Optimization of growth media for enhanced production of laccase byCryptococcus albidusand its application for bioremediation of chemicals A paper submitted to the Journal of Environmental Engineering and Science.

2009 ◽  
Vol 36 (7) ◽  
pp. 1253-1264 ◽  
Author(s):  
Anjali Singhal ◽  
Gaurav Choudhary ◽  
Indu Shekhar Thakur
Keyword(s):  
Laccase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Laccase Production ◽  
Maximum Effect ◽  
Growth Media ◽  
Cryptococcus Albidus ◽  
Native Page ◽  
Enhanced Production

Cryptococcus albidus , isolated from the sediments of Century Pulp and Paper Mill, Lalkuan, Nainital, Uttarakhand, India, produced a copper containing oxidase, laccase, that was capable of degrading environmental pollutants. Bagasse was the most efficient inducer for laccase production. The Taguchi approach was used to optimize the growth media for five factors, i.e., pH, copper sulphate, carbon, nitrogen, and the inducer at four levels using an M-16 orthogonal array. The optimum conditions for laccase production were pH (6), CuSO4(2 mmol/L), meat peptone (0.5%), glucose (0.1%), and bagasse (1.0%). After optimization, laccase production increased seven times from 32 to 219 IU/mg. The inducer (bagasse) had maximum effect on laccase production leading to 52% increase, while pH had minimum effect with 7% increase. Growth media with laccase activity (2 U/mL) was applied for the bioremediation of dyes, effluent, and chemical compounds. These experiments showed that the growth media with laccase activity (2 U/mL) produced by Cryptococcus albidus had good potential for bioremediation of toxic and recalcitrant compounds. Further, the laccase enzyme extracted from the growth media was fractionated by DEAE-cellulose ion-exchange chromatography, and the molecular weight of the enzyme determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) was found to be 64 kDa. The activity of laccase was confirmed by native PAGE, in which ABTS was used for staining gel.

Lignin-Modifying Enzymes of the White Rot Basidiomycete Ganoderma lucidum

1999 ◽  
Vol 65 (12) ◽  
pp. 5307-5313 ◽  
Author(s):  
Trevor M. D’Souza ◽  
Carlos S. Merritt ◽  
C. Adinarayana Reddy
Keyword(s):  
Ganoderma Lucidum ◽  
Laccase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mesh Size ◽  
Syringic Acid ◽  
White Rot ◽  
White Rot Fungus ◽  
Laccase Production ◽  
Malt Extract

ABSTRACT Ganoderma lucidum, a white rot basidiomycete widely distributed worldwide, was studied for the production of the lignin-modifying enzymes laccase, manganese-dependent peroxidase (MnP), and lignin peroxidase (LiP). Laccase levels observed in high-nitrogen (HN; 24 mM N) shaken cultures were much greater than those seen in low-nitrogen (2.4 mM N), malt extract, or wood-grown cultures and those reported for most other white rot fungi to date. Laccase production was readily seen in cultures grown with pine or poplar (100-mesh-size ground wood) as the sole carbon and energy source. Cultures containing both pine and poplar showed 5- to 10-fold-higher levels of laccase than cultures containing pine or poplar alone. Since syringyl units are structural components important in poplar lignin and other hardwoods but much less so in pine lignin and other softwoods, pine cultures were supplemented with syringic acid, and this resulted in laccase levels comparable to those seen in pine-plus-poplar cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of concentrated extracellular culture fluid from HN cultures showed two laccase activity bands (M r of 40,000 and 66,000), whereas isoelectric focusing revealed five major laccase activity bands with estimated pIs of 3.0, 4.25, 4.5, 4.8, and 5.1. Low levels of MnP activity (∼100 U/liter) were detected in poplar-grown cultures but not in cultures grown with pine, with pine plus syringic acid, or in HN medium. No LiP activity was seen in any of the media tested; however, probing the genomic DNA with the LiP cDNA (CLG4) from the white rot fungus Phanerochaete chrysosporium showed distinct hybridization bands suggesting the presence oflip-like sequences in G. lucidum.

scholarly journals Affinity purification and subunit structures of β-N-acetylhexosaminidases A and B from boar epididymis

1984 ◽  
Vol 219 (3) ◽  
pp. 1009-1015 ◽  
Author(s):  
H C Parkes ◽  
J L Stirling ◽  
P Calvo
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Gradient Gel Electrophoresis ◽  
Sepharose 4B ◽  
Electrophoretic Transfer ◽  
Peptide Maps

beta-N-Acetylhexosaminidase from boar epididymis was separated into two forms, A and B, on DEAE-cellulose. Both these forms were excluded from Sepharose S-200 and had apparent Mr values of 510 000 on gradient gel electrophoresis under non-denaturing conditions. Affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylam ine coupled to CNBr-activated Sepharose 4B was used to separate and purify beta-N-acetylhexosaminidases A and B that had specific activities of 115 and 380 mumol/min per mg of protein respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of denatured beta-N-acetylhexosaminidase A gave a single major component of Mr 67 000. beta-N-Acetylhexosaminidase B also had this component, and in addition had polypeptides of Mr 29 000 and 26 000. All these polypeptides were glycosylated. Antiserum to the B form precipitated form A from solution and reacted with the 67 000-Mr component or form A after electrophoretic transfer from sodium dodecyl sulphate/polyacrylamide gels to nitrocellulose sheets. The 67 000-Mr components of forms A and B yielded identical peptide maps when digested with Staphylococcus aureus V8 proteinase, and the 29 000-Mr and 26 000-Mr components in form B may be related to the 67 000-Mr polypeptide.

Biosynthesis of glucosyl monophosphoryl undecaprenol and its role in lipoteichoic acid biosynthesis

1982 ◽  
Vol 152 (2) ◽  
pp. 616-625
Author(s):  
D J Mancuso ◽  
T H Chiu
Keyword(s):  
Thin Layer ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Lipoteichoic Acid ◽  
Molar Ratio ◽  
Streptococcus Sanguis ◽  
Chromatographic Profile ◽  
Hydrolysis Of ◽  
Cellulose Column

A glucophospholipid was detected in an incubation mixture containing UDP-glucose, MgCl2, ATP, and a particulate enzyme prepared from Streptococcus sanguis. The synthesis of this lipid was inhibited strongly by UDP and moderately by UMP. The molar ratio of glucose to phosphate in the purified lipid was found to be 1:1. Glucose and glucose 1-phosphate were released by mild alkaline hydrolysis of the glucophospholipid. The lipid produced by mild acid degradation of the purified lipid yielded a thin-layer chromatographic profile similar to that of acid-treated undecaprenol. One of the minor components exhibited the same mobility as untreated undecaprenol. To characterize further the lipid moiety of the glucophospholipid, a polyisoprenol was purified from the neutral lipid of S. sanguis. The polyisoprenol was converted in the presence of ATP, UDP-glucose, and the particulate enzyme into a lipid which exhibited the same thin-layer chromatographic mobility as the glucophospholipid. The structure of the polyisoprenol was determined by nuclear magnetic resonance and mass spectrometry to be an undecaprenol with an internal cis-trans ratio of 7:2. These results indicate that the glucophospholipid is glucosyl monophosphoryl undecaprenol. The glucosyl moiety of the glucophospholipid was shown to be incorporated in the presence of the particulate enzyme into a macromolecule which was characterized as a lipoteichoic acid by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and DEAE-cellulose column chromatography. This result indicates that glucosyl monophosphoryl undecaprenol is the direct glucosyl donor in the synthesis of lipoteichoic acid.

Biocontrol of wood-rotting fungi withStreptomyces violaceusnigerXL-2

2006 ◽  
Vol 52 (9) ◽  
pp. 805-808 ◽  
Author(s):  
Nilanshu Shekhar ◽  
Debaditya Bhattacharya ◽  
Dishant Kumar ◽  
Rajinder K Gupta
Keyword(s):  
Biocontrol Agent ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Industrial Applications ◽  
Stress Factors ◽  
Antihypertensive Activity ◽  
Growth Media ◽  
Fungal Cell ◽  
Wide Range ◽  
Streptomyces Violaceusniger

During the previous decade, chitinases have received increased attention because of their wide range of applications. Chito-oligomers produced by enzymatic hydrolysis of chitin have been of interest in recent years because of their broad applications in medical, agricultural, and industrial applications, such as antibacterial, antifungal, hypo cholesterolemic, and antihypertensive activity, and as food quality enhancer. Fungal cell walls being rich in chitin also enable the use of chitinases in biocontrol of fungal pathogens, as bio-fungicides. An actinomycete was isolated from the bark of trees of Dehradun in India and was later identified as Streptomyces violaceusniger. This strain exhibits strong antagonism towards various wood-rotting fungi, such as Phanerochaete chrysosporium, Postia placenta, Coriolus versicolor, and Gloeophyllum trabeum. Further, studies showed an extracellular bioactive compound was responsible for the antagonism. The conditions for the production of this biocontrol agent were optimized, and the effects of various stress factors (like nitrogen-deficient media, carbon-deficient media, etc.) were studied. The presence of chitin in the growth media was found to be an essential factor for the active production of the biocontrol agent. The pH and temperature optima for the biocontrol agent were determined. Purification and characterization of this specific biocontrol agent was performed through anion exchange chromatography using a DEAE–cellulose column, and a single protein band was obtained on a 10% sodium dodecyl sulfate – polyacrylamide gel. The protein was later identified as a 28 kDa endo chitinase by MALDI–TOF (matrix-assisted laser desorption ionization – time of flight) and by a chitobiose activity assay.Key words: actinomycetes, biocontrol agents, Streptomyces violaceusniger, chitinase.

scholarly journals Phosphoenolpyruvate carboxylase from the crassulacean plant Bryophyllum fedtschenkoi Hamet et Perrier. Purification, molecular and kinetic properties

1978 ◽  
Vol 175 (2) ◽  
pp. 391-406 ◽  
Author(s):  
R Jones ◽  
M B Wilkins ◽  
J R Coggins ◽  
C A Fewson ◽  
A D B Malcolm
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Maximum Velocity ◽  
Single Band ◽  
Kinetic Properties ◽  
Subunit Structure

Phosphoenolpyruvate carboxylase from the Crassulacean plant Bryophyllum fedtschenkoi has been purified to homogenetity by DEAE-cellulose treatment, (NH4)2SO4 fractionation,, and chromatography on DEAE-cellulose and hydroxyapatite. Poly(ethylene glycol) is required in the extraction medium to obtain maximum enzyme activity. The purified enzyme has a specific activity of about 26 units/mg of protein at 25 degrees C. It gives a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, corresponding to a mol.wt. of 105,000, and gives a single band on non-denaturing gel electrophoresis at pH8.4. Cross-linking studies at pH8.0 indicate that the subunit structure is tetrameric but that the dimer may also be an important unit of polymerization. Gel filtration results at pH6.7 confirm that the native enzyme is tetrameric with a concentration-dependent dissociation to a dimer. The kinetic behaviour is characterized by (i) relatively small variations in maximum velocity between pH5.5 and 9.0 with a double optimum, (ii) a reversible temperature-dependent inactivation between 30 and 45 degrees C, (iii) inhibition by malate, which is pH-sensitive, and (iv) almost Michaelis-Menten behaviour with phosphoenolpyruvate as the varied ligand but sigmoidal behaviour under suitable conditions with malate as the varied ligand. The findings are related to other studies to the possible role phosphoenolpyruvate carboxylase in controlling a circadian rhythm of CO2 fixation.

Purification and characterization of enterocin MC13 produced by a potential aquaculture probiontEnterococcus faeciumMC13 isolated from the gut ofMugil cephalus

2011 ◽  
Vol 57 (12) ◽  
pp. 993-1001 ◽  
Author(s):  
R. Satish Kumar ◽  
P. Kanmani ◽  
N. Yuvaraj ◽  
K.A. Paari ◽  
V. Pattukumar ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Enterococcus Faecium ◽  
Vibrio Vulnificus ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Probiotic Bacterium ◽  
Mass Spectrometry Analysis ◽  
Native Page ◽  
Spectrometry Analysis

A bacteriocin producer strain MC13 was isolated from the gut of Mugil cephalus (grey mullet) and identified as Enterococcus faecium . The bacteriocin of E. faecium MC13 was purified to homogeneity, as confirmed by Tricine sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS–PAGE). Reverse-phase high-performance liquid chromatography (HPLC) analysis showed a single active fraction eluted at 26 min, and matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry analysis showed the molecular mass to be 2.148 kDa. The clear zone in native PAGE corresponding to enterocin MC13 band further substantiated its molecular mass. A dialyzed sample (semicrude preparation) of enterocin MC13 was broad spectrum in its action and inhibited important seafood-borne pathogens: Listeria monocytogenes , Vibrio parahaemolyticus , and Vibrio vulnificus . This antibacterial substance was sensitive to proteolytic enzymes: trypsin, protease, and chymotrypsin but insensitive to catalase and lipase, confirming that inhibition was due to the proteinaceous molecule, i.e., bacteriocin, and not due to hydrogen peroxide. Enterocin MC13 tolerated heat treatment (up to 90 °C for 20 min). Enterococcus faecium MC13 was effective in bile salt tolerance, acid tolerance, and adhesion to the HT-29 cell line. These properties reveal the potential of E. faecium MC13 to be a probiotic bacterium. Enterococcus faecium MC13 could be used as potential fish probiotic against pathogens such as V. parahaemolyticus, Vibrio harveyi , and Aeromonas hydrophila in fisheries. Also, this could be a valuable seafood biopreservative against L. monocytogenes.

scholarly journals Purification and properties of adenylyl sulphate:ammonia adenylyltransferase from Chlorella catalysing the formation of adenosine 5′-phosphoramidate from adenosine 5′-phosphosulphate and ammonia

1981 ◽  
Vol 195 (3) ◽  
pp. 545-560 ◽  
Author(s):  
Heinz Fankhauser ◽  
Jerome A. Schiff ◽  
Leonard J. Garber
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Low Ph ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Unknown Compound ◽  
Reactive Blue 2

Extracts of Chlorella pyrenoidosa, Euglena gracilis var. bacillaris, spinach, barley, Dictyostelium discoideum and Escherichia coli form an unknown compound enzymically from adenosine 5′-phosphosulphate in the presence of ammonia. This unknown compound shares the following properties with adenosine 5′-phosphoramidate: molar proportions of constituent parts (1 adenine:1 ribose:1 phosphate:1 ammonia released at low pH), co-electrophoresis in all buffers tested including borate, formation of AMP at low pH through release of ammonia, mass and i.r. spectra and conversion into 5′-AMP by phosphodiesterase. This unknown compound therefore appears to be identical with adenosine 5′-phosphoramidate. The enzyme that catalyses the formation of adenosine 5′-phosphoramidate from ammonia and adenosine 5′-phosphosulphate was purified 1800-fold (to homogeneity) from Chlorella by using (NH4)2SO4 precipitation and DEAE-cellulose, Sephadex and Reactive Blue 2–agarose chromatography. The purified enzyme shows one band of protein, coincident with activity, at a position corresponding to 60000–65000 molecular weight, on polyacrylamide-gel electrophoresis, and yields three subunits on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of 26000, 21000 and 17000 molecular weight, consistent with a molecular weight of 64000 for the native enzyme. Isoelectrofocusing yields one band of pI4.2. The pH optimum of the enzyme-catalysed reaction is 8.8. ATP, ADP or adenosine 3′-phosphate 5′-phosphosulphate will not replace adenosine 5′-phosphosulphate, and the apparent Km for the last-mentioned compound is 0.82mm. The apparent Km for ammonia (assuming NH3 to be the active species) is about 10mm. A large variety of primary, secondary and tertiary amines or amides will not replace ammonia. One mol.prop. of adenosine 5′-phosphosulphate reacts with 1 mol.prop. of ammonia to yield 1 mol.prop. each of adenosine 5′-phosphoramidate and sulphate; no AMP is found. The highly purified enzyme does not catalyse any of the known reactions of adenosine 5′-phosphosulphate, including those catalysed by ATP sulphurylase, adenosine 5′-phosphosulphate kinase, adenosine 5′-phosphosulphate sulphotransferase or ADP sulphurylase. Adenosine 5′-phosphoramidate is found in old samples of the ammonium salt of adenosine 5′-phosphosulphate and can be formed non-enzymically if adenosine 5′-phosphosulphate and ammonia are boiled. In the non-enzymic reaction both adenosine 5′-phosphoramidate and AMP are formed. Thus the enzyme forms adenosine 5′-phosphoramidate by selectively speeding up an already favoured reaction.

Rat liver ribonucleotide reductase: separation, purification, and properties of two nonidentical subunits

1982 ◽  
Vol 60 (4) ◽  
pp. 463-470 ◽  
Author(s):  
T. Youdale ◽  
J. P. MacManus ◽  
J. F. Whitfield
Keyword(s):  
Rat Liver ◽  
Ribonucleotide Reductase ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Relative Mass ◽  
Purification And Properties ◽  
Exclusion Chromatography

Two nonidentical subunits of mammalian ribonucleotide reductase, L1 and L2, from regenerating rat liver have been extensively purified for the first time. They were separated by dATP-Sepharose affinity chromatography. Subunit L1, which bound to dATP-Sepharose, was eluted with 50 mM ATP and purified to homogeneity (as demonstrated by sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis) by molecular exclusion high-pressure liquid chromatography (HPLC). This subunit had an apparent relative mass (Mr) of 45 000 and a Km of 0.9 × 10−4 for CDP. Subunit L2, which did not bind to dATP-Sepharose, was purified by pH 5.2 precipitation followed by chromatography on CM-Sephadex, molecular exclusion HPLC, and DEAE-cellulose. This subunit contained iron and had an apparent Mr of 120 000 by HPLC molecular exclusion chromatography, but showed two bands (Mr 75 000 and Mr 47 000) on SDS–polyacrylamide gel electrophoresis. Neither L1 nor L2 separately had any enzyme activity but when combined they reduced CDP to dCDP.

scholarly journals Isolation and partial characterization of canine epidermal growth factor/urogastrone

1985 ◽  
Vol 229 (3) ◽  
pp. 611-619 ◽  
Author(s):  
R Kobayashi ◽  
J R Reeve ◽  
J H Walsh
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Mouse Fibroblast ◽  
A431 Cells ◽  
Peptide Component ◽  
Epidermal Growth

Canine epidermal growth factor (EGF)/urogastrone was partially purified from dog urine by fractional precipitation with (NH4)2SO4, ion-exchange chromatography with DEAE-cellulose DE-52, gel filtration with Sephadex G-50, and a second DE-52 chromatography, to yield receptor-competing activity equivalent to 13 micrograms of standard mouse EGF/litre of starting urine. The purification was monitored by a competitive radioreceptor assay using fixed monolayers of A431 cells. The partially purified canine EGF/urogastrone demonstrated a growth-stimulating activity in 3T3 mouse fibroblast cells as potent as mouse EGF. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed one major peptide component with an Mr similar to that of mouse EGF, and two minor peptides of slightly higher Mr. The major peptide component was isolated after reduction and its amino acid composition was determined.

Molecular weight determination and partial characterization of Klebsiella pneumoniae hemolysins

1986 ◽  
Vol 32 (11) ◽  
pp. 884-888 ◽  
Author(s):  
Lucila Isabel Barberis ◽  
Alberto Jorge Eraso ◽  
Maria Cristina Pàjaro ◽  
Inès Albesa
Keyword(s):  
Klebsiella Pneumoniae ◽  
Gel Electrophoresis ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Growth Media ◽  
Molecular Weight Determination ◽  
Periodic Acid Schiff

Two thiol-activated Klebsiella pneumoniae hemolysins were purified from growth media by means of salt precipitation, gel filtration, ion-exchange chromatography, and polyacrylamide gel electrophoresis. The hemolysins peaks coincided with the protein and glycoprotein peaks as determined by chromatography and electrophoresis, The molecular weights, estimated by gel filtration, were 8400 and 19 000; by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, the values were calculated as 15 500 and 27 000. The electrophoretic bands were best detected by the periodic acid–Schiff method. Reduction of the disulfide linkages did not cause the originally larger molecule to break into 8400 and 19 000 hemolysins. However, trypsin treatment cleaved the 19 000 hemolysin into an active moiety, with an electrophoretic migration similar to the 8400 hemolysin. A naturally occurring proteolytic activity was investigated using pepstatin and antipain. When the trypsin inhibitor was added to the system, the hemolytic activity was detected only in the 19 000 hemolysin and the smaller hemolysin was absent.

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