Commercial production of laccase in plants

Details Commercial production of laccase in plants Commercial production of laccase in plants

2001-03-12

2004-10-05

Mehta, Ashwin (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C800S298000, C800S295000, C800S287000, C800S320100, C800S320000, C800S317000, C800S288000, C435S069100, C435S468000

Reexamination Certificate

active

06800792

ABSTRACT:

BACKGROUND OF THE INVENTION
Laccase is an enzyme that is a blue copper oxidase. It is believed to have been first obtained from the Japanese tree Rhus venicifera. Yshida, H., Zur Chemie des urushi-Firniss,
J. Chem. Soc
. (Tokyo) 43, 472, 1883. It has since been found in a number of higher plants, but not at high levels. The main source of laccase for commercial purposes is fungi. White rot fungi Phanerochaete chrysosporium and Trametes (Coriolus) versicolor are common commercial sources of the enzyme. Other fungi producing laccase include Polyporus, Pleurotus, Philota, Neurospora, Podospora and Aspergillus.
The enzyme has a number of uses. Examples include catalyzing the oxidation of compounds such as o,p-diphenols, aminophenols, polyphenols, polyamines and inorganic ions. (See, e.g. Yaropolov et al., “Laccase Properties, Catalytic Mechanism, and Applicability”
Applied Biochemistry and Biotechnology
49:257-280 (1994)). Its' use as a marker enzyme in enzyme immunoassay (EIA) has also been discussed, as well as its' use in oxidation of steroids and synthesis of vinblastine, a cytostatic compound used in treating malignant diseases.
The most common use of laccase, however, is in connection with the paper and pulp industry. Lignin is a rigid organic polymer and harsh physicochemical conditions must be used to attack or modify the substance. One answer in the search for means to break down lignin was found in the white rot fungi which can naturally destroy lignin, using laccase. In plants, laccase is localized in woody tissues and cell walls of herbaceous species and it is believed it participates in lignin biosynthesis. It is involved in breaking down lignin as well as creating lignin polymers. It is also especially useful as a “biological glue” when manufacturing glued wood products. Such products include construction and industrial plywood, oriented strand board, particleboard and medium density fiberboard.
Currently, the adhesive used is either a urea-formaldehyde type or a phenol-formaldehyde resin. There are disadvantages associated with use of formaldehyde in producing such products. Processing and end use monitoring are required as the levels of formaldehyde cannot exceed certain controls. Thus, there has been considerable interest in using such natural alternatives as laccase. It is reported that more than 1.2 million metric tons of adhesive resin solids are used to bond glued wood products in the United States. Which adhesive is used is driven by cost per unit of production, process compatibility and end-use durability. See, “Technical and Market Opportunities for Glued Wood Products”
Adhesive Age
May 31, 1996 V39, N6 p.609.
An example of such a process is described by Kharazipour et al in U.S. Pat. No. 5,505,772 and by Olesen et al. at U.S. Pat. No. 5,618,482. In general, fibers and chips from wood or wood-like materials are defibrated by mechanical, steam, or other process. Laccase is then brought into contact with the material in a solution which may contain various auxiliary elements. Since laccase is a large molecule, a mediator may be utilized to aid the enzyme in penetrating the wood and may be added to the solution. The mix is incubated and may then be shaped into formed boards.
A problem with using laccase produced by white-rot fungi, however, is that it is produced in relatively low amounts. Saloheimo, M. and Niku-Paavola, M-L. “Heterologous Production of a Ligninolytic Enzyme: Expression of the
Phlebia radiata
Laccase gene in
Trichoderma reesei” Bio/Technology
9:987-990 (1991). Native expression is described at about 10,000 U/liter from
Trametes versicolor
after induction, which is about 220-250 mg/liter if all was laccase I, and about 65-75 mg/liter if the laccase was laccase II. Bourbonnais et al.
Appl. Environ. Microb
. 61, no.5 pp.1876-1880 (May 1995). Other experiments have shown expression in
Myceliophthora thermophila
and in
Aspergillus oryzae
was at about 5 mg/liter. Measurements by the inventors reflect that the natural laccase expression in plants is about less than 0.001% of soluble plant protein. Attempts by Saloheimo and Niku-Paavola to improve on these levels by using heterologous expression yielded 20 mg/l secreted active laccase. Berka et al. noted that expression levels are too low for commercial purposes. Berka et al.
Applied and Environmental Microbiology
p.3151-3157 (August 1997). While others have attempted to introduce laccase-encoding nucleotide sequences into plants for the purpose of changing the lignin content of the plant in WO 98/11205 and WO97/45549, they do not teach production of laccase at commercially acceptable levels for extraction and use.
The inventors have discovered that it is possible to produce commercially acceptable quantities of laccase in plants. This results in a considerable decrease in cost of producing the enzyme. Thus it is an object of the invention to produce laccase in plants at commercially useful levels.
Finally, also provided are improvements on methods of transforming these and other plants using Agrobacterium. Modifications to selection of the bacterial strain used, and of processing the strain are provided.
These and further objectives will become apparent from the description.
All references cited herein are incorporated herein by reference.
SUMMARY OF THE INVENTION
Plants and a process of using them is described in which commercial levels of laccase are produced in plants. Improvements to Agrobacterium-mediated transformation processes are also provided.


REFERENCES:
patent: 5480801 (1996-01-01), Wahleithner et al.
patent: 5618482 (1997-04-01), Olesen et al.
patent: 5667531 (1997-09-01), Yaver et al.
patent: 5693506 (1997-12-01), Rodriguez
patent: 5770418 (1998-06-01), Yaver et al.
patent: 9752666 (1999-07-01), None
patent: WO9709431 (1997-03-01), None
patent: WO9745549 (1997-12-01), None
patent: WO9811205 (1998-03-01), None
patent: WO0005381 (2000-02-01), None
Broun et al. Science, vol. 282, pp. 131-133, 1998.*
Lazar et al. Molecular and Cellular Biology, vol. 8(3), pp. 1247-1252, 1988.*
Becker et al. Ann. Proc. Cytochem Soci. of Europe, pp. 325-331, 1993.*
Ong, Edgar, et al.; “Cloning and sequence analysis of two laccase complementary DNA's from the ligninolytic basidiomyceteTrametes versicolor.”, Gene;196:113-119 (1997).
Crestini, Claudia, et al.; “The early biodegeneration pathways of residual kraft lingin model compounds with laccase.”, ISWPC (1997).
Bourbonnais, Robert, et al.; “Enzymatic delignification of craft pulp using laccase and a mediator”, Tappi Journal, vol. 79: No. 6, Jun. (1996) p. 199-204.
Saloheimo, Markku, et al.; “Heterologous production of a ligninolytic enzyme: expression of thePhlebia radiatalaccase gene inTrichoderma reesei”, Bio/Thechnology vol. 9, Oct. 1991 p. 987-990.
Bourbonnais, Robert, et al.; “Lignin Oxidation by Laccase Isozymes fromTrametes versicolorand role of the Mediator 2,2′ -Azinobis(3-Ethylbenzthiazoline-6-Sulfonate) in Kraft Lignin Depolymerization”, Applied and Environmental Microbiology, May 1995, p. 1876-1880; vol. 61, No. 5.
Berka, Randy, et al.; “Characterization of the gene encoding an extracellular laccase ofMyceliophthora thermophilaand analysis of the recombinant enzyme expressed inAspergillus oryzae”, Applied and Environmental Microbiology, Aug. 1997, p. 3151-3157; vol. 63, No. 8.
Call, H.P., et al.; “History, overview and applications of mediated lignolytic systems, especially laccase-mediator systems (Lignozym-process)”, Journal of Biotechnology 53 (1997) P. 163-202.
Yaropolov, A.I., et al.; “Laccase: Properties, Catalytic Machanism, and Applicability”, Applied Biochemistry and Biotechnology vol. 49, 1994 p. 257-280.
Amann, et al.; “The Lignozym Process—Coming closer to the Mill”, 1997 ISWPC.
“Technical and Market Opportunities for glued wood products”, Adhesives Age May 31, 1996 p.6-9.
Archibald, F.S., et al.; “Kraft pulp bleaching and delignification byTrametes versicolor.”, Journal of Biotechnology, vol. 53 (1997) p. 215-236.
Solomon, Edward, et al.; “Multicopper Oxidases and Oxygenases”, Chem. Rev 1996,

Affiliated with

Also associated with

Rating

Commercial production of laccase in plants does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Commercial production of laccase in plants, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Commercial production of laccase in plants will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3306519