Purification Fold Research Articles

Inhibition of Partially Purified Xanthine Oxidase Activity from renal failure patients by Theophylline and Metronidazole

Xanthine oxidase (XO) was purified from sera of patients with renal failure by using ammonium sulfate and dialysis with a specific activities 2.1×10-3 and 5.6×10-3 unit/mg protein respectively. By using anion exchange chromatography techniques, One protein peak of XO activity was obtained with specific activity 78×10-3unit/mg protein and with purification fold of 134.02 compared to crude enzyme. One isoenzyme was obtained having a molecular weight of 255344 Dalton approximately.
 Inhibition of purified XO was studied by deferent concentrations of Theophylline and Metronidazole drugs. The maximal inhibitory effect was exhibited with 70 and 80µM Theophylline and 16 and 18 mM metronidazole.
 The inhibition type of XO by Theophylline was competitive. Vmax value was 0.0416 unit/ml, and Km value without inhibitor was 0.833 mM, however values were 2 and 2.85 mM, and inhibition constant Ki values were 29.57 and 23.67 mM respectively with 70 and 80µM of Theophylline.
 The inhibition type of XO by Metronidazole was non competitive. Km value 0.833 mM and Vmax value without inhibitor was 0.0416 unit/ml. However, Vmax values were 0.027 and 0.023 unit/ml and inhibition constant Ki values were 17 and 19mM by using 16 and 18 mM of Metronidazole respectively.

A quaternary amine cryogel column for chromatographic capture of l-asparaginase

L-asparaginase is a therapeutic enzyme used in therapies of acute lymphoblastic leukemia. Its purification conventionally uses several unit operations, and usual protocols lead to low yields and extensive processing times, which reduce productivity and increases process costs. In this study, we produced a supermacroporous cryogel activated with polyethyleneimine, followed by quaternization with epichlorohydrin resulting in a quaternary ammonium anion-exchanger. The anionic cryogel had a ligand density of 121.6 mmol/L and the HETP ranged from 0.18 to 0.27 cm. The activated cryogel was applied to capture L-asparaginase from filtered broth. Yield and purification fold (PF) were optimized using the response-surface methodology, resulting in optimum values of 56 % and 7.6, respectively. The effect of flow velocity over PF was also analyzed, and the results showed that PF had an average value of 8.5 and did not vary with the fluid velocity, confirming the convective mass transfer property of the activated cryogel. The results showed that the activated cryogel shows advantages in using unclarified broth without pretreatment and works at a high flow rate with the perspective of reducing unit production cost.

Purification, Characterization and anticancer activity of L-methionine γ-lyase from thermo-tolerant Aspergillus fumigatus

Purification of L-methionine γ-lyase (MGL) from A. fumigatus was sequentially conducted using heat treatment and gel filtration, resulting in 3.04 of purification fold and 73.9% of enzymatic recovery. The molecular mass of the purified MGL was approximately apparent at 46 KDa based on SDS-PAGE analysis. The enzymatic biochemical properties showed a maximum activity at pH 7 and exhibited plausible stability within pH range 5.0–7.5; meanwhile the highest catalytic activity of MGL was observed at 30–40 °C and the enzymatic stability was noted up to 40 °C. The enzyme molecule was significantly inhibited in the presence of Cu2+, Cd2+, Li2+, Mn2+, Hg2+, sodium azide, iodoacetate, and mercaptoethanol. Moreover, MGL displayed a maximum activity toward the following substrates, L-methionine < DL-methionine < Ethionine < Cysteine. Kinetic studies of MGL for L-methioninase showed catalytic activity at 20.608 mM and 12.34568 µM.min−1. Furthermore, MGL exhibited anticancer activity against cancerous cell lines, where IC50 were 243 ± 4.87 µg/ml (0.486 U/ml), and 726 ± 29.31 µg/ml (1.452 U/ml) against Hep-G2, and HCT116 respectively. In conclusion, A. fumigatus MGL had good catalytic properties along with significantly anticancer activity at low concentration which makes it a probably candidate to apply in the enzymotherapy field.

Obtaining peroxidase from Zanthoxylum armatum DC. fruit and application in detoxification of phenol wastewater

The important role of peroxidase in plant physiology and biocatalysis has attracted much attention. To evaluate the specific properties of the peroxidase from Zanthoxylum armatum DC. fruit and its performance in phenol removal and detoxification. This study obtained low-purity peroxidase from Z. armatum fruit for the first time via three-phase partitioning with a yield of 160 mg/100 g, purification fold of 12.8, and recovery rate of 83 %. Afterward, SuperTandex-75 gel was used for further purification. The result of liquid chromatography-tandem mass spectrometry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and native-polyacrylamide gel electrophoresis revealed that the enzyme contained isoenzymes with different molecular weights (46.32, 39.41, 35.03, and 9.06 kDa). Enzymatic characterization study found that compared with commercial peroxidase, the enzyme showed better pH, temperature, and metal ion stability, however, 10 mM concentrations of ascorbic acid and citric acid could inhibit its activity by more than 90 %. Regarding the phenol bioconversion, when the enzyme activity of the system is 200 U/mL, the phenol concentration of 5–10 mM can be reduced by more than 80 % under optimal conditions. The detoxification effect of low-purity peroxidase on phenol water was tested using Vigna radiata (Linn.) Wilczek. The reduced toxicity of phenol water after treatment promoted the growth of plants, indicating that the phenol water after degradation by the low-purity peroxidase can be used in agricultural production, and it also proved that the enzyme is a promising peroxidase. In addition, the present study provides data for the selection of inhibitory peroxidase activity parameters in the food processing of Z. armatum fruit.

Evaluating Khaya senegalensis for Dipeptidyl Peptidase-IV Inhibition Using in Vitro Analysis and Molecular Dynamic Simulation of Identified Bioactive Compounds.

The dipeptidyl peptidase-IV (DPP-IV) inhibitory activity of Khaya senegalensis extracts was evaluated. The DPP-IV from a rat kidney was purified to a purification fold of 2.3. Among extracts from K. senegalensis, the hexane extract had the best DPP-IV inhibitory activity, with IC50 value of 1.56±0.61 μg/mL and was fractionated to eleven fractions (A-K). Fraction I had the best DPP-IV inhibition via uncompetitive pattern. GC-MS analysis of fraction I showed that the major bioactive compounds were 3-amino-3-hydroxyimino-N-phenylpropanamide (1) and 11-(2-cyclopenten-1-yl)undecanoic acid (2), with good binding affinities toward DPP-IV, based on molecular docking,. They were then subjected to molecular dynamic simulation using WEBGRO and utilizing a GROMACS system for 100 ns. The 3-amino-3-hydroxyimino-N-phenylpropanamide-DPP-IV complex was more stable and compact than the other complex. K. senegalensis contains compounds like 1 that might be used for the design of new DPP-IV inhibitors.

Extraction, purification and characterization of papain cysteine-proteases from the leaves of Carica papaya

Papain is a globular cysteine-protease family consisting of a single polypeptide chain with three disulfide bridges and a sulfhydryl group necessary for proteolytic activity. Its application is extensive to the fields of medicine and food. The aim of this study is to extract, purify and characterize papain enzymes from the leaves of Carica papaya. Crude extracts containing the enzymes were extracted from the leaves of papaya in Mountain Top University, Nigeria, and purified in a 3-step purification using 70% NH4(SO4)2, DEAE-Cellulose column chromatography and Sephadex G-25 column chromatography to achieve a purification fold of 1.6424. The effects of temperature, pH, substrate concentration and Mg2+ concentrations on the activity of the enzymes were determined. This is a premier report on the presence of five cysteine-proteases in the leaves of papaya with different measures of binding affinity and catalytic efficiencies towards the casein-substrate. The purified enzymes from Carica papaya are optimally active from 50 °C - 59 °C and pH 4.5 – 6.6. Their activities are generally enhanced in the presence of Mg2+. The Km and Vmax values for the enzyme in the various pools ranged from 1.47mg/ml - 8.70mg/ml and 0.42µmol/ml/min - 0.4167µmol/ml/min. In this study, papain E displayed optimum activity at pH 5.5 and 50 °C in the presence of Mg2+ with the best catalytic efficiency (Kcat/Km 59.776 - pmL/mg.min) and binding affinity (Km - 0.83 mg/mL) when compared to the isolated enzymes. Values of the parameters assessed from this study could be put to use by the industry in the production of papain and also for household and medical use in storing the product.

Enhanced Biosynthesis and Purification of Proteases from Bacillus sp. AI-5 by SmF: A Green Approach for Degradation of Peptide Bonds in Complex Proteins

Proteases are theprotein degrading commercial enzymes with considerable importance for many industrial applications such as paper, leather, food industries as well as used in detergent formulation, toxic waste removal, pharmaceutical and drilling for oil. The focus of the current study was to isolate the novel protease producing bacterial sp. from environment, to optimize the submerged fermentation parameters in order to design the best supportive media for maximum enzyme yield and to purify the enzyme to increase its utilization in various industries, most importantly the pharmaceutical industry.Five pure cultures were isolated from soil of coastal area, Karachi and the bacterial strain that was proved to be the most potent producer of protease was selected for research purposeand named as Bacillus sp AI-5 after series of morphological and biochemical tests. The fermentation conditions and media composition were optimized using the starter medium that was selected among the three previously reported media for protease production. It was found that protease production from Bacillus sp AI-5 reached to maximum when media was supplemented with 0.4 gm % casein as carbon source, the combination of 0.5 gm % yeast extract and 0.5 gm % peptone as nitrogen source and 0.05 gm CaCl₂ as inducer and stabilizer of proteases. The optimum pH and temperature for maximum production of protease were found to be pH 5 and 45 and#176;C respectively after 24 hours of incubation. The protease of the Bacillus sp. AI-5 was purified to homogeneity by salt fractionation method using 60 gm % ammonium sulfate, dialysis and Sephadex G-100 Gel filtration chromatography. The specific activity of purified protease was found to be 322.25 U/mg with 13.80 fold purification as compared to crude enzyme. The purified enzyme gave a single band on Sodiumdodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE)corresponding to a molecular weight of 66 kDa.

English

Tyrosinase catalyzes formation of browning in plants, foods and vegetables. Sweet potato (Ipomea batatas) undergoes browning after harvesting or during post-harvest operations leading to spoilage and loss of economic value. The physico-chemical properties of purified tyrosinase from I. batatas are here described with a view to providing information on the suitability or otherwise of the enzyme for several industrial and biotechnological processes. The enzyme was purified using new approach resulting into final yield and purification fold of 76% and 7.1, respectively. The molecular weight (native) was 48.3 &plusmn; 2.5 kDa as estimated on Sephadex G-100. Highest tyrosinase activity was obtained at pH 6.5 while that of temperature was 50&deg;C. Kinetic parameters studies resulted to 2.5 &plusmn; 1.2 mM and 451 &plusmn; 23.7 units/mg for Michaelis constant (Km) and maximum velocity (Vmax), respectively. This led to catalytic efficiency, kcat/Km of 1.45 &times; 105 s-1 M-1. It was concluded that, tyrosinase from I. batatas possesses remarkable properties that could be exploited for biotechnological processes. &nbsp; Key words: Polyphenols, Solanaceae, biotechnology, biocatalysis.

Evaluation of Dye Decolourization ability of Laccase Produced Curvularia lunata SS17

The increasing discharge of dyes into the environment and their consequential ecological effects has necessitated the need for the ecofriendly decolourization methods. Laccases became enzymes of research interest due to their broad substrates specificities. The aim of the study was to purify, characterize and determine the decolourization ability of Curvularia lunata SS17 produced laccase. Laccase was produced using maize cob as substrate under optimized culture conditions and purified using Gel Filtration Chromatography. Biuret method was then used to estimate the protein contents of the crude and partially purified laccase. Specific activities, purification fold and yield (%) of the crude and purified laccase enzyme were also estimated. Decolourization potentials of the crude and partially purified enzyme were evaluated using four dyes namely: Congo red, Methylene blue, Bromocresol green and direct yellow. Elution profile of partially purified laccase revealed that fraction 5 had the highest laccase activity (3.654 U/mL). Optimum conditions for enzyme activity were estimated to be 35oC and pH 6. Enzyme activity of the partially purified laccase (3.654 U/mL) was observed to be higher than that of the crude laccase (1.635 U/mL). Also, the partially purified laccase had higher specific activity (1.87 U/mg) compared to that of the crude laccase (0.41 U/mg). Higher percentage dye decolourization potential was observed using the partially purified laccase compared to the crude laccase. Increase in percentage decolourization of the dyes by the partially purified laccase as well as crude laccase was observed as incubation time proceeds.

DEVELOPMENT OF BIOPROCESSES FOR PRODUCTION AND PURIFICATION OF L-ASPARAGINASE FROM STAPHYLLOCOCCUS AUREUS, AND IN VITRO EFFECACY AGAINST HUMAN BREAST CNCER CELL LINE

The present study was aimed to isolate the abundant Staphylococcus aureus produce asparaginase and evaluate the efficacy of purified L-ASNase against a breast cancer cell line. From one hundred local isolates of S. aureus which subjected to the primary screening processes for asparaginase production, thirty-seven isolates with maximum ability of hydrolysis zone in primary screening (the ratio of Z more than 10 mm) were selected for secondary screening. It has been indicated that S. aureus TG98 had the highest productivity of the enzyme (255.8 U/mg protein). The highest producing-isolate was identified according to vitek test. The optimal conditions for ASNase production by the selected isolate was performed using submerged fermentation, where the medium (5) was utilized as the best medium for production, lactose as the best carbon source, NH4Cl as the best nitrogen source, pH 8 at 37 °C after 24 hr of incubation period, the specific activity was reached to 496.99 U/mg. The enzyme was purified by gel filtration chromatography utilizing Sephadex G-150. The results indicated that there is an elevating in final purification folds 2 time with 103% yield of enzyme. Also, it was exhibited maximal activity and stability at pH 8.0. Also it was active and stable at 37°C.The highest rate of enzyme specificity found with asparagine. The breast tumor cells proliferation was significantly suppressed by the cytotoxic effect of L-ASNase comparison with normal cell line (WRL-68).

Kinetic and thermodynamic studies of novel acid phosphatase isolated and purified from Carthamus oxyacantha seedlings

Acid phosphatase (ACP) is a key enzyme in the regulation of phosphate feeding in plants. In this study, a new ACP from C. oxyacantha was isolated to homogeneity and biochemically described for the first time. Specific activity (283 nkat/mg) was found after 2573 times purification fold and (17 %) yield. Using SDS-PAGE under denaturing and nondenaturing conditions, ACP was isolated as a monomer with a molecular weight of 36 kDa. LC-MS/MS confirmed the presence of this band, suggesting that C. oxycantha ACP is a monomer. The enzyme could also hydrolyze orthophosphate monoester with an optimal pH of 5.0 and a temperature of 50 °C. Thermodynamic parameters were also determined (Ea, ΔH°, ΔG°, and ΔS°). ACP activity was further studied in the presence of cysteine, DTT, SDS, EDTA, β-ME, Triton-X-100 H2O2, and PMSF. The enzyme had a Km of 0.167 mM and an Ea of 9 kcal/mol for p-nitrophenyl phosphate. The biochemical properties of the C. oxyacantha enzyme distinguish it from other plant acid phosphatases and give a basic understanding of ACP in C. oxyacantha. The results of this investigation also advance our knowledge about the biochemical significance of ACP in C. oxyacantha. Thermal stability over a wide pH and temperature range make it more suitable for use in harsh industrial environments. However, further structural and physiological studies are anticipated to completely comprehend its important aspects in oxyacantha species.

Simultaneous purification and characterization of detergent-stable, solvent-tolerant haloextremozymes protease and lipase from Haloferax sp. strain GUBF 2.

Industrial important proteases and lipases are in increasing demand for various biotechnological applications. In the present study, the concomitantly produced protease and lipase by Haloferax sp. strain GUBF 2 were simultaneously purified as a heterogeneous lipase (45 and 66kDa) and homogeneous protease (180kDa); with 28.3 and 31.36 fold purity, respectively using Sephadex G-200. The aforementioned extremozymes were active at pH 3-13, 20-80°C, 1-5M NaCl, with optimal activity at pH 6, 70°C, and 3M NaCl, thus exhibiting attributes of true haloextremozymes. The Km and Vmax of purified lipase were 3.47mM and 16.2U/mL, while protease were 3.29mg/mL and 28.5U/mL, respectively. FTIR bands corresponding to the vibrations of amide II and amide III were detected in haloextremozymes which could perhaps be used to determine the secondary structure of the purified proteins. Furthermore, the activity of both enzymes was stimulated by Ca2+ and inhibited by 10mM Hg2+ and phenylmethyl sulphonyl fluoride (PMSF). Additionally, these haloextremozymes are stable in the presence of detergent additives and organic solvents. In addition, purified protease displayed 74.3 ± 4.85% in-vitro blood clot dissolution activity. Conclusively this study revealed the key features, unusual properties, and possible biomedical applications of detergent-stable and organic solvent-tolerant haloextremozymes from Haloferax sp. strain GUBF 2 to date unexplored.

Purification, Characterization, and Application of Alkaline Protease Enzyme from a Locally Isolated Bacillus cereus Strain

Among the microbial enzymes protease and amylase are the most valuable enzymes which have been has diversified applications and used extensively because of their capabilities in the degradation of organic wastes, application in biofuels, agricultural, pharmaceuticals, chemical and biotechnological industries. The aim of the current research work was the purification, characterization and application of alkaline proteases extracted from Bacillus cereus AUST-7. Various concentrations of ammonium sulphate were applied for enzyme precipitation. Sephadex-G 100 was used in FPLC system for separation of protease from other proteins. SDS-PAGE was used to measure the molecular weight of required alkaline protease. Relative activities were determined against different pH, temperature, and incubation period to measure the enzymes activity. Stability of pH, temperature and various metal ions and inhibiter were also studied. Purified enzymes were applied on the goat skin to explore the dehairing efficacy. A 6.5 purification fold and 1163.50 U/mg of specific activity were obtained at 70% saturation and 35. 91 purification fold and 8902 U/mg of specific activity were observed after FPLC separation. The 35 kDa molecular size of protease enzyme was exhibited on the SDS-PAGE. The purified enzyme was stable at pH 10, temperature 55 °C and 35 min of incubation period. The purified enzyme was found to be stable at pH 8–11, thermo-stability at 50 °C and phenyl methyl sulphonyl fluoride (PMSF) and di-isopropyl fluorophosphates (DFP) inhibited the enzyme activity. The enzyme has good potential as dehairing agent in leather industries.

Application of Anoxybacillus gonensins UF7 lipase as a catalyst for biodiesel production from waste frying oils

This study was carried out to purify and characterize the lipase of locally isolated bacterium Anoxybacillus gonensis UF7 (GenBank accession: OM328064), and to investigate its applicability as a catalyst for biodiesel production. After ammonium sulphate precipitation and ion-exchange chromatography steps, the lipase of A. gonensis UF7 was purified with a specific activity of 1370 U/mg protein, a purification fold of 13.1 and a recovery yield of 40.65 %. The molecular weight of the enzyme was determined as 73 kDa by SDS-PAGE. The lipase showed maximum activity and stability at pH 8.0 and 60 °C. Its activity and stability were also high in the presence of methanol, ethanol, SDS, Tween-80, Triton X-100, Tween 80, Ca2+ and Mg2+. To prepare biodiesel from waste frying oils using purified lipase, the optimum reaction conditions were determined as a reaction time of 24 h, an enzyme concentration of 3 mL and an oil:methanol ratio of 3:1 (30 mL:10 mL). Transesterification yield was calculated as 87.7 %. The main fatty acid methyl esters of the biodiesel were methyl oleate (50.8 %), methyl palmitate (20.2 %) and methyl linoleate (16.6 %). The usability of lipases of Anoxybacillus strains including A. gonensins as a catalyst in biodiesel production was investigated for the first time in the present study.

Immobilization of Recombinant Endoglucanase (CelA) from Clostridium thermocellum on Modified Regenerated Cellulose Membrane

Cellulases are being widely employed in lignocellulosic biorefineries for the sustainable production of value-added bioproducts. However, the high production cost, sensitivity, and non-reusability of free cellulase enzymes impede their commercial applications. Enzyme immobilization seems to be a potential approach to address the aforesaid complications. The current study aims at the production of recombinant endoglucanase (CelA) originated from the cellulosome of Clostridium thermocellum in Escherichia coli (E. coli), followed by immobilization using modified regenerated cellulose (RC) membranes. The surface modification of RC membranes was performed in two different ways: one to generate the immobilized metal ion affinity membranes RC-EPI-IDA-Co2+ (IMAMs) for coordination coupling and another to develop aldehyde functional group membranes RC-EPI-DA-GA (AMs) for covalent bonding. For the preparation of IMAMs, cobalt ions expressed the highest affinity effect compared to other metal ions. Both enzyme-immobilized membranes exhibited better thermal stability and maintained an improved relative activity at higher temperatures (50–90 °C). In the storage analysis, 80% relative activity was retained after 15 days at 4 °C. Furthermore, the IMAM- and AM-immobilized CelA retained 63% and 53% relative activity, respectively, after being reused five times. As to the purification effect during immobilization, IMAMs showed a better purification fold of 3.19 than AMs. The IMAMs also displayed better kinetic parameters, with a higher Vmax of 15.57 U mg−1 and a lower Km of 36.14 mg mL−1, than those of AMs. The IMAMs were regenerated via treatment with stripping buffer and reloaded with enzymes and displayed almost 100% activity, the same as free enzymes, up to 5 cycles of regeneration.

Purification, characterization and thermodynamic analysis of cellulases produced from Thermomyces dupontii and its industrial applications

Cellulases involved in the hydrolysis of cellulose and plays a vital role in different industries like textile, detergent paper and Feed industry. Cellulases have been a prospective target for research by both the academic and industrial sectors because of the intricacy of the enzyme system and the enormous industrial potential. In the present work Thermomyces dupontii, which had previously been isolated and recorded as a promising cellulase producer were used. Both endoglucanases and betaglucosidases were purified to its homogeneity by ammonium sulfate followed by anion exchange and gel filtration chromatography. The recovery and purification fold for endoglucanases and betaglucosidases were 13.7, 10.7 % and 5.9, 2.7, respectively. The molecular weight of endoglucanases and betaglucosidases were estimated as 37 and 66 kDa on SDS-PAGE. Upon kinetic analysis the purified endoglucanases and betaglucosidases showed Km 0.63; 28.56 mg/ml and Vmax 82; 80 U/ml/min, respectively. Characterization revealed that enzyme was found to be acidophilic cellulase having optimal pH of 5.5 and 70 ⁰C. Furthermore, cellulases were accelerated in the presence of Ca2+ and EDTA. The cellulases had activation energy (Ea) of −44.55; −50.02 kJ/mol for carboxy-methyl-cellulose hydrolysis and Enthalpy (ΔH) 42.20; 47.70 kJ/mol and entropy ΔS −5.1 and −5.7 kJ/mol for EG and BGL, respectively. In addition to this the enzyme had a secondary structure of protein as represented by FTIR spectrum The current study suggested that purified cellulases can be used as a detergent additive to improve washing. Furthermore, it shows the biostoning ability when applied on jean fabric.

Isolation, production and optimisation of beta-galactosidase by utilizing yeasts isolated from selected dairy products

β-galactosidase is an enzyme that converts lactose into glucose and galactose. It alleviates the issue associated with intolerance to lactose and pollution caused as a result of milk by-products (whey). This study aimed to isolate and select yeast strains that can produce β-galactosidase from fresh milk, yoghurt and locally made cheese. A total of 115 yeasts were isolated from the samples, 9 yeast isolates had the ability to produce β-galactosidase and three (3) were selected for further analysis. β-galactosidase producers were screened using Ortho-nitrophenyl-β-D-galactopyranoside (ONPG) assay. Kluyveromyces marxianus strain SLDY – 005 produced the highest β-galactosidase. It showed maximum enzyme activity (277U/mL) at temperature 30oC, pH 5.5, after an incubation period of 36 h. Glucose had a decrease in β-galactosidase production while yeast extract and urea were considered appropriate nitrogen sources for the best enzyme synthesis. Crude β-galactosidase produced by Kluyveromyces marxianus SLDY – 005 was purified. The partially purified enzyme after dialysis showed a specific activity of 165.12 U/mL and had a purification fold of 6.02 and yield of 45.91 %. The purified enzyme had an optimum temperature of 40oC and a pH 6.0. Kluyveromyces marxianus strain isolated from local cheese is a potential candidate for the production of βgalactosidase and it could be used for combating the problem of lactose intolerance.

Characterization of novel bacteriocin PB2 and comprehensive detection of the pediocin gene ped-A1 from Pediococcus pentosaceus PB2 strain isolated from a sorghum-based fermented beverage in Nigeria

Lactic acid bacteria (LAB) have been known to possess bacteriocidal activity resulting from ribosomally synthesized antimicrobial peptides called bacteriocin. This study focused on the characterization of the bactericidal activity of bacteriocin PB2 and comprehensive detection of the pediocin ped-A1 from Pediococcus pentosaceus obtained from fermented sorghum beverage, Pito, in Nigeria against Escherichia coli ATCC 25922 and Listeria monocytogenes ATCC 15313. Bacteriocin PB2 was purified in a 2-step purification using 80% NH4 (SO4)2, and Carboxymethyl-Sephadex G-50 column chromatography to achieve a 12.62% purification fold. The physicochemical properties of purified bacteriocin were characterized being treated at different temperatures (20 – 120 °C), pH (2.0 – 10.0), with different detergents and enzymes (sodium dodecyl sulphate (SDS) urea, ox-gall, and proteinase K and RNase A), organic solvents (ethanol, phenol, acetone, chloroform and isoamyl alcohol), and exposure to ultraviolet (UV) radiation (2–12 h) respectively. The molecular weight of the bacteriocin PB2 was determined to be 4.87 kDa. The antibacterial activity of bacteriocin PB2 was optimum at 40 °C and pH 5.0. The bacteriocin PB2 lost its activity on treatment with proteinase K and exposure to UV radiation (after 6 h) but was observed to have stable activity in the presence of organic solvents. Also, P. pentosaceus PB2 harbored two plasmids, 0.9 and 1.2 kb which when cured resulted in the loss of the antimicrobial activity. The mRNA transcript for pedA was detected in P. pentosaceus PB2, but not in the cured derivative, confirming the expression of the plasmid ped-A1 gene in PB2. This study validates our previous study that the PB2 strain of Pediococcus pentosaceus isolated from fermented sorghum, Pito, may be used as a probiotic toward clinically important enteropathogenic bacteria. This peptide is a potential agent for use as an alternative antibacterial agent for the treatment of drug-resistant strains of bacterial infection.

EXTRACTION, PURIFICATION AND CHARACTERIZATION OF LIPASE FROM THE DIGESTIVE DUCT OF COMMON CARP CYPRINUS CARPIO L.

This study was aimed to use digestive duct of common carp Cyprinus Carpio L. as economic source for fish lipase extraction . The enzyme was extracted using potassium phosphate buffer (pH 7 , 0.2 M ). The crude extract then precipitated at the saturation range between 40 – 80 % and dialyzed . Further purified carried out through ion exchange chromatography using DEAE-Cellulose column (2.5 × 30 cm) and gel filtration chromatography by Sephadex G-100 column (1.5 x 60 cm). The purified lipase had a molecular weight of 37.150KDa ,and optimum pH and temperature for enzyme activity about 45◦C and 6.5 using olive oil as substrate .The specific activity, number of purification folds and yield of purified Lipase were 146.48U/mg, 9.76 and 40.50% respectively.

ASSESSEMENT OF PURIFIED COLLAGENASE INHIBITION ACTIVITY FROM STAPHYLLOCOCCUS AUREUS HN77 BY SOME LOCAL PLANTS EXTRACT

This study was aimed to inhibition of purified collagenase produced from S. aureus by local plants inhibitors (fruits and leaves). Sixty two local isolates of S. aureus which were isolated and identified by biochemical test subjected to the primary and secondary screening processes to select the active S. aureus isolate for collagenase production. Among these isolates, forty nine isolates with maximum zone of hydrolysis ability in primary screening (the ratio of Z/G more than 10 mm) were selected for secondary screening. It has been found that S. aureus HN77 had the highest productivity of the enzyme (49.122 U/mg protein). The selected isolate with highest level of collagenase activity was identified as S. aureus HN77 according to vitek test. The optimum conditions of collagenase production by selected isolate using submerged fermentation were obtained with medium (D) contain collagen as the best production medium, 2% starch as the best carbon source, 2% yeast extract/peptone as nitrogen source, temperature 37 °C and pH 7, after 48 hr. of incubation period, the specific activity was reached to1502.15 U/mg. The enzyme was purified by gel filtration chromatography using Sephadex G-150 then concentration by sucrose. The results shown an increase in final purification folds 6.1 time with an enzyme yield of 85.2%.The purified enzyme was exhibited maximal activity and stability at pH 7.0. The optimal temperature for purified enzyme activity was 45°C and it was stable until 45°C.The highest rate of enzyme specificity found with collagen. Collagenase was inhibited with local plants extract (fruit and leaves). Fruit of black fig extract inhibit 90% of collagenase activity while the inhibition efficiency of collagenase with grape leaf was reached to 12%.