High production of laccase by a new basidiomycete, <Emphasis Type="Italic">Trametes</Emphasis> sp

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     

A surfactant tolerant laccase of <Emphasis Type="Italic">Meripilus giganteus</Emphasis>

A laccase (Lcc1) from the white-rot fungus Meripilus giganteus was purified with superior yields of 34% and 90% by conventional chromatography or by foam separation, respectively. Size exclusion chromatography (SEC) and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) yielded a molecular mass of 55 kDa. The enzyme possessed an isoelectric point of 3.1 and was able to oxidize the common laccase substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) at a pH of 2.0, whereas the enzyme was still able to oxidize ABTS and 2,6-dimethoxyphenol (DMP) at pH 6.0. Lcc1 exhibited low K _m values of 8 μM (ABTS) and 80 μM (DMP) and remarkable catalytic efficiency towards the non-phenolic substrate ABTS of 37,437 k _cat/k _m (s⁻¹ mM⁻¹). The laccase showed a high stability towards high concentrations of various metal ions, EDTA and surfactants indicating a considerable biotechnological potential. Furthermore, Lcc1 exhibited an increased activity as well as a striking boost of stability in the presence of surfactants. Degenerated primers were deduced from peptide fragments. The complete coding sequence of lcc1 was determined to 1,551 bp and confirmed via amplification of the 2,214 bp genomic sequence which included 12 introns. The deduced 516 amino acid (aa) sequence of the lcc1 gene shared 82% identity and 90% similarity with a laccase from Rigidoporus microporus. The sequence data may aid theoretical studies and enzyme engineering efforts to create laccases with an improved stability towards metal ions and bipolar compounds.     

Production,purification and characterization of a thermostable laccase from a tropical white-rot fungus

A thermostable laccase was isolated from a tropical white-rot fungus Polyporus sp. which produced as high as 69,738 units of laccase l⁻¹ in an optimized medium containing 20 g of malt extract l⁻¹, 2 g of yeast extract l⁻¹, 1.5 mM CuSO₄. The laccase was purified to electrophoretic purity with a final purification of 44.70-fold and a recovery yield of 21.04%. The purified laccase was shown to be a monomeric enzyme with a molecular mass of 60 kDa. The optimum temperature and pH value of the laccase were 75°C and pH 4.0, respectively, for 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS). The Michaelis–Menten constant (K _m ) of the laccase was 18 μM for ABTS substrate. The laccase was stable at pH values between 5.5 and 7.5. About 80% of the initial enzyme activity was retained after incubation of the laccase at 70°C for 2 h, indicating that the laccase was intrinsically highly thermostable and with valuable potential applications. The laccase activity was promoted by 4.0 mM of Mg²⁺, Mn²⁺, Zn²⁺ and Ca²⁺, while inhibited by 4.0 mM of Co²⁺, Al³⁺, Cu²⁺, and Fe²⁺, showing different profiles of metal ion effects.     

Cloning,characterization and expression of the chitinase gene of <Emphasis Type="Italic">Enterobacter</Emphasis> sp. NRG4

A chitinase producing bacterium Enterobacter sp. NRG4, previously isolated in our laboratory, has been reported to have a wide range of applications such as anti-fungal activity, generation of fungal protoplasts and production of chitobiose and N-acetyl D-glucosamine from swollen chitin. In this paper, the gene coding for Enterobacter chitinase has been cloned and expressed in Escherichia coli BL21(DE3). The structural portion of the chitinase gene comprised of 1686 bp. The deduced amino acid sequence of chitinase has high degree of homology (99.0%) with chitinase from Serratia marcescens. The recombinant chitinase was purified to near homogeneity using His-Tag affinity chromatography. The purified recombinant chitinase had a specific activity of 2041.6 U mg⁻¹. It exhibited similar properties pH and temperature optima of 5.5 and 45°C respectively as that of native chitinase. Using swollen chitin as a substrate, the K_m, k_cat and catalytic efficiency (k_cat/K_m) values of recombinant chitinase were found to be 1.27 mg ml⁻¹, 0.69 s⁻¹ and 0.54 s⁻¹M⁻¹ respectively. Like native chitinase, the recombinant chitinase produced medicinally important N-acetyl D-glucosamine and chitobiose from swollen chitin and also inhibited the growth of many fungi.     

The white-rot fungus Cerrena unicolor strain 137 produces two laccase isoforms with different physico-chemical and catalytic properties

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.     

Production of laccase byCurvularia sp.

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.     

Phylogenetic Analysis and Biochemical Characterization of a Thermostable Dihydropyrimidinase from Alkaliphilic Bacillus sp. TS-23

Two degenerate primers established from the alignment of highly conserved amino acid sequences of bacterial dihydropyrimidinases (DHPs) were used to amplify a 330-bp gene fragment from the genomic DNA of Bacillus sp. TS-23 and the amplified DNA was successfully used as a probe to clone a dhp gene from the strain. The open reading frame of the gene consisted of 1422 bp and was deduced to contain 472 amino acids with a molecular mass of 52 kDa. The deduced amino acid sequence exhibited greater than 45% identity with that of prokaryotic d-hydantoinases and eukaryotic DHPs. Phylogenetic analysis showed that Bacillus sp. TS-23 DHP is grouped together with Bacillus stearothermophilus d-hydantoinase and related to dihydroorotases and allantoinases from various organisms. His₆-tagged DHP was over-expressed in Escherichia coli and purified by immobilized metal affinity chromatography to a specific activity of 3.46 U mg⁻¹ protein. The optimal pH and temperature for the purified enzyme were 8.0 and 60 °C, respectively. The half-life of His₆-tagged DHP was 25 days at 50 °C. The enzyme activity was stimulated by Co²⁺ and Mn²⁺ ions. His₆-tagged DHP was most active toward dihydrouracil followed by hydantoin derivatives. The catalytic efficiencies (k_cat/K_m) of the enzyme for dihydrouracil and hydantoin were 2.58 and 0.61 s⁻¹ mM⁻¹, respectively.     

Purification and characterisation of lignin peroxidase from <Emphasis Type="Italic">Pycnoporus sanguineus</Emphasis> MTCC-137

Extracellular secretion of lignin peroxidase from Pycnoporus sanguineus MTCC-137 in the liquid culture growth medium amended with lignin containing natural sources has been shown. The maximum secretion of lignin peroxidase has been found in the presence of saw dust. The enzyme has been purified to homogeneity from the culture filtrate of the fungus using ultrafiltration and anion exchange chromatography on DEAE-cellulose. The purified lignin peroxidase gave a single protein band in sodium dodecylsulphate polyacrylamide gel electrophoresis corresponding to the molecular mass 40 kDa. The K _m, k _cat and k _cat/K _m values of the enzyme using veratryl alcohol and H₂O₂ as the substrate were 61 M, 2.13 s⁻¹, 3.5 × 10⁴ M⁻¹s⁻¹ and 71 M, 2.13 s⁻¹, 3.0 × 10⁴ M⁻¹ s⁻¹ respectively at the optimum pH of 2.5. The temperature optimum of the enzyme was 25°C.     

Effects of salts on activity of halophilic cellulase with glucomannanase activity isolated from alkaliphilic and halophilic Bacillus sp. BG-CS10

Alkaliphilic and halophilic Bacillus sp. BG-CS10 was isolated from Zabuye Salt Lake, Tibet. The gene celB, encoding a halophilic cellulase was identified from the genomic library of BG-CS10. CelB belongs to the cellulase superfamily and DUF291 superfamily, with an unknown function domain and less than 58% identity to other cellulases in GenBank. The purified recombinant protein (molecular weight: 62 kDa) can hydrolyze soluble cellulose substrates containing beta-1,4-linkages, such as carboxylmethyl cellulose and konjac glucomannan, but has no exoglucanase and β-glucosidase activities. Thus, CelB is a cellulase with an endo mode of action and glucomannanase activity. Interestingly, the enzyme activity was increased approximately tenfold with 2.5 M NaCl or 3 M KCl. Furthermore, the optimal temperatures were 55°C with 2.5 M NaCl and 35°C without NaCl, respectively. This indicates that NaCl can improve enzyme thermostability. The K _m and k _cat values of CelB for CMC with 2.5 M NaCl were 3.18 mg mL⁻¹ and 26 s⁻¹, while the K _m and k _cat values of CelB without NaCl were 6.6 mg mL⁻¹ and 2.1 s⁻¹. Thus, this thermo-stable, salt and pH-tolerant cellulase is a promising candidate for industrial applications, and provides a new model to study salt effects on the structure of protein.     

Efficient production of laccases by Trametes sp. AH28-2 in cocultivation with a Trichoderma strain

A biocontrol fungus isolated from rotting wood was identified as a Trichoderma strain (named as Trichoderma sp. ZH1) by internal transcribed spacer (ITS) sequences of rRNA genes. The laccase yield of Trametes sp. AH28-2 in cocultivation with Trichoderma sp. ZH1 reached 6,210 U l⁻¹, approximately identical to those induced by toxic aromatic inducers. Cocultures maintained 60–70 % of their highest laccase activity obtained at 5 days after inoculation of the biocontrol fungus, at least for 20 days. Furthermore, a novel laccase isozyme (LacC) was obtained through the fungal interactions. The molecular weight of LacC is about 64 kDa, and its isoelectric point is 6.6. The temperature and pH optimum for LacC to oxidize guaiacol are 55 °C and 5.0, respectively. LacC is stable both at 60 °C and pH 4.0–8.0. Furthermore, the K _m values of LacC for various substrates were also determined. Our work demonstrates a safe strategy for the production of industrial laccases, instead of the traditional method of chemical induction.     

Gene cloning,expression and characterization of a neoagarotetraose-producing β-agarase from the marine bacterium <Emphasis Type="Italic">Agarivorans</Emphasis> sp. HZ105

A β-agarase gene hz2 with 2,868 bp was cloned from the marine agarolytic bacterium Agarivorans sp. HZ105. It encoded a mature agarase HZ2 of 102,393 Da (920 amino acids). Based on the amino acid sequence similarity, agarase HZ2 was assigned to the glycoside hydrolase family 50. The β-agarase shared a gene sequence identity of 98.6% with the reported but much less characterized β-agarase agaB from Vibrio sp. JT0107. Its recombinant agarase rHZ2 was produced in E. coli cells and purified to homogeneity. The agarase rHZ2 degraded agarose and neoagarooligosaccharides with degrees of polymerization above four, to yield neoagarotetraose as the dominant product, which was different from β-agarase agaB of Vibrio sp. JT0107. The agarose hydrolysis pattern suggested that rHZ2 was an endo-type β-agarase. Beta-mercaptoethanol (90 mM) and dithiothreitol (9 mM) increased the agarase activity of rHZ2 by 72.9% and 17.3% respectively, while SDS (9 mM) inhibited the activity completely. The agarase activity was independent of Na⁺, K⁺, Mg²⁺ and Ca²⁺. The maximal enzyme activity was observed at 40°C and pH 7. The kinetic parameters K _m, V _max, K _cat, and K _cat/K _m values toward agarose of agarase rHZ2 were 5.9 mg ml⁻¹, 235 U mg⁻¹, 401 s⁻¹ and 6.8 × 10⁵ M⁻¹ s⁻¹, respectively. Agarase rHZ2 could have a potential application in the production of bioactive neoagarotetraose.     

Secretory expression and characterization of a soluble laccase from the <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> strain 7071-9 in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Laccases are strong oxidizing enzymes that oxidize chlorinated phenols, synthetic dyes, pesticides, polycyclic aromatic hydrocarbons as well as a very wide range of other compounds with high redox potential. Based on the bias of genetic codons between fungus and yeast, we synthesized a laccase gene GlLCCI, originated from Ganoderma lucidum using optimized codons and a PCR-based two-step DNA synthesis method. The recombinant laccase, GlLCCI was successfully over-expressed in yeast, Pichia pastoris, with an alcohol oxidase1 promoter. The recombinant GlLCCI has a molecular mass of approximately 58 kDa. The K _m values of GlLCCI for 2-2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and guaiacol were 0.9665, and 1.1122 mM, respectively. The V _max of GlLCCI for both substrates was 3,024 and 82.13 μM mg⁻¹ min⁻¹. When ABTS was used as a substrate, the enzyme had an optimal temperature of approximately 55°C. The enzyme was detected over pH values from 2 to 8. The enzyme was strongly activated by K⁺, Na⁺, Cu²⁺ and mannitol. Six amino acids (alanine, histidine, glycine, arginine, aspartate and phenylalanine) increased the catalytic ability of the enzyme. The activity of laccase was obviously inhibited by Fe²⁺, Fe³⁺, sodium hydrosulphite, and sodium azide. Additionally, under optimal conditions, GlLCCI decolorized 37.62 mg l⁻¹ of azo dye methyl orange (MO) in cultural medium. With a high MO degradation ability, GlLCCI may have potential in the treatment of industrial effluent containing azo dye MO.     

Purification and Characterization of an Extracellular Chitinase from the Antifungal Biocontrol Agent <Emphasis Type="Italic">Streptomyces halstedii</Emphasis>

An extracellular chitinase from Streptomyces halstedii AJ-7, a broad spectrum antifungal biocontrol agent, was characterized and purified. The apparent molecular weight of the purified protein was 55 kDa, K_m value and V_max of the protein for colloidal chitin were 3.2 mg ml⁻¹ and 118 μmol h⁻¹, respectively. The growth and chitinase activity of S. halstedii AJ−7 were enhanced by adding of 0.1% killed mycelium of Fusarium oxysporium in a medium containing 0.2% colloidal chitin.     

Properties of native and hydrophobic laccases immobilized in the liquid-crystalline cubic phase on electrodes

Both native Trametes hirsuta laccase and the same laccase modified with palmytic chains to turn it more hydrophobic were prepared and studied with cyclic voltammetry and Raman spectroscopy. Native laccase immobilized in the monoolein cubic phase was characterized with resonance Raman spectroscopy, which demonstrated that the structure at the “blue” copper site of the protein remained intact. The diamond-type monoolein cubic phase prevents denaturation of enzymes on the electrode surface and provides contact of the enzyme with the electrode either directly or through the mediation by electroactive probes. Direct electron transfer for both laccases incorporated into a lyotropic liquid crystal was obtained under anaerobic conditions, whereas bioelectrocatalytic activity was shown only for the native enzyme. The differences in electrochemical behavior of native and hydrophobic laccase as well as possible mechanisms of direct and mediated electron transfers are discussed. The Michaelis constant for 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS²⁻), K _M^app, and the maximal current, I _max, for the native enzyme immobilized onto the electrode were estimated to be 0.24 mM, and 5.3 μA, respectively. The maximal current density and the efficiency of the catalysis, I _max/K _M^app, were found to be 73 μA cm⁻² and 208.2 μA cm⁻² mM⁻¹, respectively, and indicated a high efficiency of oxygen electroreduction by the enzyme in the presence of ABTS²⁻ in the cubic-phase environment. Rate constants were calculated to be 7.5 × 10⁴ and 3.6 × 10⁴ M⁻¹ s⁻¹ for native and hydrophobic laccase, respectively.     

Kinetics and thermodynamics of peroxidase- and laccase-catalyzed oxidation of N-substituted phenothiazines and phenoxazines

N -substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4–10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k _cat/K _m) varied from 1 ×10⁷M⁻¹s⁻¹to 2.6×10⁸M⁻¹s⁻¹ at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable groups with pK _a values of 4.9–5.7 and 7.7–9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecular rate constants varied from 1.8×10⁵M⁻¹s⁻¹ to 2.0×10⁷M⁻¹s⁻¹ at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO, fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6×10⁸M⁻¹s⁻¹ and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0×10⁷M⁻¹s⁻¹ and 0.29 eV. Received: 20 September 1999 / Accepted: 24 February 2000     

A new esterase showing similarity to putative dienelactone hydrolase from a strict marine bacterium, <Emphasis Type="Italic">Vibrio</Emphasis> sp. GMD509

Vibrio sp. GMD509, a marine bacterium isolated from eggs of the sea hare, exhibited lipolytic activity on tributyrin (TBN) plate, and the gene representing lipolytic activity was cloned. As a result, an open reading frame (ORF) consisting of 1,017 bp (338 aa) was found, and the deduced amino acid sequence of the ORF showed low similarity (<20%) to α/β hydrolases such as dienelactone hydrolases and esterase/lipase with G–X₁–S–X₂–G sequence conserved. Phylogenetic analysis suggested that the protein belonged to a new family of esterase/lipase together with various hypothetical proteins. The enzyme was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme (Vlip509) showed the best hydrolyzing activity toward p-nitrophenyl butyrate (C₄) among various p-nitrophenyl esters (C₂ to C₁₈), and optimal activity of Vlip509 occurred at 30°C and pH 8.5, respectively. Kinetic parameters toward p-nitrophenyl butyrate were determined as K _m (307 μM), k _cat (5.72 s⁻¹), and k _cat/K _m (18.61 s⁻¹ mM⁻¹). Furthermore, Vlip509 preferentially hydrolyzed the S-enantiomer of racemic ofloxacin ester. Despite its sequence homology to dienelactone hydrolase, Vlip509 showed no dienelactone hydrolase activity. This study represents the identification of a novel lipolytic enzyme from marine environment.     

Effects of manganese on the growth,photosystem II and SOD activity of the dinoflagellate <Emphasis Type="Italic">Amphidinium</Emphasis> sp.

Experimental ecology methods and chlorophyll fluorescence technology were used to study the effects of different concentrations of manganese (10⁻¹²– 10⁻⁴ mol L⁻¹) on the growth, photosystem II and superoxide dismutase (SOD) activity of Amphidinium sp. MACC/D31. The results showed that manganese had a significant effect on the growth rate, fluorescence parameters (maximal photochemical efficiency of PSII (F _v /F _m ), photochemical quenching (qP) and non-photochemical quenching (NPQ)) in the exponential stage (days 1–3) and SOD activity of Amphidinium sp. (P < 0.05). F _v/F _m in the exponential stage in 10⁻¹² mol L⁻¹ manganese concentration was significantly lower whilst qP and NPQ significantly higher than those in the other concentrations. F _v /F _m (days 6–9) in 10⁻⁴ mol L⁻¹ manganese was significantly higher than those in the other concentrations. F _v /F _m (days 3–6) increased with increased concentration of manganese from 10⁻¹² to 10⁻⁴ mol L⁻¹. The values of qP and NPQ decreased with decreased concentrations of manganese, except for those in days 4–6. F _v /F _m under each concentration increased earlier and decreased later with culture stage whilst NPQ decreased earlier and increased later. The SOD activity increased with increased concentration of manganese from 10⁻¹² to 10⁻⁸ mol L⁻¹. The SOD activity in 10⁻⁴ mol L⁻¹ manganese was significantly higher than those in the other concentrations and in 10⁻¹² mol L⁻¹ manganese, it was significantly lower than those in the other concentrations.     

Purification and characterization of an ADP-dependent phosphofructokinase from Thermococcus zilligii

The ADP-dependent phosphofructokinase (PFK) from Thermococcus zilligii has been purified 950 fold; it had a specific activity of 190 U mg⁻¹. The enzyme required Mg²⁺ ions for optimal activity and was specific for ADP. The forward reaction kinetics were hyperbolic for both cosubstrates (pH optimum of 6.4), and the apparent K _m values for ADP and fructose-6-phosphate were 0.6 mM (apparent V _max of 243 U mg⁻¹) and 1.47 mM (apparent V _max of 197 U mg⁻¹), respectively. Significantly, the enzyme is indicated to be nonallosteric but was slightly activated by some monovalent cations including Na⁺ and K⁺. The protein had a subunit size of 42.2 kDa and an estimated native molecular weight of 66 kDa (gel filtration). Maximal reaction rates for the reverse reaction were attained at pH 7.5–8.0, and the apparent K _m values for fructose-1,6-bisphosphate and AMP were 0.56 mM (apparent V _max of 2.9 U mg⁻¹) and 12.5 mM, respectively. The biochemical characteristics of this unique ADP-dependent enzymatic activity are compared to ATP and pyrophosphate-dependent phosphofructokinases. Received: August 14, 1998 / Accepted: December 2, 1998     

Biochemical and molecular genetic characterisation of a novel laccase produced by the aquatic ascomycete <Emphasis Type="Italic">Phoma</Emphasis> sp. UHH 5-1-03

A laccase from the aquatic ascomycete Phoma sp. UHH 5-1-03 (DSM 22425) was purified upon hydrophobic interaction and size exclusion chromatography (SEC). Mass spectrometric analysis of the laccase monomer yielded a molecular mass of 75.6 kDa. The enzyme possesses an unusual alkaline isoelectric point above 8.3. The Phoma sp. laccase undergoes pH-dependent dimerisation, with the dimer (∼150 kDa, as assessed by SEC) predominating in a pH range of 5.0 to 8.0. The enzyme oxidises common laccase substrates still at pH 7.0 and 8.0 and is remarkably stable at these pH values. The laccase is active at high concentrations of various organic solvents, all together indicating a considerable biotechnological potential. One laccase gene (lac1) identified at the genomic DNA level and transcribed in laccase-producing cultures was completely sequenced. The deduced molecular mass of the hypothetical protein and the predicted isoelectric point of 8.1 well agree with experimentally determined data. Tryptic peptides of electrophoretically separated laccase bands were analysed by nano-liquid chromatography–tandem mass spectrometry. By using the nucleotide sequence of lac1 as a template, eight different peptides were identified and yielded an overall sequence coverage of about 18%, thus confirming the link between lac1 and the expressed laccase protein.     

Properties of two endoglucanases from a mutant strain <Emphasis Type="Italic">Trichoderma</Emphasis> sp. M<Subscript>7</Subscript> with potential application in the paper industry

Two endoglucanases were purified to electrophoretic homogeneity from the culture filtrate of a mutant strain Trichoderma sp. M₇. EG-III and EG-IV had Mr of 49.7 and 47.5 kDa, and estimated pi values of 3.7 and 6.35, respectively. The optimal pH and temperature values were determined to be pH 5.0 and 60°C for the first cellulase, whereas pH 5.2 and 50 °C were optimal for the other. Endoglucanases exhibited typical Michaelis-Menten kinetics with K _m and V values of 2.9 mg ml⁻¹ and 60498.5 μmol min⁻¹ mg⁻¹ for EG-III and 3.8 mg ml⁻¹ and 22650.9 μmol min⁻¹ mg⁻¹ for EG-IV, respectively. Mn²⁺, Cu²⁺ and Pd²⁺ strongly inhibited the enzymes. EC-IV catalyzed the hydrolysis of Na-CMC and hydroxyethyl cellulose (HEC) only, whereas EG-III displayed high activity towards xylans, also. Different preferences towards cellulosic substrates and their regions define a different role of the investigated enzymes in the degradation of plant biomass. Published in Russian in Prikladnaya Biokhimiya i Mikrobiologiya, 2009, Vol. 45, No. 2, pp. 171–175. The article is published in the original.