U.S. patent number 10,385,346 [Application Number 16/157,692] was granted by the patent office on 2019-08-20 for xylosidase having improved enzymatic activity.
This patent grant is currently assigned to DONGGUAN APAC BIOTECHNOLOGY CO., LTD.. The grantee listed for this patent is Dongguan APAC Biotechnology CO., Ltd.. Invention is credited to Chun-Chi Chen, Ya-Shan Cheng, Rey-Ting Guo, Jian-Wen Huang, Ting-Yung Huang, Hui-Lin Lai, Cheng-Yen Lin, I-Hsuan Lin, Tzu-Hui Wu, Cheng-Bin Zheng.
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United States Patent |
10,385,346 |
Cheng , et al. |
August 20, 2019 |
Xylosidase having improved enzymatic activity
Abstract
A xylosidase having improved enzymatic activity is disclosed.
The amino acid sequence of the xylosidase is a modified amino acid
sequence of SEQ ID NO: 2, wherein the modification is a
substitution of phenylalanine at position 35 with glutamate, and/or
a substitution of glutamine at position 41 with histidine.
Inventors: |
Cheng; Ya-Shan (New Taipei,
TW), Wu; Tzu-Hui (New Taipei, TW), Lin;
Cheng-Yen (New Taipei, TW), Lai; Hui-Lin (New
Taipei, TW), Zheng; Cheng-Bin (New Taipei,
TW), Huang; Ting-Yung (New Taipei, TW),
Lin; I-Hsuan (New Taipei, TW), Huang; Jian-Wen
(New Taipei, TW), Chen; Chun-Chi (New Taipei,
TW), Guo; Rey-Ting (New Taipei, TW) |
Applicant: |
Name |
City |
State |
Country |
Type |
Dongguan APAC Biotechnology CO., Ltd. |
DongGuan |
N/A |
CN |
|
|
Assignee: |
DONGGUAN APAC BIOTECHNOLOGY CO.,
LTD. (Dongguan, CN)
|
Family
ID: |
63256074 |
Appl.
No.: |
16/157,692 |
Filed: |
October 11, 2018 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20190119687 A1 |
Apr 25, 2019 |
|
Foreign Application Priority Data
|
|
|
|
|
Oct 25, 2017 [TW] |
|
|
106136658 A |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N
9/2402 (20130101); C12N 15/52 (20130101); C12Y
302/01037 (20130101); C12N 15/102 (20130101); C12Y
302/01177 (20130101); C12Y 302/01072 (20130101) |
Current International
Class: |
C12N
9/24 (20060101); C12N 15/52 (20060101); C12N
15/10 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
PIR Accession No. S55893, published Mar. 19, 1997 (Year: 1997).
cited by examiner.
|
Primary Examiner: Ekstrom; Richard C
Attorney, Agent or Firm: Kirton McConkie Witt; Evan R.
Claims
What is claimed is:
1. A xylosidase consisting of a modified amino acid sequence of SEQ
ID NO: 2, wherein the modification is a substitution of
phenylalanine at position 35 with glutamate, and a substitution of
glutamine at position 41 with histidine.
2. A xylosidase consisting of a modified amino acid sequence of SEQ
ID NO: 2, wherein the modification is a substitution of
phenylalanine at position 35 with glutamate.
3. A xylosidase consisting of a modified amino acid sequence of SEQ
ID NO: 2, wherein the modification is a substitution of glutamine
at position 41 with histidine.
Description
FIELD OF THE INVENTION
The present invention relates to a xylosidase, and more
particularly to a xylosidase having improved enzymatic
activity.
BACKGROUND OF THE INVENTION
Xylan is hemicellulose, which is the major component in plant cell
wall, and also the second most abundant polysaccharides on earth.
Therefore, the hydrolytic enzymes that degrade xylan are highly
attractive and widely applied in many industries for a long time.
Xylan is a long chain polysaccharide, which is composed of many
pentose xylose units linked by .beta.-1,4-glycosidic bond as a
backbone of xylan. In nature, xylan is complex and highly branched
heteropolysaccharide which can be decorated by methyl group or
acetyl group, even branched by other sugar molecules to form
various structures of xylan. Because of this complicated
architecture of xylan, the complete degradation of xylan requires
different xylanolytic enzymes to work together for decomposing
xylan into simple sugars that can be used by organisms.
In general, xylanolytic enzymes can be divided into several groups
including endo-.beta.-D-xylanase, .beta.-1,4-xylosidase, arabinase,
acetylxylan esterase and .alpha.-glucuronidase. Among these
enzymes, .beta.-1,4-xylosidase (EC 3.2.1.37) is a crucial enzyme
for complete degradation of xylan. It is an exoglucosidase that can
hydrolyze the non-reducing ends of xylooligosaccharides into simple
sugar xylose.
Since the xylosidase works together with the endo-xylanase and
other xylanolytic enzymes, these enzymes can be cooperatively used
in many different industries, such as bleaching process in paper
industry, dough quality and juice clearance in food industry,
animal nutrition in feed industry, even in biofuel production.
According to different industrial needs, xylosidase is required to
be suitable for different appropriate working conditions. In
addition to the protein properties of enzyme, its catalytic
efficiency is also the key point for improving industrial enzyme.
Higher enzymatic activity represents the cost reduction in the
industrial process and further enhances the commercial profit.
Currently, many researches try to obtain better enzymes by either
screening in nature or modifying present enzymes. In the present
invention, xylosidase is modified by rational design to increase
its enzymatic activity, so as to further increase its application
potential and economic value in industry.
SUMMARY OF THE INVENTION
An object of the present invention is to modify a xylosidase by
means of structural analysis and site-directed mutagenesis for
improving the enzymatic activity of the xylosidase and further
increasing its application potential and economic value in
industry.
According to an aspect of the present invention, there is provided
a xylosidase comprising a modified amino acid sequence of SEQ ID
NO: 2, wherein the modification is a substitution of phenylalanine
at position 35 with glutamate, and a substitution of glutamine at
position 41 with histidine. The gene encoding the amino acid
sequence of SEQ ID NO: 2 is Hixy143A gene isolated from Humicola
insolens. The xylosidase has a full length amino acid sequence of
SEQ ID NO: 10.
According to another aspect of the present invention, there is
provided a xylosidase comprising a modified amino acid sequence of
SEQ ID NO: 2, wherein the modification is a substitution of
phenylalanine at position 35 with glutamate. The gene encoding the
amino acid sequence of SEQ ID NO: 2 is Hixy143A gene isolated from
Humicola insolens. The xylosidase has a full length amino acid
sequence of SEQ ID NO: 6.
According to an additional aspect of the present invention, there
is provided a xylosidase comprising a modified amino acid sequence
of SEQ ID NO: 2, wherein the modification is a substitution of
glutamine at position 41 with histidine. The gene encoding the
amino acid sequence of SEQ ID NO: 2 is Hixy143A gene isolated from
Humicola insolens. The xylosidase has a full length amino acid
sequence of SEQ ID NO: 8.
The above objects and advantages of the present invention will
become more readily apparent to those ordinarily skilled in the art
after reviewing the following detailed description and accompanying
drawings, in which:
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the nucleotide sequence and the amino acid sequence of
the wild type xylosidase Hixy143A;
FIG. 2 shows the mutagenic primer sequences for site-directed
mutagenesis;
FIG. 3 shows the nucleotide sequence and the amino acid sequence of
the F35E mutant;
FIG. 4 shows the nucleotide sequence and the amino acid sequence of
the Q41H mutant;
FIG. 5 shows the nucleotide sequence and the amino acid sequence of
the F35E/Q41H mutant; and
FIG. 6 shows the enzymatic activity analysis of the wild type
Hixy143A and the three mutants.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention will now be described more specifically with
reference to the following embodiments. It is to be noted that the
following descriptions of preferred embodiments of this invention
are presented herein for purpose of illustration and description
only; it is not intended to be exhaustive or to be limited to the
precise form disclosed.
The xylosidase employed in the present invention is encoded by
Hixy143A gene isolated from the themophilic fungus Humicola
insolens Y1. According to previous studies, the optimal working
condition of this xylosidase is at 50.degree. C., pH 6.8. In the
present invention, the Hixy143A gene was cloned into a vector and
transformed into Pichia pastoris for protein expression. For
improving the industrial application value of this xylosidase, the
present invention analyzed its protein structure and chose some
potential amino acids for modifications by site-directed
mutagenesis so as to improve the enzymatic activity of the
xylosidase. Based on the structural analysis, Phe35 and Gln41,
which are both located in the active site of the xylosidase, were
chosen for further modifications. By site-directed mutagenesis,
Phe35 was singly mutated to glutamate as F35E mutant, while Gln41
was singly mutated to histidine as Q41H mutant. These two mutation
sites were even combined into F35E/Q41H double mutant. The above
mutations all successfully improved the enzymatic activity of the
xylosidase.
The enzyme modification processes and the resulted xylosidase are
described in detail as follows.
FIG. 1 shows the nucleotide sequence and the amino acid sequence of
the wild type xylosidase Hixy143A, wherein the Hixy143A gene
includes 978 base pairs (the nucleotide sequence was numbered as
SEQ ID NO: 1) and encodes 326 amino acids (the amino acid sequence
was numbered as SEQ ID NO: 2). First, the Hixy143A gene was cloned
into pPICZ.alpha.A vector by EcoRI and NotI. The plasmid DNA was
linearized by PmeI and then transformed into Pichia pastoris. The
transformants were selected by YPD plate with 0.1 mg/ml zeocin at
30.degree. C. for 2 days. The selected clones were individually
inoculated in YPD medium at 30.degree. C. overnight and then
amplified in BMGY medium at 30.degree. C. overnight. Finally, the
amplified cells were transferred to BMMY medium containing 0.5%
methanol to induce the protein expression. The supernatants with
induced proteins were collected by centrifugation for following
analysis.
The three mutated genes of Hixy143A were obtained by site-directed
mutagenesis. Particularly, these mutated sequences were obtained by
polymerase chain reaction method using the wild type Hixy143A gene
as the template and using the mutagenic primers shown in FIG. 2.
F35E means the phenylalanine at position 35 was substituted with
glutamate, and the mutagenic primer F35E was numbered as SEQ ID NO:
3. Q41H means the glutamine at position 41 was substituted with
histidine, and the mutagenic primer Q41H was numbered as SEQ ID NO:
4. Therefore, the three mutated genes of Hixy143A obtained by
site-directed mutagenesis in the present invention were F35E, Q41H
and F35E/Q41H.
FIGS. 3 to 5 show the nucleotide sequences and the amino acid
sequences of the three mutants. FIG. 3 shows the nucleotide
sequence and the amino acid sequence of the F35E mutant, wherein
the nucleotide sequence was numbered as SEQ ID NO: 5, the amino
acid sequence was numbered as SEQ ID NO: 6, and the phenylalanine
at position 35 was substituted with glutamate. FIG. 4 shows the
nucleotide sequence and the amino acid sequence of the Q41H mutant,
wherein the nucleotide sequence was numbered as SEQ ID NO: 7, the
amino acid sequence was numbered as SEQ ID NO: 8, and the glutamine
at position 41 was substituted with histidine. FIG. 5 shows the
nucleotide sequence and the amino acid sequence of the F35E/Q41H
mutant, wherein the nucleotide sequence was numbered as SEQ ID NO:
9, the amino acid sequence was numbered as SEQ ID NO: 10, and the
phenylalanine at position 35 was substituted with glutamate and the
glutamine at position 41 was substituted with histidine.
The original DNA template was removed by DpnI at 37.degree. C. The
three mutated genes were individually transformed into E. coli. The
success of gene mutation was confirmed by DNA sequencing. Finally,
the three successful mutated genes were separately transformed into
P. pastoris and then induced for expressing the mutated proteins by
the same method mentioned above. Afterwards, the wild type protein
and the mutated proteins were further analyzed for their enzymatic
activity.
The xylosidase activity analysis was determined by the measurement
of released nitrophenol that is a chromogenic product from the
hydrolysis of the substrate p-nitrophenyl-.beta.-D-xylopyranoside
by xylosidase and further calculated to determine the enzymatic
activity of xylosidase. Basically, the reaction mixture composed of
diluted protein sample and 5 mM
p-nitrophenyl-.beta.-D-xylopyranoside was incubated at 50.degree.
C. for 10 min. The reaction was then stopped by using 2 M
Na.sub.2CO.sub.3. Finally, the absorption of OD410 nm was detected
to determine the activity of xylosidase.
FIG. 6 shows the enzymatic activity analysis of the wild type
Hixy143A and the three mutants. As shown in FIG. 6, under the same
protein concentrations of these proteins, the single mutants F35E
and Q41H both showed higher activities than did the wild type
protein. When compared to the wild type protein, F35E mutant
significantly increased the activity of nearly 90% while Q41H
mutant increased the activity of about 20%. The double mutant
F35E/Q41H showed notably increased activity to about 250%, which is
much higher than the wild type protein and the single mutants.
Besides, the protein expression levels of the mutants were similar
to that of the wild type protein. Therefore, the total activities
of these mutants F35E, Q41H and F35E/Q41H were also higher than
that of the wild type protein, and the double mutant F35E/Q41H had
the highest activity. It is clear that the three mutants provided
in the present invention all have higher activities when compared
to the wild type protein Hixy143A, that means the production cost
of the xylosidase can be reduced, and thus the mutants have higher
economic value of industrial application.
In conclusion, to improve the enzymatic activity of the xylosidase
Hixy143A, the present invention chose some potential amino acids
according to its structural analysis and further modified this
enzyme by rational design. As a result, the three mutants including
F35E, Q41H and F35E/Q41H all showed higher enzymatic activities
compared to the wild type protein, and even had 2.5-fold increase.
Therefore, the present invention successfully improves the
enzymatic activity of the xylosidase and further increases its
economic value of industrial application.
Nowadays, the application fields of the xylosidase and its related
xylanase in industry are widespread. For feed industry, xylanolytic
enzymes can be mixed with feed to help the digestion and absorption
of the monogastric animals like pig and chicken by degrading feed
materials with hemicellulose. As for paper and pulp industry, the
enzymes also can reduce or replace the traditional toxic chemical
method to reach the same result of bleaching. Besides, the enzymes
provide assistances in the juice clearance and the saccharification
step of brewing industry. As for biofuel production, xylosidase can
degrade substrates to produce single sugars that can be utilized in
fermentation by microorganisms. Thus, xylosidase can be widely used
in various industries and has high economic value. The present
invention modifies the xylosidase by genetic engineering, and the
modified enzymes have significantly improved enzymatic activity, so
the production cost of the xylosidase can be reduced to further
improve the economic value of industrial application.
While the invention has been described in terms of what is
presently considered to be the most practical and preferred
embodiments, it is to be understood that the invention needs not be
limited to the disclosed embodiment. On the contrary, it is
intended to cover various modifications and similar arrangements
included within the spirit and scope of the appended claims which
are to be accorded with the broadest interpretation so as to
encompass all such modifications and similar structures.
SEQUENCE LISTINGS
1
101978DNAHumicola insolens 1gctcctttga ttactaacat ctatactgct
gatccttcag ctcatgtttt taacggaaag 60ttgtacatat atccttcaca tgatagagaa
actgatattc agtttaacga taacggagat 120caatacgata tggctgatta
ccatgtattc tccttggatt ccttggatcc accatccgaa 180gttactgatc
atggtgttgt tttgaaggtt gaagatattc catgggtttc taagcaattg
240tgggctccag atgctgctac taaggatgga aagtactact tgtactttcc
agctagagat 300aaggaaggta tttttagaat tggagttgct gtttccgata
agccagaagg accattcact 360ccagatcctg aacctattaa gggttcttac
tctattgatc cagctgtttt tgttgatgat 420gatggttccg cttacatgta
ctttggtgga ttgtggggag gtcaattgca atgttaccaa 480aagggtaaca
acatttttga tgctgaatgg tcaggaccaa aggaaccttc aggttccggt
540gctaaggctt tgggacctag agttgctaag ttgactgatg atatgagaca
atttgctgaa 600gaagttagag aaattgttat tttggctcca gaaactggtg
aacctttggc tgctgatgat 660catgatagaa gattctttga agctgcttgg
atgcataagt acaacggaaa gtactacttt 720tcctactcaa ctggagatac
tcattacttg gtttacgctg ttggagattc accatacggt 780ccgttcactt
acggaggtag aattttggaa cctgttttgg gatggactac tcatcattct
840attgttgaat ttcaaggtag atggtggttg tttcatcatg attgtgaatt
gtccaaggga 900gttgatcatt tgagatccgt taaggttaag gaaatttggt
acgataagga tggcaaaatt 960gttactgaaa agccagaa 9782326PRTHumicola
insolens 2Ala Pro Leu Ile Thr Asn Ile Tyr Thr Ala Asp Pro Ser Ala
His Val1 5 10 15Phe Asn Gly Lys Leu Tyr Ile Tyr Pro Ser His Asp Arg
Glu Thr Asp 20 25 30Ile Gln Phe Asn Asp Asn Gly Asp Gln Tyr Asp Met
Ala Asp Tyr His 35 40 45Val Phe Ser Leu Asp Ser Leu Asp Pro Pro Ser
Glu Val Thr Asp His 50 55 60Gly Val Val Leu Lys Val Glu Asp Ile Pro
Trp Val Ser Lys Gln Leu65 70 75 80Trp Ala Pro Asp Ala Ala Thr Lys
Asp Gly Lys Tyr Tyr Leu Tyr Phe 85 90 95Pro Ala Arg Asp Lys Glu Gly
Ile Phe Arg Ile Gly Val Ala Val Ser 100 105 110Asp Lys Pro Glu Gly
Pro Phe Thr Pro Asp Pro Glu Pro Ile Lys Gly 115 120 125Ser Tyr Ser
Ile Asp Pro Ala Val Phe Val Asp Asp Asp Gly Ser Ala 130 135 140Tyr
Met Tyr Phe Gly Gly Leu Trp Gly Gly Gln Leu Gln Cys Tyr Gln145 150
155 160Lys Gly Asn Asn Ile Phe Asp Ala Glu Trp Ser Gly Pro Lys Glu
Pro 165 170 175Ser Gly Ser Gly Ala Lys Ala Leu Gly Pro Arg Val Ala
Lys Leu Thr 180 185 190Asp Asp Met Arg Gln Phe Ala Glu Glu Val Arg
Glu Ile Val Ile Leu 195 200 205Ala Pro Glu Thr Gly Glu Pro Leu Ala
Ala Asp Asp His Asp Arg Arg 210 215 220Phe Phe Glu Ala Ala Trp Met
His Lys Tyr Asn Gly Lys Tyr Tyr Phe225 230 235 240Ser Tyr Ser Thr
Gly Asp Thr His Tyr Leu Val Tyr Ala Val Gly Asp 245 250 255Ser Pro
Tyr Gly Pro Phe Thr Tyr Gly Gly Arg Ile Leu Glu Pro Val 260 265
270Leu Gly Trp Thr Thr His His Ser Ile Val Glu Phe Gln Gly Arg Trp
275 280 285Trp Leu Phe His His Asp Cys Glu Leu Ser Lys Gly Val Asp
His Leu 290 295 300Arg Ser Val Lys Val Lys Glu Ile Trp Tyr Asp Lys
Asp Gly Lys Ile305 310 315 320Val Thr Glu Lys Pro Glu
325336DNAArtificial SequenceSynthetically generated primer
3gagaaactga tattcaggaa aacgataacg gagatc 36441DNAArtificial
SequenceSynthetically generated primer 4gtttaacgat aacggagatc
attacgatat ggctgattac c 415978DNAArtificial SequenceSynthetically
generated DNA encoding a modified enzyme 5gctcctttga ttactaacat
ctatactgct gatccttcag ctcatgtttt taacggaaag 60ttgtacatat atccttcaca
tgatagagaa actgatattc aggaaaacga taacggagat 120caatacgata
tggctgatta ccatgtattc tccttggatt ccttggatcc accatccgaa
180gttactgatc atggtgttgt tttgaaggtt gaagatattc catgggtttc
taagcaattg 240tgggctccag atgctgctac taaggatgga aagtactact
tgtactttcc agctagagat 300aaggaaggta tttttagaat tggagttgct
gtttccgata agccagaagg accattcact 360ccagatcctg aacctattaa
gggttcttac tctattgatc cagctgtttt tgttgatgat 420gatggttccg
cttacatgta ctttggtgga ttgtggggag gtcaattgca atgttaccaa
480aagggtaaca acatttttga tgctgaatgg tcaggaccaa aggaaccttc
aggttccggt 540gctaaggctt tgggacctag agttgctaag ttgactgatg
atatgagaca atttgctgaa 600gaagttagag aaattgttat tttggctcca
gaaactggtg aacctttggc tgctgatgat 660catgatagaa gattctttga
agctgcttgg atgcataagt acaacggaaa gtactacttt 720tcctactcaa
ctggagatac tcattacttg gtttacgctg ttggagattc accatacggt
780ccgttcactt acggaggtag aattttggaa cctgttttgg gatggactac
tcatcattct 840attgttgaat ttcaaggtag atggtggttg tttcatcatg
attgtgaatt gtccaaggga 900gttgatcatt tgagatccgt taaggttaag
gaaatttggt acgataagga tggcaaaatt 960gttactgaaa agccagaa
9786326PRTArtificial SequenceSequence synthetically translated from
SEQ ID NO 5 6Ala Pro Leu Ile Thr Asn Ile Tyr Thr Ala Asp Pro Ser
Ala His Val1 5 10 15Phe Asn Gly Lys Leu Tyr Ile Tyr Pro Ser His Asp
Arg Glu Thr Asp 20 25 30Ile Gln Glu Asn Asp Asn Gly Asp Gln Tyr Asp
Met Ala Asp Tyr His 35 40 45Val Phe Ser Leu Asp Ser Leu Asp Pro Pro
Ser Glu Val Thr Asp His 50 55 60Gly Val Val Leu Lys Val Glu Asp Ile
Pro Trp Val Ser Lys Gln Leu65 70 75 80Trp Ala Pro Asp Ala Ala Thr
Lys Asp Gly Lys Tyr Tyr Leu Tyr Phe 85 90 95Pro Ala Arg Asp Lys Glu
Gly Ile Phe Arg Ile Gly Val Ala Val Ser 100 105 110Asp Lys Pro Glu
Gly Pro Phe Thr Pro Asp Pro Glu Pro Ile Lys Gly 115 120 125Ser Tyr
Ser Ile Asp Pro Ala Val Phe Val Asp Asp Asp Gly Ser Ala 130 135
140Tyr Met Tyr Phe Gly Gly Leu Trp Gly Gly Gln Leu Gln Cys Tyr
Gln145 150 155 160Lys Gly Asn Asn Ile Phe Asp Ala Glu Trp Ser Gly
Pro Lys Glu Pro 165 170 175Ser Gly Ser Gly Ala Lys Ala Leu Gly Pro
Arg Val Ala Lys Leu Thr 180 185 190Asp Asp Met Arg Gln Phe Ala Glu
Glu Val Arg Glu Ile Val Ile Leu 195 200 205Ala Pro Glu Thr Gly Glu
Pro Leu Ala Ala Asp Asp His Asp Arg Arg 210 215 220Phe Phe Glu Ala
Ala Trp Met His Lys Tyr Asn Gly Lys Tyr Tyr Phe225 230 235 240Ser
Tyr Ser Thr Gly Asp Thr His Tyr Leu Val Tyr Ala Val Gly Asp 245 250
255Ser Pro Tyr Gly Pro Phe Thr Tyr Gly Gly Arg Ile Leu Glu Pro Val
260 265 270Leu Gly Trp Thr Thr His His Ser Ile Val Glu Phe Gln Gly
Arg Trp 275 280 285Trp Leu Phe His His Asp Cys Glu Leu Ser Lys Gly
Val Asp His Leu 290 295 300Arg Ser Val Lys Val Lys Glu Ile Trp Tyr
Asp Lys Asp Gly Lys Ile305 310 315 320Val Thr Glu Lys Pro Glu
3257978DNAArtificial SequenceSynthetically generated DNA encoding a
modified enzyme 7gctcctttga ttactaacat ctatactgct gatccttcag
ctcatgtttt taacggaaag 60ttgtacatat atccttcaca tgatagagaa actgatattc
agtttaacga taacggagat 120cattacgata tggctgatta ccatgtattc
tccttggatt ccttggatcc accatccgaa 180gttactgatc atggtgttgt
tttgaaggtt gaagatattc catgggtttc taagcaattg 240tgggctccag
atgctgctac taaggatgga aagtactact tgtactttcc agctagagat
300aaggaaggta tttttagaat tggagttgct gtttccgata agccagaagg
accattcact 360ccagatcctg aacctattaa gggttcttac tctattgatc
cagctgtttt tgttgatgat 420gatggttccg cttacatgta ctttggtgga
ttgtggggag gtcaattgca atgttaccaa 480aagggtaaca acatttttga
tgctgaatgg tcaggaccaa aggaaccttc aggttccggt 540gctaaggctt
tgggacctag agttgctaag ttgactgatg atatgagaca atttgctgaa
600gaagttagag aaattgttat tttggctcca gaaactggtg aacctttggc
tgctgatgat 660catgatagaa gattctttga agctgcttgg atgcataagt
acaacggaaa gtactacttt 720tcctactcaa ctggagatac tcattacttg
gtttacgctg ttggagattc accatacggt 780ccgttcactt acggaggtag
aattttggaa cctgttttgg gatggactac tcatcattct 840attgttgaat
ttcaaggtag atggtggttg tttcatcatg attgtgaatt gtccaaggga
900gttgatcatt tgagatccgt taaggttaag gaaatttggt acgataagga
tggcaaaatt 960gttactgaaa agccagaa 9788326PRTArtificial
SequenceSequence synthetically translated from SEQ ID NO 7 8Ala Pro
Leu Ile Thr Asn Ile Tyr Thr Ala Asp Pro Ser Ala His Val1 5 10 15Phe
Asn Gly Lys Leu Tyr Ile Tyr Pro Ser His Asp Arg Glu Thr Asp 20 25
30Ile Gln Phe Asn Asp Asn Gly Asp His Tyr Asp Met Ala Asp Tyr His
35 40 45Val Phe Ser Leu Asp Ser Leu Asp Pro Pro Ser Glu Val Thr Asp
His 50 55 60Gly Val Val Leu Lys Val Glu Asp Ile Pro Trp Val Ser Lys
Gln Leu65 70 75 80Trp Ala Pro Asp Ala Ala Thr Lys Asp Gly Lys Tyr
Tyr Leu Tyr Phe 85 90 95Pro Ala Arg Asp Lys Glu Gly Ile Phe Arg Ile
Gly Val Ala Val Ser 100 105 110Asp Lys Pro Glu Gly Pro Phe Thr Pro
Asp Pro Glu Pro Ile Lys Gly 115 120 125Ser Tyr Ser Ile Asp Pro Ala
Val Phe Val Asp Asp Asp Gly Ser Ala 130 135 140Tyr Met Tyr Phe Gly
Gly Leu Trp Gly Gly Gln Leu Gln Cys Tyr Gln145 150 155 160Lys Gly
Asn Asn Ile Phe Asp Ala Glu Trp Ser Gly Pro Lys Glu Pro 165 170
175Ser Gly Ser Gly Ala Lys Ala Leu Gly Pro Arg Val Ala Lys Leu Thr
180 185 190Asp Asp Met Arg Gln Phe Ala Glu Glu Val Arg Glu Ile Val
Ile Leu 195 200 205Ala Pro Glu Thr Gly Glu Pro Leu Ala Ala Asp Asp
His Asp Arg Arg 210 215 220Phe Phe Glu Ala Ala Trp Met His Lys Tyr
Asn Gly Lys Tyr Tyr Phe225 230 235 240Ser Tyr Ser Thr Gly Asp Thr
His Tyr Leu Val Tyr Ala Val Gly Asp 245 250 255Ser Pro Tyr Gly Pro
Phe Thr Tyr Gly Gly Arg Ile Leu Glu Pro Val 260 265 270Leu Gly Trp
Thr Thr His His Ser Ile Val Glu Phe Gln Gly Arg Trp 275 280 285Trp
Leu Phe His His Asp Cys Glu Leu Ser Lys Gly Val Asp His Leu 290 295
300Arg Ser Val Lys Val Lys Glu Ile Trp Tyr Asp Lys Asp Gly Lys
Ile305 310 315 320Val Thr Glu Lys Pro Glu 3259978DNAArtificial
SequenceSynthetically generated DNA encoding a modified enzyme
9gctcctttga ttactaacat ctatactgct gatccttcag ctcatgtttt taacggaaag
60ttgtacatat atccttcaca tgatagagaa actgatattc aggaaaacga taacggagat
120cattacgata tggctgatta ccatgtattc tccttggatt ccttggatcc
accatccgaa 180gttactgatc atggtgttgt tttgaaggtt gaagatattc
catgggtttc taagcaattg 240tgggctccag atgctgctac taaggatgga
aagtactact tgtactttcc agctagagat 300aaggaaggta tttttagaat
tggagttgct gtttccgata agccagaagg accattcact 360ccagatcctg
aacctattaa gggttcttac tctattgatc cagctgtttt tgttgatgat
420gatggttccg cttacatgta ctttggtgga ttgtggggag gtcaattgca
atgttaccaa 480aagggtaaca acatttttga tgctgaatgg tcaggaccaa
aggaaccttc aggttccggt 540gctaaggctt tgggacctag agttgctaag
ttgactgatg atatgagaca atttgctgaa 600gaagttagag aaattgttat
tttggctcca gaaactggtg aacctttggc tgctgatgat 660catgatagaa
gattctttga agctgcttgg atgcataagt acaacggaaa gtactacttt
720tcctactcaa ctggagatac tcattacttg gtttacgctg ttggagattc
accatacggt 780ccgttcactt acggaggtag aattttggaa cctgttttgg
gatggactac tcatcattct 840attgttgaat ttcaaggtag atggtggttg
tttcatcatg attgtgaatt gtccaaggga 900gttgatcatt tgagatccgt
taaggttaag gaaatttggt acgataagga tggcaaaatt 960gttactgaaa agccagaa
97810326PRTArtificial SequenceSequence synthetically translated
from SEQ ID NO 9 10Ala Pro Leu Ile Thr Asn Ile Tyr Thr Ala Asp Pro
Ser Ala His Val1 5 10 15Phe Asn Gly Lys Leu Tyr Ile Tyr Pro Ser His
Asp Arg Glu Thr Asp 20 25 30Ile Gln Glu Asn Asp Asn Gly Asp His Tyr
Asp Met Ala Asp Tyr His 35 40 45Val Phe Ser Leu Asp Ser Leu Asp Pro
Pro Ser Glu Val Thr Asp His 50 55 60Gly Val Val Leu Lys Val Glu Asp
Ile Pro Trp Val Ser Lys Gln Leu65 70 75 80Trp Ala Pro Asp Ala Ala
Thr Lys Asp Gly Lys Tyr Tyr Leu Tyr Phe 85 90 95Pro Ala Arg Asp Lys
Glu Gly Ile Phe Arg Ile Gly Val Ala Val Ser 100 105 110Asp Lys Pro
Glu Gly Pro Phe Thr Pro Asp Pro Glu Pro Ile Lys Gly 115 120 125Ser
Tyr Ser Ile Asp Pro Ala Val Phe Val Asp Asp Asp Gly Ser Ala 130 135
140Tyr Met Tyr Phe Gly Gly Leu Trp Gly Gly Gln Leu Gln Cys Tyr
Gln145 150 155 160Lys Gly Asn Asn Ile Phe Asp Ala Glu Trp Ser Gly
Pro Lys Glu Pro 165 170 175Ser Gly Ser Gly Ala Lys Ala Leu Gly Pro
Arg Val Ala Lys Leu Thr 180 185 190Asp Asp Met Arg Gln Phe Ala Glu
Glu Val Arg Glu Ile Val Ile Leu 195 200 205Ala Pro Glu Thr Gly Glu
Pro Leu Ala Ala Asp Asp His Asp Arg Arg 210 215 220Phe Phe Glu Ala
Ala Trp Met His Lys Tyr Asn Gly Lys Tyr Tyr Phe225 230 235 240Ser
Tyr Ser Thr Gly Asp Thr His Tyr Leu Val Tyr Ala Val Gly Asp 245 250
255Ser Pro Tyr Gly Pro Phe Thr Tyr Gly Gly Arg Ile Leu Glu Pro Val
260 265 270Leu Gly Trp Thr Thr His His Ser Ile Val Glu Phe Gln Gly
Arg Trp 275 280 285Trp Leu Phe His His Asp Cys Glu Leu Ser Lys Gly
Val Asp His Leu 290 295 300Arg Ser Val Lys Val Lys Glu Ile Trp Tyr
Asp Lys Asp Gly Lys Ile305 310 315 320Val Thr Glu Lys Pro Glu
325
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