Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1995-06-06
2002-02-12
Zeman, Mary K. (Department: 1631)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S069100, C435S201000, C435S209000, C435S252300, C435S252310, C435S267000, C435S278000, C435S832000, C424S094610, C530S350000, C530S370000
Reexamination Certificate
active
06346407
ABSTRACT:
The invention relates to a new xylanase. The invention also relates to the methods for preparing this xylanase, to the uses of the latter and to compositions comprising it.
The invention also relates to a new strain of microorganisms producing this xylanase and to a DNA molecule comprising the nucleotide sequence which codes for this xylanase. The invention also relates to vectors containing this DNA molecule and to strains transformed by these vectors.
The invention also relates to the promoter derived from the gene which codes for
Bacillus pumilus
PRL B12 xylanase and the presequence which codes for the signal peptide of
Bacillus pumilus
PRL B12 xylanase. The invention also relates to vectors which contain this promoter and this presequence, and also to the DNA molecule comprising the nucleotide sequence which codes for the mature portion of the xylanase of the invention. The invention also relates to strains transformed by these vectors.
Thermostable xyalanases which are active over a wide pH range are known, such as, in particular, xylanases produced by strains of alkalophilic bacillus (Gupta et al. Biotechnology Letters, 1992, 14 (11), pages 1045-1046 and International Patent Application WO 94/04664). However, despite these properties, these enzymes would appear to be poorly effective in bleaching paper pulp.
Consequently, there is at present a need for a xylanase which can be used in the treatment of paper pulp, which is very stable and also very active over a wide range of temperature and of basic and acid pH.
The object of the present invention is to provide a new xylanase which is active over a wide pH range, both at alkaline pH and at acid pH.
The object of the present invention is also to identify, isolate and provide a strain, especially a Bacillus strain, which produces the said xylanase naturally.
The object of the present invention is also to isolate and provide a DNA molecule comprising a nucleotide sequence which codes for the said xylanase.
The object of the present invention is also to prepare and provide an expression vector containing the nucleotide sequence coding for the said xylanase.
The object of the present invention is also to prepare and provide an integration vector containing the nucleotide sequence coding for the said xylanase.
The object of the present invention is also to prepare and provide the promoter drived from the gene which codes for
Bacillus pumilus
PRL B12 xylanase. The object of the present invention is also to prepare and provide the presequence which codes for the signal peptide of
Bacillus pumilus
PRL B12 xylanase. The vectors which comprise this promoter and/or this presequence also contain the DNA molecule comprising the nucleotide sequence which codes for the mature portion of the xylanase of the invention. The strains transformed by these vectors produce the xylanase of the invention heterologously.
The object of the present invention is also to prepare and provide a Bacillus host transformed with the expression vector which contains the DNA molecule comprising the nucleotide sequence of the Bacillus strain coding for the said xylanase.
The object of the present invention is also to prepare and provide a Bacillus host transformed with the expression vector which contains the DNA molecule comprising the nucleotide sequence of the Bacillus strain coding for the said xylanase [sic].
The object of the present invention is also to prepare and provide a composition containing this xylanase.
The object of the present invention is also to prepare and provide a xylanase which can be used in the treatment of paper pulp, and pulps having a basic, neutral or acid pH, and in particular pulps having an especially basic pH and paper pulps of various origins, such as the pulps originating from coniferous trees, the pulps originating from broad-leaved trees and especially eucalyptus pulp.
To this end, the invention relates to a xylanase originating from a Bacillus, and more especially from an aerobic and non-thermophilic microorganism.
It is preferable to use Bacillus sp. strain 720/1 or a derivative or mutant of this strain. The xylanase of the invention is derived from (naturally produced by) Bacillus sp. strain 720/1. Xylanase is classified in the international system under the EC number 3.2.1.8. It is an endo-1,4-beta-xylanase.
Preferably, the isolated and purified xylanase consists of a single type of polypeptide having a molecular weight of approximately 25 kDa.
The invention relates to an isolated and purified xylanase comprising the amino acid sequence from 1 to 221 amino acids (SEQ ID NO:3) or a modified sequence derived from this sequence. The amino acid sequence and the nucleotide sequence (SEQ ID NO:1) coding for the mature xylanase, together with its translation into amino acids (SEQ ID NO:2), is given in
FIG. 1
(
FIGS. 1
a
and
1
b
).
The xylanase of the invention is synthesized in the form of a precursor. The precursor contains 248 amino acids: (SEQ ID NO:6). The nucleotide sequence SEQ ID NO:4) coding for the xylanase precursor, as well as its translation into amino acids (SEQ ID NO:5), are identified.
The precursor contains the sequence of 221 amino acids (SEQ ID NO:3) of the mature xylanase and the sequence of 27 amino acids (SEQ ID NO:9) of the presequence.
The mature xylanase sequence is preceded by a presequence. The latter is an additional sequence of 27 amino acids (SEQ ID NO:9). The corresponding nucleotide sequence (SEQ ID NO:7), as well as its translation into amino acids (SEQ ID NO:8), are identified. This presequence codes for the signal peptide of the xylanase of the invention.
As a special preference, the said xylanase has a determined isoelectric point of between approximately 9.5 and approximately 9.7.
The xylanase according to the invention is thermostable and active over a wide pH range. Preferably, the xylanase according to the invention is alkaline.
The xylanase according to the invention possesses, moreover, all appropriate properties compatible with the actual industrial conditions of enzyme treatment of paper pulp. According to the numerous steps of the various treatments of paper pulp employed industrially, these properties are good stability with respect to pH and temperature, and enzyme activity over a wide range of pH and temperature, such as, in particular, a pH of between approximately 5 and 10 and a temperature of between approximately 50 and 80° C.
The xylanase of the invention is active over a range of pH above or equal to approximately 5. The xylanase of the invention is active over a range of pH lower or equal to approximately 11. The xylanase develops an enzyme activity of more than 50% of the maximal activity, measured at a temperature of approximately 50° C. and in the presence of xylan, over a range of pH above or equal to approximately 5.0. The xylanase develops an enzyme activity of more than 50% of the maximal activity, measured at a temperature of approximately 50° C. and in the presence of xylan, over a pH range below approximately 10.5.
The xylanase of the invention is active over a range of temperature above or equal to approximately 50° C. The xylanase of the invention is active over a range of temperature below or equal to approximately 80° C. The xylanase develops an enzyme activity of more than 50% of the maximal activity, measured at a pH of approximately 9 and in the presence of xylan, over a range of temperature above or equal to approximately 50° C. The xylanase develops an enzyme activity of more than 50% of the maximal activity, measured at a pH of approximately 9 and in the presence of xylan, over a temperature range below approximately 80° C.
The invention also relates to a modified xylanase, that is to say an enzyme whose amino acid sequence differs from that of the wild-type enzyme by at least one amino acid. These modifications may be obtained by standard mutagenesis techniques on the DNA, such as exposure to ultraviolet radiation or to chemical products such as ethyl methanesulphonate (EMS), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), sodium nitr
De Buyl Eric
Detroz Rene
Lahaye Andrée
Ledoux Pierre
Genencor International Inc.
Genencor International Inc.
Zeman Mary K.
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