Mechanisms of redox interactions between lignin peroxidase and cellobiose:quinone oxidoreductase.

The mechanism of redox interactions between the heme-enzyme, lignin peroxidase (LiP), and the FAD-enzyme, cellobiose:quinone oxidoreductase (CBQ) (EC 1.1.5.1), was investigated under various conditions. Veratryl alcohol oxidation by LiP was inhibited by CBQ in the presence of cellobiose. Lineweaver-Burk plots at various CBQ concentrations suggest that this inhibition is non-competitive. The oxidation rate of the reduced CBQ (FADH2) by LiP plus H2O2 increased significantly only in the presence of veratryl alcohol. Furthermore, the cation radical derived from 1,2,4,5-tetramethoxybenzene was reduced by CBQ in the presence of cellobiose. It is concluded from these results that CBQ can reduce aromatic cation radicals and that veratryl alcohol acts as a radical mediator of the redox interactions between LiP and CBQ.

Laccase is essential for lignin degradation by the white-rot fungus Pycnoporus cinnabarinus.

The white-rot fungus, Pycnoporus cinnabarinus, provides an excellent model organism to elucidate the controversial role of laccase in lignin degradation. P. cinnabarinus produces laccase in one isoform as the predominant phenoloxidase in ligninolytic cultures, and neither LiP nor MnP are secreted. Yet, P. cinnabarinus degrades lignin very efficiently. In the present work, we show that laccase-less mutants of P. cinnabarinus were greatly reduced in their ability to metabolize 14C ring-labeled DHP. However, 14CO2 evolution in these mutant cultures could be restored to levels comparable to those of the wild-type cultures by addition of purified P. cinnabarinus laccase. This clearly indicates that laccase is absolutely essential for lignin degradation by P. cinnabarinus.

Cellobiose oxidase from Phanerochaete chrysosporium. Stopped-flow spectrophotometric analysis of pH-dependent reduction.

Cellobiose oxidase (CBO) from Phanerochaete chrysosporium can utilize dichlorphenol-indophenol (Cl2Ind) and cytochrome c as effective electron acceptors for the oxidation of cellobiose. However, the pH dependencies of activity for these electron acceptors are significantly different. Both compounds act as effective electron acceptors at pH 4.2, whereas only dichlorophenol-indophenol is active at pH 5.9. To explain this discrepancy, the pH dependencies of the reduction rates of FAD and heme, respectively, in CBO by cellobiose have been investigated by stopped-flow spectrophotometry. Both FAD and heme are reduced with a high rate constant at pH 4.2. In contrast, at pH 5.9, only FAD reduction is fast, while the reduction of the heme is extremely slow. As a conclusion, the reduction of cytochrome c by CBO is dependent on heme, which functions at a lower pH range compared to reduction of FAD.

Release of the FAD domain from cellobiose oxidase by proteases from cellulolytic cultures of Phanerochaete chrysosporium.

Evidence has previously suggested that cellobiose:quinone oxidoreductase (CBQ) in cellulolytic cultures of Phanerochaete chrysosporium might be produced from cellobiose oxidase (CBO) by proteolytic cleavage. This study demonstrates that the ratio of CBO activity to (CBO + CBQ) activity declines with decreasing culture pH, while protease activity increases. Furthermore, we demonstrate that endogenous P. chrysosporium proteases can only cleave CBO when the enzyme is bound to cellulose. This is the first demonstration that the proteases produced in cellulolytic cultures of P. chrysosporium can release the FAD domain from CBO.

Laccase-mediated formation of the phenoxazinone derivative, cinnabarinic acid.

The phenoxazinone chromophore occurs in a variety of biological systems, including numerous pigments and certain antibiotics. It also appears to form as part of a mechanism to protect mammalian tissue from oxidative damage. During cultivation of the basidiomycete, Pycnoporus cinnabarinus, a red pigment was observed to accumulate in the culture medium. It was identified as the phenoxazinone derivative, cinnabarinic acid (CA). Laccase was the predominant extracellular phenoloxidase activity in P. cinnabarinus cultures. In vitro studies showed that CA was formed after oxidation of the precursor, 3-hydroxyanthranilic acid (3-HAA), by laccases. Moreover, oxidative coupling of 3-HAA to form CA was also demonstrated for the mammalian counterpart of laccase, the blue copper oxidase, ceruloplasmin.

A fungal metabolite mediates degradation of non-phenolic lignin structures and synthetic lignin by laccase.

Lignin peroxidase is generally considered to be a primary catalyst for oxidative depolymerization of lignin by white-rot fungi. However, some white-rot fungi lack lignin peroxidase. Instead, many produce laccase, even though the redox potentials of known laccases are too low to directly oxidize the non-phenolic components of lignin. Pycnoporus cinnabarinus is one example of a laccase-producing fungus that degrades lignin very efficiently. To overcome the redox potential barrier, P. cinnabarinus produces a metabolite, 3-hydroxyanthranilate that can mediate the oxidation of how non-phenolic substrates by laccase. This is the first description of how laccase might function in a biological system for the complete depolymerization of lignin.

Advances in microbial delignification.

Microbial delignification is a new field of applied research. The progress will therefore run parallel to the development of new basic knowledge on the physiological demands of white-rot fungi to degrade lignin and on new knowledge on enzyme mechanisms involved in lignin degradation. In the last few years both basic and applied research on microbial conversion of lignocellulosic materials have vastly expanded. In certain areas, such as microbial delignification, considerable progress has recently been made. Basidiospores from Sporotrichum pulverulentum and some CEL(-) mutants have been obtained. Crossing of mycelium from single basidiospore cultures of wild-type and CEL(-) mutants will eventually give rise to much better CEL(-) mutants than those which have been used in the past. An understanding of which enzymes are the most important for lignin degradation to take place is also beginning to develop. This review discusses present knowledge and future possibilities in this field.

Microorganisms as an alternative source of protein.

Demand for human food and animal feed proteins from nonconventional sources has increased, particularly in developing countries. Microbial protein is one such source. It is desirable because it is amenable to controlled intensive cultivation and is less dependent on variations in climate, weather, and soil. Microbial proteins must be evaluated for nutritive value, safety, and economic considerations before mass production is undertaken.

Concluding remarks: where do we stand and where are we going? Lignin biodegradation and practical utilization.

The progress made in the efforts to develop biotechnology based on lignocellulosic materials is discussed in some detail. It is appreciated that biotechnical conversion of lignocellulosics means production of inexpensive products on a large scale and is therefore a more difficult task than development of biotechnology in medicine and pharmacology, i.e. production of expensive products on a small scale. However, the massive efforts devoted over the past few decades to a better understanding of the enzyme mechanisms involved in degradation of wood components have not been in vain. The literature base so essential for successful application of biotechnology to conversion of lignocellulosic materials is now in place. The article presents a summary of our knowledge of the enzyme mechanisms involved in the degradation of the three main lignocellulosic components. It also tries to evaluate in which important areas we lack the necessary information to apply biotechnology, particularly in the pulp and paper industry.

Production of succinate from glucose, cellobiose, and various cellulosic materials by the ruminal anaerobic bacteria Fibrobacter succinogenes and Ruminococcus flavefaciens.

The production of organic acids by two anaerobic ruminal bacteria Fibrobacter succinogenes S85 and Ruminococcus flavefaciens FD-1, was compared with glucose, cellobiose, microcrystalline cellulose, Walseth cellulose (acid swollen cellulose), pulped paper, and steam-exploded yellow poplar as substrates. The major end product produced by F. succinogenes from each of these substrates was succinate (69.5-83%), the principal secondary product was acetate (16-30.5%). Maximum succinate productivity ranged from 14.1 mg/L.h for steam-exploded yellow Poplar to 59.7 mg/L.h for pulped paper. For R. flavefaciens, the major end product from cellobiose, microcrystalline cellulose, and acid-swollen Walseth cellulose was acetate (39-46%), pulped paper and steam-exploded yellow poplar yielded succinate (42-54%) as the major product. Maximum succinate productivity by R. flavefaciens ranged from 9.21 mg/L.h for cellobiose to 43.1 mg/L.h for pulped paper. In general, much less succinate was produced at a lower maximum productivity by R. flavefaciens than by F. succinogenes under similar fermentation conditions. The maximum succinate productivities by these two organisms are comparable to the previously reported value of 59 mg/L.h for Anderobiospirillum succiniciproducens grown on glucose and corn steep liquor.

From the Big Bang to sustainable societies.

A series of events in the history of cosmos has created the prerequisites for life on Earth. With respect to matter, the earth is a closed system. However, it receives light from the sun and emits infrared radiation into space. The difference in thermodynamic potential between these two flows has provided the physical conditions for self-organization. The transformation of lifeless matter into modern life forms, with their high degree of order and complexity, has occurred in the context of the earth's natural cycles, including the water cycle and the biochemical cycles between plants and animals. Primary production units, the cells of green plants, can use the thermodynamic potential of the energy balance in a very direct way, i.e. in photosynthesis. Plant cells are unique in their ability to synthesize more structure than is broken down elsewhere in the biosphere. The perpetuation of this process requires the recycling of wastes. However, modern industrial societies are obsessed with the supply side, ignoring the principle of matter's conservation and neglecting to plan for the entire material flow. As a result there has been an accumulation of both visible and invisible garbage (pollution), which disturbs the biosphere and reduces stocks of natural resources. Furthermore, due to complexity and delay mechanisms, we usually cannot predict time parameters for the resulting socio-economic consequences or the development of disease. To continue along this path of folly is not compatible with the maintenance of wealth, nor with the health of humans or the biosphere. Rather than address the millions of environmental problems one at a time, we need to approach them at the systemic level. It is essential to convert to human life-styles and forms of societal organization that are based on cyclic processes compatible with the earth's natural cycles. The challenge to the developed countries is not only to decrease their own emissions of pollutants but to develop the cyclic technology and life styles needed by the entire human community.

Enzymic hydrolysis of lignocellulosic materials: I. Models for the hydrolysis process--a theoretical study.

At the end of an enzymic hydrolysis process involving a solid lignocellulosic substrate, enzymes are found both in solution and absorbed to the substrate residue. Removal of residue from the system will result in loss of some of the enzymes, the extent of which will depend on the design of the process. To minimize enzyme loss, a study has been conducted in which six process models have been formulated and an enzyme loss function derived for each model based on the total amount of enzymes lost through residue removal. Model 1 is a reference model, characterized by an uninterrupted hydrolysis throughout the entire hydrolysis period. The residue is then washed in order to recover both sugar and adsorbed enzymes before the residue is discarded. Models 2-6 are all characterized by the removal of hydrolysate three times during the process, recirculation of dissolved and adsorbed enzymes to various points in the process and selection of a stage at which the residue is removed. The following conclusions could be drawn from the derived enzyme loss functions: Increased enzyme adsorption leads to increased enzyme loss.The enzyme loss decreases if the solid residue is removed late in the process.Both adsorbed and dissolved enzymes should be introduced at the starting point of the process. This is particularly important for dissolved enzymes. Three models were chosen for experimental studies, which are reported in a second, accompanying article. The experimental results obtained are compared with the theoretical study reported here.