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 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 Purificación Parcial y Caracterización de Alfa Amilasa de granos germinados de Chenopodium quinoa (Quinua)

2018 ◽  
pp. 52-58
Keyword(s):  
Specific Activity ◽  
Chenopodium Quinoa ◽  
Maximum Activity ◽  
Alpha Amylase ◽  
Partial Purification ◽  
Chemical Hydrolysis ◽  
Germination Process ◽  
Sds Page ◽  
Global Population

Purificación Parcial y Caracterización de Alfa Amilasa de granos germinados de Chenopodium quinoa (Quinua) Partial Purification and Characterization of Alpha Amylase from germinated grains from Chenopopdium quinoa (Quinua) Melissa Bedón Gómez, Oscar Nolasco Cárdenas, Carlos Santa Cruz C. y Ana I. F. Gutiérrez Román Universidad Nacional Federico Villarreal, Facultad de Ciencias Naturales y Matemática, Laboratorio de Bioquímica y Biología Molecular, Jr. Río Chepén S/N, El Agustino. Telefax: 362 - 3388 DOI: https://doi.org/10.33017/RevECIPeru2013.0007/ Resumen Las alfa amilasas son las enzimas más estudiadas e importantes en el campo biotecnológico e industrial; ya que han reemplazado por completo la hidrólisis química del almidón. Estas enzimas son imprescindibles en la elaboración de productos alimenticios, combustibles, medicamentos y detergentes con la finalidad de optimizar procesos y conservar el medio ambiente. La α-amilasa puede ser purificada de diferentes organismos como plantas, animales, hongos y bacterias; actualmente un gran número de α-amilasas bacterianas en especial del género Bacillus están disponibles comercialmente y son las más utilizadas en las industrias. Sin embargo, la producción de éstas no satisfacen los requerimientos industriales en el mundo; ya que, la demanda de esta enzima se ha incrementado en los últimos dos años y el empleo de α-amilasas bacterianas ha provocado alergias afectando al 15% de la población a nivel mundial. . En este estudio, como fuente de α-amilasa se emplearon semillas de Chenopodium quinoa (quinua) var hualhuas blanca durante el proceso de germinación; esta enzima fue parcialmente purificada por precipitación con sulfato de amonio obteniendo una actividad específica final de 35.60U/mg y un grado de purificación de 5 veces. La purificación fue confirmada por SDS-PAGE, encontrando un peso molecular de 44kDa. La actividad enzimática se evaluó mediante el método de Miller mostrando máxima actividad a pH 7 y a temperatura de 37ºC. La linealización de Lineweaver-Burk nos dio un Km de 16mg/mL y Vmax de 100µM de maltosa/min. Por lo tanto, esta caracterización reúne los pre-requisitos necesarios para la aplicación en la industria. Descriptores: Chenopodium quinoa, alfa amilasa, germinación, purificación parcial. Abstract The alpha amylases are the enzymes most studied and important in biotechnology and industry; because they have completely replaced the starch’s chemical hydrolysis. These enzymes are essential in the food production, medicines and detergents in order to optimize processes and conserve the environment. The α-amylase can be isolated from different organisms such as plants, animals, fungi and bacteria, now a large number of bacterial α-amylases especially from genus Bacillus are commercially available and they are the most used in industry. However, the production of these do not meet industry requirements in the world, because the demand for this enzyme has increased in the last two years and the use of bacterial α-amilase has caused allergies affecting the 15% of the global population. In this study, as a source of α-amylase used the seeds from Chenopodium quinoa (quinoa). Var. white hualhuas during the germination process, this enzyme was partially purified by ammonium sulfate precipitation to obtain a final specific activity of 35.60U/mg, and a grade of purification of 5 times. The purification was confirmed by SDS-PAGE, where the molecular weight was 44kDa. The enzyme activity was evaluated by Miller method showing maximum activity at pH 7 and 37ºC. The Lineweaver-Burk linearization shows a Km of 16mg/mL and Vmax of 100μM the maltose / min. Therefore, these characterizations meet the prerequisites need for industry. Keywords: Chenopodium quinoa; alpha amylase; germination; partial purification

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.

Electrophoretic characterization of endo-(1,4)-β-glucanases secreted during assimilative growth and antheridiol-induced branching inAchlya ambisexualis

1996 ◽  
Vol 42 (6) ◽  
pp. 557-561 ◽  
Author(s):  
Terry W. Hill
Keyword(s):  
Enzyme Activity ◽  
Sodium Dodecyl Sulfate ◽  
Cell Walls ◽  
Sodium Dodecyl ◽  
Achlya Ambisexualis ◽  
Dodecyl Sulfate ◽  
Exclusion Chromatography ◽  
Molecular Masses

Secreted endo-(1,4)-β-glucanases ("cellulases") of Achlya ambisexualis were analyzed by a technique that permits visualization of enzyme activity in situ after electrophoresis in gels containing sodium dodecyl sulfate. Catalytic polypeptides with molecular masses of about 97, 74, 36, 29, and 25 kDa were observed in media from young cultures, though progressively fewer bands were observed as cultures aged. Based on size estimations of native enzymes with gel exclusion chromatography, the 97- and 36-kDa polypeptides were concluded to be subunits of a 245-kDa holoenzyme and the 25-kDa polypeptides were concluded to be subunits of a second holoenzyme of about 92 kDa. The data were insufficient to allow similar assignments for the more ephemeral 74- and 29-kDa polypeptides. The endoglucanases secreted during branch induction by antheridiol or 0.2% peptone comigrated in electrophoretic gels with enzymes secreted during normal assimilative growth. No endoglucanases specific to induced branching were observed.Key words: oomycetes, cell walls, endoglucanases, cellulases, antheridiol.

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 Purification and Characterization of a Novel 5-Oxoprolinase (without ATP-Hydrolyzing Activity) fromAlcaligenes faecalis N-38A

1999 ◽  
Vol 65 (2) ◽  
pp. 712-717 ◽  
Author(s):  
Atsuhisa Nishimura ◽  
Yasunori Ozaki ◽  
Hiroshi Oyama ◽  
Takashi Shin ◽  
Sawao Murao
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Alcaligenes Faecalis ◽  
Cell Extract ◽  
Homogeneous State ◽  
Amino Terminal ◽  
A Cell ◽  
Terminal Amino

ABSTRACT A novel type of 5-oxoprolinase was found in a cell extract of strain N-38A, which was later identified as Alcaligenes faecalis. The enzyme in the cell extract was purified to a homogeneous state with a yield of 16.6%. The molecular weight of the purified enzyme was estimated to be 47,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, suggesting that the enzyme is a monomeric protein. The enzyme specifically catalyzed a decyclization of l-pyroglutamate without hydrolyzing ATP and also without any requirements for metal ions such as Mg2+ and K+. The optimal pH for the decyclization was 7.4. The reaction was reversible. The equilibrium constant of the reaction, K eq = [l-glutamate]/[l-pyroglutamate], was evaluated to be approximately 0.035, which indicates that the reaction tends to form l-pyroglutamate. The amino-terminal amino acid sequence of the enzyme was H-Glu-Pro-Arg-Leu-Asp-Thr-Ser-Gln-Leu-Tyr-Ala-Asp-Val-His-Phe-. No protein with a similar sequence was found in the DNASIS database. Based on these data, it was strongly suggested that the enzyme described here is a novel type of 5-oxoprolinase.

Purification and Partial Characterization of Rat Liver Lipoxygenase

1987 ◽  
Vol 42 (10) ◽  
pp. 1343-1348 ◽  
Author(s):  
Pedro Macias ◽  
M. Carmen Pinto
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Rat Liver ◽  
Polymorphonuclear Leukocytes ◽  
Fluorescence Spectra ◽  
Nordihydroguaiaretic Acid ◽  
Maximum Activity ◽  
Absorption And Fluorescence Spectra ◽  
Lipoxygenase Inhibitor

Abstract Lipoxygenase was purified from rat liver cytosolic fraction by a method involving two successive chromatographic steps on Sephacryl S-200 and Phenyl Sepharose CL-4B. The enzyme has a molecular weight of 96 Kdal and it seems to be composed of two identical subunits. Chromatofocusing of the enzyme revealed a single band of activity at pi 6.3. The enzyme activity of the purified fraction showed maximum activity at pH 7.0 with a Km for linoleic acid of 1.4 μM and is competitively inhibited by the specific lipoxygenase inhibitor nordihydroguaiaretic acid. The purified enzyme shows absorption and fluorescence spectra similar to those of lipoxygenase from other sources. However, the molecular weight of lipoxygenase purified from liver is found to be different from that of the enzyme from polymorphonuclear leukocytes. It is suggested that there are different isoenzymes of lipoxygenases in mammals.

scholarly journals Purification and characterization of a multicatalytic high-molecular-mass proteinase from rat skeletal muscle

1985 ◽  
Vol 228 (1) ◽  
pp. 161-170 ◽  
Author(s):  
B Dahlmann ◽  
L Kuehn ◽  
M Rutschmann ◽  
H Reinauer
Keyword(s):  
Skeletal Muscle ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Maximum Activity ◽  
Disc Electrophoresis ◽  
Polyacrylamide Gels ◽  
Rat Skeletal Muscle ◽  
Muscle Extract

A proteolytic enzyme was purified from the post-myofibrillar fraction of rat skeletal muscle. The purification procedure consisted of fractionation of the muscle extract by (NH4)2SO4, chromatography on DEAE-Sephacel, fast protein liquid chromatography on Mono Q and gel filtration on Sepharose 6B. The enzyme preparation appeared to be homogeneous as judged by disc electrophoresis in polyacrylamide gels and by immunoelectrophoresis. The isoelectric point of the proteinase is at 5.1-5.2. The enzyme has an Mr of about 650 000 and dissociates into eight subunits of Mr 25 000-32 000 when subjected to electrophoresis in sodium dodecyl sulphate/polyacrylamide gels. The proteinase contains hydrolytic activity against N-blocked tripeptide 4-methyl-7-coumarylamide substrates with an arginine or phenylalanine residue adjacent to the leaving group. Maximum activity with the first group of substrates was at pH 10.5, and this activity was inhibited by leupeptin, chymostatin and Ca2+. Maximum activity with the latter group of substrates was at pH 7.5, and was also inhibited by the two microbial inhibitors, but was activated by Ca2+ ions. By using [14C]methylcasein as a substrate, maximum activity was observed at pH9.0, and this proteolytic activity was not affected by leupeptin, was enhanced by chymostatin and inhibited by Ca2+. Similar effects were observed when benzyloxycarbonyl-Leu-Leu-Glu 2-naphthylamide was used as a substrate. These enzymic activities were abolished by p-hydroxymercuribenzenesulphonic acid or mersalyl acid, whereas a small activation was observed with cysteine or dithiothreitol.

scholarly journals Characterization of a Thermostable d-Stereospecific Alanine Amidase from Brevibacillus borstelensis BCS-1

2003 ◽  
Vol 69 (2) ◽  
pp. 980-986 ◽  
Author(s):  
Dae Heoun Baek ◽  
Seok-Joon Kwon ◽  
Seung-Pyo Hong ◽  
Mi-Sun Kwak ◽  
Mi-Hwa Lee ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Optimum Temperature ◽  
Gene Encoding ◽  
Ph Range

ABSTRACT A gene encoding a new thermostable d-stereospecific alanine amidase from the thermophile Brevibacillus borstelensis BCS-1 was cloned and sequenced. The molecular mass of the purified enzyme was estimated to be 199 kDa after gel filtration chromatography and about 30 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme could be composed of a hexamer with identical subunits. The purified enzyme exhibited strong amidase activity towards d-amino acid-containing aromatic, aliphatic, and branched amino acid amides yet exhibited no enzyme activity towards l-amino acid amides, d-amino acid-containing peptides, and NH2-terminally protected amino acid amides. The optimum temperature and pH for the enzyme activity were 85°C and 9.0, respectively. The enzyme remained stable within a broad pH range from 7.0 to 10.0. The enzyme was inhibited by dithiothreitol, 2-mercaptoethanol, and EDTA yet was strongly activated by Co2+ and Mn2+. The k cat/Km for d-alaninamide was measured as 544.4 ± 5.5 mM−1 min−1 at 50°C with 1 mM Co2+.

scholarly journals The deoxyribonucleic acid polymerases from the diatom Cylindrotheca fusiformis. Partial purification and characterization of four distinct activities

1977 ◽  
Vol 167 (3) ◽  
pp. 601-610 ◽  
Author(s):  
T W Okita ◽  
B E Volcani
Keyword(s):  
Deoxyribonucleic Acid ◽  
Maximum Activity ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Cylindrotheca Fusiformis ◽  
High K ◽  
Zinc Chelator ◽  
Relationship Of ◽  
The Relationship

Four extramitochondrial DNA polymerases from the marine photosynthetic diatom Cylindrotheca fusiformis were isolated and purified more than 1200-fold by chromatography on DNA-cellulose and DEAE-Sephadex. The enzymes were equally susceptible to inhibition by the thiol-blocking agents N-ethylmaleimide and p-chloromercuribenzoate, the zinc chelator o-phenathroline, and the nucleic acid interchelators ethidium bromide and acriflavin; they displayed similar pH optima, preferred activated DNA, and had strict dependence on high K+ for maximum activity. They were differentiated on the basis of their kinetic parameters, template-primer utilization and salt requirements. The four activities varied with growth stage of C. fusiformis. Activities of polymerases A and D doubled in exponential-phase cells as compared with those in stationary-phase cells, and the increase in polymerase B and chloroplast activity C was 20-40%. The relationship of the diatom polymerases to the complements in other organisms is discussed.

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