CN113249357B - Rhamnosidase TpeRhha-H570A mutant and preparation method and application thereof - Google Patents
Rhamnosidase TpeRhha-H570A mutant and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a rhamnosidase TpeRhha-H570A mutant and a preparation method and application thereof, relates to the technical field of biological medicine, and aims to provide a mutant with an amino acid sequence shown as SEQ ID NO: 2 to alanine at position 570 of the TpeRha enzyme. The rhamnosidase TpeRhha-H570A mutant shows high specificity in the application of catalyzing and hydrolyzing icariin to generate icariside I, and the product conversion rate is improved by 15.63 times compared with the wild TpeRhha enzyme. The rhamnosidase TpeRhha-H570A mutant can be applied to biological processing to prepare epimedium extracts with different component contents, and has short enzymolysis time and high conversion rate.
Description
Technical Field
The invention relates to the technical field of biological medicines, and in particular relates to a rhamnosidase TpeRhha-H570A 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 rational design of the alpha-L-rhamnosidase TpeRha by adopting the site-directed mutagenesis technology to obtain the high-efficiency rhamnosidase TpeRha mutant which can hydrolyze icariin to generate icariside I 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 TpeRhha-H570A 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 TpeRhha-H570A mutant.
The invention also aims to provide application of the rhamnosidase TpeRhha-H570A mutant in preparation of the icariside I composition.
One of the purposes of the invention is realized by adopting the following technical scheme:
a rhamnosidase TpeRhha-H570A mutant is prepared by converting an amino acid sequence shown as SEQ ID NO: 2, histidine (His, H) at position 570 of the TpeRha enzyme is mutated to alanine (Ala, a).
Further, the amino acid sequence of the TpeRhha-H570A mutant is shown as SEQ ID NO: 4, respectively.
Further, the nucleotide sequence of the gene encoding the TpeRhha-H570A mutant is shown as SEQ ID NO: 3, respectively.
Further, the TpeRha enzyme is derived from thermotoga petrosella DSM 13995.
The second purpose of the invention is realized by adopting the following technical scheme:
a method for preparing a rhamnosidase TpeRhha-H570A mutant comprises the following steps:
s1, connecting the TpeRhha enzyme 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 TpeRha-H570A mutant expression strain, and performing induced expression to obtain a rhamnosidase TpeRha-H570A mutant.
Further, the nucleotide sequence of the TpeRhha gene is shown as SEQ ID NO: 1 is shown.
Further, the sequence of the mutant primer is shown as SEQ ID NO: 11 and SEQ ID NO: shown at 12.
Further, the host cell is Escherichia coli.
The third purpose of the invention is realized by adopting the following technical scheme:
an application of a rhamnosidase TpeRhha-H570A mutant in preparing icariside I composition.
Further, the icariside I composition is prepared by catalyzing and converting multicomponent flavone glycoside in epimedium total flavone by using a rhamnosidase TpeRha-H570A mutant.
Compared with the prior art, the invention has the beneficial effects that:
the rhamnosidase TpeRhha-H570A mutant is a combined mutant of alpha-L-rhamnosidase TpeRhha-H570A constructed by a site-directed mutagenesis technology, and is used for preparing an icariside I composition by catalytically converting multi-component flavonoid glycoside in total flavonoids of epimedium, and the enzymolysis time is short, and the conversion rate is high. The 570 th histidine (H570) position of rhamnosidase TpeRha is related to the size of the active pocket, and on the basis, the histidine is mutated into alanine, so that the space of the active pocket is enlarged, the icariin molecule can be contained more favorably, and the binding effect of a substrate is enhanced. The molecular docking result shows that the mutant TpeRha-H570A and the icariin molecules are superior to the wild TpeRha and icariin molecules in interaction force and complex conformation stability; the product conversion rate of the mutant enzyme is improved by 15.63 times compared with that of the wild TpeRhha enzyme, and high specificity is displayed.
The preparation method of the rhamnosidase TpeRhha mutant provided by the invention analyzes the active pocket region of the alpha-L-rhamnosidase TpeRhha derived from Thermotoga petrosella DSM 13995 by a computer-aided protein engineering enzyme molecular design means, and reasonably designs a new enzyme, so that the efficiency of hydrolyzing icariin (Icarin) to generate Icariside I (Icariside I) is greatly improved.
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 model diagram of the three-dimensional structure of the TpeRha enzyme;
FIG. 2 is a diagram showing an alignment of the structures of key amino acid positions of the TpeRhha enzyme and the DtRha enzyme;
FIG. 3 is a view of the position of H570 in the active pocket; wherein, a is the position condition of the wild TpeRhha enzyme, and b is the position condition of the mutant TpeRhha-H570A;
FIG. 4 is a graph showing the results of molecular docking simulations of wild-type TpeRha enzyme and mutant TpeRha-H570A with icariin, respectively; wherein, a is a docking simulation diagram of a wild type TpeRhha enzyme, and b is a docking simulation diagram of a mutant enzyme TpeRhha-H570A;
FIG. 5 is a graph showing the standard curve of icariside I content;
FIG. 6 is a graph showing HPLC results after reaction of TpeRhha enzyme with each mutant enzyme;
FIG. 7 is a graph comparing the conversion efficiency of TpeRha enzyme and the mutant enzyme TpeRha-H570A for different substrates;
FIG. 8 is a HPLC result chart of the contents of the components 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.
In the following examples, the reaction reagents mentioned in the examples are commercially available according to the usual experimental conditions or the experimental conditions recommended by the manufacturers, unless otherwise specified. The molecular biological experiment method not specifically described in this example can be referred to "molecular cloning Experimental Manual".
Example 1
Establishing a TpeRha enzyme tertiary structure model and determining mutation sites:
1. establishing a three-dimensional structure model of TpeRha
Carrying out homologous modeling on the TperRha by using a homologous modeling tool MODELLER, and evaluating the model to obtain a reliable three-dimensional structure model according to a scoring standard, wherein the result is shown in figure 1.
2. Determination of the site of mutation
The three-dimensional structure model of the TpeRhha obtained by homologous modeling is established according to the three-dimensional structure model of the alpha-L-rhamnosidase DtRha (PDB ID: 6i 60) of streptococcus thermophilus (Dictyoglusthermophium). Studies have shown that 11 amino acid residues interacting with rhamnosyl group are involved in crystal structure models of DtRha enzyme and glycoside ligands, D473, R477, E479, W483, W538, Y587, W590, E782, R783, M792 and H797, respectively. Performing conformation comparison on the three-dimensional structure models of the TpeRha enzyme and the DtRha enzyme, determining an active pocket of the tpeRha enzyme, and positioning corresponding 10 directly related amino acids: d456, R460, E462, W466, W521, W573, E746, R747, M756 and H761. Among these, as shown in fig. 2, studies of the DtRha enzyme indicate that E479 and E782 are key catalytic centers, and correspondingly, that the key residues in the TpeRha model are E462 and E746, respectively, and in order to investigate the roles of these two residues, preferably, these two residues are mutated to alanine, the importance of which is investigated by testing the activity of the mutant enzyme. Specifically, as shown in fig. 3a, the amino acid corresponding to Y610 of DtRha enzyme in TpeRha enzyme is H570, and this amino acid is located at the active pocket luminal edge, having some influence on the active pocket size, and thus is a target amino acid for rational design.
Example 2
Molecular docking simulation predicts the effect of H570A:
1. construction of three-dimensional Structure model of mutant enzyme TpeRhha-H570A
As shown in fig. 3b, the 570 th histidine was virtually mutated to alanine by using the protein structure analysis tool Swiss-PdbViewer based on the TpeRha three-dimensional structure model constructed in example 1, to obtain a TpeRha-H570A three-dimensional structure model, which was mutated to alanine with the shortest branched chain, which can effectively enlarge the size of the active pocket.
2. Molecular docking simulation
The center of the active pocket of the model is positioned by using molecular simulation software Discovery Studio, the center coordinates are (-18.5, -56.9, 32.1), the wild type TpeRhha enzyme and the mutant enzyme TpeRhha-H570A are respectively docked with the icariin molecules by using molecular docking software AutodockVina, the size of the box is set to be 20 x 20, 20 docking conformations are set, and each docking complex is analyzed by using a protein conformation analysis tool PyMOL.
3. Analysis of docking results
As shown in FIG. 4, the binding energy of the complex after the wild-type TpeRha enzyme was docked with the icariin molecule was-5.7 kj/mol, while the binding energy of the complex after the mutant enzyme TpeRha-H570A was docked with the icariin molecule was-7.2 kj/mol, with lower binding energy and more stable conformation and higher response to the reaction. Meanwhile, the interaction force between the substrate and the enzyme is analyzed, 5 interaction forces are formed by the wild TpeRhha enzyme and rhamnosyl in the icariin, and 4 interaction forces are formed by the wild TpeRhha enzyme and aglycone and glucosyl parts; the mutant enzyme TpeRhha-H570A forms 6 interaction forces with rhamnosyl group in icariin and 5 interaction forces with aglycone and glucosyl group. The mutant enzyme TpeRha-H570A acts more strongly on icariin than the wild type TpeRha enzyme on the interaction between the substrate and the enzyme. Therefore, the mutant enzyme TpeRhha-H570A was analyzed from the molecular docking level for better catalytic efficiency and specificity for icariin than the wild-type TpeRhha enzyme.
Example 3
Obtaining a recombinant wild enzyme strain BL21 (DE 3)/pET-28 a-TpeRhha:
the alpha-L-rhamnosidase gene from Thermotoga petroselinus DSM 13995 is synthesized in a whole gene way and optimized by the codon preference of Escherichia coli, and the nucleotide sequence of the alpha-L-rhamnosidase gene is shown as SEQ ID NO: 1, it was ligated to a plasmid pET-28a, the resulting recombinant plasmid was named pET-28a-TpeRha, this plasmid was transformed into E.coli BL21 (DE 3), and the recombinant strain was named BL21 (DE 3)/pET-28 a-TpeRha. The amino acid sequence of the wild type TpeRhha enzyme expressed by the recombinant wild enzyme strain is shown as SEQ ID NO: 2, respectively.
Example 4
Obtaining of TpeRha mutant strain:
1. 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.
The primer pair E462A-F and E462A-R is used for obtaining the mutant enzyme TpeRha-E462A, and the amino acid sequence of the mutant enzyme TpeRha-E462A is SEQ ID NO: 5.
the primer pair E746A-F and E746A-R is used for obtaining the mutant enzyme TpeRha-E746A, and the amino acid sequence of the mutant enzyme TpeRha-E746A is SEQ ID NO: 6.
the primer pair H570A-F and H570A-R is used for obtaining the mutant enzyme TpeRha-H570A, and the amino acid sequence of the mutant enzyme TpeRha-H570A is SEQ ID NO: 4.
PCR amplification procedure: pre-denaturation at 98 ℃ for 3 min; and (3) cycle setting: denaturation at 98 deg.C for 10 s, annealing at 62 deg.C for 15 s, extension at 72 deg.C for 3min, and 30 cycles; finally, extending for 10 min 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.
2. Sequencing verification of 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.
Example 5
Whole-cell catalytic reaction of TpeRha key mutase:
1. induction of TpeRha recombinant strain and mutant enzyme recombinant strain and preparation of whole-cell enzyme solution
(1) BL21 (DE 3)/pET-28 a-TpeRha, BL21 (DE 3)/pET-28 a-TpeRha-E462A, BL21 (DE 3)/pET-28 a-TpeRha-E746A and BL21 (DE 3)/pET-28 a-TpeRha-H570A strains are taken and streaked and activated 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 ℃.
2. Whole cell reaction
(1) Using the whole cell suspensions of the wild type TpeRha enzyme and the mutant TpeRha-E462A enzyme, TpeRha-E746A enzyme, and TpeRha-H570A enzyme obtained in the above experiment as enzyme solutions, reaction systems shown in Table 4 were prepared:
(2) the reaction system is reacted for 1d at 55 ℃. After the reaction is finished, adding 2 times volume of DMSO to terminate the reaction, fully whirling, removing cell thalli by 12000 rpm and centrifuging for 1 min, and filtering a reaction product by a 0.45-micrometer organic filter head and then carrying out HPLC detection.
3. 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:
4. preparation of icariside I standard curve
Icariside I control (98.6% HPLC) was weighed with DMSO as a solvent to prepare standard solutions of 0, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, and 0.4 mg/mL, respectively, and the solutions were tested under the above HPLC test conditions, and the standard curve is shown in FIG. 5.
5. Analysis of results
As shown in FIG. 6, the results of whole-cell catalytic reactions showed that the conversion of icariside I by wild-type TpeRha enzyme reaction of reaction 1d at 55 ℃ and pH 4.6 was only 3.73. + -. 0.09%, whereas icariside I was not produced by both mutant TpeRha-E462A enzyme and TpeRha-E746A enzyme reactions, and that mutations at E462 and E746 sites play an important role in the hydrolysis of alpha-1 glycosidic bond by icariin catalyzed by TpeRha. The conversion rate of the product of icariside I generated by the mutant TpeRha-H570A enzyme reaction reaches up to 58.24 +/-2.86 percent, which is 15.63 times higher than that of the wild type.
Example 6
Evaluation of specificity of mutant enzyme TpeRhha-H570A
Taking whole-cell enzyme solution of wild type TpeRhha enzyme and mutant enzyme TpeRhha-H570A, changing substrate icariin into naringin, hesperidin, rutin and naringin dihydrochalcone respectively according to the reaction system and conditions of example 5, shortening the reaction time to 4H, keeping the other conditions unchanged, and carrying out comparative analysis according to the peak areas of corresponding hydrolysis products naringenin monoglucoside, hesperetin monoglucoside, isoquercitrin and trilobatin.
As shown in FIG. 7, the mutant enzyme TpeRha-H570A has a higher hydrolysis efficiency than the wild-type TpeRha enzyme only when icariin is used as a substrate, and has a different reduction in hydrolysis efficiency than the wild-type TpeRha enzyme when naringin, hesperidin, rutin and naringin dihydrochalcone are used as substrates, which indicates that the mutant enzyme TpeRha-H570A has an improved specificity for icariin.
Example 7
Application of mutant bacterium BL21 (DE 3)/pET-28 a-TpeRhha-H570A in biological processing of epimedium herb extract
100 mL of TB medium with pH of 5 was used as the fermentation medium, and the medium formulation was 1.2% (w/v) tryptone, 2.4% (w/v) yeast extract powder, 0.4% (v/v) glycerol, 17 mM KH2PO4，72 mM KH2PO41g of epimedium extract (containing 20% epimedin C and icariin) is added. Inoculating 2% mutant strain BL21 (DE 3)/pET-28 a-TpeRhha-H570A, fermenting at 37 ℃ and 200rpm for 1d, adjusting the fermentation temperature to 55 ℃, continuing to ferment for 1d, adding 5 times volume of DMSO into the fermentation liquor, fully mixing, centrifuging, taking supernatant, and carrying out HPLC detection, wherein the result is shown in figure 8.
Referring to fig. 8, the epimedium extract components before and after fermentation are obviously changed, the epimedin C (retention time is 6.382 min) content after fermentation is reduced, the icariin (retention time is 7.209 min) content and the icariside i (retention time is 14.145 min) content with high biological activity are obviously improved, the icariside ii (retention time is 15.683 min) is also generated, the content of the main component icaritin (retention time is 23.128 min) of the anti-tumor new medicine is also improved, and the specific component changes are shown in table 6 below.
The mutant enzyme TpeRhha-H570A is shown to convert epimedin C into icariin, icariside I, icariside II and icaritin, and the conversion efficiency is high.
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 TpeRhha-H570A mutant and preparation method and application thereof
<130> SEQ ID No. 1-12
<160> 12
<170> PatentIn version 3.3
<210> 1
<211> 2628
<212> DNA
<213> Thermotoga petrophila DSM 13995
<400> 1
atgatccagg catgtgatct gcgttgtgaa tatctgacca gcccggttct gggtctggat 60
gttattccgc gttttagctg gcgtctgaaa ggtaatggca aaaaacagac ccgctataaa 120
attattgtga gcgataattt cgacgatatt gaacgcggca ttggtaatgt gtgggaaagc 180
gaaaaagata gcagtaaaaa tctgaatatc gagtatgaag gcccgaaact gaaagcatat 240
aaaggctatt attggcgtgt gaaactgtgg gatgaaaaag aaaatggtcc gtggagcgaa 300
accgcatatt ttgaaatggg cccgctggaa gattggcgtg gtaaatggat taccatgccg 360
agtccgctga gctttaaaga tccggcccat cgtcatgaac tgttttatgc catgtatttt 420
cgtaaagaat ttctgctgaa caaggaagtg gaaaaagccc gtgtttatgt tagtggtctg 480
ggtgtttatg aactgcatct gaatggtaaa cgcgtgggca ataatgtgct ggaccctgca 540
ccgaccgatt ataataaggt tgccctgtat agcacctatg atgtgaccca gtatctgacc 600
accggtaaaa ataccattgg cgttattctg ggcaatggtc gtcatattcg tgattatggt 660
tatagtaaac cgaaactgta tctgcagctg ctggttttct ataaagatgg tagccgcgag 720
tttatttgta gcgatgaaac ctggaaagtg agtcatggtc cgctgaaaga aaatggcatc 780
tattttggtg aagtttatga tgcccgtgat gaaattagcg gttgggatag cccgggtttt 840
gatgatcgca attggagtga agtggaaatt gttgaaggtc cgagcctgaa agcccagctg 900
attccggtta ttcgtgtgtg tgaagttatt aagccgaaac gtctgtggct gagcagccgc 960
ggcaccttta ttgttgattt tggcaaaaat atcagcggtt gggttaaact gcgcgtgaat 1020
aatggtaaac gtggtgaaaa aattatcatc cgttatgcag aagtgctgga ccctagtatg 1080
gatcgtctgg atacccgtaa tctgcgcctg gcccgcgcaa ccgatgaata tattctgaaa 1140
ggtcagggtg tggaaatcta tgaaccgcgt tttacctatc atggttttcg ctatgttgaa 1200
gttgaagatt atccgggtac cctgaccagc gataatattg aagcaatgtt tgttcatacc 1260
gatgttgaaa aagtgggtga ctttgcatgc agcagcgaac tgctgaataa gattcatagt 1320
tgcgtggtga atagccagct ggcaaatctg atgggtattc cgaccgattg cccgcagcgt 1380
gatgaacgta tgggctggct gggtgacgcc cagctgaccg tggaagaagc catgtataat 1440
tttgatatgg ccgcctttta taccaaatat ctgatggata ttaagctgag tcagaaagaa 1500
gatggtagta ttagtgatgt ggcaccgccg tattggaaac gctatccgag cgatccggcc 1560
tggggtaccg cctatgcaac cattctgtgg tatctgtatt tcttttatga ggatcgccgc 1620
gttctggaag aacattatga tagtctgaaa cgctatgtgg aatttctgcg taaaaatagc 1680
ccgaatcatc tgaccaaact gggtcagcat ggtgactggt gtccgccggg tgacaaattt 1740
ccgaaacgta ccccgctgat tctgaccagt acctggtatt attatcatga taccctgatt 1800
ctgagtgaaa ttgccaaaat tctgggtaaa aaagaagatg aacacgaata tcgtaagctg 1860
gccggtgaaa ttaaggaagc ctttaatcgt cattttctgc gcaaagtgga agatcatacc 1920
ggtcgtattg tttgttttta tcgcggcatt aagctgagcc cgaaagatcg cattccgacc 1980
acccagacct gtaatgtgct gccgctgtgg aataagatgg ttccggaaga atgtcgcgaa 2040
gatgttttta aagttctgga acgcctgatt gaagttgata atgataccca ttttgacacc 2100
ggcattgtgg gcacccgcta tattctggaa gttctgagcg aaaatggtcg caaagatctg 2160
gccctgaaac tgctgctgaa agaagattat cctagctttg gttatatgat taagaacggt 2220
gccaccaccc tgtgggaacg ctgggaaaaa ctggaaggca ccggtatgaa tagccataat 2280
catgttatgc tgggtagcgt tgatacctgg ttttataaat atctgagcgg cattaagccg 2340
gttgcaccgg gctggaaaaa gattcgtatt gaaccgtatt ttgcagatca gattgatttt 2400
gtgagtgcaa aaattaagac cccgaatggc agcctggaag tgagttggaa aaaacagaat 2460
aaggaatatg agatccagat tattatcccg gtgaataccg ttggtatttt tgcagtgccg 2520
gaaagtttta aagttagtgc aattaatagc aagcaggtta gttatccgag tgaatttgaa 2580
ctggaaccgg gtgcctataa tattgtgctg gaacgcgttc gcgaatgt 2628
<210> 2
<211> 876
<212> PRT
<213> Thermotoga petrophila DSM 13995
<400> 2
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> 3
<211> 2628
<212> DNA
<213> Artificial Synthesis
<400> 3
atgatccagg catgtgatct gcgttgtgaa tatctgacca gcccggttct gggtctggat 60
gttattccgc gttttagctg gcgtctgaaa ggtaatggca aaaaacagac ccgctataaa 120
attattgtga gcgataattt cgacgatatt gaacgcggca ttggtaatgt gtgggaaagc 180
gaaaaagata gcagtaaaaa tctgaatatc gagtatgaag gcccgaaact gaaagcatat 240
aaaggctatt attggcgtgt gaaactgtgg gatgaaaaag aaaatggtcc gtggagcgaa 300
accgcatatt ttgaaatggg cccgctggaa gattggcgtg gtaaatggat taccatgccg 360
agtccgctga gctttaaaga tccggcccat cgtcatgaac tgttttatgc catgtatttt 420
cgtaaagaat ttctgctgaa caaggaagtg gaaaaagccc gtgtttatgt tagtggtctg 480
ggtgtttatg aactgcatct gaatggtaaa cgcgtgggca ataatgtgct ggaccctgca 540
ccgaccgatt ataataaggt tgccctgtat agcacctatg atgtgaccca gtatctgacc 600
accggtaaaa ataccattgg cgttattctg ggcaatggtc gtcatattcg tgattatggt 660
tatagtaaac cgaaactgta tctgcagctg ctggttttct ataaagatgg tagccgcgag 720
tttatttgta gcgatgaaac ctggaaagtg agtcatggtc cgctgaaaga aaatggcatc 780
tattttggtg aagtttatga tgcccgtgat gaaattagcg gttgggatag cccgggtttt 840
gatgatcgca attggagtga agtggaaatt gttgaaggtc cgagcctgaa agcccagctg 900
attccggtta ttcgtgtgtg tgaagttatt aagccgaaac gtctgtggct gagcagccgc 960
ggcaccttta ttgttgattt tggcaaaaat atcagcggtt gggttaaact gcgcgtgaat 1020
aatggtaaac gtggtgaaaa aattatcatc cgttatgcag aagtgctgga ccctagtatg 1080
gatcgtctgg atacccgtaa tctgcgcctg gcccgcgcaa ccgatgaata tattctgaaa 1140
ggtcagggtg tggaaatcta tgaaccgcgt tttacctatc atggttttcg ctatgttgaa 1200
gttgaagatt atccgggtac cctgaccagc gataatattg aagcaatgtt tgttcatacc 1260
gatgttgaaa aagtgggtga ctttgcatgc agcagcgaac tgctgaataa gattcatagt 1320
tgcgtggtga atagccagct ggcaaatctg atgggtattc cgaccgattg cccgcagcgt 1380
gatgaacgta tgggctggct gggtgacgcc cagctgaccg tggaagaagc catgtataat 1440
tttgatatgg ccgcctttta taccaaatat ctgatggata ttaagctgag tcagaaagaa 1500
gatggtagta ttagtgatgt ggcaccgccg tattggaaac gctatccgag cgatccggcc 1560
tggggtaccg cctatgcaac cattctgtgg tatctgtatt tcttttatga ggatcgccgc 1620
gttctggaag aacattatga tagtctgaaa cgctatgtgg aatttctgcg taaaaatagc 1680
ccgaatcatc tgaccaaact gggtcaggca ggtgactggt gtccgccggg tgacaaattt 1740
ccgaaacgta ccccgctgat tctgaccagt acctggtatt attatcatga taccctgatt 1800
ctgagtgaaa ttgccaaaat tctgggtaaa aaagaagatg aacacgaata tcgtaagctg 1860
gccggtgaaa ttaaggaagc ctttaatcgt cattttctgc gcaaagtgga agatcatacc 1920
ggtcgtattg tttgttttta tcgcggcatt aagctgagcc cgaaagatcg cattccgacc 1980
acccagacct gtaatgtgct gccgctgtgg aataagatgg ttccggaaga atgtcgcgaa 2040
gatgttttta aagttctgga acgcctgatt gaagttgata atgataccca ttttgacacc 2100
ggcattgtgg gcacccgcta tattctggaa gttctgagcg aaaatggtcg caaagatctg 2160
gccctgaaac tgctgctgaa agaagattat cctagctttg gttatatgat taagaacggt 2220
gccaccaccc tgtgggaacg ctgggaaaaa ctggaaggca ccggtatgaa tagccataat 2280
catgttatgc tgggtagcgt tgatacctgg ttttataaat atctgagcgg cattaagccg 2340
gttgcaccgg gctggaaaaa gattcgtatt gaaccgtatt ttgcagatca gattgatttt 2400
gtgagtgcaa aaattaagac cccgaatggc agcctggaag tgagttggaa aaaacagaat 2460
aaggaatatg agatccagat tattatcccg gtgaataccg ttggtatttt tgcagtgccg 2520
gaaagtttta aagttagtgc aattaatagc aagcaggtta gttatccgag tgaatttgaa 2580
ctggaaccgg gtgcctataa tattgtgctg gaacgcgttc gcgaatgt 2628
<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 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> 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 Ala 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> 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 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 Ala 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> 30
<212> DNA
<213> Artificial Synthesis
<400> 7
cgtgatgcac gtatgggctg gctgggtgac 30
<210> 8
<211> 30
<212> DNA
<213> Artificial Synthesis
<400> 8
catacgtgca tcacgctgcg ggcaatcggt 30
<210> 9
<211> 30
<212> DNA
<213> Artificial Synthesis
<400> 9
ctgtgggcac gctgggaaaa actggaaggc 30
<210> 10
<211> 30
<212> DNA
<213> Artificial Synthesis
<400> 10
ccagcgtgcc cacagggtgg tggcaccgtt 30
<210> 11
<211> 30
<212> DNA
<213> Artificial Synthesis
<400> 11
ggtcaggcag gtgactggtg tccgccgggt 30
<210> 12
<211> 30
<212> DNA
<213> Artificial Synthesis
<400> 12
gtcacctgcc tgacccagtt tggtcagatg 30
Claims (10)
1. A rhamnosidase TpeRhha-H570A mutant is characterized in that the amino acid sequence is shown as SEQ ID NO: 2 to alanine at position 570 of the TpeRha enzyme.
2. The mutant of rhamnosidase TpeRha-H570A of claim 1, wherein the amino acid sequence of the TpeRha-H570A mutant is as shown in SEQ ID NO: 4, respectively.
3. The rhamnosidase TpeRha-H570A mutant according to claim 2, wherein the nucleotide sequence encoding the TpeRha-H570A mutant gene is as shown in SEQ ID NO: 3, respectively.
4. The mutant of rhamnosidase TpeRhha-H570A according to claim 1, wherein the TpeRhha enzyme is derived from Thermotoga petroselinum DSM 13995.
5. A method for preparing the rhamnosidase TpeRhha-H570A mutant according to any one of claims 1 to 4, comprising the following steps:
s1, connecting the TpeRhha enzyme 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 TpeRha-H570A mutant expression strain, and performing induced expression to obtain a rhamnosidase TpeRha-H570A mutant.
6. The method for preparing the mutant of the rhamnosidase TpeRha-H570A of claim 5, wherein the nucleotide sequence of the TpeRha enzyme gene is shown in SEQ ID NO: 1 is shown.
7. The method for preparing the rhamnosidase TpeRhha-H570A mutant according to claim 5, wherein the sequence of the mutation primer is shown in SEQ ID NO: 11 and SEQ ID NO: shown at 12.
8. The method for preparing the mutant of rhamnosidase TpeRhha-H570A of claim 5, wherein the host cell is Escherichia coli.
9. The use of a mutant of rhamnosidase tpeRhha-H570A as claimed in any one of claims 1 to 4 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 glycosides in epimedium total flavonoids using a rhamnosidase TpeRha-H570A mutant.
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