Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1991-02-01
2002-11-26
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S183000, C435S171000, C435S254100, C435S071100
Reexamination Certificate
active
06485952
ABSTRACT:
BACKGROUND OF THE INVENTION
Enzymes for degrading aromatic compounds have potential commercial application in the pulp and paper industry, the production of fuels and chemicals from lignocellulose, the enhancement of livestock feeds, and the bioremediation of aromatic hazardous wastes.
Lignin is a complex polymer of phenyl propanoid units with a variety of interunit linkages forming a nonlinear, random structure. Lignin comprises 10-35% of the dry weight of lignocellulose-rich materials such as wood, straw, and corn stover. Lignin is resistant to biological destruction, although it is enzymatically degraded by various higher order fungi. In nature, the basidiomycetes that cause white-rot wood decay are major degraders of lignocellulose. White-rot fungi oxidize lignin completely to carbon dioxide. Extracellular enzyme complexes secreted by these fungi catalize oxidative reactions of the lignin structure. White-rot fungi have also been shown to oxidize and degrade a wide range of other aromatic structures including a variety of man-made, toxic aromatic compounds. The term “white-rot fungi” as used herein is intended to include fungi having enzymes capable of oxidizing and thereby degrading aromatic compounds.
There are an estimated 1700 species of white-rot fungi. However, research on enzymatic lignin degradation has concentrated on one organism:
Phanerochaete chrysosporium.
Lignin-degrading enzymes from this organism have been purified and characterized. A large volume of research literature describes processes for growing
P. chrysosporium
in liquid media for lignin degradation or production of lignin-degrading enzymes. The conventional production of lignin-degrading enzymes in liquid media occurs during secondary metabolism and is initiated by nitrogen or glucose starvation. For instance, in U.S. Pat. No. 4,554,075, Chang et al. describe a process for growing white-rot fungi by carrying growth into secondary metabolism wherein nitrogen starvation occurs. See also Ming Tien in an article in
CRC Critical Reviews in Microbiology,
titled “Properties of Ligninase From Phanerochaete Chrysosporium and Their Possible Applications”, Volume 15, Issue 2 (1987) at p. 143 and U.S. Pat. No. 4,891,230 to Aust et al.
The slow growth rates and low cell mass production associated with starved cultures results in long growth times and low yields thus making this impractical for commercially producing enzymes for pretreating wood pulp in paper making processes, for in situ treatment of toxic waste, or for enhancing lignocellulose for livestock feed. Tien notes on page 144 in the same article listed above that scale-up from liquid culture grown in flasks has proven difficult.
To overcome the low cell mass production, the art has suggested growing several species of white-rot fungi using solid culture media in solid state reactors. In these instances, the fungus grows on a substrate of moist solid lignocellulose-containing materials. Straw, several types of wood, and milled corn cob have been disclosed as substrates in the literature. These materials have been selected as culture substrates primarily because they are typical of the materials degraded by the white-rot fungi in nature. They have a relatively high lignin content of 10-35%, low nitrogen levels, and limited access to cellulose as a carbon source. White-rot fungi can be grown in such solid-state cultures, but obtaining lignin-degrading enzymes in cell and solids free extracts of such cultures has proved an elusive task as the enzyme activity remains bound to the substrate.
Several patents as well as other literature disclose processes for preparing ligninase in solid cultures including U.S. Pat. No. 4,711,787 to Odakra, which describes using okra as a substrate for the production of livestock feed. Rolz, et al., in an article in
Applied Microbiology and Biotechnology
titled, “White-Rot Fungal Growth on Sugarcane Lignocellulosic Residue”, Volume 25 (1987) pp. 535-541, report using sugarcane residue as a substrate. In U.S. Pat. No. 4,891,320, Aust et al. list as typical materials used to grow white-rot fungi for use in degradation of aromatic compounds shredded paper, wood shavings, sawdust, corn cobs, and humus. None of these references discloses the production of enzymes during the primary metabolic growth phase or the production of cell-free extracts of the culture containing lignin-degrading enzymes.
It is believed that the reason why extracting cell-free enzymes is difficult in conventional solid state processes for producing enzymes is that the enzymes are absorbed into the lignocellulosic substrate materials. Thus, when using substrates of the type normally associated in nature with white-rot fungi, lignin-degrading enzymes are difficult to extract or purify in active form. These substrates typically have a high lignin content and low protein content. On the other hand, small amounts of cell-free enzymes are present in liquid cultures, presumably because there are no surfaces for enzyme absorption.
Both liquid and solid substrate cultures of white-rot fungi have been the subject of at least 15 years of intensive research in numerous laboratories, as evidenced by the volume of research literature and patents granted in this field. However, the problems of producing enzymes during the primary metabolic growth phase, of producing cell-free enzymes from solid culture and of producing lignin-degrading enzyme preparations with commercially useful enzyme concentrations remain unsolved.
SUMMARY OF INVENTION
This invention pertains to a process for growing white-rot fungus in a solid state culture using sugar beet pulp as a substrate. The fungus can be grown for the production of by-products of fungal growth such as enzymes for degrading aromatic compounds such as lignin. The process advantageously permits the production of lignin-degrading enzymes during the primary metabolic growth phase of the fungus rather than during secondary metabolism, and the easy separation of lignin-degrading enzymes from the substrate material for the production of cell-free enzymes preparations.
According to the process, white-rot fungi are grown under growth supportive conditions on a sugar beet pulp substrate. A substrate of sugar beet pulp for growth of white-rot fungi may be prepared by, e.g., sterilizing the raw pulp as by autoclaving and then cooling and adding water or nutrient solutions. The substrate then is inoculated with what may be a conventional white-rot fungi inoculum. The inoculated substrate is then placed in a solid state reactor for growing fungi and the mixture is aerated to enhance growth.
The method of the invention is particularly advantageous for the production of lignin-degrading enzymes. The growth of white-rot fungi on sugar beet pulp substrate results in production of lignin-degrading enzymes during the primary metabolic growth phase of the fungi when an abundance of nutrients are available. This is in contrast to conventional culture systems where production takes place during secondary metabolism when nitrogen or carbon may be restricted.
At the conclusion of the growing period, the substrate can be washed with water to bring enzymes into solution. The lignin-degrading enzymes are not irreversibly bound to the substrate using this process. The enzyme-rich solution can be centrifuged and filtered to provide an active, cell-free liquid enzyme preparation containing lignin-degrading enzymes that have been removed from the substrate.
The ability to commercially produce lignin-degrading enzymes during the primary metabolic growth phase and further to produce cell-free lignin-degrading enzymes is an advantage of this invention over conventional solid state or liquid culture process used to produce these enzymes using white-rot fungi.
DETAILED DESCRIPTION OF THE INVENTION
Sugar beet pulp is used as the substrate material for fungal growth in accordance with this invention. Sugar beet pulp is produced in large amounts and is readily available for high-volume, commercial applications for growing white-rot fungi.
Sugar beet pulp has
Black William E.
Bradley Clifford A.
Kearns Robert D.
Wood Pauline P.
Emerald BioAgriculture Corporation
Marx Irene
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