Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1995-11-02
1998-01-13
Wax, Robert A.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435196, C12N 918, C12N 916
Patent
active
057078475
DESCRIPTION:
BRIEF SUMMARY
This application is a 371 of PCT/DK94/00173, filed Apr. 28, 1994, which included the US as a designated state and claimed foreign priority benefits of DK 0487/93, filed Apr. 30, 1993 and DK 1217/93, filed Oct. 28, 1993.
FIELD OF INVENTION
The present invention relates to an enzyme with pectin methylesterase (PME) activity, a DNA construct encoding the enzyme, a method of producing the enzyme, an enzyme preparation containing the enzyme and various uses of the enzyme.
BACKGROUND OF THE INVENTION
Pectin polymers are important constituents of plant primary cell walls. They are mainly composed of chains of 1,4-1inked .alpha.-D-galacturonic acid and methylated as well as acetylated derivatives thereof. The use of pectin-degrading enzymes is important in the food industry, primarily for fruit and vegetable processing Such as fruit juice production or wine making, where their ability to catalyse the degradation of the backbone of the pectin polymer is utilised.
An assortment of different pectin degrading enzymes is known to be present in various microorganisms such as Aspergillus niger. Of these, pectin methylesterase catalyses the removal of methanol from pectin, resulting in the formation of pectic acid (polygalacturonic acid). Pectate lyase cleaves glycosidic bonds in polygalacturonic acid by .beta.-elimination, pectin lyase cleaves the glycosidic bonds of highly methylated pectins by .beta.-elimination, and polygalacturonase hydrolyses the glycosidic linkages in the polygalacturonic acid chain.
The nucleotide and derived amino acid sequence of an A. niger pectin esterase cDNA sequence is disclosed by Khanh et al. (1990). EP 388 593 discloses the recombinant production of an A. niger pectin esterase in A. awamori or A. niger. Khanh et al. (1992) disclose the effect, on enzyme yield, of using various promoters in the expression of an A. niger pectin methyl esterase in A. niger.
Markovic and Jornvall (1992) have analyzed disulfide bridges in a tomato pectin esterase and suggest the number and location of disulphide bridges in other known and distantly related pectin esterases from A. niger, Erwinia chrysanthemi and Pseudomonas solanacearum. Furthermore, various amino acid residues conserved between the various enzymes are identified and a possible location of the active site of the enzymes is suggested.
van Rijssel et al. (1993) disclose a protein complex isolated from Clostridium thermosaccharolyticum which has pectin methylesterase activity. WO 93/13212 discloses a tomato pectin esterase cDNA sequence.
WO 93/09683 discloses the use of a purified A. niger pectin esterase in the production of juice from fruits and vegetables.
SUMMARY OF THE INVENTION
It is an object of the present invention to prepare a single-component pectin methylesterase (PME).
Accordingly, the present invention relates to an enzyme exhibiting PME activity, which enzyme PME derived from Aspergillus aculeatus, CBS 101.43, and/or or a sequence homologous thereto encoding an enzyme exhibiting PME activity, and/or homologous with said sequence.
In the present context, the term "derived from" as used in connection with property a) is intended not only to indicate a PME produced by strain CBS 101.43, but also a PME encoded by a DNA sequence isolated from strain CBS 101.43 and produced in a host organism transformed with said DNA sequence.
In a further aspect, the invention relates to an enzyme exhibiting PME activity, which enzyme is encoded by a DNA sequence comprising the following partial sequence ##STR1## or a sequence homologous thereto encoding a polypeptide with PME activity.
In the present context, the term "homologous" is intended to indicate a DNA which hybridizes to the same probe as the DNA coding for the PME enzyme under certain specified conditions (such as presoaking in 5xSSC and prehybridizing for 1 h at .about.40.degree. C. in a solution of 5xSSC, 5x Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50 .mu.g of denatured sonicated calf thymus DNA, followed by hybridization in the same solution supplemented with 50
REFERENCES:
patent: 4200694 (1980-04-01), Ishii et al.
patent: 5413937 (1995-05-01), Bridges et al.
Dialog, file 55: Biosis Preview.
Khan, N., et al. Gene., vol. 106, pp. 71-77 (1991).
Markovic, O., et al., Biologia, vol. 44, No. 12, pp. 1185-1189 (1989).
Abstract -PCT WO 93/13212.
Abstract -PCT WO 93/09683.
Van Rijssel, M., et al., Applied and Environmental Microbiology, vol. 59, No. 3, pp. 828-836 (1993).
Khanh, N., et al., Nucleic Acids Research, vol. 18, No. 14, p. 4262 (1990).
Khanh, N., et al., Biotechnology Letters, vol. 14, No. 11, pp. 1047-1052, (1992).
Markovic, O., et al., Protein Science, vol. 1, pp. 1288-1292, (1992).
Andersen Lene Nonboe
Budolfsen Gitte
Christgau Stephan
Dalb.o slashed.ge Henrik
Heldt-Hansen Hans Peter
Gregg, Esq. Valeta A.
Novo Nordisk A S
Slobodyarisky Elizabeth
Wax Robert A.
Zelson Esq. Steve T.
LandOfFree
Enzyme exhibiting pectin methylesterase activity does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Enzyme exhibiting pectin methylesterase activity, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Enzyme exhibiting pectin methylesterase activity will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-325529