Molecular characterization of laccase genes from the basidiomycete Coprinus cinereus and heterologous expression of the laccase lcc1.

A laccase from Coprinus cinereus is active at alkaline pH, an essential property for some potential applications. We cloned and sequenced three laccase genes (lcc1, lcc2, and lcc3) from the ink cap basidiomycete C. cinereus. The lcc1 gene contained 7 introns, while both lcc2 and lcc3 contained 13 introns. The predicted mature proteins (Lcc1 to Lcc3) are 58 to 80% identical at the amino acid level. The predicted Lcc1 contains a 23-amino-acid C-terminal extension rich in arginine and lysine, suggesting that C-terminal processing may occur during its biosynthesis. We expressed the Lcc1 protein in Aspergillus oryzae and purified it. The Lcc1 protein as expressed in A. oryzae has an apparent molecular mass of 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and absorption maxima at 278 and 614 nm. Based on the N-terminal protein sequence of the laccase, a 4-residue propeptide was processed during the maturation of the enzyme. The dioxygen specificity of the laccase showed an apparent K(m) of 21 +/- 2 microM and a catalytic constant of 200 +/- 10 min(-1) for O(2) with 2, 2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) as the reducing substrate at pH 5.5. Lcc1 from A. oryzae may be useful in industrial applications. This is the first report of a basidiomycete laccase whose biosynthesis involves both N-terminal and C-terminal processing.     

Purification, characterization, molecular cloning, and expression of two laccase genes from the white rot basidiomycete Trametes villosa.

Two laccases have been purified to apparent electrophoretic homogeneity from the extracellular medium of a 2,5-xylidine-induced culture of the white rot basidiomycete Trametes villosa (Polyporus pinsitus or Coriolus pinsitus). These proteins are dimeric, consisting of two subunits of 63 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and have typical blue laccase spectral properties. Under nondenaturing conditions, the two purified laccases have different pIs; purified laccase forms 1 and 3 have pIs of 3.5 and 6 to 6.5, respectively. A third purified laccase form 2 has the same N terminus as that of laccase form 3, but its pI is in the range of 5 to 6. The laccases have optimal activity at pH 5 to 5.5 and pH < or = 2.7 with syringaldazine and ABTS [2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)] as substrates, respectively. The genes lcc1 and lcc2 coding for the two purified laccases (forms 1 and 3) have been cloned, and their nucleotide sequences have been determined. The genes for lcc1 and lcc2 have 8 and 10 introns, respectively. The predicted proteins are 79% identical at the amino acid level. From Northern (RNA) blots containing total RNA from both induced and uninduced cultures, expression of lcc1 is highly induced, while the expression of lcc2 appears to be constitutive. Lcc1 has been expressed in Aspergillus oryzae, and the purified recombinant protein has the same pI, spectral properties, stability, and pH profiles as the purified native protein.     

芳香族化合物对斑玉蕈菌丝生物量、漆酶活性及其转录水平的影响

芳香族化合物适当时间适当浓度添加到培养基中,可提高真菌漆酶活性,有助于增强其对木质纤维素的利用效率。为了增强斑玉蕈漆酶活性,本文研究了8种芳香族化合物对其酶活的影响及其与菌丝生物量的相关性。研究发现在无诱导物条件下,斑玉蕈漆酶活性和菌丝生物量相关系数r为0.9956,说明它们呈正相关,但是整个培养过程漆酶活性相对较低;供试的芳香族化合物对漆酶活性都有不同程度的诱导作用,其中添加0.1mmol/L的愈创木酚对斑玉蕈漆酶活性诱导作用最大,达到3倍以上,同时提高了斑玉蕈菌丝生长速度和菌丝生物量;而随着添加时间的延长,部分化合物对漆酶活性和菌丝生物量都产生不同程度的抑制作用,这可能因为化合物对菌丝毒性的延长导致菌丝生长变慢或死亡;进一步研究发现,斑玉蕈3个漆酶同工酶基因lcc2、lcc3和lcc4在诱导剂愈创木酚的影响下转录水平都不同程度地上调。研究结果表明诱导漆酶活性可以提高斑玉蕈菌丝生长速度和生物量,暗示可能通过提高漆酶活性的方法,提高斑玉蕈的培养基利用效率。     

Effects of aromatic compounds on hemicellulose-degrading enzymes in Aspergillus japonicus

Aspergillus japonicus was grown in the presence of various aromatic compounds at 0.1 and 1 mg/mL, and extracellular xylanase and arabanase activities were measured. Some of the aromatic compounds tested, especially at the higher concentration, suppressed the appearance of hemicellulase activities, expressed as xylanase or arabanase. Vanillin at 1 mg/mL in the presence of either xylan or araban completely suppressed growth, and guaiacol and p-coumaric acid markedly inhibited the growth of A. japonicus. The effects of the aromatic compounds on the activity of crude enzyme preparations were also ascertained. In vitro arabanase activity was affected more than xylanase activity.     

Symbiotic fungi produce laccases potentially involved in phenol degradation in fungus combs of fungus-growing termites in Thailand

Fungus-growing termites efficiently decompose plant litter through their symbiotic relationship with basidiomycete fungi of the genus Termitomyces. Here, we investigated phenol-oxidizing enzymes in symbiotic fungi and fungus combs (a substrate used to cultivate symbiotic fungi) from termites belonging to the genera Macrotermes, Odontotermes, and Microtermes in Thailand, because these enzymes are potentially involved in the degradation of phenolic compounds during fungus comb aging. Laccase activity was detected in all the fungus combs examined as well as in the culture supernatants of isolated symbiotic fungi. Conversely, no peroxidase activity was detected in any of the fungus combs or the symbiotic fungal cultures. The laccase cDNA fragments were amplified directly from RNA extracted from fungus combs of five termite species and a fungal isolate using degenerate primers targeting conserved copper binding domains of basidiomycete laccases, resulting in a total of 13 putative laccase cDNA sequences being identified. The full-length sequences of the laccase cDNA and the corresponding gene, lcc1-2, were identified from the fungus comb of Macrotermes gilvus and a Termitomyces strain isolated from the same fungus comb, respectively. Partial purification of laccase from the fungus comb showed that the lcc1-2 gene product was a dominant laccase in the fungus comb. These findings indicate that the symbiotic fungus secretes laccase to the fungus comb. In addition to laccase, we report novel genes that showed a significant similarity with fungal laccases, but the gene product lacked laccase activity. Interestingly, these genes were highly expressed in symbiotic fungi of all the termite hosts examined.     

Characterization of a gene encoding Trametes versicolor laccase A and improved heterologous expression in Saccharomyces cerevisiae by decreased cultivation temperature

Laccase can be used for enzymatic detoxification of lignocellulosic hydrolysates. A Saccharomyces cerevisiae strain with enhanced resistance to phenolic inhibitors and thereby improved ability to ferment lignocellulosic hydrolysates would presumably be obtained by heterologous expression of laccase. Sequencing of the cDNA for the novel laccase gene lcc2 from the lignin-degrading basidiomycete Trametes versicolor showed that it encodes an isoenzyme of 499 amino-acid residues preceded by a 21-residue signal peptide. By comparison with Edman degradation data, it was concluded that lcc2 encodes an isoenzyme corresponding to laccase A. The gene product of lcc2 displays 71% identity with the previously characterized T. versicolor lcc1 gene product. An alignment of laccase sequences revealed that the T. versicolor isoenzymes in general are more closely related to corresponding isoenzymes from other white-rot fungi than to the other T. versicolor isoenzymes. The multiplicity of laccase is thus a conserved feature of T. versicolor and related species of white-rot fungi. When the T. versicolor lcc2 cDNA was expressed in S. cerevisiae, the production of active enzyme was strongly dependent on the temperature. After 3 days of incubation, a 16-fold higher laccase activity was found when a positive transformant was kept at 19 °C instead of 28 °C. Similar experiments with Pichia pastoris expressing the T. versicolor laccase gene lcc1 also showed that the expression level was favoured considerably by lower cultivation temperature, indicating that the observation made for the S. cerevisiae expression system is of general significance. Received: 8 December 1998 / Received revision: 9 April 1999 / Accepted: 16 April 1999     

Molecular Characterization of Laccase Genes from the Basidiomycete Coprinus cinereus and Heterologous Expression of the Laccase Lcc1

A laccase from Coprinus cinereus is active at alkaline pH, an essential property for some potential applications. We cloned and sequenced three laccase genes (lcc1, lcc2, and lcc3) from the ink cap basidiomycete C. cinereus. The lcc1 gene contained 7 introns, while both lcc2 and lcc3 contained 13 introns. The predicted mature proteins (Lcc1 to Lcc3) are 58 to 80% identical at the amino acid level. The predicted Lcc1 contains a 23-amino-acid C-terminal extension rich in arginine and lysine, suggesting that C-terminal processing may occur during its biosynthesis. We expressed the Lcc1 protein in Aspergillus oryzae and purified it. The Lcc1 protein as expressed in A. oryzae has an apparent molecular mass of 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and absorption maxima at 278 and 614 nm. Based on the N-terminal protein sequence of the laccase, a 4-residue propeptide was processed during the maturation of the enzyme. The dioxygen specificity of the laccase showed an apparent K_m of 21 ± 2 μM and a catalytic constant of 200 ± 10 min⁻¹ for O₂ with 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) as the reducing substrate at pH 5.5. Lcc1 from A. oryzae may be useful in industrial applications. This is the first report of a basidiomycete laccase whose biosynthesis involves both N-terminal and C-terminal processing.     

Metabolism of cinnamic, p-coumaric, and ferulic acids by Streptomyces setonii

Streptomyces setonii strain 75Vi2 was grown at 45 degrees C in liquid media containing yeast extract and trans-cinnamic acid, p-coumaric acid, ferulic acid, or vanillin. Gas chromatography, thin-layer chromatography, and mass spectrometry showed that cinnamic acid was catabolized via benzaldehyde, benzoic acid, and catechol; p-coumaric acid was catabolized via p-hydroxybenzaldehyde, p-hydroxybenzoic acid, and protocatechuic acid; ferulic acid was catabolized via vanillin, vanillic acid, and protocatechuic acid. When vanillin was used as the initial growth substrate, it was catabolized via vanillic acid, guaiacol, and catechol. The inducible ring-cleavage dioxygenases catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase were detected with an oxygen electrode in cell-free extracts of cultures grown in media with aromatic growth substrates and yeast extract.     

Interaction of reactive and inert chemicals in the presence of oxidoreductases: Reaction of the herbicide bentazon and its metabolites with humic monomers

The herbicide bentazon (3-isopropyl-1H-2,1,3-benzothiadiazine-4(3 H)-one-2,2-dioxide), a relatively inert chemical, and some of its metabolites were incubated with a laccase or a peroxidase in the presence or absence of humic monomers to evaluate the incorporation of the herbicide and its metabolites into humic material by oxidative enzymes. Guaiacol and ferulic acid were used as representative electron donor co-substrates in most of the oxidative coupling reactions. Bentazon and its metabolites, with the exception of hydroxy metabolites, underwent little or no transformation by the two enzymes in the absence of guaiacol and ferulic acid,but in the presence of these co-substrates transformation occurred. The reaction of bentazon with guaiacol in the presence of the laccase or a peroxidase was almost complete in30 min. 6-Hydroxy- and 8-hydroxy-bentazon were completely transformed by each enzyme both with and with out co-substrates. At pH 3.0 and in the presence of laccase and guaiacol, the concentrations of bentazon and its metabolites2-amino-N-isopropyl-benzamide (AIBA), des-isopropyl-bentazon and 8-chloro-bentazon decreased by 27, 57, 20 and 4%,respectively. The corresponding levels of transformation with peroxidase at pH 3.0 were 9, 70, 30 and 5%, respectively. The extent of transformation decreased with increasing pH. At low pH, the hydroxy-bentazons were completely transformed,followed by (in order of percentage transformation) AIBA,des-isopropyl-bentazon, bentazon and 8-chloro-bentazon. Transformation of bentazon by the laccase increased with increasing guaiacol concentration. In the presence of the peroxidase, the most effective co-substrates for transformation of bentazon were (in decreasing order) catechol, vanillicacid, protocatechuic acid, syring aldehyde and caffeic acid,while in the presence of the laccase, catechol was most effective, followed by caffeic acid, protocatechuic acid and syringaldehyde. This revised version was published online in August 2006 with corrections to the Cover Date.