Partial purification and characterisation of cytosolic Fructose-1, 6-bisphosphatase from Drymaria cordata

Drymaria cordata is an important ethnomedicinal plant from which many important secondary metabolites have been reported. Partial purification of the enzyme, fructose 1,6-bisphosphatase was carried out following the methods of homogenization, streptomycin sulphate precipitation, ammonium sulphate cut and molecular sieve chromatography through Bio-Gel A-0.5m column. Biochemical characterization experiments were performed by standard methods with the enzyme preparation as purified from the column. Cytosolic fructose 1,6-bisphosphatase from the leaves of Drymaria cordata was purified to about 27-fold with 77% of recovery over homogenate fraction. The enzyme was highly specific to D-fructose-1,6-bisphosphate. With increase of protein concentration upto 300µg and incubation time upto120 minutes, the enzyme activity increased linearly. The metal ions Mg2+ or Mn2+ strongly stimulated the enzyme activity on the other hand Li+, Hg2+ and Zn2+ were potent inhibitors. The D. cordata enzyme showed temperature maxima at 40˚C while the optimum pH was at 8.0. The Km value of the enzyme for its substrate, Fructose 1,6-bisphosphate was 1.11µM proving its strong affinity.

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Gluconeogenic fructose-1,6-bisphosphatase from the mature sporocarps of common aquatic ferns: partial purification and basic characterization of this enzyme from Marsilea minuta (Polypodiopsida)

Ukrainian Botanical Journal ◽

10.15407/ukrbotj77.05.386 ◽

2020 ◽

Vol 77 (5) ◽

pp. 386-397

Author(s):

S.S. Ghosh ◽

◽

M. Das ◽

S. Basu ◽

J. Adhikari ◽

...

Keyword(s):

Partial Purification ◽

Light Illumination ◽

Azolla Pinnata ◽

Fructose 1,6 Bisphosphatase ◽

Salvinia Natans ◽

Km Value ◽

Gluconeogenic Enzyme ◽

Ph Range ◽

Fructose 1,6 Bisphosphate

The present communication reports substantial activity of gluconeogenic fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) in three common heterosporous aquatic ferns (Marsilea minuta, Salvinia natans, and Azolla pinnata) and also describes a protocol for its partial purification from mature sporocarps of Marsilea minuta. The cytosolic FBPase, obtained from Marsilea minuta, Salvinia natans, and Azolla pinnata was recognized as gluconeogenic enzyme due to its drastic catabolic inactivation in presence of externally administered glucose and its insensitivity towards photosynthetic light illumination. Cytosolic gluconeogenic FBPase was partially purified from mature sporocarps of Marsilea minuta to about 22-fold over homogenate following low-speed centrifugation (11, 400 × g), 30–80% ammonium sulfate fractionation followed by subsequent chromatography using matrices like CM-Cellulose, Sephadex G-200, and Ultrogel AcA 34. The profile of partially purified FBPase in PAGE under non-denaturing condition was recorded. The enzyme activity increased linearly with respect to protein concentration to about 100 µg and with respect to time up to 75 minutes. Temperature optimum was found at 35 °C. The effect of substrate concentration and kinetic analyses for FBPase were carried out using D-fructose-1,6-bisphosphate (D-FBP, the substrate) in the range of 0.0 to 1.0 mM at an interval of 0.1 mM concentration. The Km value for D-FBP of FBPase was 0.06129 mM and Vmax was 4525 nmole Pi released (mg)-1 protein h-1 as determined by nonlinear regression kinetics using Prism 8 software (Graph Pad). The enzyme was functional in a constricted pH range of 7.0 to 8.0, giving maxima at pH 7.5. This cytosolic enzyme was significantly stimulated by Mg2+ and strongly inhibited by Hg2+, Cu2+ and Zn2+.

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Partial purification and biochemical characterization of amylase from Aeromonas caviaeNK1isolated from Industrial waste of India

Journal of Biology and Life Science ◽

10.5296/jbls.v7i1.8361 ◽

2016 ◽

Vol 7 (1) ◽

pp. 85

Author(s):

Kalyani Khanra ◽

Indranil Choudhuri ◽

Sudipta Panja ◽

Nandan Bhattacharyya

Keyword(s):

Enzyme Activity ◽

Industrial Waste ◽

Biochemical Characterization ◽

Partial Purification ◽

Ammonium Sulfate Precipitation ◽

Operational Conditions ◽

Industrial Utilization ◽

Optimum Ph ◽

Optimum Ph And Temperature

A partial purification and biochemical characterization of theamylase from AeromonascaviaeNK1 were carried out in this study. The extracellular extract was concentrated using ammonium sulfate precipitation and optimum operational conditions for the enzyme activity from the strain were evaluated. The optimum pH and temperature were observed 11.5 and370C respectively. Ca2+, Mn2+, Mg2+, Zn2+, Pb2+, Co2+ were found to have effect on amylase activity.Furthermore, the analysis of kinetic showed that the enzyme has 𝐾𝑚 of 2.4mg/mL and 𝑉max of 21853.0𝜇mol/min/mg for starch. The results indicate that the enzyme reflects their potentiality towards industrial utilization.

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Partial purification and characterization of an endo-α-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242

Biochemical Journal ◽

10.1042/bj2880475 ◽

1992 ◽

Vol 288 (2) ◽

pp. 475-482 ◽

Cited By ~ 30

Author(s):

I Ishii-Karakasa ◽

H Iwase ◽

K Hotta ◽

Y Tanaka ◽

S Omura

Keyword(s):

Enzyme Activity ◽

Culture Medium ◽

Gel Filtration ◽

Partial Purification ◽

Gastric Mucus ◽

Purification And Characterization ◽

Streptomyces Sp ◽

Optimum Ph ◽

New Type

For the purification of a new type of endo-alpha-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242 (endo-GalNAc-ase-S) [Iwase, Ishii, Ishihara, Tanaka, Omura & Hotta (1988) Biochem. Biophys. Res. Commun. 151, 422-428], a method for assaying enzyme activity was established. Using purified pig gastric mucus glycoprotein (PGM) as the substrate, oligosaccharides liberated from PGM were pyridylaminated, and the reducing terminal sugars of oligosaccharides larger than Gal beta 1-3GalNAc were analysed by h.p.1.c. The crude enzyme of endo-GalNAc-ase-S was prepared as an 80% (w/v) ammonium sulphate precipitate from the concentrated culture medium. The enzyme was partially purified by gel chromatofocusing and subsequent DEAE-Toyopearl chromatography. Endo-enzyme activity eluted around pI 4.8 on a gel chromatofocusing column and eluted with 0.19-0.25 M-NaCl on a DEAE-Toyopearl column. In the enzyme fraction obtained, no exo-glycosidases or proteases could be detected. The molecular mass of the enzyme was estimated as 105 kDa by gel filtration, and the optimum pH was 5.5. Endo-GalNAc-ase-S hydrolysed the O-glycosidic linkage between GalNAc and Ser (Thr) in 3H-labelled and unlabelled asialofetuin, liberating both the disaccharide (Gal beta 1-3GalNAc) and the tetrasaccharide [Gal beta 1-3 (Gal beta 1-4GlcNAc beta 1-6)GalNAc]. When endo-alpha-N-acetylgalactosaminidase from Alcaligenes sp. (endo-GalNac-ase-A) was incubated with 3H-labelled and unlabelled asialofetuin, only the disaccharide (Gal beta 1-3GalNAc) was liberated.

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Optimization of Growth Conditions for Purification and Production of L-Asparaginase bySpirulina maxima

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2016/1785938 ◽

2016 ◽

Vol 2016 ◽

pp. 1-7 ◽

Cited By ~ 12

Author(s):

Hanaa H. Abd El Baky ◽

Gamal S. El Baroty

Keyword(s):

Enzyme Activity ◽

Gel Filtration ◽

Growth Conditions ◽

Partial Purification ◽

Specific Enzyme ◽

Purification And Characterization ◽

Spirulina Maxima ◽

Optimum Ph ◽

Biomass Yields

L-asparaginase (L-AsnA) is widely distributed among microorganisms and has important applications in medicine and in food technology sectors. Therefore, the ability of the production, purification, and characterization of AsnA fromSpirulina maxima(SM) were tested. SM cultures grown in Zarrouk medium containing different N2(in NaNO3form) concentrations (1.25, 2.50, and 5.0 g/L) for 18 days contained a significant various quantity of dry biomass yields and AsnA enzyme levels. MS L-AsnA activity was found to be directly proportional to the N2concentration. The cultures of SM at large scales (300 L medium, 5 g/L N2) showed a high AsnA enzyme activity (898 IU), total protein (405 mg/g), specific enzyme activity (2.21 IU/mg protein), and enzyme yield (51.28 IU/L) compared with those in low N2cultures. The partial purification of crude MS AsnA enzyme achieved by 80% ammonium sulfate AS precipitated and CM-Sephadex C-200 gel filtration led to increases in the purification of enzyme with 5.28 and 10.91 times as great as that in SM crude enzymes. Optimum pH and temperature of purified AsnA for the hydrolyzate were 8.5 and 37 ± 0.2°C, respectively. To the best of our knowledge, this is the first report on L-asparaginase production inS. maxima.

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Development and properties of fructose 1,6-bisphosphatase in the endosperm of castor-bean seedlings

Biochemical Journal ◽

10.1042/bj1540647 ◽

1976 ◽

Vol 154 (3) ◽

pp. 647-652 ◽

Cited By ~ 12

Author(s):

R J. Youle ◽

A H. C. Huang

Keyword(s):

Castor Bean ◽

Ricinus Communis ◽

Developmental Pattern ◽

Marker Enzyme ◽

Animal Tissues ◽

Optimal Activity ◽

Neutral Ph ◽

Fructose 1,6 Bisphosphatase ◽

Km Value ◽

Fructose 1,6 Bisphosphate

1. The activity of fructose 1,6-bisphosphatase (EC 3.1.3.11) in the fatty endosperm of castor bean (Ricinus communis) increases 25-fold during germination and then declines. The developmental pattern follows that of catalase, a marker enzyme for gluconeogenesis in this tissue. 2. The enzyme at its peak of development was partially purified, and its properties were studied. It has an optimal activity at neutral pH (7.0-8.0). The apparent Km value for fructose 1,6-bisphosphate is 3.8 × 10-5 M. The activity is inhibited by AMP allosterically with an apparent Ki value of 2.2 × 10-4 M. The enzyme hydrolyses fructose 1,6-bisphosphate and not ribulose 1,5-bisphosphate or sedoehptulose 1,7-bisphosphate. 3. Treatment of the partially purified enzyme with acid leads to an 80% decrease in activity. The remaining activity is insensitive to AMP and has optimal activity at pH 6.7 and a high apparent Km value (2.5 × 10-4 M) for fructose 1.6-bisphosphate. Enzyme extracted from the tissue with water instead of buffer has a similar modification. The effect of acid explains the discrepancies between this report and previous ones on the properties of the enzyme in this tissue. 4. The storage tissues of various fatty seedlings all contain a ‘neutral’ fructose 1,6-bisphosphatase. The activities of the enzyme from some of the tissues are inhibited by AMP. 5. The properties of the enzyme in fatty seedlings and in green leaves are discussed in comparison with that in animal tissues.

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Partial Purification and Characterization of a Heat Stable α-Amylase from a Thermophilic Actinobacteria, Streptomyces sp. MSC702

Enzyme Research ◽

10.1155/2014/106363 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 6

Author(s):

Renu Singh ◽

Vijay Kumar ◽

Vishal Kapoor

Keyword(s):

Enzyme Activity ◽

Biochemical Characterization ◽

Enzyme Stability ◽

Potato Starch ◽

Tween 80 ◽

Partial Purification ◽

Amylolytic Enzyme ◽

Operational Conditions ◽

Heat Stable

A partial purification and biochemical characterization of the α-amylase from Streptomyces sp. MSC702 were carried out in this study. The optimum operational conditions for enzyme substrate reaction for amylolytic enzyme activity from the strain were evaluated. The optimum pH, temperature, and incubation period for assaying the enzyme were observed to be 5.0, 55°C, and 30 min, respectively. The extracellular extract was concentrated using ammonium sulfate precipitation. It was stable in the presence of metal ions (5 mM) such as K+, Co2+, and Mo2+, whereas Pb2+, Mn2+, Mg2+, Cu2+, Zn2+, Ba2+, Ca2+, Hg2+, Sn2+, Cr3+, Al3+, Ag+, and Fe2+ were found to have inhibitory effects. The enzyme activity was also unstable in the presence of 1% Triton X-100, 1% Tween 80, 5 mM sodium lauryl sulphate, 1% glycerol, 5 mM EDTA, and 5 mM denaturant urea. At temperature 60°C and pH 5.0, the enzyme stability was maximum. α-amylase retained 100% and 34.18% stability for 1 h and 4 h, respectively, at 60°C (pH 7.0). The enzyme exhibited a half-life of 195 min at 60°C temperature. The analysis of kinetic showed that the enzyme has Km of 2.4 mg/mL and Vmax of 21853.0 μmol/min/mg for soluble potato starch. The results indicate that the enzyme reflects their potentiality towards industrial utilization.

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Kinetics Properties of Polyphenol Oxidase in Hawthorn (Crataegus spp)

International Letters of Natural Sciences ◽

10.18052/www.scipress.com/ilns.25.29 ◽

2014 ◽

Vol 25 ◽

pp. 29-38 ◽

Cited By ~ 1

Author(s):

Shahriar Saeidian

Keyword(s):

Enzyme Activity ◽

Polyphenol Oxidase ◽

Thermal Inactivation ◽

Kojic Acid ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Optimum Ph ◽

Km Value ◽

Ph Range ◽

Menten Constant

Polyphenol oxidase (PPO) from hawthorn was extracted and partially purified through (NH4)2SO4 precipitation, dialysis and ion exchange chromatography. The activity of polyphenol oxidase was investigated in Crataegus spp. Spectrophotometric method was used to assay the enzyme activity and the kinetic constants - maximum enzyme velocity (Vmax) and Michealis - Menten constant (Km). Of the substrates tested, catechol was the best substrate for PPO with a Km value of 2.2 mM. The optimum pH for PPO activity was found to be 7. The enzyme showed high activity over a broad pH range of 4 - 8. The optimal pH and temperature for enzyme activity were found to be 7 and 40-45 °C, respectively. km value for hawthorn PPO is calculated 22 mM for catechol and 6.7 mM for pyrogallol and 9.7 mM for L-dopa. As can be seen, affinity of PPOs for various substrates varies widely. The enzyme showed a broad activity over a broad pH and temperature range. The thermal inactivation studies showed that the enzyme is heat resistant. The enzyme showed the highest activity toward pyrogallol and no activity toward tyrosine. Of the inhibitors tested, the most potent inhibitors were kojic acid, cysteine and glycine , respectively

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Molecular and Biochemical Characterization of a Distinct Type of Fructose-1,6-Bisphosphatase from Pyrococcus furiosus

Journal of Bacteriology ◽

10.1128/jb.184.12.3401-3405.2002 ◽

2002 ◽

Vol 184 (12) ◽

pp. 3401-3405 ◽

Cited By ~ 23

Author(s):

Corné H. Verhees ◽

Jasper Akerboom ◽

Emile Schiltz ◽

Willem M. de Vos ◽

John van der Oost

Keyword(s):

Biochemical Characterization ◽

Pyrococcus Furiosus ◽

Distinct Type ◽

Sequence Motifs ◽

Conserved Sequence ◽

New Family ◽

Fructose 1,6 Bisphosphatase ◽

Conserved Sequence Motifs ◽

Fructose 1,6 Bisphosphate

ABSTRACT The Pyrococcus furiosus fbpA gene was cloned and expressed in Escherichia coli, and the fructose-1,6-bisphosphatase produced was subsequently purified and characterized. The dimeric enzyme showed a preference for fructose-1,6-bisphosphate, with a Km of 0.32 mM and a V max of 12.2 U/mg. The P. furiosus fructose-1,6-bisphosphatase was strongly inhibited by Li+ (50% inhibitory concentration, 1 mM). Based on the presence of conserved sequence motifs and the substrate specificity of the P. furiosus fructose-1,6-bisphosphatase, we propose that this enzyme belongs to a new family, class IV fructose-1,6-bisphosphatase.

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Isolation and Partial Purification of Polyphenol Oxidase from Seed of Melon (Cucumeropsis edulis)

Biointerface Research in Applied Chemistry ◽

10.33263/briac112.90859096 ◽

2020 ◽

Vol 11 (2) ◽

pp. 9085-9096

Keyword(s):

Metal Ions ◽

Polyphenol Oxidase ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Partial Purification ◽

Inhibitory Effects ◽

Optimum Ph ◽

Km Value ◽

Menten Constant ◽

Diphenolase Activity

Polyphenol oxidase (PPO) from Cucumeropsis edulis was extracted and partially purified through (NH4)2SO4 precipitation, dialysis, and ion-exchange chromatography on DEAE-Sephadex-A50. The spectrophotometric method was used to assay the enzyme activity in C. edulis using L-DOPA as substrate, the physicochemical properties such as the effect of pH and temperature, substrate specificity, kinetic constants - maximum enzyme velocity (Vmax), and Michaelis - Menten constant (Km) for three substrates namely, L-Dopa catechol and tyrosine were determined. The effects of inhibitors and metal ions on PPO activity were also investigated. The optimum pH and temperature values were found to be pH 6.5 and 50 °C, and the inhibitory effects of inhibitors such as ascorbic acid, EDTA, SDS, and metal ions were enhanced positively with increased concentration except with divalent metals such as Cu2+, Fe2+, and Zn2+ reflecting an activating effect on C. edulis PPO. Moreover, the enzyme solution showed both monophenolase and diphenolase activity with L-DOPA having the highest Vmax/Km value. However, the data obtained in this research provided a theoretical basis for the prevention of enzymatic browning of C. edulis during processing.

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Phosphofructokinases A and B from Mycobacterium tuberculosis Display Different Catalytic Properties and Allosteric Regulation

International Journal of Molecular Sciences ◽

10.3390/ijms22031483 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1483

Author(s):

Jan Snášel ◽

Iva Machová ◽

Veronika Šolínová ◽

Václav Kašička ◽

Marcela Krečmerová ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Biochemical Characterization ◽

Metabolic Enzyme ◽

Kinetic Properties ◽

Reaction Products ◽

Hypoxic Conditions ◽

Glycolytic Activity ◽

Gene And Protein Expression ◽

Fructose 1,6 Bisphosphatase ◽

Fructose 1,6 Bisphosphate

Tuberculosis (TB) remains one of the major health concerns worldwide. Mycobacterium tuberculosis (Mtb), the causative agent of TB, can flexibly change its metabolic processes during different life stages. Regulation of key metabolic enzyme activities by intracellular conditions, allosteric inhibition or feedback control can effectively contribute to Mtb survival under different conditions. Phosphofructokinase (Pfk) is one of the key enzymes regulating glycolysis. Mtb encodes two Pfk isoenzymes, Pfk A/Rv3010c and Pfk B/Rv2029c, which are differently expressed upon transition to the hypoxia-induced non-replicating state of the bacteria. While pfkB gene and protein expression are upregulated under hypoxic conditions, Pfk A levels decrease. Here, we present biochemical characterization of both Pfk isoenzymes, revealing that Pfk A and Pfk B display different kinetic properties. Although the glycolytic activity of Pfk A is higher than that of Pfk B, it is markedly inhibited by an excess of both substrates (fructose-6-phosphate and ATP), reaction products (fructose-1,6-bisphosphate and ADP) and common metabolic allosteric regulators. In contrast, synthesis of fructose-1,6-bisphosphatase catalyzed by Pfk B is not regulated by higher levels of substrates, and metabolites. Importantly, we found that only Pfk B can catalyze the reverse gluconeogenic reaction. Pfk B thus can support glycolysis under conditions inhibiting Pfk A function.

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