Partial Purification Research Articles

Metagenomic discovery of lipases with predicted structural similarity to Candida antarcticalipase B.

Here we employed sequence-based and structure-based screening for prospecting lipases that have structural homolog to Candida antarctica lipase B (CalB). CalB, a widely used biocatalyst, was used as structural template reference because of its enzymatic properties. Structural homolog could aid in the discovery of novel wild-type enzymes with desirable features and serve as a scaffold for further biocatalyst design. The available metagenomic data isolated from various environments was leveraged as a source for bioprospecting. We identified two bacteria lipases that showed high structural similarity to CalB with <40% sequence identity. Partial purification was conducted. In comparison to CalB, the enzymatic characteristics of two potential lipases were examined. A candidate exhibited optimal pH of 8 and temperature of 50°C similar to CalB. The second lipase candidate demonstrated an optimal pH of 8 and a higher optimal temperature of 55°C. Notably, this candidate sustained considerable activity at extreme conditions, maintaining high activity at 70°C or pH 9, contrasting with the diminished activity of CalB under similar conditions. Further comprehensive experimentation is warranted to uncover and exploit these novel enzymatic properties for practical biotechnological purposes.

Production, partial purification and characterization of intracellular Azoreductase from bacterial isolates during biodecolorization of textile dye Acid Maroon V

Azoreductase is an important enzyme for the reduction of the azo linkage of dye during the biodecolorization process. In the present study, the production of intracellular azoreductase from bacterial cultures Enterobacter dissolvens AGYP1 (ED) and Pseudomonas aeruginosa AGYP2 (PA) was achieved during biodecolorization of synthetic textile dye Acid Maroon V. When compared with PA, the enzyme activity of ED was 2.31 times greater. The partial purification of azoreductase revealed specific activity of the dialyzed enzyme 51.72 U mg-1 protein (ED) and 33.06 U mg1 protein (PA). Methyl Red served as the best substrate for the azoreductase enzyme with superior activity at optimal pH 7 and temperature 30 °C. 75-80% of azoreductase enzyme activity was maintained when Mg+2 and Ca+2 were present. The azoreductase activity was considerably increased with NADH as the most suitable electron donor. The kinetic study showed consequent Michaelis-Menten constant (Km) and maximal velocity (Vmax) values of 50 μM and 2222 U ml-1 (ED) and 250 μM and 1000 U ml-1 (PA), respectively

Extracellular pectinase production from a novel Yarrowia phangngaensis XB3 grown on banana waste and its application in fruit juice clarification

Pectinases are important enzymes used in several industries, including food and beverage. Yarrowia phangngaensis, a yeast with pectinase-producing potentials, was investigated for its ability to produce copious pectinase using agro-wastes as substrates. Culture optimization studies were conducted, followed by partial purification by ethanol precipitation and application in apple juice clarification. The optimum pH and temperature for the pectinase activity were studied, and the suitable parameters for apple juice clarification were determined. Culture optimization remarkably influenced pectinase production by Yarrowia phangngaensis. Banana peels elicited the highest pectinase production compared to potato, pawpaw, orange and lemon peels. Yeast extract and ammonium sulphate proved best for pectinase synthesis. The optimum condition for pectinase production was 5% banana peel, pH 7.5, 1.4 × 104 cells ml−1 and incubation for 36 h. Culture optimization increased the pectinase yield by 182%. Ethanol precipitation (50%) resulted in a 5.31-fold increase in the specific activity of the pectinase. The optimum juice clarification was achieved by treating 10 ml apple juice with 7.03 IU ml−1 pectinase for 120 min. The turbidity and clarity reduction percentages were 39.33% and 59.84%, respectively. The pectinase from Yarrowia phangngaensis is applicable in food and other industries.

Stigmasterol- an Acetylcholinesterase Inhibitor from Phormidium retzii with relevance to Alzheimer's disease Therapy.

This study observed in vitro screening, purification and identification of cholinesterase inhibitors from the microalgae Phormidium retzii. Mixed microalgal culture was screened from freshwater samples for Phormidium sp. Single colony was purified and authenticated as P. retzii. Acetylcholinesterase (AChE) enzyme was purified from hRBC ghost. Sequential extraction of P. retzii was performed using organic solvents. Cholinesterase enzyme activity and its inhibition by various extracts were then tested. The active fractions were then subjected to partial purification and characterization. Petroleum ether extract of P. retzii showed maximum inhibition of 68.6 % against AChE while other solvent extracts showed no inhibition. Seven fractions were obtained from the active extract using thin layer chromatography. Among which fraction no. 5 showed maximum inhibition of 86.37 % towards AChE. Fraction no. 5 when subjected to GC-MS led to determination of the active principle as stigmasterol. The maximum inhibition of stigmasterol (0.45µM) was 81.2±0.08% with IC50 value of 0.214. Stigmasterol from P. retzii inhibited AChE projecting itself as safer drug for Alzheimer's disease with minimal side effects.

Foliar application of growth regulators and nutrients for enhanced yield attributes of guava cv Lalit

Azoreductase is an important enzyme for the reduction of the azo linkage of dye during the biodecolorization process. In the present study, the production of intracellular azoreductase from bacterial cultures Enterobacter dissolvens AGYP1 (ED) and Pseudomonas aeruginosa AGYP2 (PA) was achieved during biodecolorization of synthetic textile dye Acid Maroon V. When compared with PA, the enzyme activity of ED was 2.31 times greater. The partial purification of azoreductase revealed specific activity of the dialyzed enzyme 51.72 U mg-1 protein (ED) and 33.06 U mg1 protein (PA). Methyl Red served as the best substrate for the azoreductase enzyme with superior activity at optimal pH 7 and temperature 30 °C. 75-80% of azoreductase enzyme activity was maintained when Mg+2 and Ca+2 were present. The azoreductase activity was considerably increased with NADH as the most suitable electron donor. The kinetic study showed consequent Michaelis-Menten constant (Km) and maximal velocity (Vmax) values of 50 μM and 2222 U ml-1 (ED) and 250 μM and 1000 U ml-1 (PA), respectively

Partial Purification of Protease from Bovine Spleen

Partial Purification of Protease from Bovine Spleen

Extraction, Partial Purification and Utilization of Milk Coagulating Enzyme from Kiwifruit (Actinidia Deliciosa) in Fresh Cheesemaking

The study aimed to determine the potential of kiwifruit milk clotting enzyme in cheesemaking. The kiwifruit crude enzyme, extracted with sodium phosphate buffer (pH 7.0), was partially purified by 30-80% ammonium sulfate precipitation. 50% ammonium sulfate saturation exhibited maximum milk clotting activity (MCA), along with 1.56 purification fold, and 78.84% activity recovery. From SDS-PAGE analysis, the partially purified protease showed two bands with a molecular mass of 24 kDa and 23 kDa respectively. The optimum conditions (temperature and pH of milk) for a minimum time of coagulation (TOC) and maximum MCA were determined by response surface methodology (RSM). From the numerical optimization study, the optimum conditions for cheesemaking were pH 6.5 and temperature 55oC, having 0.94 desirability. The cheese prepared by kiwifruit protease had significantly (p&lt;0.05) higher moisture, ash, calcium content, and yield than rennet cheese, while significantly (p&lt;0.05) lower fat content and acidity were observed in kiwifruit protease coagulated cheese. However, a non-significant (p&gt;0.05) difference in protein content was obtained between both cheeses. This study highlighted that kiwifruit protease has the ability to be used as an efficient milk clotting enzyme in fresh cheesemaking.

Investigation of Pharmaceuticals in Sakarya Sewage Wastewater

Active substances of drugs can cause various adverse effects by accumulating in the ecosystem. Many medications are resistant to biodegradation, given the recipient media in conventional wastewater treatment plants, and are thus released into the environment after only partial purification or no purification at all. The study focuses on 13 different pharmaceutical compounds belonging to drug classes of anti-depressants, antiepileptic’s, anti-inflammatories, beta-blockers, lidocaine, and stimulants. These compounds were selected with reference to the literature as the ones most commonly encountered in domestic wastewater, surface, and groundwater. The presence of these compounds in the wastewater samples from Sakarya sewage and wastewater treatment plant was investigated. For this purpose, composite samples were taken at various sampling points, and duly analyzed. The analysis revealed the presence of the pharmaceutical residues in the sewage waters from Sakarya Municipality. Some of them were still present in the effluent of the treatment plant. On the other hand, fluoxetine, propranolol, and metoprolol drug active ingredients were not detected at any sampling point. Among all the compounds examined, the highest percentage of residues were observed in the case of active caffeine and paracetamol. In the light of these findings, advanced treatment units such as high-pressure membrane systems (including ozonizing, ultrafiltration, and reverse osmosis) can help adsorption rates at the treatment plant, increasing removal efficiency regarding drug compounds.

Partial Purification and Characterization of Polyphenol Oxidase Enzyme from Common-Morning Glory (Ipomoea purpurea)

This study aimed to purify and biochemically characterize polyphenol oxidase (PPO) enzyme from the plant Ipomoea purpurea (I. purpurea) for the first time. For this purpose, the crude extract sample obtained from the extraction of in vitro cultured plant leaves under optimum conditions (25 mgmL-1 Polyvinylpolypyrrolidone, pH 7.0) was subjected to three-phase partitioning, and the PPO enzyme was 10.5-fold purified with a 57% activity recovery. The optimum pH and temperature values were determined as 7.0 and 30°C, respectively. Laccase, peroxidase, and catechol oxidase activities were observed after activity staining of partially purified enzyme. From stability tests, it was noted that more than 75% and 65% of its original activity were maintained at temperatures 20℃-40℃ and pH 7.0-9.0, respectively.

Media Optimization and Partial Purification of Anti-Candida Compound of Probiotics Cultures

Fungal infections in humans are mostly due to Candida albicans, an opportunistic commensal yeast that causes systemic and invasive candidiasis. Conventional treatment of candidiasis using antifungal compounds such as polyenes and azoles can cause various side effects. Besides, treatment becomes difficult as the organism acquires resistance. Probiotics with anti-candida activity have been projected as a safe and alternative solutions to treat candidiasis. Probiotic Bacillus isolates previously reported showed wonderful activity against pathogenic Candida species. This study reports on media optimization and partial purification of anti-candida compounds from probiotic Bacillus isolates MW3, MW9, MW19, and MW27. Maximum growth and anti-candida activity (2.9 cm inhibition zone) by the isolate culture was observed when media had been optimized with dextrose, casein and peptone. The media composition numbers 18, 4 and 19 showed the best growth for MW3, MW9, MW19 and MW27. Fractions obtained by solvent precipitation using acetone and methanol showed activity against the pathogen, and the fractions were further purified by ultra-filtration. After ultra-filtration, it was found that the size of the active compound was between 10 to 30 kDa. The compound lost its activity after treatment with proteinase K.

Biological Study of protease produced by clinical isolates of Staphylococcus aureus

The bacterial isolates were obtained from Al-Kindi Hospital were diagnosed by the Vitek-2 system and re confirm by 16srRNA gene as S. aurous, the results were shown 20 isolates (66.7%) out of 30 isolates were positive to protease production. All bacterial isolates (100%) were sensitive to Gentamicin and Levofloxacin. but resistant (100%) to aztreonam. The best temperature for enzyme production from bacteria was 37 °C, and the best pH for enzyme production was 7. Partial purification of the bacterial enzyme (protease) was carried out using short steps included ammonium sulfate 65% saturation, ion exchange using DEAE- cellulose column and then applied on gel filtration chromatography using Sephadex G-200 column. The enzymatic activity was determined for each purification step. The specific fold and yield of the purified enzyme were 5.91 and 38.3 % respectively. The molecular weight of the purified enzyme was 37 kDa , it was determined by SDS-PAGE. After being exposed to high concentrations of the protease enzyme (800-1000 µg/ml), the proliferation of a breast cancer cell line (MCF-7) was seen to be suppressed, however the inhibitory effect gradually diminished as the concentration of the enzyme decreased. 200–400 µg/ml is the recommended concentration.

Optimization, Partial Purification, and Characterization of Bioactive Peptides of Lactobacillus paracasei Isolated from Traditional Egyptian Cheese

Background: Bacteriocins are small peptides which are ribosomally synthesized and have been shown to have wide range of antimicrobial activity. The aim of this study was to optimize the production of L. paracasei MG847589 bacteriocin. Furthermore, the potential antibacterial properties of the novel bacteriocins were characterized and evaluated against Staphylococcus aureus. Methods: The present study optimized the growth media constituents of Lactobacillus paracasei MG847589 to improve bacteriocin yield by applying One-Factor-at-a-Time (OFAT) and Response Surface Methodology (RSM) methods. Results: At OFAT, two-fold activity increased against Staphylococcus aureus in the presence of whey (22.5 g/L) as nitrogen source and sucrose (30 g/L) as carbon source. RSM tool was performed with media compounds using design expert 12.0.1.0. Whey (22.5 g/L), sucrose (30 g/L), temperature (30 ºC), and pH (6.5) condition yielded 25,600 AU/ml of bacteriocin against S. aureus. Bacteriocin was stable at pH range of 2.0 to 8.0 for one h and at 60 ºC for 15 min. The produced antimicrobial peptide is a novel bacteriocin with molecular mass of 2,611.122 Da. Conclusion: Bacteriocin of L. paracasei MG847589 isolated from traditional Egyptian cheese (Kareish) showed great antimicrobial activity and could be applied as food preservative in food manufacturing.

Secondary metabolites produced by endophytic fungi, Alternaria alternata, as potential inhibitors of the human immunodeficiency virus.

Antiretroviral treatment has significantly reduced human immunodeficiency virus infection and mortality. However, the current treatment regimen is limited by adverse side effects, the emergence of drug resistance, and the inability to eliminate viral reservoirs. Here, fifteen endophytic fungi were isolated from Sclerocarya birrea and Hypoxis plants. Crude extracts of Alternaria alternata (strain ID PO4PR1, PO4PR2, and PO2PL1) of the fifteen isolate's crude extracts showed anti-HIV-1 activity in TZM-bl cell line at inhibitory concentration (IC50) values ranging from 0.017 to 1.170μg/ml. The three crude extracts also maintained the virus replication inhibition profile on PBMCs and CD4+ T cells at concentrations ranging from 0.3 to 50.2ng/ml. Partial purification using the solid phase extraction and analysis with Gas Chromatography-Mass spectrophotometry showed a diverse profile. The bioactive compounds were identified based on peak area, retention time, similarity index. The major compounds from GC-MS analysis of A. Alternata revealed the existence of cyclotrisiloxane octamethyl (22.92%); Propaninitrile (16,67%); Pyrrolol[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methyl propyl) (10.42%); Silane, diethylethoxy(2-ethoxyethyloxy) (4.17%); Coumarin, 3,4-dihydro-4,5,7-trimethyl- 4,5,7-Trimethyl-2-chromanone (13.7%) and 1,2-Cyclobutanedicarbonitrile (2.08%) with previously reported biological activities such as antimicrobial, anti-inflammatory and antioxidant properties. Therefore, these bioactive compounds from A. alternata fungal endophytes could be repurposed as potential anti-HIV agents. This study showed the potential of endophytic fungi, Alternaria alternata from S. birrea, and Hypoxis species as producers of anti-HIV compounds.

Sequential extraction process and physicochemical characterization of R-phycoerythrin and agar from red macroalgae Gracilaria birdiae

This study evaluated the sequential extraction of R-phycoerythrin (R-PE) and agar from the red macroalgae Gracilaria birdiae and the effects on yield, recovery, purity index and physicochemical characterization of both biomolecules. Three R-PE extraction and partial purification strategies were evaluated and the three most promising pigmented fractions were F25/35, F35/45 and F25/45 with purity/yield indexes 1.64/0.17, 0.71/0.44 and 0.56/ 1.20 mg.g−1 dw, respectively. In addition to the purity index, yield and recovery, optimizations 1 and 2 resulted in obtaining pigments with high intensity of red color, confirmed by the values of parameter a*: 78.30 ± 0.01, 64.07 ± 0.03 and 60.96 ± 0.02, for fractions 25/35, 35/45 and 25/45, in that order. The agar obtained from the residue of the extraction of R-PE (ARES-Gb) showed a yield of 31.64 %, higher than those reported in the literature when extracted from integral wet or dry macroalgae. In addition, RES-Gb had physicochemical and rheological properties similar to commercial agar. Therefore, the development of the biotechnological process proposed contributes to the maximum use of G. birdiae, to the valorization of this renewable source and to the blue economy.

Partial purification and characterization of keratinase produced by keratinolytic fungi, Earliella scabrosa A2 and Aspergillus flavus A11

Chicken feather waste originating from the poultry industry and its processing is a potential source of protein and amino acids that can still be used as functional compounds. This study aimed to study the keratinolytic activity of local fungal isolates in vitro as a keratin-degrading agent. Keratinase secreted by keratinolytic fungal isolates were produced and characterized in 1% (w/v) Feather Meal Broth (FMB) composed of pure chicken feathers. Fungal isolates used in this study were A2 and A11 which were later identified based on ITS-rDNA genetic similarity to Earliella scabrosa and Aspergillus flavus, respectively. Crude enzymes were precipitated using an increasing ammonium sulphate salt gradient. The enzyme activity of Earliella scabrosa A2 and Aspergillus flavus A11 were 19 and 7.5 U/mL respectively. The crude enzymes showed an increase of activity during 80% of precipitation for isolate A2 with 10 U/mL and for isolate A11 with 18.6 U/mL. The specific activity after dialysis were 61.99 U/mg for isolate A2 and 75.11 U/mg for isolate A11. The optimum condition of keratinase activity for isolate A2 were at pH 7 and 30°C while isolate A11 showed its optimum activity at pH 8 and 45°C.

A novel cyclic dipeptide from Lactiplantibacillus plantarum MC39 inhibits proliferation of multidrug-resistant Klebsiella pneumoniae W8 and Enterobacter hormaechei U25

The present study pays special attention to lactic acid bacteria products, cyclic dipeptides (CDPs), as an effective prevention strategy that can be used to eradicate the threats posed by multidrug-resistant (MDR) Enterobacteriaceae. Herein, Lactiplantibacillus plantarum MC39, a strain isolated from an artisanal Egyptian cheese, was a producer of a novel CDP that could strongly inhibit, in vitro, MDR Klebsiella pneumoniae W8 and Enterobacter hormaechei U25, for the first time. Following partial purification with 80% ammonium sulphate precipitation and Sephadex G-100 chromatography, this biomolecule was identified as the cyclic dipeptide cyclo(Ala–Glu) using IR, HNMR, and LC–MS. Cyclo(Ala–Glu) was thermostable up to 121 °C, insensitive to pepsin and α-chymotrypsin, tolerant to organic solvents, active at acidic pH, and stable for 45 days of storage. Thus, it shows clear evidence to be usable as a bio-preservative in dairy products and as a potential therapeutic agent, controlling MDR K. pneumoniae and E. hormaechei.

Study of some Biochemical Parameters and Partial Purification of amylase from diabetic patients

The aim of the study was to evaluate the level of amylase in the serum of diabetic patients compared to the level of healthy controls, in glucose, lipids, lipoproteins, thyroid hormones, electrolytes (calcium, potassium, and sodium) and transporter enzymes were also studied. (AST), (ALT),(ALP). the amylase enzyme was purified and the association between the enzyme and the previously mentioned variants was studied. it was studied amylase and other biochemical variables and it was found that there was a significant positive correlation when comparing the enzyme level with the variants (TG, VLDL, ALP) and there was no significant correlation. between enzyme activity and other biochemical variables. Where one peak was obtained in the fourth part of the purification, the degree of purification was 16.1; the result of the enzyme was 108.2% and the specific activity (0.189 ng/ mg, kinetic studies of the partially purified enzyme. The substrat concentration was 10 ng/mg, and the value of Michaelis Menten’s constant km (5.55 ng) and maximum velocity Vmax was (0.98 ng/ml) the optimum temperature of the enzyme were reached (37 °C) while the optimum pH was (7.5).

Screening of peroxidase from various vegetables sources, partial purification, some biochemical properties and its antimicrobial activity

The peroxidase enzyme was extracted from four different vegetables which were radish, turnip, cabbage and green pepper. The extracted enzymes were partially purified by ammonium sulphate and DEAE cellulose (Diethylaminoethyl cellulose) chromatography. The effect of amount of guaiacol, hydrogen peroxide, pH and temperature were investigated. The optimum pH for peroxidase activity was in the range (6.5-7.0), while optimum temperature for extracted enzymes was 30⁰c. The extracted enzyme from selected vegetables was tested for their antimicrobial activity against different species of bacteria. The enzymes show weak antibacterial activity towards the tested species of bacteria.

Mycobacterial MenG: Partial Purification, Characterization, and Inhibition.

Menaquinone (MK) is an essential component of the electron transport chain (ETC) in the gram-variable Mycobacterium tuberculosis and many Gram-positive pathogens. Three genes in the M. tuberculosis genome were annotated as methyltransferases involved in lipoquinone synthesis in mycobacteria. Heterologous expression of Rv0558 complemented an ubiE (the quinone C-methyltransferase involved in ubiquinone and menaquinone synthesis) deletion in Escherichia coli, and expression in a wild-type E. coli strain increased quinone C-methyltransferase specific activity by threefold. Rv0558 encodes a canonical C-methyltransferase or, more specifically, a S-adenosylmethionine/demethylmenaquinol methyltransferase. Partially purified recombinant protein catalyzed the formation of MK from demethylmenaquinone (DMK), although the activity of the recombinant protein was low and appeared to require a cofactor or intact membrane structure for activity. Membrane preparations from irradiated M. tuberculosis also showed poor activity; however, membrane preparations from wild-type Mycobacterium smegmatis showed robust, substrate-dependent activity. The apparent Km values for demethylmenaquinone and SAM were 14 ± 5.0 and 17 ± 7.0 μM, respectively. Interestingly, addition of dithiothreitol, dithionite, NADH, or other substrates of primary dehydrogenases to reaction mixtures containing membrane preparations stimulated the activity. Thus, these observations strongly suggest that demethylmenaquinol is the actual substrate of MenG. Ro 48-8071, previously reported to inhibit mycobacterial MK synthesis and growth, inhibited Rv0558 activity with an IC50 value of 5.1 ± 0.5 μM, and DG70 (GSK1733953A), first described as a respiration inhibitor in M. tuberculosis, inhibits MenG activity with an IC50 value of 2.6 ± 0.6 μM.

Isolation, Characterization, and Effect on Biofilm Formation of Bacteriocin Produced by Lactococcus lactis F01 Isolated from Cyprinus carpio and Application for Biopreservation of Fish Sausage.

The aim of this work was the screening of bacteriocin-producing LABs isolated from fish, the selection of promising/prominent strain(s), the characterization of the bacteriocin produced, and the evaluation of its potential to be used as biopreservative(s). Amplification and sequencing of the 16S rRNA gene of the bacteriocin-producing strain was performed. Then a partial purification of the produced bacteriocin, using a combination of ammonium sulfate and chloroform-methanol precipitation, was done. Its molecular weight was determined by SDS-PAGE. In addition, the action spectrum, the hemolysis test, and its ability to inhibit biofilm formation were analyzed. A total of 88 isolates of lactic acid bacteria (LAB) including one bacteriocin producer, which was identified as Lactococcus lactis F01, were collected. The bacteriocin was partially purified with an estimated yield of 40%. Regarding the SDS-PAGE profile, the secreted bacteriocin has molecular weight of about 3.5 kDa and was identified as class I bacteriocin. The antimicrobial test showed that the bacteriocin inhibits pathogenic and/or spoilage bacteria, 10 Gram-positive and 16 Gram-negative bacterial species. Moreover, it can inhibit biofilm formation from 1.3% (Escherichia coli) to 63.92% (Pseudomonas aeruginosa ATCC15692) depending on the strain. The hemolytic activity of novel bacteriocin was observed at the concentration of 10 μg/ml of bacteriocin crude extract, which was 0.7 ± 0.0029%. In addition, it exhibited good thermal and pH stability with retained antibacterial activity of 85.25% after treatment at 121°C for 20 min, as well as at a pH range between 2.0 and 10.0. Moreover, this bacteriocin showed the ability to inhibit the growth of bacterial culture load in fish sausage stored at 8°C for 28 days. Considering the results obtained, bacteriocin could be potentially exploited as an alternative to chemical preservatives or as a substitute for antibiotics.