Degradation of nonphenolic lignin by the laccase/1-hydroxybenzotriazole system

Srebotnik, Ewald; Hammel, Kenneth E.

doi:10.1016/s0168-1656(00)00303-5

Cited by 141 publications

(91 citation statements)

References 22 publications

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“…Laccases are monomeric glycoproteins (23) that catalyze the fourelectron reduction of oxygen to water with one-electron oxidation of substrate without producing peroxide (27,28). Important applications for laccase are pulp bleaching and delignification for the paper industry (4,7,10,53) and biosensors for phenols or oxygen (22,36). Degradation of polycyclic aromatic hydrocarbons (29,39) and the cathodic reaction in biofuel cells (31) are also potential applications.…”

Section: Et Al Demonstrated Expression Of Laccase Frommentioning

confidence: 99%

Functional Expression of a Fungal Laccase in Saccharomyces cerevisiae by Directed Evolution

Bülter

Alcalde

Sieber

et al. 2003

Appl Environ Microbiol

257

170

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Laccase from Myceliophthora thermophila (MtL) was expressed in functional form in Saccharomyces cerevisiae. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together with a 22-fold increase in k cat , the total activity was enhanced 170-fold. Specific activities of MtL mutants toward 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations. The most effective mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host. The C terminus is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold. Mutations accumulated during nine generations of evolution for higher activity decreased enzyme stability. Screening for improved stability in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity. The molecular mass of MtL expressed in S. cerevisiae is 30% higher than that of the same enzyme expressed in M. thermophila (110 kDa versus 85 kDa). Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible for this increase. This S. cerevisiae expression system makes MtL available for functional tailoring by directed evolution.Directed evolution by random mutagenesis and recombination followed by screening or selection is a valuable tool for the engineering of enzymes (2,3,16,61). Functional gene expression in a suitable host is a prerequisite for directed evolution. Considering transformation efficiency, stability of plasmid DNA, and growth rate, Escherichia coli and Saccharomyces cerevisiae are best suited for these experiments. Heterologous expression in these hosts, however, is often limited by differences in the expression systems from the native organism (50). Different codon usage, missing chaperones, and posttranslational modifications such as disulfide bridges or glycosylation can all cause low expression levels and misfolded proteins that are degraded or driven into inclusion bodies (20). Finding the bottlenecks of a specific expression system requires consideration of many possibilities whose impact is hard to predict. Some incompatibilities between the expressed gene and heterologous host, such as codon usage or the recognition of signal sequences, can be overcome by changing the gene sequence. Thus, achieving functional expression is a good target for directed evolution (13,42,43).Laccases, like other ligninolytic enzymes, are notoriously difficult to express in nonfungal systems. The laccase from Myceliophthora thermophila (MtL) used in this work was previously expressed only in Aspergillus oryzae (6). Expression in S. cerevisiae has been reported for other laccase genes (11,33,34,60). Kojima et al. demonstrated expression of...

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Section: Et Al Demonstrated Expression Of Laccase Frommentioning

confidence: 99%

Functional Expression of a Fungal Laccase in Saccharomyces cerevisiae by Directed Evolution

Bülter

Alcalde

Sieber

et al. 2003

Appl Environ Microbiol

257

170

View full text Add to dashboard Cite

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“…The nature of these wood constituents is still unknown. As already mentioned, lipids comprising unsaturated fatty acids (22,23,24), but also thiols (4,43) or aromatic mediators, which have been shown to expand laccase activities in vitro (2,42), should be taken into consideration.…”

Section: Discussionmentioning

confidence: 99%

Conversion of Milled Pine Wood by Manganese Peroxidase from Phlebia radiata

Hofrichter

Lundell

Hatakka

2001

Appl Environ Microbiol

103

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“…The introduction of the laccase-mediator concept, based on the use of low-molecular weight redox mediators that generate stable radicals, has enlarged the potential of laccases in different applications including the paper pulp industry [10]. These mediators enhance the oxidative capabilities of the enzyme enabling oxidation of non-phenolic lignin [11]. During last decade, much research has focused on the laccase-mediator system for pulp bleaching including understanding the action mechanisms [12][13][14] and description of more suitable mediators [15][16][17][18].…”

Section: Introductionmentioning

confidence: 99%

Efficient bleaching of non-wood high-quality paper pulp using laccase-mediator system

Camarero

García

Vidal

et al. 2004

Enzyme and Microbial Technology

176

106

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High-quality flax pulp was bleached in a totally-chlorine-free (TCF) sequence using a laccase-mediator system. Three fungal laccases (from Pycnoporus cinnabarinus, Trametes versicolor and Pleurotus eryngii) and two mediators, 2,2 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and 1-hydroxybenzotriazole (HBT), were compared. P. cinnabarinus and T. versicolor laccases in the presence of HBT gave the best results in terms of high brightness and low lignin content (kappa number). The former laccase also resulted in the best preservation of cellulose and the largest removal of residual lignin as revealed by analytical pyrolysis, and was selected for subsequent TCF bleaching. Up to 90% delignification and strong brightness increase were attained after a laccase-mediator treatment followed by H 2 O 2 bleaching. This TCF sequence was further improved by applying H 2 O 2 under pressurized O 2 . In this way, we obtained up to 82% ISO brightness (compared with 37% in the initial pulp, and 60% in the peroxide-bleached control) and very low kappa number (near 1). Good results were also found when the laccase-mediator treatment was performed in a bioreactor under pressurized oxygen. The pulp properties obtained, which could not be attained by conventional TCF bleaching of flax pulp, demonstrate the feasibility of enzymatic bleaching to substitute chlorine-containing reagents in manufacturing of these high-price paper pulps.

show abstract

Degradation of nonphenolic lignin by the laccase/1-hydroxybenzotriazole system

Cited by 141 publications

References 22 publications

Functional Expression of a Fungal Laccase in Saccharomyces cerevisiae by Directed Evolution

Functional Expression of a Fungal Laccase in Saccharomyces cerevisiae by Directed Evolution

Conversion of Milled Pine Wood by Manganese Peroxidase from Phlebia radiata

Efficient bleaching of non-wood high-quality paper pulp using laccase-mediator system

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