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

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

<p class="jbls">A partial purification and biochemical characterization of theamylase from <em>Aeromonascaviae</em>NK1 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 and37<sup>0</sup>C respectively. Ca<sup>2+</sup>, Mn<sup>2+</sup>, Mg<sup>2+</sup>, Zn<sup>2+</sup>, Pb<sup>2+</sup>, Co<sup>2+</sup> were found to have effect on amylase activity.Furthermore, the analysis of kinetic showed that the enzyme has 𝐾<sub>𝑚</sub> of 2.4mg/mL and 𝑉<sub>max</sub> of 21853.0𝜇mol/min/mg for starch. The results indicate that the enzyme reflects their potentiality towards industrial utilization.</p>

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

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
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.

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

1992 ◽  
Vol 288 (2) ◽  
pp. 475-482 ◽  
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.

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

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
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.

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

2018 ◽  
Vol 7 (4) ◽  
pp. 2126
Author(s):  
Niki Doma Sherpa ◽  
Raksha Mukhia ◽  
Dhani Raj Chhetri
Keyword(s):  
Enzyme Activity ◽  
Protein Concentration ◽  
Biochemical Characterization ◽  
Partial Purification ◽  
Streptomycin Sulphate ◽  
Fructose 1,6 Bisphosphatase ◽  
Ethnomedicinal Plant ◽  
Optimum Ph ◽  
Km Value ◽  
Fructose 1,6 Bisphosphate

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.

scholarly journals Partial purification and biochemical characterization of a new highly acidic NYSO laccase from Alcaligenes faecalis

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Soad A. Abdelgalil ◽  
Ahmad R. Attia ◽  
Reyed M. Reyed ◽  
Nadia A. Soliman
Keyword(s):  
Enzyme Activity ◽  
Sodium Dodecyl ◽  
Alcaligenes Faecalis ◽  
Maximum Activity ◽  
Sodium Oxalate ◽  
Partial Purification ◽  
Bromophenol Blue ◽  
Bromocresol Purple ◽  
Laccase Enzyme

Abstract Background Due to the multitude industrial applications of ligninolytic enzymes, their demands are increasing. Partial purification and intensive characterization of contemporary highly acidic laccase enzyme produced by an Egyptian local isolate designated Alcaligenes faecalis NYSO were studied in the present investigation. Results Alcaligenes faecalis NYSO laccase has been partially purified and intensively biochemically characterized. It was noticed that 40–60% ammonium sulfate saturation showed maximum activity. A protein band with an apparent molecular mass of ~ 50 kDa related to NYSO laccase was identified through SDS-PAGE and zymography. The partially purified enzyme exhibited maximum activity at 55 °C and pH suboptimal (2.5–5.0). Remarkable activation for enzyme activity was recognized after 10-min exposure to temperatures (T) 50, 60, and 70 °C; time elongation caused inactivation, where ~ 50% of activity was lost after a 7-h exposure to 60 °C. Some metal ions Cu2+, Zn2+, Co2+, Ni2+, Mn2+, Cd2+, Cr2+, and Mg2+ caused strong stimulation for enzyme activity, but Fe2+ and Hg2+ reduced the activity. One millimolar of chelating agents [ethylenediamine tetraacetic acid (EDTA), sodium citrate, and sodium oxalate] caused strong activation for enzyme activity. Sodium dodecyl sulfate (SDS), cysteine-HCl, dithiothreitol (DTT), β-mercaptoethanol, thioglycolic acid, and sodium azide caused strong inhibition for NYSO laccase activity even at low concentration. One millimolar of urea, imidazole, kojic acid, phenylmethylsulfonyl fluoride (PMSF), H2O2, and Triton X-100 caused activation. The partially purified NYSO laccase had decolorization activity towards different dyes such as congo red, crystal violet, methylene blue, fast green, basic fuchsin, bromophenol blue, malachite green, bromocresol purple eriochrome black T, and Coomassie Brilliant Blue R-250 with various degree of degradation. Also, it had a vast range of substrate specificity including lignin, but with high affinity towards p-anisidine. Conclusion The promising properties of the newly studied laccase enzyme from Alcaligenes faecalis NYSO strain would support several industries such as textile, food, and paper and open the possibility for commercial use in water treatment. It will also open the door to new applications due to its ligninolytic properties in the near future.

scholarly journals Partial purification and characterization of human erythrocyte phosphoglycollate phosphatase

1980 ◽  
Vol 191 (1) ◽  
pp. 117-124 ◽  
Author(s):  
R Zecher ◽  
H U Wolf
Keyword(s):  
Specific Activity ◽  
Total Activity ◽  
Partial Purification ◽  
Half Maximum ◽  
Purification And Characterization ◽  
Dimeric Molecule ◽  
Maximum Inhibition ◽  
Optimum Ph ◽  
Triton X

Human erythrocytes contain a phosphatase that is highly specific for phosphoglycollate. It shows optimum pH of 6.7 and has Km 1 mM for phosphoglycollate. The molecular weight appears to be about 72000. The enzyme is a dimeric molecule having subunits of mol. wt. about 35000. It could be purified approx. 4000-fold up to a specific activity of 5.98 units/mg of protein. The activity of the enzyme is Mg2+-dependent. Co2+, and to a smaller extent Mn2+, may substitute for Mg2+. Half-maximum inhibition of the phosphatase by 5,5′-dithiobis-(2-nitrobenzoate), EDTA and NaF is obtained at 0.5 microM, 1 mM and 4 mM respectively. Moreover, it needs a univalent cation for optimum activity. Phosphoglycollate phosphatase is a cytoplasmic enzyme. Approx. 5% of its total activity is membrane-associated. This part of activity can be approx. 70% solubilized by freezing, thawing and treatment with 0.25% Triton X-100.

scholarly journals Biochemical Characterization of Laboratory Mutants of Extended-Spectrum β-Lactamase TEM-60

2004 ◽  
Vol 48 (9) ◽  
pp. 3579-3582 ◽  
Author(s):  
Bibiana Caporale ◽  
Nicola Franceschini ◽  
Mariagrazia Perilli ◽  
Bernardetta Segatore ◽  
Gian Maria Rossolini ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Extended Spectrum

ABSTRACT Three mutants of the extended-spectrum β-lactamase TEM-60, the P51L, K104E, and S164R mutants, were constructed by site-directed mutagenesis. The kinetic parameters of the mutated enzymes and interactions of inhibitors were significantly different from those of TEM-60, revealing that the L51P mutation plays an important role in enzyme activity and stability in the TEM-60 background.

scholarly journals Produksi dan karakterisasi lakase Omphalina sp. Production and characterization of Omphalina sp. laccase

2016 ◽  
Vol 75 (2) ◽  
Author(s):  
. SISWANTO ◽  
. SUHARYANTO ◽  
Rossy FITRIA
Keyword(s):  
Oil Palm ◽  
Laccase Activity ◽  
Research Result ◽  
White Rot Fungi ◽  
White Rot ◽  
Partial Purification ◽  
Empty Fruit Bunches ◽  
Optimum Ph ◽  
Laccase Enzyme

SummaryOmphalina sp. a white-rot fungi (WRF)originated from oil palm plantation has abilityto degrade empty fruit bunches of oil palm(EFBOP) so that it is expected to producelaccase with high activity. The ability ofOmphalina sp. to produce laccase enzyme onliquid fermentation will be studied. The enzymewill also be partially purified andcharacterized. The research result showed thatthe highest enzyme activity (1.162 U/mL) wasobtained using glucose malt yeast (GMY)medium at room temperature for four days.The addition of 2,5-xylidine as an inducerproduced laccase earlier i.e two days, but theactivity of laccase was less active afterprolonged incubation compared to that ofcontrol. The laccase produced on mediumcontaining 2% EFBOP reached optimumactivity as much as 0.38 U/mL after 10 th daysof incubation. Partial purification of laccaseon Sephacryl S-200 HR column resulted58.23% of yield recovery with twice purity thanbefore. The optimum pH of laccase was 4.5.Laccase activity was stable even after heatedon 50 o C for 30 minutes, but then decreasedwhen heated until 60 o C. The laccase has K Mand V max as much as 0.15 mM and 0.56 U/mLrespectively.RingkasanOmphalina sp., adalah fungi pelapuk putih(FPP) hasil isolasi dari kebun kelapa sawityang diketahui mampu mendegradasi tandankosong kelapa sawit (TKKS) dengan cepatsehingga diharapkan mampu menghasilkanlakase dengan aktivitas tinggi. KemampuanOmphalina sp. menghasilkan enzim lakasepada fermentasi cair akan dipelajari. Selain itu,lakase yang dihasilkan akan dimurnikan secaraparsial serta dilakukan karakterisasi pH, suhu,dan konsentrasi substrat optimum. Hasilpenelitian menunjukkan bahwa Omphalina sp.menghasilkan lakase dengan aktivitas tertinggi(1,162 U/mL) pada medium glucose malt yeast(GMY) yang diinkubasikan pada suhu ruangselama empat hari. Penambahan 2,5-xilidinsebagai induser mempercepat produksi lakaselebih awal yaitu dalam waktu dua hari, namunaktivitasnya masih lebih rendah dibandingkandengan kontrol pada inkubasi lebih lanjut.Lakase dari Omphalina sp. juga dapatdiproduksi pada medium yang mengandung2% TKKS dan aktivitasnya mencapai0,38 U/mL yang diinkubasi dalam suhu ruangselama 10 hari. Pemurnian parsial pada kolomSephacryl S-200 HR menghasilkan rendemensebesar 58,23% dengan kemurnian dua kalinya.Aktivitas lakase optimum pada pH 4,5 dantetap stabil setelah pemanasan selama 30 menitpada suhu ruang hingga 50 o C dan menuruntajam pada suhu 60 o C. Lakase Omphalina sp.menghasilkan nilai K M dan V maks masing-masing sebesar 0,15 mM dan 0,56 U/mL.

scholarly journals Partial Purification and Characterization of Inulinase Produced from a local isolate of Aspergillus niger J3

2010 ◽  
Vol 4 (2) ◽  
pp. 78-85
Author(s):  
Jasim M. Awdaa
Keyword(s):  
Aspergillus Niger ◽  
Gel Filtration ◽  
Partial Purification ◽  
Mercury Chloride ◽  
Purification And Characterization ◽  
Original Activity ◽  
Local Isolate ◽  
Optimum Ph ◽  
Final Purification

Inulinase was produced from local isolate of Aspergillus niger J3. The inulinase was purified by two steps included precipitation by amonium sulphate at (30-80) % sa-turation and gel filtration on sephadex G100. The final purification folds and the yield of the enzyme were 3.15 times and 28.24%, respectively. The purified enzyme has the following characteristics: The optimum pH of the enzyme activity was 5.5. The enzyme was most stable at pH (4.5 - 6). The optimum temperature for its activity was 45c. The enzyme retained its original activity when incubates at (30-55) c for 20 minutes. Mercury chloride inhibited the enzyme completely at concentration of 10mM, cupper sulphate and calisium chloride inhibited the enzyme at concentrations of 85% and 7% respectively. It was revealed that the enzyme had the efficiency to hydrolyze 87% of 5% inulin solution when treated at 45c for 120 min.

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