scholarly journals Expression and characterization of Trichoderma reesei endoglucanase II in Pichia pastoris under the regulation of the GAP promoter

2020 ◽  
Vol 25 (2) ◽  
pp. 127
Author(s):  
Kezia Abib Yerah Tjandra ◽  
Kartika Sari Dewi ◽  
Asrul Muhamad Fuad ◽  
Trisanti Anindyawati
Keyword(s):  
Pichia Pastoris ◽  
Trichoderma Reesei ◽  
Specific Activity ◽  
Catalytic Efficiency ◽  
Endoglucanase Activity ◽  
Gap Promoter ◽  
Sds Page ◽  
Optimum Ph ◽  
High Concentrations

Trichoderma reesei is known to be one of the organisms capable for producing various types of cellulase in high concentrations. Among these cellulases, the highest catalytic efficiency of endoglucanases II (EGII, EC 3.2.1.4) are considered important for industrial application. The characterization of the EGII is necessary since it is widely used in high-temperature reactions in the industries. In this study, the recombinant EGII protein was expressed in Pichia pastoris and it has a molecular mass of approximately 52 kDa. Recombinant EGII was purified using Ni-NTA affinity chromatography and characterized by SDS-PAGE and western blot analyses. The enzyme activity of recombinant EGII was measured using the Nelson Somogyi method to determine its optimum pH and temperature. The result showed that the maximum EGII expression was achieved after 72 h of culture incubation. The crude enzyme has optimum activity at pH 5.0, resulting in 16.3 U/mL and 14.6 U/mL activity at 40 °C and 50 °C, respectively. While the purified enzyme gave the specific activity of 115.7 U/mg under the optimum condition. Finally, our study demonstrated that recombinant EGII could retain the endoglucanase activity for 89% and 80% at 40 °C and 50 °C, respectively.

scholarly journals Peroxidase from Coleus Forskohlii: Purification and Biochemical Characterization

2020 ◽  
Vol 5 (1) ◽  
pp. 9-20
Author(s):  
Yaaser Q. Almulaiky ◽  
Yaaser Q. Almulaiky
Keyword(s):  
Ion Exchange ◽  
Peroxidase Activity ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Gel Filtration ◽  
Catalytic Efficiency ◽  
Industrial Applications ◽  
Coleus Forskohlii ◽  
Sds Page ◽  
Optimum Ph

In this study, a peroxidase from new source was purified using ion exchange and gel filtration techniques. The recovery for peroxidase activity was 19% with 11-fold purification and specific activity of 749 unit/mg protein. Purified peroxidase demonstrated a molecular mass of 39 kDa using gel filtration and was confirmed as a single band on SDS-PAGE. The purified peroxidase revealed a broad optimum pH activity at 6.0-6.5 and 50°C temperature. The kinetic parameters for purified peroxidase toward H2O2 and guaiacol as substrates were found to be Km = 3.355, 5.395 mM, Kcat = 99.52, 79.56 s-1 and Vmax =1.531, 1.242 µmole ml-1 min-1, respectively. The catalytic efficiency (kcat/Km) of the purified peroxidase was 14.75 and 29.66 s−1 mM−1 for guaiacol and H2O2, respectively. Peroxidase activity was observed to be enhanced by Cu2+, Co2+, Ni2+ and inhibited in the presence of Sn2+, Al3+, Hg2+, NaN3, EDTA and urea. Characterization showed that peroxidase purified from C. forskohlii has the ability to be used for food industrial applications.

Expression of Xylanase Gene from Pyromyces finnis in Pichia pastoris and Characterization of Recombinant Protein

2019 ◽  
Vol 35 (4) ◽  
pp. 24-32 ◽  
Author(s):  
L.N. Borschevskaya ◽  
T.L. Gordeeva ◽  
S.P. Sineoky
Keyword(s):  
Pichia Pastoris ◽  
Specific Activity ◽  
Ministry Of Education ◽  
Kurchatov Institute ◽  
Putative Signal Peptide ◽  
Neocallimastix Patriciarum ◽  
Optimum Ph ◽  
Sequence Encoding ◽  
Positive Effect

The heterologous expression and characteristics of a new xylanase from Pyromyces finnis have been described. The endo-l,4-β-xylanase XylP (EC 3.2.1.8) consists of 223 amino acids and 19 residues of a putative signal peptide in the N-terminal region. The amino acid sequence of the mature protein has the greatest homology with the sequence of the native catalytic N-terminal domain of Neocallimastix patriciarum endo-l,4-β-xylanase (84%). A synthetic nucleotide sequence encoding a mature XylP protein was expressed in Pichia pastoris. The purified recombinant enzyme showed activity with birch xylan and arabinoxylan. When using birch xylan as a substrate, the optimum pH for the enzyme was 5.0, and the optimum temperature was 50 °C. The specific activity of the xylanase was 4700 U/mg protein, and Km and Vmax were equal to 0.51 mg/mL and 7395.3 umol/(min∙mg), respectively. The recombinant XylP protein showed moderate thermal stability and high pH stability, resistance to digestive enzymes and protein inhibitors of grain xylanases. It was also shown that the Mg2+, Co2+ and Li+ ions have a positive effect on the enzyme activity. xylanase, xylan, feed enzyme, Pichia pastoris, Pyromyces finnis The work was performed with the financial support of the Ministry of Education and Science of Russia (Unique Project Identifier RFMEFI60717X0180) using the Unique Scientific Installation -National Bioresource Center «All-Russian Collection of Industrial Microorganisms», NRC «Kurchatov Institute» - GOSNIIGENETIKA

scholarly journals Biosynthesis, purification, characterization and immobilization of laccase from Lithothelium sp.

Chemija ◽  
2020 ◽  
Vol 31 (3) ◽  
Author(s):  
Ingrida Radveikienė ◽  
Ingrida Pilotaitė ◽  
Rimgailė Dainytė ◽  
Regina Vidžiūnaitė
Keyword(s):  
Residual Activity ◽  
Catalytic Efficiency ◽  
Hydrophobic Interaction Chromatography ◽  
Metal Salts ◽  
Biotechnological Applications ◽  
Affinity Constants ◽  
Sds Page ◽  
Wide Range ◽  
Optimum Ph ◽  
Sulphate Salts

Novel fungal laccase isoenzymes (namely L95-1 and L95-2) produced by the Ascomycete Lithothelium sp. isolated from the forest soil were purified. However, only one of them was characterized, because the other isoenzyme lost its activity during purification. Extracellular L95-1 laccase was purified 30-fold using ion-exchange and hydrophobic interaction chromatography, with an overall yield of 88%. The molecular mass of purified L95-1 was estimated to be 85 kDa by SDS-PAGE analysis. L95-1 laccase was stable at temperature 4–22°C and pH 6.0–6.5. The substrate specificity of L95-1 laccase was examined with various compounds. Determined affinity constants (KM) varied in a wide range of 3.7–2020.0 µM, whereas catalytic efficiency constants (kcat/KM) covered a range of 0.008–1.9 µM–1 s–1. The optimum pH for most substrates varied in a range from pH 5.0 to 6.0. Sodium azide and fluoride strongly inhibited L95-1 activity, whereas sulphate salts inhibited weakly. The laccase was immobilized on the Fe3O4 nanoparticles and characterized. Residual activity remained at 20% after ten cycles of ABTS oxidation reaction. The immobilized laccase showed higher tolerance to various metal salts. The properties of L95-1 laccase make it potentially useful in the biotechnological applications.

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

2017 ◽  
Vol 18 (2) ◽  
pp. 1-10 ◽  
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 60°C.

scholarly journals Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium, Halomonas elongata

1999 ◽  
Vol 181 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Hisayo Ono ◽  
Kazuhisa Sawada ◽  
Nonpanga Khunajakr ◽  
Tao Tao ◽  
Mihoko Yamamoto ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Mass ◽  
Optimum Temperature ◽  
Sds Page ◽  
Halomonas Elongata ◽  
Optimum Ph

ABSTRACT 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic β-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with l-glutamate. This enzyme required pyridoxal 5′-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25°C and had Km s of 9.1 mM forl-glutamate and 4.5 mM for dl-ASA. DABA acetyltransferase catalyzed acetylation of DABA to γ-N-acetyl-α,γ-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0.77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30°C.

scholarly journals Purification and characterization of cytosolic pyruvate kinase from banana fruit

2000 ◽  
Vol 352 (3) ◽  
pp. 875-882 ◽  
Author(s):  
William L. TURNER ◽  
William C. PLAXTON
Keyword(s):  
Pyruvate Kinase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Banana Fruit ◽  
Ph Optimum ◽  
Cytosolic Ph ◽  
Pep Carboxylase ◽  
Sds Page ◽  
Optimal Efficiency

Cytosolic pyruvate kinase (PKc) from ripened banana (Musa cavendishii L.) fruits has been purified 543-fold to electrophoretic homogeneity and a final specific activity of 59.7µmol of pyruvate produced/min per mg of protein. SDS/PAGE and gel-filtration FPLC of the final preparation indicated that this enzyme exists as a 240kDa homotetramer composed of subunits of 57kDa. Although the enzyme displayed a pH optimum of 6.9, optimal efficiency in substrate utilization [in terms of Vmax/Km for phosphoenolpyruvate (PEP) or ADP] was equivalent at pH6.9 and 7.5. PKc activity was absolutely dependent upon the presence of a bivalent and a univalent cation, with Mg2+ and K+ respectively fulfilling this requirement. Hyperbolic saturation kinetics were observed for the binding of PEP, ADP, Mg2+ and K+ (Km values of 0.098, 0.12, 0.27 and 0.91mM respectively). Although the enzyme utilized UDP, IDP, GDP and CDP as alternative nucleotides, ADP was the preferred substrate. L-Glutamate and MgATP were the most effective inhibitors, whereas L-aspartate functioned as an activator by reversing the inhibition of PKc by L-glutamate. The allosteric features of banana PKc are compared with those of banana PEP carboxylase [Law and Plaxton (1995) Biochem. J. 307, 807Ő816]. A model is presented which highlights the roles of cytosolic pH, MgATP, L-glutamate and L-aspartate in the co-ordinate control of the PEP branchpoint in ripening bananas.

scholarly journals Purification and Characterization of Endoglucanase from local isolate of Aspergillus flavus

2013 ◽  
Vol 10 (3) ◽  
pp. 844-853
Author(s):  
Baghdad Science Journal
Keyword(s):  
Aspergillus Flavus ◽  
Specific Activity ◽  
Gel Filtration ◽  
Temperature Stability ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Original Activity ◽  
A Value ◽  
Optimum Ph

Endoglucanase produced from Aspergillus flavus was purified by several steps including precipitation with 25 % ammonium sulphate followed by Ion –exchange chromatography, the obtained specific activity was 377.35 U/ mg protein, with a yield of 51.32 % .This step was followed by gel filtration chromatography (Sepharose -6B), when a value of specific activity was 400 U/ mg protein, with a yield of 48 %. Certain properties of this purified enzyme were investigated, the optimum pH of activity was 7 and the pH of its stability was 4.5, while the temperature stability was 40 °C for 60 min. The enzyme retained 100% of its original activity after incubation at 40 °C for 60 min; the optimum temperature for enzyme activity was 40 °C.

scholarly journals Purification and characterization of a thermostable ADP-glucose pyrophosphorylase from Thermus caldophilus GK-24

1996 ◽  
Vol 319 (3) ◽  
pp. 977-983 ◽  
Author(s):  
Jeong Heon KO ◽  
Cheorl Ho KIM ◽  
Dae-Sil LEE ◽  
Yu Sam KIM
Keyword(s):  
Molecular Mass ◽  
Specific Activity ◽  
Gel Filtration ◽  
Higher Plant ◽  
Sds Page ◽  
Thermus Caldophilus ◽  
Internal Peptide ◽  
Adp Glucose Pyrophosphorylase ◽  
Fructose 1,6 Bisphosphate

An extremely thermostable ADP-glucose pyrophosphorylase (AGPase) has been purified from Thermus caldophilus GK-24 to homogeneity by chromatographic methods, including gel filtration and ion-exchange and affinity chromatography. The specific activity of the enzyme was enriched 134.8-fold with a recovery of 10.5%. The purified enzyme was a single band by SDS/PAGE with a molecular mass of 52 kDa. The homotetrameric structure of the native enzyme was determined by gel filtration analysis, which showed a molecular mass of 230 kDa on a Superose-12 column, indicating that the structure of the enzyme is different from the heterotetrameric structures of higher-plant AGPases. The enzyme was most active at pH 6.0. The activity was maximal at 73–78 °C and its half-life was 30 min at 95 °C. Kinetic and regulatory properties were characterized. It was found that AGPase activity could be stimulated by a number of glycolytic intermediates. Fructose 6-phosphate, fructose 1,6-bisphosphate, phenylglyoxal and glucose 6-phosphate were effective activators, of which fructose 1,6-bisphosphate was the most effective. The enzyme was inhibited by phosphate, AMP or ADP. ATP and glucose 1-phosphate gave hyperbolic-shaped rate-concentration curves in the presence or absence of activator. A remarkable aspect of the amino acid composition was the existence of the hydrophobic and Ala+Gly residues. The N-terminal and internal peptide sequences were determined and compared with known sequences of various sources. It was apparently similar to those of AGPases from other bacterial and plant sources, suggesting that the enzymes are structurally related.

The assay and partial characterization of 3β-hydroxysteroid sulfotransferase of the hamster epididymis

1985 ◽  
Vol 63 (1) ◽  
pp. 71-76 ◽  
Author(s):  
M. Bouthillier ◽  
G. Bleau ◽  
A. Chapdelaine ◽  
K. D. Roberts
Keyword(s):  
Organic Phase ◽  
Enzyme Preparation ◽  
Enzymatic Reaction ◽  
Partial Characterization ◽  
Optimal Conditions ◽  
Steroid Nucleus ◽  
Optimum Ph ◽  
High Concentrations

Using a partially purified enzyme preparation obtained from hamster epididymis, a simple assay has been developed to measure the sulfurylation of dehydroisoandrosterone (DHA) and desmosterol in the presence of 3′-phosphoadenosine 5′-phospho[35S]sulfate ([35S]PAPS). After stopping the enzymatic reaction with methanol and KCl, the 35S-labelled steroid sulfates are readily extracted into an organic phase. Optimal conditions for the sulfurylation of the two steroids were compared; optimum pH is 8.7 for DHA and 9.8 for desmosterol. Sulfoconjugation of desmosterol increases with magnesium concentrations up to 6 mM, while 40 mM concentrations of the divalent ion are required for the optimal sulfurylation of DHA. Maximum sulfurylation of these steroids requires the presence of 15 mM cysteine. Michaelis–Menten kinetics are observed with DHA which has an apparent Km of 32 μM, while desmosterol inhibits sulfotransferase activity at high concentrations. Saturation of the enzyme with PAPS results in an allosteric behaviour. Only the 3β-hydroxyl function of the steroid nucleus appears to be an appropriate sulfate acceptor for the epididymal hydroxysteroid sulfotransferase.

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