CN113512542B - Rhamnosidase mutant and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a rhamnosidase mutant and a preparation method and application thereof, relating to the technical field of biological medicine, which comprises the following steps of preparing a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1 is mutated into alanine, wherein the mutation site is a combination of more than two of 512 th tryptophan, 570 th histidine and 579 th lysine. According to the rhamnosidase mutant, the combined mutant of the alpha-L-rhamnosidase TpeRha is constructed by the site-directed mutagenesis technology, and multi-component flavonoid glycoside in epimedium total flavone is catalytically converted to prepare the icariside I composition, so that the enzymolysis time is short, and the conversion rate is high.
Description
Technical Field
The invention relates to the technical field of biological medicines, and particularly relates to a rhamnosidase mutant and a preparation method and application thereof.
Background
The epimedium is a plant of the genus Epimedium of the family berberidaceae, is a traditional Chinese medicine collected in Chinese pharmacopoeia, has the effects of benefiting vital essence, strengthening bones and muscles, tonifying waist and knees, strengthening heart force and the like, has the main active ingredient of epimedium flavonoid glycoside containing 2-3 glycosyl groups, and has the effects of resisting cancer, resisting osteoporosis, resisting aging, regulating immunity, tonifying kidney yang and the like. A large number of researches show that icariin protoglycosides such as epimedin C and icariin are poorly absorbed in the small intestine, and are mainly absorbed in the form of intestinal metabolites (secondary glycosides or aglycones) to exert curative effects. Therefore, the method has important significance for enhancing the drug synthesis and increasing the bioavailability to improve the drug effect. Icariside I is a high-activity substance in epimedium flavone secondary glycoside, shows potential enhancement of host immune function and anticancer activity through flora adjustment, and has good anticancer drug development prospect. However, the content of icariside I in epimedium is extremely low, and the market demand of the icariside I is more and more increased along with the continuous excavation of the active function of the icariside I. The traditional plant extraction method has extremely low content and high difficulty in later purification, and the epimedium extract has relatively high content of protoglycosides, such as epimedin C (accounting for 20.8 percent of total flavonoids in the epimedium) and icariin (accounting for 21.9 percent of the total flavonoids in the epimedium), and the preparation of the icariside I by using the protoglycosides as precursor substances and utilizing alpha-L-rhamnosidase is the most effective preparation method at present.
alpha-L-rhamnosidase (EC 3.2.1.40) is a glycoside hydrolase with wide application prospect, widely exists in plants, animals and microorganisms, can break the glycosidic bond formed by the reaction of alcoholic hydroxyl and hemiacetal in an exo-or endo-mode, and plays an important role in the synthesis and hydrolysis processes of glycoconjugates and saccharides in organisms. alpha-L-rhamnosidase has wide application value in the industries of food, medicine and the like, and a great amount of reports have shown that the alpha-L-rhamnosidase can effectively and directionally hydrolyze natural active substances or natural medicines containing rhamnoside, such as ginsenoside, naringin, hesperidin and rutin, and improve the biological activity and bioavailability of the original substances, and is a hydrolase commonly used in industrial production.
However, a large technical barrier still exists when the alpha-L-rhamnosidase is applied in a large scale, the activity of the natural alpha-L-rhamnosidase is insufficient, and the reaction environment of the natural alpha-L-rhamnosidase often cannot meet the optimal conditions of the natural enzyme. With the gradual maturity of related technologies such as molecular biology, genetic engineering and the like, the protein sequence is edited and modified, so that the reading of an enzyme catalysis mechanism can be strengthened and the design of the enzyme can be guided. The protein engineering is mainly designed by using site-directed mutagenesis technology, and the protein is purposefully designed and modified according to the understanding of the relationship between the structure and the function of the protein, so that the performance of the protein can achieve the expected effect. At present, a great deal of research and success are carried out on the modification of the catalytic activity, the substrate specificity, the thermal stability, the allosteric effect of the enzyme and the like of the natural enzyme by using a rational design technology. However, rational design methods require a deeper understanding of the three-dimensional structure of the enzyme and the structural and functional interrelationships. With the development of computational biology, methods for guiding enzyme modification by computer-aided design are becoming mature. A computer simulation method is utilized to establish a three-dimensional structure model of the enzyme, analyze the conformation relation of the enzyme and the substrate, quickly locate a region related to catalytic reaction, reduce the capacity of a mutant library, efficiently obtain related targets and provide guidance for protein engineering modification.
The patent number is CN201710448315.6, which is a Chinese patent named a glycosidase composition and a method for preparing icariin by an enzyme method, the preparation of the icariin by the composition of alpha-L-rhamnosidase and beta-glucosidase has higher conversion rate of the icariin, but the content of icariin I in the icariin is unknown. The patent number is CN201710332977.7, entitled method for preparing icariin by converting total flavonoids of epimedium by enzyme method, a two-enzyme system consisting of heat-resistant alpha-L-rhamnosidase and heat-resistant beta-glucosidase synergistically converts multicomponent flavonoid glycoside in total flavonoids of epimedium to generate the icariin, and icariside I can not be obtained by high-efficiency catalytic conversion. Therefore, the efficient rhamnosidase mutant which can hydrolyze icariin to generate icariside I is obtained by rationally designing the alpha-L-rhamnosidase TpeRha by adopting the site-directed mutagenesis technology, and is a significant research direction.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a rhamnosidase mutant which is used for preparing an icariside I composition and has high conversion rate.
The invention also aims to provide a preparation method of the rhamnosidase mutant.
The invention also aims to provide application of the rhamnosidase mutant in preparing the icariside I composition.
One of the purposes of the invention is realized by adopting the following technical scheme:
a rhamnosidase mutant is prepared by using a polypeptide with an amino acid sequence shown as SEQ ID NO: 1 to alanine (Ala, A), wherein the mutation site is a combination of two or more of tryptophan (Trp, W) at position 512, histidine (His, H) at position 570 and lysine (Lys, K) at position 579.
Further, the amino acid sequence is as shown in SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: shown at 9.
Further, the polypeptide comprises a nucleotide sequence encoding an amino acid sequence shown as SEQ ID NO: 6, and the nucleotide sequence of the coding gene is shown as SEQ ID NO: shown at 10.
The second purpose of the invention is realized by adopting the following technical scheme:
the preparation method of the rhamnosidase mutant comprises the following steps:
s1, connecting the rhamnosidase gene to a plasmid to obtain a recombinant plasmid;
s2, designing a mutation primer, carrying out PCR amplification by adopting the mutation primer and taking the recombinant plasmid as a template, and carrying out enzyme digestion to remove template DNA to obtain a mutation product;
s3, transforming the mutation product into a host cell, screening to obtain a rhamnosidase mutant expression strain, and performing induced expression to obtain a rhamnosidase mutant.
Further, the nucleotide sequence of the rhamnosidase gene is shown as SEQ ID NO: 2, respectively.
Further, the sequence of the mutant primer is shown as SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15 and SEQ ID NO: shown at 16.
Further, the host cell is saccharomyces cerevisiae or escherichia coli.
Further, the specific parameters of the PCR amplification are: pre-denaturation at 98 ℃ for 3 min; and (3) cycle setting: denaturation at 98 deg.C for 10s, annealing at 62 deg.C for 15s, extension at 72 deg.C for 3min, and 30 cycles; finally, extending for 10min at 72 ℃; the reaction was complete.
The third purpose of the invention is realized by adopting the following technical scheme:
an application of rhamnosidase mutant in preparing icariside I composition is provided.
Further, the icariside I composition is prepared by catalyzing and converting multicomponent flavone glycoside in epimedium total flavone by using rhamnosidase mutant.
Compared with the prior art, the invention has the beneficial effects that:
according to the rhamnosidase mutant, the combined mutant of the alpha-L-rhamnosidase TpeRha is constructed by the site-directed mutagenesis technology, and multi-component flavonoid glycoside in epimedium total flavone is catalytically converted to prepare the icariside I composition, so that the enzymolysis time is short, and the conversion rate is high.
The preparation method of the rhamnosidase mutant disclosed by the invention is characterized in that the alpha-L-rhamnosidase TpeRha from Thermotoga petrosella DSM 13995 is rationally designed by a site-directed mutagenesis technology, transformed into a host cell and then induced to express to obtain the high-efficiency rhamnosidase mutant.
The invention discloses an application of a rhamnosidase mutant in preparation of an icariside I composition, which is to prepare the composition containing 0.01-99% of the icariside I by utilizing the rhamnosidase mutant to catalyze and convert multi-component flavone glycoside in total flavonoids of epimedium.
Drawings
FIG. 1 is a graph showing the standard curve of icariside I content;
FIG. 2 is a bar graph comparing the conversion rates of TpeRha enzyme and each mutant enzyme to icariside I;
FIG. 3 is a diagram of the pESE-HIS-WHK plasmid vector;
FIG. 4 is a graph showing HPLC results after the reaction of TpeRha enzyme and TpeRha-WHK enzyme;
FIG. 5 is HPLC result chart of the content of each component before and after processing herba Epimedii extract.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific embodiments, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict. The following are specific examples of the present invention, and raw materials, equipments and the like used in the following examples can be obtained by purchasing them unless otherwise specified.
Example 1
Obtaining of recombinant Escherichia coli wild strain BL21 (DE 3)/pET-28 a-TpeRhha:
the alpha-L-rhamnosidase gene from Thermotoga petroselinus DSM 13995 is optimized by the codon bias of Saccharomyces cerevisiae, and the nucleotide sequence of the gene is shown as SEQ ID NO: 2 (GenBank: ABQ 47687.1), connecting it to a pET-28a plasmid, obtaining a recombinant plasmid named as pET-28a-TpeRha, transforming the plasmid into escherichia coli BL21 (DE 3), expressing the amino acid sequence of the obtained alpha-L-rhamnosidase as shown in SEQ ID NO: 1, the recombinant strain is named as BL21 (DE 3)/pET-28 a-TpeRha.
Example 2
Obtaining of TpeRha mutant strain:
(I) construction of mutant enzyme vector by whole plasmid PCR
1. Extracting the recombinant plasmid pET-28a-TpeRha in a small amount;
2. designing mutation primer, the primer has 15 bp overlap region and 15 bp extension region, and designing mutation site in the overlap region. The plasmid pET-28a-TpeRha is taken as a template to carry out PCR full plasmid amplification, and the PCR system is shown in the table 1:
the primers Primer-F and Primer-R are PCR upstream primers and PCR downstream primers designed according to different mutation sites, and specific Primer information is shown in Table 2.
Primer pairs W512A-F and W512A-R are used for obtaining TpeRha mutase TpeRha-W512A, TpeRha-WH, TpeRha-WK and TpeRha-WHK, and the amino acid sequences of the primer pairs are respectively SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6.
primer pairs H570A-F and H570A-R were used to obtain the TpeRha mutases TpeRha-H570A, TpeRha-WH, TpeRha-HK and TpeRha-WHK, whose amino acid sequences are SEQ ID NO: 7. SEQ ID NO: 4. SEQ ID NO: 8 and SEQ ID NO: 6.
the primer pair K579A-F and K579A-R is used for obtaining the TpeRha mutase TpeRha-K579A, TpeRha-WK, TpeRha-HK and TpeRha-WHK, and the amino acid sequences of the primer pair are respectively SEQ ID NO: 9. SEQ ID NO: 5. SEQ ID NO: 8 and SEQ ID NO: 6.
PCR amplification procedure: pre-denaturation at 98 ℃ for 3 min; and (3) cycle setting: denaturation at 98 deg.C for 10s, annealing at 62 deg.C for 15s, extension at 72 deg.C for 3min, and 30 cycles; finally, extending for 10min at 72 ℃; after the reaction is finished, the PCR product is recovered by using the kit.
3. And (3) performing enzyme digestion to remove template DNA, and performing enzyme digestion on the PCR recovered product, wherein an enzyme digestion system is shown in Table 3:
and (3) digesting the enzyme digestion system in a metal bath at 37 ℃ for 1 h, and recovering the enzyme digestion product by using a kit after the reaction is finished.
(II) sequencing verification of the successful construction of mutant enzyme strain
And (3) transforming the enzyme digestion recovery product into an escherichia coli BL21 (DE 3) competent cell, inverting the cell at 37 ℃ for overnight culture, and selecting a quasi-positive transformant for sequencing verification to successfully obtain a mutant enzyme expression strain. Construction of the multiple site combinatorial mutants the procedure described in example 2 was repeated with single site mutants or double site mutant site templates.
Example 3
Whole-cell catalytic reaction of each mutant enzyme of TpeRha:
(I) induction of wild enzyme recombinant strain and mutant recombinant strain and preparation of whole cell enzyme solution
1. Taking a wild enzyme recombinant strain and a mutant enzyme expression strain, and streaking and activating on an LB plate containing Kan (100 mu g/mL). After being subjected to inverted culture at 37 ℃ overnight, a single colony is selected and inoculated into 5 mL of LB liquid medium containing Kan, and shaking culture is carried out at 37 ℃ and 200 r/min for 12-16 h. Inoculating the seed solution cultured overnight according to the inoculation amount of 1% into 20 mL of fresh TB liquid culture medium containing Kan, carrying out shake culture at 37 ℃ at 200 r/min for 2-3 h until the OD600 is 0.6-0.8, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.5 mM, placing the culture solution at 37 ℃ and carrying out shake culture at 200 r/min for 16 h, and carrying out induced expression on the protein.
2. After the induction, the cells were collected by centrifugation at 8000 rpm for 8 min at 4 ℃ and washed once with a citric acid-phosphoric acid buffer (pH 4.6), and then resuspended in an appropriate amount of citric acid-phosphoric acid buffer (pH 4.6) according to the wet weight of the cells to obtain a cell suspension with a whole cell concentration of 200 g/mL, all on ice or at 4 ℃.
(II) Whole cell reaction
1. Using the whole cell suspension of the wild type TpeRhha enzyme and each mutant enzyme obtained in the above experiment as an enzyme solution, a reaction system was prepared as shown in Table 4:
2. the reaction system is reacted for 1d at 55 ℃. After the reaction is finished, 10 times volume of DMSO is added to stop the reaction, cell thalli are removed by centrifugation at 12000 rpm for 1 min after full vortex, and a reaction product is filtered by a 0.45-micron organic filter head and then subjected to HPLC detection.
Method for detecting icariside I
Quantitative analysis of icariside I was performed by HPLC, and the chromatographic conditions were as follows:
high performance liquid chromatograph: agilent 1100 Series
A chromatographic column: diamond C18 (250 mm X4.6 mm X5 μm)
A detector: VWD detector with detection wavelength of 270 nm
Mobile phase ratio and elution conditions: the flow rate is 1 mL/min; the column temperature is 30 ℃; the sample volume is 10 mu L; the gradient elution system is shown in table 5:
preparation of (IV) icariside I standard curve
Using DMSO as a solvent, icariside i control (98.6% HPLC) was weighed to prepare 0, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, and 0.4 mg/mL standard solutions, and the solutions were tested under the above HPLC test conditions, as shown in fig. 1, in a coordinate system of icariside i concentration and peak area, the standard curve was y =22083x +46.44, and R is R =22083x +46.442=0.9998。
(V) analysis of results
The result of the whole-cell catalytic reaction is shown in figure 2, and the reaction time is 1d under the reaction condition of 55 ℃ and pH 4.6, and the result shows that the conversion rate of the product for generating the icariside I by using the wild type TpeRha enzyme reaction is only 3.73 +/-0.09%, and the conversion rates of the icariside I are respectively improved by 4.73 times, 15.63 times and 9.83 times by using the single-point mutant enzymes TpeRha-W512A enzyme, TpeRha-H570A enzyme and TpeRha-K579A enzyme; the double-point combined mutant enzyme TpeRha-WH enzyme, TpeRha-WK enzyme and TpeRha-HK enzyme are used for reaction, and the conversion rate of icariside I is respectively improved by 13.96 times, 10.07 times and 20.96 times; the three-point combined mutant enzyme TpeRhha-WHK is used for reaction, the conversion rate of icariside I is improved by 21.43 times, and the conversion rate is up to 79.85 +/-0.61%.
Example 4
Constructing mutant enzyme recombinant saccharomyces cerevisiae engineering bacteria:
construction of mutant enzyme Saccharomyces cerevisiae vector
1. Extracting the recombinant plasmid pET-28 a-TpeRhha-WHK and the plasmid of a saccharomyces cerevisiae expression vector pESC-HIS in a small quantity; the nucleotide sequence of the coding gene of the mutant enzyme TpeRhha-WHK is shown as SEQ ID NO: shown at 10.
2. Designing an amplification primer, wherein the primer has a 17 bp homologous arm, and carrying out PCR amplification by using a plasmid pET-28 a-TpeRhha-WHK as a template, wherein a PCR system is shown in a table 6:
the specific primer information of the primers pESC-WHK-F and pESC-WHK-R is shown in Table 7.
PCR amplification procedure: pre-denaturation at 98 ℃ for 3 min; and (3) cycle setting: denaturation at 98 ℃ for 10s, annealing at 52 ℃ for 15s, extension at 72 ℃ for 90 s, and 30 cycles; finally, extending for 10min at 72 ℃; after the reaction, the PCR product was recovered using the kit to obtain whk gene fragment.
3. The linearized expression vector pESC-HIS was digested as shown in Table 8:
and (3) placing the enzyme digestion system in a metal bath at 37 ℃ for enzyme digestion linearization for 45 min, and recovering the enzyme digestion product by using a gel recovery kit after the reaction is finished.
4. The whk gene fragment and the pESC-HIS linearized vector fragment were ligated, and the fragment and the vector were ligated by using a seamless cloning kit, in the system shown in Table 9:
the connection system is placed in a metal bath at 37 ℃ for connection for 30 min, after the connection is finished, escherichia coli Top 10 is transformed, and then the escherichia coli Top 10 is coated on an LB solid culture medium containing Amp and is inversely cultured at 37 ℃ overnight.
5. The transformant was successfully constructed by colony PCR and sequencing to identify the vector, and the results are shown in FIG. 3.
(II) mutant enzyme vector transformation Saccharomyces cerevisiae BY4742
Extracting a recombinant expression vector pESC-HIS-WHK with correct sequencing from escherichia coli Top 10 in a plasmid form, preparing competent cells of saccharomyces cerevisiae BY4742 BY using sorbitol, transforming the plasmid vector into the competent cells of saccharomyces cerevisiae BY4742 BY combining an electric shock transformation method, performing screening culture BY using an SD-HIS histidine defective culture medium according to auxotrophy of the saccharomyces cerevisiae, and performing inverted culture at 30 ℃ for 2 d. And (3) carrying out colony PCR identification on the transformant BY using the identification primer GAL-F/CYC-R in the table 2, determining that the vector is successfully converted, and successfully constructing the recombinant saccharomyces cerevisiae engineering bacteria BY 4742/pESC-HIS-WHK.
Example 5
Yeast fermentation and whole cell catalytic reaction of mutant enzyme
Fermentation of recombinant yeast BY4742/pESC-HIS-WHK
1. And (4) taking the recombinant saccharomyces cerevisiae storage strain, and streaking and activating on an SD-HIS (SD-HIS) plate. After being subjected to inversion culture at 30 ℃ overnight, a single colony is picked and inoculated into 15 mL of SD-HIS liquid culture medium, and shaking culture is carried out at 30 ℃ and 200 r/min for 48 h. After shaking culture, centrifuging at 4 deg.C and 6000 rpm for 5 min to collect Saccharomyces cerevisiae cells, transferring into 50 mL SD-HIS induction culture medium (with the original formula of SD-HIS glucose being changed into galactose, and the addition amount being unchanged), and fermenting and culturing at 30 deg.C and 200 r/min for 4 d.
2. After the fermentation was completed, the cells were collected by centrifugation at 8000 rpm for 8 min at 4 ℃ and washed once with a citric acid-phosphoric acid buffer (pH 4.6), and then resuspended in an appropriate amount of citric acid-phosphoric acid buffer (pH 4.6) according to the wet weight of the cells to obtain a cell suspension having a whole cell concentration of 200 g/mL, all on ice or at 4 ℃.
(II) Whole cell reaction
The whole-cell catalytic reaction of Saccharomyces cerevisiae was performed in the same manner as in example 3.
(III) product detection and results
After the reaction is carried out for 1d under the conditions of 55 ℃ and pH 4.6, the HPLC result of the icariside I generated by the whole-cell catalytic hydrolysis of icariin by the saccharomyces cerevisiae is shown in figure 4, the conversion rate is 95.54 +/-0.04%, and the HPLC result is more advantageous than the whole-cell catalytic result of escherichia coli, and in figure 4, Sc/TpeRha-WHK is mutant enzyme TpeRha-WHK in a saccharomyces cerevisiae system; the Ec/TpeRha-WHK is mutase TpeRha-WHK in an escherichia coli system, and the TpeRha is wild-type alpha-L-rhamnosidase; ICA is icariin.
Example 6
The application of mutant enzyme recombinant saccharomyces cerevisiae BY4247/pESC-HIS-WHK in the epimedium extract:
50 mL of SD-HIS medium (pH 5) was used as a fermentation medium, and 1g of Epimedium extract (containing 20% epimedin C and icariin) was added thereto. Inoculating 1% mutant enzyme recombinant Saccharomyces cerevisiae BY4247/pESC-HIS-WHK, fermenting at 30 deg.C and 200rpm for 1d, adding galactose (final concentration of 20 g/L), and fermenting for 4 d. The fermentation temperature was then increased to 55 ℃ and fermentation continued for 1 d. The fermentation broth was added to twice the volume of DMSO and mixed well and centrifuged to take the supernatant for HPLC assay, the results are shown in FIG. 5.
As shown in FIG. 5, the epimedium extract components before and after fermentation changed significantly, the epimedin C (retention time at 6.520 min) content after fermentation was significantly reduced, the difference in icariin (retention time at 7.283 min) content was not great, the icariside I (retention time at 13.918 min) content with high biological activity was significantly increased, and the specific component changes are shown in Table 10 below.
As can be seen from FIG. 5 and Table 10, the mutant enzyme recombinant Saccharomyces cerevisiae BY4247/pESC-HIS-WHK can effectively convert epimedin C into icariside I after fermentation, and has short conversion time and high specificity.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
SEQUENCE LISTING
<110> Guangdong Jinjunkang Biotech Ltd
<120> rhamnosidase mutant and preparation method and application thereof
<130> 16
<160> 16
<170> PatentIn version 3.3
<210> 1
<211> 876
<212> PRT
<213> Thermotoga petrophila DSM 13995
<400> 1
Met Ile Gln Ala Cys Asp Leu Arg Cys Glu Tyr Leu Thr Ser Pro Val
1 5 10 15
Leu Gly Leu Asp Val Ile Pro Arg Phe Ser Trp Arg Leu Lys Gly Asn
20 25 30
Gly Lys Lys Gln Thr Arg Tyr Lys Ile Ile Val Ser Asp Asn Phe Asp
35 40 45
Asp Ile Glu Arg Gly Ile Gly Asn Val Trp Glu Ser Glu Lys Asp Ser
50 55 60
Ser Lys Asn Leu Asn Ile Glu Tyr Glu Gly Pro Lys Leu Lys Ala Tyr
65 70 75 80
Lys Gly Tyr Tyr Trp Arg Val Lys Leu Trp Asp Glu Lys Glu Asn Gly
85 90 95
Pro Trp Ser Glu Thr Ala Tyr Phe Glu Met Gly Pro Leu Glu Asp Trp
100 105 110
Arg Gly Lys Trp Ile Thr Met Pro Ser Pro Leu Ser Phe Lys Asp Pro
115 120 125
Ala His Arg His Glu Leu Phe Tyr Ala Met Tyr Phe Arg Lys Glu Phe
130 135 140
Leu Leu Asn Lys Glu Val Glu Lys Ala Arg Val Tyr Val Ser Gly Leu
145 150 155 160
Gly Val Tyr Glu Leu His Leu Asn Gly Lys Arg Val Gly Asn Asn Val
165 170 175
Leu Asp Pro Ala Pro Thr Asp Tyr Asn Lys Val Ala Leu Tyr Ser Thr
180 185 190
Tyr Asp Val Thr Gln Tyr Leu Thr Thr Gly Lys Asn Thr Ile Gly Val
195 200 205
Ile Leu Gly Asn Gly Arg His Ile Arg Asp Tyr Gly Tyr Ser Lys Pro
210 215 220
Lys Leu Tyr Leu Gln Leu Leu Val Phe Tyr Lys Asp Gly Ser Arg Glu
225 230 235 240
Phe Ile Cys Ser Asp Glu Thr Trp Lys Val Ser His Gly Pro Leu Lys
245 250 255
Glu Asn Gly Ile Tyr Phe Gly Glu Val Tyr Asp Ala Arg Asp Glu Ile
260 265 270
Ser Gly Trp Asp Ser Pro Gly Phe Asp Asp Arg Asn Trp Ser Glu Val
275 280 285
Glu Ile Val Glu Gly Pro Ser Leu Lys Ala Gln Leu Ile Pro Val Ile
290 295 300
Arg Val Cys Glu Val Ile Lys Pro Lys Arg Leu Trp Leu Ser Ser Arg
305 310 315 320
Gly Thr Phe Ile Val Asp Phe Gly Lys Asn Ile Ser Gly Trp Val Lys
325 330 335
Leu Arg Val Asn Asn Gly Lys Arg Gly Glu Lys Ile Ile Ile Arg Tyr
340 345 350
Ala Glu Val Leu Asp Pro Ser Met Asp Arg Leu Asp Thr Arg Asn Leu
355 360 365
Arg Leu Ala Arg Ala Thr Asp Glu Tyr Ile Leu Lys Gly Gln Gly Val
370 375 380
Glu Ile Tyr Glu Pro Arg Phe Thr Tyr His Gly Phe Arg Tyr Val Glu
385 390 395 400
Val Glu Asp Tyr Pro Gly Thr Leu Thr Ser Asp Asn Ile Glu Ala Met
405 410 415
Phe Val His Thr Asp Val Glu Lys Val Gly Asp Phe Ala Cys Ser Ser
420 425 430
Glu Leu Leu Asn Lys Ile His Ser Cys Val Val Asn Ser Gln Leu Ala
435 440 445
Asn Leu Met Gly Ile Pro Thr Asp Cys Pro Gln Arg Asp Glu Arg Met
450 455 460
Gly Trp Leu Gly Asp Ala Gln Leu Thr Val Glu Glu Ala Met Tyr Asn
465 470 475 480
Phe Asp Met Ala Ala Phe Tyr Thr Lys Tyr Leu Met Asp Ile Lys Leu
485 490 495
Ser Gln Lys Glu Asp Gly Ser Ile Ser Asp Val Ala Pro Pro Tyr Trp
500 505 510
Lys Arg Tyr Pro Ser Asp Pro Ala Trp Gly Thr Ala Tyr Ala Thr Ile
515 520 525
Leu Trp Tyr Leu Tyr Phe Phe Tyr Glu Asp Arg Arg Val Leu Glu Glu
530 535 540
His Tyr Asp Ser Leu Lys Arg Tyr Val Glu Phe Leu Arg Lys Asn Ser
545 550 555 560
Pro Asn His Leu Thr Lys Leu Gly Gln His Gly Asp Trp Cys Pro Pro
565 570 575
Gly Asp Lys Phe Pro Lys Arg Thr Pro Leu Ile Leu Thr Ser Thr Trp
580 585 590
Tyr Tyr Tyr His Asp Thr Leu Ile Leu Ser Glu Ile Ala Lys Ile Leu
595 600 605
Gly Lys Lys Glu Asp Glu His Glu Tyr Arg Lys Leu Ala Gly Glu Ile
610 615 620
Lys Glu Ala Phe Asn Arg His Phe Leu Arg Lys Val Glu Asp His Thr
625 630 635 640
Gly Arg Ile Val Cys Phe Tyr Arg Gly Ile Lys Leu Ser Pro Lys Asp
645 650 655
Arg Ile Pro Thr Thr Gln Thr Cys Asn Val Leu Pro Leu Trp Asn Lys
660 665 670
Met Val Pro Glu Glu Cys Arg Glu Asp Val Phe Lys Val Leu Glu Arg
675 680 685
Leu Ile Glu Val Asp Asn Asp Thr His Phe Asp Thr Gly Ile Val Gly
690 695 700
Thr Arg Tyr Ile Leu Glu Val Leu Ser Glu Asn Gly Arg Lys Asp Leu
705 710 715 720
Ala Leu Lys Leu Leu Leu Lys Glu Asp Tyr Pro Ser Phe Gly Tyr Met
725 730 735
Ile Lys Asn Gly Ala Thr Thr Leu Trp Glu Arg Trp Glu Lys Leu Glu
740 745 750
Gly Thr Gly Met Asn Ser His Asn His Val Met Leu Gly Ser Val Asp
755 760 765
Thr Trp Phe Tyr Lys Tyr Leu Ser Gly Ile Lys Pro Val Ala Pro Gly
770 775 780
Trp Lys Lys Ile Arg Ile Glu Pro Tyr Phe Ala Asp Gln Ile Asp Phe
785 790 795 800
Val Ser Ala Lys Ile Lys Thr Pro Asn Gly Ser Leu Glu Val Ser Trp
805 810 815
Lys Lys Gln Asn Lys Glu Tyr Glu Ile Gln Ile Ile Ile Pro Val Asn
820 825 830
Thr Val Gly Ile Phe Ala Val Pro Glu Ser Phe Lys Val Ser Ala Ile
835 840 845
Asn Ser Lys Gln Val Ser Tyr Pro Ser Glu Phe Glu Leu Glu Pro Gly
850 855 860
Ala Tyr Asn Ile Val Leu Glu Arg Val Arg Glu Cys
865 870 875
<210> 2
<211> 2628
<212> DNA
<213> Thermotoga petrophila DSM 13995
<400> 2
atgattcagg cctgtgactt gagatgcgag tacttgactt ctccagtctt gggtcttgac 60
gtcatcccaa gattctcttg gagactgaaa ggtaacggta agaagcagac caggtacaag 120
atcatcgtgt ccgataactt cgacgacatc gagagaggta tcggtaacgt ttgggaatct 180
gaaaaggact cctccaagaa cctgaacatc gaatacgagg gtccaaagct gaaggcctac 240
aagggttact attggagagt taagctgtgg gacgagaaag agaacggtcc ttggtctgaa 300
actgcctact ttgagatggg tccattggag gattggcgtg gtaagtggat tactatgcca 360
tctccactgt ccttcaagga cccagctcat agacacgagt tgttctacgc catgtacttc 420
agaaaagagt tcctgctgaa caaagaggtc gagaaggcca gagtttacgt ttccggtttg 480
ggtgtttacg agttgcacct gaacggaaag agagtcggta acaacgtttt ggatccagct 540
ccaaccgact acaacaaggt tgccttgtac tccacttacg acgtcaccca atacttgacc 600
actggtaaga acaccatcgg tgtcatcctt ggaaacggta gacacatcag agactacggt 660
tactccaagc caaagttgta cttgcagctg ctggtgttct acaaggacgg ttcccgtgag 720
ttcatttgtt ctgacgagac ttggaaggtt tcccacggtc cattgaaaga aaacggtatc 780
tacttcggtg aggtctacga cgctagagat gagatttctg gttgggactc tccaggtttc 840
gatgacagaa actggtctga ggttgagatc gttgaaggtc catctttgaa ggcccagttg 900
atcccagtta tcagagtttg cgaggtcatc aagcctaaga gattgtggtt gtcctccaga 960
ggaaccttca tcgttgactt cggaaagaac atctccggtt gggtcaagct gagagtcaac 1020
aatggtaaga gaggtgagaa gatcatcatc agatacgccg aggtcttgga tccatccatg 1080
gacagattgg acaccagaaa cttgagattg gctagagcta ccgacgagta catcttgaaa 1140
ggtcagggtg ttgagatcta cgagccaaga ttcacctacc acggtttcag atacgttgag 1200
gtcgaagatt acccaggtac tttgacctcc gacaacatcg aggctatgtt cgttcacact 1260
gacgttgaga aggttggtga cttcgcttgt tcttccgagc tgttgaacaa gatccactcc 1320
tgtgttgtca actcccagtt ggctaacttg atgggtatcc caactgactg tccacaaagg 1380
gacgaaagaa tgggttggtt gggtgatgct cagttgactg ttgaggaagc catgtacaac 1440
ttcgacatgg ctgctttcta caccaagtac ctgatggaca tcaagctgtc ccaaaaagag 1500
gatggttcca tctctgacgt tgctccacca tactggaaga gatacccttc tgatccagct 1560
tggggtactg cttacgctac tatcttgtgg tacttgtact ttttctacga ggacaggcgt 1620
gtgttggagg aacactacga ttccttgaag aggtacgtcg agttcctgag aaagaactcc 1680
ccaaaccact tgaccaagtt gggtcagcat ggtgattggt gtccaccagg tgataagttc 1740
ccaaagagaa ccccattgat cttgacctcc acctggtact actaccacga cactttgatc 1800
ttgtccgaga tcgctaagat cctgggtaag aaagaagatg agcacgagta cagaaagctg 1860
gccggtgaaa tcaaagaggc tttcaacaga cacttcctga ggaaggttga ggaccacact 1920
ggtagaatcg tgtgcttcta cagaggtatc aagttgtccc caaaggacag aatcccaacc 1980
actcagacct gtaacgtttt gccattgtgg aacaagatgg tcccagagga atgtagagag 2040
gacgttttca aggttttgga gaggttgatc gaggtggaca acgatactca cttcgacact 2100
ggtatcgtcg gtactcgtta catcttggag gtcttgtctg agaacggtag aaaggacttg 2160
gccttgaagt tgctgctgaa agaggactac ccatccttcg gttacatgat caagaacggt 2220
gctaccacct tgtgggaaag atgggaaaag ttggaaggta ctggtatgaa ctcccacaac 2280
cacgttatgt tgggttccgt tgatacctgg ttctacaaat acctgtccgg tatcaagcca 2340
gttgctccag gttggaagaa gatcagaatc gagccatact tcgctgacca gatcgacttc 2400
gtttccgcca agattaagac tccaaacggt tccttggaag tctcctggaa gaagcaaaac 2460
aaagagtacg agatccagat tatcatcccc gtcaacaccg ttggtatctt cgctgttcca 2520
gagtctttta aggtgtccgc cattaactcc aagcaagtct cttacccatc cgagttcgaa 2580
ttggaaccag gtgcctacaa catcgtcttg gaaagggtta gagaatgt 2628
<210> 3
<211> 876
<212> PRT
<213> Artificial Synthesis
<400> 3
Met Ile Gln Ala Cys Asp Leu Arg Cys Glu Tyr Leu Thr Ser Pro Val
1 5 10 15
Leu Gly Leu Asp Val Ile Pro Arg Phe Ser Trp Arg Leu Lys Gly Asn
20 25 30
Gly Lys Lys Gln Thr Arg Tyr Lys Ile Ile Val Ser Asp Asn Phe Asp
35 40 45
Asp Ile Glu Arg Gly Ile Gly Asn Val Trp Glu Ser Glu Lys Asp Ser
50 55 60
Ser Lys Asn Leu Asn Ile Glu Tyr Glu Gly Pro Lys Leu Lys Ala Tyr
65 70 75 80
Lys Gly Tyr Tyr Trp Arg Val Lys Leu Trp Asp Glu Lys Glu Asn Gly
85 90 95
Pro Trp Ser Glu Thr Ala Tyr Phe Glu Met Gly Pro Leu Glu Asp Trp
100 105 110
Arg Gly Lys Trp Ile Thr Met Pro Ser Pro Leu Ser Phe Lys Asp Pro
115 120 125
Ala His Arg His Glu Leu Phe Tyr Ala Met Tyr Phe Arg Lys Glu Phe
130 135 140
Leu Leu Asn Lys Glu Val Glu Lys Ala Arg Val Tyr Val Ser Gly Leu
145 150 155 160
Gly Val Tyr Glu Leu His Leu Asn Gly Lys Arg Val Gly Asn Asn Val
165 170 175
Leu Asp Pro Ala Pro Thr Asp Tyr Asn Lys Val Ala Leu Tyr Ser Thr
180 185 190
Tyr Asp Val Thr Gln Tyr Leu Thr Thr Gly Lys Asn Thr Ile Gly Val
195 200 205
Ile Leu Gly Asn Gly Arg His Ile Arg Asp Tyr Gly Tyr Ser Lys Pro
210 215 220
Lys Leu Tyr Leu Gln Leu Leu Val Phe Tyr Lys Asp Gly Ser Arg Glu
225 230 235 240
Phe Ile Cys Ser Asp Glu Thr Trp Lys Val Ser His Gly Pro Leu Lys
245 250 255
Glu Asn Gly Ile Tyr Phe Gly Glu Val Tyr Asp Ala Arg Asp Glu Ile
260 265 270
Ser Gly Trp Asp Ser Pro Gly Phe Asp Asp Arg Asn Trp Ser Glu Val
275 280 285
Glu Ile Val Glu Gly Pro Ser Leu Lys Ala Gln Leu Ile Pro Val Ile
290 295 300
Arg Val Cys Glu Val Ile Lys Pro Lys Arg Leu Trp Leu Ser Ser Arg
305 310 315 320
Gly Thr Phe Ile Val Asp Phe Gly Lys Asn Ile Ser Gly Trp Val Lys
325 330 335
Leu Arg Val Asn Asn Gly Lys Arg Gly Glu Lys Ile Ile Ile Arg Tyr
340 345 350
Ala Glu Val Leu Asp Pro Ser Met Asp Arg Leu Asp Thr Arg Asn Leu
355 360 365
Arg Leu Ala Arg Ala Thr Asp Glu Tyr Ile Leu Lys Gly Gln Gly Val
370 375 380
Glu Ile Tyr Glu Pro Arg Phe Thr Tyr His Gly Phe Arg Tyr Val Glu
385 390 395 400
Val Glu Asp Tyr Pro Gly Thr Leu Thr Ser Asp Asn Ile Glu Ala Met
405 410 415
Phe Val His Thr Asp Val Glu Lys Val Gly Asp Phe Ala Cys Ser Ser
420 425 430
Glu Leu Leu Asn Lys Ile His Ser Cys Val Val Asn Ser Gln Leu Ala
435 440 445
Asn Leu Met Gly Ile Pro Thr Asp Cys Pro Gln Arg Asp Glu Arg Met
450 455 460
Gly Trp Leu Gly Asp Ala Gln Leu Thr Val Glu Glu Ala Met Tyr Asn
465 470 475 480
Phe Asp Met Ala Ala Phe Tyr Thr Lys Tyr Leu Met Asp Ile Lys Leu
485 490 495
Ser Gln Lys Glu Asp Gly Ser Ile Ser Asp Val Ala Pro Pro Tyr Ala
500 505 510
Lys Arg Tyr Pro Ser Asp Pro Ala Trp Gly Thr Ala Tyr Ala Thr Ile
515 520 525
Leu Trp Tyr Leu Tyr Phe Phe Tyr Glu Asp Arg Arg Val Leu Glu Glu
530 535 540
His Tyr Asp Ser Leu Lys Arg Tyr Val Glu Phe Leu Arg Lys Asn Ser
545 550 555 560
Pro Asn His Leu Thr Lys Leu Gly Gln His Gly Asp Trp Cys Pro Pro
565 570 575
Gly Asp Lys Phe Pro Lys Arg Thr Pro Leu Ile Leu Thr Ser Thr Trp
580 585 590
Tyr Tyr Tyr His Asp Thr Leu Ile Leu Ser Glu Ile Ala Lys Ile Leu
595 600 605
Gly Lys Lys Glu Asp Glu His Glu Tyr Arg Lys Leu Ala Gly Glu Ile
610 615 620
Lys Glu Ala Phe Asn Arg His Phe Leu Arg Lys Val Glu Asp His Thr
625 630 635 640
Gly Arg Ile Val Cys Phe Tyr Arg Gly Ile Lys Leu Ser Pro Lys Asp
645 650 655
Arg Ile Pro Thr Thr Gln Thr Cys Asn Val Leu Pro Leu Trp Asn Lys
660 665 670
Met Val Pro Glu Glu Cys Arg Glu Asp Val Phe Lys Val Leu Glu Arg
675 680 685
Leu Ile Glu Val Asp Asn Asp Thr His Phe Asp Thr Gly Ile Val Gly
690 695 700
Thr Arg Tyr Ile Leu Glu Val Leu Ser Glu Asn Gly Arg Lys Asp Leu
705 710 715 720
Ala Leu Lys Leu Leu Leu Lys Glu Asp Tyr Pro Ser Phe Gly Tyr Met
725 730 735
Ile Lys Asn Gly Ala Thr Thr Leu Trp Glu Arg Trp Glu Lys Leu Glu
740 745 750
Gly Thr Gly Met Asn Ser His Asn His Val Met Leu Gly Ser Val Asp
755 760 765
Thr Trp Phe Tyr Lys Tyr Leu Ser Gly Ile Lys Pro Val Ala Pro Gly
770 775 780
Trp Lys Lys Ile Arg Ile Glu Pro Tyr Phe Ala Asp Gln Ile Asp Phe
785 790 795 800
Val Ser Ala Lys Ile Lys Thr Pro Asn Gly Ser Leu Glu Val Ser Trp
805 810 815
Lys Lys Gln Asn Lys Glu Tyr Glu Ile Gln Ile Ile Ile Pro Val Asn
820 825 830
Thr Val Gly Ile Phe Ala Val Pro Glu Ser Phe Lys Val Ser Ala Ile
835 840 845
Asn Ser Lys Gln Val Ser Tyr Pro Ser Glu Phe Glu Leu Glu Pro Gly
850 855 860
Ala Tyr Asn Ile Val Leu Glu Arg Val Arg Glu Cys
865 870 875
<210> 4
<211> 876
<212> PRT
<213> Artificial Synthesis
<400> 4
Met Ile Gln Ala Cys Asp Leu Arg Cys Glu Tyr Leu Thr Ser Pro Val
1 5 10 15
Leu Gly Leu Asp Val Ile Pro Arg Phe Ser Trp Arg Leu Lys Gly Asn
20 25 30
Gly Lys Lys Gln Thr Arg Tyr Lys Ile Ile Val Ser Asp Asn Phe Asp
35 40 45
Asp Ile Glu Arg Gly Ile Gly Asn Val Trp Glu Ser Glu Lys Asp Ser
50 55 60
Ser Lys Asn Leu Asn Ile Glu Tyr Glu Gly Pro Lys Leu Lys Ala Tyr
65 70 75 80
Lys Gly Tyr Tyr Trp Arg Val Lys Leu Trp Asp Glu Lys Glu Asn Gly
85 90 95
Pro Trp Ser Glu Thr Ala Tyr Phe Glu Met Gly Pro Leu Glu Asp Trp
100 105 110
Arg Gly Lys Trp Ile Thr Met Pro Ser Pro Leu Ser Phe Lys Asp Pro
115 120 125
Ala His Arg His Glu Leu Phe Tyr Ala Met Tyr Phe Arg Lys Glu Phe
130 135 140
Leu Leu Asn Lys Glu Val Glu Lys Ala Arg Val Tyr Val Ser Gly Leu
145 150 155 160
Gly Val Tyr Glu Leu His Leu Asn Gly Lys Arg Val Gly Asn Asn Val
165 170 175
Leu Asp Pro Ala Pro Thr Asp Tyr Asn Lys Val Ala Leu Tyr Ser Thr
180 185 190
Tyr Asp Val Thr Gln Tyr Leu Thr Thr Gly Lys Asn Thr Ile Gly Val
195 200 205
Ile Leu Gly Asn Gly Arg His Ile Arg Asp Tyr Gly Tyr Ser Lys Pro
210 215 220
Lys Leu Tyr Leu Gln Leu Leu Val Phe Tyr Lys Asp Gly Ser Arg Glu
225 230 235 240
Phe Ile Cys Ser Asp Glu Thr Trp Lys Val Ser His Gly Pro Leu Lys
245 250 255
Glu Asn Gly Ile Tyr Phe Gly Glu Val Tyr Asp Ala Arg Asp Glu Ile
260 265 270
Ser Gly Trp Asp Ser Pro Gly Phe Asp Asp Arg Asn Trp Ser Glu Val
275 280 285
Glu Ile Val Glu Gly Pro Ser Leu Lys Ala Gln Leu Ile Pro Val Ile
290 295 300
Arg Val Cys Glu Val Ile Lys Pro Lys Arg Leu Trp Leu Ser Ser Arg
305 310 315 320
Gly Thr Phe Ile Val Asp Phe Gly Lys Asn Ile Ser Gly Trp Val Lys
325 330 335
Leu Arg Val Asn Asn Gly Lys Arg Gly Glu Lys Ile Ile Ile Arg Tyr
340 345 350
Ala Glu Val Leu Asp Pro Ser Met Asp Arg Leu Asp Thr Arg Asn Leu
355 360 365
Arg Leu Ala Arg Ala Thr Asp Glu Tyr Ile Leu Lys Gly Gln Gly Val
370 375 380
Glu Ile Tyr Glu Pro Arg Phe Thr Tyr His Gly Phe Arg Tyr Val Glu
385 390 395 400
Val Glu Asp Tyr Pro Gly Thr Leu Thr Ser Asp Asn Ile Glu Ala Met
405 410 415
Phe Val His Thr Asp Val Glu Lys Val Gly Asp Phe Ala Cys Ser Ser
420 425 430
Glu Leu Leu Asn Lys Ile His Ser Cys Val Val Asn Ser Gln Leu Ala
435 440 445
Asn Leu Met Gly Ile Pro Thr Asp Cys Pro Gln Arg Asp Glu Arg Met
450 455 460
Gly Trp Leu Gly Asp Ala Gln Leu Thr Val Glu Glu Ala Met Tyr Asn
465 470 475 480
Phe Asp Met Ala Ala Phe Tyr Thr Lys Tyr Leu Met Asp Ile Lys Leu
485 490 495
Ser Gln Lys Glu Asp Gly Ser Ile Ser Asp Val Ala Pro Pro Tyr Ala
500 505 510
Lys Arg Tyr Pro Ser Asp Pro Ala Trp Gly Thr Ala Tyr Ala Thr Ile
515 520 525
Leu Trp Tyr Leu Tyr Phe Phe Tyr Glu Asp Arg Arg Val Leu Glu Glu
530 535 540
His Tyr Asp Ser Leu Lys Arg Tyr Val Glu Phe Leu Arg Lys Asn Ser
545 550 555 560
Pro Asn His Leu Thr Lys Leu Gly Gln Ala Gly Asp Trp Cys Pro Pro
565 570 575
Gly Asp Lys Phe Pro Lys Arg Thr Pro Leu Ile Leu Thr Ser Thr Trp
580 585 590
Tyr Tyr Tyr His Asp Thr Leu Ile Leu Ser Glu Ile Ala Lys Ile Leu
595 600 605
Gly Lys Lys Glu Asp Glu His Glu Tyr Arg Lys Leu Ala Gly Glu Ile
610 615 620
Lys Glu Ala Phe Asn Arg His Phe Leu Arg Lys Val Glu Asp His Thr
625 630 635 640
Gly Arg Ile Val Cys Phe Tyr Arg Gly Ile Lys Leu Ser Pro Lys Asp
645 650 655
Arg Ile Pro Thr Thr Gln Thr Cys Asn Val Leu Pro Leu Trp Asn Lys
660 665 670
Met Val Pro Glu Glu Cys Arg Glu Asp Val Phe Lys Val Leu Glu Arg
675 680 685
Leu Ile Glu Val Asp Asn Asp Thr His Phe Asp Thr Gly Ile Val Gly
690 695 700
Thr Arg Tyr Ile Leu Glu Val Leu Ser Glu Asn Gly Arg Lys Asp Leu
705 710 715 720
Ala Leu Lys Leu Leu Leu Lys Glu Asp Tyr Pro Ser Phe Gly Tyr Met
725 730 735
Ile Lys Asn Gly Ala Thr Thr Leu Trp Glu Arg Trp Glu Lys Leu Glu
740 745 750
Gly Thr Gly Met Asn Ser His Asn His Val Met Leu Gly Ser Val Asp
755 760 765
Thr Trp Phe Tyr Lys Tyr Leu Ser Gly Ile Lys Pro Val Ala Pro Gly
770 775 780
Trp Lys Lys Ile Arg Ile Glu Pro Tyr Phe Ala Asp Gln Ile Asp Phe
785 790 795 800
Val Ser Ala Lys Ile Lys Thr Pro Asn Gly Ser Leu Glu Val Ser Trp
805 810 815
Lys Lys Gln Asn Lys Glu Tyr Glu Ile Gln Ile Ile Ile Pro Val Asn
820 825 830
Thr Val Gly Ile Phe Ala Val Pro Glu Ser Phe Lys Val Ser Ala Ile
835 840 845
Asn Ser Lys Gln Val Ser Tyr Pro Ser Glu Phe Glu Leu Glu Pro Gly
850 855 860
Ala Tyr Asn Ile Val Leu Glu Arg Val Arg Glu Cys
865 870 875
<210> 5
<211> 876
<212> PRT
<213> Artificial Synthesis
<400> 5
Met Ile Gln Ala Cys Asp Leu Arg Cys Glu Tyr Leu Thr Ser Pro Val
1 5 10 15
Leu Gly Leu Asp Val Ile Pro Arg Phe Ser Trp Arg Leu Lys Gly Asn
20 25 30
Gly Lys Lys Gln Thr Arg Tyr Lys Ile Ile Val Ser Asp Asn Phe Asp
35 40 45
Asp Ile Glu Arg Gly Ile Gly Asn Val Trp Glu Ser Glu Lys Asp Ser
50 55 60
Ser Lys Asn Leu Asn Ile Glu Tyr Glu Gly Pro Lys Leu Lys Ala Tyr
65 70 75 80
Lys Gly Tyr Tyr Trp Arg Val Lys Leu Trp Asp Glu Lys Glu Asn Gly
85 90 95
Pro Trp Ser Glu Thr Ala Tyr Phe Glu Met Gly Pro Leu Glu Asp Trp
100 105 110
Arg Gly Lys Trp Ile Thr Met Pro Ser Pro Leu Ser Phe Lys Asp Pro
115 120 125
Ala His Arg His Glu Leu Phe Tyr Ala Met Tyr Phe Arg Lys Glu Phe
130 135 140
Leu Leu Asn Lys Glu Val Glu Lys Ala Arg Val Tyr Val Ser Gly Leu
145 150 155 160
Gly Val Tyr Glu Leu His Leu Asn Gly Lys Arg Val Gly Asn Asn Val
165 170 175
Leu Asp Pro Ala Pro Thr Asp Tyr Asn Lys Val Ala Leu Tyr Ser Thr
180 185 190
Tyr Asp Val Thr Gln Tyr Leu Thr Thr Gly Lys Asn Thr Ile Gly Val
195 200 205
Ile Leu Gly Asn Gly Arg His Ile Arg Asp Tyr Gly Tyr Ser Lys Pro
210 215 220
Lys Leu Tyr Leu Gln Leu Leu Val Phe Tyr Lys Asp Gly Ser Arg Glu
225 230 235 240
Phe Ile Cys Ser Asp Glu Thr Trp Lys Val Ser His Gly Pro Leu Lys
245 250 255
Glu Asn Gly Ile Tyr Phe Gly Glu Val Tyr Asp Ala Arg Asp Glu Ile
260 265 270
Ser Gly Trp Asp Ser Pro Gly Phe Asp Asp Arg Asn Trp Ser Glu Val
275 280 285
Glu Ile Val Glu Gly Pro Ser Leu Lys Ala Gln Leu Ile Pro Val Ile
290 295 300
Arg Val Cys Glu Val Ile Lys Pro Lys Arg Leu Trp Leu Ser Ser Arg
305 310 315 320
Gly Thr Phe Ile Val Asp Phe Gly Lys Asn Ile Ser Gly Trp Val Lys
325 330 335
Leu Arg Val Asn Asn Gly Lys Arg Gly Glu Lys Ile Ile Ile Arg Tyr
340 345 350
Ala Glu Val Leu Asp Pro Ser Met Asp Arg Leu Asp Thr Arg Asn Leu
355 360 365
Arg Leu Ala Arg Ala Thr Asp Glu Tyr Ile Leu Lys Gly Gln Gly Val
370 375 380
Glu Ile Tyr Glu Pro Arg Phe Thr Tyr His Gly Phe Arg Tyr Val Glu
385 390 395 400
Val Glu Asp Tyr Pro Gly Thr Leu Thr Ser Asp Asn Ile Glu Ala Met
405 410 415
Phe Val His Thr Asp Val Glu Lys Val Gly Asp Phe Ala Cys Ser Ser
420 425 430
Glu Leu Leu Asn Lys Ile His Ser Cys Val Val Asn Ser Gln Leu Ala
435 440 445
Asn Leu Met Gly Ile Pro Thr Asp Cys Pro Gln Arg Asp Glu Arg Met
450 455 460
Gly Trp Leu Gly Asp Ala Gln Leu Thr Val Glu Glu Ala Met Tyr Asn
465 470 475 480
Phe Asp Met Ala Ala Phe Tyr Thr Lys Tyr Leu Met Asp Ile Lys Leu
485 490 495
Ser Gln Lys Glu Asp Gly Ser Ile Ser Asp Val Ala Pro Pro Tyr Ala
500 505 510
Lys Arg Tyr Pro Ser Asp Pro Ala Trp Gly Thr Ala Tyr Ala Thr Ile
515 520 525
Leu Trp Tyr Leu Tyr Phe Phe Tyr Glu Asp Arg Arg Val Leu Glu Glu
530 535 540
His Tyr Asp Ser Leu Lys Arg Tyr Val Glu Phe Leu Arg Lys Asn Ser
545 550 555 560
Pro Asn His Leu Thr Lys Leu Gly Gln His Gly Asp Trp Cys Pro Pro
565 570 575
Gly Asp Ala Phe Pro Lys Arg Thr Pro Leu Ile Leu Thr Ser Thr Trp
580 585 590
Tyr Tyr Tyr His Asp Thr Leu Ile Leu Ser Glu Ile Ala Lys Ile Leu
595 600 605
Gly Lys Lys Glu Asp Glu His Glu Tyr Arg Lys Leu Ala Gly Glu Ile
610 615 620
Lys Glu Ala Phe Asn Arg His Phe Leu Arg Lys Val Glu Asp His Thr
625 630 635 640
Gly Arg Ile Val Cys Phe Tyr Arg Gly Ile Lys Leu Ser Pro Lys Asp
645 650 655
Arg Ile Pro Thr Thr Gln Thr Cys Asn Val Leu Pro Leu Trp Asn Lys
660 665 670
Met Val Pro Glu Glu Cys Arg Glu Asp Val Phe Lys Val Leu Glu Arg
675 680 685
Leu Ile Glu Val Asp Asn Asp Thr His Phe Asp Thr Gly Ile Val Gly
690 695 700
Thr Arg Tyr Ile Leu Glu Val Leu Ser Glu Asn Gly Arg Lys Asp Leu
705 710 715 720
Ala Leu Lys Leu Leu Leu Lys Glu Asp Tyr Pro Ser Phe Gly Tyr Met
725 730 735
Ile Lys Asn Gly Ala Thr Thr Leu Trp Glu Arg Trp Glu Lys Leu Glu
740 745 750
Gly Thr Gly Met Asn Ser His Asn His Val Met Leu Gly Ser Val Asp
755 760 765
Thr Trp Phe Tyr Lys Tyr Leu Ser Gly Ile Lys Pro Val Ala Pro Gly
770 775 780
Trp Lys Lys Ile Arg Ile Glu Pro Tyr Phe Ala Asp Gln Ile Asp Phe
785 790 795 800
Val Ser Ala Lys Ile Lys Thr Pro Asn Gly Ser Leu Glu Val Ser Trp
805 810 815
Lys Lys Gln Asn Lys Glu Tyr Glu Ile Gln Ile Ile Ile Pro Val Asn
820 825 830
Thr Val Gly Ile Phe Ala Val Pro Glu Ser Phe Lys Val Ser Ala Ile
835 840 845
Asn Ser Lys Gln Val Ser Tyr Pro Ser Glu Phe Glu Leu Glu Pro Gly
850 855 860
Ala Tyr Asn Ile Val Leu Glu Arg Val Arg Glu Cys
865 870 875
<210> 6
<211> 876
<212> PRT
<213> Artificial Synthesis
<400> 6
Met Ile Gln Ala Cys Asp Leu Arg Cys Glu Tyr Leu Thr Ser Pro Val
1 5 10 15
Leu Gly Leu Asp Val Ile Pro Arg Phe Ser Trp Arg Leu Lys Gly Asn
20 25 30
Gly Lys Lys Gln Thr Arg Tyr Lys Ile Ile Val Ser Asp Asn Phe Asp
35 40 45
Asp Ile Glu Arg Gly Ile Gly Asn Val Trp Glu Ser Glu Lys Asp Ser
50 55 60
Ser Lys Asn Leu Asn Ile Glu Tyr Glu Gly Pro Lys Leu Lys Ala Tyr
65 70 75 80
Lys Gly Tyr Tyr Trp Arg Val Lys Leu Trp Asp Glu Lys Glu Asn Gly
85 90 95
Pro Trp Ser Glu Thr Ala Tyr Phe Glu Met Gly Pro Leu Glu Asp Trp
100 105 110
Arg Gly Lys Trp Ile Thr Met Pro Ser Pro Leu Ser Phe Lys Asp Pro
115 120 125
Ala His Arg His Glu Leu Phe Tyr Ala Met Tyr Phe Arg Lys Glu Phe
130 135 140
Leu Leu Asn Lys Glu Val Glu Lys Ala Arg Val Tyr Val Ser Gly Leu
145 150 155 160
Gly Val Tyr Glu Leu His Leu Asn Gly Lys Arg Val Gly Asn Asn Val
165 170 175
Leu Asp Pro Ala Pro Thr Asp Tyr Asn Lys Val Ala Leu Tyr Ser Thr
180 185 190
Tyr Asp Val Thr Gln Tyr Leu Thr Thr Gly Lys Asn Thr Ile Gly Val
195 200 205
Ile Leu Gly Asn Gly Arg His Ile Arg Asp Tyr Gly Tyr Ser Lys Pro
210 215 220
Lys Leu Tyr Leu Gln Leu Leu Val Phe Tyr Lys Asp Gly Ser Arg Glu
225 230 235 240
Phe Ile Cys Ser Asp Glu Thr Trp Lys Val Ser His Gly Pro Leu Lys
245 250 255
Glu Asn Gly Ile Tyr Phe Gly Glu Val Tyr Asp Ala Arg Asp Glu Ile
260 265 270
Ser Gly Trp Asp Ser Pro Gly Phe Asp Asp Arg Asn Trp Ser Glu Val
275 280 285
Glu Ile Val Glu Gly Pro Ser Leu Lys Ala Gln Leu Ile Pro Val Ile
290 295 300
Arg Val Cys Glu Val Ile Lys Pro Lys Arg Leu Trp Leu Ser Ser Arg
305 310 315 320
Gly Thr Phe Ile Val Asp Phe Gly Lys Asn Ile Ser Gly Trp Val Lys
325 330 335
Leu Arg Val Asn Asn Gly Lys Arg Gly Glu Lys Ile Ile Ile Arg Tyr
340 345 350
Ala Glu Val Leu Asp Pro Ser Met Asp Arg Leu Asp Thr Arg Asn Leu
355 360 365
Arg Leu Ala Arg Ala Thr Asp Glu Tyr Ile Leu Lys Gly Gln Gly Val
370 375 380
Glu Ile Tyr Glu Pro Arg Phe Thr Tyr His Gly Phe Arg Tyr Val Glu
385 390 395 400
Val Glu Asp Tyr Pro Gly Thr Leu Thr Ser Asp Asn Ile Glu Ala Met
405 410 415
Phe Val His Thr Asp Val Glu Lys Val Gly Asp Phe Ala Cys Ser Ser
420 425 430
Glu Leu Leu Asn Lys Ile His Ser Cys Val Val Asn Ser Gln Leu Ala
435 440 445
Asn Leu Met Gly Ile Pro Thr Asp Cys Pro Gln Arg Asp Glu Arg Met
450 455 460
Gly Trp Leu Gly Asp Ala Gln Leu Thr Val Glu Glu Ala Met Tyr Asn
465 470 475 480
Phe Asp Met Ala Ala Phe Tyr Thr Lys Tyr Leu Met Asp Ile Lys Leu
485 490 495
Ser Gln Lys Glu Asp Gly Ser Ile Ser Asp Val Ala Pro Pro Tyr Ala
500 505 510
Lys Arg Tyr Pro Ser Asp Pro Ala Trp Gly Thr Ala Tyr Ala Thr Ile
515 520 525
Leu Trp Tyr Leu Tyr Phe Phe Tyr Glu Asp Arg Arg Val Leu Glu Glu
530 535 540
His Tyr Asp Ser Leu Lys Arg Tyr Val Glu Phe Leu Arg Lys Asn Ser
545 550 555 560
Pro Asn His Leu Thr Lys Leu Gly Gln Ala Gly Asp Trp Cys Pro Pro
565 570 575
Gly Asp Ala Phe Pro Lys Arg Thr Pro Leu Ile Leu Thr Ser Thr Trp
580 585 590
Tyr Tyr Tyr His Asp Thr Leu Ile Leu Ser Glu Ile Ala Lys Ile Leu
595 600 605
Gly Lys Lys Glu Asp Glu His Glu Tyr Arg Lys Leu Ala Gly Glu Ile
610 615 620
Lys Glu Ala Phe Asn Arg His Phe Leu Arg Lys Val Glu Asp His Thr
625 630 635 640
Gly Arg Ile Val Cys Phe Tyr Arg Gly Ile Lys Leu Ser Pro Lys Asp
645 650 655
Arg Ile Pro Thr Thr Gln Thr Cys Asn Val Leu Pro Leu Trp Asn Lys
660 665 670
Met Val Pro Glu Glu Cys Arg Glu Asp Val Phe Lys Val Leu Glu Arg
675 680 685
Leu Ile Glu Val Asp Asn Asp Thr His Phe Asp Thr Gly Ile Val Gly
690 695 700
Thr Arg Tyr Ile Leu Glu Val Leu Ser Glu Asn Gly Arg Lys Asp Leu
705 710 715 720
Ala Leu Lys Leu Leu Leu Lys Glu Asp Tyr Pro Ser Phe Gly Tyr Met
725 730 735
Ile Lys Asn Gly Ala Thr Thr Leu Trp Glu Arg Trp Glu Lys Leu Glu
740 745 750
Gly Thr Gly Met Asn Ser His Asn His Val Met Leu Gly Ser Val Asp
755 760 765
Thr Trp Phe Tyr Lys Tyr Leu Ser Gly Ile Lys Pro Val Ala Pro Gly
770 775 780
Trp Lys Lys Ile Arg Ile Glu Pro Tyr Phe Ala Asp Gln Ile Asp Phe
785 790 795 800
Val Ser Ala Lys Ile Lys Thr Pro Asn Gly Ser Leu Glu Val Ser Trp
805 810 815
Lys Lys Gln Asn Lys Glu Tyr Glu Ile Gln Ile Ile Ile Pro Val Asn
820 825 830
Thr Val Gly Ile Phe Ala Val Pro Glu Ser Phe Lys Val Ser Ala Ile
835 840 845
Asn Ser Lys Gln Val Ser Tyr Pro Ser Glu Phe Glu Leu Glu Pro Gly
850 855 860
Ala Tyr Asn Ile Val Leu Glu Arg Val Arg Glu Cys
865 870 875
<210> 7
<211> 876
<212> PRT
<213> Artificial Synthesis
<400> 7
Met Ile Gln Ala Cys Asp Leu Arg Cys Glu Tyr Leu Thr Ser Pro Val
1 5 10 15
Leu Gly Leu Asp Val Ile Pro Arg Phe Ser Trp Arg Leu Lys Gly Asn
20 25 30
Gly Lys Lys Gln Thr Arg Tyr Lys Ile Ile Val Ser Asp Asn Phe Asp
35 40 45
Asp Ile Glu Arg Gly Ile Gly Asn Val Trp Glu Ser Glu Lys Asp Ser
50 55 60
Ser Lys Asn Leu Asn Ile Glu Tyr Glu Gly Pro Lys Leu Lys Ala Tyr
65 70 75 80
Lys Gly Tyr Tyr Trp Arg Val Lys Leu Trp Asp Glu Lys Glu Asn Gly
85 90 95
Pro Trp Ser Glu Thr Ala Tyr Phe Glu Met Gly Pro Leu Glu Asp Trp
100 105 110
Arg Gly Lys Trp Ile Thr Met Pro Ser Pro Leu Ser Phe Lys Asp Pro
115 120 125
Ala His Arg His Glu Leu Phe Tyr Ala Met Tyr Phe Arg Lys Glu Phe
130 135 140
Leu Leu Asn Lys Glu Val Glu Lys Ala Arg Val Tyr Val Ser Gly Leu
145 150 155 160
Gly Val Tyr Glu Leu His Leu Asn Gly Lys Arg Val Gly Asn Asn Val
165 170 175
Leu Asp Pro Ala Pro Thr Asp Tyr Asn Lys Val Ala Leu Tyr Ser Thr
180 185 190
Tyr Asp Val Thr Gln Tyr Leu Thr Thr Gly Lys Asn Thr Ile Gly Val
195 200 205
Ile Leu Gly Asn Gly Arg His Ile Arg Asp Tyr Gly Tyr Ser Lys Pro
210 215 220
Lys Leu Tyr Leu Gln Leu Leu Val Phe Tyr Lys Asp Gly Ser Arg Glu
225 230 235 240
Phe Ile Cys Ser Asp Glu Thr Trp Lys Val Ser His Gly Pro Leu Lys
245 250 255
Glu Asn Gly Ile Tyr Phe Gly Glu Val Tyr Asp Ala Arg Asp Glu Ile
260 265 270
Ser Gly Trp Asp Ser Pro Gly Phe Asp Asp Arg Asn Trp Ser Glu Val
275 280 285
Glu Ile Val Glu Gly Pro Ser Leu Lys Ala Gln Leu Ile Pro Val Ile
290 295 300
Arg Val Cys Glu Val Ile Lys Pro Lys Arg Leu Trp Leu Ser Ser Arg
305 310 315 320
Gly Thr Phe Ile Val Asp Phe Gly Lys Asn Ile Ser Gly Trp Val Lys
325 330 335
Leu Arg Val Asn Asn Gly Lys Arg Gly Glu Lys Ile Ile Ile Arg Tyr
340 345 350
Ala Glu Val Leu Asp Pro Ser Met Asp Arg Leu Asp Thr Arg Asn Leu
355 360 365
Arg Leu Ala Arg Ala Thr Asp Glu Tyr Ile Leu Lys Gly Gln Gly Val
370 375 380
Glu Ile Tyr Glu Pro Arg Phe Thr Tyr His Gly Phe Arg Tyr Val Glu
385 390 395 400
Val Glu Asp Tyr Pro Gly Thr Leu Thr Ser Asp Asn Ile Glu Ala Met
405 410 415
Phe Val His Thr Asp Val Glu Lys Val Gly Asp Phe Ala Cys Ser Ser
420 425 430
Glu Leu Leu Asn Lys Ile His Ser Cys Val Val Asn Ser Gln Leu Ala
435 440 445
Asn Leu Met Gly Ile Pro Thr Asp Cys Pro Gln Arg Asp Glu Arg Met
450 455 460
Gly Trp Leu Gly Asp Ala Gln Leu Thr Val Glu Glu Ala Met Tyr Asn
465 470 475 480
Phe Asp Met Ala Ala Phe Tyr Thr Lys Tyr Leu Met Asp Ile Lys Leu
485 490 495
Ser Gln Lys Glu Asp Gly Ser Ile Ser Asp Val Ala Pro Pro Tyr Trp
500 505 510
Lys Arg Tyr Pro Ser Asp Pro Ala Trp Gly Thr Ala Tyr Ala Thr Ile
515 520 525
Leu Trp Tyr Leu Tyr Phe Phe Tyr Glu Asp Arg Arg Val Leu Glu Glu
530 535 540
His Tyr Asp Ser Leu Lys Arg Tyr Val Glu Phe Leu Arg Lys Asn Ser
545 550 555 560
Pro Asn His Leu Thr Lys Leu Gly Gln Ala Gly Asp Trp Cys Pro Pro
565 570 575
Gly Asp Lys Phe Pro Lys Arg Thr Pro Leu Ile Leu Thr Ser Thr Trp
580 585 590
Tyr Tyr Tyr His Asp Thr Leu Ile Leu Ser Glu Ile Ala Lys Ile Leu
595 600 605
Gly Lys Lys Glu Asp Glu His Glu Tyr Arg Lys Leu Ala Gly Glu Ile
610 615 620
Lys Glu Ala Phe Asn Arg His Phe Leu Arg Lys Val Glu Asp His Thr
625 630 635 640
Gly Arg Ile Val Cys Phe Tyr Arg Gly Ile Lys Leu Ser Pro Lys Asp
645 650 655
Arg Ile Pro Thr Thr Gln Thr Cys Asn Val Leu Pro Leu Trp Asn Lys
660 665 670
Met Val Pro Glu Glu Cys Arg Glu Asp Val Phe Lys Val Leu Glu Arg
675 680 685
Leu Ile Glu Val Asp Asn Asp Thr His Phe Asp Thr Gly Ile Val Gly
690 695 700
Thr Arg Tyr Ile Leu Glu Val Leu Ser Glu Asn Gly Arg Lys Asp Leu
705 710 715 720
Ala Leu Lys Leu Leu Leu Lys Glu Asp Tyr Pro Ser Phe Gly Tyr Met
725 730 735
Ile Lys Asn Gly Ala Thr Thr Leu Trp Glu Arg Trp Glu Lys Leu Glu
740 745 750
Gly Thr Gly Met Asn Ser His Asn His Val Met Leu Gly Ser Val Asp
755 760 765
Thr Trp Phe Tyr Lys Tyr Leu Ser Gly Ile Lys Pro Val Ala Pro Gly
770 775 780
Trp Lys Lys Ile Arg Ile Glu Pro Tyr Phe Ala Asp Gln Ile Asp Phe
785 790 795 800
Val Ser Ala Lys Ile Lys Thr Pro Asn Gly Ser Leu Glu Val Ser Trp
805 810 815
Lys Lys Gln Asn Lys Glu Tyr Glu Ile Gln Ile Ile Ile Pro Val Asn
820 825 830
Thr Val Gly Ile Phe Ala Val Pro Glu Ser Phe Lys Val Ser Ala Ile
835 840 845
Asn Ser Lys Gln Val Ser Tyr Pro Ser Glu Phe Glu Leu Glu Pro Gly
850 855 860
Ala Tyr Asn Ile Val Leu Glu Arg Val Arg Glu Cys
865 870 875
<210> 8
<211> 876
<212> PRT
<213> Artificial Synthesis
<400> 8
Met Ile Gln Ala Cys Asp Leu Arg Cys Glu Tyr Leu Thr Ser Pro Val
1 5 10 15
Leu Gly Leu Asp Val Ile Pro Arg Phe Ser Trp Arg Leu Lys Gly Asn
20 25 30
Gly Lys Lys Gln Thr Arg Tyr Lys Ile Ile Val Ser Asp Asn Phe Asp
35 40 45
Asp Ile Glu Arg Gly Ile Gly Asn Val Trp Glu Ser Glu Lys Asp Ser
50 55 60
Ser Lys Asn Leu Asn Ile Glu Tyr Glu Gly Pro Lys Leu Lys Ala Tyr
65 70 75 80
Lys Gly Tyr Tyr Trp Arg Val Lys Leu Trp Asp Glu Lys Glu Asn Gly
85 90 95
Pro Trp Ser Glu Thr Ala Tyr Phe Glu Met Gly Pro Leu Glu Asp Trp
100 105 110
Arg Gly Lys Trp Ile Thr Met Pro Ser Pro Leu Ser Phe Lys Asp Pro
115 120 125
Ala His Arg His Glu Leu Phe Tyr Ala Met Tyr Phe Arg Lys Glu Phe
130 135 140
Leu Leu Asn Lys Glu Val Glu Lys Ala Arg Val Tyr Val Ser Gly Leu
145 150 155 160
Gly Val Tyr Glu Leu His Leu Asn Gly Lys Arg Val Gly Asn Asn Val
165 170 175
Leu Asp Pro Ala Pro Thr Asp Tyr Asn Lys Val Ala Leu Tyr Ser Thr
180 185 190
Tyr Asp Val Thr Gln Tyr Leu Thr Thr Gly Lys Asn Thr Ile Gly Val
195 200 205
Ile Leu Gly Asn Gly Arg His Ile Arg Asp Tyr Gly Tyr Ser Lys Pro
210 215 220
Lys Leu Tyr Leu Gln Leu Leu Val Phe Tyr Lys Asp Gly Ser Arg Glu
225 230 235 240
Phe Ile Cys Ser Asp Glu Thr Trp Lys Val Ser His Gly Pro Leu Lys
245 250 255
Glu Asn Gly Ile Tyr Phe Gly Glu Val Tyr Asp Ala Arg Asp Glu Ile
260 265 270
Ser Gly Trp Asp Ser Pro Gly Phe Asp Asp Arg Asn Trp Ser Glu Val
275 280 285
Glu Ile Val Glu Gly Pro Ser Leu Lys Ala Gln Leu Ile Pro Val Ile
290 295 300
Arg Val Cys Glu Val Ile Lys Pro Lys Arg Leu Trp Leu Ser Ser Arg
305 310 315 320
Gly Thr Phe Ile Val Asp Phe Gly Lys Asn Ile Ser Gly Trp Val Lys
325 330 335
Leu Arg Val Asn Asn Gly Lys Arg Gly Glu Lys Ile Ile Ile Arg Tyr
340 345 350
Ala Glu Val Leu Asp Pro Ser Met Asp Arg Leu Asp Thr Arg Asn Leu
355 360 365
Arg Leu Ala Arg Ala Thr Asp Glu Tyr Ile Leu Lys Gly Gln Gly Val
370 375 380
Glu Ile Tyr Glu Pro Arg Phe Thr Tyr His Gly Phe Arg Tyr Val Glu
385 390 395 400
Val Glu Asp Tyr Pro Gly Thr Leu Thr Ser Asp Asn Ile Glu Ala Met
405 410 415
Phe Val His Thr Asp Val Glu Lys Val Gly Asp Phe Ala Cys Ser Ser
420 425 430
Glu Leu Leu Asn Lys Ile His Ser Cys Val Val Asn Ser Gln Leu Ala
435 440 445
Asn Leu Met Gly Ile Pro Thr Asp Cys Pro Gln Arg Asp Glu Arg Met
450 455 460
Gly Trp Leu Gly Asp Ala Gln Leu Thr Val Glu Glu Ala Met Tyr Asn
465 470 475 480
Phe Asp Met Ala Ala Phe Tyr Thr Lys Tyr Leu Met Asp Ile Lys Leu
485 490 495
Ser Gln Lys Glu Asp Gly Ser Ile Ser Asp Val Ala Pro Pro Tyr Trp
500 505 510
Lys Arg Tyr Pro Ser Asp Pro Ala Trp Gly Thr Ala Tyr Ala Thr Ile
515 520 525
Leu Trp Tyr Leu Tyr Phe Phe Tyr Glu Asp Arg Arg Val Leu Glu Glu
530 535 540
His Tyr Asp Ser Leu Lys Arg Tyr Val Glu Phe Leu Arg Lys Asn Ser
545 550 555 560
Pro Asn His Leu Thr Lys Leu Gly Gln Ala Gly Asp Trp Cys Pro Pro
565 570 575
Gly Asp Ala Phe Pro Lys Arg Thr Pro Leu Ile Leu Thr Ser Thr Trp
580 585 590
Tyr Tyr Tyr His Asp Thr Leu Ile Leu Ser Glu Ile Ala Lys Ile Leu
595 600 605
Gly Lys Lys Glu Asp Glu His Glu Tyr Arg Lys Leu Ala Gly Glu Ile
610 615 620
Lys Glu Ala Phe Asn Arg His Phe Leu Arg Lys Val Glu Asp His Thr
625 630 635 640
Gly Arg Ile Val Cys Phe Tyr Arg Gly Ile Lys Leu Ser Pro Lys Asp
645 650 655
Arg Ile Pro Thr Thr Gln Thr Cys Asn Val Leu Pro Leu Trp Asn Lys
660 665 670
Met Val Pro Glu Glu Cys Arg Glu Asp Val Phe Lys Val Leu Glu Arg
675 680 685
Leu Ile Glu Val Asp Asn Asp Thr His Phe Asp Thr Gly Ile Val Gly
690 695 700
Thr Arg Tyr Ile Leu Glu Val Leu Ser Glu Asn Gly Arg Lys Asp Leu
705 710 715 720
Ala Leu Lys Leu Leu Leu Lys Glu Asp Tyr Pro Ser Phe Gly Tyr Met
725 730 735
Ile Lys Asn Gly Ala Thr Thr Leu Trp Glu Arg Trp Glu Lys Leu Glu
740 745 750
Gly Thr Gly Met Asn Ser His Asn His Val Met Leu Gly Ser Val Asp
755 760 765
Thr Trp Phe Tyr Lys Tyr Leu Ser Gly Ile Lys Pro Val Ala Pro Gly
770 775 780
Trp Lys Lys Ile Arg Ile Glu Pro Tyr Phe Ala Asp Gln Ile Asp Phe
785 790 795 800
Val Ser Ala Lys Ile Lys Thr Pro Asn Gly Ser Leu Glu Val Ser Trp
805 810 815
Lys Lys Gln Asn Lys Glu Tyr Glu Ile Gln Ile Ile Ile Pro Val Asn
820 825 830
Thr Val Gly Ile Phe Ala Val Pro Glu Ser Phe Lys Val Ser Ala Ile
835 840 845
Asn Ser Lys Gln Val Ser Tyr Pro Ser Glu Phe Glu Leu Glu Pro Gly
850 855 860
Ala Tyr Asn Ile Val Leu Glu Arg Val Arg Glu Cys
865 870 875
<210> 9
<211> 876
<212> PRT
<213> Artificial Synthesis
<400> 9
Met Ile Gln Ala Cys Asp Leu Arg Cys Glu Tyr Leu Thr Ser Pro Val
1 5 10 15
Leu Gly Leu Asp Val Ile Pro Arg Phe Ser Trp Arg Leu Lys Gly Asn
20 25 30
Gly Lys Lys Gln Thr Arg Tyr Lys Ile Ile Val Ser Asp Asn Phe Asp
35 40 45
Asp Ile Glu Arg Gly Ile Gly Asn Val Trp Glu Ser Glu Lys Asp Ser
50 55 60
Ser Lys Asn Leu Asn Ile Glu Tyr Glu Gly Pro Lys Leu Lys Ala Tyr
65 70 75 80
Lys Gly Tyr Tyr Trp Arg Val Lys Leu Trp Asp Glu Lys Glu Asn Gly
85 90 95
Pro Trp Ser Glu Thr Ala Tyr Phe Glu Met Gly Pro Leu Glu Asp Trp
100 105 110
Arg Gly Lys Trp Ile Thr Met Pro Ser Pro Leu Ser Phe Lys Asp Pro
115 120 125
Ala His Arg His Glu Leu Phe Tyr Ala Met Tyr Phe Arg Lys Glu Phe
130 135 140
Leu Leu Asn Lys Glu Val Glu Lys Ala Arg Val Tyr Val Ser Gly Leu
145 150 155 160
Gly Val Tyr Glu Leu His Leu Asn Gly Lys Arg Val Gly Asn Asn Val
165 170 175
Leu Asp Pro Ala Pro Thr Asp Tyr Asn Lys Val Ala Leu Tyr Ser Thr
180 185 190
Tyr Asp Val Thr Gln Tyr Leu Thr Thr Gly Lys Asn Thr Ile Gly Val
195 200 205
Ile Leu Gly Asn Gly Arg His Ile Arg Asp Tyr Gly Tyr Ser Lys Pro
210 215 220
Lys Leu Tyr Leu Gln Leu Leu Val Phe Tyr Lys Asp Gly Ser Arg Glu
225 230 235 240
Phe Ile Cys Ser Asp Glu Thr Trp Lys Val Ser His Gly Pro Leu Lys
245 250 255
Glu Asn Gly Ile Tyr Phe Gly Glu Val Tyr Asp Ala Arg Asp Glu Ile
260 265 270
Ser Gly Trp Asp Ser Pro Gly Phe Asp Asp Arg Asn Trp Ser Glu Val
275 280 285
Glu Ile Val Glu Gly Pro Ser Leu Lys Ala Gln Leu Ile Pro Val Ile
290 295 300
Arg Val Cys Glu Val Ile Lys Pro Lys Arg Leu Trp Leu Ser Ser Arg
305 310 315 320
Gly Thr Phe Ile Val Asp Phe Gly Lys Asn Ile Ser Gly Trp Val Lys
325 330 335
Leu Arg Val Asn Asn Gly Lys Arg Gly Glu Lys Ile Ile Ile Arg Tyr
340 345 350
Ala Glu Val Leu Asp Pro Ser Met Asp Arg Leu Asp Thr Arg Asn Leu
355 360 365
Arg Leu Ala Arg Ala Thr Asp Glu Tyr Ile Leu Lys Gly Gln Gly Val
370 375 380
Glu Ile Tyr Glu Pro Arg Phe Thr Tyr His Gly Phe Arg Tyr Val Glu
385 390 395 400
Val Glu Asp Tyr Pro Gly Thr Leu Thr Ser Asp Asn Ile Glu Ala Met
405 410 415
Phe Val His Thr Asp Val Glu Lys Val Gly Asp Phe Ala Cys Ser Ser
420 425 430
Glu Leu Leu Asn Lys Ile His Ser Cys Val Val Asn Ser Gln Leu Ala
435 440 445
Asn Leu Met Gly Ile Pro Thr Asp Cys Pro Gln Arg Asp Glu Arg Met
450 455 460
Gly Trp Leu Gly Asp Ala Gln Leu Thr Val Glu Glu Ala Met Tyr Asn
465 470 475 480
Phe Asp Met Ala Ala Phe Tyr Thr Lys Tyr Leu Met Asp Ile Lys Leu
485 490 495
Ser Gln Lys Glu Asp Gly Ser Ile Ser Asp Val Ala Pro Pro Tyr Trp
500 505 510
Lys Arg Tyr Pro Ser Asp Pro Ala Trp Gly Thr Ala Tyr Ala Thr Ile
515 520 525
Leu Trp Tyr Leu Tyr Phe Phe Tyr Glu Asp Arg Arg Val Leu Glu Glu
530 535 540
His Tyr Asp Ser Leu Lys Arg Tyr Val Glu Phe Leu Arg Lys Asn Ser
545 550 555 560
Pro Asn His Leu Thr Lys Leu Gly Gln His Gly Asp Trp Cys Pro Pro
565 570 575
Gly Asp Ala Phe Pro Lys Arg Thr Pro Leu Ile Leu Thr Ser Thr Trp
580 585 590
Tyr Tyr Tyr His Asp Thr Leu Ile Leu Ser Glu Ile Ala Lys Ile Leu
595 600 605
Gly Lys Lys Glu Asp Glu His Glu Tyr Arg Lys Leu Ala Gly Glu Ile
610 615 620
Lys Glu Ala Phe Asn Arg His Phe Leu Arg Lys Val Glu Asp His Thr
625 630 635 640
Gly Arg Ile Val Cys Phe Tyr Arg Gly Ile Lys Leu Ser Pro Lys Asp
645 650 655
Arg Ile Pro Thr Thr Gln Thr Cys Asn Val Leu Pro Leu Trp Asn Lys
660 665 670
Met Val Pro Glu Glu Cys Arg Glu Asp Val Phe Lys Val Leu Glu Arg
675 680 685
Leu Ile Glu Val Asp Asn Asp Thr His Phe Asp Thr Gly Ile Val Gly
690 695 700
Thr Arg Tyr Ile Leu Glu Val Leu Ser Glu Asn Gly Arg Lys Asp Leu
705 710 715 720
Ala Leu Lys Leu Leu Leu Lys Glu Asp Tyr Pro Ser Phe Gly Tyr Met
725 730 735
Ile Lys Asn Gly Ala Thr Thr Leu Trp Glu Arg Trp Glu Lys Leu Glu
740 745 750
Gly Thr Gly Met Asn Ser His Asn His Val Met Leu Gly Ser Val Asp
755 760 765
Thr Trp Phe Tyr Lys Tyr Leu Ser Gly Ile Lys Pro Val Ala Pro Gly
770 775 780
Trp Lys Lys Ile Arg Ile Glu Pro Tyr Phe Ala Asp Gln Ile Asp Phe
785 790 795 800
Val Ser Ala Lys Ile Lys Thr Pro Asn Gly Ser Leu Glu Val Ser Trp
805 810 815
Lys Lys Gln Asn Lys Glu Tyr Glu Ile Gln Ile Ile Ile Pro Val Asn
820 825 830
Thr Val Gly Ile Phe Ala Val Pro Glu Ser Phe Lys Val Ser Ala Ile
835 840 845
Asn Ser Lys Gln Val Ser Tyr Pro Ser Glu Phe Glu Leu Glu Pro Gly
850 855 860
Ala Tyr Asn Ile Val Leu Glu Arg Val Arg Glu Cys
865 870 875
<210> 10
<211> 2628
<212> DNA
<213> Artificial Synthesis
<400> 10
atgattcagg cctgtgactt gagatgcgag tacttgactt ctccagtctt gggtcttgac 60
gtcatcccaa gattctcttg gagactgaaa ggtaacggta agaagcagac caggtacaag 120
atcatcgtgt ccgataactt cgacgacatc gagagaggta tcggtaacgt ttgggaatct 180
gaaaaggact cctccaagaa cctgaacatc gaatacgagg gtccaaagct gaaggcctac 240
aagggttact attggagagt taagctgtgg gacgagaaag agaacggtcc ttggtctgaa 300
actgcctact ttgagatggg tccattggag gattggcgtg gtaagtggat tactatgcca 360
tctccactgt ccttcaagga cccagctcat agacacgagt tgttctacgc catgtacttc 420
agaaaagagt tcctgctgaa caaagaggtc gagaaggcca gagtttacgt ttccggtttg 480
ggtgtttacg agttgcacct gaacggaaag agagtcggta acaacgtttt ggatccagct 540
ccaaccgact acaacaaggt tgccttgtac tccacttacg acgtcaccca atacttgacc 600
actggtaaga acaccatcgg tgtcatcctt ggaaacggta gacacatcag agactacggt 660
tactccaagc caaagttgta cttgcagctg ctggtgttct acaaggacgg ttcccgtgag 720
ttcatttgtt ctgacgagac ttggaaggtt tcccacggtc cattgaaaga aaacggtatc 780
tacttcggtg aggtctacga cgctagagat gagatttctg gttgggactc tccaggtttc 840
gatgacagaa actggtctga ggttgagatc gttgaaggtc catctttgaa ggcccagttg 900
atcccagtta tcagagtttg cgaggtcatc aagcctaaga gattgtggtt gtcctccaga 960
ggaaccttca tcgttgactt cggaaagaac atctccggtt gggtcaagct gagagtcaac 1020
aatggtaaga gaggtgagaa gatcatcatc agatacgccg aggtcttgga tccatccatg 1080
gacagattgg acaccagaaa cttgagattg gctagagcta ccgacgagta catcttgaaa 1140
ggtcagggtg ttgagatcta cgagccaaga ttcacctacc acggtttcag atacgttgag 1200
gtcgaagatt acccaggtac tttgacctcc gacaacatcg aggctatgtt cgttcacact 1260
gacgttgaga aggttggtga cttcgcttgt tcttccgagc tgttgaacaa gatccactcc 1320
tgtgttgtca actcccagtt ggctaacttg atgggtatcc caactgactg tccacaaagg 1380
gacgaaagaa tgggttggtt gggtgatgct cagttgactg ttgaggaagc catgtacaac 1440
ttcgacatgg ctgctttcta caccaagtac ctgatggaca tcaagctgtc ccaaaaagag 1500
gatggttcca tctctgacgt tgctccacca tacgcgaaga gatacccttc tgatccagct 1560
tggggtactg cttacgctac tatcttgtgg tacttgtact ttttctacga ggacaggcgt 1620
gtgttggagg aacactacga ttccttgaag aggtacgtcg agttcctgag aaagaactcc 1680
ccaaaccact tgaccaagtt gggtcaggct ggtgattggt gtccaccagg tgatgcgttc 1740
ccaaagagaa ccccattgat cttgacctcc acctggtact actaccacga cactttgatc 1800
ttgtccgaga tcgctaagat cctgggtaag aaagaagatg agcacgagta cagaaagctg 1860
gccggtgaaa tcaaagaggc tttcaacaga cacttcctga ggaaggttga ggaccacact 1920
ggtagaatcg tgtgcttcta cagaggtatc aagttgtccc caaaggacag aatcccaacc 1980
actcagacct gtaacgtttt gccattgtgg aacaagatgg tcccagagga atgtagagag 2040
gacgttttca aggttttgga gaggttgatc gaggtggaca acgatactca cttcgacact 2100
ggtatcgtcg gtactcgtta catcttggag gtcttgtctg agaacggtag aaaggacttg 2160
gccttgaagt tgctgctgaa agaggactac ccatccttcg gttacatgat caagaacggt 2220
gctaccacct tgtgggaaag atgggaaaag ttggaaggta ctggtatgaa ctcccacaac 2280
cacgttatgt tgggttccgt tgatacctgg ttctacaaat acctgtccgg tatcaagcca 2340
gttgctccag gttggaagaa gatcagaatc gagccatact tcgctgacca gatcgacttc 2400
gtttccgcca agattaagac tccaaacggt tccttggaag tctcctggaa gaagcaaaac 2460
aaagagtacg agatccagat tatcatcccc gtcaacaccg ttggtatctt cgctgttcca 2520
gagtctttta aggtgtccgc cattaactcc aagcaagtct cttacccatc cgagttcgaa 2580
ttggaaccag gtgcctacaa catcgtcttg gaaagggtta gagaatgt 2628
<210> 11
<211> 33
<212> DNA
<213> Artificial Synthesis
<400> 11
ccatacgcga agagataccc ttctgatcca gct 33
<210> 12
<211> 33
<212> DNA
<213> Artificial Synthesis
<400> 12
tctcttcgcg tatggtggag caacgtcaga gat 33
<210> 13
<211> 30
<212> DNA
<213> Artificial Synthesis
<400> 13
ggtcaggctg gtgattggtg tccaccaggt 30
<210> 14
<211> 30
<212> DNA
<213> Artificial Synthesis
<400> 14
atcaccagcc tgacccaact tggtcaagtg 30
<210> 15
<211> 30
<212> DNA
<213> Artificial Synthesis
<400> 15
ggtgatgcgt tcccaaagag aaccccattg 30
<210> 16
<211> 30
<212> DNA
<213> Artificial Synthesis
<400> 16
tgggaacgca tcacctggtg gacaccaatc 30
Claims (10)
1. A rhamnosidase mutant is characterized in that the amino acid sequence is shown as SEQ ID NO: 1 is mutated into alanine, wherein the mutation site is a combination of more than two of 512 th tryptophan, 570 th histidine and 579 th lysine.
2. The rhamnosidase mutant of claim 1, characterized in that its amino acid sequence is as shown in SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: shown at 9.
3. The rhamnosidase mutant according to claim 2, which comprises a sequence encoding an amino acid sequence as shown in SEQ ID NO: 6, and the nucleotide sequence of the coding gene is shown as SEQ ID NO: shown at 10.
4. A method for preparing a rhamnosidase mutant of any of claims 1-3, characterized in that it comprises the following steps:
s1, connecting the rhamnosidase gene to a plasmid to obtain a recombinant plasmid;
s2, designing a mutation primer, carrying out PCR amplification by adopting the mutation primer and taking the recombinant plasmid as a template, and carrying out enzyme digestion to remove template DNA to obtain a mutation product;
s3, transforming the mutation product into a host cell, screening to obtain a rhamnosidase mutant expression strain, and performing induced expression to obtain a rhamnosidase mutant.
5. The method for preparing the rhamnosidase mutant according to claim 4, wherein the nucleotide sequence of the rhamnosidase gene is shown in SEQ ID NO: 2, respectively.
6. The method for preparing the rhamnosidase mutant according to claim 4, wherein the sequence of the mutation primer is shown as SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15 and SEQ ID NO: shown at 16.
7. The method for preparing rhamnosidase mutants according to claim 4, wherein the host cell is Saccharomyces cerevisiae or Escherichia coli.
8. The method for preparing rhamnosidase mutants according to claim 4, wherein the specific parameters for PCR amplification are: pre-denaturation at 98 ℃ for 3 min; and (3) cycle setting: denaturation at 98 deg.C for 10s, annealing at 62 deg.C for 15s, extension at 72 deg.C for 3min, and 30 cycles; finally, extending for 10min at 72 ℃; the reaction was complete.
9. Use of a rhamnosidase mutant as claimed in any of claims 1-3 in the preparation of icariside I compositions.
10. The use as claimed in claim 9, wherein the icariside I composition is prepared by catalytic conversion of multicomponent flavonoid glycoside in total flavonoids of epimedium herb using rhamnosidase mutants.
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104762281A (en) * | 2015-03-09 | 2015-07-08 | 南京林业大学 | Alpha-L-rhamnosidase and preparing method and applications thereof |
| CN104825479A (en) * | 2015-05-20 | 2015-08-12 | 佛山市金骏康健康科技有限公司 | Icariside derivatives as well as preparation method and application thereof in promoting human cells to generate interferon-gamma and treating disease |
| CN106119268A (en) * | 2016-08-05 | 2016-11-16 | 集美大学 | A kind of method improving α L rhamnosidase r Rha1 heat stability |
| CN106995829A (en) * | 2017-05-12 | 2017-08-01 | 南京林业大学 | A kind of method that enzymatic conversion method barren wort total chromocor prepares epimedium aglucone |
| CN107641621A (en) * | 2017-06-14 | 2018-01-30 | 江苏康缘药业股份有限公司 | The method that a kind of glucosides enzymatic compositions and enzyme process prepare epimedium aglucone |
| CN108467858A (en) * | 2018-02-05 | 2018-08-31 | 南京林业大学 | A kind of alpha-L-Rhamnosidase and its application |
| CN109369747A (en) * | 2018-10-29 | 2019-02-22 | 广东金骏康生物技术有限公司 | Icariside I compound and its derivative, pharmaceutical composition and its preparation method and application |
| CN110066760A (en) * | 2019-05-23 | 2019-07-30 | 江南大学 | It is a kind of express alpha-L-Rhamnosidase recombination bacillus coli and its application |
| CN111575304A (en) * | 2020-05-29 | 2020-08-25 | 江西省科学院微生物研究所 | Encoding gene of alpha-L-rhamnosidase mutant and expression vector thereof |
| CN112210548A (en) * | 2020-09-07 | 2021-01-12 | 佛山市汇腾生物技术有限公司 | Pichia pastoris for expressing alpha-L-rhamnosidase and preparation method and application thereof |
-
2021
- 2021-06-22 CN CN202110692714.3A patent/CN113512542B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104762281A (en) * | 2015-03-09 | 2015-07-08 | 南京林业大学 | Alpha-L-rhamnosidase and preparing method and applications thereof |
| CN104825479A (en) * | 2015-05-20 | 2015-08-12 | 佛山市金骏康健康科技有限公司 | Icariside derivatives as well as preparation method and application thereof in promoting human cells to generate interferon-gamma and treating disease |
| CN106119268A (en) * | 2016-08-05 | 2016-11-16 | 集美大学 | A kind of method improving α L rhamnosidase r Rha1 heat stability |
| CN106995829A (en) * | 2017-05-12 | 2017-08-01 | 南京林业大学 | A kind of method that enzymatic conversion method barren wort total chromocor prepares epimedium aglucone |
| CN107641621A (en) * | 2017-06-14 | 2018-01-30 | 江苏康缘药业股份有限公司 | The method that a kind of glucosides enzymatic compositions and enzyme process prepare epimedium aglucone |
| CN108467858A (en) * | 2018-02-05 | 2018-08-31 | 南京林业大学 | A kind of alpha-L-Rhamnosidase and its application |
| CN109369747A (en) * | 2018-10-29 | 2019-02-22 | 广东金骏康生物技术有限公司 | Icariside I compound and its derivative, pharmaceutical composition and its preparation method and application |
| CN110066760A (en) * | 2019-05-23 | 2019-07-30 | 江南大学 | It is a kind of express alpha-L-Rhamnosidase recombination bacillus coli and its application |
| CN111575304A (en) * | 2020-05-29 | 2020-08-25 | 江西省科学院微生物研究所 | Encoding gene of alpha-L-rhamnosidase mutant and expression vector thereof |
| CN112210548A (en) * | 2020-09-07 | 2021-01-12 | 佛山市汇腾生物技术有限公司 | Pichia pastoris for expressing alpha-L-rhamnosidase and preparation method and application thereof |
Non-Patent Citations (6)
| Title |
|---|
| ACCESSION NO：WP_011944094,alpha-L-rhamnosidase [Thermotoga petrophila];NA;《Genbank》;20190923;Features and Origin部分 * |
| Biochemical characterization of a novel hyperthermophilic α-Lrhamnosidase from Thermotoga petrophila and its application in production of icaritin from epimedin C with a thermostable β-glucosidase;Xie J et al.;《Process Biochemistry》;20200404;第93卷;第115-124页 * |
| Site-directed mutagenesis of α-L-rhamnosidase from Alternaria sp. L1 to enhance synthesis yield of reverse hydrolysis;Li Xu et al.;《Appl Microbiol Biotechnol》;20160628;第100卷(第24期);第10385-10394页 * |
| α-L-鼠李糖苷酶基因的重组表达及其应用;叶德晓 等;《生物化工》;20200425;第6卷(第02期);第103-105、111页 * |
| 源自嗜热菌的α-L-鼠李糖苷酶交联聚集体的制备及应用;许锦 等;《林业工程学报》;20200324;第5卷(第02期);第122-129页 * |
| 真核α-L-鼠李糖苷酶的碳水化合物结合结构域功能架构分析;蔡雨晨 等;《现代食品科技》;20200120;第36卷(第01期);第156-162页 * |
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