scholarly journals Purification, Characterization and De-Staining Potentials of a Thermotolerant Protease Produced by Fusarium oxysporum

2019 ◽  
Vol 64 (4) ◽  
pp. 539-547
Author(s):  
Mohammed Inuwa Ja’afaru ◽  
Konjerimam Ishaku Chimbekujwo ◽  
Obinna Markraphael Ajunwa
Keyword(s):  
Enzyme Activity ◽  
Fusarium Oxysporum ◽  
Treatment Time ◽  
Specific Activity ◽  
Total Yield ◽  
Tween 20 ◽  
Optimum Temperature ◽  
Industrial Enzymes ◽  
Sds Page ◽  
Optimum Ph

Proteases are important industrial enzymes and fungi prove to be good sources of such enzymes. Purification techniques are however necessary for increased specificity in activity and better industrial value. Based on this, a protease produced by a Fusarium oxysporum was purified to homogeneity by Sephadex G-200 column and α–casein agarose chromatography. The enzyme had a molecular weight of 70 kDa in SDS-PAGE. Purified Fusarium oxysporum protease had a specific activity of 93.88 U/mg protein. The purification magnitude was 7.7 and the total yield was 20 %. Purified protease had an optimum pH of 5.0 while the optimum temperature was 40 °C. The enzyme was also thermotolerant (approximately 100 % at 40 °C for 2 h). The enzyme activity was stimulated by surfactants and metal ions like, Tween-20 and Mg2+. Enzyme activity was inhibited in presence of PMSF and EDTA. Casein was found to be the best substrate for protease activity of Fusarium oxysporum FWT1. Protease were tested upon blood stain for de-clotting of blood and was found to exhibit good de-clotting and de-staining activity after 15 minutes treatment time.

Molecular characterization of an endo-type chitosanase from the fish pathogenRenibacteriumsp. QD1

2014 ◽  
Vol 94 (4) ◽  
pp. 681-686 ◽  
Author(s):  
Peichuan Xing ◽  
Dan Liu ◽  
Wen-Gong Yu ◽  
Xinzhi Lu
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Specific Activity ◽  
Fermentation Broth ◽  
Maximal Activity ◽  
Optimum Temperature ◽  
Sds Page ◽  
Ph Range ◽  
Tlc Analysis

Renibacteriumsp. QD1, a bacteria strain capable of hydrolysing chitosan, was isolated from the homogenate of small crabs. An extracellular chitosanase, Csn-A, was purified from the QD1 fermentation broth. The enzyme was purified to homogeneity, with a yield of eight-fold, 67% recovery and a specific activity of 1575 U/mg proteins. The molecular weight of Csn-A was estimated to be 26.1 kDa by SDS-PAGE. Unlike other chitosanases, the purified Csn-A displayed maximal activity at a pH range of 5.3–6.5, and it was stable in a broad pH range of 5.0–10.0. The optimum temperature for chitosanlytic activity was 55°C. The enzyme activity was strongly stimulated by Mn2+but inhibited by Fe3+, Cu2+, Al3+, Zn2+and SDS. TLC analysis demonstrated that Csn-A hydrolysed N-deacetylated polymeric glucosamines into chito-biose and -triose in an endo-type manner. The amino acid seuquence of Csn-A showed close identity with an uncharacterized chitosanase of strain ATCC33209.

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 Studies on the production of glucose isomerase by Bacillus licheniformis

2015 ◽  
Vol 17 (3) ◽  
pp. 84-88 ◽  
Author(s):  
Ogbonnaya Nwokoro
Keyword(s):  
Enzyme Activity ◽  
Bacillus Licheniformis ◽  
Organic Nitrogen ◽  
Specific Activity ◽  
Inorganic Nitrogen ◽  
Glucose Isomerase ◽  
Culture Conditions ◽  
Enzyme Productivity ◽  
Carbon Substrates ◽  
Optimum Ph

Abstract This work reports the effects of some culture conditions on the production of glucose isomerase by Bacillus licheniformis. The bacterium was selected based on the release of 3.62 mg/mL fructose from the fermentation of glucose. Enzyme was produced using a variety of carbon substrates but the highest enzyme activity was detected in a medium containing 0.5% xylose and 1% glycerol (specific activity = 6.88 U/mg protein). Media containing only xylose or glucose gave lower enzyme productivies (specific activities= 4.60 and 2.35 U/mg protein respectively). The effects of nitrogen substrates on glucose isomerase production showed that yeast extract supported maximum enzyme activity (specific activity = 5.24 U/mg protein). Lowest enzyme activity was observed with sodium trioxonitrate (specific activity = 2.44 U/mg protein). In general, organic nitrogen substrates supported higher enzyme productivity than inorganic nitrogen substrates. Best enzyme activity was observed in the presence of Mg2+ (specific activity = 6.85 U/mg protein) while Hg2+ was inhibitory (specific activity = 1.02 U/mg protein). The optimum pH for best enzyme activity was 6.0 while optimum temperature for enzyme production was 50ºC.

scholarly journals Isolation, Purification, and Characterization of Fungal Laccase from Pleurotus sp.

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Sunil S. More ◽  
Renuka P. S. ◽  
Pruthvi K. ◽  
Swetha M. ◽  
S. Malini ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Laccase Activity ◽  
Gel Filtration ◽  
Industrial Enzymes ◽  
Purification And Characterization ◽  
Sds Page ◽  
Concomitant Reduction ◽  
The One ◽  
Inhibited Enzyme

Laccases are blue copper oxidases (E.C. 1.10.3.2 benzenediol: oxygen oxidoreductase) that catalyze the one-electron oxidation of phenolics, aromatic amines, and other electron-rich substrates with the concomitant reduction of O2 to H2O. They are currently seen as highly interesting industrial enzymes because of their broad substrate specificity. A positive strain was isolated and characterized as nonspore forming Basidiomycetes Pleurotus sp. Laccase activity was determined using ABTS as substrate. Laccase was purified by ionexchange and gel filtration chromatography. The purified laccase was a monomer showed a molecular mass of 40±1 kDa as estimated by SDS-PAGE and a 72-fold purification with a 22% yield. The optimal pH and temperature were 4.5 and 65°C, respectively. The Km and Vmax⁡ values are 250 (mM) and 0.33 (μmol/min), respectively, for ABTS as substrate. Metal ions like CuSO4, BaCl2, MgCl2, FeCl2, ZnCl2 have no effect on purified laccase whereas HgCl2 and MnCl2 moderately decrease enzyme activity. SDS and sodium azide inhibited enzyme activity, whereas Urea, PCMB, DTT, and mercaptoethanol have no effect on enzyme activity. The isolated laccase can be used in development of biosensor for detecting the phenolic compounds from the effluents of paper industries.

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 Isolation and caracterization of ficin enzyme from Ficus septica Burm F stem latex

2017 ◽  
Vol 20 (2) ◽  
pp. 161
Author(s):  
Sri Wahyuni ◽  
R. Susanti ◽  
Retno Sri Iswari
Keyword(s):  
Enzyme Activity ◽  
Column Chromatography ◽  
Temperature Treatment ◽  
Optimum Temperature ◽  
Optimum Ph ◽  
Incubation Temperatures ◽  
Joint Treatment ◽  
Plant Latex

This research aims to isolate and characterize the fcin enzyme from Ficus septica stem latex. Ficin from Ficus septica stem latex was isolated using column chromatography. Then enzyme activity was tested at different temperature (40oC, 50oC, 60oC, 70oC) and pH (6.0, 7.0, 8.0) levels. Ficin enzyme activity of joint treatment with variations in temperature and pH was analyzed using two-way ANOVA with a factorial pattern followed by Least Signifcant Difference (LSD) test. The results showed that temperature treatment signifcantly affects enzyme activity. However, the treatment of pH and the interaction between temperature and pH did not signifcantly affect the fcin enzyme activity. There was no signifcant difference in fcin activity at the incubation temperatures of 40oC and 50oC, as well as 60oC and 70oC. However, comparing the incubation temperatures of 40oC and 50oC with treatment 60°C and 70°C showed a signifcant difference in fcin enzyme activity. In the treatment of incubation at pH 6, 7 and 8 for fcin enzyme activity showed no signifcant difference. We concluded that the Ficus septica plant latex contained fcin enzyme with an optimum temperature of 60°C and optimum pH of 6, 7, and 8.

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.

scholarly journals Cyclodextrin production by Bacillus lehensis isolated from cassava starch: Characterisation of a novel enzyme

2014 ◽  
Vol 32 (No. 1) ◽  
pp. 48-53 ◽  
Author(s):  
K.C. Blanco ◽  
F.F. de Moraes ◽  
N.S. Bernardi ◽  
M.H.P.B. Vettori ◽  
R. Monti ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Maximum Velocity ◽  
Temperature Stability ◽  
Cassava Starch ◽  
Optimum Temperature ◽  
Cyclodextrin Glycosyltransferase ◽  
Sds Page ◽  
Optimum Ph ◽  
Menten Constant ◽  
Cyclodextrin Production

The properties of a previously unknown enzyme, denominated cyclodextrin glycosyltransferase, produced from Bacillus lehensis, were evaluated using affinity chromatography for protein purification. Enzyme characteristics (optimum pH and temperature; pH and temperature stability), the influence of substances on the enzyme activity, enzyme kinetics, and cyclodextrin production were analysed. Cyclodextrin glycosyltransferase was purified up to 320.74-fold by affinity chromatography using β-cyclodextrin as the binder and it exhibited 8.71% activity recovery. This enzyme is a monomer with a molecular weight of 81.27 kDa, as estimated by SDS-PAGE. Optimum temperature and pH for cyclodextrin glycosyltransferase were 55°C and 8.0, respectively. The Michaelis-Menten constant was 8.62 g/l during maximum velocity of 0.858 g/l.h.  

scholarly journals ISOLASI DAN KARAKTERISASI EKSTRAK KASAR ENZIM BROMELIN DARI BATANG NANAS (Ananas comusus L.merr)

2006 ◽  
Vol 12 (1) ◽  
pp. 75-77
Author(s):  
Nuniek Herdyastuti
Keyword(s):  
Enzyme Activity ◽  
Crude Extract ◽  
Soluble Protein ◽  
Food Industry ◽  
Optimum Temperature ◽  
Ph Variation ◽  
Isolation And Characterization ◽  
Optimum Ph

Brommelain is an enzyme hydrolyze most soluble protein easily and efficiently. This enzyme is used in many industry like food industry. This research aimed to isolation and characterization crude extract brommelain. This enzyme has been extracted from the stems of pineapples to produce crude extract, precipitated with amonium sulfat, and enzyme activity to decided with Bergmeyer methode. The higher activity was 1,996 U/ml in precipitate 40-60 percent amonium sulfat. Optimum temperature and pH to decided temperature and pH variation was detected based on enzyme activity. Characterization to indicate that bromelain has an optimum temperature at 55°C, optimum pH of 7, KM = 5.074 mg/ml and Vmax = 0.666 mg/ml.second.

Screening of the Bacterium for Chlorobenzene Degradation and its Enzymatic Properties

2012 ◽  
Vol 610-613 ◽  
pp. 404-408
Author(s):  
Xian Niu ◽  
Cheng Ding ◽  
Jin Long Yan ◽  
Bai Ren Yang
Keyword(s):  
Enzyme Purification ◽  
Extracellular Enzymes ◽  
Ph Value ◽  
Morphological Characteristics ◽  
Enzymatic Properties ◽  
Purification Process ◽  
Optimum Temperature ◽  
16S Rdna Sequence ◽  
Sds Page ◽  
Optimum Ph

A dominant bacterium (LW13) for the degradation of chlorobenzene was selected from maturity sludge in a novel combined bio-filter polluted by chlorobenzene gas. Based on the morphological characteristics observation, physio-biochemical characteristics and 16S rDNA sequence homology analysis, strain LW13 was identified as Lysinibacillus fusiformis. Crude enzyme from the fermentation was extracted and their enzymatic properties were also investigated. Results showed that the degradation enzyme produced by the bacteria belong to extracellular enzymes. The purity of the enzyme was determined by SDS-PAGE gel electrophoresis and the molecular weight was found to be 52 kDa. The optimum pH value was about 8.0 with the optimum temperature of 45° C. Throughout the purification process, 85-fold of enzyme purification was achieved with the recovery of 20.69%.

Sign in / Sign up

Export Citation Format

Share Document