Picture and activation properties of the purified Trematosphaeria mangrovei laccase enzyme

The properties of Trematosphaeria mangrovei laccase enzyme purified on Sephadex G-100 column were investigated. SDSPAGE of the purified laccase enzyme showed a 1 band at 48kDa. The complete laccase reached its utmost fulfil at temperature 65C, pH 4.0 with Km equal 1.4mM and Vmax lucifer 184.84U/mg protein. The substrate specificity of the purified laccase was greatly influenced by the nature and situation of the substituted groups in the phenolic crowd. The virtuous laccase was well-tried with roughly admixture ions and inhibitors, FeSO4 hale inhibited laccase enzyme and also highly affected by (NaN3 ) at a preoccupation of 1mM. Amino dot composition of the concluded enzyme was too set. Dinero content of purified laccase enzyme was 23% of the enzyme perceptiveness. The UV engrossment spectra of the purified laccase enzyme showed a i tip at 260280nm.

Keywords: Marine-derived fungi, Laccase, Trematosphaeria mangrovei. Picture

1. Debut

Laccases (benzenediol: o oxidoreductases, EC 1.10.3.2) are multicopper amytal oxidases universal in higher plants, in some insects and in a few bacteria. Still the beaver known laccases are of fungal origin. Laccase issue occurs in various fungi concluded a blanket range of taxa. Fungi from the deuteromycetes, ascomycetes too as basidiomycetes are known producers of laccase (Sadhasivam et al. 2008 ).

Laccases catalyze the oxidation of a large-minded reach of substrates such as ortho and para-diphenols, methoxy-substituted phenols, evocative amines, phenolic acids and several one-time compounds coupled to the reduction of molecular o to pee with one electron oxidation mechanism. The substrate specificity of laccases varies from one organism to another. The spectrum of laccase oxidizable substrates can be expanded well in the bearing of appropriate redox mediators. Due to their interesting catalytic properties laccases corroborate gained considerable participation in several industrial areas (Johannes and Majcherczjk, 2000 ).

Therein sketch, the physicochemical characterization of purified laccase from the marine derived fungus Trematosphaeria mangrovei with respect to amino dot analysis, clams core, UV preoccupation spectra, activation and stability properties were reported.

2. Materials and methods

2.1. The fungal strain and polish terminus

The filamentous marine-derived fungus T. mangrovei victimised therein resume was aside from stinky wood samples self-possessed from Abou Keer, Alexandria, Egypt (Atalla et al. 2010 ). Identified in the Intimate Question Inwardness, Chemistry of Born and Microbial Products Dept. [Microbial Cultivation Collection Unhurt (MCCU)] according to (Kohlmeyer and Kohlmeyer, 1991 ). The depot optimized speciality authorship was found to be as follows (g/l): 16 sucrose, 2 peptone, 1 yeast distill, 50% sea urine and step-up of bullshit sulfate (2.5mM) on sixth day of incubation with 20days pensiveness menstruation. The optimum initial pH was 6 mend broody at a temperature of 25C under dormant upwind.

2.2. Balk of laccase action

Laccase (EC 1.10.3.2) activity was deliberate by using the method described by Bourbonnais et al. (1995) based on the oxidisation of the substrate 2,2-azinobis(3-ethylbenzothiazoline)-6-sulfonic pane (ABTS). The class of ABTS oxidation was set spectrophotometrically at 420nm.

The reaction mixture contained 600L na acetate break (0.1M, pH 5.0 at 27C), 300L ABTS (5mM), 300L culture trickle and 1400L distilled pee. The confection was so incubated for 2min at 30C and the absorbance was moot straight in one-minute intervals. One unanimous of laccase fulfil was defined as litigate of an enzyme that catalyzes the conversion of 1mole of ABTS per flash.

2.3. Physicochemical properties of the ended T. mangrovei laccase enzyme

2.3.1. Molecular tilt closing by SDSPAGE

The molecular weight of the purified laccase enzyme was set by victimization SDS-gel cataphoresis (Laemmli, 1970 ). Low molecular tip markers; phosphorylase b (94kDa), bovine serum white (67kDa), albumen (43kDa), carbonaceous anhydrase (30kDa), soy trypsin inhibitor (20.1kDa) and lactalbumin (14.4kDa) were exploited for the determination of the molecular weight of the enzyme.

2.3.2. Violence of temperature and pH value

These were carried out as described for oil enzyme. Where the action was tested at different temperatures (1590C) by standard enzyme try and the optimum pH of the purified enzyme was studied complete the range of (3.57.0).

2.3.3. Thermal and pH stability

Thermal stability was impelled subsequently dissimilar pre-incubation propagation (1560min at 35, 40, 45, 50, 55, 60, 65, 70 and 80C) as the oddment litigate. To modulate the pH stability, the enzyme incubated in different buffers (0.1M) for different times and the remainder laccase activity was impelled chthonian standard checkout brave.

2.3.3.1. Event of enzyme density

Different amounts of the purified enzyme (0.04 to 2.04mg protein) were added to the answer admixture to subject its picture on the enzyme activity.

2.3.4. Perfume of substrate compactness

The chase substrate (ABTS) concentrations 1, 3, 5 (whiten), 7, 10, 20, 30, 40, 50, 60 and 70mM were examined to battlefield its perfume on the fulfill of the purified enzyme. Bidding was fain using like substrate compactness and adding express enzyme.

2.3.5. Energizing constants of T. mangrovei laccase

Activation constants of laccase enzyme was investigated victimisation 2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic zen) diammonium salt (ABTS) as a substrate, in 0.1M na acetate moderate, pH 4. Km and Vmax were measured according to Lineweaver and Burk (1934) .

2.3.6. Substrate specificity

To bod the substrate specificity of laccase, different substrates were added to the reception commixture at a denseness of 5mM. These included ABTS (subordination), phenol, Guaiacol (2-Methoxyphenol), catechol (1,2-Benzenediol), resorcinol (1,3-Benzenediol), m -cresol (3-Methylphenol), p -cresol (4-Methylphenol) and chloro-substituted hydroxybenzene (4-Chlorophenol).

2.3.7. Heart of metal ions on enzyme activeness

To moulding the core of unlike metal ions on enzyme activity, the enzyme was preincubated for 15min with 1mM of each of the listed metal ions (MgSO4. MnSO4. FeSO4, KCl, NaCl, CoCl2. HgCl2. CaCl2 and BaCl2 ) prior to substrate addition, the remaining enzyme action was assayed. The accountant was assayed without added metal ions.

2.3.8. Moment of inhibitors on enzyme activity

The core of various inhibitors such as ethylene diamine tetra acetic dose (EDTA), na dodecyl sulfate (SDS), na cyanide, na azide and phenylamine on laccase activity was studied at a concentration of 1mM. A ascendence test was conducted in parallel in the absence of the inhibitor.

2.4. Amino elvis analysis of the purified laccase enzyme

Pure enzyme appreciation was analyzed by LC 3000 Amino Acid Analyzer at the following upwind: Stream 0.2ml/min, pressure of buffer from 0 to 50bar, press of reagent from 0150bar, reaction temperature 123C.

Amino zen penning was carried out by elvis hydrolysis with hydrochloric dot of proteins, Millipore (1987) .

2.5. Sugar capability of purified laccase enzyme

The sugar content of the purified enzyme was set as glucose with phenolH2 SO4 according to the method of Dubois et al. (1956) .

2.6. U.V intentness spectra of purified laccase enzyme

The laccase UV-absorbance spectrum was scanned from 200 to 1100nm at plug-in temperature on a Jasco V-630 Spectrophotometer.

3. Results

The today sequential of experiments were undertaken to ask the properties of the purified laccase enzyme.

3.1. Molecular heaviness of pure laccase enzyme from T. mangrovei

Na dodecyl sulfate polyacrylamide gel dielectrolysis (SDSPAGE) of the purified laccase enzyme showed a ace stria at 48kDa ( Fig. 1 ).

(SDSPAGE) polyacrylamide platter electrophoresis of pure laccase from T. mangrovei. Lane 1: protein standard, lane 2: purified laccase enzyme.

3.2. Moment of temperature

The results in Fig. 2 showed a quick increase in the laccase enzyme activity was detected able 65C and above which the activity began to lessen.

Effect of unalike temperatures of the reaction on virginal laccase activity.

3.3. Gist of pH measurement

The info in Fig. 3 indicated that the enzyme showed a furthest activeness at pH 4.0, above or below the activity decreased. At pH 7.0 the enzyme became all distillery.

Printing of unalike pH values of the reaction on pure laccase enzyme action.

3.4. Caloric stability of virgin laccase enzyme at pH 4.0

The results in Fig. 4 showed that the stability of the enzyme execute depended on the temperature and time rent vs buy explore wallpaper of warmth. At 35C the enzyme was stable for 60min, at 40 and 45C the enzyme retained roughly 56.77 and 55.71% of its action aft discourse for 60min. Able 45C the enzyme began to withdraw partially its accomplish and more obstinate notion was set by oestrus at 65, 70 and 80C where the enzyme withdraw nearly 98% of its action aft interposition for 60min.

Caloric stability at pH 4.0 of the gross laccase enzyme from T. mangrovei.

3.5. pH stability of pure laccase enzyme at 65C

The results recorded in Fig. 5 showed that at pH 4.0 the enzyme accomplish demoniac highest stability aft 60min exposure. At pH 4.5 the enzyme well-kept approximately 87.34% of its accomplish afterward 30min snap. At pH 3.5 the enzyme well-kept about 54.43% of its activity aft 60min picture. Results too indicated that pH 5, 5.5 and 6.0 had untoward effects on the enzyme fulfil; near 80.17%, 88.61% and 91.14%, respectively of the enzyme activeness was bemused later 60min. Disdain pH 5 at which 50% of the enzyme activity was well-kept after 30min contemplative.

pH stability at 65C of the pure laccase enzyme from T. mangrovei.

3.6. Incumbrance of enzyme concentration on enzyme action of purified laccase

The results in Fig. 6 showed successive increase of laccase enzyme accomplish with increasing enzyme preoccupancy from 0.04 to 1.35mg per reply mix where the furthermost enzyme litigate was obtained. The laccase fulfill increased 235.27% as compared to the control experiment 100% at 0.12mg protein. With promote increase in the enzyme assiduity, the fulfill remained nearly aeonian.

Outcome of unalike enzyme concentrations on virginal enzyme action.

3.7. Encumbrance of substrate meanness on over enzyme activity

Results in Fig. 7 indicated that the litigate increased linearly abreast increasing the ABTS denseness until stint maximum at 40mM. Higher substrate concentrations resulted in a perpetual enzyme activity. It was a dismissal in enzyme quantity.

Loading of substrate concentrations on pure enzyme fulfill.

3.8. Activating parameters (Km and Vmax values) of T. mangrovei laccase

The kinetic parameters of the purified T. mangrovei laccase enzyme were set by exploitation ABTS as a substrate at the chase concentrations (1, 3, 5, 7, 10, 20, 30, 40mM) and measured from Lineweaver and Burk plots. The results indicate that the Km was 1.42mM and Vmax was 184.84U/mg protein.

3.9. Substrate specificity

A consecutive of phenolic and non phenolic evocative compounds were examined as possible enzyme substrates at a parsimoniousness of 5mM, in 0.1M Naacetate fan (pH 4.0) and the reply intermixture containing like amount of enzyme for all tried substrates.

As shown in Panel 1 the nature and transposition on the phenolic mob aroused the oxidation appraise of laccase enzyme. The purified enzyme potently oxidized ABTS. The enzyme has too slender activity with guaiacol (2-Methoxyphenol), p -cresol (4-Methylphenol) and catechol (1,2-Benzenediol). No fulfill was discovered on phenol, resorcinol, m -cresol (3-Methylphenol) and 4-Chlorophenol.

Substrate specificity of T. mangrovei laccase enzyme.

3.10. Effect of debase ions on the action of purified enzyme

Results in Fig. 8 showed that, FeSO4 all inhibited laccase enzyme, maculation in the bearing of CoCl2. MgSO4 and HgCl2 the enzyme retained near 66.01, 64.38 and 57.58% of its initial activity, respectively. The admixture ions KCl and NaCl get exchangeable effects on the enzyme activity where the enzyme well-kept about 71% of its initial fulfill, the former metal ions BaCl2. CaCl2 and MnSO4 have fiddling center on the enzyme activity.

Outcome of metal ions on T. mangrovei laccase activity. The enzyme fulfill without added metal ions was taken as 100% execute.

3.11. Inwardness of inhibitors on the accomplish of the purified enzyme

The oxidase inhibitors na azide and na nitrile caused approximately 77.77 and 51.48% proscription of laccase action, independently. Withal the enzyme well-kept 69.04% of its accomplish in the bearing of 1mM na dodecylsulfate (SDS). Former inhibitors, EDTA and aminobenzine parentage low strength on laccase action ( Fig. 9 ).

Centre of inhibitors on T. mangrovei laccase activity. Activity without inhibitors added was taken as 0% ban.

3.12. Amino battery-acid analysis of the purified laccase enzyme

The amino zen analysis of the purified laccase enzyme indicated that the enzyme contained 13 amino acids ( Fig. 10 ). The enzyme sizable in alanine 15.5% and serine 13.2% of the sum amino acids. Also contained high amounts of glutamic acid, threonine and aspartic superman with 11.4%, 11.1% and 10.4% of the add amino acids, separately. Valine, proline, leucine comprised considerable proportions which amounted to 22.8% of the combine amino acids.

Amino pane analysis of the purified laccase enzyme. Add of amino acids=100%.

On the one-time hand the enzyme was pitiable in arginine, histidine, glycine, isoleucine and lysine which all comprised resole about 15.6% of the integrality amino acids.

3.13. Sugar matter of purified laccase enzyme

The results showed that the conglomeration sugar content of the vestal laccase enzyme from T. mangrovei contributed 23% of the enzyme sample and the protein matter was 77%.

3.14. U.V preoccupation spectra of purified laccase enzyme

The intentness spectra of the purified laccase enzyme was recorded ‘between 200 and 300nm. The spectrophotometric crease of the purified enzyme Fig. 11 exhibited a farthermost engrossment at 260280nm.

UV acculturation spectra of purified laccase enzyme.

4. Countersign

The portraying and katharsis of the crying laccase enzyme obtained from the threadlike marine-derived fungus T. mangrovei was described in the one-time resume (Atalla et al. 2010 ).

SDS-gel ionophoresis of the purified laccase enzyme showed a ace hoop at 48kDa. This answer agreed with that reported roughly the molecular mass of Ganoderma lucidum laccases (40 and 66kDa) (DSouza et al. 1999 ).

The general properties of the purified laccase enzyme were studied. Optimum temperature of the reply of the purified enzyme was name to be 65C. These results do not resist from otc laccases with optima temperature between 50 and 70C (Baldrian, 2006 ). This ending to approximately extent was wish thereto reported by Liers et al. (2007) who found that the highest fulfill of Xylaria polymorpha laccase enzyme was ascertained ‘between 55 and 60C. Laccase from Galerina sp. exhibited optimal fulfil on substrate 2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic battery-acid) (ABTS) at 60C (Ibrahim et al. 2011 ).

Dias et al. (2003) reported that Phanerochaete chrysosporium ATCC 24725 laccase enzyme displayed utmost activity at 5060C with the strain ABTS try. The enzyme proved to be almost wide-cut alert between 45 and 70C but lonely 37% of maximum execute was achieved at 25C. The results obtained in the salutation study, concurred with these observations.

The optimum pH of the purified enzyme with ABTS as the substrate was embed to be 4.0. This terminus is interchangeable thereto obtained by ula et al. (2007) who establish that the optimum pH of Dichomitus squalens purified laccase enzyme was 4.0. Studies with laccases from Coriolus hirsutus. Trichoderma atroviride. Chalara (syn. Thielaviopsis) paradoxa CH 32 and Cerrena unicolor 059 showed that the optimal pH orbit for fungal laccase was from 4.0 to 6.0 (Holker et al. 2002 and Robles et al. 2002 ).

Stability of the enzyme with obeisance to temperature was too studied. At 35C the enzyme was stable for 60min. at 40 and 45C the enzyme well-kept about 56.77 and 55.71% of its action aft treatment for 60min. Able 45C the enzyme began to retreat contribution its litigate and more untoward aftermath was implant by rut at 65, 70 and 80C where the enzyme lose around 98% of its fulfill aft treatment for 60min. These results are to about extent interchangeable thereto reported by Forootanfar et al. (2011) who indicated that at 50C the purified enzyme well-kept 50% of its initial fulfil aft 1h. Still, it was some altogether inactive at higher temperatures.

Detail the germ of the microorganism, thermic stability varied at unlike temperatures. E.g., the laccase from T. harzianum well-kept 70% of its initial execute after 1h musing at 55C (Sadhasivam et al. 2008 ), whereas the laccase from Daedalea quercina was indistinguishable stable below 45C (Baldrian, 2004 ).

Our study showed that the enzyme below pH 4.0 and above pH 4.5 was partially perilous. This issue is standardized thereto reported up the wide stability of T. atroviride laccase in slenderly acid pH values (Chakroun et al. 2010 ). This stability is idealistic among fungal laccases, e.g. laccases from Melanocarpus albomyces (Kiiskinen et al. 2002 ), Pleurotus ostreatus (Palmieri et al. 2003 ) and Perenniporia tephropora (Ben Younes et al. 2007 ) let good stability at neutral and slimly alkaline pH values.

The center of enzyme niggardness on enzyme action was carried out under standard endure with varying amounts of enzyme (mg protein) added. The results showed that, the maximum laccase activity was obtained with 1.35mg protein of the purified enzyme per reception classification. On the nonprescription hand, 5.429mg protein of the raw enzyme per reaction mix was the beaver concentration for the maximum enzyme fulfill. This dissolver indicated that the raw sample contains high sum of protein with low laccase litigate but the pure try contains low protein with soaring enzyme fulfil.

The inwardness of substrate (ABTS) concentration on the activity of the purified laccase enzyme was studied. Results indicated that the fulfill increased linearly abreast increasing the ABTS concentration to scope maximum at 40mM and higher substrate concentrations than 40mM resulted in a incessant enzyme action.

The activating parameters (Km and Vmax values) of the purified T. mangrovei laccase enzyme was compulsive by using ABTS as a substrate and metric from Lineweaver and Burk plots (1934). The results indicated that the Km tax was 1.42mM and Vmax value was 184.84U/mg protein. This resolvent is lower than that reported by Chakroun et al. (2010) who found that the Km order of T. atroviride laccase enzyme was 2.5mM towards ABTS.

Robles et al. (2002) reported that the Km of laccase from Chalara (syn. Thielaviopsis) paradoxa CH 32 by exploitation ABTS as a substrate at pH 4.5 was 0.77mM. Dapple the Km 1.6mM was foot with Ceriporiopsis subvermispora at pH 3.5 exploitation Guaiacol (Fukushima and Kirk, 1995 ). This observance of the dissimilar Km values confirms the mesmerism of Banerjee and Vohra (1991) who attributed these differences to be due to qualifying of culture brave, pH and substrate put-upon.

Substrate specificity of T. mangrovei laccase enzyme was studied on unlike phenolic and non phenolic aromatic compounds. The results indicated that, purified enzyme potently oxidized ABTS. The enzyme has likewise slight accomplish with Guaiacol (2-Methoxyphenol), p -cresol (4- Methylphenol) and catechol (1,2-Benzenediol). No execute was observed on phenol, resorcinol, 3-Methylphenol (m -cresol) and 4-Chlorophenol. The enzyme showed higher buy research paper relationship and action toward ABTS than other substrates. These results are standardised thereto reported by Zouari-Mechichi et al. (2006) about the substrate specificity of both laccases obtained from Trametes trogii and Laccase from Galerina sp. showed the highest activity toward ABTS, a non-phenolic heterocycle, eyepatch the fulfill with the phenolic substrates was oftentimes lower (Ibrahim et al. 2011 ).

The opinion of different metal ions on the activity of laccase enzyme was investigated. Results showed that all the metal ions ill-used exhibited banning effects to the enzyme action but with dissimilar degrees. FeSO4 showed expeditiousness forbiddance heart on laccase enzyme fleck in the mien of CoCl2. MgSO4 and HgCl2 the enzyme well-kept around 66.01, 64.38 and 57.58% of its initial activeness, singly. The metal ions KCl and NaCl abide less effect on the enzyme execute where the enzyme retained about 71% of its initial activeness, the erstwhile metal ions BaCl2. CaCl2 and MnSO4 expect fiddling incumbrance on the enzyme action.

Sadhasivam et al. (2008) showed that the metals such as Co, Hg, Fe, K, Mg, Mn, Na, Ba and Ca at a density of 1mM had no hearty gist terminated Trichoderma harzianum WL1 laccase action overleap Hg, which caused 17.2% quelling. On the otc paw the purified laccase from the eatable mushroom Lentinula edodes was inhibited in the breast of 1mM Sn 2+ (99%), Ca 2+ (70%), Zn 2+ (64%), Hg 2+ (55%), K + (54%) and Cd 2+ (45%) (Nagai et al. 2002 ). The observations indicated that the effect of metal ions on laccase activity was exceedingly detail its base and the case of metals used, which had a heavy configuration on the catalytic activeness of the enzyme.

Heart of a orbit of substantial laccase inhibitors on the enzyme execute was tested. The oxidase inhibitors na azide and na nitrile caused around 77.77 and 51.48% prohibition of laccase activeness, separately. Stillness the enzyme well-kept 69.04% of its activity in the bearing of 1mM na dodecylsulfate (SDS). Otc inhibitors, EDTA and phenylamine sustain low load on laccase activity, similar to T. harzianum laccase which was mildly inhibited by the admixture chelator EDTA 1mM concentration (16.8% inhibition) Sadhasivam et al. (2008). T. atroviride laccase was potently inhibited by the typical laccase inhibitor na azide, but it was not raw to EDTA and SDS (Chakroun et al. 2010 ).

Robles et al. (2002) stated that na azide and k nitril caused complete ban of Chalara (syn. Thielaviopsis) paradoxa CH 32 laccase activity at 2mM terminal concentration. Also inhibited by the chelating agent EDTA and na dodecylsulfate caused cop quelling of laccase accomplish at a net tightness of 0.1%.

Laccase activity was entirely inhibited by the pleb metalloenzyme inhibitor, na azide (NaN3 ) at the parsimoniousness of 20M. It was reported that the dressing of na azide to the types 2 and 3 cu sites affects inside electron transfer, thus inhibiting the fulfill of Sclerotium rolfsii laccase Ryan et al. (2003) .

The amino dot analysis of the purified laccase enzyme indicated that the enzyme contained 13 amino acids. Alanine 15.5% and serine 13.2% were the major amino acids. Glutamic zen, threonine and aspartic dot comprised considerable proportions which amounted to 11.4%, 11.1% and 10.4% of the tally amino acids, separately. On the old paw the enzyme was poor in arginine, histidine, glycine, isoleucine and lysine which all comprised but round 15.6% of the sum amino acids. These results were standardized thereto reported some the amino zen composition of Trametes sp. AH282 laccase (Xiao et al. 2004 ).

Sugar gist of purified laccase enzyme was estimated to be 23% of the enzyme sample, bandage it contained 77% protein. This settlement is likewise thereto showed by Xiao et al. (2004) who indicated that Trametes sp. AH282 laccase B was a monomeric glycoprotein with 25% bread content. This grasp with that is known approximately fungal laccases where laccase typically contains 1530% boodle, which may contribute to the high stability of the enzyme.

U.V dousing spectra of purified laccase enzyme was recorded between a range of 200300nm. The spectrophotometric cut of the purified enzyme exhibited a furthermost compactness at 260280nm. This conclusion is standardized to the preoccupation spectra of the purified laccase enzymes (LA and LB) obtained from P. ostreatus NRRL (0366) torture where LA exhibited a utmost preoccupancy at 260280nm and 260275nm for LB enzyme (Hala, 1996 ). Alike Liers et al. (2007) plant that UV concentration spectra of X. polymorpha showed characteristic immersion maxima at 280nm.

It can be terminated that, the marinederived fungus T. mangrovei laccase enzyme exhibited interesting enzymatic properties, such as caloric stability at 35, 40 and 45C (1h), has all-inclusive substrate specificity, showed identical low quelling with Mn, Ca, Ba, K, Na, Mg, phenylamine, EDTA and SDS.

Reader chthonic duty of Fag Saud University.

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