Sephadex G-100 Gel Filtration Research Articles

Isolation, Identification and in-Silico Characterization of Bioactive Peptide from the Venom Sac of Conus inscriptus

Nature has been the source of new medications since ancient times, and at least half of all commercialised pharmaceuticals have been made from renewable resources to treat various illnesses, including pain, inflammation, and infection. In recent years, interest has risen in bioprospecting of new bioactive peptides, especially for medical and health-related applications. Therefore, the production of functional meals is starting to get recognition for its potential to enhance quality of life when combined with a healthy lifestyle. In view of this, the current work aimed to isolate, purify, and identify bioactive peptides from Conus inscriptus venom sac protein extracts, with an emphasis on antioxidant and anti-inflammatory properties. The findings reveal that the DPPH radical scavenging activity, albumin denaturation, and HRBC membrane stabilisation inhibition activities of Conus inscriptus venom sac crude protein extracts are 23.76±2.4, 66.23±3.1, and 57.32±2.6% respectively. Secondary screening of purified protein fractions obtained through sephadex G100 gel filtration exhibited increased anti-inflammatory efficacy from fractions 19 to 23. Electrophoresis and PMF analysis revealed that the purified anti-inflammatory peptide was homogenous with a molecular weight of 12 KDa, and it was identified as a conotoxin MI15b precursor. The 3D model of conotoxin exhibits the highest similarity with template c6nk9A with a confidence of 54.1%, and its function was predicted as a potassium ion channel inhibitor. The findings of the Insilco characterisation of the conotoxin MI15b precursor concluded that this protein might serve as an anti-inflammatory agent and may be responsible for therapeutic actions in the medical management of many inflammatory-related disorders.

Purification and characterization of L-asparaginase produced from Bacillus sp.

The objective of this study was to isolate and identify the asparaginase-producing bacteria, then purify and characterize the enzyme in order to investigate their properties in the future. Fifteen local bacterial isolates were isolated from various sites in the city of Baghdad, identified by conventional morphological and biochemical procedures, and confirmed using vitek 2 methods, and submitted to primary screening processes for asparaginase production. For secondary screening, eight isolates with the greatest yellow zone ability on a specific solid medium were chosen. Bacillus sp. was reported to have the highest enzyme production (7.5 U/mg proteins). After 24 hours of incubation, submerged fermentation yielded optimal conditions for the production of L-asparaginase (L-ASNase) by the chosen isolate, with medium (2) serving as the optimal medium for production and fructose serving as the optimal source of carbon. In pH 6 at 40°C, Sephadex G-150 gel filtration chromatography was used to purify the enzyme. The final purification folds were increased by 2.5 times, resulting in an enzyme yield of 93.7%. It also showed the highest purified enzyme activity and stability was at 37°C. Also it revealed the highest activity and stability at pH 7.0 and pH 8.0 respectively. Enzyme lost activity when exposed to several metallic ions at concentrations of 1, 5, and 10 mM.

Characterization of lipases and preliminary purification of tributyrinesterases from lactic acid bacteria strains belonging to the Enterococcus and Lactococcus genera

The objective of this study is to characterize and attempt to purify lipolytic enzymes derived from strains of Enterococcus and Lactococcus genera isolated from various environments. For this purpose, ten strains of lactic acid bacteria were studied for their lipolytic and esterasic activities on an MRS (Man, Rogosa, and Sharpe) medium containing natural and/or artificial lipid substrates. Among them, two strains: Lactococcus lactis subsp lactis and Lactococcus lactis subsp cremoris showed maximum extracellular lipolytic activity in the presence of 1% olive oil. It was observed that both strains exhibited higher lipolytic activity at pH 7 and 8, with an optimal temperature of 30 °C and 37 °C, respectively. Glucose was found to be the best carbon source for both tested strains. The study of growth kinetics and fatty acid production over time revealed that fatty acid production begins during the exponential phase and reaches its maximum during the stationary phase. The purification of tributyrinesterases was carried out on two strains of Enterococcus genus: Enterococcus feacium and Enterococcus durans through ammonium sulfate precipitation and Sephadex G-100 gel filtration chromatography allowed the estimation of the molecular weight of the tributyrinesterases as 32.09 kDa and 38.49 kDa for both strains, respectively.

Bioprospecting of a Thermostable L-Methioninase from Alcaligenes aquatilis BJ-1 in Agro-Industrial Waste

L-methioninase is an enzyme that has recently gained significant interest in the scientific community because of its potential as a targeted therapy for cancer. This study aims to isolate and identify extremophilic bacteria that could produce L-methioninase and to access the enzymatic potential of isolated bacteria under stress conditions, specifically in agro-industrial waste. In this study, a rare marine bacterium, Alcaligenes aquatilis BJ-1, exhibited the highest specific activity of 4.61 U/mg at an optimum pH of 8.3. The L-methioninase was purified 4.3-fold and 7.15-fold by acetone precipitation and Sephadex G-100 gel filtration chromatography, which revealed a molecular weight of 46 kDa. In addition, agriculture waste materials such as cottonseed oil cake had the highest L-methioninase production. Moreover, A. aquatilis BJ-1 can tolerate and produce enzymes in the presence of 10% NaCl, 6% KCl, and 4% MgSO4. Similarly, substrates such as L-asparagine, L-glutamine, L-alanine, and L-tyrosine were found suitable to increase enzyme production. The strain produced L-methioninase in the presence of various heavy metals. Maximum enzyme activity was found in Zn2+ at 0.1% (2.52 U/mL), Li2+ at 0.03% (2.90 U/mL), and Ni2+ at 0.01% (2.78 U/mL), as compared to the control (2.23 U/mL) without metal. Enzyme production was also observed at a high temperature (60 °C), with the produced enzymes possessing antioxidant properties. In addition, no hemolytic activity was observed. The results indicate that A. aquatilis BJ-1 is an appropriate bacterium for metal bioremediation procedures in unfavorable circumstances.

Exploring the Antimicrobial Potential and Biofilm Inhibitory Properties of Hemocyanin from Hemifusus pugilinus (Born, 1778).

The seafood industry plays a huge role in the blue economy, exploiting the advantage of the enriched protein content of marine organisms such as shrimps and molluscs, which are cultured in aquafarms. Diseases greatly affect these aquatic organisms in culture and, hence, there is need to study, in detail, their innate immune mechanisms. Hemocyanin is a non-specific innate defense molecule present in the blood cells of several invertebrates, especially molluscs, arthropods, and annelids. It is concerned with oxygen transport, blood clotting, and immune enhancement. In the present study, this macromolecular metalloprotein was isolated from the hemolymph of the marine snail Hemifusus pugilinus (Born, 1778) using Sephadex G-100 gel filtration column chromatography. It occurred as a single band (MW 80 kDa) on SDS-PAGE. High-performance liquid chromatography (HPLC) of the purified hemocyanin showed a single peak with a retention time of 4.3 min. The secondary structure and stability of the protein were detected using circular dichroism (CD), and the spectra demonstrated negative ellipticity bands close to 208 nm and 225 nm, indicating β-sheets. Further exploration of the purified hemocyanin revealed remarkable antimicrobial and antibiofilm activities against Gram-positive (Enterococcus faecalis and Staphylococcus aureus) and Gram-negative bacteria (Pseudomonas aeruginosa and Proteus vulgaris) at a concentration of 1-5 μg/mL. Spectrophotometric and in situ microscopic analyses (CLSM) unveiled the potential of the purified hemocyanin to inhibit biofilm formation in these bacteria with a minimal inhibitory concentration of 40 μg/mL. Furthermore, H. pugilinus hemocyanin (10 μg/mL concentration) displayed antifungal activity against Aspergillus niger. The purified hemocyanin was also assessed for cytotoxicity against human cancer cells using cell viability assays. Altogether, the present study shows that molluscan hemocyanin is a potential antimicrobial, antibiofilm, antifungal, anticancer, and immunomodulatory agent, with great scope for application in the enhancement of the immune system of molluscs, thereby facilitating their aquaculture.

Extracellular β-d-fructofuranosidase from a novel Aspergillus sp. DHE1 with high potential for biotechnological applications: Purification and biochemical characterization

The current work focused on the purification and biochemical characterization of an extracellular β-d-fructofuranosidase from a novel fungus identified genetically using 18 S rRNA molecular sequencing, as Aspergillus sp. DHE1. The fungus was cultured under solid state fermentation using the Cotton (Gossypium herbaceum) seed waste as substrate. β-d-fructofuranosidase was purified through fractionation by ammonium sulphate salt, ion-exchange chromatography on DEAE-cellulose, and Sephadex G-100 gel filtration, yielding a 13.3-purification fold, 22.6% recovery and 192.9 U/mg protein specific activity. The apparent molecular mass of Aspergillus sp. DHE1 β-d-fructofuranosidase was 78 kDa as estimated on sodium dodecyl sulphate polyacrylamide gel electrophoresis. Optimal pH and temperature were observed at 6.0 and 50 °C, respectively, and the enzyme was quite thermostable at neutral/alkalophilic conditions, retaining 72% of its original activity after 1 h of incubation at 60 °C. The activity of the purified Aspergillus sp. DHE1 β-d-fructofuranosidase was stimulated by 1 mM of KCl (138.5%), NaCl (125.9%), CoCl2 (121.2%), and CaCl2 (112.3%), whereas the same concentration of AgCl, FeCl3, HgCl2, and CuCl2 inhibited the activity by 48.7%, 30.5%, 29.9%, and 26.2%, respectively. The kinetic studies using d-sucrose as the substrate were investigated using a Lineweaver-Burk plot, with Km and Vmax of 0.85 mM and 47.62 U/mL, respectively, indicating the high substrate affinity of β-d-fructofuranosidase. The ability of Aspergillus sp. DHE1 β-d-fructofuranosidase to tolerate ethanol suggests that it may have numerous uses in the food industry, including the production of alcoholic beverages.

Extraction and Purification of Pullulanase from Local Mushrooms

Pullulanase is defined as an extracellular carbohydrase enzyme responsible for the hydrolysis of pullulan into maltotriose. Pullulanase can be extracted from Agaricus bisporus edible mushroom and concentrated by dialysis tube using Tris-HCl buffer. This enzyme was purified by different ratios of ammonium sulphate; the optimal ratio was 30%. It can be purified by ion exchange using a DEAE-cellulose column with a final yield of 5.25 and a purification fold of 4.38. Following Sephadex G-100 gel filtration with a yield of 2.60 and purification fold of 5.0, the total activity of the enzyme reached 160IU and the specific activity was 14.5 U/mg. This study was concerned with estimating the optimum pH and temperature of pullulanase, and it recorded an optimal pH of 7, and the optimal temperature was 60°C. The enzyme was more stable at pH 8 and 70°C; the incubation period was also determined, and it appeared the most appropriate period was 30 minutes. CaCl2 and ZnCl2 were activator metal ions. Mercaptoethanol in different concentrations was the greatest inhibitor of pullulanase. One of the problems arising in the surrounding area is that much of the environmental pollution consists of starchy food waste. Pullulanase can degrade the glycosidic linkage of pullulan or starch present in the starch waste. The objective of this study is to produce enzymes like pullulanases to participate in minimizing environmental pollution through the degradation of starch waste as a biotechnological application. The conclusion of this study is that pullulanase can be produced from readily available, safe, and low-cost sources such as mushrooms.

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.

The Tannase from red yeast Rhodotorula glutinis: purification and characterization

The tannase enzyme was successfully purified to homogeneity from the culture broth of red yeast strain Rhodotorula glutinis DB2 in a three-tandem step involving ultrafiltration (5 kDa and 100 kDa systems), Sephadex G-200 gel filtration chromatography, and DEAE Sepharose CL-4B anion exchange chromatography. The purified tannase appeared to be homogeneous on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). The purified tannase had a specific activity of 3.33 U mg−1, with a 1.3% recovery and overall purification of 302-fold. The molecular mass of the tannase estimated by SDS-PAGE was about 73 kDa. The tannase had an optimum pH of 6.0 and an optimum temperature of 40 °C. The most stable pH was 7.0, and the enzyme was stable up to 40 °C. One mmol L−1 of Fe3+, Sr2+, Na+, and Pb2+ were found to promote tannase activity, whilst 1.0 mmol L−1 of Ba2+, Ca2+, Mg2+, Zn2+, Hg+, Ag+, Co2+, Fe2+, Mn2+, Cu2+, Cd2+, Al3+, K+, Ni2+, and Li+ inhibited tannase activity.

Production and biochemical characterization of partially purified cellulase-free, thermo-acidophilic endoxylanase from Lysinibacillus fusiformis strain TB7 using kolanut husk as feedstock

Xylanases have become very important enzymes in many industrial processes for the valorization of xylan-rich lignocellulosic wastes. Here, some physicochemical and kinetic properties of a purified endoxylanase produced on kolanut husk-based medium by Lysinibacillus fusiformis are presented. The crude enzyme solution was first subjected to precipitation with solid ammonium sulphate and further purified on DEAE-Sephadex A-50 anion-exchange and Sephadex G-100 gel filtration columns chromatography prior to biochemical characterization. The purified endoxylanase was 21 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and was thermostable, exhibiting optimum activity at 60 °C and pH 5.0. The Km and Vmax were respectively estimated to be 29.5 mg/ml and 125 μmol/min/ml using Birchwood xylan as substrate. Activity of the enzyme was enhanced by Na+, Ca2+, Mn2+, Mg2+ and K+ at concentration of 5 mM but inhibited by Hg2+, Cu2+, Pb2+, Fe3+, EDTA, SDS and Urea. The purified endoxylanase showed high hydrolytic activity on Birchwood xylan and kolanut husk but extremely poor or no activity on carboxymethyl cellulose, starch or pectin. This L. fusiformis strain TB7 endoxylanase has desirable properties useful for biotechnological applications in laundry, fuels, feeds, paper and pulp industries.

Antigenic components, identification, and characterization of whole worm extract of Platynosomum illiciens

The antigenic components of adult Platynosomum illiciens were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from cats naturally infected with P. illiciens, Dipylidium caninum, Toxocara cati and uninfected cat sera. The whole worm extract (WWE) of P. illiciens was fractionated by Sephadex G-200 gel filtration chromatography. The results showed that WWE fraction and F2 were highly antigenic as well as F1 and F3, which were moderately antigenic. For SDS-PAGE and immunoblotting, the antigenic molecules of WWE and all three fractions were mostly at molecular weights (MW) ranging from 11 to 150 kDa. Four antigenic proteins of 11, 18, 27 and 75 kDa detected in WWE and F1-F3 were found to give a reaction with sera from P. illiciens infected cats, and these proteins were also identified using liquid chromatography-mass spectrometry (LC-MS/MS). For immunolocalization observation, it was revealed that the P. illiciens antigen was present in high concentration in the cytoplasm of vitelline cells in the vitelline glands, the shell of the eggs and the eggs within the uterus, but not in other organs, i.e., tegument, muscle, parenchymal cells, testes and oral and ventral suckers of adult fluke. This finding indicates that these proteins may be potential antigen candidates for the immunodiagnosis of feline platynosomosis caused by P. illiciens.

A glycoprotein from mountain cultivated ginseng: Insights into their chemical characteristics and intracellular antioxidant activity

A glycoprotein (MGP2) from mountain-cultivated ginseng (MCG) was purified by Tris-HCl extraction followed by DEAE-52 ion exchange chromatography and Sephadex G-100 gel filtration chromatography. The approximate molecular weight (27.0 kDa) and monomeric nature were determined by reduced and non-reduced SDS-PAGE. The structure of MGP2 was characterized by a practical and reliable “protein-polysaccharide analyzed by spectroscopy combined with chemical analysis” strategy. The results showed that MGP2 belonged to Arabinogalactan proteins (AGPs) which contained high amount of Glc (35.1 %). The hemagglutination test concluded that MGP2 was not a lectin. In addition, the MGP2 exhibited antioxidant activity by scavenging radical capacity tests and the ability to protect human erythrocytes and RAW264.7 cells from oxidative damage induced by AAPH. Therefore, these results suggested that glycoprotein MGP2 could be used as a natural antioxidant in drug and food industry.

Microbiological degradation of food dyes and amylase production, immobilization by Iraqi isolate Anoxybacillus rupiensis strain Ir3 (JQ912241)

Amylases are employed in the starch processing industries for the hydrolysis of polysaccharides such as starch into simple sugar constituents. With the advent of new frontiers in biotechnology, the spectrum of amylase applications has expanded into many new fields such as clinical, medicinal, and analytical chemistry. Α-Amylase from Anoxybacillus rupiensis strain Ir3 (JQ912241) isolated from hydrocarbon soil of Iraq was purified using ammonium sulphate precipitation and Sephadex G-200 gel filtration chromatography. Partially purified enzyme with 189U/ml activity was used for immobilization study by sodium alginate and Agar, The immobilized enzyme was very effective increase the activity of the enzyme to be (10.7, 14.8 U/ml) respectively for the sodium alginate and agar. This immobilized enzyme can be used commercially as a replacement of free enzyme because it has shown greater operational flexibility and higher enzymatic activity. The A. rupiensis strain Ir3 shows high decolorization (99%) of food dyes in the concentration 0.25 mg/ml for three days while (98%) decolorization the concentration 0.5 mg/ml for ten days.

Structural and bioactive characterization of purified polysaccharide from deep-freeze pretreated Sorghum bicolor L. leaf sheath

This study determined the effect of deep-freeze pretreatment of sorghum leaf sheath on the extraction yield of ultrasound-extracted purified polysaccharides (DFUP-1), and confirmed the physicochemical, structural and bioactive characteristics of DFUP-1. Deep-freeze pretreatment induced breaks in the cell walls and resulted in a higher yield (14.35%) of crude polysaccharide extract than without pretreatment (9.23%). The crude extract was purified using DEAE-52 cellulose ion exchange and Sephadex G-100 gel-filtration chromatography to obtain DFUP-1. The 1H and 13C NMR spectra of DFUP-1 showed the presence of β-glycosidic linkages and a substitution at C6 of the sugars which may be O-methyl substitution or due to β (1−6) linkages. Peaks for α-anomeric linkages were not detected. The FTIR spectrum of DFUP-1 confirmed its carbohydrate functional groups. Periodate oxidation and formic acid analysis of DFUP-1 showed 1-, 1–6, 1–2, 1–3 and 1–4 linkages. GC-MS analysis of monosaccharide residues of DFUP-1 showed presence of glucose, galactose, arabinose, mannose and xylose. Congo red assay showed that DFUP-1 was not of triple-helix configuration. The ABTS+ and DPPH radical scavenging assay showed an IC50 of 0.10 mg/mL and 1.19 mg/mL for DFUP-1 respectively. At 0.2 mg/mL DFUP-1 showed anti-inflammatory (anti-egg denaturation) activity of 41.52%. The DFUP-1 sample showed lower inhibitory effect against proliferation of leukemia cells at an IC50 of 37.43 µg/mL compared to curcumin with IC50 of 0.98 µg/mL. Taken together, deep-freeze pretreatment coupled with ultrasound extraction increases yield of sorghum leaf sheath polysaccharides with potential nutraceutical and pharmaceutical applications.

Evaluation of Antioxidant and Antibacterial Activities of the Polysaccharides from Mycelium and the Culture Medium of Pleurotus Eryngii Strain UN-1

Pleurotus eryngii was one of the most precious and ordinary edible mushrooms, and its polysaccharides were the major active substances. In this study, P. eryngii strain UN-1 was cultured in a liquid medium at 25℃ under a shaking of 80 rpm for 7 days. Two crude polysaccharides named CPN and CPNM were obtained from the mycelium of P. eryngii UN-1 and its broth culture medium, respectively. After partial purification by cellulose DEAE-52 column chromatography and Sephadex G-100 gel-filtration, two fractions (PNP-1 and PNP-2) from CPN and one (PNMP-1) from CPNM were eluted and subjected to physicochemical properties, antioxidant, and antibacterial activities, in addition, the crude polysaccharides. The in vitro antioxidant assay revealed that all samples showed moderate scavenging capacities on DPPH radical (6.31-80.92%), ABTS radical (9.33-67.26%), and hydroxyl radical (11.21-79.53%) at different concentrations (0-2000 μg/mL), and PNP-2 exhibited the highest scavenging activity for all tested free radicals. Moreover, the antibacterial activity of the most active fraction (PNP-2) showed a significant effect against Escherichia coli, Bacillus Subtillis, Salmonella enterica, and Staphylococcus aureus subsp. Aureus strains with Minimum Inhibitory Concentration (MIC) values (2.5, 5, 2.5 and 5 mg/mL, respectively). Notably, the UV-vis Spectrophotometer confirmed that multiple active known compounds such as uremic acid were detected in all tested fractions, which confirmed the antioxidant and antimicrobial activities obtained in this study. Conclusively, the polysaccharides obtained from P. eryngii UN-1 have the potential to act as a functional food ingredient or a preventive drug against human pathogenic bacteria.

The mechanism of interaction between lotus rhizome polyphenol oxidase and ascorbic acid: Inhibitory activity, thermodynamics, and conformation analysis.

In this study, the interaction between lotus rhizome polyphenol oxidase (PPO) and ascorbic acid (AA) was discussed from the aspects of inhibitory activity, thermodynamics, and conformation. Results showed that PPO was purified from lotus rhizome by DEAE-52 anion exchange chromatography and Sephadex G-100 gel filtration chromatography, with its optimum substrate being determined as pyrogallic acid. Spectrophotometric and polarographic assays demonstrated that AA exhibited strong inhibitory activity against PPO. Thermodynamics, fluorescence, and circular dichroism spectral analysis showed that hydrophobic interactions caused the formation of AA-PPO complex, leading to the remarkable fluorescence quenching and conformational change of PPO. Atomic force microscopic analysis revealed that binding to AA induced significant changes in the surface morphology and molecular aggregation of PPO molecules. In this study, the interaction mechanism between PPO and AA was proposed for the first time, which provided a theoretical basis for AA to inhibit lotus rhizome browning. PRACTICAL APPLICATIONS: Lotus rhizome, an aquatic vegetable, is prone to enzymatic browning in processing operations, which leads to a decrease in market value and economic loss. At present, ascorbic acid (AA) is widely used in industries as an excellent antioxidant because of its good antibrowning effect and relatively low cost. However, the interaction between the enzymatic browning-related polyphenol oxidase (PPO) from lotus rhizome and ascorbic acid has not been clearly studied. Understanding the mechanism of inhibiting PPO will help to prevent vegetable browning, especially fresh-cut products. The inhibitory effect of AA on PPO in lotus rhizome favors simultaneous use with other types of PPO inhibitors because of their likely synergistic effects.

Purification and characterization of chitinase produced by thermophilic fungi Thermomyces lanuginosus

Background In the past few years, the production of shrimp shell waste from the seafood processing industries has confronted a significant surge. Furthermore, insignificant dumping of waste has dangerous effects on both nature and human well-being. This marine waste contains a huge quantity of chitin which has several applications in different fields. The chitinase enzyme can achieve degradation of chitin, and the chitin itself can be used as the substrate as well for production of chitinase. In the current study, the chitinase enzyme was produced by Thermomyces lanuginosus. The extracellular chitinase was purified from crude extract using ammonium sulfate precipitation followed by DEAE-cellulose ion-exchange chromatography and Sephadex G-100 gel filtration chromatography. The stability and activity of chitinase with different pH, temperature, different times for a reaction, in the presence of different metal ions, and different concentration of enzyme and substrate were analyzed. Result The chitinase activity was found to be highest at pH 6.5, 50 °C, and 60 min after the reaction began. and the chitinase showed the highest activity and stability in the presence of β-mercaptoethanol (ME). The SDS-PAGE of denatured purified chitinase showed a protein band of 18 kDa. Conclusion The characterization study concludes that Cu2+, Hg2+, and EDTA have an inhibitory effect on chitinase activity, whereas β-ME acts as an activator for chitinase activity. The utilization of chitin to produce chitinase and the degradation of chitin using that chitinase enzyme would be an opportunity for bioremediation of shrimp shell waste.

Production, Optimization, Purification, Kinetic analysis and applications of alkaline proteases produced from Bacillus subtilis through solid state fermentation of agricultural byproducts

Proteases have gained more commercial value to date due to multiple applications in different industrial sectors. Current research was aimed to use the cheaper agricultural waste for optimal protease production. Maximum level of protease production was achieved at 37 °C, incubation period of 24 h, pH 9.0, inoculum size 3%, 1.5 g sucrose as a carbon source and 30% moisture content by using solid-state fermentation. Among the various inorganic and organic nitrogen sources, ammonium nitrate and yeast extract tremendously increased the production of protease. Among metal ions and surfactants tested, Ca2+ and Tween 40 showed the optimal protease production. The purification of protease was carried out by ammonium salt precipitation followed by sephadex G-100 gel filtration chromatography. The purification resulted in 1.3 fold of purified protease with a specific activity of 51.5 and a total yield of 37.5 %. Molecular weight of purified protease was predicted upon SDS-PAGE with a single band of ~36 kDa. The protease was quite stable over a temperature range of 35-45 °C and pH 7-9 with maximal activity at 40 °C and pH 9. The kinetic parameters Vmax (maximum rate) and Km (Michaelis-Menten constant) were calculated as 0.307 U/g and 11.2 mg/mL, respectively. The alkaline protease significantly de-hair the goat skin and successfully removed the animal blood stain from cotton cloth.

IN VITRO ANTIOXIDANT ACTIVITY OF C-PHYCOCYANIN PURIFIED FROM Spirulina platensis DRY BIOMASS

C-phycocyanin (C-PC) was a phycobiliprotein found exclusively in cyanobacteria as Spirulina and exhibited a variety of pharmacological properties, which were not yet studied for any purpose in commercial applications in the pharmaceutical and cosmetic industry in Vietnam. At present, there are no other sources of natural phycocyanin than Spirulina, it is the sole efficient source of natural phycocyanin production under photoautotrophic conditions. The present research was conducted to determine an efficient extraction method from C-PC from Spirulina platensis dry biomass. Antioxidant activity of purified C-PC was investigated. Extractions were carried out using distilled water and Na-phosphate buffer pH 7.0 as solvents and freeze\thaw and ultrasonication techniques were applied to optimize the extraction process. Extraction using the freeze\thaw method proved to be the most efficient method. Phycocyanin purification from crude extracts was carried out by a combination of methods such as ammonium sulfate (NH4)2SO4 65% precipitation, dialysis filter, and Sephadex G-100 gel filtration chromatography, to give 121.63 ± 0.03 mg\g C-PC content and 3.52 ± 0.04 purity. Antioxidant activity in vitro was determined by nitric oxide (NO) scavenging activity, percentage inhibition of NO by the C-PC in concentration 10 μg\mL to 100 μg\mL which significantly scavenged 55.89% to 91.54% of NO radicals, respectively. They exhibited strong NO radical scavenging capacity, the C-PC and vitamin C were able to reduce the stable NO radical to 50% reduction with EC50 of 4.53 and 3.66 µg\mL. Research results showed that S. platenis dry biomass provided by Dalitra was potential for obtaining C-phycocyanin (12.16% of dry biomass) and it was evaluated as an antioxidant in vitro able to scavenge nitric oxide. C-PC (12.16% of dry biomass) and was evaluated as an antioxidant in vitro able to scavenge nitric oxide.

In vitro and in silico enzyme inhibition effects of some metal ions and compounds on glutathione S-transferase enzyme purified from Vaccinium arctostapylous L.

Glutathione s-transferase (GST) is a class of enzymes that performs a wide array of biological functions. However, GST enzymes are most famously known for their roles in catalyzing the conjugation of reduced glutathione (GSH) to electrophilic centers on a wide variety of substrates to induce water-solubility to compounds as a protective antioxidant mechanism against toxic substances. In the present study, in vitro inhibition effects of coumarin, ascorbic acid, sodium sulfide, sodium azide, citric acid compounds, and Cd2+, Cu2+, Ni2+, Mg2+ metal ions against GST enzyme were determined. For this aim, the GST enzyme was purified from Vaccinium arctostapylous L. using the glutathione-agarose affinity chromatography and Sephadex G-100 gel filtration steps. The respective metals and chemical compounds were used at different concentrations for measuring their in vitro GST activity effects. The Ki values of these agents were determined as 0.450 ± 0.13, 15.05 ± 7.05, 0.009 ± 0.001, 0.022 ± 0.006, 0.120 ± 0.36, 0.150 ± 0.06, 0.223 ± 0.03, 0.002 ± 0.0003, and 0.136 ± 0.06 mM, respectively. Finally, the molecular docking interactions of the compounds with the GST target enzyme were evaluated using Autodock Tools-1.5.6. The effective molecular interactions of coumarin, citric acid, ascorbic acid, and sodium sulfide with GST target enzyme were found with their binding lowest energy affinities −4.62, −3.04, −2.53, and −1.67 kcal/mol, respectively. Communicated by Ramaswamy H. Sarma