Xylose isomerase with improved properties

Details Xylose isomerase with improved properties Xylose isomerase with improved properties

2000-04-07

2002-11-05

Patterson, Jr., Charles L. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Isomerase

C435S254200, C435S254210, C435S254220, C435S254230, C435S320100, C435S161000, C536S023200

Reexamination Certificate

active

06475768

ABSTRACT:

TECHNICAL FIELD
This invention relates to nucleic acid sequences encoding novel modified xylose isomerases, to vectors carrying the sequences, to the polypeptides encoded by these sequences, to the production of a polypeptide encoded by these sequences and to the use of the isomerase in a process of ethanol production. The present invention further provides novel modified xylose isomerases that advantageously can be applied in the production of fructose syrups, in particular high fructose corn syrups.
BACKGROUND ART
D-xylose is a five-carbon sugar. It is present in nature, for example as xylan polymer in plant hemicellulose. As a carbon source, D-xylose has been reported to be utilised widely by bacteria and to a lesser extent by fungi. D-xylose is first converted to its keto isomer, D-xylulose, which is then phosphorylated to D-xylulose-5-phosphate, a normal pentose phosphate pathway intermediate.
There are two possible routes for the isomerisation of D-xylose: (a) direct conversion to D-xylulose by an isomerase; or (b) an oxidoreductive pathway, in which D-xylose is first reduced to xylitol and in a second reaction xylitol is oxidised to D-xylulose. The enzymes involved in this oxidoreductive pathway require coenzymes. The direct isomerisation is reported from bacteria and plant, while the oxidoreductive pathway is the main route for D-xylose to D-xylulose conversion in fungi.
Xylose isomerase catalyses the direct isomerisation of D-xylose to D-xylulose and vice versa, with the direction dependent on the relative concentration of the aldo and keto forms and the reaction conditions. Xylose isomerase also catalyses of interconversion of the 6carbon aldose sugar D-glucose and its keto isomer D-fructose. Thus, bacterial xylose isomerases are used industrially to produce D-fructose from D-glucose.
The economic use of lignocellulosic biomass as a renewable energy source is strongly dependent on the fermentation of xylose to ethanol. Since the best ethanol-producing organism,
Saccharomyces cerevisiae
is not able to utilise xylose, major efforts have been taken to develop recombinant strains possessing the necessary enzymes. The xylose reductase and xylitol dehydrogenase from
Pichia stipitis
were cloned and expressed in
Saccharomyces cerevisiae.
The resulting strains were able to ferment xylose, however the yields were far from the theoretical due to the xylitol excretion (Tantirungkij et al. (1993)).
Several attempts to circumvent this problem by introducing the alternative direct isomerisation pathway have been made. The xylose isomerases form two clusters based on the sequence homology, the actinomycetic genes showing less than 40% homology to the others. From the latter subgroup the
Bacillus subtilis
(Amore et al. (1989)) and the
Escherichia coli
(Sarthy et al. (1987)) xylose isomerases were expressed inactively in
Saccharomyces cerevisiae,
while introduction of the
Clostridium thermosulfurogenes
(Moes et al. (1996)) gene did not result in a protein that cross-reacted with a specific antibody. From the first subgroup the
Actinoplanes missouriensis
(Amore et al. (1989)) gene was cloned in yeast but the production of xylose isomerase failed due to an aberrant transcription start.
The
Thermus thermophilus
xylose isomerase only recently has been actively expressed in
Saccharomyces cerevisiae
(Walfridsson et al. (1996)). This gene is closely related to the actinomycetic genes showing approximately 65% homology. The enzyme, however, has only trace activity at 30° C., because of its high (95° C.) temperature optimum. The other important factor for the poor performance of the strain was the formation of xylitol, probably by the unspecific NADPH linked aldose reductase described by (Kuhn et al. (1995)), which is coded by the gene GRE3. The xylitol formation has a dual effect on the ethanol yield; it does not only lead to carbon loss, but it also competitively inhibits the xylose isomerase (Yamanaka (1969)). By increasing intracellular xylitol concentration the apparent affinity of the xylose isomerase towards xylose decreases and more xylose is channelled into xylitol, until the NADPH pool of the cell is depleted. In the presence of active xylose isomerase some xylitol can be formed also by the endogenous xylitol dehydrogenase described by (Richard et al. (1999)). To prevent the xylitol impairing the xylose isomerase and thereby blocking the xylose metabolism of the recombinant yeast, it is advantegous to have a xylose isomerase expressed which is less sensitive to the presence of xylitol.
DISCLOSURE OF THE INVENTION
In light of the previously described problems, there is a need to find a xylose isomerase being actively expressed in
Saccharomyces cerevisiae
and being more active at a broader range of pH values and temperatures.
In the present invention, a series of novel xylose isomerases have been isolated. They were modified from the
Thermus thermophilus
enzyme by random mutagenesis and identified as having elevated activity at mesophilic (<40° C.) temperatures, compared to the wild type enzyme. Expressed either in procaryotic or eucaryotic cells, the specific enzyme activity in the mutants is at least 10% higher than that of the wildtype enzyme. They are, therefore, expected to be more suitable for expression in yeast. The present invention thus comprises any isolated nucleic acid sequence which upon expression in a procaryotic or eucaryotic host cell codes for a polypeptide having at least 10% higher specific xylose isomerase activity, than the wild type xylose isomerase which nucleic acid sequence is shown in SEQ. ID. No. 2. The specific activity of the enzyme is to be determined as described in example 3, wherein one unit of xylose (glucose) isomerase activity is defined as the amount of crude enzyme needed to produce 1 &mgr;mol of product per minute under the assay conditions. The specific activity (U min
−1
mg
−1
) is herein determined from the activity and the protein concentration of the crude enzyme preparations.
The present invention further comprises any isolated nucleic acid sequence that is derived and shows at least one nucleic acid modification from the nucleic acid sequence shown in SEQ. ID. No. 2.
Furthermore, the mutants show a higher thermostability than the wildtype enzyme. One embodiment of the invention thus comprises an isolated nucleic acid sequence which upon expression in a procaryotic or eucaryotic host cell codes for a polypeptide having at least 10% higher specific xylose isomerase activity at temperatures above 80° C. such as 81° C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C., 88° C., 89° C., 90° C., 91° C., 92° C., 93° C., 94° C., 95° C., 96° C., 97° C., 98° C., 99° C. and/or 100° C., than the wild type xylose isomerase which nucleic acid sequence is shown in SEQ. ID. No. 2.
In another embodiment of the invention, the mutants also show a higher acceptance for xylitol, which is an inhibitor to xylose isomerase, compared to the wildtype enzyme.
In a further embodiment of the invention, the mutated xylose isomerases have a broader pH range than the wildtype, even though it may have the same pH optimum at ~7.0. The present invention therefor also comprises any isolated nucleic acid sequence which upon expression in a procaryotic or eucaryotic host cell codes for a polypeptide having at least 10% higher specific xylose isomerase activity at acidic pH values i.e. lower than pH 7.5 such as pH 7.4, pH 7.3, pH 7.2, pH 7.1, pH 7.0, pH 6.9, pH 6.8, pH 6.7, pH 6.6, pH 6.5, pH 6.4, pH 6.3, pH 6.2, pH 6.1, pH 6.0, pH 5.9, pH 5.8, pH 5.7, pH 5.6, pH 5.5, pH 5.4, pH 5.3, pH 5.2, pH 5.1, pH 5.0, pH 4.5 and/or pH 4.0 than the wild type xylose isomerase which nucleic acid sequence is shown in SEQ. ID. No. 2.
In a preferred embodiment, the present invention provides:
An isolated nucleic acid sequence which codes upon expression in a procaryotic or eucaryotic host cell for a polypeptide having xylose isomerase activity at mesophilic temperatures, which nucleic acid sequence is selected from
a) nucleic acid sequences shown in SEQ. I

Affiliated with

Also associated with

Rating

Xylose isomerase with improved properties does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Xylose isomerase with improved properties, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Xylose isomerase with improved properties will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2914628