Kaichang Li

Redox Chemistry in Laccase-Catalyzed Oxidation of N-Hydroxy Compounds

ABSTRACT 1-Hydroxybenzotriazole, violuric acid, andN-hydroxyacetanilide are three N-OH compounds capable of mediating a range of laccase-catalyzed biotransformations, such as paper pulp delignification and degradation of polycyclic hydrocarbons. The mechanism of their enzymatic oxidation was studied with seven fungal laccases. The oxidation had a bell-shaped pH-activity profile with an optimal pH ranging from 4 to 7. The oxidation rate was found to be dependent on the redox potential difference between the N-OH substrate and laccase. A laccase with a higher redox potential or an N-OH compound with a lower redox potential tended to have a higher oxidation rate. Similar to the enzymatic oxidation of phenols, phenoxazines, phenothiazines, and other redox-active compounds, an “outer-sphere” type of single-electron transfer from the substrate to laccase and proton release are speculated to be involved in the rate-limiting step for N-OH oxidation.

Chemical Modification of Soy Protein for Wood Adhesives

Mussel protein is a strong and water-resistant adhesive, but is expensive and not readily available. Soy protein is inexpensive, abundant, and annually renewable, but suffers from low adhesive strengths and low water resistance of the bonded products. This study reveals that introducing a key functional group from the marine adhesive protein to soy protein converts the soy protein to a strong and water-resistant wood adhesive.

Purification and characterization of laccase from wood-degrading fungus Trichophyton rubrum LKY-7

A new wood-degrading fungus Trichophyton rubrum LKY-7 secretes a high level of laccase in a glucose-peptone liquid medium. The production of laccase by the fungus was barely induced by 2,5-xylidine. The laccase has been purified to homogeneity through three chromatography steps in an overall yield of 40%. The molecular mass of the purified laccase was about 65 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE). The purified laccase had the distinct blue color and the basic spectroscopic features of a typical blue laccase: two absorption maxima at 278 and 610 nm and a shoulder at 338 nm. The N-terminus of the laccase has been sequenced, revealing high homology to laccases from wood-degrading white-rot fungi such as Ceriporiopsis subvermispora, but little similarity to laccases from non-wood-degrading fungi such as Agaricus bisporus and Cryptococcus neoformans. The enzyme had a low redox potential of ∼0.5 V, yet it was one of the most active laccases in oxidizing a series of representative substrates/mediators. Compared with other fungal laccases, the laccase has a very low Km value with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as a substrate and a very high Km value with violuric acid as a substrate. The laccase had the isoelectric point of 4.0. The laccase had very acidic optimal pH values (3–4), although it was more stable at neutral pH than at acidic pH. The laccase oxidized hydroquinone faster than catechol and pyrogallol. The oxidation of tyrosine by the laccase was not detectable under the reaction conditions. The laccase was strongly inhibited by sodium azide and sodium fluoride.

Mechanistic studies of the oxidation of a non‐phenolic lignin model compound by the laccase/1‐hydroxybenzotriazole redox system

The use of a laccase‐mediator system is one of the promising possibilities for an environmentally benign pulp‐bleaching process. Thus a better understanding of the mechanism for the oxidation of lignin by a laccasemediator system would facilitate the commercialization of such a system for pulp bleaching. By using 1‐hydroxybenzotriazole (1‐HBT) as a mediator, it was found to be partly regenerated during the oxidation of a lignin dimer, 1‐(3, 4‐dimethoxyphenyl)‐2‐(2‐methoxyphenoxy)propan‐1, 3‐diol (I). A free radical of 1‐HBT, generated by laccase, was probably responsible for the oxidation of I. The free radical of 1‐HBT was, however, transformed to benzotriazole, which could not mediate the oxidation of I. The hypothesis that 1‐HBT acted as an activator or a co‐substrate for laccase was not confirmed. Active oxygen species such as hydroxyl free radical (HO•) and superoxide (O2•‐) were also found not to be involved in the oxidation of I.

Electricity generation from polyalcohols in single-chamber microbial fuel cells.

Six polyalcohols derived from lignocellulosic carbohydrates were investigated as carbon sources for electricity generation in single-chamber mediator-less microbial fuel cells (MFCs) for the first time. Electricity was directly generated from all polyalcohols tested, including pentitols (xylitol, arabitol, and ribitol) and hexitols (galactitol, mannitol, and sorbitol). Bacterial cultures initially enriched using acetate could be adapted to these substrates with varied adaptation times. The resultant maximum power density ranged from 1490+/-160 mW/m(2) to 2650+/-10 mW/m(2) at current densities between 0.58 mA/cm(2) and 0.78 mA/cm(2). Galactitol generated the highest maximum power density, while mannitol resulted in the lowest one. The estimated maximum voltage output at an external resistance of 120 Omega ranged between 0.24 V and 0.34 V with half saturation kinetic constants varied from 298 mg/L to 753 mg/L. The removal of chemical oxygen demand (COD) was above 91% for all polyalcohols except sorbitol (71%). Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene segments of the anode biofilms showed the influence of substrates (polyalcohols) on the anode microbial populations.

Investigation of the role of 3-hydroxyanthranilic acid in the degradation of lignin by white-rot fungus Pycnoporus cinnabarinus.

An aminophenol, 3-hydroxyanthranilic acid (3-HAA), has been proposed to play important roles in lignin degradation. Production of 3-HAA in Pycnoporus cinnabarinus was completely inhibited by a combination of tryptophan and S-(2-aminophenyl)-L-cysteine S,S-dioxide (APCD) while the fungus grew well and produced high amounts of laccase. The biosynthesis of 3-HAA is mainly through the metabolism of tryptophan in the kynurenine pathway. A minor pathway for 3-HAA synthesis is through the hydroxylation of anthranilic acid during the biosynthesis of tryptophan in the shikimic acid pathway. Through UV irradiation of wild-type P. cinnabarinus (WT-Pc) spores, a 3-HAA-less mutant was produced. Both WT-Pc, under the inhibitory culture condition, and the 3-HAA-less mutant were found to degrade lignin in unbleached kraft pulp as efficiently as the WT-Pc, which unambiguously demonstrated that 3-HAA does not play an important role in the fungal degradation of lignin.

Degradation of non-phenolic lignin by the white-rot fungus Pycnoporus cinnabarinus

Abstract. High-molecular-weight lignin was methylated with diazomethane. The lignin (i.e., phenolic lignin) and methylated lignin (i.e., non-phenolic lignin) were mixed with fully bleached softwood pulp. Degradation of the lignin preparations by the white rot fungus Pycnoporus cinnabarinus was studied. After a 3-month incubation with the fungus, over 40% of the non-phenolic lignin and about 70% the phenolic lignin were degraded. The presence of phenolic hydroxyl groups in lignin greatly enhanced the degradation rate of lignin. This study reveals that P. cinnabarinus, an exclusively laccase-producing fungus, is capable of oxidatively degrading both phenolic and non-phenolic lignins. The ability of the fungus to degrade non-phenolic lignin suggests that a laccase/mediator system is involved in the complete degradation of lignin. After the fungal degradation of lignins, the content of carboxylic acids substantially increased for both phenolic and non-phenolic lignins.

Investigation of chitosan–phenolics systems as wood adhesives

Chitosan-phenolics systems were investigated as wood adhesives. Adhesion between two pieces of wood veneer developed only when all three components-chitosan, a phenolic compound, and laccase-were present. For the adhesive systems containing a phenolic compound with only one phenolic hydroxyl group, adhesive strengths were highly dependent upon the chemical structures of phenolic compounds used in the system and the relative oxidation rates of the phenolic compounds by laccase. The adhesive strengths were also directly related to the viscosity of the adhesive systems. However, for the adhesive systems containing a phenolic compound with two or three phenolic hydroxyl groups adjacent to each other, no correlations among adhesive strengths, relative oxidation rates of the phenolic compounds by laccase, and viscosities were observed. The adhesion mechanisms of these chitosan-phenolics systems were proposed to be similar to those of mussel adhesive proteins.

Laccase-less mutants of the white-rot fungus Pycnoporus cinnabarinus cannot delignify kraft pulp

The white-rot fungus Pycnoporus cinnabarinus produces only one isoform of laccase and traces of a peroxidase which is neither manganese peroxidase (MnP) nor lignin peroxidase (LiP). To test the impact of laccase on pulp bleaching we performed studies with P. cinnabarinus wild type (WT) and its laccase-less mutants on softwood kraft pulp (SWKP) in both static and shake flask cultures. In the static pulp cultures (at 20% pulp consistency in water), after 25 days incubation, P. cinnabarinus wild type increased the pulp brightness from 35 to 59% ISO, and decreased the kappa number from 15 to 5.7. However P. cinnabarinus laccase-less mutants 51 and 85 increased the brightness to only 40.6 and 40.7, and decreased the kappa number to 12 and 13.4, respectively. In the shake flask cultures with pulp suspended in water (at 2% consistency), the wild type increased pulp brightness from 38 to 52.4% ISO, and decreased the kappa number from 12 to 5.9. The laccase-less mutants 51, and 85 increased the brightness only to 43.8 and 40, and decrease the kappa number to 9.2 and 10.6, respectively. These results showed that both in the static and shaking cultures, the laccase-less mutants were very inefficient or unable to bleach pulp which is a clear indication of the importance of laccase for lignin degradation and pulp bleaching by P. cinnabarinus.

Investigation of hydroxamic acids as laccase-mediators for pulp bleaching.

Abstract A number of hydroxamic acids have been synthesized and investigated as laccase-mediators for pulp bleaching. As compared with N-hydroxyacetanilide (NHA), one of the most effective laccase-mediators reported so far, N-(4-cyanophenyl)acetohydroxamic acid (NCPA), resulted in the highest brightness and lowest kappa number of hardwood kraft pulp of all the laccase-mediators studied. The bleaching efficacy of a laccase/7-cyano-4-hydroxy-2H-1,4-benzoxazin-3-one system was also comparable with that of a laccase/NHA system. A laccase/NCPA system was further studied for the bleaching of unbleached softwood kraft pulp. The effects of pulp consistency, laccase dosage, NCPA dosage, incubation time, and oxygen pressure on the bleaching efficacy of a laccase/NCPA system were studied.