U.S. patent application number 14/019263 was filed with the patent office on 2014-10-23 for beta-mannanase having improved enzymatic activity.
This patent application is currently assigned to Dongguan APAC Biotechnology CO., Ltd.. The applicant listed for this patent is Dongguan APAC Biotechnology CO., Ltd.. Invention is credited to Ya-Shan Cheng, Rey-Ting Guo, Jian-Wen Huang, Ting-Yung Huang, Hui-Lin Lai, Cheng-Yen Lin, Tzu-Hui Wu.
Application Number | 20140315273 14/019263 |
Document ID | / |
Family ID | 51702264 |
Filed Date | 2014-10-23 |
United States Patent
Application |
20140315273 |
Kind Code |
A1 |
Guo; Rey-Ting ; et
al. |
October 23, 2014 |
BETA-MANNANASE HAVING IMPROVED ENZYMATIC ACTIVITY
Abstract
A .beta.-mannanase having increased enzymaic activity is
disclosed. The .beta.-mannanase has a modified amino acid sequence
of SEQ ID NO: 2, wherein the modification is a substitution of
Tyrosine at position 216 with Tryptophan.
Inventors: |
Guo; Rey-Ting; (Taipei,
TW) ; Huang; Jian-Wen; (Taipei, TW) ; Cheng;
Ya-Shan; (Taipei, TW) ; Wu; Tzu-Hui; (Taipei,
TW) ; Lai; Hui-Lin; (Taipei, TW) ; Lin;
Cheng-Yen; (Taipei, TW) ; Huang; Ting-Yung;
(Taipei, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dongguan APAC Biotechnology CO., Ltd. |
DongGuan |
|
CN |
|
|
Assignee: |
Dongguan APAC Biotechnology CO.,
Ltd.
DongGuan
CN
|
Family ID: |
51702264 |
Appl. No.: |
14/019263 |
Filed: |
September 5, 2013 |
Current U.S.
Class: |
435/200 ;
435/320.1; 536/23.2 |
Current CPC
Class: |
C12N 9/2491 20130101;
C12Y 302/01025 20130101 |
Class at
Publication: |
435/200 ;
536/23.2; 435/320.1 |
International
Class: |
C12N 9/24 20060101
C12N009/24 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 17, 2013 |
TW |
102113688 |
Claims
1. A .beta.-mannanase comprising a modified amino acid sequence of
SEQ ID NO: 2, wherein the modification is a substitution of
Tyrosine at position 216 with Tryptophan.
2. The .beta.-mannanase according to claim 1 wherein the amino acid
sequence of SEQ ID NO: 2 is encoded by ManBK gene isolated from
Aspergillus niger BK01.
3. The .beta.-mannanase according to claim 1 being an acidic and
thermotolerant mannanase.
4. The .beta.-mannanase according to claim 1 having a full length
amino acid sequence of SEQ ID NO: 4.
5. (canceled)
6. (canceled)
7. The .beta.-mannanase according to claim 1 wherein the
.beta.-mannanase is used in a food industry, a feed industry, or a
paper pulp industry.
8. The .beta.-mannanase according to claim 2 wherein the
.beta.-mannanase is used in a food industry, a feed industry, or a
paper pulp industry.
9. The .beta.-mannanase according to claim 3 wherein the
.beta.-mannanase is used in a food industry, a feed industry, or a
paper pulp industry.
10. The .beta.-mannanase according to claim 4 wherein the
.beta.-mannanase is used in a food industry, a feed industry, or a
paper pulp industry.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a .beta.-mannanase, and
more particularly to a .beta.-mannanase having improved enzymatic
activity.
BACKGROUND OF THE INVENTION
[0002] .beta.-1,4 Mannans are major components of hemicellulose in
plant cell wall of softwood, plant seeds and beans. Four types of
polysaccharides including linear mannan, galactomannan,
glucomannan, galactoglucomannan that are linked via
.beta.-1,4-glycosidic bonds compose mannans. Mannan hydrolysis
provides wide array of biotechnological applications, such as feed
manufacture, pulp and paper industries, and hydrolyzing coffee
extract to reduce viscosity. A set of enzymes are required for
complete degradation of mannans, including
endo-.beta.-1,4-mannanase (.beta.-mannanase, EC 3.2.1.78),
exo-.beta.-mannosidase (EC 3.2.1.25) to cleave the main chain, and
.beta.-glucosidase (EC 3.2.1.21), .alpha.-galactosidase (EC
3.2.1.22), and acetyl mannan esterase to remove side chain
decoration. Among them, .beta.-mannanase which catalyzes random
hydrolysis of manno-glycosidic bonds in the main chain is the key
enzyme. More recently, major products of .beta.-mannanase,
mannotriose and mannobiose (mannooligosaccharides, MOS), have been
proved beneficial as animal nutrition additive due to its prebiotic
properties.
[0003] .beta.-Mannanases are derived from various organisms
including bacteria, yeasts, and filamentous fungi. According to the
amino acid sequence homology, .beta.-mannanases are mostly
classified to glycoside hydrolase (GH) families 5, 26 and 113.
These families share the same (.beta./.alpha.).sub.8 folding and
catalytic machinery, that two glutamate residues at active site
serve as general acid/base and nucleophile to catalyze the cleavage
of glycosidic bonds via a retaining double displacement mechanisms.
Since industrial process is usually carried out at high
temperatures, stable enzyme usage under a broad range of
temperature is highly desirable. Therefore, .beta.-mannanase needs
to be modified to meet the requirement for different industrial
usages. There are two ways to achieve these goals, one way is to
screen suitable genes in nature, and the second way is modifying
current enzyme genes based on their 3-D structural information.
[0004] In the present invention, the crystal structure of
.beta.-mannanase is analyzed and the enzyme activity of
.beta.-mannanase is improved by site-directed mutagenesis of the
gene.
SUMMARY OF THE INVENTION
[0005] An object of the present invention is to modify
.beta.-mannanase by means of structural analysis and site-directed
mutagenesis to efficiently increase the enzyme activity, and
improve its economic value of industrial application.
[0006] According to an aspect of the present invention, there is
provided a .beta.-mannanase having increased enzymaic activity. The
.beta.-mannanase has a modified amino acid sequence of SEQ ID NO:
2, wherein the modification is a substitution of Tyrosine at
position 216 with Tryptophan.
[0007] In an embodiment, the amino acid sequence of SEQ ID NO: 2 is
encoded by ManBK gene isolated from Aspergillus niger BK01, and the
.beta.-mannanase is an acidic and thermotolerant mannanase.
[0008] In an embodiment, the .beta.-mannanase has a full length
amino acid sequence of SEQ ID NO: 4.
[0009] According to another aspect of the present invention, there
is provided a nucleic acid encoding the aforesaid .beta.-mannanase,
and a recombinant plasmid comprising the aforesaid nucleic
acid.
[0010] According to an additional aspect of the present invention,
there is provided an industrial use of the aforesaid
.beta.-mannanase, wherein the industrial use comprises uses in food
industry, feed industry, and paper pulp industry.
[0011] 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
[0012] FIG. 1 shows the gene sequence and the amino acid sequence
of the wild-type ManBK;
[0013] FIG. 2 shows the protein structure of the wild-type ManBK,
which was superimposed with Trichoderma reesei mannanase in complex
with mannobiose;
[0014] FIG. 3 shows the sequence of the mutagenic primer for the
Y216W mutant;
[0015] FIG. 4 shows the gene sequence and the amino acid sequence
of the Y216W mutant;
[0016] FIG. 5 shows the .beta.-mannanase activity analysis of the
wild-type ManBK and the Y216W mutant; and
[0017] FIG. 6 shows the kinetic analysis of the wild-type ManBK and
the Y216W mutant.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0018] 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.
[0019] In the present invention, a gene of the .beta.-mannanase
ManBK was isolated from Aspergillus niger BK01, and ManBK is an
acidic and thermotolerant .beta.-mannanase. In order to improve the
industrial application value of this enzyme, the protein structure
of the apo-form ManBK was solved by X-ray crystallography, and the
solved structure was superimposed with Trichoderma reesei mannanase
(having 57% similarity in protein sequence compared with ManBK) in
complex with mannobiose. Then, based on the structural information
of the enzyme, the important amino acid residues in the active site
were selected for site-directed mutagenesis to improve the
enzymatic activity. The enzyme modification process of ManBK and
the resulted mannanase protein are described in detail as
follows.
[0020] First, the ManBK gene was obtained from Aspergillus niger
BK01 (GenBank accession no. FJ268574), and as shown in FIG. 1, the
full length of sequence of the ManBK gene is 1038 base pairs (SEQ
ID NO: 1), which encodes a protein of 345 amino acids (SEQ ID NO:
2). The ManBK gene was constructed into pPICZ.alpha.A vector by
using EcoRI and NotI sites. The primers for polymerase chain
reaction were 5'-GGTATTGAGGGTCGCGCGGCGGC
GGCGGCGATGTCCTTCGCTTCCACTTCCG-3' (forward primer) and
5'-AGAGGAGAGTTAGAGCCTTAAGCGGAACCGATAGCAGC-3' (reverse primer). The
constructed plasmid was transformed into a competent cell as a
wild-type expression vector.
[0021] To solve the protein structure of ManBK by X-ray
crystallography, the protein crystal was obtained by using sitting
drop vapor diffusion method at room temperature by Hampton screen
kit. The protein crystal of ManBK in apo form was prepared by
mixing 2 .mu.l mannanase solution (10 mg/ml in 25 mM Tris-HCl, pH
7.5) with equal amounts of mixture solution and mother liquor, and
equilibrating with 500 .mu.l of the mother liquor at room
temperature. The wild-type ManBK crystal was obtained by a
condition composed of 0.1M Bis-Tris pH 5.5, 0.4M magnesium
chloride, and 29% PEG3350. The molecular replacement method was
used for phasing X-ray diffraction data, and the protein structure
of ManBK was subsequently determined by crystallographic
software.
[0022] FIG. 2 shows the protein structure of ManBK solved by X-ray
crystallography, and the solved structure was superimposed with
Trichoderma reesei mannanase in complex with mannobiose in subsites
+1 and +2. The protein structure of ManBK has
(.beta./.alpha.).sub.8 barrel fold, wherein 8 .mu.-sheets are
located in the interior and 8 .alpha.-helixes pack around the
exterior. By studying the structural information of ManBK, 30 amino
acid residues were selected to be modified. Particularly, Tyr216 is
located in the active site of the enzyme and may be important to
the catalytic reaction of ManBK, and thus is targeted for
site-directed mutagenesis, and it is found that the mutation of
Tyr216 improves the enzymatic activity of ManBK, while other
mutations do not show significant effects and are not redundantly
described here. The following describes the processes for
site-directed mutagenesis, protein expression and activity assay of
Y216W mutant.
[0023] The Y216W mutant was prepared by using QuikChange
site-directed mutagenesis kit with ManBK gene as a template. The
sequence of the primer for Y216W mutant was shown in FIG. 3,
wherein Y216W means Tyrosine at position 216 was mutated into
Tryptophan; in other words, the modification is a substitution of
Tyrosine at position 216 with Tryptophan. The original template was
removed via DpnI digestion under 37.degree. C., and then the
plasmid with mutated gene was transformed into E. coli and screened
with Ampicillin. Finally, the mutated gene was confirmed by DNA
sequencing. Therefore, the Y216W mutant was constructed, and as
shown in FIG. 4, the gene sequence was numbered as SEQ ID NO: 3,
and the amino acid sequence was numbered as SEQ ID NO: 4.
[0024] The wild-type and mutant ManBK were expressed in Pichia.
First, the plasmid DNA was linearized by PmeI and transformed into
the P. pastoris X33 strain by electroporation. The transformants
were selected on YPD (1% yeast extract, 2% peptone, 2% glucose, 2%
agar) plates containing 100 .mu.g/mL Zeocin and incubated at
30.degree. C. for 2 days. The picked colonies were inoculated into
5 ml YPD medium at 30.degree. C. overnight and further amplified
into 50 ml BMGY medium at 30.degree. C. overnight. After that, the
cultured medium was changed to 20 ml BMMY with 0.5% methanol to
induce the target protein expression. The samples were collected at
different time points for every 24 hours, and meanwhile, the
methanol was added into the flask to the final concentration of
0.5%. After induction for 4 days, the cells were harvested by
centrifugation at 3500 rpm and the supernatant was collected for
further purification.
[0025] The supernatant was purified by FPLC (fast protein liquid
chromatography) using Ni.sup.2+ column and DEAE column. Finally,
the wild-type and mutant ManBK peoteins, which had above 95%
purity, were concentrated up to 5 mg/ml in protein buffer (25 mM
Tris and 150 mM NaCl, pH 7.5) and then stored at -80.degree. C.
[0026] To verify the difference between the wild-type and mutant
ManBK, the .beta.-mannanase activity assay and the kinetic analysis
were performed. The .beta.-mannanase activity was determined by
dinitrosalicylic acid (DNS) method using mannose as a standard. The
reaction was started by mixing 0.2 mL appropriately diluted enzyme
sample with 1.8 mL of 3 mg/L locust bean gum (LBG) in 0.05 M
citrate acid, pH 5.3. After 5-min incubation at 50.degree. C., the
reaction was stopped by adding 3 ml of DNS-reagent and boiled for 5
min to remove residual enzyme activity. After cooling in cold water
bath for 5 min, the 540 nm absorbance of the reaction solution was
measured. One unit of .beta.-mannanase activity was defined as the
amount of enzyme releasing 1 .mu.mol of mannose equivalents per
minute per mg of total soluble proteins under the assay
conditions.
[0027] FIG. 5 shows the .beta.-mannanase activity analysis of the
wild-type ManBK and the Y216W mutant. The specific activity of the
wild-type ManBK and the Y216W mutant are 646 and 784 U/mg. These
results indicated that the specific activity of enzyme was
increased 19% when Tyr216 was mutated to Tryptophan.
[0028] For the kinetic analysis, optimal protein concentration was
first determined by using a series of 0.4-3.6 .mu.g/ml protein
solutions and 10 mg/ml LBG. The enzyme activity was then measured
by using the optimal level of protein and a series of 0.5-10 mg/ml
LBG solutions. Based on these data, the kinetic parameters were
obtained by using the Michaelis-Menten model and curve-fitting
analysis with a computer.
[0029] FIG. 6 shows the kinetic analysis of the wild-type ManBK and
the Y216W mutant. The Y216W mutant had a higher catalytic rate
(k.sub.cat) than the wild-type enzyme while the K.sub.m value of
the Y216W mutant was also slightly higher than that of the
wild-type enzyme. Higher K.sub.m of an enzyme indicates lower
affinity to the substrate. However, it also indicates faster
substrate release rate. Therefore, with the presence of sufficient
substrate in general industrial application, the specific activity
of Y216W mutant was higher than that of the wild-type enzyme.
[0030] From the above, in order to improve the enzymatic activity
of ManBK, the present invention solved the protein structure of the
apo-form ManBK by X-ray crystallography, and the ManBK structure
was superimposed with Trichoderma reesei mannanase complex
structure. According to the superimposed structure, Tyr216 which is
located in the active site is selected for site-directed
mutagenesis and the tyrosine at position 216 was mutated into
tryptophan to construct the Y216W mutant. From the .beta.-mannanase
activity assay and the kinetic analysis, the Y216W mutant exhibited
significantly increased specific activity when compared to the
wild-type, so it can reduce the production cost and will has more
industrial applications. In addition, since ManBK has
thermostability and can be applied to many industries with thermal
processes, once the enzymatic activity thereof is increased, the
production cost will be reduced and the profit will be increased.
Therefore, the present invention successfully modified ManBK to
improve the enzymatic activity thereof, and thus, the present
invention possesses high industrial value.
[0031] 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 CWU 1
1
411038DNAAspergillus niger BK01 1tccttcgctt ccacttccgg attgcagttc
actattgacg gtgagactgg ttacttcgct 60ggaactaact cctactggat cggtttcttg
actgacaacg ctgacgttga cttggttatg 120ggtcacttga agtcctccgg
tttgaagatc ttgagagttt ggggtttcaa cgacgttact 180tcccaaccat
cctccggtac tgtttggtat caattgcacc aggacggaaa gtccactatc
240aacactggtg ctgacggatt gcagagattg gactacgttg tttcctccgc
tgagcagcac 300gacatcaagc ttatcatcaa cttcgttaac tactggactg
actacggtgg tatgtccgct 360tacgtttctg cttatggtgg ttctggtgag
actgacttct acacttccga cactatgcag 420tccgcttacc agacttacat
caagactgtt gttgagagat actccaactc ctccgctgtt 480ttcgcttggg
aattggctaa cgagccaaga tgtccttcct gtgacacttc cgtcttgtac
540aactggatcg aaaagacttc caagttcatc aagggtttgg acgctgacag
aatggtctgt 600attggtgacg agggtttcgg tttgaacatt gactctgacg
gttcctaccc ataccaattc 660tccgagggtt tgaacttcac tatgaacttg
gacatcgaca ctatcgactt cggtacattg 720cacttgtacc cagactcttg
gggtacttct gatgattggg gtaacggttg gatcactgct 780catggtgctg
cttgtaaggc tgctggtaag ccatgtttgt tggaagagta cggtgttact
840tccaaccact gttctgttga gggtgcttgg caaaagactg ctttgtccac
tactggtgtt 900ggtgctgact tgttctggca atacggtgac gacttgtcca
ctggtaagtc tccagatgac 960ggtaacacta tctactacgg tacttccgac
taccagtgtt tggttactga ccacgttgct 1020gctatcggtt ccgcttaa
10382345PRTAspergillus niger BK01 2Ser Phe Ala Ser Thr Ser Gly Leu
Gln Phe Thr Ile Asp Gly Glu Thr 1 5 10 15 Gly Tyr Phe Ala Gly Thr
Asn Ser Tyr Trp Ile Gly Phe Leu Thr Asp 20 25 30 Asn Ala Asp Val
Asp Leu Val Met Gly His Leu Lys Ser Ser Gly Leu 35 40 45 Lys Ile
Leu Arg Val Trp Gly Phe Asn Asp Val Thr Ser Gln Pro Ser 50 55 60
Ser Gly Thr Val Trp Tyr Gln Leu His Gln Asp Gly Lys Ser Thr Ile65
70 75 80Asn Thr Gly Ala Asp Gly Leu Gln Arg Leu Asp Tyr Val Val Ser
Ser 85 90 95Ala Glu Gln His Asp Ile Lys Leu Ile Ile Asn Phe Val Asn
Tyr Trp 100 105 110Thr Asp Tyr Gly Gly Met Ser Ala Tyr Val Ser Ala
Tyr Gly Gly Ser 115 120 125 Gly Glu Thr Asp Phe Tyr Thr Ser Asp Thr
Met Gln Ser Ala Tyr Gln 130 135 140Thr Tyr Ile Lys Thr Val Val Glu
Arg Tyr Ser Asn Ser Ser Ala Val145 150 155 160Phe Ala Trp Glu Leu
Ala Asn Glu Pro Arg Cys Pro Ser Cys Asp Thr 165 170 175Ser Val Leu
Tyr Asn Trp Ile Glu Lys Thr Ser Lys Phe Ile Lys Gly 180 185 190Leu
Asp Ala Asp Arg Met Val Cys Ile Gly Asp Glu Gly Phe Gly Leu 195 200
205 Asn Ile Asp Ser Asp Gly Ser Tyr Pro Tyr Gln Phe Ser Glu Gly Leu
210 215 220 Asn Phe Thr Met Asn Leu Asp Ile Asp Thr Ile Asp Phe Gly
Thr Leu225 230 235 240 His Leu Tyr Pro Asp Ser Trp Gly Thr Ser Asp
Asp Trp Gly Asn Gly 245 250 255 Trp Ile Thr Ala His Gly Ala Ala Cys
Lys Ala Ala Gly Lys Pro Cys 260 265 270Leu Leu Glu Glu Tyr Gly Val
Thr Ser Asn His Cys Ser Val Glu Gly 275 280 285Ala Trp Gln Lys Thr
Ala Leu Ser Thr Thr Gly Val Gly Ala Asp Leu 290 295 300Phe Trp Gln
Tyr Gly Asp Asp Leu Ser Thr Gly Lys Ser Pro Asp Asp305 310 315
320Gly Asn Thr Ile Tyr Tyr Gly Thr Ser Asp Tyr Gln Cys Leu Val Thr
325 330 335Asp His Val Ala Ala Ile Gly Ser Ala 340 345
31038DNAArtificial SequenceSynthetically generated DNA encoding a
modified enzyme 3tccttcgctt ccacttccgg attgcagttc actattgacg
gtgagactgg ttacttcgct 60ggaactaact cctactggat cggtttcttg actgacaacg
ctgacgttga cttggttatg 120ggtcacttga agtcctccgg tttgaagatc
ttgagagttt ggggtttcaa cgacgttact 180tcccaaccat cctccggtac
tgtttggtat caattgcacc aggacggaaa gtccactatc 240aacactggtg
ctgacggatt gcagagattg gactacgttg tttcctccgc tgagcagcac
300gacatcaagc ttatcatcaa cttcgttaac tactggactg actacggtgg
tatgtccgct 360tacgtttctg cttatggtgg ttctggtgag actgacttct
acacttccga cactatgcag 420tccgcttacc agacttacat caagactgtt
gttgagagat actccaactc ctccgctgtt 480ttcgcttggg aattggctaa
cgagccaaga tgtccttcct gtgacacttc cgtcttgtac 540aactggatcg
aaaagacttc caagttcatc aagggtttgg acgctgacag aatggtctgt
600attggtgacg agggtttcgg tttgaacatt gactctgacg gttcctggcc
ataccaattc 660tccgagggtt tgaacttcac tatgaacttg gacatcgaca
ctatcgactt cggtacattg 720cacttgtacc cagactcttg gggtacttct
gatgattggg gtaacggttg gatcactgct 780catggtgctg cttgtaaggc
tgctggtaag ccatgtttgt tggaagagta cggtgttact 840tccaaccact
gttctgttga gggtgcttgg caaaagactg ctttgtccac tactggtgtt
900ggtgctgact tgttctggca atacggtgac gacttgtcca ctggtaagtc
tccagatgac 960ggtaacacta tctactacgg tacttccgac taccagtgtt
tggttactga ccacgttgct 1020gctatcggtt ccgcttaa 10384345PRTArtificial
SequenceSequence synthetically translated from SEQ ID NO 3 4Ser Phe
Ala Ser Thr Ser Gly Leu Gln Phe Thr Ile Asp Gly Glu Thr 1 5 10 15
Gly Tyr Phe Ala Gly Thr Asn Ser Tyr Trp Ile Gly Phe Leu Thr Asp 20
25 30 Asn Ala Asp Val Asp Leu Val Met Gly His Leu Lys Ser Ser Gly
Leu 35 40 45 Lys Ile Leu Arg Val Trp Gly Phe Asn Asp Val Thr Ser
Gln Pro Ser 50 55 60 Ser Gly Thr Val Trp Tyr Gln Leu His Gln Asp
Gly Lys Ser Thr Ile65 70 75 80Asn Thr Gly Ala Asp Gly Leu Gln Arg
Leu Asp Tyr Val Val Ser Ser 85 90 95Ala Glu Gln His Asp Ile Lys Leu
Ile Ile Asn Phe Val Asn Tyr Trp 100 105 110Thr Asp Tyr Gly Gly Met
Ser Ala Tyr Val Ser Ala Tyr Gly Gly Ser 115 120 125 Gly Glu Thr Asp
Phe Tyr Thr Ser Asp Thr Met Gln Ser Ala Tyr Gln 130 135 140Thr Tyr
Ile Lys Thr Val Val Glu Arg Tyr Ser Asn Ser Ser Ala Val145 150 155
160Phe Ala Trp Glu Leu Ala Asn Glu Pro Arg Cys Pro Ser Cys Asp Thr
165 170 175Ser Val Leu Tyr Asn Trp Ile Glu Lys Thr Ser Lys Phe Ile
Lys Gly 180 185 190Leu Asp Ala Asp Arg Met Val Cys Ile Gly Asp Glu
Gly Phe Gly Leu 195 200 205 Asn Ile Asp Ser Asp Gly Ser Trp Pro Tyr
Gln Phe Ser Glu Gly Leu 210 215 220 Asn Phe Thr Met Asn Leu Asp Ile
Asp Thr Ile Asp Phe Gly Thr Leu225 230 235 240 His Leu Tyr Pro Asp
Ser Trp Gly Thr Ser Asp Asp Trp Gly Asn Gly 245 250 255 Trp Ile Thr
Ala His Gly Ala Ala Cys Lys Ala Ala Gly Lys Pro Cys 260 265 270Leu
Leu Glu Glu Tyr Gly Val Thr Ser Asn His Cys Ser Val Glu Gly 275 280
285Ala Trp Gln Lys Thr Ala Leu Ser Thr Thr Gly Val Gly Ala Asp Leu
290 295 300Phe Trp Gln Tyr Gly Asp Asp Leu Ser Thr Gly Lys Ser Pro
Asp Asp305 310 315 320Gly Asn Thr Ile Tyr Tyr Gly Thr Ser Asp Tyr
Gln Cys Leu Val Thr 325 330 335Asp His Val Ala Ala Ile Gly Ser Ala
340 345
* * * * *