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1.
The main physicochemical characteristics of the major isoform of the laccase secreted by the fungus Trametes hirsuta 072 were studied. The enzyme belongs to the group of high redox potential laccases ( E T1 0 790 ± 5), and it oxidizes with high efficiency various substrates of phenolic nature. The gene of this isoform was cloned, and its nucleotide sequence was determined. The length of the complete gene is 2134 bp. It comprises 11 exons and 10 introns. Analysis of the amino acid sequence of T. hirsuta 072 laccase demonstrated a high homology to the other laccases secreted by fungi of the genus Trametes. 相似文献
3.
AIM: To produce high laccase activities from the white-rot fungus Trametes hirsuta in an in-house air-lift bioreactor (ALB). METHODS AND RESULTS: Trametes hirsuta was grown in a 6-l ALB. A fed-batch strategy with glycerol as an addition resulted in maximum laccase activity of 19,400 U l(-1), which was the highest reported from the fungus. CONCLUSION: The ALB configuration with additional glycerol resulted in high laccase activities. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides useful information on how to produce high concentrations of laccase. 相似文献
4.
Trametes hirsuta and a purified laccase from this organism were able to degrade triarylmethane, indigoid, azo, and anthraquinonic dyes. Initial decolorization velocities depended on the substituents on the phenolic rings of the dyes. Immobilization of the T. hirsuta laccase on alumina enhanced the thermal stabilities of the enzyme and its tolerance against some enzyme inhibitors, such as halides, copper chelators, and dyeing additives. The laccase lost 50% of its activity at 50 mM NaCl while the 50% inhibitory concentration (IC 50) of the immobilized enzyme was 85 mM. Treatment of dyes with the immobilized laccase reduced their toxicities (based on the oxygen consumption rate of Pseudomonas putida) by up to 80% (anthraquinonic dyes). Textile effluents decolorized with T. hirsuta or the laccase were used for dyeing. Metabolites and/or enzyme protein strongly interacted with the dyeing process indicated by lower staining levels (K/S) values than obtained with a blank using water. However, when the effluents were decolorized with immobilized laccase, they could be used for dyeing and acceptable color differences (ΔE*) below 1.1 were measured for most dyes. 相似文献
5.
The alanine racemase (EC 5.1.1.1) gene of a thermophilic bacterium, Bacillus stearothermophilus, was cloned and expressed in Escherichia coli C600 with vector plasmid pICR301, which was constructed from pBR322 and the L-alanine dehydrogenase gene derived from B. stearothermophilus. A coupled assay method with L-alanine dehydrogenase and tetrazolium salts was used to detect visually the alanine racemase activity in the clones. Alanine racemase overproduced in a clone carrying the plasmid pICR4, 12 kilobases of DNA, was purified from cell extracts about 340-fold to homogeneity by five steps including heat treatment. The overproduced enzyme was confirmed to originate from B. stearothermophilus by an immunochemical cross-reaction with the enzyme of B. stearothermophilus. The purified enzyme has a molecular weight of about 78 000 and consists of two identical subunits of Mr of 39 000. At the optimum temperature (50 degrees C), the enzyme has a specific activity of 1800 units/mg (Vmax, D- to L-alanine). Resolution and reconstitution experiments together with the absorption spectrum of the enzyme clearly indicate that alanine racemase of B. stearothermophilus is a pyridoxal 5'-phosphate enzyme. 相似文献
6.
Chicken DNase was purified to apparent homogeneity from the pancreas extract. It showed two isoforms, A and B forms, on cation-exchange chromatography. On SDS-PAGE it was a 30-kDa protein. When analyzed on an electrospray-mass analyzer, form A showed a major mass peak of 30859, and form B, 30882. The enzyme was bound to concanavalin A, indicating its glycoprotein nature. The carbohydrate side chain could be removed by endoglycosidase F. Chicken DNase was activated by metal ions and for half-maximum activation, Mn 2+ and Mg 2+ required were 1 mM and 4 mM, respectively. The pH optimum was between 7 and 8 depending on the metal ions used. In the presence of Cu 2+, it was almost completely inactivated by 0.1 M iodoacetate within 1 min. In the absence of Ca 2+ at pH 8, chicken DNase resisted to the trypsin or -mercaptoethenol inactivation. When the purified enzyme was subjected to protein sequencing, 93% of the sequence was established. Based on the amino acid sequence, the cDNA of chicken DNase was amplified, cloned and sequenced. The cDNA sequence consisted of 1079 nucleotides in which 67 were of the 5-untranslated region and 166 of the 3 and, in the 5-untranslated region, two types of sequences occurred. The polypeptide chain of 282 amino acids, translated from the open reading frame, was composed of the mature protein of 262 amino acids and a putative signal peptide of 20 amino acids. As compared with mammalian DNases, chicken DNase had an overall 58 ± 61% sequence identity, one less potential N-glycosylation site, and one extra disulfide. The cDNA was cloned into the pET15b expression vector. When induced, active recombinant chicken DNase was expressed in Escherichia coli strain BL21(DE3)pLysS and was present in the insoluble fraction of cell lysates. 相似文献
7.
A gene encoding an esterase ( estO) was identified and sequenced from a gene library screen of the psychrotolerant bacterium Pseudoalteromonas arctica. Analysis of the 1,203 bp coding region revealed that the deduced peptide sequence is composed of 400 amino acids with a
predicted molecular mass of 44.1 kDa. EstO contains a N-terminal esterase domain and an additional OsmC domain at the C-terminus
(osmotically induced family of proteins). The highly conserved five-residue motif typical for all α/β hydrolases (G × S × G)
was detected from position 104 to 108 together with a putative catalytic triad consisting of Ser 106, Asp 196, and His 225. Sequence comparison showed that EstO exhibits 90% amino acid identity with hypothetical proteins containing similar esterase
and OsmC domains but only around 10% identity to the amino acid sequences of known esterases. EstO variants with and without
the OsmC domain were produced and purified as His-tag fusion proteins in E. coli. EstO displayed an optimum pH of 7.5 and optimum temperature of 25°C with more than 50% retained activity at the freezing
point of water. The thermostability of EstO (50% activity after 5 h at 40°C) dramatically increased in the truncated variant
(50% activity after 2.5 h at 90°C). Furthermore, the esterase displays broad substrate specificity for esters of short-chain
fatty acids (C 2–C 8). 相似文献
8.
In order to save energy during the pulp making process, we tried to use white-rot basidiomycete, Trametes hirsuta, which degrades lignin efficiently. But a decrease in paper strength caused by cellulolytic activity ruled this out for practical application. Since the cellulolytic activity of the fungus must be decreased, we purified and characterized a cellobiose dehydrogenase (CDH) that was reported to damage pulp fiber. The CDH in the culture filtrate of C. hirsutus was purified by freeze-thawing and chromatographic methods. The pI of the enzyme was 4.2 and its molecular weight was 92 kDa. The optimal temperature and pH of the enzyme were 60-70 degrees C and 5.0 respectively. Since the purified CDH decreased the viscosity of pulp in the presence of Fe(III) and cellobiose, it was shown that the suppression of CDH should be an effective way to reduce cellulose damage. 相似文献
9.
NAD(P)H-flavin oxidoreductases (flavin reductases) from luminous bacteria catalyze the reduction of flavin by NAD(P)H and are believed to provide the reduced form of flavin mononucleotide (FMN) for luciferase in the bioluminescence reaction. By using an oligonucleotide probe based on the partial N-terminal amino acid sequence of the Vibrio harveyi NADPH-FMN oxidoreductase (flavin reductase P), a recombinant plasmid, pFRP1, was obtained which contained the frp gene encoding this enzyme. The DNA sequence of the frp gene was determined; the deduced amino acid sequence for flavin reductase P consists of 240 amino acid residues with a molecular weight of 26,312. The frp gene was overexpressed, apparently through induction, in Escherichia coli JM109 cells harboring pFRP1. The cloned flavin reductase P was purified to homogeneity by following a new and simple procedure involving FMN-agarose chromatography as a key step. The same chromatography material was also highly effective in concentrating diluted flavin reductase P. The purified enzyme is a monomer and is unusual in having a tightly bound FMN cofactor. Distinct from the free FMN, the bound FMN cofactor showed a diminished A375 peak and a slightly increased 8-nm red-shifted A453 peak and was completely or nearly nonfluorescent. The Kms for FMN and NADPH and the turnover number of this flavin reductase were determined. In comparison with other flavin reductases and homologous proteins, this flavin reductase P shows a number of distinct features with respect to primary sequence, redox center, and/or kinetic mechanism. 相似文献
10.
Six putative lignin peroxidase (LIP) genes were isolated from a lambda EMBL3 phage library of the white-rot fungus, Trametes versicolor, using the Phanerochaete chrysosporium LIP cDNA CLG5 as the probe. Sequence analysis of one of the genes, VLG1, showed that its coding region is interrupted by six small introns (49-64 bp) and that it encodes a mature LIP protein (341 aa; Mr: 36,714) that is preceded by a 25 aa signal sequence. This protein has a relatively high degree of aa homology to the N-termini of the LIP proteins purified from T. versicolor and has an aa homology of 55-60% to the LIP proteins of P. chrysosporium, which is comparable to that found between P. chrysosporium and Phlebia radiata LIP proteins. 相似文献
11.
In this study, two laccase isoenzymes (Lac1 and Lac2) from the culture supernatant of Trametes hirsuta MX2 were purified, and the genes (Lac1 and Lac2) coding the isoenzymes were cloned. Both Lac1 and Lac2 contained an open reading frame of 1563 bp with an identity of 79%. The two isoenzymes showed significant biochemical differences. The maximal activities of Lac1 and Lac2 were at pH 2.5 with 2-2′-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS), and the optimal temperatures for the activities of Lac1 and Lac2 were 60 and 50 °C, respectively. Lac1 exhibited excellent resistance to acidic conditions and retained 62.17% of its initial activity at pH 2.5 after a 72-h incubation. Lac2 was more thermostable than Lac1 with half-lives (t1/2) of 9.58 and 3.12 h at 50 and 60 °C, respectively; the t1/2 of Lac1 were only 4.19 and 0.88 h, respectively. Both Lac1 and Lac2 isoenzymes have a strong tolerance to Mg2+, Mn2+, Cu2+, and EDTA (50 mM). At a low concentration of 0.05 U mL?1, the enzymes could decolorize towards Remazol Brilliant Blue R, Acid Red 1, Crystal Violet, and Neutral Red in the presence of ABTS. These unusual properties demonstrated that the two laccases have strong potential for specific industrial applications. 相似文献
12.
Endo-beta-mannosidase is a novel endoglycosidase that hydrolyzes the Manbeta1-4GlcNAc linkage in the trimannosyl core structure of N-glycans. This enzyme was partially purified and characterized in a previous report (Sasaki, A., Yamagishi, M., Mega, T., Norioka, S., Natsuka, S., and Hase, S. (1999) J. Biochem. 125, 363-367). Here we report the purification and molecular cloning of endo-beta-mannosidase. The enzyme purified from lily flowers gave a single band on native-PAGE and three bands on SDS-PAGE with molecular masses of 42, 31, and 28 kDa. Amino acid sequence information from these three polypeptides allowed the cloning of a homologous gene, AtEBM, from Arabidopsis thaliana. AtEBM was engineered for expression in Escherichia coli, and the recombinant protein comprised a single polypeptide chain with a molecular mass of 112 kDa corresponding to the sum of molecular masses of three polypeptides of the lily enzyme. The recombinant protein hydrolyzed pyridylamino derivatives (PA) of Manalpha1-6Manbeta1-4Glc-NAcbeta1-4GlcNAc into Manalpha1-6Man and GlcNAcbeta1-4Glc-NAc-PA, showing that AtEBM is an endo-beta-mannosidase. AtEBM hydrolyzed Man(n)Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc-PA (n = 0-2) but not PA-sugar chains containing Manalpha1-3Manbeta or Xylosebeta1-2Manbeta as for the lily endo-beta-mannosidase. AtEBM belonged to the clan GH-A of glycosyl hydrolases. Site-directed mutagenesis experiments revealed that two glutamic acid residues (Glu-464 and Glu-549) conserved in this clan were critical for enzyme activity. The amino acid sequence of AtEBM has distinct differences from those of the bacterial, fungal, and animal exo-type beta-mannosidases. Indeed, AtEBM-like genes are only found in plants, indicating that endo-beta-mannosidase is a plant-specific enzyme. The role of this enzyme in the processing and/or degradation of N-glycan will be discussed. 相似文献
13.
The white rot fungus Trametes hirsuta produced ethanol from a variety of hexoses: glucose, mannose, cellobiose and maltose, with yields of 0.49, 0.48, 0.47 and 0.47 g/g of ethanol per sugar utilized, respectively. In addition, this fungus showed relatively favorable xylose consumption and ethanol production with a yield of 0.44 g/g. T. hirsuta was capable of directly fermenting starch, wheat bran and rice straw to ethanol without acid or enzymatic hydrolysis. Maximum ethanol concentrations of 9.1, 4.3 and 3.0 g/l, corresponding to 89.2%, 78.8% and 57.4% of the theoretical yield, were obtained when the fungus was grown in a medium containing 20 g/l starch, wheat bran or rice straw, respectively. The fermentation of rice straw pretreated with ball milling led to a small improvement in the ethanol yield: 3.4 g ethanol/20 g ball-milled rice straw. As T. hirsuta is an efficient microorganism capable of hydrolyzing biomass to fermentable sugars and directly converting them to ethanol, it may represent a suitable microorganism in consolidated bioprocessing applications. 相似文献
14.
The finding of heat-stable enzymes or the engineering of moderately thermostable enzymes into more stable ones by random or site-directed mutagenesis has become a main priority of modern biotechnology. We report here for the first time a heat-stable phytase able to withstand temperatures up to 100 degrees C over a period of 20 min, with a loss of only 10% of the initial enzymatic activity. The gene (phyA) encoding this heat-stable enzyme has been cloned from Aspergillus fumigatus and overexpressed in Aspergillus niger. The enzyme showed high activity with 4-nitrophenyl phosphate at a pH range of 3 to 5 and with phytic acid at a pH range of 2.5 to 7.5. 相似文献
15.
We obtained structural and functional characterization of a recombinant Laccase from Rigidoporus lignosus (formerly Rigidoporus microporus), a white‐rot basidiomycete, by means of circular dichroism (CD) spectra, cyclic voltammetry (CV) and biochemical assays. Here we report the optimization of expression and purification procedures of a recombinant Laccase expressed in supercompetent Escherichia coli cells. We amplified the coding sequence of Laccase using PCR from cDNA and cloned into a bacterial expression system. The resulting expression plasmid, pET‐28b, was under a strong T7/Lac promoter induced by IPTG (isopropyl‐β‐ d‐thiogalactoipyranoside). We obtained purification by fast protein liquid chromatography (FPLC) method. We recorded the variation of the current of a solution containing purified Laccase with increasing Syringaldazine (SGZ) concentration using a potentiometer as proof of principle, showing its compatibility with the development of a new enzymatic biosensor for medical purposes, as described in Part II. J. Cell. Biochem. 114: 599–605, 2013. © 2012 Wiley Periodicals, Inc. 相似文献
16.
Gene 1 of bacteriophage T4 has been cloned into a lambda pL expression vector, resulting in the overproduction of deoxynucleotide kinase. A procedure that includes affinity chromatography on Cibacron Blue F3GA-agarose has been used to purify milligram quantities of enzymes from a 2-liter culture. The enzyme has been partially characterized in vitro and in vivo, and it appears to be identical to the deoxynucleotide kinase isolated from T4-infected Escherichia coli. These results prove the earlier contention that the phosphorylation of three dissimilar deoxynucleotides (5-hydroxymethyldeoxycytidylate, dTMP, and dGMP), to the exclusion of most others, is catalyzed by a single protein. 相似文献
17.
The medicinal mushroom Agaricus blazei produced high amounts of laccase (up to 5,000 units l –1) in a complex, agitated liquid medium based on tomato juice, while only traces of the enzyme (<100 units l –1) were detected in synthetic glucose-based medium. Purification of the enzyme required three chromatographic steps, including anion and cation exchanging. A. blazei laccase was expressed as a single protein with a molecular mass of 66 kDa and an isoelectric point of 4.0. Spectroscopic analysis of the purified enzyme confirmed that it belongs to the blue copper oxidases. The enzymes pH optimum for 2,6-dimethoxyphenol (DMP) and syringaldazine was pH 5.5; but for 2,2-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS) no distinct pH optimum was observed (highest activity at the lowest pH tested). Purified laccase was stable at 20°C, pH 7.0 and pH 3.0, but rapidly lost its activity at 40°C or pH 10. Sodium chloride strongly inhibited the enzyme activity, although the inhibition was completely reversible. The following kinetic constants were determined ( Km, kcat): 63 M, 21 s –1 for ABTS, 4 M, 5 s –1 for syringaldazine, 1,026 M, 15 s –1 for DMP and 4307 M, 159 s –1 for guaiacol. The results show that—in addition to the wood-colonizing white-rot fungi—the typical litter-decomposing basidiomycetes can also produce high titers of laccase in suitable liquid media. 相似文献
18.
The gene encoding proline dehydrogenase (ProDH) from Pseudomonas fluorescence was isolated using PCR amplification and cloned into pET23a expression vector. The expression of the recombinant target enzyme was induced by addition of IPTG. The produced His-fusion enzyme was purified and its kinetic properties were studied. The 3D structure modeling was also performed to identify key amino acids involved in FAD-binding and catalysis. The PCR product contained a 1033 bp open reading frame encoding 345 amino acid residue polypeptide chain. SDS-PAGE analysis revealed a MW of 40 kDa, whereas the native enzyme exhibited a MW of 40 kDa suggesting a monomeric protein. The K(m) and V(max) values of the P. fluorescence ProDH were estimated to be 35 mM and 116 micromol/min, respectively. ProDH activity was stable at alkaline pH and the highest activity was observed at 30 degrees C and pH 8.5. The modeling analysis of the three dimensional structure elucidated that Lys-173 and Asp-202, which were oriented near the hydroxyl group of the substrate, were essential residues for the ProDH activity. This study, to our knowledge, is the first data on the cloning and biochemical and structural properties of P. fluorescence ProDH. 相似文献
19.
The Helicobacter pylori NCTC 11637 alanine racemase gene, alr1, was cloned based on a putative alanine racemase gene, alr, of H. pylori 26695. The protein, Alr1, was purified to homogeneity from Escherichia coli MB2795 cells harboring the alr1 gene. The protein exclusively catalyzes the conversion of l-alanine to the d-isomer with K(m) and V(max) values of 100 mM and 909 mumol min(-1) mg(-1), respectively. The values are 16-fold higher than those for the reaction in the reverse direction. The molecular weight of Alr1 is 42,000 by SDS-PAGE, and 68,000 by gel-filtration analysis. The optimal pH and temperature are pH 8.3 and 37 degrees C, respectively, in good accordance with the characteristics shown by the alanine racemase purified from H. pylori NCTC 11637 cells. Pyridoxal 5'-phosphate was suggested to be the cofactor. The physiological function of Alr1 is discussed regarding energy production in the microbial cells. 相似文献
20.
The aryl tetralin lignans are synthesized by Podophyllum sps. and are in great demand worldwide due to their use in synthesis of topoisomerase inhibitors. However, the sustained production of these aryl tetralin lignans requires large-scale harvesting from the natural environments, which has resulted in the plant-endangered status. In view of the difficulties in their total chemical synthesis, cultivation and failure of metabolic engineering approaches, there is a need to search for alternative sources of production of aryl tetralin lignans. We unequivocally established the methodology for isolation, identification, and characterization of a novel fungal endophyte (Trametes hirsuta) that produces aryl tetralin lignans consistently as shown by HPLC, LC-MS, LC/MS-MS and (1)H NMR. The lignans produced by the microorganism are biologically active, and exhibit potent antioxidant, anticancer and radioprotective properties. This strategy promises to improve the production of these therapeutically important compounds at lower costs. 相似文献
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