Novel Thermotolerant Amylase from Bacillus licheniformis Strain LB04: Purification, Characterization and Agar-Agarose

This study analyzed the thermostability and effect of calcium ions on the enzymatic activity of α-amylase produced by Bacillus licheniformis strain LB04 isolated from Espinazo Hot springs in Nuevo Leon, Mexico. The enzyme was immobilized by entrapment on agar-agarose beads, with an entrapment yield of 19.9%. The identification of the bacteria was carried out using 16s rDNA sequencing. The enzyme was purified through ion exchange chromatography (IEX) in a DEAE-Sephadex column, revealing a protein with a molecular weight of ≈130 kDa. The enzyme was stable at pH 3.0 and heat stable up to 80 °C. However, the optimum conditions were reached at 65 °C and pH 3.0, with a specific activity of 1851.7 U mg−1 ± 1.3. The agar-agarose immobilized α-amylase had a hydrolytic activity nearly 25% higher when compared to the free enzyme. This study provides critical information for the understanding of the enzymatic profile of B. licheniformis strain LB04 and the potential application of the microorganisms at an industrial level, specifically in the food industry.

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Two acid RNases from Dactylis glomerata seeds. Purification, properties and effect of polyamines and lectins on their activity

Acta Societatis Botanicorum Poloniae ◽

10.5586/asbp.1985.024 ◽

2014 ◽

Vol 54 (3) ◽

pp. 241-253 ◽

Cited By ~ 1

Author(s):

Janina Wiśniowska ◽

Bronisława Morawiecka

Keyword(s):

Specific Activity ◽

Gel Filtration ◽

Deae Cellulose ◽

Dactylis Glomerata ◽

Hydrolytic Activity ◽

Relative Activity ◽

Exchange Chromatography ◽

Lentil Lectin ◽

Optimum Ph ◽

Rnase Ii

Two glycoproteidic acid RNases (RNase I and RNase II) were obtained and purified from the seeds of Dactylis glomerata by extraction with acetate buffer, fractionation with ammonium sulfate, ion-exchange chromatography on DEAE-cellulose, DEAE-Sphadex, affinity chromatography on Con A-Sepharose and gel filtration on Bio-Gel P60. RNase I with a specific activity of 2582 U•mg-1 protein and an optimum pH of 4.9 and RNase II with a specific activity of 1928 U• mg-1 protein and optimum pH of 4.6, were isolated. They lacked nuclease, phosphodi- and monoesterase activities. Both forms of the enzyme hydrolyzed pyrimidine homopolymers with a preference for poly U and exhibited a low specificity for purine homopolymers (poly G and poly A). RNase I acted with a 3-fold higher hydrolytic activity on poly C homopolymer than RNase IL The hydrolytic activity of both enzymes was inhibited by Zn+2, Fe+2, Cu+2 ions when yeast RNA was the substrate. The amines spermine, spermidine and tyramine at a concentration of 0.1 mM increased the enzymatic activity of both RNases by 20 to 60% of the relative activity. The hydrolytic activity of RNases I and II was stimulated by the presence of lentil lectin (LL), soybean lectin (SBA) and potato lectin (STA), and inhibited by the presence of concanavalin A. The 20-200% stimulation and 40-60% inhibition depended on the proportion, on a weight basis, of enzyme to lectin and were reversible in the presence of receptor sugars.

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Purification and characterization of the extracellular proteinase ofPseudomonas fluorescensNCDO 2085

Journal of Dairy Research ◽

10.1017/s0022029900025073 ◽

1986 ◽

Vol 53 (3) ◽

pp. 457-466 ◽

Cited By ~ 10

Author(s):

David J. Fairbairn ◽

Barry A. Law

Keyword(s):

Specific Activity ◽

Gel Filtration ◽

Fold Increase ◽

Exchange Chromatography ◽

Purification And Characterization ◽

Extracellular Proteinase ◽

Original Activity ◽

Heat Stable ◽

D Values

SUMMAEYPseudomonas fluorescensNCDO 2085 produced a single heat-stable extracellular proteinase in Na caseinate medium at 20 °C and pH 7·0. The proteinase was purified to electrophoretic homogeneity using chromatofocusing, gel filtration and ion-exchange chromatography. The purification procedure resulted in a 158-fold increase in the specific activity and a yield of 3·5% of the original activity. The enzyme is a metalloproteinase containing Zn and Ca, with an isoelectric point at 5·40±0·05 and a mol. wt of 40200±2100. It is heat-stable having D-values at 74 and 140 °C of 1·6 and 1·0 min respectively; 40 and 70% of the original activity remained after HTST (74 °C/17 s) and ultra high temperature (140°C/4 s) treatments respectively. The amino acid composition of the proteinase was determined and compared with those from otherPseudomonasspp.

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Purification and Properties of the Thrombin-Like Principle of Agkistrodon acutus Venom and Its Comparison with Bovine Thrombin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653671 ◽

1971 ◽

Vol 26 (02) ◽

pp. 224-234 ◽

Cited By ~ 12

Author(s):

Chaoho Ouyang ◽

Jau-Shyong Hong ◽

Che-Ming Teng

Keyword(s):

Specific Activity ◽

Ph Value ◽

Activate Factor ◽

Clot Retraction ◽

Crude Venom ◽

Sephadex Column ◽

Bovine Thrombin ◽

Heat Stable ◽

Purification And Properties ◽

Microzone Electrophoresis

SummaryBy means of DEAE-Sephadex column chromatography, Agkistrodon acutus venom was separated into 12 fractions. The thrombin-like activity was concentrated in Fr.10. This fraction was rechromatographed on Sephadex G-200. A single band was found on microzone electrophoresis at pH 7.4. A single boundary with S20, w of 3.82 S, which was almost symmetrical, was observed by ultracentrifugation. The estimated molecular weight is 33,500. The chemical analysis shows that the thrombin-like principle of the venom is a glycoprotein. The specific activity is 13 times higher than that of the crude venom. The optimal pH value of the thrombin-like principle (pH 7.5) of the venom is almost identical with that of the bovine thrombin (pH 7.2). The thrombinlike principle of the venom is not affected by heparin, while the clotting activity of the bovine thrombin is inhibited by heparin. The thrombin-like principle of the venom is much more heat stable than the bovine thrombin.No clot retraction was found with the thrombin-like principle of the venom, while the marked clot retraction occurred after plasma was coagulated with thrombin.Thrombin can activate factor XIII (Fibrin stabilizing factor), while the thrombinlike principle can not.

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Production and Partial Purification of Heat-Stable Enterotoxin (A) Produced by Enterotoxigenic Escherichia coli

The Iraqi Journal of Veterinary Medicine ◽

10.30539/iraqijvm.v34i1.668 ◽

2010 ◽

Vol 34 (1) ◽

pp. 122-129

Author(s):

Rajiha I. Al-Nuaimy

Keyword(s):

Large Scale ◽

Specific Activity ◽

Exchange Chromatography ◽

Enterotoxigenic Escherichia Coli ◽

Partial Purification ◽

Scale Production ◽

E Coli ◽

Standard Curve ◽

Heat Stable ◽

Large Scale Production

A total of (25) stool samples were collected from children and adults (2- 4) years oldsuffering from diarrhea to isolate E. coli strains that produce heat-stable enterotoxin a (STa),and after performing microscopic examination, cultural characterization and biochemicalidentification only (11) isolates showed positive E. coli. STa activity was estimated by usingsuckling mouse assay (SMA) and from these (11) isolates only (5) showed STa activity andthe one with the highest STa activity was selected for large scale production of STa, whichwas followed by partial purification using ion-exchange chromatography (normal phase)using DEAE sephadex A-50 column. After purification and determination of proteinconcentration by using the standard curve of bovine serum albumin, the concentration oftoxin-protein was estimated as (1.08) mg/ml. The specific activity varied from (350) U/mgprotein at the first step of purification to (2366.6) U/mg protein at the final step, while thefinal purification of the toxin was about (6.76) fold and with a yield of (18.25) %.

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Kinetic and Thermodynamic Characterization of Lignin Peroxidase Isolated from Agaricus bitorqus A66

International Journal of Chemical Reactor Engineering ◽

10.1515/1542-6580.2837 ◽

2012 ◽

Vol 10 (1) ◽

Author(s):

Ismat Bibi ◽

Haq Nawaz Bhatti

Keyword(s):

Lignin Peroxidase ◽

Thermal Inactivation ◽

Specific Activity ◽

Gel Filtration ◽

Veratryl Alcohol ◽

Exchange Chromatography ◽

Different Temperatures ◽

Scale Experiment ◽

Menten Kinetic

This study deals with purification and characterization of lignin peroxidase (LiP) isolated from Agaricus bitorqus A66 during decolorization of NOVASOL Direct Black dye. A laboratory scale experiment was conducted for maximum LiP production under optimal conditions. Purification & fractionation of LiP was performed on DEAE-Sepharose ion exchange chromatography followed by Sephadex G-50 gel filtration. The purified LiP has a specific activity of 519 U/mg with 6.73% activity recover. The optimum pH and temperature of purified LiP for the oxidation of veratryl alcohol were 6.8 and 45 °C, respectively. Michaelis-Menten kinetic constants (Vmax and Km) were determined using different concentrations of veratryl alcohol (1-35 mM). The Km and Vmax were 16.67 mM and 179.2 U/mL respectively, for veratryl alcohol oxidation as determined from the Lineweaver-Burk plot. Thermal inactivation studies were carried out at different temperatures to check the thermal stability of the enzyme. Enthalpy of activation decreased where Free energy of activation for thermal denaturation increased at higher temperatures. A possible explanation for the thermal inactivation of LiP at higher temperatures is also discussed.

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Isolation, partial purification and characterization of phospholipase A2 from Naja Katiensis venom

Bayero Journal of Pure and Applied Sciences ◽

10.4314/bajopas.v13i2.14 ◽

2021 ◽

Vol 13 (2) ◽

pp. 107-112

Author(s):

C.F. Okechukwu ◽

P.L. Shamsudeen ◽

R.K. Bala ◽

B.G. Kurfi ◽

A.M. Abdulazeez

Keyword(s):

Ion Exchange ◽

Phospholipase A2 ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Partial Purification ◽

Crude Venom

The most effective and acceptable therapy for snakebite victims is the immediate administration of antivenin which is limited by problems of hypersensitivity reactions in some individuals and its inability to resolve the local effects of the venom. The aim of this study was to isolate, partially purify and characterize phospholipase A2 from Naja Katiensis venom. Phospholipase A2 was partially purified via a two-step process: gel filtration on Sephadex G-75 and ion exchange chromatography using CM Sephadex, and subjected to SDS-PAGE analysis. From the results, the specific activity of the partially purified PLA2 decreased from 0.67μmol/min/mg in crude venom to 0.29μmol/min/mg after ion exchange chromatography with a yield of 5% and purification fold of 0.43. The optimum temperature of the purified PLA2 was found to be 35ºC and optimum p.H of 7. velocity studies for the determination of kinetic constants using L-a-lecithin as substrate revealed a Km of 1.47mg/ml and Vmax of 3.32μ moles/min/mg. The sodium dodecyl sulphate polyacrylamide gel electrophoresis of the purified PLA2 showed a distinct band with molecular weight estimated to be 14KDa. In conclusion, the present study shows that phospholipase A2 was isolated, purified and characterized. This may serve as a promising candidate for future development of a novel anti-venin drug.

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A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

10.20944/preprints202009.0390.v1 ◽

2020 ◽

Author(s):

Cecy Xi ◽

Arianna Arianna Di Fazio ◽

Naveed Nadvi ◽

Karishma Patel ◽

Michelle Xiang ◽

...

Keyword(s):

Affinity Chromatography ◽

High Purity ◽

Specific Activity ◽

Specific Binding ◽

Exchange Chromatography ◽

Cell Surface Receptor ◽

Spike Protein ◽

High Yield ◽

Purification Procedure ◽

Dipeptidyl Peptidase 4

Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29-766) produced in insect cells. Purification used differential ammonium sulfate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor binding domain (RBD) were measured using surface plasmon resonance. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity >30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.

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Optimization of Metal Ions in Sugarcane Bagasse Fermenting Medium for the Production of Streptokinase by Streptococcus equisimilis

Science Letters ◽

10.47262/sl/9.2.132021003 ◽

2021 ◽

Vol 9 (2) ◽

pp. 24-30

Keyword(s):

Metal Ions ◽

Sugarcane Bagasse ◽

Specific Activity ◽

Gel Filtration ◽

Value Added ◽

Cost Effective ◽

Fermentation Medium ◽

Exchange Chromatography ◽

Sds Page ◽

The Cost

Streptokinase is a fibrinolytic enzyme and a product of β-hemolytic Streptococci strains. This enzyme is used as a medication to break down clots in some cases of heart disease. Streptococcus equisimilis, a species of group C Streptococci, is widely used for the production of streptokinase by fermentation technology. In this study, the sugarcane bagasse fermentation medium was optimized for metal ions (KH2PO4, MgSO4.7H2O, CaCO3 and NaHCO3) at various levels to attain the maximal production of streptokinase. Sugarcane bagasse was used due to its profuse availability and as an ideal substrate for microbial processes for the manufacturing of value-added products. The results showed that maximal streptokinase production was found at 0.04% KH2PO4, 0.04% MgSO4.7H2O, 0.15% NaHCO3 and 0.04% CaCO3. Finally, the optimized medium resulted in 84.75 U/mg specific activity and 74.5% recovery. The purification process was carried out simultaneously using ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. Finally, a purified sample of streptokinase was run on SDS-PAGE and resolute 47 kDa molecular weight. The use of β-hemolytic Streptococci to obtain streptokinase is not free from health risks and is related to anaphylaxis. This study provides a way forward for the cost-effective ways to obtain streptokinase for the treatment of thrombosis.

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PURIFICATION AND PARTIAL CHARACTERIZATION OF L-ASPARAGINASE ENZYME PRODUCED BY NEWLY ISOLATE BACILLUS SP.

IIUM Engineering Journal ◽

10.31436/iiumej.v18i2.654 ◽

2017 ◽

Vol 18 (2) ◽

pp. 1-10 ◽

Cited By ~ 3

Author(s):

Dzun Noraini Jimat ◽

Intan Baizura Firda Mohamed ◽

Azlin Suhaida Azmi ◽

Parveen Jamal

Keyword(s):

Specific Activity ◽

Hot Spring ◽

Gel Filtration ◽

Maximum Activity ◽

Exchange Chromatography ◽

Bacillus Sp ◽

Sds Page ◽

Fast Flow ◽

Microbial Strain

A newly bacterial producing L-asparaginase was successful isolated from Sungai Klah Hot Spring, Perak, Malaysia and identified as Bacillus sp. It was the best L-asparaginase producer as compared to other isolates. Production of L-asparaginase from the microbial strain was carried out under liquid fermentation. The crude enzyme was then centrifuged and precipitated with ammonium sulfate before further purified with chromatographic method. The ion exchange chromatography HiTrap DEAE-Sepharose Fast Flow column followed by separation on Superose 12 gel filtration were used to obtain pure enzyme. The purified enzyme showed 10.11 U/mg of specific activity, 50.07% yield with 2.21 fold purification. The purified enzyme was found to be dimer in form, with a molecular weight of 65 kDa as estimated by SDS-PAGE. The maximum activity of the purified L-asparaginase was observed at pH 9 and temperature of 60Â°C.

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Characterisation and molecular dynamic simulations of J15 asparaginase from Photobacterium sp. strain J15.

Acta Biochimica Polonica ◽

10.18388/abp.2014_1840 ◽

2014 ◽

Vol 61 (4) ◽

Cited By ~ 10

Author(s):

Mohd Adilin Yaacob ◽

Wan Atiqah Najiah Wan Hasan ◽

Mohd Shukuri Mohamad Ali ◽

Raja Noor Zaliha Raja Abdul Rahman ◽

Abu Bakar Salleh ◽

...

Keyword(s):

Molecular Dynamic ◽

Conformational Changes ◽

Specific Activity ◽

3D Structure ◽

Coli Strain ◽

Exchange Chromatography ◽

Molecular Dynamic Simulations ◽

Type I ◽

Enzymatic Reactions ◽

Dynamic Simulations

Genome mining revealed a 1011 nucleotide-long fragment encoding a type I L-asparaginase (J15 asparaginase) from the halo-tolerant Photobacterium sp. strain J15. The gene was overexpressed in pET-32b (+) vector in E. coli strain Rosetta-gami B (DE3) pLysS and purified using two-step chromatographic methods: Ni(2+)-Sepharose affinity chromatography and Q-Sepharose anion exchange chromatography. The final specific activity and yield of the enzyme achieved from these steps were 20 U/mg and 49.2%, respectively. The functional dimeric form of J15-asparaginase was characterised with a molecular weight of ~70 kDa. The optimum temperature and pH were 25°C and pH 7.0, respectively. This protein was stable in the presence of 1 mM Ni(2+) and Mg(2+), but it was inhibited by Mn(2+), Fe(3+) and Zn(2+) at the same concentration. J15 asparaginase actively hydrolysed its native substrate, l-asparagine, but had low activity towards l-glutamine. The melting temperature of J15 asparaginase was ~51°C, which was determined using denatured protein analysis of CD spectra. The Km, Kcat, Kcat/Km of J15 asparaginase were 0.76 mM, 3.2 s(-1), and 4.21 s(-1) mM(-1), respectively. Conformational changes of the J15 asparaginase 3D structure at different temperatures (25°C, 45°C, and 65°C) were analysed using Molecular Dynamic simulations. From the analysis, residues Tyr₂₄ , His₂₂, Gly₂₃, Val₂₅ and Pro₂₆ may be directly involved in the 'open' and 'closed' lid-loop conformation, facilitating the conversion of substrates during enzymatic reactions. The properties of J15 asparaginase, which can work at physiological pH and has low glutaminase activity, suggest that this could be a good candidate for reducing toxic effects during cancer treatment.

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