Cell-wall degrading enzyme variants

Details Cell-wall degrading enzyme variants Cell-wall degrading enzyme variants

2003-03-31

2004-10-26

Nashed, Nashaat T. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Lyase

C435S253600, C435S264000, C510S300000, C536S023100, C536S023200

Reexamination Certificate

active

06808915

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to variants of microbial cell-wall degrading enzymes, more specifically to variants of enzymes having a pectinase structure similar to that of
Bacillus licheniformis
enzymes exhibiting pectate lyase activity as their major enzymatic activity in the neutral and alkaline pH ranges; to a method of producing such enzymes; and to methods for using such enzymes in the textile, detergent and cellulose fiber processing industries. The enzyme variants of the invention may exhibit increased thermostability as compared to the parent enzyme.
2. Description of Related Art
Plant cell walls consist of a complicated network of fibrous materials. The composition of the cell walls varies considerably, depending on the source of the vegetable material. However, in general its composition can be summarized as mainly comprising non-starch polysaccharides. These polysaccharides can be found in various forms: cellulose, hemicellulose and pectins.
The composition of a plant cell wall is both complex and variable. Polysaccharides are mainly found in the form of long chains of cellulose (the main structural component of the plant cell wall), hemicellulose (comprising e.g. various beta-xylan chains) and pectin. The occurrence, distribution and structural features of plant cell wall polysaccharides are determined by: 1. Plant species; 2. Variety; 3. Tissue type; 4. Growth conditions; and 5. Ageing (Chesson (1987), Recent Advances in Animal Food Nutrition, Haresign on Cole, eds.). Butterworth, London, 71-89).
Basic differences exist between monocotyledons (e.g. cereals and grasses) and dicotyledons (e.g. clover, rapeseed and soybean) and between the seed and vegetative parts of the plant (Carre'and Brillouet (1986), Science and Food Agric. 37, 341-351). Monocotyledons are characterized by the presence of an arabinoxylan complex as the major hemicellulose backbone. The main structure of hemicellulose in dicotyledons is a xyloglucan complex. Moreover, higher pectin concentrations are found in dicotyledons than in monocotyledons. Seeds are generally very high in pectic substances, but relatively low in cellulosic material. Three more or less interacting polysaccharide structures can be distinguished in the cell wall:
1. The middle lamella forms the exterior cell wall. It also serves as the point of attachment for the individual cells to one another within the plant tissue matrix. The middle lamella consists primarily of calcium salts of highly esterified pectins;
2. The primary wall is situated just inside the middle lamella. It is a well-organized structure of cellulose microfibrils embedded in an amorphous matrix of pectin, hemicellulose, phenolic esters and proteins;
3. The secondary wall is formed as the plant matures.
During the plant's growth and ageing phase, cellulose microfibrils, hemicellulose and lignin are deposited.
There is a high degree of interaction between cellulose, hemicellulose and pectin in the cell wall. The enzymatic degradation of these rather intensively cross-linked polysaccharide structures is not a simple process. A large number of enzymes are known to be involved in the degradation of plant cell walls. They can broadly be subdivided in cellulases, hemicellulases and pectinases (Ward and Young (1989), CRC Critical Rev. in Biotech. 8, 237-274).
Cellulose is the major polysaccharide component of plant cell walls. It consists of beta 1,4 linked glucose polymers.
Cellulose can be broken down by cellulases, also called cellulolytic enzymes. Cellulolytic enzymes have been divided traditionally into three classes: endoglucanases, exoglucanases or cellobichydrolases and beta-glucosidases (Knowles, J., et al. (1987), TIBTECH 5, 255-261). Like all cell wall degrading enzymes they can be produced by a large number of bacteria, yeasts and fungi. Apart from cellulases degrading beta-1,4 glucose polymers, endo-1,3/1,4 beta-glucanases and xyloglucanases should be mentioned (Ward and Young op. cit.).
Pectins are major constituents of the cell walls of edible parts of fruits and vegetables. The middle lamella which are situated between the cell walls are mainly built up from protopectin which is the insoluble form of pectin. Pectins are considered as intracellular adhesives and due to their colloidal nature they also have an important function in the water regulation system of plants. The amount of pectin can be very high. For example, lemon peels are reported to contain pectin at up to 30% of their dry weight, orange peels contain from 15-20% and apple peels about 10% (Norz, K. (1985). Zucker und Susswaren Wirtschaft 38, 5-6).
Pectins are composed of a rhamno-galacturonan backbone in which 1,4-linked (alpha-D-galacturonan chains are interrupted at intervals by the insertion of 1,2-linked (alpha-L-rhamnopyranosyl residues (Pilnik, W. and A. Voragen (1970), In: The Biochemistry of fruits and their products, vol. 1, Chapter 3, p. 53. Acad. Press). Other sugars, such as D-galactose, L-arabinose and D-xylose, are present as side chains. A large part of the galacturonan residues is esterified with methyl groups at the C2 and C3 position.
A large number of enzymes are known to degrade pectins. Examples of such enzymes are pectin esterase, pectin lyase (also called pectin transeliminase), pectate lyase, and endo- or exo-polygalacturonase (Pilnik and Voragen (1990). Food Biotech 4, 319-328). Apart from enzymes degrading smooth regions, enzymes degrading hairy regions such as rhamnogalacturonase and accesory enzymes have also been found (Schols et al. (1990), Carbohydrate Res. 206, 105-115; Searle Van Leeuwen et al. (1992). Appl. Microbiol. Biotechn. 38, 347-349).
Pectinases can be classified according to their preferential substrate, highly methyl-esterified pectin or low methyl-esterified pectin and polygalacturonic acid (pectate), and their reaction mechanism, beta-elimination or hydrolysis. Pectinases can be mainly endo-acting, cutting the polymer at random sites within the chain to give a mixture of oligomers, or they may be exo-acting, attacking from one end of the polymer and producing monomers or dimers. Several pectinase activities acting on the smooth regions of pectin are included in the classification of enzymes provided by the Enzyme Nomenclature (1992) such as pectate lyase (EC 4.2.2.2), pectin lyase (EC 4.2.2.10), polygalacturonase (EC 3.2.1.15), exo-polygalacturonase (EC 3.2.1.67), exo-polygalacturonate lyase (EC 4.2.2.9) and exo-poly-alpha-galacturonosidase (EC 3.2.1.82).
Pectate lyases degrade un-methylated (polygalacturonate) or low-methylated pectin by beta-elimination of the alpha-1,4-glycosidic bond. The enzymes are generally characterized by an alkaline pH optimum, an absolute requirement for Ca
2+
(though its role in binding and catalysis is unknown) and good temperature stability.
Pectate lyases have been cloned from different bacterial genera such as Bacillus, Erwinia, Pseudomonas, Klebsiella and Xanthomonas.
U.S. patent application Ser. No. 09/073,684, which is hereby incorporated by reference in its entirety, discloses a cloned
Bacillus licheniformis
pectate lyase. The DNA sequence encoding this
B. licheniformis
pectate lyase and the deduced amino acid sequence are provided in SEQ ID NOS: 1 and 2, respectively.
The crystal structures of pectate lyases of
Bacillus subtilis
(1BN8
1
(and an R279K mutant, 2BSP
2
)), of
Erwinia chrysanthemi
(PeIC: 2PEC
3
/1AIR
4
; PeIC (R218K) in complex with substrate: ref 10; and PeIE: 1PCL
5
), of
Erwinia carotovora
(PeIC: 1PLU
6
), and of Bacillus sp. strain 2 KSM-P15 (1EE6) have been published. In addition, the crystal structures of the structurally very similar pectin lyases from
Aspergillus niger
(PlyA:1IDJ
7
/1IDK
7
and PlyB:1QCX
8
) and of the polygalacturonase from
Erwinia carotovora
(1 BHE
9
) are also known.
1: R. Pickersgill, J. Jenkins, G. Harris, W. Nasser, J. Robert-Baudrouy;
Nat. Struct Biol
. 1994, 1, 717;
2: R. Pickersgill, K. Worboys, M. Scott, N. Cummings, A. Cooper, J. Jenkins, D. Smith To Be Published;
3: M.

Affiliated with

Also associated with

Rating

Cell-wall degrading enzyme variants does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Cell-wall degrading enzyme variants, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Cell-wall degrading enzyme variants will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3289143