnP) from G anoderma Lucidum and Its Application in Fruit Juice Clarification

Manganese peroxidase (MnP) is necessary in fruit juice clarification for the degradation of phenolic compounds. In the present study, MnP was produced using basal medium supplemented with tamarind shell (1% w/v), ethanol (2% v/v) and gallic acid (1 mM) to a maximum activity of 65 U/mL. A 43.0 kDa MnP was purified by ammonium sulfate fractionation, Sephadex G-100 and DEAE-cellulose column chromatography with 6.5-fold and 13% yield. The optimal activity of the purified MnP was achieved at pH 5.5 and temperature of 45C with 2,6-dimethoxyphenol as substrate. The inhibitors sodium azide, cyanide and EDTA inhibited the MnP activity up to 100, 93 and 84%, respectively. MnP showed high activity and stability in the presence of different metal ions, surfactants and organic solvents. Clarification of different fruit juices with MnP showed its potential application in the food and beverage industry. Practical Applications Manganese peroxidase (MnP) is extensively used in various applications, such as pulp delignification, dye decoloration, biotransformation, biosensor applications and fruit juice clarification. Ganoderma lucidum-derived purified MnP is a solvent-tolerant metalloenzyme that showed its potential for industrial application in clarification of various fruit juices. The stability of MnP in the presence of various divalent cations, surfactants and solvents suggests that it could be used in industrial effluent treatments as well as the food and beverage industry.

Assessment of alkaline cholesterol oxidase purified from Rhodococcus sp. PKPD-CL for its halo tolerance, detergent and organic solvent stability

The novel bacterium, Rhodococcus sp. PKPD-CL was isolated and identified from the ‘Chilika Lake’ located at Odisha state of India, which is a largest brackish water habitat in Asia. Rhodococcus sp. PKPD-CL produces extracellular halo tolerant, detergent and organic solvent stable alkaline cholesterol oxidase. It has apparent molecular weight of 60kDa and was purified 59 fold by using 60% saturated ammonium sulfate fractionation, anion exchange followed by size exclusion chromatographic techniques with 37% recovery. It showed substrate specificity for 3β-hydroxysteroids with Km of 1.1×10−4M for cholesterol. The pH, 8.0 and the temperature, 37°C were required for its optimum activity. Enzyme is considerably stable at pH 6.0–8.5 and temperature up to 50°C. At 4 and 30°C it maintained its 100% activity up to 60days. The isoelectric point of the enzyme was 9.5. It showed 80% residual activity with 20% NaCl (3.42M) and 83% relative activity with 12% NaCl (2.05M) concentration. The metal ions like Zn2+, Cu2+, Ag+, Fe3+, Ba2+ inhibited the enzyme activity >60% while Hg2+ served a potent inhibitor whereas Mg2+ found to be a good enhancer for it. The enzyme was stable in presence of chemical reagents (NaN3, EDTA), detergents (Tween-80, Tween-20, Triton X-100, sodium cholate) and various organic solvents (isopropanol, ethanol, benzene, chloroform, methanol, toluene, ethyl acetate, butanol and dimethylsulfoxide). Such a multi stress tolerant and versatile enzyme produced by Rhodococcus sp. PKPD-CL may serve as a good choice for industrial applications.

Identification of Toxoplasma gondii protein fractions induce immune response against melanoma in mice.

Dendritic cells (DCs) play a crucial role in the initiation of adaptive immune responses against tumor cells. We recently found that protein components of Toxoplasmagondii (T.gondii) could mature DCs efficiently. Therefore, in this study, we aimed to find the most effective protein components of T.gondii which are able to mature DCs and consequently instruct immune responses in tumor-bearing mice. Soluble tachyzoite antigens (STAgs) were fractionated by ammonium sulfate precipitation and subsequently by anion-exchange HPLC. Immature DCs (iDCs) were treated by these protein fractions and were monitored for IL-12p70 and IL-10 production. Moreover, the capacity of mature DCs (mDCs) to induce lymphocyte proliferation was investigated. Ultimately, we analyzed the ability of mDCs in instructing immune responses in tumor-bearing mice. We found that ammonium sulfate fraction one (A1) matured-DCs produced higher IL-12 level and IL-12/IL-10 ratio; therefore, this fraction was selected for further fractionation by anion-exchange HPLC. The results showed that anion-exchange HPLC fraction 14 (C14) matured-DCs secrete higher levels of IL-12p70 and IL-12p70/IL-10 ratio. Survival of the mice matured by A1 fraction increased significantly compared to other groups. Moreover, SDS-PAGE electrophoresis showed that different obtained fractions have distinct proteins based on their size. These results demonstrate that two protein fractions of T.gondii are able to mature DCs more efficient.

Purification and biochemical characterization of a novel fibrinolytic enzyme, PSLTro01, from a medicinal animal Porcellio scaber Latreille

A novel protease, named PSLTro01, with fibrinolytic and anticoagulant activity was isolated from Porcellio scaber Latreille and was purified by a combination of hollow fibre membrane molecular weight cut-off (MWCO), ammonium sulfate fractionation, gel filtration and ion-exchange chromatography. PSLTro01 is a single-chain protein with a molecular mass of 38,497Da as estimated by non-reduced SDS-PAGE and MALDI-TOF MS spectrometry, and its N-terminal 15 amino acid sequence was determined as DINGGGATLPQPLYQ. PSLTro01 is stable in the range of 20–40°C and pH 6.0–10.0, with a maximum fibrinolytic activity at 40°C and pH 7.0. The PSLTro01-induced fibrinolytic activity was not influenced by K+ or Na+ but was slightly increased by Mg2+ and completely inhibited by aprotinin and pepstatin A. Fibrin plate assays revealed that PSLTro01 could not directly degrade fibrin but was a plasminogen activator. PSLTro01 exhibited high specificity for the substrate S-2251 for plasmin, followed by S-2238 for thrombin and S-2444 for urokinase. Moreover, the fibrinogenolysis pattern of PSLTro01 was Aα-chains>Bβ-chains>γ-chain. Tail-thrombus of the enzyme treated group was significantly shorter than the physiological saline treated group and the thrombus decrement was correlated with the enzyme dose. PSLTro01 prolongs both thrombin time (TT) and activated partial thromboplastin time (APTT). These results indicate that PSLTro01 may have potential applications in the prevention and treatment of thrombosis.

A novel keratinase from Bacillus tequilensis strain Q7 with promising potential for the leather bating process

The present paper reports on the purification and characterization of an extracellular keratinase (KERQ7) newly purified from Bacillus tequilensis Q7. Pure protein was obtained after ammonium sulfate fractionation (30–60%), followed by Mono S Sepharose cation-exchange chromatography. MALDI-TOF/MS analysis revealed that the purified enzyme was a monomer with a molecular mass of 28,355.07-Da. The sequence of the 21 N-terminal residues of KERQ7 showed high homology with those of Bacillus keratinases. Optimal activity was achieved at pH 7 and 30°C. KERQ7 was completely inhibited by PMSF and DFP, which suggests that it belongs to the serine keratinase family. KERQ7 displayed higher levels of hydrolysis and catalytic efficiency than Basozym® CS 10, Koropon® SC 5K, and Pyrase® 250 MP. The kerQ7 gene encoding KERQ7 was cloned, sequenced, and expressed in Escherichia coli BL21(DE3)pLysS. The biochemical properties of the extracellular purified recombinant enzyme (rKERQ7) were similar to those of native KERQ7. The deduced amino acid sequence showed strong homology with other Bacillus keratinases. The highest sequence identity value (97%) was obtained with KERUS from Brevibacillus brevis US575, with only 7 aa of difference. These properties make KERQ7 a potential promising and eco-friendly enzymatically enhanced process for animal hide bating in the leather processing industry.

Two-year comparison of the biochemical properties of polyphenol oxidase from Turkish Alyanak apricot (Prunus armenica L.)

Polyphenol oxidase (PPO) was extracted and purified from Turkish Alyanak apricot variety and its characteristics were studied in two consecutive years (2008 and 2009). Three isoenzymes (isoenzyme A1, A2 and B) were obtained upon ammonium sulfate fractionation, ion exchange chromatography using DEAE-Toyopearl 650M and gel filtration chromatography using Sephadex G-100. The isoenzymes exhibited different kinetic properties. Furthermore, year-to-year variability in Km and Vmax values was significant. The pH optimum for enzyme activity was 4.98 for isoenzymes A1 and A2, and 5.8 for isoenzyme B. The isoenzymes A1 and B had optimum temperature at 30°C in both years whereas isoenzyme A2 had maximum activity at 40°C in 2008 and 30°C in 2009. The inactivation kinetics parameters and the effects of inhibitors tested exhibited significant year-to-year variation.

Purification and characterization of a new cold active lipase, EnL A from Emericella nidulans NFCCI 3643

A mesophilic fungi producing an extracellular cold-active lipase was isolated from the soil samples of palm oil mill effluent dump sites, Pedavegi, West Godavari Dist, A.P. India and was identified as Emericella nidulans . The enzyme was purified by ammonium sulfate fractionation followed by hydrophobic interaction chromatography using phenyl sepharose. The enzyme was 35 fold pure compared to crude with a specific activity of 1494.51 U/mg. SDS PAGE analysis revealed that the protein is monomeric with a MW of ˜54 kDa and zymogram analysis showed that the purified protein was active. Characterization studies revealed that the temperature optimum was at 30°C and an optimum pH of 5. The K m and V max values were found to be 0.61 mM and 322.58 mM/min.mg, respectively. Sequencing of the purified protein by MALDI TOF-MS analysis followed by BLAST P analysis indicated that the protein is a putative secretary lipase from E. nidulans. Search of lipase engineering data base (LED) revealed that this protein belongs to a newly introduced super family of Candida antarctica lipase A like and to the homologous family of Aspergillus lipase like. Key words : Cold active lipase, Emericella nidulans, hydrophobic interaction chromatography, Candida antarctica lipase A like.

Characterization and PCR Detection Of Binary, Pir-Like Toxins from Vibrio parahaemolyticus Isolates that Cause Acute Hepatopancreatic Necrosis Disease (AHPND) in Shrimp.

Unique isolates of Vibrio parahaemolyticus (VPAHPND) have previously been identified as the causative agent of acute hepatopancreatic necrosis disease (AHPND) in shrimp. AHPND is characterized by massive sloughing of tubule epithelial cells of the hepatopancreas (HP), proposed to be induced by soluble toxins released from VPAHPND that colonize the shrimp stomach. Since these toxins (produced in broth culture) have been reported to cause AHPND pathology in reverse gavage bioassays with shrimp, we used ammonium sulfate precipitation to prepare protein fractions from broth cultures of VPAHPND isolates for screening by reverse gavage assays. The dialyzed 60% ammonium sulfate fraction caused high mortality within 24–48 hours post-administration, and histological analysis of the moribund shrimp showed typical massive sloughing of hepatopancreatic tubule epithelial cells characteristic of AHPND. Analysis of the active fraction by SDS-PAGE revealed two major bands at marker levels of approximately 16 kDa (ToxA) and 50 kDa (ToxB). Mass spectrometry analysis followed by MASCOT analysis revealed that both proteins had similarity to hypothetical proteins of V. parahaemolyticus M0605 (contig034 GenBank accession no. JALL01000066.1) and similarity to known binary insecticidal toxins called 'Photorhabdus insect related' proteins A and B (Pir-A and Pir-B), respectively, produced by the symbiotic, nematode bacterium Photorhabdus luminescens. In in vivo tests, it was shown that recombinant ToxA and ToxB were both required in a dose dependent manner to cause AHPND pathology, indicating further similarity to Pir-A and -B. A single-step PCR method was designed for detection of the ToxA gene and was validated using 104 bacterial isolates consisting of 51 VPAHPND isolates, 34 non-AHPND VP isolates and 19 other isolates of bacteria commonly found in shrimp ponds (including other species of Vibrio and Photobacterium). The results showed 100% specificity and sensitivity for detection of VPAHPND isolates in the test set.

Purification and biochemical characterization of two detergent-stable serine alkaline proteases from Streptomyces sp. strain AH4

Streptomyces sp. strain AH4 exhibited a high ability to produce two extracellular proteases when cultured on a yeast malt-extract (ISP2)-casein-based medium. Pure proteins were obtained after heat treatment (30 min at 70 °C) and ammonium sulphate fractionation (30-60 %), followed by size exclusion HPLC column. Matrix assisted laser desorption ionization-time of flight mass spectrometry analysis revealed that the purified enzymes (named SAPS-P1 and SAPS-P2) were monomers with molecular masses of 36,417.13 and 21,099.10 Da, respectively. Their identified N-terminal amino acid displayed high homologies with those of Streptomyces proteases. While SAPS-P1 was optimally active at pH 12.0 and 70 °C, SAPS-P2 showed optimum activity at pH 10.0 and 60 °C. Both enzymes were completely stable within a wide range of temperature (45-75 °C) and pH (8.0-11.5). They were noted to be completely inhibited by phenylmethanesulfonyl fluoride and diisopropyl fluorophosphates, which confirmed their belonging to the serine proteases family. Compared to SAPS-P2, SAPS-P1 showed high thermostability and excellent stability towards bleaching, denaturing, and oxidizing agents. Both enzymes displayed marked stability and compatibility with a wide range of commercial laundry detergents and significant catalytic efficiencies compared to Subtilisin Carlsberg and Protease SG-XIV. Overall, the results indicated that SAPS-P1 and SAPS-P2 can be considered as potential promising candidates for future application as bioadditives in detergent formulations.

Characterization of a novel endoglucanase from Ganoderma lucidum.

We evaluated the production and characterization of endoglucanase from Ganoderma lucidum using different lignocellulose biomasses. We purified a novel carboxymethyl cellulose (CMC) hydrolyzing endoglucanase from the white-rot fungus G. lucidum when the medium was supplemented with 1% (w/v) wheat bran. Endoglucanase was purified 12.5-fold via ammonium sulfate fractionation, Sephadex G-100, and Q-Sepharose column chromatography with a final yield of 15%. SDS-PAGE analysis revealed that the endoglucanase had a molecular mass of 64.0 kDa. The optimal activity of purified endoglucanase was at pH 5.0 and 35 °C, though it was stable between pH 4.0-7.0 and temperatures of 30-60 °C. The purified enzyme was specific to CMC as a suitable substrate. The metal ions Hg(2+), Fe(2+), and Cr(2+) inhibited enzyme activity, while Ca(2+), Mg(2+), and Mn(2+) enhanced enzyme activity. The endoglucanase showed high activity and stability in the presence of different surfactants and non-polar hydrophobic organic solvents. This endoglucanase is tolerant to high temperature, metal ions, surfactants, and solvents, suggesting that it is appropriate for use in biomass conversion for biofuel production under harsh environmental conditions.

Purification and biochemical characterization of a puromycin-sensitive aminopeptidase from black carp muscle

Exopeptidases such as aminopeptidases and carboxypeptidases are believed to contribute to the formation of free amino acids during postmortem storage and the processing of meat. In order to understand the role of aminopeptidases in the generation of amino acids and formation of flavor compounds in Chinese traditional salted fish, an aminopeptidase was purified and characterized from black carp muscle. The peptide mass fingerprinting of this enzyme suggested that it was a puromycin-sensitive aminopeptidase purified to homogeneity by ammonium sulfate fractionation and three chromatographies. Puromycin was further confirmed as a competitive inhibitor with Ki value of 0.25μM. The 100-kDa enzyme preferentially hydrolyzed substrate Lys-MCA with optimum temperature and pH at 40°C and 7.5, respectively. At the concentration of 3.2% NaCl and 5% ethanol, the enzyme remained 58.5% and 87.5% of its initial activity, respectively. The aminopeptidase(s) activity decreased slowly and remained 41.1% of its initial activity even after 12 days salting of black carp muscle. These results suggest the possible contribution of fish aminopeptidases to free amino acid formation and flavor generation in Chinese traditional salted fish.

Kinetics Study of Extracellular Detergent Stable Alkaline Protease from Rhizopus oryzae

ABSTRACT In this study, extracellular alkaline protease was produced from Rhizopus oryzae in submerged fermentation using dairy waste (whey) as a substrate. Fermentation kinetics was studied and various parameters were optimized. The strain produced maximum protease at initial medium pH of 6.0 medium depth of 26 mm, inoculum size of 2% at incubation temperature of 35 o C for 168 h of fermentation. Alkaline protease was purified to homogeneity by ammonium sulphate fractionation followed by sephadex G-100 chromatography. The molecular mass of alkaline protease was 69 kDa determined by 10% SDS-PAGE. The optimum pH and temperature of alkaline protease was 9.0 and 40 o C, respectively. Metal profile of the enzyme showed that the enzyme was non-metallic in nature. The K m , K cat , V max and K cat /K m values of purified protease were 7.0 mg/mL, 3.8 x10 2 S -1 , 54.30 µmol/min and 54.28 s -1 mg -1 .mL respectively, using casein as substrate. The purified alkaline protease had stability with commercial detergents.

Effects of physicochemical factors and in vitro gastrointestinal digestion on antioxidant activity of R‐phycoerythrin from red algae Bangia fusco‐purpurea

SummaryR‐phycoerythrin (R‐PE) was purified from the red algae Bangia fusco‐purpurea after 35–50% ammonium sulphate fractionation followed by ion‐exchange column chromatography on DEAE‐Sepharose, resulting in a purity (A565/A280) ratio of 5.1. The circular dichroism spectroscopy results suggested that the structure of R‐PE is predominately helical. The antioxidant activity of R‐PE was studied and revealed changes in conformation and antioxidant activity at different temperatures and pH values. After in vitro‐simulated gastrointestinal (GI) digestion of R‐PE, the scavenging activity of ABTS radical (EC50, 769.9 μg mL−1), DPPH radical (EC50, 421.9 μg mL−1), hydroxyl radical (EC50, 32.4 μg mL−1) and reducing power (A700 = 0.5, 625.8 μg mL−1) were measured. Gel filtration chromatography analysis showed that the molecular weight distribution of the final GI digest that still contained high antioxidant activity was <3 kDa. Our present results indicate that digestion‐resistant antioxidant peptides of R‐PE may be obtained by in vitro GI proteinases degradation.