scholarly journals Purification and characterization of a novel laccase from the mushroom Pleurotus nebrodensis.

2012 ◽  
Vol 59 (3) ◽  
Author(s):  
Guo-Ting Tian ◽  
Guo-Qing Zhang ◽  
He-Xiang Wang ◽  
Tzi Bun Ng
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Slight Reduction ◽  
Pleurotus Nebrodensis ◽  
Precipitous Decline ◽  
Ph Range ◽  
Purification Protocol

A novel laccase with a molecular mass of 64 kDa and the N-terminal sequence AIGPDDTINF was isolated from fresh fruiting bodies of the mushroom Pleurotus nebrodensis. The purification protocol comprised ion exchange chromatography on DEAE-cellulose, CM-cellulose, and Q-Sepharose, and gel filtration on Superdex 75. The laccase was adsorbed on DEAE-cellulose and Q-Sepharose, but not on CM-cellulose. It demonstrated an optimal temperature of 70°C. The enzyme activity increased steadily over the temperature range 20°C-70°C. There was only a slight reduction in activity at 80°C. However, all activity disappeared following exposure to 100°C for 10 minutes. The enzyme activity changed only slightly over the pH range 3-5, with the optimum at pH 5, but underwent a precipitous decline when the pH was elevated to 6, and was undetectable at pH 8 and pH 9.

scholarly journals Purification and characterization of acid phosphatase from a germinating black gram (Vigna mungo L.) seedling

2011 ◽  
Vol 63 (3) ◽  
pp. 747-756 ◽  
Author(s):  
A.K.M. Asaduzzaman ◽  
Habibur Rahman ◽  
Tanzima Yeasmin
Keyword(s):  
Acid Phosphatase ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Inhibitory Effect ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Black Gram ◽  
Km Value

An acid phosphatase has been isolated and purified from an extract of a germinating black gram seedling. The method was accomplished by gel filtration of a germinating black gram seedling crude extract on sephadex G-75 followed by ion exchange chromatography on DEAE cellulose. The acid phosphatase gave a single band on SDS-polyacrylamide slab gel electrophoresis. The molecular weight of the acid phosphatase determined by SDS-polyacrylamide slab gel electrophoresis was estimated to be 25 kDa. The purified enzyme showed maximum activity at pH 5 and at temperature of 55?C. Mg2+, Zn2+ and EDTA had an inhibitory effect on the activity of the acid phosphatase. Black gram seedling acid phosphatase was activated by K+, Cu2+ and Ba2+. The Km value of the enzyme was found to be 0.49 mM for pNPP as substrate.

scholarly journals PURIFICATION AND CHARACTERIZATION OF SALIVARY AMYLASE INHIBITOR EXTRACTED FROM BARLEY

2016 ◽  
Vol 47 (4) ◽  
Author(s):  
Abood & Hakeem
Keyword(s):  
Ammonium Sulfate ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Alpha Amylase ◽  
Ionic Exchange ◽  
Inhibition Activity ◽  
Amylase Inhibitors

Amylase inhibitors were purified by many sequential steps included concentration by gradual addition of ammonium sulfate at  saturation ratios. ranged from 0 to 90% . The best ratio of saturation was found to be 70% as the specific activity and inhibition activity toward Human alpha-amylase(HAS)  were the highest ( 8 U/mg and 6 U/ml respectively as compared to those of the rest ratios, the ratio of saturation with ammonium sulfate 60 % and then 50%, (5.8 ,5.5  )U/ml and( 7.7 ،7 )U/mg respectively for inhibition activity and specific activity and for  40% ,30%20%  saturation  the inhibition activity and specific activity were(5 ،4.8 ،4 ) u/ml (6.6 ،6 ،5.8) u/mg respectively .The precepitation step was followed by ionic exchange chromatography technique by DEAE-cellulose column( 3×11 )cm and the results showed that there was one peak with inhibition activity toward (HAS). Further  purification steps were conducted using gel filtration on Sephacryl S-200 column    (1.5  ×  60)cm; the purification folds was5.59 times with outcome of 46.5%.The results of alpha-amylase inhibitors characterization showed that the molecular weight was about 23.44 and 22.9  kDa  as determined by electrophoresis and gel filteration respectively.                                         

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 Purification and Characterization of a Prolyl Aminopeptidase from Debaryomyces hansenii

2003 ◽  
Vol 69 (1) ◽  
pp. 227-232 ◽  
Author(s):  
Tomás Bolumar ◽  
Yolanda Sanz ◽  
M.-Concepción Aristoy ◽  
Fidel Toldrá
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Debaryomyces Hansenii ◽  
Cysteine Proteases ◽  
Anion Exchange Chromatography ◽  
Cell Extract ◽  
Exchange Chromatography ◽  
Prolyl Aminopeptidase ◽  
Ph Range ◽  
Meat Fermentation

ABSTRACT A prolyl aminopeptidase (PAP) (EC 3.4.11.5) was isolated from the cell extract of Debaryomyces hansenii CECT12487. The enzyme was purified by selective fractionation with protamine and ammonium sulfate, followed by two chromatography steps, which included gel filtration and anion-exchange chromatography. The PAP was purified 248-fold, with a recovery yield of 1.4%. The enzyme was active in a broad pH range (from 5 to 9.5), with pH and temperature optima at 7.5 and 45°C. The molecular mass was estimated to be around 370 kDa. The presence of inhibitors of serine and aspartic proteases, bestatin, puromycin, reducing agents, chelating agents, and different cations did not have any effect on the enzyme activity. Only iodoacetate, p-chloromercuribenzoic acid, and Hg2+, which are inhibitors of cysteine proteases, markedly reduced the enzyme activity. The Km for proline-7-amido-4-methylcoumarin was 40 μM. The enzyme exclusively hydrolyzed N-terminal-proline-containing substrates. This is the first report on the identification and purification of this type of aminopeptidase in yeast, which may contribute to the scarce knowledge about D. hansenii proteases and their possible roles in meat fermentation.

scholarly journals Purification and characterization of amylase from local isolate Pseudomonas sp.SPH4

2012 ◽  
Vol 6 (1) ◽  
pp. 69-79
Author(s):  
Hala M. Ali ◽  
Ghazi M. Aziz
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Silver Ions ◽  
Enzyme Characterization ◽  
Maximum Activity ◽  
Mercury Ions ◽  
Local Isolate ◽  
Optimum Ph

The amylase produced from local isolate Pseudomonas sp. SPH4 was purified by precipitation with 30% saturation ammonium sulphate, followed by ion-exchange chromotography using DEAE-cellulose column, and Gel filtration using Sephacryl S-300 column.The two iso-enzymes (a, b) were purified to (2.83, 3.47) times in the last step with an enzymes yields of (32.36, 76.34)% respectively. Enzyme characterization of the two iso-enzymes indicated that the optimum pH for the two iso-enzymes a and b were (7, 7.5) respectively, while the optimum pH for the iso-enzymes stability were (6.5, 7) respectively. The maximum activity for iso-enzymes (a, b) appeared at 45ºC and stable for 15 min at 30-50ºC and lost approximately 50% of it's activity at rang above 75ºC. Enzyme characterization results showed that the chlorides of silver and mercury had inhibitory effect on enzyme activity, the remaining enzyme activity for the iso-enzymes (a, b) were (46.66, 36.36)% for silver ions and (41.33, 33.63)% for mercury ions at 5 mM respectively, and (28, 28.18)% for silver ions and (25.33, 19.09)% for mercury ions at 10 mM respectively. The iso-enzymes a and b were affected by chelating agent ethylene diamine tetra acetic acid (EDTA) at concentration 2mM the remaining activity (45.33, 43.63)% respectively, and 5mM the remaining activity (28, 28.18)% respectivily, and these iso-enzymes (a, b) refered to metalloenzymes. The iso-enzymes (a, b) were kept their activity when treated by reducing agent (2-mercaptoethanol) at 2 mM the remaining activity (92, 92.72)% respectively, and 5 mM the remaining activity (85.3, 89.09)% respectivily. The iso-enzymes (a, b) were kept their activity when treated by phenyl methyl sulphonyl fluoride (PMSF) at concentration 1mM the remaining activity (93.33, 90.90)% respectivily,and 5 mM the remaining activity (90.66, 87.27)% respectivily, and these indicated that these iso-enzymes didnot referred to serineamylases group.

Purification and characterization of chymosin and pepsin from kid

2006 ◽  
Vol 73 (1) ◽  
pp. 49-57 ◽  
Author(s):  
Ekaterini E Moschopoulou ◽  
Ioannis G Kandarakis ◽  
Efstathios Alichanidis ◽  
Emmanouil M Anifantakis
Keyword(s):  
Gel Filtration ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Temperature Dependent ◽  
Molecular Weights ◽  
Indigenous Breed ◽  
Increased Sensitivity ◽  
Pepstatin A

The objective of this work was to study the characteristics of the gastric aspartic proteinases chymosin and pepsin which are constituents of the kid rennet. The two enzymes were extracted from abomasal tissue of one kid from a local indigenous breed, separated from each other by DEAE-cellulose chromatography and then were purified by gel filtration and anion-exchange chromatography. The molecular weights of the purified kid chymosin and pepsin as determined by gel filtration were 36 kDa and 40 kDa respectively. The isoelectric point of kid chymosin was as multiple forms of 3–6 zones at pH 4·6–5·1, while that of kid pepsin was at pH [les ]3·0. Kid pepsin contained 0·37 molecules phosphorous per molecule and was totally inhibited by 5 μM pepstatin A, being more sensitive than kid chymosin. Both enzymes were almost equally as proteolytic as calf chymosin on total casein at pH 5·6. Kid pepsin activity was more pH and temperature dependent than kid chymosin activity. In comparison with the calf chymosin temperature sensitivity, the order of increased sensitivity was: calf chymosin <kid chymosin <kid pepsin.

scholarly journals Purification and Characterization of a Lectin fromPhaseolus vulgaris cv.(Anasazi Beans)

2009 ◽  
Vol 2009 ◽  
pp. 1-9 ◽  
Author(s):  
Arishya Sharma ◽  
Tzi Bun Ng ◽  
Jack Ho Wong ◽  
Peng Lin
Keyword(s):  
Sequence Similarity ◽  
Gel Filtration ◽  
Peritoneal Macrophages ◽  
Exchange Chromatography ◽  
Hemagglutinating Activity ◽  
Murine Splenocytes ◽  
Ph Range ◽  
Hiv 1 ◽  
Mcf 7

A lectin has been isolated from seeds of thePhaseolus vulgaris cv.“Anasazi beans” using a procedure that involved affinity chromatography on Affi-gel blue gel, fast protein liquid chromatography (FPLC)-ion exchange chromatography on Mono S, and FPLC-gel filtration on Superdex 200. The lectin was comprised of two 30-kDa subunits with substantial N-terminal sequence similarity to otherPhaseoluslectins. The hemagglutinating activity of the lectin was stable within the pH range of 1–14 and the temperature range of 0–80∘C. The lectin potently suppressed proliferation of MCF-7 (breast cancer) cells with anIC50of 1.3 μM, and inhibited the activity of HIV-1 reverse transcriptase with anIC50of 7.6 μM. The lectin evoked a mitogenic response from murine splenocytes as evidenced by an increase in [3H-methyl]-thymidine incorporation. The lectin had no antifungal activity. It did not stimulate nitric oxide production by murine peritoneal macrophages. Chemical modification results indicated that tryptophan was crucial for the hemagglutinating activity of the lectin.

scholarly journals Biochemical properties and positional specificity of Lipase from hepatopancreas of Tra catfish (Pangasius)

2013 ◽  
Vol 16 (4) ◽  
pp. 85-91
Author(s):  
Thy Bao Vuong ◽  
Lam Bich Tran ◽  
Duan Luu
Keyword(s):  
Ion Exchange ◽  
Lipase Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Biochemical Properties ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Positional Specificity ◽  
Tra Catfish ◽  
Ph Range

Lipase from the hepatopancreas of Tra catfish (Pangasius) was precipitated by ammonium sulfate fractionation, purified by ion-exchange chromatography on DEAE cellulose and gel filtration on Sephadex G- 75. On substrate triolein, purified lipase has Km= 1.381 mM and Vmax= 0.063 mM/min. The lipase was stable at a pH range of 7.0- 9.0 and in temperatures of 35-50°C. At 500C the enzyme loosed 44,7% activity after 120 min. The enzyme was specific for the α- positions (1, 3) of triglyceride. In bile salt solution of 0.015M NaTC, lipase activity of the enzyme increased in 3.08 folds in comparison of sample without NaTC.

scholarly journals A novel alkaline protease from wild edible mushroom Termitomyces albuminosus.

2011 ◽  
Vol 58 (2) ◽  
Author(s):  
Suyue Zheng ◽  
Hexiang Wang ◽  
Guoqing Zhang
Keyword(s):  
Gel Filtration ◽  
Deae Cellulose ◽  
Tween 80 ◽  
Lima Bean ◽  
Edible Mushroom ◽  
Exchange Chromatography ◽  
Bean Trypsin Inhibitor ◽  
Triton X ◽  
Wild Edible Mushroom ◽  
Purification Protocol

A protease with a molecular mass of 30 kDa and the N-terminal sequence of GLQTNAPWGLARSS, was isolated from fresh fruiting bodies of the wild edible mushroom Termitomyces albuminosus. The purification protocol included ion exchange chromatography on DEAE-cellulose, Q-Sepharose, SP-Sepharose and FPLC-gel filtration on Superdex 75. The protein was unadsorbed on DEAE-cellulose and Q-Sepharose, but adsorbed on SP-Sepharose. The optimal pH and temperature of the purified enzyme were 10.6 and 60 °C, respectively. The enzyme was stable in the presence of 2 % (v/v) Tween 80 and 4 M urea. More than 80 % of the enzyme activity was retained in 2 % (v/v) Triton X 100, 54 % in 10 mM EDTA and 31 % in 2 % (w/v) SDS. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), but not inhibited by dithiothreitol (DTT), pepstatin or lima bean trypsin inhibitor suggesting that it was a serine protease but not a trypsin-like one. The protease was inhibited by Hg(2+), Cu(2+), and Fe(3+) ions. The K(m) and V(max) values of the purified enzyme for casein were 8.26 mg ∙ ml(-1) and 0.668 mg ∙ ml(-1) ∙ min(-1), respectively.

scholarly journals Purification and Characterization of Carboxymethyl Cellulase from Bacillus sp. Isolated from a Paddy Field

2012 ◽  
Vol 61 (1) ◽  
pp. 51-55 ◽  
Author(s):  
PONNUSWAMY VIJAYARAGHAVAN ◽  
S.G. PRAKASH VINCENT
Keyword(s):  
Paddy Field ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Relative Molecular Mass ◽  
Bacillus Sp ◽  
Ammonium Sulphate Precipitation ◽  
Sds Page ◽  
Ph Range

A microorganism hydrolyzing carboxymethyl cellulose was isolated from a paddy field and identified as Bacillus sp. Production of cellulase by this bacterium was found to be optimal at pH 6.5, 37 degrees C and 150 rpm of shaking. This cellulase was purified to homogeneity by the combination of ammonium sulphate precipitation, DEAE cellulose, and sephadex G-75 gel filtration chromatography. The cellulase was purified up to 14.5 fold and had a specific activity of 246 U/mg protein. The enzyme was a monomeric cellulase with a relative molecular mass of 58 kDa, as determined by SDS-PAGE. The enzyme exhibited its optimal activity at 50 degrees C and pH 6.0. The enzyme was stable in the pH range of 5.0 to 7.0 and its stability was maintained for 30 min at 50 degrees C and its activity got inhibited by Hg2+, Cu2+, Zn2+, Mg2+, Na2+, and Ca2+.

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