Purification and Characterization of Melanogenic Enzyme Tyrosinase from Button Mushroom

Melanogenesis is a biosynthetic pathway for the formation of the pigment melanin in human skin. A key enzyme, tyrosinase, catalyzes the first and only rate-limiting steps in melanogenesis. Since the discovery of its melanogenic properties, tyrosinase has been in prime focus and microbial sources of the enzyme are sought. Agaricus bisporus widely known as the common edible mushroom, it’s taking place in high amounts of proteins, enzyme, carbohydrates, fibers, and low fat contents are frequently cited in the literature in relation to their nutritional value. In the present study tyrosinase from Agaricus bisporus was purified by ammonium sulphate precipitation, dialysis followed by gel filtration chromatography on Sephadex G-100, and ion exchange chromatography on DEAE-Cellulose; the enzyme was purified, 16.36-fold to give 26.6% yield on total activity in the crude extract and final specific activity of 52.19 U/mg. The SDS-PAGE electrophoresis showed a migrating protein band molecular weight of 95 kDa. The purified tyrosinase was optimized and the results revealed that the optimum values are pH 7.0 and temperature 35°C. The highest activity was reported towards its natural substrate, L-DOPA, with an apparent Km value of 0.933 mM. This indicated that tyrosinase purified from Agaricus bisporus is a potential source for medical applications.

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PURIFICATION AND CHARACTERIZATION OF SALIVARY AMYLASE INHIBITOR EXTRACTED FROM BARLEY

IRAQI JOURNAL OF AGRICULTURAL SCIENCES ◽

10.36103/ijas.v47i4.536 ◽

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.

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Purification and characterization of a plasma membrane ferricyanide-utilizing NADH dehydrogenase from Ehrlich tumour cells

Biochemical Journal ◽

10.1042/bj3140587 ◽

1996 ◽

Vol 314 (2) ◽

pp. 587-593 ◽

Cited By ~ 11

Author(s):

Antonio del CASTILLO-OLIVARES ◽

Miguel A. MEDINA ◽

Ignacio NÚÑEZ de CASTRO ◽

Javier MÁRQUEZ

Keyword(s):

Plasma Membrane ◽

Molecular Mass ◽

Nadh Dehydrogenase ◽

Specific Activity ◽

Gel Filtration ◽

Maximum Activity ◽

Exchange Chromatography ◽

Tumour Cells ◽

Ammonium Sulphate Precipitation ◽

Apparent Homogeneity

A ferricyanide-utilizing NADH dehydrogenase (NADH-ferricyanide oxidoreductase) from the plasma membrane of Ehrlich ascites tumour cells has been purified about 1500-fold to apparent homogeneity. The method comprises the isolation of an enriched plasma membrane fraction, solubilization with Triton X-100, ion-exchange chromatography, ammonium sulphate precipitation, Cibacron Blue chromatography and fast-protein liquid chromatography with a Superose-6 gel filtration column. The specific activity of the final pool was more than 61 units/mg protein. The pure enzyme examined by SDS/PAGE displayed only one type of subunit with an apparent molecular mass of 32.0 kDa. The molecular mass of the native protein (117.0 kDa) was estimated by gel filtration; these results suggest a protein composed of four subunits of identical molecular mass. The enzyme was stable in the pH interval between 6 and 9, with maximum activity at pH values from 7.5 to 8.5. The purified enzyme showed Michaelis–Menten kinetics for the substrates, with apparent Km values of 4.3×10-5 M and 6.7×10-5 M for NADH and ferricyanide respectively. The isolated protein was strongly inhibited by Zn2+ and the thiol-specific reagents mersalyl and p-chloromercuribenzenesulphonic acid.

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PURIFICATION OF β-GLUCOSIDASE PRODUCED FROM Trichoderma viride USING COW DUNG AS CARBON SOURCE

FUDMA Journal of Sciences ◽

10.33003/fjs-2020-0402-210 ◽

2020 ◽

Vol 4 (2) ◽

Author(s):

S. T. Tyohemba ◽

S. Aliyu ◽

N. N. Ndukwe ◽

G. G. Memi ◽

U. O. Edem

Keyword(s):

Specific Activity ◽

Trichoderma Viride ◽

Gel Filtration ◽

Exchange Chromatography ◽

Broad Peak ◽

Cow Dung ◽

Elution Profile ◽

Ammonium Sulphate Precipitation ◽

Sds Page ◽

Peak Fraction

β-glucosidases have characteristics of biotechnological interest and have thus become important industrial enzymes.In this study, β-glucosidase produced by Trichoderma viride from cow dung was subjected to a three step purification process involving ammonium sulphate precipitation, gel filtration by Sephadex G-100 and ion exchange chromatography by DEAE-Sephadex A-25. The elution profile on Sephadex G-100 resulted in a single broad peak (fractions 9-21) which had a yield of 3.7% and a purification fold of 4.29 with a specific activity of 25.70 µmol/min/mg proteins while the elution profile on DEAE-Sephadex A-25 resulted in a single broad peak (fraction 8-14) which had a yield of 2.76% and a purification fold of 22.14 with a specific activity of 132.41µmol/min/mg of protein. The purified enzyme was obtained as a single band and had a molecular mass of 51.8 kDa on SDS-PAGE. This results provide support for further studies of this enzyme towards revealing its potential biotechnological applications.

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Two acid RNases from Dactylis glomerata seeds. Purification, properties and effect of polyamines and lectins on their activity

Acta Societatis Botanicorum Poloniae ◽

10.5586/asbp.1985.024 ◽

2014 ◽

Vol 54 (3) ◽

pp. 241-253 ◽

Cited By ~ 1

Author(s):

Janina Wiśniowska ◽

Bronisława Morawiecka

Keyword(s):

Specific Activity ◽

Gel Filtration ◽

Deae Cellulose ◽

Dactylis Glomerata ◽

Hydrolytic Activity ◽

Relative Activity ◽

Exchange Chromatography ◽

Lentil Lectin ◽

Optimum Ph ◽

Rnase Ii

Two glycoproteidic acid RNases (RNase I and RNase II) were obtained and purified from the seeds of Dactylis glomerata by extraction with acetate buffer, fractionation with ammonium sulfate, ion-exchange chromatography on DEAE-cellulose, DEAE-Sphadex, affinity chromatography on Con A-Sepharose and gel filtration on Bio-Gel P60. RNase I with a specific activity of 2582 U•mg-1 protein and an optimum pH of 4.9 and RNase II with a specific activity of 1928 U• mg-1 protein and optimum pH of 4.6, were isolated. They lacked nuclease, phosphodi- and monoesterase activities. Both forms of the enzyme hydrolyzed pyrimidine homopolymers with a preference for poly U and exhibited a low specificity for purine homopolymers (poly G and poly A). RNase I acted with a 3-fold higher hydrolytic activity on poly C homopolymer than RNase IL The hydrolytic activity of both enzymes was inhibited by Zn+2, Fe+2, Cu+2 ions when yeast RNA was the substrate. The amines spermine, spermidine and tyramine at a concentration of 0.1 mM increased the enzymatic activity of both RNases by 20 to 60% of the relative activity. The hydrolytic activity of RNases I and II was stimulated by the presence of lentil lectin (LL), soybean lectin (SBA) and potato lectin (STA), and inhibited by the presence of concanavalin A. The 20-200% stimulation and 40-60% inhibition depended on the proportion, on a weight basis, of enzyme to lectin and were reversible in the presence of receptor sugars.

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Study on Isolation and Partial Purification of Lactase (β-galactosidase) Enzyme from Lactobacillus Bacteria Isolated from Yogurt

Journal of Scientific Research ◽

10.3329/jsr.v4i1.8478 ◽

2011 ◽

Vol 4 (1) ◽

pp. 239 ◽

Cited By ~ 1

Author(s):

N. H. M. R. Mozumder ◽

M. Akhtaruzzaman ◽

M. A. Bakr ◽

F. Tuj Zohra

Keyword(s):

Specific Activity ◽

Culture Media ◽

Deae Cellulose ◽

Chemical Properties ◽

Total Activity ◽

Partial Purification ◽

Anion Exchange Column ◽

Ammonium Sulphate Precipitation ◽

Agar Media ◽

Modified Media

Lactase has many applications in dairy industry including for the treatment of lactose intolerance. The present study was conducted to identify the activity of lactase enzyme produced by Lactobacillus bacteria isolated from yogurts available in Dhaka city. The strains were identified to be gram positive, catalase negative, fermentative and lactase producer when cultured on selective MRS agar media by using standard bacteriological procedures and techniques. The study revealed that enzymes produced by lactobacilli were capable to produce glucose from substrate lactose in lactose modified media using lactase assay Kit Glu IB and their highest protein concentration (17.25 mg/ml) was observed in the supernatant of culture media isolated from L. lactis. Highest total activity (850.69 U/l) and specific activity (50.04 U/mg) of lactase enzyme was observed in the strain of L. bulgaricus. The crude extract which showed highest activity was further purified by ammonium sulphate precipitation followed by anion exchange column chromatography (DEAE cellulose). Final specific activity and fold purification of lactase enzyme reached to 62.80 U/mg and 1.47 respectively. The highest physic-chemical properties (Effect of pH and temperature) of lactase enzyme were observed at PH 6.0 which was 43.98 U/mg of protein and at 70°c temperature which was 111.11 U/mg of protein.Keywords: β-Galactosidase; Specific activity; DEAE cellulose; Fold purification; Yogurt.© 2012 JSR Publications. ISSN: 2070-0237 (Print); 2070-0245 (Online). All rights reserved.doi: http://dx.doi.org/10.3329/jsr.v4i1.8478J. Sci. Res. 4 (1), 239-249 (2012)

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Purification and partial characterization of glutamate synthase from Rhodospirillum rubrum grown under nitrogen-fixing conditions

Biochemical Journal ◽

10.1042/bj2790151 ◽

1991 ◽

Vol 279 (1) ◽

pp. 151-154 ◽

Cited By ~ 12

Author(s):

I Carlberg ◽

S Nordlund

Keyword(s):

Metabolic Regulation ◽

Rhodospirillum Rubrum ◽

Specific Activity ◽

Gel Filtration ◽

Glutamate Synthase ◽

Photosynthetic Bacterium ◽

Exchange Chromatography ◽

Ammonia Assimilation ◽

Key Enzyme ◽

Molecular Masses

Glutamate synthase, a key enzyme in ammonia assimilation, has been purified from the photosynthetic bacterium Rhodospirillum rubrum. The purification procedure involves ion-exchange chromatography, affinity chromatography and gel filtration. The recovery in the procedure is high (62%) and the specific activity is 21 mumol of NADPH oxidized/min per mg. The enzyme is specific for its substrates, and no activity was demonstrated with NADH or NH4+ ions substituting for NADPH and glutamine respectively. The enzyme is composed of two dissimilar subunits with molecular masses of 53 and 152 kDa, and it is shown that Cl- ions have an effect on the aggregation of the enzyme. Km values for the substrates are: NADPH, 16 microM; 2-oxoglutarate, 10 microM; and glutamine, 65 microM. The enzyme is inhibited by amidotransferase inhibitors at micromolar concentrations. The role of the enzyme in the metabolic regulation of nitrogenase is discussed.

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A novel alkaline protease from wild edible mushroom Termitomyces albuminosus.

Acta Biochimica Polonica ◽

10.18388/abp.2011_2277 ◽

2011 ◽

Vol 58 (2) ◽

Cited By ~ 14

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.

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A biological study of chitinase produced by clinical isolates of Pseudomonas aeruginosa and detection of ChiA responsible gene

International Journal of Research in Pharmaceutical Sciences ◽

10.26452/ijrps.v11i2.1989 ◽

2020 ◽

Vol 11 (2) ◽

pp. 1318-1330

Author(s):

Tahreer Hadi Saleh ◽

SabaTalib Hashim ◽

Raghad Abdulatif Abdulrazaq Al-Obaidi ◽

Bahaa Abdullah Laftaah AL-Rubaii

Keyword(s):

Liver Cell ◽

Cytotoxic Effect ◽

Specific Activity ◽

Morphological Changes ◽

Gel Filtration ◽

Deae Cellulose ◽

Exchange Chromatography ◽

Clinical Isolates ◽

Biological Study ◽

Variable Degree

All isolates in this study were diagnosed as P. aeroginosa according to the 16srRNA gene. Only two isolates were produced chitinase on chitin agar medium and were positive to the chiA gene. Most of the isolates exhibited high sensitivity (95%) and (90%) to Imipenem and Carbenicillin, respectively, and the resistance to Amoxicillin +Clavulanic acid was shown (80%), while revealed a variable degree in their response to others antibiotics. The crude extract activity and specific activity for extracted chitinase enzymes were 33 U/ml and 18.644U/mg, respectively. The enzyme was purified by different steps include: precipitating with saturation 70% of ammonium sulfate and then applied on ion-exchange chromatography using DEAE- cellulose column and then employ the Sephadex G-200 column for gel filtration chromatography. The purification fold and yield was 28.5%. The molecular weight of the purified enzyme was determined by SDS-PAGE method, and it appeared at 50 kDa. The results of the MTT assay showed that the chitinase has a cytotoxic effect on cancer liver cell lines at a concentration of 100 µg /ml and increased gradually at a concentration of 600 µg /ml, while it showed no or less cytotoxic effect on normal embryonic liver cell line (WRL-68). Chitinase enzyme appeared a higher antibacterial activity at concentration 600 µg/ml and a lower activity at concentration 400 towards clinical isolates of S. aurous and E. coli. The study of histopathological effects was exhibited little morphological changes on cells of liver tissues.

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Caldolase, a chelator-insensitive extracellular serine proteinase from a Thermus spp

Biochemical Journal ◽

10.1042/bj2620409 ◽

1989 ◽

Vol 262 (2) ◽

pp. 409-416 ◽

Cited By ~ 16

Author(s):

G A Saravani ◽

D A Cowan ◽

R M Daniel ◽

H W Morgan

Keyword(s):

Specific Activity ◽

Gel Filtration ◽

Deae Cellulose ◽

Serine Proteinase ◽

Gel Permeation Chromatography ◽

Exchange Chromatography ◽

Acetate Buffer ◽

Ph Optimum ◽

Low Concentrations ◽

Low Ionic Strength

An extracellular alkaline serine proteinase from Thermus strain ToK3 was isolated and purified to homogeneity by (NH4)2SO4 precipitation followed by ion-exchange chromatography on DEAE-cellulose and QAE-Sephadex, affinity chromatography on N alpha-benzyloxycarbonyl-D-phenylalanyl-triethylenetetraminyl-Sepha rose 4B and gel-filtration chromatography on Sephadex G-75. The purified enzyme had a pI of 8.9 and an Mr determined by gel-permeation chromatography of 25,000. The specific activity was about 37,700 proteolytic units/mg with casein as substrate, and the pH optimum was 9.5. Proteolytic activity was inhibited by low concentrations of di-isopropyl phosphorofluoridate and phenylmethanesulphonyl fluoride, but was unaffected by EDTA, EGTA, o-phenanthroline, N-ethyl-5-phenylisoxazolium-3′-sulphonate, N alpha-p-tosyl-L-phenylalanylchloromethane, N alpha-p-tosyl-L-lysylchloromethane, trypsin inhibitors and pepstatin A. The enzyme contained approx. 10% carbohydrate and four disulphide bonds. No Ca2+, Zn2+ or free thiol groups were detected. It hydrolysed several native and dye-linked proteins and synthetic chromogenic peptides and esters. The enzyme was very thermostable (half-life values were 840 min at 80 degrees C, 45 min at 90 degrees C and 5 min at 100 degrees C). The enzyme was unstable at low ionic strength: after 60 min at 75 degrees C in 0.1 M-Tris/acetate buffer, pH 8, only 20% activity remained, compared with no loss in 0.1 M-Tris/acetate buffer, pH 8, containing 0.4 M-NaCl.

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Purification and characterization of tonin

Canadian Journal of Biochemistry ◽

10.1139/o76-113 ◽

1976 ◽

Vol 54 (9) ◽

pp. 788-795 ◽

Cited By ~ 37

Author(s):

S. Demassieux ◽

R. Boucher ◽

C. Grisé ◽

J. Genest

Keyword(s):

Analytical Ultracentrifugation ◽

Gel Filtration ◽

Deae Cellulose ◽

Potassium Phosphate ◽

Kinetic Studies ◽

Exchange Chromatography ◽

Sedimentation Equilibrium ◽

Angiotensin I ◽

Ph Optimum ◽

Ammonium Sulphate Precipitation

Tonin was purified from rat submaxillary glands by differential centrifugation, ammonium sulphate precipitation, gel filtration on Sephadex G150, and by ion-exchange chromatography on DEAE-cellulose, phospho-cellulose, SP-Sephadex C25, and SP-Sephadex C50. Purified tonin was shown to be homogeneous by analytical electrophoresis and by analytical ultracentrifugation analysis. Purified tonin was very stable when stored in buffers of low pH values or when incubated at high temperatures in neutral solutions. The molecular weight estimated by sedimentation equilibrium was 28 700. The pH optimum was near 6.8 in a 0.1 M potassium phosphate buffer. The Michaelis–Menten constant for tonin using angiotensin I as substrate was about 4 × 10−5 M. Tonin activity was strongly inhibited by plasma. Kinetic studies using angiotensin I as substrate showed that the inhibition of tonin by plasma was of the non-competitive type.

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