Expression of the pyranose 2-oxidase from Trametes pubescens in Escherichia coli and characterization of the recombinant enzyme

cDNA-encoding pyranose 2-oxidase (P2O) from Trametes pubescens was sequenced and cloned into Escherichia coli strain BL21/DE3 on a multicopy plasmid under the control of trc promoter. The synthesis of P2O was studied in a batch culture in M9-based mineral medium: the enzyme was synthesized constitutively at 28 °C in amount corresponding to 8% of the cell soluble protein (0.6 U mg⁻¹). Only small portion of P2O (11%) was in the form of non-active inclusion bodies. Purified recombinant enzyme has similar physico-chemical and kinetic parameters with other P2Os. When compared to the expression of p2o of Trametes ochracea, a ratio of the mature enzyme to inclusion bodies found in the same E. coli host at 28 °C is as much as nine times higher. The finding makes the enzyme from T. pubescens preferable for the large-scale production by recombinant bacteria. The difference in amino acid sequences of the P2O from T. ochracea and T. pubescens may explain the favourable trait of the latter enzyme regarding protein folding.     

Gene cloning and heterologous expression of pyranose 2-oxidase from the brown-rot fungus, <Emphasis Type="Italic">Gloeophyllum</Emphasis><Emphasis Type="Italic">trabeum</Emphasis>

A pyranose 2-oxidase gene from the brown-rot basidiomycete Gloeophyllum trabeum was isolated using homology-based degenerate PCR. The gene structure was determined and compared to that of several pyranose 2-oxidases cloned from white-rot fungi. The G. trabeum pyranose 2-oxidase gene consists of 16 coding exons with canonical promoter CAAT and TATA elements in the 5′UTR. The corresponding G. trabeum cDNA was cloned and contains an ORF of 1,962 base pairs encoding a 653 amino acid polypeptide with a predicted molecular weight of 72 kDa. A Hisx6 tagged recombinant G. trabeum pyranose 2-oxidase was generated and expressed heterologously in Escherichia coli yielding 15 U enzyme activity per ml of induced culture. Structural alignment and phylogenetic analysis were performed and are discussed.     

Batch production of Pyranose 2-oxidase from <Emphasis Type="Italic">Trametes versicolor</Emphasis> (ATCC 11235) in medium with a lignocellulosic substrate and enzymatic bleaching of cotton fabrics

The aim of this work was to determine new, different and low-cost substrates that can be used for enzyme production from the white rot fungus Trametes versicolor (ATCC 11235) by taking advantage of the broad substrate specificity of pyranose 2-oxidase. In this report, we investigated the production of pyranose 2-oxidase from T. versicolor (ATCC 11235) using ten different agricultural residues such as clover straw, almond shells, hazelnut cobs, grass and others. Pyranose 2-oxidase activity was determined as 2.332 U/g at the 9th day in a submerged culture containing clover straw and tap water shaken at 150 rpm and 26°C, and the optimum clover straw concentration was determined to be 12 g/l. The effects of different glucose, nitrogen and phosphate sources on the production of pyranose 2-oxidase were studied in the clover straw medium. Analyses of biomass, protein, reduced sugar and nitrogen concentrations were also monitored in a clover straw medium that did not contain carbon or nitrogen and phosphate sources under the parameters determined. The produced pyranose 2-oxidase was used for improving the properties of cotton fabrics.     

六种白腐菌预处理对毛白杨木材酶水解效果的影响

通过化学分析和酶水解试验,研究了不同的白腐菌对毛白杨的预处理效果及不同组分的降解对酶水解的影响。毛白杨木片经6种白腐菌预处理30d后,各组分都发生了降解,其中半纤维素的损失最为显著,Trametes ochracea C6888引起半纤维素降解率高达47.19%,其次是纤维素和酸不溶木素的降解。在后续酶水解过程中,6种白腐菌处理后的样品显示出不同的水解模式,菌株Trametes ochracea C6888、T. pubescens C7571和T. versicolor C6915预处理效果最为显著,还原糖得率在整个酶水解过程中一直高于对照,其中T. ochracea C6888在水解96h后还原糖得率达到15.93%,比未处理样品提高了25%。分析酸不溶木素降解率及半纤维素降解率与还原糖得率的关系发现,不同菌株在作用同一种基质时,预处理效果差异显著,木质素和半纤维素的脱除都会影响木质纤维素的酶水解。     

Rapid cultivation of stable aerobic phenol-degrading granules using acetate-fed granules as microbial seed

cDNA-encoding pyranose 2-oxidase (P2O) from Trametes pubescens was sequenced and cloned into Escherichia coli strain BL21/DE3 on a multicopy plasmid under the control of trc promoter. The synthesis of P2O was studied in a batch culture in M9-based mineral medium: the enzyme was synthesized constitutively at 28 °C in amount corresponding to 8% of the cell soluble protein (0.6 U mg⁻¹). Only small portion of P2O (11%) was in the form of non-active inclusion bodies. Purified recombinant enzyme has similar physico-chemical and kinetic parameters with other P2Os. When compared to the expression of p2o of Trametes ochracea, a ratio of the mature enzyme to inclusion bodies found in the same E. coli host at 28 °C is as much as nine times higher. The finding makes the enzyme from T. pubescens preferable for the large-scale production by recombinant bacteria. The difference in amino acid sequences of the P2O from T. ochracea and T. pubescens may explain the favourable trait of the latter enzyme regarding protein folding.     

基于ITS序列的栓菌属部分种的分子分类初步研究

栓菌属 Trametes 的一些近缘种宏观和微观形态学非常相近,传统分类学方法难于对其进行准确分类定位。测定了 34 个分类单元的 ITS(包括 5.8SrDNA)序列,并对得到的 43 个分类单元的 ITS 序列进行系统发生分析,构建了聚类分析树状图。该树状图显示,栓菌属类群与其他属类群明显分开,Trametes versicolor 聚类到一个高支持率的独立分支。形态学上定名为 T. hirsuta 和 T. pubescens 物种聚类到同一高支持率的独立分支,试验分析表明这两个种应视为同一物种。     

Identification of the covalent flavin adenine dinucleotide-binding region in pyranose 2-oxidase from Trametes multicolor

We present the first report on characterization of the covalent flavinylation site in flavoprotein pyranose 2-oxidase. Pyranose 2-oxidase from the basidiomycete fungus Trametes multicolor, catalyzing C-2/C-3 oxidation of several monosaccharides, shows typical absorption maxima of flavoproteins at 456, 345, and 275 nm. No release of flavin was observed after protein denaturation, indicating covalent attachment of the cofactor. The flavopeptide fragment resulting from tryptic/chymotryptic digestion of the purified enzyme was isolated by anion-exchange and reversed-phase high-performance liquid chromatography. The flavin type, attachment site, and mode of its linkage were determined by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy of the intact flavopeptide, without its prior enzymatic degradation to the central aminoacyl moiety. Mass spectrometry identified the attached flavin as flavin adenine dinucleotide (FAD). Post-source decay analysis revealed that the flavin is covalently bound to histidine residue in the peptide STHW, consistent with the results of N-terminal amino acid sequencing by Edman degradation. The type of the aminoacyl flavin covalent link was determined by NMR spectroscopy, resulting in the structure 8alpha-(N(3)-histidyl)-FAD.     

Evaluation of different expression systems for the heterologous expression of pyranose 2-oxidase from <Emphasis Type="Italic">Trametes multicolor</Emphasis> in <Emphasis Type="Italic">E. coli</Emphasis>

The heterologous production of the industrially relevant fungal enzyme pyranose 2-oxidase in the prokaryotic host E. coli was investigated using 3 different expression systems, i.e. the well-studied T7 RNA polymerase based pET21d⁺, the L-arabinose inducible pBAD and the pCOLD system. Preliminary experiments were done in shaking flasks at 25°C and optimized induction conditions to compare the productivity levels of the different expression systems. The pET21d⁺ and the pCOLD system gave 29 U/L·h and 14 U/L·h of active pyranose 2-oxidase, respectively, whereas the pBAD system only produced 6 U/L·h. Process conditions for batch fermentations were optimized for the pET21d⁺ and the pCOLD systems in order to reduce the formation of inactive inclusion bodies. The highest productivity rate with the pET21d⁺ expression system in batch fermentations was determined at 25°C with 32 U/L·h. The pCOLD system showed the highest productivity rate (19 U/L·h) at 25°C and induction from the start of the cultivation. Using the pCOLD system in a fed batch fermentation at 25°C with a specific growth rate of μ = 0.15 h^-1resulted in the highest productivity rate of active pyranose oxidase with 206 U/L·h.     

Glucose 1- and 2-oxidases from fungal strains: isolation and production of monoclonal antibodies.

Monoclonal antibodies (Mabs) against purified glucose 2-oxidase (EC 1.1.3.10) from Coriolus versicolor were raised by hybridoma technology using Sp2/0 myeloma cells as a fusion partner. Hybrid growth was observed in 42% of culture wells and 30% of these (i.e. 30 culture wells) contained anti-glucose 2-oxidase activity. Three positive wells containing hybrid cell lines were selected and cloned twice by the limiting dilution method and two hybridoma clones (E1A5 and E1A6) secreting Mabs were selected at random for purification and characterisation purposes. Both cell lines secreted Mabs of IgM class which were purified by gel filtration chromatography on a Sephacryl S-200 column with a final recovery of 80% and a purification factor of 16. The purified preparations were apparently homogeneous on native PAGE running with a M(r) of 950 kDa. Mabs were highly specific for glucose 2-oxidase as determined by Western blotting. These Mabs also crossreacted with glucose 1- and 2-oxidases from other fungal sources (Phanerochaeta chrysosporium, Penicillium amagasakiense and Aspergillus niger) as determined by Western blotting and by ELISA. Both glucose 1- and 2-oxidases from C. versicolor, P. chrysosporium, P. amagasakiense and A. niger were purified by hydrophobic interaction chromatography on Sepharose 4B-triazine dye with a recovery of enzyme activity in the range 85-92%. Purified preparations of glucose oxidases from fungal strains were apparently homogeneous on native PAGE. Glucose 2-oxidases were more hydrophobic than glucose 1-oxidases as determined by their chomatographic behaviour on Sepharose 4B-Cibacron Red G-E which could be used to study their roles in lignin biodegradation.     

Silver stained polyacrylamide gels and fluorescence-based automated capillary electrophoresis for detection of amplified fragment length polymorphism patterns obtained from white-rot fungi in the genus Trametes

Silver stained denaturing polyacrylamide gels (PAGEs) and fluorescent denaturing automated capillary electrophoresis (CE) were used to detect amplified fragment length polymorphism (AFLP) patterns obtained from white-rot fungi belonging to the genus Trametes. AFLP fingerprinting detected by the fluorescence-based method as well as by silver staining showed a high discriminatory power in differentiating nine strains of Trametes ochracea, nine strains of Trametes hirsuta and ten isolates of Trametes versicolor. UPGMA dendrograms derived from fluorescently labelled and silver stained AFLP patterns were similar, but a few differences were detected especially in the clustering of T. ochracea and T. hirsuta strains. Compared to silver-stained AFLP, detection of fluorescent AFLP was fast, reliable and easy to perform and it facilitated surveying with a computerized analysis system. Fluorescent CE seems to be well suited for studying similarity between Trametes species.     

A method for identifying a proposed carbohydrate-binding motif of proteins.

An examination of the binding sites of four carbohydrate binding proteins (Escherichia coli lactose repressor, E. coli arabinose-binding protein, yeast hexokinase A and Concanavalin A) revealed certain similarities of amino acid sequences and residues forming hydrogen bonds and hydrophobic interactions with the bound carbohydrate. These were: (i) Asx-Asx, hydrogen bonding to the pyranose ring oxygen and anomeric-OH group; (ii) Arg-X-X-X-(Ser/Thr), or the reverse sequence, with the Arg hydrogen bonding to the pyranose ring oxygen; (iii) Lys-(Ser/Thr)-X-X-Asp, or the reverse sequence and with interchange of the Lys-(Ser/Thr) positions, with hydrogen bonding of either or both the Lys and Asp residues to the -OH groups at carbons 2, 3, 4 or 6; (iv) a diaromatic sequence with possible hydrophobic interactions to the faces of the pyranose ring structure. An algorithm was devised to search the amino acid sequences of a large number of proteins, those known to bind carbohydrates as well as those without known carbohydrate-binding activities, for the four amino acid sequence criteria. The algorithm incorporated a weighted distance value (WDV) to assess the approximate distance between any two criteria, with the WDV being based on the predicted secondary structure of the protein amino acid sequence. When the algorithm using criteria 1 and 2 plus the WDV was applied to the sequences of 125 proteins, the method indicated the presence of the potential carbohydrate-binding site motif for 42% of proteins with known carbohydrate binding, only 8% of proteins were predicted as false positives, and the accuracy of the method was calculated to be 61.6%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning, sequencing, and expression of a fibrinolytic serine-protease gene from the earthworm Lumbricus rubellus

The full-length cDNA of the lumbrokinase fraction 6 (F6) protease gene of Lumbricus rubellus was amplified using an mRNA template, sequenced and expressed in E. coli cells. The F6 protease gene consisted of pro- and mature sequences by gene sequence analysis, and the protease was translated and modified into active mature polypeptide by N-terminal amino acid sequence analysis of the F6 protease. The pro-region of F6 protease consisted of the 44 residues from methionine-1 to lysine-44, and the mature polypeptide sequence (239 amino acid residues and one stop codon; 720 bp) started from isoleucine-45 and continued to the terminal residue. F6 protease gene clones having pro-mature sequence and mature sequence produced inclusion bodies in E. coli cells. When inclusion bodies were orally administrated rats, generated thrombus weight in the rat's venous was reduced by approximately 60 % versus controls. When the inclusion bodies were solubilized in pepsin and/or trypsin solutions, the solubilized enzymes showed hemolytic activity in vitro. It was concluded the F6 protease has hemolytic activity, and that it is composed of pro- and mature regions.     

Comparison of physico-chemical characteristics of four laccases from different basidiomycetes

New strains of basidiomycetes producing extracellular laccases (Trametes ochracea 92-78, and Trametes hirsuta 56) have been found by screening of isolates of Trametes fungi. The laccases from T. hirsuta 56 and T. ochracea 92-78 as well as two laccases from previously found and described strains of basidiomycetes, namely Cerrena maxima and Coriolopsis fulvocinerea, were purified to homogeneity. The standard redox potentials of type 1 copper in the enzymes were determined and found to be 780, 790, 750, and 780 mV, respectively. The spectral and biochemical studies showed that the enzymes had no significant differences between the structures of their active sites (T1, T2, and T3). In spite of this fact, the basic biochemical properties as well as the redox potentials of the T1 sites of the enzymes were found to be different. The molecular weights of the laccases range from 64 to 70 kDa, and their pI values range from 3.5 to 4.7. The pH-optima are in the range 3.5-5.2. The temperature optimum for activity is about 50 degrees C. The thermal stabilities of the enzymes were studied. The catalytic and Michaelis constants for catechol, guaiacol, hydroquinone, sinapinic acid, and K(4)Fe(CN)(6) were determined. Based on these results as well as results obtained by comparing with published properties of several laccases, it could be concluded that T. hirsuta and Cerrena maxima laccases have some superior characteristics such as high stability, high activity, and low carbohydrate content, making them attractive objects for further investigations as well as for application in different areas of biotechnology.     

Isolation of cDNAs encoding for two distinct isoforms of the TATA-Box binding proteins from common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

The TATA-box binding protein (TBP) is one of the 4 DNA-binding proteins that has been shown to associate with the proximal promoter region (−295) of the gene for bean seed storage protein phaseolin. The −295 promoter is essential for spatial and temporal control of the phaseolin gene expression. We designed a pair of degenerated primers based on the highly conserved sequence of the carboxyl-terminal domain of yeast TBP and used PCR to amplify the corresponding sequence from the bean cDNA. By using the amplified fragment as a probe, we screened a cDNA library derived from poly A(+) RNA from developing bean seeds and isolated 2 nearly full-length cDNA clones (813 and 826 bp long). The cDNAs encode 2 distinct isoforms of bean TBP, PV1 and PV2, each with an open reading frame of 200 amino acid residues. The 2 cDNA sequences share an 85.8% overall nucleotide sequence identity, with the coding region showing a higher degree of identity (94.4%) than the 5′- and 3′-untranslated regions (69%). The deduced amino acid sequence of the bean TBP isoforms differ in only 3 amino acid residues at positions 5, 9, and 16, all located in the amino-terminal region. The carboxyl-terminal domain of 180 amino acid residues shows a high degree (>82%) of evolutionary sequence conservation with the TBP sequences from other eukaryotic species. This domain possesses the 3 highly conserved structural motifs, namely the 2 direct repeat sequences, a central basic region rich in basic amino acid residues, and a region similar to the sigma factor of prokaryote. On the basis of this and other findings, we suggest that higher plants in general may have at least 2 copies of TBP gene, presumably resulting from the global duplication of the genome. Accession numbers AF015784 and AF015785 at the GenBank.     

Oxidation of Phe454 in the Gating Segment Inactivates Trametes multicolor Pyranose Oxidase during Substrate Turnover

The flavin-dependent enzyme pyranose oxidase catalyses the oxidation of several pyranose sugars at position C-2. In a second reaction step, oxygen is reduced to hydrogen peroxide. POx is of interest for biocatalytic carbohydrate oxidations, yet it was found that the enzyme is rapidly inactivated under turnover conditions. We studied pyranose oxidase from Trametes multicolor (TmPOx) inactivated either during glucose oxidation or by exogenous hydrogen peroxide using mass spectrometry. MALDI-MS experiments of proteolytic fragments of inactivated TmPOx showed several peptides with a mass increase of 16 or 32 Da indicating oxidation of certain amino acids. Most of these fragments contain at least one methionine residue, which most likely is oxidised by hydrogen peroxide. One peptide fragment that did not contain any amino acid residue that is likely to be oxidised by hydrogen peroxide (DAFSYGAVQQSIDSR) was studied in detail by LC-ESI-MS/MS, which showed a +16 Da mass increase for Phe454. We propose that oxidation of Phe454, which is located at the flexible active-site loop of TmPOx, is the first and main step in the inactivation of TmPOx by hydrogen peroxide. Oxidation of methionine residues might then further contribute to the complete inactivation of the enzyme.     

Development of a recombinant bacterial expression system for the active form of a human transforming growth factor beta type II receptor ligand binding domain

Expression systems have been designed to test the suitability of expressing the high cysteine containing extracellular domain (residues 1-136) of human transforming growth factor beta type II receptor (TbetaRII). Receptor expressed using a baculovirus system was functional following both enzymatic deglycosylation and elimination of the N-terminal 22 amino acids by protease degradation. Bacterial expression of a TbetaRII lacking the 26 N-terminal amino acids retained the ability to bind its ligand, TGF-beta1. Receptor expressed in bacteria was sensitive to proteolytic degradation at residue Lys98 but a K98T mutation eliminated degradation and did not disrupt binding. Although several different forms of TbetaRII were expressed, only a fusion with glutathione S-transferase gave soluble TbetaRII, which was purified at a yield of 0.1 mg/10 L of bacterial growth. N-Terminal truncations of TbetaRII (residues 22-136 or 27-136) could be refolded from inclusion bodies and purified to an active form with an efficiency of 10%.     

High-level expression of a fungal pyranose oxidase in high cell-density fed-batch cultivations of Escherichia coli using lactose as inducer

Expression of a recombinant pyranose oxidase (P2O) from the basidiomycete Trametes ochracea has been increased 10-fold in shaking flask cultures of Escherichia coli BL21(DE3) harboring plasmid pSE33 by optimizing the composition of the culture medium using an experimental design approach. Inexpensive lactose was used as a medium component and inducer of expression of the P2O gene, which is under the control of a trc promoter. The expression system was studied in detail in batch and fed-batch cultivations with the aim to improve the expression level of active recombinant protein and to minimize the formation of inclusion bodies. In batch cultivations, the highest specific P2O activity of 0.9 U (mg of soluble protein)(-1) was measured in oxygen-limited cultures grown at 25 degrees C. The highest overall volumetric productivity of 33 mg of active P2O per liter and hour (corresponding to 345U (L h)(-1)) has been determined in a high-density fed-batch process with a feed-forward exponential feeding strategy. During the fed-batch process, lactose was added intermittently to the culture. A final biomass concentration of 33 g L(-1) (based on cell dry weight) was obtained. Compared to shaking flask cultures in not optimized culture media, the overall volumetric P2O productivity has been improved by a factor of 110 using the fed-batch strategy and the optimized culture medium. Recombinant P2O was expressed in the cytoplasm with 9% of the total soluble protein being active P2O. In terms of physical and enzyme kinetic properties, the purified recombinant P2O was found to be similar to the previously published data of P2O isolated from its original host.