Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
1997-10-10
2001-03-13
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S195000, C435S200000, C435S274000, C435S278000, C435S839000, C426S018000, C426S028000, C426S053000, C426S054000, C426S496000
Reexamination Certificate
active
06200797
ABSTRACT:
This invention relates to proteins with xylanase activity derived from bacteria, and in particular to xylanases which are free of any significant cellulase activity and which are active at high temperature and at neutral to alkaline pH. Xylanases having these characteristics are particularly useful in the bleaching of wood pulps, such as kraft pulps.
BACKGROUND OF THE INVENTION
Enzymes are proteins present in all living cells, where apart from controlling metabolic processes, they break down food materials into simpler compounds. The enzymes are catalysts which speed up processes which would otherwise proceed very slowly, or not at all. Moreover, enzymes are very specific, breaking down only one type of compound.
Xylan is a polysaccharide found in most plant cell walls, consisting of D-xylose units linked by &bgr;-1-4 glycosidic bonds. It occurs with another polysaccharide, cellulose and an amorphous binding polymer, lignin. Xylan forms a major component of plant hemicelluloses, and varies in the nature of substituents on the sugar groups, depending on the origin. For example, xylans derived from hardwoods typically consist of a backbone of O-acetyl-4-O-methylglucuronoxylan, in which about 10% of the xylose units carry 4-O-methylglucuronic acid side chains linked via &agr;-1,2 bonds, and 70% of the xylose residues are acetylated at C-2 or C-3. In contrast, xylans derived from softwoods are usually arabino-4-O-methylglucuronoxylans in which over 10% of the xylose sub-units carry arabinofuranose residues linked via &agr;-1,3 bonds. Enzymes which are able to degrade xylan are called xylanases (endo-1,4-&bgr;-D-xylanases; International enzyme nomenclature EC 3.2.1.8).
Commercial preparations of xylanase, often in combination with other cell wall degrading enzymes, have been used in the extraction or liquefaction of plant material. For example, in the food industry, the mashing process for the production of juices can be made to produce higher yields and better processing with the application of cell wall degrading enzymes, which include xylanase.
The primary source of cellulose for paper manufacture is wood, and may be either hardwood or softwood. The initial step in paper manufacture is the reduction of wood to the fibre state, which may be achieved by mechanical or chemical pulping methods. Chemical pulping involves the “cooking” of woodchips with chemical reagents in order to separate the cellulose fibres from the other wood components, and to break down the lignin and other extraneous compounds so that the cellulose is left in tact in its fibrous form. The most common process is the kraft or sulphate process, which can be applied to almost any timber species. The active ingredients are sodium hydroxide and sodium sulphide in a strongly alkaline solution.
During the kraft pulping process, xylan in the wood is initially dissolved in the pulping liquor, but with time, reprecipitates on to the resulting pulp. Wood lignin is modified and dissolved by the pulping liquors. However, about 10% of the lignin remains in the kraft pulp. To brighten the pulp, the lignin must be removed by bleaching chemicals, such as chlorine, which generate environmentally hazardous wastes.
More recently, commercial xylanase preparations have been used as an aid to the bleaching of kraft wood pulps. A program of cooperation between research institutes and the pulping industry has shown that treating the unbleached kraft pulp with xylanase results in a reduciton in the amount of bleaching chemicals required to obtain a full brightness pulp. It is believed that xylanase acts as a bleaching aid (bleach booster) by releasing some trapped residual lignin within the pulp matrix and giving better access to bleaching chemicals. It is widely believed that xylanase breaks down reprecipitated xylan which forms a coating on the pulp, thus releasing trapped residual lignin from within the pulp matrix, and allowing better access of bleaching chemicals to this matrix. Thus xylanase acts as a bleaching aid or bleach booster.
In the kraft process, the pulp is typically handled at high temperatures and neutral to alkaline pH. Commercial xylanases typically have a temperature optimum of about 50° C. and a pH optimum of about 5, and are thus subject to rapid denaturation under process conditions. Thus there is a need for xylanases which are able to act optimally on the kraft pulp without any requirement to adjust the temperature or pH. In order to be useful as a bleaching aid, the xylanase must also be free of any significant cellulase activity, since cellulase would cause an undesirable loss of cellulose fibre.
We have screened microorganisms newly isolated from a range of environments in order to identify those which produce high levels of xylanases with high temperature optima and which are active at neutral to alkaline pH. A previously unidentified bacterium isolated from white-rotted wood, produces such a xylanase in high yield and free of significant cellulase activity. Thus bacterium is a strain of
Bacillus subtilis
which we have designated B230.
SUMMARY OF THE INVENTION
According to one aspect, the invention provides a bacterium, isolatable from wood compost, having the following characteristics:
A. Ability to grow at a temperature between 20° and 45°;
B. Ability to grow in the pH range of 5 to 9.5;
C. Ability to grow on Luria-Bertani agar at 37°;
D. Ability to grow under solid state or submerged culture conditions; and
E. Constitutive production and/or extracellular release of at least one protein with xyalanse activity having an associated cellulase activity of less than 0.1, said at least one protein having a molecular weight of about 28 kD.
Preferably the bacterium is isolated such that a biologically pure culture exits.
Preferably xylanase production is enhanced by growth in the presence of xylan or of lignocellulose substrates, or degradation products, including xylose and xylitol, derived from such substrates.
More preferably the xylanase has at least one characteristic selected from the group consisting of activity at about pH between 4.5 and 9.5, a thermal activity range up to 70° C., and high thermal stability up to 65° C. Most preferably the xylanase produced by the bacterium is effective on both soluble and insoluble xylans.
In a particularly preferred embodiment, the bacterium has the characteristics of the bacterial isolate designated B230, as deposited under the provisions of the Budapest Treaty in the Australian Government Analytical Laboratories, PO Box 385, Pymble, New South Wales 2073, Australia, on Sep. 6, 1994, under Accession No. N94/41262, or a mutant or derivative thereof having the ability to produce a xylanase as described above. The term “mutant or derivative” thereof includes naturally occurring and artificially induced mutants which retain their ability to digest xylans. Production of such mutants or derivatives will be well known by those skilled in the art.
According to a second aspect, the invention provides a process for producing at least one protein with xylanase activity said process comprising cultivating a bacterium under conditions and for a time sufficient to produce said protein and collecting culture medium wherein said bacterium has the following characteristics:
A. Ability to grow at a temperature between 20 and 45°;
B. Ability to grow in the pH range of 5 to 9.5;
C. Ability to grow on Luria-Berani agar at 37°;
D. Ability to grow under solid state or submerged culture conditions; and
E. Constitutive production and/or extracellular release of at least one protein with xylanase activity, said protein having an associated cellulase activity of <0.1%.
Preferably the bacterium used is strain B230 or a mutant, variant or derivative thereof.
Preferably the bacterium is grown under optimal conditions for extracellular production of said at least one protein. Still more preferably the production of said at least one protein is induced by the addition of xylitol to the culture medium. Preferably xylitol is added in an amount of 0.01 to 2% of the culture medium which is preferab
Ball Diane
Dunlop Robert William
Falk Cedric John
Roullo Alexander Buhisan
Wang Bin
Biotech International Limited
Larson & Taylor PLC
Prats Francisco
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