scholarly journals Biotransformation of Aromatic Methyl Groups to Aldehyde Groups Using Laccase of Gloephyllum Stratum MTCC-1117

2020 ◽  
Author(s):  
RAM SAHAY
Keyword(s):  
White Rot Fungi ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
White Rot ◽  
Show Absorption Band ◽  
Yellow Colour ◽  
Sds Page ◽  
Coir Dust ◽  
Laccase Enzyme ◽  
Aldehyde Groups

Abstract Laccases has been produced by white rot fungi are involved in lignin containing natural substrates wheat-straw, bagasse, saw-dust, corn cob and coir dust particle on the production of laccase enzyme in the aqueous cultivation medium of Gloephyllum stratum MTCC1117. The approach involved concentration of aqueous filtrate by ultrafiltration and anion exchange chromatography on DEAE (diethyl aminoethyl cellulose). From SDS-PAGE analysis the molecular mass of the purified enzyme is 57 kDa. The Km and kcat values of the laccase are found to be 18 μM and 0.34 s-1 using 2,6-dimethoxyphenol as the substrate, giving a kcat/Km value is 1.70 x 103 M-1 s-1. The pH and temperature optimum were 4.5 and 40 °C respectively. The purified enzyme has yellow colour and does not show absorption band around 610 nm found in blue laccases. Moreover the conversion of methylbenzene to benzaldehyde in the lack of mediator molecules, property exhibited by yellow laccases.

scholarly journals Isolation and Physicochemical Characterization of Laccase fromGanoderma lucidum-CDBT1 Isolated from Its Native Habitat in Nepal

2016 ◽  
Vol 2016 ◽  
pp. 1-10 ◽  
Author(s):  
Prabin Shrestha ◽  
Bishnu Joshi ◽  
Jarina Joshi ◽  
Rajani Malla ◽  
Lakshmaiah Sreerama
Keyword(s):  
Lentinula Edodes ◽  
Culture Media ◽  
Physicochemical Characterization ◽  
White Rot Fungi ◽  
Anion Exchange Chromatography ◽  
Fungal Species ◽  
Exchange Chromatography ◽  
White Rot ◽  
Laccase Production ◽  
Native Habitat

At present, few organisms are known to and capable of naturally producing laccases and white rot fungi are one such group. In the present study, three fungal species, namely,Ganoderma lucidum-CDBT1, Ganoderma japonicum,andLentinula edodes, isolated from their native habitat in Nepal were screened for laccase production, andG. lucidum-CDBT1 was found to express highest levels of enzyme (day 10 culture media showed 0.92 IU/mg total protein or 92 IU/mL laccase activity with ABTS as substrate). Lignin extracted from rice straw was used in Olga medium for laccase production and isolation fromG. lucidum-CDBT1. Presence of lignin (5 g/L) and copper sulfate (30 μM) in the media increased the extracellular laccase content by 111% and 114%, respectively. The laccase enzyme produced byG. lucidum-CDBT1 was fractionated by ammonium sulfate and purified by DEAE Sepharose anion exchange chromatography. The purified enzyme was found to have a molecular mass of 43 kDa and exhibits optimal activity at pH 5.0 and 30°C. The isolated laccase was thermally stable for up to 70°C for 1 h and exhibited broad pH stability. The kinetic constants,Km,Vmax, andKcat, determined using 2,2′-azinobis-(-3-ethylbenzothiazoline-6-sulfonic acid) as substrate were found to be 110 μM, 36 μmol/min/mg, and 246 min−1, respectively. The isolated thermostable laccase will be used in future experiments for delignification process.

scholarly journals Coal Depolymerising Activity and Haloperoxidase Activity of Mn Peroxidase fromFomes durissimusMTCC-1173

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Sunil Kumar Singh ◽  
Meera Yadav ◽  
Sudha Yadava ◽  
Kapil Deo Singh Yadav
Keyword(s):  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Sodium Lactate ◽  
Low Ph ◽  
Exchange Chromatography ◽  
Fungal Strain ◽  
Mn Peroxidase ◽  
Sds Page ◽  
Catalytic Rate ◽  
Temperature Optima

Mn peroxidase has been purified to homogeneity from the culture filtrate of a new fungal strainFomes durissimusMTCC-1173 using concentration by ultrafiltration and anion exchange chromatography on diethylaminoethyl (DEAE) cellulose. The molecular mass of the purified enzyme has been found to be 42.0 kDa using SDS-PAGE analysis. The values using MnSO4and H2O2as the variable substrates in 50 mM lactic acid-sodium lactate buffer pH 4.5 at were 59 μM and 32 μM, respectively. The catalytic rate constants using MnSO4and H2O2were 22.4 s−1and 14.0 s−1, respectively, giving the values of 0.38 μM−1s−1and 0.44 μM−1s−1, respectively. The pH and temperature optima of the Mn peroxidase were 4 and , respectively. The purified MnP depolymerises humic acid in presence of H2O2. The purified Mn peroxidase exhibits haloperoxidase activity at low pH.

Production of PEGylated GCSF from Non-classical Inclusion Bodies Expressed in Escherichia coli

2021 ◽  
Author(s):  
Nguyen Thi My Trinh ◽  
Tran Linh Thuoc ◽  
Dang Thi Phuong Thao
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Insoluble Fraction ◽  
Exchange Chromatography ◽  
Native Page ◽  
E Coli ◽  
Sds Page ◽  
Stimulating Factor

Background: The recombinant human granulocyte colony stimulating factor con-jugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that Granulocyte Colony Stimula-ting Factor (GCSF) could be expressed as non-classical Inclusion Bodies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, in this study, a simple process was developed to produce PEGylated GCSF from ncIBs. Methods: BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 L bioreactor and the expression of GCSF was induced by adding 0.5 mM IPTG. After 24 hr of fermentation, cells were collected, resuspended, and disrupted. The insoluble fraction was obtained from cell lysates and dissolved in 0.1% N-lauroylsarcosine solution. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used for the con-jugation with 5 kDa PEG. The PEGylated GCSF was purified using two purification steps, including anion exchange chromatography and gel filtration chromatography. Results: PEGylated GCSF was obtained with high purity (~97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 kDa PEG molecule (monoPEG-GCSF). Conclusion: These results clearly indicate that the process developed in this study might be a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in Escherichia coli (E. coli).

Antithrombin Milano, Single Amino Acid Substitution at the Reactive Site, Arg393 to Cys

1988 ◽  
Vol 60 (03) ◽  
pp. 471-475 ◽  
Author(s):  
H Erdjument ◽  
D A Lane ◽  
H Ireland ◽  
V Di Marzo ◽  
M Panico ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Fast Atom Bombardment ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Reducing Conditions ◽  
Sds Page ◽  
Two Peaks

SummaryAntithrombin Milano is an unusual antithrombin variant, exhibiting an abnormal, fast moving component on crossed immunoelectrophoresis (in the absence of heparin). Antithrombin isolated from the propositus could be resolved into two peaks on anion-exchange chromatography; anti thrombin Milano peak 1 of Mr ∼60,000 which could inhibit thrombin, and antithrombin Milano peak 2 of Mr ∼120,000 which was inactive. The latter component also reacted with antisera to both antithrombin and albumin on immunoblotting. Under reducing conditions, the ∼120,000 Mr component migrated on SDS-PAGE as two distinct bands with Mr ∼60,000, one of which reacted with antiserum to antithrombin and the other (of slower mobility) of which reacted with antiserum to albumin only. These and other results established the ∼120,000 Mr component to be an inactive, disulphide-linked variant antithrombin and albumin complex. The variant antithrombin was isolated, following reduction and S-carboxy-methylation, by reverse-phase HPLC and then it was fragmented with CNBr. A major CNBr pool containing the sequence Gly339-Met423 was treated with trypsin, followed by V8 protease. The resulting peptides were analysed by fast atom bombardment mass spectrometry (Fab-MS) mapping. A peptide of molecular mass 1086, corresponding to the normal sequence Ala382-Arg393, was almost absent from the mass spectrum, but an additional peptide of mass number 1772 was present. These results are almost identical to those found in another variant antithrombin, North-wick Park (Erdjument et al., J Biol Chem, 262: 13381, 1987; Erdjument et al., J Biol Chem, 263: 5589-5593, 1988), indicating the same single amino acid substitution of Arg393 to Cys.

scholarly journals Glutathione S-transferases of the yeast Yarrowia lipolytica have unusually large molecular mass

1998 ◽  
Vol 333 (3) ◽  
pp. 839-845 ◽  
Author(s):  
Vivienne FOLEY ◽  
David SHEEHAN
Keyword(s):  
Molecular Mass ◽  
Yarrowia Lipolytica ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Post Translational Modification ◽  
Native Molecular Mass ◽  
Sds Page ◽  
Glutathione S Transferases ◽  
Yeast Yarrowia Lipolytica

Two similar glutathione S-transferases (GSTs), which do not bind to glutathione– or S-hexylglutathione–agarose affinity resins, have been purified from the yeast Yarrowia lipolytica. An approx. 400-fold purification was obtained by a combination of DEAE-Sephadex, phenyl-Sepharose, hydroxyapatite and Mono-Q anion-exchange chromatography. The native molecular mass of both proteins was estimated as approx. 110 kDa by both Superose-12 gel-filtration chromatography and non-denaturing electrophoresis. SDS/PAGE indicated a subunit mass of 50 kDa. Reverse-phase HPLC of purified proteins gave a single, well-resolved, peak, suggesting that the proteins are homodimers. Identical behaviour on HPLC, native electrophoresis and SDS/PAGE, N-terminal sequencing, sensitivity to a panel of inhibitors and identical specific activities with 1-chloro-2,4-dinitrobenzene as substrate suggest that the two isoenzymes are very similar. The enzymes do not immunoblot with antisera to any of the main GST classes, and N-terminal sequencing suggests no clear relationship with previously characterized enzymes, such as that of the fungus, Phanerochaete chrysosporium [Dowd, Buckley and Sheehan (1997) Biochem. J. 324, 243–248]. It is possible that the two isoenzymes arise as a result of post-translational modification of a single GST isoenzyme.

Production, purification, and characterization of an extracellular endo-β-1, 3-glucanase from a monokaryon of Schizophyllum commune ATCC 38548 defective in exo-β-1, 3-glucanase formation

1994 ◽  
Vol 40 (1) ◽  
pp. 18-23 ◽  
Author(s):  
Andreas Prokop ◽  
Peter Rapp ◽  
Fritz Wagner
Keyword(s):  
Molecular Mass ◽  
Schizophyllum Commune ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Glucanase Activity ◽  
Sds Page ◽  
S 100 ◽  
Reduced Activity

Production of extracellular β-1, 3-glucanase activity by a monokaryotic Schizophyllum commune strain was monitored and results indicated that the β-glucanase activity consisted of an endo- β-1, 3-glucanase activity, besides a negligible amount of β-1, 6-glucanase and β-glucosidase activity. Unlike the β-1, 3-glucanase production of the dikaryotic parent strain S. commune ATCC 38548, the β-1, 3-glucanase formation of the monokaryon was not regulated by catabolite repression. The endo- β-1, 3-glucanase of the monokaryon was purified from the culture filtrate by lyophilization, anion exchange chromatography on Mono Q, and gel filtration on Sephacryl S-100. It appeared homogeneous on SDS-PAGE with a molecular mass of 35.5 kDa and the isoelectric point was 3.95. The enzyme was only active toward glucans containing β-1, 3-linkages, including lichenan, a β-1, 3-1, 4-D-glucan. It attacked laminarin in an endo-like fashion to form laminaribiose, laminaritriose, and high oligosaccharides. While the extracellular β-glucanases from the dikaryotic S. commune ATCC 38548 degraded significant amounts of schizophyllan, the endo- β-1, 3-glucanase from the monokaryon showed greatly reduced activity toward this high molecular mass β-1, 3-/β-1, 6-glucan. The Km of the endoglucanase, using laminarin as substrate, was 0.28 mg/mL. Optimal pH and temperature were 5.5 and 50 °C, respectively. The enzyme was stable between pH 5.5 and 7.0 and at temperatures below 50 °C. The enzyme was completely inhibited by 1 mM Hg2+. Growth of the monokaryotic S. commune strain was not affected by its constitutive endo- β-1, 3-glucanase formation.Key words: endo- β-1, 3-glucanase, Schizophyllum commune, monokaryon, constitutive endo- β-1, 3-glucanase formation.

scholarly journals Effect of Physisporinus vitreus on wood properties of Norway spruce. Part 2: Aspects of microtensile strength and chemical changes

Holzforschung ◽  
2011 ◽  
Vol 65 (5) ◽  
Author(s):  
Christian Lehringer ◽  
Bodo Saake ◽  
Vjekoslav Živković ◽  
Klaus Richter ◽  
Holger Militz
Keyword(s):  
Norway Spruce ◽  
Lignin Content ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
White Rot ◽  
White Rot Fungus ◽  
Wall Structure ◽  
Fungal Activity ◽  
Selective Delignification ◽  
Simultaneous Degradation

AbstractThe biotechnological application of the white rot fungusPhysisporinus vitreusnamed “bioincising” is currently being investigated for permeability improvement of Norway spruce (Picea abies(L.) Karst.) wood. During short-term (<9 weeks) incubation, fungal activity induces degradation of pit membranes and a simultaneous alteration of the tracheid cell wall structure. In Part 1 of this article series, the occurrence of selective delignification and simultaneous degradation was shown by UV-microspectrophotometry (UMSP). Moreover, significant reduction of Brinell hardness was recorded after 7 and 9 weeks incubation. For a better understanding of the chemical alterations in the wood constituents and the corresponding changes of mechanical properties due to fungal activity, we applied microtensile tests on thin strips that were prepared from the surface of incubated Norway spruce wood. Indications for the occurrence of selective delignification and simultaneous degradation were evident. Determination of lignin content and carbohydrate analysis by borate anion exchange chromatography confirmed the results. The present study verifies the findings from Part 1 of this article series and from previously conducted microscopic investigations. Now, the degradation characteristics ofP. vitreusare established and the bioincising process can be further optimized with higher reliability.

scholarly journals Produksi dan karakterisasi lakase Omphalina sp. Production and characterization of Omphalina sp. laccase

2016 ◽  
Vol 75 (2) ◽  
Author(s):  
. SISWANTO ◽  
. SUHARYANTO ◽  
Rossy FITRIA
Keyword(s):  
Oil Palm ◽  
Laccase Activity ◽  
Research Result ◽  
White Rot Fungi ◽  
White Rot ◽  
Partial Purification ◽  
Empty Fruit Bunches ◽  
Optimum Ph ◽  
Laccase Enzyme

SummaryOmphalina sp. a white-rot fungi (WRF)originated from oil palm plantation has abilityto degrade empty fruit bunches of oil palm(EFBOP) so that it is expected to producelaccase with high activity. The ability ofOmphalina sp. to produce laccase enzyme onliquid fermentation will be studied. The enzymewill also be partially purified andcharacterized. The research result showed thatthe highest enzyme activity (1.162 U/mL) wasobtained using glucose malt yeast (GMY)medium at room temperature for four days.The addition of 2,5-xylidine as an inducerproduced laccase earlier i.e two days, but theactivity of laccase was less active afterprolonged incubation compared to that ofcontrol. The laccase produced on mediumcontaining 2% EFBOP reached optimumactivity as much as 0.38 U/mL after 10 th daysof incubation. Partial purification of laccaseon Sephacryl S-200 HR column resulted58.23% of yield recovery with twice purity thanbefore. The optimum pH of laccase was 4.5.Laccase activity was stable even after heatedon 50 o C for 30 minutes, but then decreasedwhen heated until 60 o C. The laccase has K Mand V max as much as 0.15 mM and 0.56 U/mLrespectively.RingkasanOmphalina sp., adalah fungi pelapuk putih(FPP) hasil isolasi dari kebun kelapa sawityang diketahui mampu mendegradasi tandankosong kelapa sawit (TKKS) dengan cepatsehingga diharapkan mampu menghasilkanlakase dengan aktivitas tinggi. KemampuanOmphalina sp. menghasilkan enzim lakasepada fermentasi cair akan dipelajari. Selain itu,lakase yang dihasilkan akan dimurnikan secaraparsial serta dilakukan karakterisasi pH, suhu,dan konsentrasi substrat optimum. Hasilpenelitian menunjukkan bahwa Omphalina sp.menghasilkan lakase dengan aktivitas tertinggi(1,162 U/mL) pada medium glucose malt yeast(GMY) yang diinkubasikan pada suhu ruangselama empat hari. Penambahan 2,5-xilidinsebagai induser mempercepat produksi lakaselebih awal yaitu dalam waktu dua hari, namunaktivitasnya masih lebih rendah dibandingkandengan kontrol pada inkubasi lebih lanjut.Lakase dari Omphalina sp. juga dapatdiproduksi pada medium yang mengandung2% TKKS dan aktivitasnya mencapai0,38 U/mL yang diinkubasi dalam suhu ruangselama 10 hari. Pemurnian parsial pada kolomSephacryl S-200 HR menghasilkan rendemensebesar 58,23% dengan kemurnian dua kalinya.Aktivitas lakase optimum pada pH 4,5 dantetap stabil setelah pemanasan selama 30 menitpada suhu ruang hingga 50 o C dan menuruntajam pada suhu 60 o C. Lakase Omphalina sp.menghasilkan nilai K M dan V maks masing-masing sebesar 0,15 mM dan 0,56 U/mL.

scholarly journals Adding value in barley malt rootlets as a source of 5’-phosphodiesterase

2020 ◽  
Vol 15 (1-2) ◽  
pp. 27-37
Author(s):  
Sunčica Beluhan ◽  
Ivana Karmelić ◽  
Mirela Ivančić Šantek
Keyword(s):  
Half Life ◽  
Thermal Inactivation ◽  
Specific Activity ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Barley Malt ◽  
First Order Kinetics ◽  
Unit Fractions ◽  
Sds Page ◽  
Phosphodiesterase 5

A thermostable 5’-phosphodiesterase (5’-PDE, EC 3.1.4.1) was extracted from barley (Hordeum distichum var. Rex) malt rootlets. The purification procedure comprised acetone precipitation, S-Sepharose cation-exchange and DEAE-Sepharose anion-exchange chromatography. The enzyme was purified 101-fold with a recovery of 22% and a specific activity of 81.9 U mg-1 protein, Optimum enzyme activity was obtained at 70 °C, and pH 8.9. The SDS-PAGE profiling of the purified protein exhibited molecular weight of 116 kDa and revealed three sub-unit fractions of 26, 43, and 56 kDa making up its active configuration. The kinetic constants Km and Vmax were determined as 0.25 mM and 0.816 mmol min-1, respectively. Thermodynamic studies showed that the thermal inactivation of purified barley malt rootlets 5’-PDE followed the first-order kinetics, indicating inactivation energy (Ed) of 134 kJ mol-1. The half-life (t1/2) at 70 °C was estimated as 169 min. Thermodynamic parameters ΔH*, ΔS* and ΔG* were determined as a function of temperature and were 131.15 kJ mol-1, 37.01 kJ mol-1 K-1 and 118.4 kJ mol-1, respectively. The purified enzyme has long half-life with 11 days at 0 °C, 37 hours at 4 °C and 11 hours at room temperature. These results provide useful information about the factors that affects the activity of barley malt rootlets 5’-PDE and suggests a good indication for application of this enzyme in pharmaceutical and food industry.

scholarly journals A New Laccase of Lac 2 from the White Rot Fungus Cerrena unicolor 6884 and Lac 2-Mediated Degradation of Aflatoxin B1

Toxins ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 476 ◽  
Author(s):  
Zhimin Zhou ◽  
Renkuan Li ◽  
Tzi Bun Ng ◽  
Yunyun Lai ◽  
Jie Yang ◽  
...  
Keyword(s):  
Aflatoxin B1 ◽  
Environmental Pollutants ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Mass Spectrometry Data ◽  
White Rot ◽  
White Rot Fungus ◽  
Multicopper Oxidases ◽  
Cerrena Unicolor

Aflatoxin B1 (AFB1) is a known toxic human carcinogen and can be detoxified by laccases, which are multicopper oxidases that convert several environmental pollutants and toxins. In this study, a new laccase that could catalyze AFB1 degradation was purified and identified from the white-rot fungus Cerrena unicolor 6884. The laccase was purified using (NH4)2SO4 precipitation and anion exchange chromatography, and then identified as Lac 2 through zymogram and UHPLC-MS/MS based on the Illumina transcriptome analysis of C. unicolor 6884. Six putative laccase protein sequences were obtained via functional annotation. The lac 2 cDNA encoding a full-length protein of 512 amino acids was cloned and sequenced to expand the fungus laccase gene library for AFB1 detoxification. AFB1 degradation by Lac 2 was conducted in vitro at pH 7.0 and 45 °C for 24 h. The half-life of AFB1 degradation catalyzed by Lac 2 was 5.16 h. Acetosyringone (AS), Syrinagaldehyde (SA) and [2,2′ -azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] (ABTS) at 1 mM concentration seemed to be similar mediators for strongly enhancing AFB1 degradation by Lac 2. The product of AFB1 degradation catalyzed by Lac 2 was traced and identified to be Aflatoxin Q1 (AFQ1) based on mass spectrometry data. These findings are promising for a possible application of Lac 2 as a new aflatoxin oxidase in degrading AFB1 present in food and feeds.

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