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1.
An inducible form of extracellular laccase (EC 1.14.18.1) was isolated from the basidiomycete Coriolus hirsutus. The induction was performed with 0.11 microM syringaldazine, a substrate of laccase. The inducible form of the enzyme consisted of two isoforms, laccase I1 and laccase I2, whose molecular weights were 69 +/- 2 and 67 +/- 2 kDa, respectively. The isoelectric points of these isoenzymes were found to be 3.5 and 4.2, respectively. The optimum pH range for both laccases was 4.4-4.6, and the optimum temperature was 50 degrees C. The thermal stability of these isoenzymes was examined, and KM values for the substrates syringaldazine and pyrocatechol were determined. Our biochemical and physicochemical studies demonstrated that inducible laccase isoforms differed from constitutive forms in molecular weight, IP, KM, and thermal stability. However, their optimum pH ranges and temperatures were identical. 相似文献
2.
Michniewicz A Ullrich R Ledakowicz S Hofrichter M 《Applied microbiology and biotechnology》2006,69(6):682-688
Cerrena unicolor secreted two laccase isoforms with different characteristics during the growth in liquid media. In a synthetic low-nutrient
nitrogen glucose medium (Kirk medium), high amounts of laccase (4,000 U l−1) were produced in response to Cu2+. Highest laccase levels (19,000 U l−1) were obtained in a complex tomato juice medium. The isoforms (Lacc I, Lacc II) were purified to homogeneity with an overall
yield of 22%. Purification involved ultrafiltration and Mono Q separation. Lacc I and II had M
w of 64 and 57 kDa and pI of 3.6 and 3.7, respectively. Both isoforms had an absorption maximum at 608 nm but different pH optima and thermal stability.
Optimum pH ranged from 2.5 to 5.5 depending on the substrate. The pH optima of Lacc II were always higher than those of Lacc
I. Both laccases were stable at pH 7 and 10 but rapidly lost activity at pH 3. Their temperature optimum was around 60°C,
and at 5°C they still reached 30% of the maximum activity. Lacc II was the more thermostable isoform that did not lose any
activity during 6 months storage at 4°C. Kinetic constants (K
m, k
cat) were determined for 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS), 2,6-dimethoxyphenol and syringaldazine. 相似文献
3.
Studies on the <Emphasis Type="Italic">Pycnoporus sanguineus</Emphasis> CCT-4518 laccase purified by hydrophobic interaction chromatography 总被引:1,自引:0,他引:1
A laccase from Pycnoporus sanguineus was purified by two steps using phenyl-Sepharose columm. A typical procedure provided 54.1-fold purification, with a yield
of 8.37%, using syringaldazine as substrate. The molecular weight of the purified laccase was 69 and 68 kDa as estimated by
12% (w/v) SDS-PAGE gel and by gel filtration, respectively. The K
m values for the substrates ABTS, syringaldazine, and guaiacol were 58, 8.3, and 370 μM, respectively. The enzyme’s pH optimum
for syringaldazine was 4.2 and optimal activity was 50°C. The enzyme showed to be thermostable because when kept at 50°C for
24 and 48 h it retained 93 and 76% activity. This laccase was inhibited by l-cysteine, β-mercaptoethanol, NaN3, NaF, and HgCl2. 相似文献
4.
Two isoforms of laccase produced from the culture supernatant of Pycnoporus sanguineus were partially purified by phenyl-Sepharose chromatography. Molecular masses of the enzymes were 80 kDa (Lac I) and 68 kDa
(Lac II). Optimum activity of Lac I was at pH 4.8 and 30 °C, and Lac II was at pH 4.2 and 50 °C over 5 min reaction. The Km values of enzymes toward syringaldazine were 10 μm (Lac I) and 8 μm (Lac II). Sodium azide inhibited Lac I (85%) and Lac II (75%) activities.
Revisions requested 30 November 2005; Revisions received 26 January 2006 相似文献
5.
ACurvularia sp. isolated from soil was found to contain laccase activity toward guaiacol as substrate. The organism produced an extracellular
laccase in a medium containing yeast extract, peptone and dextrose. Initial medium pH 4.0 and cultivation temperature 30°C
were found to be most suitable for maximum enzyme production. The optimum pH and temperature for laccase activity were found
to be 5.2 and 50°C, respectively. Under optimum conditions, the enzyme had aK
m (guaiacol) of 0.75 mmol/L and aV of 1.50 CU min−1 ml−1. Some divalent metal ions inhibited laccase activity at very low concentrations. 相似文献
6.
Martin C Pecyna M Kellner H Jehmlich N Junghanns C Benndorf D von Bergen M Schlosser D 《Applied microbiology and biotechnology》2007,77(3):613-624
Myrioconium sp. strain UHH 1-13-18-4 is an ascomycete anamorph isolated from the river Saale, Central Germany. An extracellular, monomeric,
and glycosylated laccase with a molecular mass of 72.7 kDa as determined by matrix-assisted laser desorption/ionization-time
of flight-mass spectrometry and an isoelectric point below 2.8 was purified from CuSO4 and vanillic acid amended liquid fungal cultures grown in malt extract medium. The catalytic efficiencies (k
cat/K
m) for the oxidation of syringaldazine, 2,6-dimethoxyphenol, and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate) were 67.3,
46.9, and 28.2 s−1 mM−1, respectively, with K
m values of 4.2, 67.8, and 104.9 μM. After pre-incubation at different pH values and temperatures for 1 h, more than 80% of
the initial laccase activity was retained between pH 4 to 6 and 15°C. The laccase-encoding gene was identified and sequenced
at both the genomic and complementary DNA (cDNA) level, and corresponding structural characteristics and putative regulatory
elements of the promoter region are reported. The identification of two tryptic peptides of the purified enzyme by mass spectrometry
confirmed the identity of the functional laccase protein with the translated genomic sequence of the Myrioconium sp. laccase. Myrioconium sp. laccase shows the highest degree of identity with laccases from ascomycetes belonging to the family Sclerotiniaceae, order Helotiales. 相似文献
7.
Murugesan K Arulmani M Nam IH Kim YM Chang YS Kalaichelvan PT 《Applied microbiology and biotechnology》2006,72(5):939-946
An extracellular laccase was isolated and purified from Pleurotus sajor-caju grown in submerged culture in a bioreactor, and used to investigate its ability to decolorize three azo dyes. The extracellular laccase production was enhanced up to 2.5-fold in the medium amended with xylidine (1 mM). Purification was carried out using ammonium sulfate (70% w/v), DEAE-cellulose, and Sephadex G-100 column chromatography. The enzyme was purified up to 10.3-fold from the initial protein preparation with an overall yield of 53%. The purified laccase was monomeric with an apparent molecular mass of 61.0 kDa. The purified enzyme exerted its optimal activity with 2,2-azino–bis(3-ethylbenzo-thiazoline-6-sulfonate (ABTS) and oxidized various lignin-related phenols. The catalytic efficiencies k
cat/K
m determined for ABTS and syringaldazine were 9.2×105 and 8.7×105, respectively. The optimum pH and temperature for the purified enzyme was 5.0 and 40 °C, respectively. Sodium azide completely inhibited the laccase activity. The absorption spectrum revealed type 1 and type 3 copper signals. The purified enzyme decolorized azo dyes such as acid red 18, acid Black 1, and direct blue 71 up to 90, 87, and 72%, respectively. Decolorization ability of P. sajor-caju laccase suggests that this enzyme could be used for decolorization of industrial effluents. 相似文献
8.
A strain of Pleurotus ostreatus was grown in tomato pomace as sole carbon source for production of laccase. The culture of P. ostreatus revealed a peak of laccase activity (147 U/L of fermentation broth) on the 4th day of culture with a specific activity of
2.8 U/mg protein. Differential chromatographic behaviour of laccase was investigated on affinity chromatographic matrices
containing either urea, acetamide, ethanolamine or IDA as affinity ligands. Laccase exhibited retention on such affinity matrices
and it was purified on a Sepharose 6B—BDGE—urea column with final enzyme recoveries of about 60%, specific activity of 6.0
and 18.0 U/mg protein and purification factors in the range of 14–46. It was also possible to demonstrate that metal-free
laccase did not adsorb to Sepharose 6B—BDGE—urea column which suggests that adsorption of native laccase on this affinity
matrix was apparently due to the specific interaction of carbonyl groups available on the matrix with the active site Cu (II)
ions of laccase. The kinetic parameters (V
max, K
m
, K
cat, and K
cat/K
m
) of the purified enzyme for several substrates were determined as well as laccase stability and optimum pH and temperature
of enzyme activity. This is the first report describing the production of laccase from P. ostreatus grown on tomato pomace and purification of this enzyme based on affinity matrix containing urea as affinity ligand. 相似文献
9.
Baldrian P 《Applied microbiology and biotechnology》2004,63(5):560-563
The white-rot fungus Daedalea quercina produced the ligninolytic enzymes laccase and Mn-dependent peroxidase. Laccase was purified using anionexchange and size-exclusion chromatographies. SDS-PAGE showed the purified laccase to be a monomeric protein of 69 kDa (71 kDa using gel filtration) with an isoelectric point near 3.0. The optimum pH for activity was bellow 2.0 for 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (Km=38 M), 4.0 for 2,6-dimethoxyphenol (Km=48 M), 4.5 for guaiacol (Km=93 M) and 7.0 for syringaldazine (Km=131 M). The temperature optimum was between 60 and 70 °C depending on the pH and buffer used. The enzyme was stable up to 45 °C, and stability was higher at alkaline pH. Enzyme activity was increased by the addition of Cu2+ and inhibited by Mn2+, sodium azide, dithiothreitol, and cysteine. Laccase from Daedalea quercina was able to decolorize the synthetic dyes Chicago sky blue, poly B-411, remazol brilliant blue R, trypan blue and reactive blue 2. 相似文献
10.
The detail understanding of physiological/biochemical characteristics of individual laccase isoenzymes in fungi is necessary for fundamental and application purposes, but our knowledge is still limited for most of fungi due to difficult to express laccases heterologously. In this study, two novel laccase genes, named lac3 and lac4, encoding proteins of 547 and 532-amino acids preceded by 28 and 16-residue signal peptides, respectively, were cloned from the edible basidiomycete Coprinus comatus. They showed 70% identity but much lower homology with other fungal laccases at protein level (less than 58%). Two novel laccase isoenzymes were successfully expressed in Pichia pastoris by fusing an additional 10 amino acids (Thr-Pro-Phe-Pro-Pro-Phe-Asn-Thr-Asn-Ser) tag at N-terminus, and the volumetric activities could be dramatically enhanced from undetectable level to 689 and 1465 IU/l for Lac3 and Lac4, respectively. Both laccases possessed the lowest K
m and highest k
cat/K
m value towards syringaldazine, followed by ABTS, guaiacol and 2,6-dimethylphenol similar as the low redox potential laccases from other microorganisms. Lac3 and Lac4 showed resistant to SDS, and retained 31.86% and 43.08% activity in the presence of 100 mM SDS, respectively. Lac3 exhibited higher decolorization efficiency than Lac4 for eleven out of thirteen different dyes, which may attribute to the relatively higher catalytic efficiency of Lac3 than Lac4 (in terms of k
cat/K
m) towards syringaldazine and ABTS. The mild synergistic decolorization by two laccases was observed for triphenylmethane dyes but not for anthraquinone and azo dyes. 相似文献
11.
Lu L Zhao M Zhang BB Yu SY Bian XJ Wang W Wang Y 《Applied microbiology and biotechnology》2007,74(6):1232-1239
The white rot fungus Pycnoporus sanguineus produced high amount of laccase in the basal liquid medium without induction. Laccase was purified using ultrafiltration,
anion-exchange chromatography, and gel filtration. The molecular weight of the purified laccase was estimated as 61.4 kDa
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme oxidized typical substrates of laccases including
2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate), 2,6-dimethoxyphenol, and syringaldazine. The optimum pH and temperature for the purified
laccase were 3.0 and 65°C, respectively. The enzyme was stable up to 40°C, and high laccase activity was maintained at pH 2.0–5.0.
Sodium azide, l-cysteine, and dithiothreitol strongly inhibited the laccase activity. The purified enzyme efficiently decolorized Remazol
Brilliant Blue R in the absence of added redox mediators. The high production of P. sanguineus laccase as well as its decolorization ability demonstrated its potential applications in dye decolorization. 相似文献
12.
Marc Dubernet Pascal Ribereau-Gayon Henri R. Lerner Eitan Harel Alfred M. Mayer 《Phytochemistry》1977,16(2):191-193
The partial purification of an extracellular laccase from Botrytis cinerea is described. Specificity of the enzyme, its Km for a number of substrates and sensitivity to some inhibitors are described. The enzyme is a typical laccase but has an exceptionally low pI and great stability to acid pH. On gel electrophoresis two isoenzymes could be detected. 相似文献
13.
Two isoenzymes of chorismate mutase (EC 5.4.99.5), designated as CM-1 and CM-2, were isolated and partially purified from suspension-cultured cells of Ruta gravelens by DEAE-sephacel chromatography and gel filtration. 60–72% of the total activity measured after DEAE-sephacel chromatography were obtained as CM-1 and 28–40% were CM-2 activity. CM-1 was inhibited by phenylalanine (K1 = 4 · 10?6 M) and tyrosine (K1 = 8. 10?6M) and activated by tryptophan. In contrast, CM-2 was not influenced by these three amino acids. The molecular weights estimated by gel filtration on SEPHADEX G-150 were 56000 for CM-1 and 45000 for CM-2, respectively. Both isoenzymes were stable at ?20°C, but exhibited different behaviour during thermal inactivation and different optima of reaction temperature. CM-1 catalysed the reaction at a pH optimum of pH 7.8 and CM-2 showed a broad optimum between 6–10. The Km-values for chorismic acid were determined to be 1.1 mM for CM-1 and 0.5 mM for CM-2. The isoenzymes showed different behaviour to inhibitors of sulfhydryl groups. There were no differences in all parameters of chorismate mutase examined for two various cell lines of Ruta graveolens. 相似文献
14.
High production of laccase by a new basidiomycete, <Emphasis Type="Italic">Trametes</Emphasis> sp 总被引:1,自引:0,他引:1
A new basidiomycete, Trametes sp. 420, produced laccase at 6,810 U l−1 (268 mg, 25.4 U mg−1 protein for guaiacol) in glucose medium and 7,870 U l−1 (310 mg) in cellobiose medium with induction by 0.5 mM Cu2+ and 6 mM o-toluidine. Laccase isozyme E (LacE) was the sole laccase in the fermentation products. It was stable at pH 5–9 and below
70°C over 30 min. The K
m values of LacE for four substrates (guaiacol ABTS, 2,6-dimethoxyphenol and syringaldazine) varied from 5 to 245 μM. The activity
of LacE was strongly inhibited by NaN3 but not by EDTA or dimethylsulfoxide. LacE at 0.5 U l−1 could decolorize industrial dyes. The open reading frame of the lacE gene was 2,130 bp and was interrupted by 10 introns. It displayed a high homology to laccases from other fungi.
Pingui Tong and Yuzhi Hong contributed equally to the study 相似文献
15.
Preparation and characterization of epoxy-functionalized magnetic chitosan beads: laccase immobilized for degradation of reactive dyes 总被引:1,自引:0,他引:1
Gulay Bayramoglu Meltem Yilmaz M. Yakup Arica 《Bioprocess and biosystems engineering》2010,33(4):439-448
Cross-linked magnetic chitosan beads were prepared by phase-inversion technique in the presence of epichlorohydrin under alkaline
condition, and used for covalent immobilization of laccase. The activity of the immobilized laccase on the magnetic chitosan
was about 260 U (g/dry beads) with an enzyme loading of about 16.33 ± 0.39 mg [(g/dry beads) mg/g]. Kinetic parameters, V
max and K
m values were determined as 21.7 U/mg protein and 9.4 μM for free enzyme, and 15.6 U/mg protein and 19.7 μM for the immobilized
laccase, respectively. The operational and thermal stabilities of the immobilized laccase were improved compared to free counterpart.
The immobilized laccase was operated in a batch reactor for the decolorization of reactive dyes from aqueous solution. The
laccase immobilized on magnetic chitosan beads was very effective for removal of textile dyes from aqueous solution which
creates an important environmental problem in the discharged textile dying solutions. 相似文献
16.
6-Phosphogluconate dehydrogenase (6PG) was purified from rat small intestine with 36% yield and a specific activity of 15 U/mg.
On SDS/PAGE, one band with a mass of 52 kDa was found. On native PAGE three protein and two activity bands were observed.
The pH optimum was 7.35. Using Arrhenius plots, Ea, ΔH, Q10 and Tm for 6PGD were found to be 7.52 kcal/mol, 6.90 kcal/mol, 1.49 and 49.4°C, respectively. The enzyme obeyed “Rapid Equilibrium
Random Bi Bi” kinetic model with Km values of 595 ± 213 μM for 6PG and 53.03±1.99 μM for NADP. 1/Vm versus 1/6PG and 1/NADP plots gave a Vm value of 8.91±1.92 U/mg protein. NADPH is the competitive inhibitor with a Ki of 31.91±1.31 μM. The relatively small Ki for the 6PGD:NADPH complex indicates the importance of NADPH in the regulation of the pentose phosphate pathway through G6PD
and 6PGD. 相似文献
17.
Ashoka Sreedhara Ragnar Flengsrud Thor Langsrud Purnima Kaul Vishweshwaraiah Prakash Gerd Elisabeth Vegarud 《Biometals》2010,23(6):1159-1170
Apo and holo forms of lactoferrin (LF) from caprine and bovine species have been characterized and compared with regard to
the structural stability determined by thermal denaturation temperature values (T
m), at pH 2.0–8.0. The bovine lactoferrin (bLF) showed highest thermal stability with a T
m of 90 ± 1°C at pH 7.0 whereas caprine lactoferrin (cLF) showed a lower T
m value 68 ± 1°C. The holo form was much more stable than the apo form for the bLF as compared to cLF. When pH was gradually
reduced to 3.0, the T
m values of both holo bLF and holo cLF were reduced showing T
m values of 49 ± 1 and 40 ± 1°C, respectively. Both apo and holo forms of cLF and bLF were found to be most stable at pH 7.0.
A significant loss in the iron content of both holo and apo forms of the cLF and bLF was observed when pH was decreased from
7.0 to 2.0. At the same time a gradual unfolding of the apo and holo forms of both cLF and bLF was shown by maximum exposure
of hydrophobic regions at pH 3.0. This was supported with a loss in α-helix structure together with an increase in the content
of unordered (aperiodic) structure, while β structure seemed unchanged at all pH values. Since LF is used today as fortifier
in many products, like infant formulas and exerts many biological functions in human, the structural changes, iron binding
and release affected by pH and thermal denaturation temperature are important factors to be clarified for more than the bovine
species. 相似文献
18.
Youshuang Zhu Haibo Zhang Mingle Cao Zhenzhen Wei Feng Huang Peiji Gao 《Biotechnology and Bioprocess Engineering》2011,16(5):1027-1035
Production of laccase using a submerged culture of Trametes versicolor sdu-4 was optimized using a central composite design of the Response Surface Methodology. Optimized conditions gave a laccase
yield of 4,213 U/L which was approximately three times of that in basal medium. The laccase was purified to homogeneity using
a three-step process. The overall yield of the purification was 58%, with a purification fold of 11.4 and a specific activity
of 1320.7 U/mg protein. The molecular mass of the laccase was 60 kDa. The optimum pH values of the enzyme were 2.2, 3.7, and
7 for the oxidations of ABTS, DMP, and syringaldazine, respectively. The enzyme had adaptability to a broad pH range and high
temperature and wsa stable at pH 3.0 ∼ 10.0. The half-life of this laccase at 70°C was 2.2 h. Methyl red, 2-bromophenol, and
4-bromophenol were oxidized by the purified laccase in the absence of mediators. Purified laccase was effective in the decolorization
of several dyes and was not inhibited by Cu2+, Mn2+, Zn2+, Na+, K+, Mg2+, Ba2+, and Ca2+ at 5 mM. These excellent characteristics made it a highly attractive candidate for industrial use. 相似文献
19.
L. G. Vasil’chenko O. V. Koroleva E. V. Stepanova E. O. Landesman M. L. Rabinovich 《Applied Biochemistry and Microbiology》2000,36(4):353-362
—Samples of South Vietnamese soils intensely treated with Agent Orange defoliant were tested for the presence of fungi and
actinomycetes with an elevated phenol oxidase activity. As a result, a fast-growing nonsporulating strain producing neutral
phenol oxidases was isolated and identified asMycelia sterilia INBI2-26. The strain formed extracellular phenol oxidases during surface growth on a liquid medium in the presence of guayacol
and copper sulfate, as well as during submerged cultivation in liquid medium containing wheat bran and sugar beet pulp. Isoelectric
focusing of the culture liquid revealed two major catechol oxidases (PO1 and PO2) with pI 3.5 and 8, respectively. The enzymes
were purified by Ultrafiltration, ion exchange chromatography, and exclusion HPLC. Both were stable between pH 3 and 8. At
pH 8 and 40°C., they retained at least 50% of activity after incubation for 50 h. At 50°C., PO2 was more stable and retained
40% of activity after 50 h, whereas PO1 was inactivated in 3–6 h. The pH-optimutns for PO1 and PO2 toward catechol were 6
and 6.5; and theK
m values were 1.5±0.35 and 1.25±0.2 mM, respectively. PO1 and PO2 most optimally oxidized 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic
acid) at pH 3 withK
m values 1.6±0.18 and 0.045±0.01 mM, respectively, but displayed no activity toward tyrosine. The PO2 absorbance spectrum had
a peak at 600 nm, thus indicating the enzyme to be a member of the laccase family. 相似文献
20.
Cloning and characterization of a new laccase from Bacillus licheniformis catalyzing dimerization of phenolic acids 总被引:1,自引:0,他引:1
Koschorreck K Richter SM Ene AB Roduner E Schmid RD Urlacher VB 《Applied microbiology and biotechnology》2008,79(2):217-224
A new laccase gene (cotA) was cloned from Bacillus licheniformis and expressed in Escherichia coli. The recombinant protein CotA was purified and showed spectroscopic properties, typical for blue multi-copper oxidases. The
enzyme has a molecular weight of ~65 kDa and demonstrates activity towards canonical laccase substrates 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic
acid) (ABTS), syringaldazine (SGZ) and 2,6-dimethoxyphenol (2,6-DMP). Kinetic constants K
M and k
cat for ABTS were of 6.5 ± 0.2 μM and 83 s−1, for SGZ of 4.3 ± 0.2 μM and 100 s−1, and for 2,6-DMP of 56.7 ± 1.0 μM and 28 s−1. Highest oxidizing activity towards ABTS was obtained at 85°C. However, after 1 h incubation of CotA at 70°C and 80°C, a
residual activity of 43% and 8%, respectively, was measured. Furthermore, oxidation of several phenolic acids and one non-phenolic
acid by CotA was investigated. CotA failed to oxidize coumaric acid, cinnamic acid, and vanillic acid, while syringic acid
was oxidized to 2,6-dimethoxy-1,4-benzoquinone. Additionally, dimerization of sinapic acid, caffeic acid, and ferulic acid
by CotA was observed, and highest activity of CotA was found towards sinapic acid. 相似文献