Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1999-09-03
2002-04-02
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S183000, C435S320100, C435S252300, C536S023100, C536S023200, C530S350000
Reexamination Certificate
active
06365390
ABSTRACT:
BACKGROUND OF THE INVENTION
The field of the present invention is the area of enzymes which degrade plant cell walls, and certain other substrates, in particular, the phenolic acid esterases, feruloyl esterases and/or coumaroyl esterase, nucleotide sequences encoding them and recombinant host cells and methods for producing them.
Plant cell wall material is one of the largest sources of renewable energy on earth. Plant cell walls are composed mainly of cellulose, hemicelluloses, lignin and pectin. Arabinoxylan is one of the main constituents of hemicelluloses. It is composed of a chain of &bgr;(1→4) linked xylose units that are substituted by arabinose, acetate, and glucuronic acid. The arabinose has ester linked ferulic and p-coumaric acids [Borneman et al. (1993) In: Hemicellulose and Hemicellulases, Coughlan and Hazlewood, Eds., pp. 85-102]. Ferulic acid has been shown to link hemicellulose and lignin [Ralph et al. (1995)
Carbohydrate Research
275:167-178]. Feruloyl esterases are involved in breaking the bond between the arabinose and ferulic acid, thus releasing the covalently bound lignin from hemicelluloses. Feruloyl esterases have been found in many bacteria as well as fungi, but have not been extensively studied nor is there much sequence data available [Christov and Prior (1993)
Enzyme. Microb. Technol
. 15(6):460-75].
Clostridium thermocellum
is a gram-positive bacterium that produces a multienzymatic structure termed the cellulosome. The cellulosome is one of the most active cellulose degrading complexes described to date. The cellulosome has a multi-polypeptide structure, including a scaffolding subunit which has nine cohesins binding to nine catalytic subunits, a dockerin domain for attachment to the cell wall, and a cellulose binding domain [Felix and Ljungdahl (1993)
Annu. Rev. Microbiol
. 47:791-819]. The catalytic subunits include endoglucanase, cellobiohydrolase, lichenase, and xylanase, many of which have been cloned and sequenced. They all have multidomain structures that include at least a dockerin domain for binding to the scaffolding domain, a linker, and a catalytic domain. They may also contain cellulose binding domains and fibronectin-like domains. There are reports that some enzymatic components may have more than one catalytic domain. Two of these are xylanase Y [XynY, Fontes et al. (1995)
Biochem. J
. 307: 151-158] and xylanase Z [XynZ, Grépinet et al. (1988)
J. Bacteriol
. 170(10):4582-8]. XynY has a C-terminal domain whereas XynZ N-terminal domain without any functions determined. Although enzymes with dual catalytic domains (xylanase and &bgr;-glucanase) have been found in other bacteria [Flint et al. (1993)
J. Bacteriol
. 175:2943-2951] neither phenolic acid esterase nor bifunctional enzymes have been found in
C thermocellum.
There is a need in the art for phenolic acid esterases, feruloyl esterases and/or coumaroyl esterases in pure form which degrade plant cell wall materials, and certain other substrates, and for DNA encoding these enzymes to enable methods of producing ferulic acid and/or coumaric acid as well as facilitating degradation of plant cell wall materials.
SUMMARY OF THE INVENTION
The present invention provides novel phenolic acid esterases, having feruloyl esterase and coumaroyl esterase activities, and coding sequences for same.
One phenolic acid esterase of the present invention corresponds to a domain of previously unknown function from xylanase Y of
Clostridium thermocellum
. The recombinantly expressed domain polypeptide is active and has an amino acid sequence as given in
FIG. 1
as “XynY_Clotm.” The nucleotide sequence encoding the esterase polypeptide is given in Table 5, nucleotides 2383-3219, exclusive of translation start and stop signals. See also SEQ ID NOs:11 and 12.
A second phenolic acid esterase of the present invention corresponds to a domain of previously unknown function of xylanase Z from C. thermocellum. The amino acid sequence of the esterase domain, which also is active when expressed as a recombinant polypeptide, is given in
FIG. 1
as “XynZ_Clotm.” The nucleotide sequence encoding this polypeptide is given in Table 6, nucleotides 58-858. The present invention further provides a phenolic acid esterase polypeptide further comprising a cellulose binding domain. A specifically identified cellulose binding domain has an amino acid sequence as given in Table 6, 289-400, with a corresponding coding sequence as given in Table 6, nucleotides 867-1200. See also SEQ ID NOs:13 and 14.
An additional object of the present invention is a phenolic acid esterase (i.e., a feruloyl esterase) derived from a previously uncharacterized portion of a Ruminococcus xylanase (See FIG.
1
). The coding (nucleotides 2164-2895, exclusive of translation start and stop signals) and deduced amino acid sequences (amino acids 546-789) are given in Table 10. See also SEQ ID NOs: 15 and 16.
The present invention further provides a feruloyl (phenolic acid) esterase from the anaerobic fungus Orpinomyces PC-2. The coding sequence and deduced amino acid sequences of the mature esterase protein are given in Table 9, and the purification of the Orpinomyces enzyme is described herein below. See also SEQ ID NOs: 17 and 18.
A further aspect of the present invention methods for the recombinant production of the phenolic (especially ferulic) acid esterases of the present invention.
Escherichia coli, Bacillus subtilis
, Streptomyces sp.,
Saccharomyces cerevisiae, Aureobasidium pullulans, Pichia pastoris
, Trichoderma,
Aspergillus nidulans
or any other host cell suitable for the production of a heterologous protein can be transfected or transformed with an expression vector appropriate for the chosen host. Compatible combinations of vectors and host cells are well known in the art as are appropriate promoters to be used to direct the expression of a particular coding sequence of interest. The recombinant host cells are cultured under conditions suitable for growth and expression of the phenolic acid esterase and the recombinant esterase is then collected or the recombinant host cells in which the esterase has been produced are collected. The coding sequence of the esterase can be operably linked to a nucleotide sequence encoding a signal peptide which is known in the art and functional in the desired host cell if secretion of the esterase into the culture medium is desired. In that case, the culture medium serves as the source of esterase after growth of the host cells.
It is recognized by those skilled in the art that the DNA sequences may vary due to the degeneracy of the genetic code and codon usage. All DNA sequences which encode a phenolic acid esterase polypeptide having a specifically exemplified amino acid sequence are included in this invention, including DNA sequences encoding them having an ATG preceding the coding region for the mature protein and a translation termination codon (TAA, TGA or TAG) after the coding sequence.
Additionally, it will be recognized by those skilled in the art that allelic variations may occur in the phenolic acid esterase polypeptide coding sequences which will not significantly change activity of the amino acid sequences of the polypeptides which the DNA sequences encode. All such equivalent DNA sequences are included within the scope of this invention and the definition of a phenolic acid esterase. The skilled artisan will understand that the amino acid sequence of an exemplified phenolic acid esterase polypeptide and signal peptide(s) can be used to identify and isolate additional, nonexemplified nucleotide sequences which will encode functional equivalents to the polypeptides defined by the amino acid sequences given herein or an amino acid sequence of greater than 40% identity thereto and having equivalent biological activity. All integer percents between 40 and 100 are encompassed by the present invention. DNA sequences having at least about 75% homology to any of the ferulic acid esterases coding sequences presented
Blum David L.
Kataeva Irina
Li Xin-Liang
Ljungdahl Lars G.
Greenlee Winner and Sullivan PC
Hudson Richard
Prouty Rebecca E.
University of Georgia Research Foundation Inc.
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