This project concerns the structure, functions and potential applications of two distinct groups of plant enzymes that catalyse the oxidation of phenolic compounds: oxidases (especially laccases) and peroxidases. Typical reactions catalysed by these two groups of enzymes can be summarised as follows:
oxidases (laccases): 4 F-H + 02 >> F-F + 2 H20
peroxidases: 2 F-H + H202 >> F-F + 2 H20
where F represents an aromatic substance such as the precursors of lignin in wood or certain coloured pollutants. Pairs of aromatic molecules are thereby cross linked to give a wide diversity of products. The oxidases contain copper and the peroxidases contain iron. For this and many other reasons they are quite distinct groups of enzymes, although the end products of their action may be identical. It is therefore an important challenge, and the basic aim of the present collaborative project, to define their respective structures, enzymological properties, expression, biological roles and applicability in industry. These aims are being met through implementation of a research programme which has been divided into a number of tasks as described below.
Task 1 is the purification and characterisation of specific isoenzymes of laccase and peroxidase, the cloning of the corresponding genes and a study of their expression. Enzymes studied will be:
I. Arabidopsis peroxidase isoenzymes. Arabidopsis is an appropriate species as it is related to horseradish, traditionally the subject of peroxidase studies.
II. Xylem-specific isoperoxidases from poplar. This source is likely to be enriched in those isoenzymes principally involved in lignin biosynthesis.
III. Laccases from poplar and Arabidopsis.
Task 2 is a study of the catalytic properties of the purified isoenzymes. The results will provide information on the reactions catalysed so that the enzymes physiological roles and potential for exploitation can be assessed. Substrate specificity and reaction products will be determined. Spectroscopic thermodynamic and electrochemical studies will probe the catalytic mechanisms with special reference to copper centers.
Task 3 is the production of transgenic plants that over - or under - express specific isoperoxidases and laccases. Transformants with altered phenotype could be of value agriculturally and also for our basic understanding of the in-vivo roles of the enzymes. "Molecular farming" - the production of large amounts of specific isoenzymes in a stable form in the plants storage compartment - could be a valuable source of the interesting enzymes.
Task 4 is the testing of large quantities of specific laccases and peroxidases for industrial applications. The isoenzymes will be produced in bulk in microbial and/or higher plant heterologous systems. The ability of these enzymes to catalyse the degradation of aromatic pollutants in waste water and the decoloration of unwanted dyes, and their use in immunoassays will be explored. The stability of the enzymes under conditions encountered industrially will be tested. It is hoped that this work will allow European industry to exploit these two important but poorly understood enzymes.
Studies of laccase have resulted in discovery of a novel 90 kDa oxidase from poplar cell walls which has been isolated and partially characterised and found capable of oxidatively polymerising coniferyl alcohol. Cell cultures have been obtained from Arabidopsis and used as a source of peroxidase, which has been purified and partially sequenced.
The only published laccase cDNA sequence (from sycamore) has been compared with new laccase cDNAs from poplar and tobacco. Sequence analysis indicated a relatively high degree of homology between the laccase from sycamore, poplar and tobacco. Similar work with peroxidase has resulted in a cDNA clone from poplar which encodes a xylem specific isoperoxidase (Pxp). Work is under way to produce a construct containing a 'tag' sequence, which will be used in studies of the timing and location of expression of Pxp. Three new cDNAs coding for Arabidopsis isoperoxidases have been obtained. One of these is 95% identical to horseradish peroxidase (HRP), another is able to hybridise with a presumptive lignifying and suberising peroxidase from tobacco and a third is 88% identical to HRP. Clones for five different Arabidopsis peroxidases have also been obtained.
Peroxidase expression studies:
It has been found that in transgenic poplars defective in the penultimate enzymic step of lignin biosynthesis (cinnamyl alcohol dehydrogenase), one group of acidic peroxidases was strongly reduced in activity, suggesting the involvement of this group in lignin biosynthesis. So far, the polypeptides of several Arabidopsis isoperoxidases have been expressed in high yields in the bacteria Erischia coli. A basic Arabidopsis isoperoxidase has also been expressed in baculovirus.
A robust, HPLC based protocol for the determination of reaction rates of purified peroxidases and laccases against a wide battery of cell wall related electron donor substrates has been established. In addition, conditions have been defined for the production of sufficient Rigidoporus lignosus laccase to enable physicochemical characterisation, including investigations of the copper (catalytic) sites, and the protein's redox properties.
The work previously described (Item NFI/098), dealing with phenol oxidases (especially laccases) and peroxidases from higher plants, has continued as anticipated in the original proposal. The nine participating laboratories have made good progress in meeting the main objectives. Several of the aspects reported as aims in the last year report have, essentially, been completed enabling new goals to be approached. With the finishing of previous work and the initiation of new areas of study, there are seven main areas of endeavour to report, as follows.
Work and Achievements
a. Protein discovery. The group has now essentially completed the isolation and purification of peroxidases, laccases and a newly discovered laccase like protein.
b. Transformation of plants. The group has begun the construction of antisense transformant plants carrying chimaeric genes including the laccase sequence and peroxidase.
c. Expression studies. Studies of the expression of peroxidases and of the functionally related enzyme CAD have been initiated.
d. Enzymology. Exploration of the enzymological characteristics of the oxidases and peroxidases, especially of the metal centres of laccase, and the chemical structure of their natural substrates has continued.
e. Heterologous expression in E coli. Work has now successfully begun on expression and folding of recombinant plant peroxidases in E coli.
f. Large scale laccase production. The preparation and purification of Rigidoporus lignosus laccase has been achieved by one of the participating industrial laboratories.
g. Industrial applicability. Work has continued to explore the usefulness of laccases and peroxidases in industrial bleaching and in laboratory assay kits.
h. Cloning. Several novel peroxidases have been cloned, completely sequenced and expressed in a eukaryotic expression system.
The main accomplishments of the year have included (a) isolation and purification of peroxidases, laccases and the newly discovered laccase like proteins; (b) construction of antisense transformant plants carrying chimaeric genes including the laccase sequence and peroxidase; (c) study of the expression of peroxidases and the related enzyme CAD; (d) exploitation of the enzymological characteristics of the oxidases and peroxidases and the chemical structure of their natural substrates; (e) expression and folding of recombinant peroxidases in E. coli; (f) large scale preparation and purification of Rigidoporus lignosus laccase; (h) testing their usefulness in industrial processes and enzyme linked immunoassays (EIAs)
Laccases and peroxidases are enzymes involved in the oxidation of phenolic compounds. Such compounds fill many roles in plants. They are constituents of lignin, provide pigmentation and are involved in a number of protective mechanisms including the response to wounding and viral infection. In the agro-food industry they are important as the major cause of biological colour in sugar and starch crops. The mechanisms of colour formation in paper pulping is also, in part, similar. In effect, the breaking of cells or cell walls releases both substrate and enzymes, resulting in coloured products. This project aims to obtain a better understanding of these enzymes through isolation, characterisation and expression in fermentation systems, as well as to look at potential applications in effluent decolorisation and other industrial applications such as enzyme-linked immunoassays.
Funding SchemeCSC - Cost-sharing contracts
1017 K Copenhagen