Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1997-11-18
2002-04-09
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C536S023100, C536S023200, C435S320100, C435S252300, C435S325000, C435S197000
Reexamination Certificate
active
06368833
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention is directed to a novel esterolytic enzyme, novel genetic material encoding that enzyme and esterolytic proteins developed therefrom. In particular, the present invention provides an esterase derived from Aspergillus, a DNA encoding that esterase, vectors comprising that DNA, host cells transformed with that DNA and a protein product produced by such host cells.
Xylan, next to cellulose, is the most abundant renewable polysaccharide in nature. It is the major hemicellulosic component in plants and is located predominantly in the secondary cell walls of angiosperms and gymnosperms. The composition and structure of xylan are more complicated than that of cellulose and can vary quantitatively and qualitatively in various woody plant species, grasses, and cereals. Xylan is a heteropolymer in which the constituents are linked together not only by glycosidic linkages but also by ester linkages. Ferulic acid is the most abundant hydroxycinnamic acid found in plants and is known to be esterified to arabinose in wheat bran, wheat flour, barley straw, maize, sugar-cane bagasse, rice straw and other monocotyledons and also found esterified to galactose residues in pectins of sugar beet, spinach and other dicotyledons. p-Coumaric acid is also linked in a similar fashion in monocots. The presence of these phenolic acids has been shown to limit cell-wall biodegradation and play significant roles in cell wall extension and stabilization through cross-linking heteroxylan chains by forming phenolic dimers via plant peroxidases and/or photodimerization initiated by sunlight. Further, phenolic acids have been shown to function as cross-links between cell wall polysaccharides and the phenylpropanoid lignin polymer. The covalent attachment of lignin to wall polysaccharides and the crosslinking of xylan chains within hemicellulose limit overall polysaccharide bioavailability resulting in significant amounts of undigested fiber in animal feedstuffs, poor bioconversion of agricultural residue into useful products and incomplete processing of grains.
Enzyme hydrolysis of xylan to its monomers requires the participation of several enzymes with different functions. These are classified in two groups based on the nature of the linkages that they cleave. The first group of enzymes is hydrolases (EC 3.2.1) involved in the hydrolysis of the glycosidic bonds of xylan. These include endo-xylanases (EC 3.2.1.8) which randomly dismember the xylan backbone into shorter xylooligosaccharides; &bgr;-xylosidase (EC 3.2.1.37) which cleave the xylooligosaccharides in an exo-manner producing xylose; &agr;-L-arabinofuranosidase (EC 3.2.1.55); and &agr;-glucoronidase (EC 3.2.1.1) which remove the arabinose and 4-O-methylglucuronic acid substituents, respectively, from the xylan backbone. The second group includes enzymes that hydrolyze the ester linkages (esterase, EC 3.1.1) between xylose units of the xylan polymer and acetyl groups (acetyl xylan esterase, EC 3.1.1.6) or between arabinosyl groups and phenolic moieties such as ferulic acid (feruloyl esterase) and p-coumaric acid (coumaroyl esterase).
Faulds et al., reported two forms of ferulic acid esterase isolated from
Aspergillus niger
. The different esterases were distinguished on the basis of molecular weight and substrate specificity (Faulds et al., Biotech. Appl. Biochem., vol. 17, pp. 349-359 (1993)). Brezillon et al. disclosed the existence of at least two cinnamoyl esterases which were believed to be distinct from the ferulic acid esterases shown in the prior art (Brezillon et al., Appl. Microb. Biotechnol., vol. 45, pp. 371-376 (1996)). A ferulic acid esterase called FAE-III was isolated from
Aspergillus niger
CBS 120.49 and shown to act together with xylanase to eliminate nearly all of the ferulic acid and low molecular mass xylooligosaccharides in a wheat bran preparation; ferulic acid was also removed without the addition of xylanase, albeit at a lower level. Faulds et al. further isolated and partially characterized FAE-III from
Aspergillus niger
CBS 120.49 grown on oat spelt xylan (Faulds et al., Microbiology, vol. 140, pp. 779-787 (1994)) and showed it to have a pl of 3.3, a molecular weight of 36 kD (SDS-PAGE) and 14.5 kD (Gel Filtration method), a pH optimum of 5 and a temperature optimum of 55-60° C.; microcrystalline cellulose binding was also detected. The authors theorized that FAE-II may be a proteolytically modified FAE-III. Recently, the various known ferulic acid esterases derived from
Aspergillus niger
have been distinguished based on their distinct substrate specificity and it was noted that FAE-II and FAE-III were unable to release ferulic acid from sugar beet pulp (Brezillon et al., supra).
Nonetheless, despite the characterization work which has been directed to
Aspergillus niger
esterases, the art remains in need of additional esterases for its various applications. Further, those of skill in the art have thus far failed to discover a nucleotide sequence which can be used to produce more efficient genetically engineered organisms capable of expressing such esterases in large quantities suitable for industrial production. However, a pressing need exists for the development of an esterase expression system via genetic engineering which will enable the purification and utilization of working quantities of relatively pure enzyme.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide for novel esterase proteins and DNA encoding such proteins.
It is an object of the present invention to provide for a method of isolating DNA from many different species, which DNA encodes protein having esterase activity.
It is a further object of the present invention to provide for an esterase which is produced by a suitable host cell which has been transformed by the DNA encoding the esterolytic activity.
The present invention provides for a purified 38 kD esterase which is derived from
Aspergillus niger
. Further, a DNA sequence coding for the 38 kD esterase comprising a DNA as shown in
FIG. 4
(SEQ. ID NO: 27); a DNA which encodes the amino acid sequence also shown in
FIG. 4
(SEQ. ID NO: 28); a DNA which encodes an esterase which comprises an amino acid segment which differs from the sequence in
FIG. 4
, provided that the DNA encodes a derivative of the 38 kD esterase specifically described herein; and a DNA which encodes an esterase that comprises an amino acid segment which differs from the sequence in
FIG. 4
, provided that the DNA hybridizes under low-stringency conditions and/or standard stringency conditions, as defined below, with a DNA comprising all or part of the DNA in
FIG. 4
are provided. The present invention further encompasses vectors which include the DNA sequences described above, host cells which have been transformed with such DNA or vectors, fermentation broths comprising such host cells and esterase proteins encoded by such DNA which are expressed by the host cells. Preferably, the DNA of the invention is in substantially purified form and is used to prepare a transformed host cell capable of producing the encoded protein product thereof. Additionally, polypeptides which are the expression product of the DNA sequences described above are within the scope of the present invention.
The enzyme of the instant invention has application as a supplement to an animal feed; in a process for treating fabric; to improve the mechanical properties of dough and the end product of baking of foods; in the modification of polysaccharides to give novel properties, e.g., gums; and in the processing grains. Further, the enzyme also has application in processing of plant materials for the release of free phenolic groups for use as an antioxidant, photoprotector, anti-inflammatory and/or anti-microbial agent which find use in personal care products such as cosmetics and as an aid in the conversion of chemical feed stocks to valuable specialty chemicals, food additives and flavorings.
An advantage of the present invention is that a DNA has been isolated which provides t
Borneman William S.
Bower Benjamin S.
Genencor International Inc.
Genencor International Inc.
Prouty Rebecca E.
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