CN115125229A - Lysine decarboxylase mutant for synthesizing pentanediamine - Google Patents

Lysine decarboxylase mutant for synthesizing pentanediamine Download PDF

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CN115125229A
CN115125229A CN202110320357.8A CN202110320357A CN115125229A CN 115125229 A CN115125229 A CN 115125229A CN 202110320357 A CN202110320357 A CN 202110320357A CN 115125229 A CN115125229 A CN 115125229A
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黄玉红
李壮
薛雅鞠
张锁江
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Abstract

The invention relates to a lysine decarboxylase mutant for synthesizing pentanediamine, the amino acid sequence of the mutant is mutated from a sequence shown in SEQ ID NO.1, and one or more groups of amino acid residues are mutated to form disulfide bonds: 91 bits/445 bits, 128 bits/163 bits, 233 bits/628 bits, 250 bits/395 bits. The invention provides a gene and a protein sequence of the lysine decarboxylase mutant, a constructed expression vector and a genetic engineering strain and application of the lysine decarboxylase mutant in the synthesis of bio-based pentamethylene diamine. The lysine decarboxylase is induced and expressed by constructing an expression vector and a genetic engineering bacterium, and the bio-based pentamethylene diamine is synthesized. The lysine decarboxylase mutant developed by the invention has the highest catalytic activity under the condition of pH 8, the residual activity of the mutant is still more than 50 percent after the mutant is placed in a pH 8 buffer solution for 312 hours, the half-life period is as long as 330 hours, the mutant has excellent alkali resistance, is beneficial to efficiently synthesizing high-concentration pentamethylene diamine, and has industrial application prospect.

Description

Lysine decarboxylase mutant for synthesizing pentanediamine
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a lysine decarboxylase mutant for synthesizing pentanediamine, which comprises an amino acid sequence and a nucleotide sequence of the lysine decarboxylase mutant, an expression vector and a recombinant engineering strain constructed by the lysine decarboxylase mutant, and application of the lysine decarboxylase mutant, the expression vector and the recombinant engineering strain in production of the pentanediamine.
Background
Nylon Polyamides (PAs) are widely used in automotive, engineering plastics, textile and other industries, and the demand of the nylon market is driven by the growing population and the huge manufacturers of secondary processed products. PA 66 is one of the most important and widely used varieties of nylon, and has a very high dielectric resistance in hot pressing and mechanical stress forming. However, the synthesis technology of adiponitrile, a precursor of hexamethylenediamine, a core monomer, is monopolized by foreign enterprises all the time, and is mainly derived from petroleum, so that the resource is short and the cost fluctuation is huge.
Bio-based nylon PA5X product has received much attention due to its similar properties and sustainable production. Pentanediamine and diacid can synthesize a series of nylon 5X products, such as nylon 52, 5T, 54, 56, 510, 516, 518 and the like. The synthesis of the bio-based nylon 5X can not only reduce the dependency on petroleum resources, but also break through monopoly of transnational enterprises on the output and technology of hexamethylenediamine products, and has wide prospects in the fields of national defense, aerospace and the like. The bio-based nylon PA56 is reported to have remarkable performances of light weight, good hygroscopicity, strong heat resistance, high tensile strength, low dyeing temperature, high elasticity, large flame retardance and the like, and can be developed into various high-end applications.
The key of the bio-based nylon 5X is the efficient synthesis of the core monomer of the bio-based nylon 5X. The highly efficient and stable lysine decarboxylase is the core of the synthesis of bio-based pentanediamine. In the process of catalytically producing the pentamethylene diamine with high concentration, the pH value of the solution is obviously increased along with the accumulation of products, and the cost is increased and inorganic salt waste is generated by adding a large amount of acid for neutralizing to maintain the catalytic activity of the enzyme. Lysine decarboxylase is a decamer consisting of 5 dimers, has a complex structure, is derived from escherichia coli, has the optimum pH of 5.5, and has the enzyme activity which is reduced when the pH is close to neutral and almost completely lost when the pH is 8.0.
CN106148373A, EP3118312B1 and US7189543 were used to sequence an inducible lysine decarboxylase CadA of Escherichia coli, and the Japanese Ajinomoto corporation selected mutants with higher heat stability by directed evolution of CadA, and the Mitsui chemical corporation also disclosed mutants with 10-20% improved activity in its patent. Mutant F14C/K44C/L7M/N8G designed by Hong et al at the lysine decarboxylase poly interface increased the half-life of CadA 216-fold at 60 ℃. The cata mutant was screened by the grand guest team of the institute of biotechnology and industry of Tianjin, academy of sciences, to obtain mutant T88S, and the residual activity of the enzyme still reached 78% after incubation for 10h under alkaline conditions of pH 8. The modification of alkali resistance reported so far increases the reaction pH of the enzyme, but the alkali resistance is lower than the results of the invention.
Disclosure of Invention
The invention aims to provide a lysine decarboxylase mutant for synthesizing pentanediamine, which comprises an amino acid sequence of lysine decarboxylase, a nucleotide sequence for coding the lysine decarboxylase, a gene expression vector and a recombinant engineering strain.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a lysine decarboxylase mutant for synthesizing pentamethylene diamine, which catalyzes the production of pentamethylene diamine from L-lysine, the lysine decarboxylase having any one of the amino acid sequences shown in (I), (II), or (III):
(I) the amino acid sequence is obtained by mutating the sequence shown in SEQ ID NO.1, and one or more amino acid residues selected from the following group are mutated: positions 91, 128, 163, 233, 250, 395, 445 and 628, and the amino acid residue is mutated to Cys;
(II) the lysine decarboxylase has an amino acid sequence which is not less than 95% identical to the amino acid sequence of (I), preferably 99% identical to the amino acid sequence of (I);
(III) the lysine decarboxylase is an amino acid sequence formed by adding or deleting 1-35 amino acids, preferably 1-6 amino acid residues on the C terminal and/or N terminal of the amino acid sequence of (I).
The lysine decarboxylase mutant preferred in the present invention is preferably characterized in that the amino acid of the lysine decarboxylase is mutated to an amino acid residue shown below: cys at position 91/445, and/or Cys at position 128/163, and/or Cys at position 233/628, and/or Cys at position 250/395; and/or two or more amino acid residue mutations at the above positions.
Preferably, the lysine decarboxylase is mutated from 233/628 to Cys and from 250/395 to Cys.
Preferably, the amino acid sequence of the lysine decarboxylase is SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.
In a second aspect, a nucleotide encoding a lysine decarboxylase as defined in claim 1 or 2, preferably the nucleotide sequence of the lysine decarboxylase as set forth in SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No. 5; more preferably, the nucleotide sequence shown as SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO. 9.
In a third aspect, the invention also provides a gene expression vector. The gene expression vector includes: a nucleotide sequence encoding an amino acid sequence as described in the first aspect or a nucleotide sequence as described in the second aspect.
The expression vector may be any of various expression vectors commonly used in the art for expressing a gene of interest in E.coli. Preferably, the gene expression vector is a pET plasmid, preferably a petdue plasmid.
Preferably, the gene expression vector further comprises a nucleotide sequence encoding lysine pentanediamine antiporter.
In a fourth aspect, the present invention also provides a method for constructing a gene expression vector as described in the third aspect, the method comprising the steps of:
inserting a nucleotide sequence encoding the amino acid sequence according to the first aspect or a nucleotide sequence according to the second aspect between restriction enzyme sites of a plasmid to obtain the gene expression vector.
The construction method also comprises the operation of inserting a lysine pentanediamine antiporter gene.
Preferably, the lysine pentanediamine antiporter gene also comprises a signal peptide.
Preferably, the signal peptide comprises an E.coli periplasmic space secretion signal peptide.
The expression vector is exemplified by pelB signal peptide, but not limited to the signal peptide, and can be common in Escherichia coli, such as dsbA, hlyA, lamB, malE, ompA, ompF, ompT, phoA, and the like.
Illustratively, the construction method specifically includes the following steps:
the lysine decarboxylase mutant gene delta ldc and the gene cadB for coding lysine pentanediamine antiporter are respectively inserted between restriction sites NcoI/SacI and Bgl II/Pac I of pETDuet plasmid to construct plasmid pETDuet-delta ldc-cadB, pelB is introduced in front of the cadB sequence and is connected through NdeI/Bgl II restriction sites, and finally, the constructed expression vector is pETDuet-delta ldc-pelB-cadB.
The lysine decarboxylase mutant is named as M61, the mutation site is Cys at position 91/position 445, and the nucleotide sequence is shown as SEQ ID NO. 6; the lysine decarboxylase mutant is named as M62, the mutation site is Cys at 128 bit/163 bit, and the nucleotide sequence is shown as SEQ ID NO. 7; the lysine decarboxylase mutant is named as M63, the mutation site is Cys at the 233/628 sites, and the nucleotide sequence is shown in SEQ ID NO. 8; the lysine decarboxylase mutant is named as M64, the mutation site is Cys at 250 th/395 th, and the nucleotide sequence is shown as SEQ ID NO. 9.
In a fifth aspect, a recombinant engineered bacterium for synthesizing pentanediamine comprises the gene expression vector of the third aspect and/or the nucleotide encoding lysine decarboxylase of the first aspect and/or the nucleotide of the second aspect. The engineering bacterium can be E.coli BL21(DE 3).
In a sixth aspect, the invention also provides a method for preparing pentamethylene diamine by using the recombinant engineering bacteria in the fifth aspect, wherein the method comprises the following steps:
and centrifuging and resuspending the bacterial liquid obtained after the culture and induction of the recombinant engineering bacteria to obtain bacterial suspension, performing protein purification after ultrasonic crushing to obtain free lysine decarboxylase, mixing and reacting the free enzyme with buffer solution containing lysine hydrochloride and pyridoxal phosphate, and centrifuging to obtain the pentanediamine.
In a preferred embodiment of the present invention, the reaction temperature is 35 to 65 ℃, for example, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃ or 65 ℃, and the time is 0.5 to 24 hours, for example, 0.5 hour, 1 hour, 1.5 hour, 2 hours, 2.5 hours, 3 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 15 hours, 16 hours, 18 hours, 20 hours or 24 hours, preferably 1 to 4 hours.
Preferably, the oscillation rate during the reaction is 400 to 800rpm, such as 400rpm, 500rpm, 550rpm, 600rpm, 650rpm, 700rpm or 800 rpm.
Preferably, the rotation speed during centrifugation is 8000-12000 rpm, such as 8000rpm, 8500rpm, 9000rpm, 10000rpm, 10500rpm, 11000rpm or 12000rpm, and the time is 1-3 min, such as 1min, 1.5min, 2min, 2.5min or 3 min.
Preferably, the molarity of lysine hydrochloride in the buffer is 0.01-3M, such as 0.01M, 0.1M, 0.5M, 0.8M, 1M, 1.2M, 1.5M, 1.8M, 2M, 2.5M or 3M.
Preferably, the molar concentration of PLP in the buffer is 0.1-0.5 mM, and may be, for example, 0.1mM, 0.15mM, 0.2mM, 0.25mM, 0.3mM, 0.35mM, 0.4mM, 0.45mM, or 0.5 mM.
Preferably, the buffer comprises any one of a sodium acetate buffer, a phosphate buffer, a Tris-HCl buffer or a sodium carbonate buffer, preferably a Tris-HCl buffer.
Preferably, the buffer solution is added with 0.9% of NaCl by mass fraction.
Preferably, the pH of the buffer is 5-11, for example, 5, 6, 6.4, 7, 7.2, 7.5, 8, 9, 10 or 11, preferably 6-10; the buffer was the same as the solution used in resuspension.
Preferably, after the induced bacterial liquid is centrifuged, the method further comprises a cryopreservation operation, wherein the cryopreservation operation comprises the following steps: freezing and storing at-80 deg.C for more than 1 h.
Preferably, the method for preparing pentamethylene diamine comprises the following steps:
(1) centrifuging a bacterial solution obtained after culturing and inducing the recombinant engineering bacteria, performing cryopreservation, then performing resuspension to obtain a bacterial suspension, performing lysis on the bacterial suspension by using an ultrasonic cell disruptor, and centrifuging at 12000rpm for 20min to obtain a crude enzyme solution;
(2) separating and purifying lysine decarboxylase in the crude enzyme solution by using an AKTA protein purifier, and quantifying the free enzyme by using a BCA protein quantification method;
(3) mixing the free enzyme with a buffer solution containing lysine hydrochloride and pyridoxal phosphate, wherein the molar concentration of the lysine hydrochloride in the buffer solution is 0.1-3M, the molar concentration of the pyridoxal phosphate is 0.1-0.5 mM, the buffer solution is a phosphate buffer solution, and the pH value is 6-10;
(4) oscillating and reacting at 35-65 ℃ at 400-800 rpm for 1-4 h, and centrifuging at 8000-12000 rpm for 1-3 min to obtain the pentanediamine.
In the invention, after the synthesis of the pentamethylene diamine, the method for detecting the lysine and the pentamethylene diamine comprises the following steps:
(1) to the reaction system were added 600. mu.L of 50mM pH 9 boric acid buffer, 200. mu.L of methanol, 60. mu.L of diluted sample, and 130. mu.L of ddH 2 Placing O and 10 mu L of 1M diethyl ethoxymethylenemalonate (DEEMM) at room temperature for reaction for 10min, transferring to water bath at 60-80 ℃ for 1-2 h, and stopping the reaction;
(2) detecting by using a reversed-phase high performance liquid chromatography ultraviolet detector, wherein a mobile phase A is 100% acetonitrile; the mobile phase B is 25mM sodium acetate buffer solution with pH 4.8, and the flow rate is 0.5 mL/min; the detection column is C18; column detection temperature: 35 ℃; sample introduction amount: 2-10 mu L; wavelength: 284 nm.
Gradient elution was used: 0min A: B is 20: 80; 2min A: B is 25: 75; 22min A: B is 48.4: 51.6; 22.01min A: B is 20: 80; 27min A: B is 20: 80; 27.01min, and the gradient elution is not limited to the gradient elution ratio.
In a seventh aspect, the invention also provides a use of the lysine decarboxylase mutant as described in the first aspect, the nucleotide as described in the second aspect, the gene expression vector as described in the third aspect or the recombinant engineered strain as described in the fifth aspect in the synthesis of bio-based pentanediamine.
The recitation of numerical ranges herein includes not only the above-recited values, but also any values between any of the above-recited numerical ranges not recited, and for brevity and clarity, is not intended to be exhaustive of the specific values encompassed within the range.
Compared with the prior art, the invention has at least the following beneficial effects:
(1) the invention provides a type lysine decarboxylase mutant capable of efficiently synthesizing pentanediamine, which can be obtained by inducing expression after constructing an expression vector and a genetic engineering bacterium, and can be kept stable at the pH value of 5-8 and the temperature of 40-50 ℃ when lysine decarboxylase mutant free enzyme catalyzes lysine hydrochloride to synthesize the pentanediamine, and the catalysis efficiency is higher;
(2) the lysine decarboxylase mutant provided by the invention can realize that the residual activity of the mutant is still more than 50% after the mutant is placed in an alkaline environment with the pH value of 8 for 312h, the half-life period is as long as 330h, and the mutant has excellent alkali resistance; and when the pH value is 8 and the temperature is 55 ℃, the conversion rate of the pentamethylene diamine is highest, the alkali resistance of the pentamethylene diamine is obviously higher than that of the Escherichia coli CadA and mutants thereof reported in the prior art, the pentamethylene diamine can be synthesized efficiently, and the industrial application prospect is very high.
Drawings
FIG. 1 shows the construction of lysine decarboxylase mutant M63 plasmid.
FIG. 2 shows the pH tolerance of lysine decarboxylase mutant M63.
FIG. 3 shows the pH tolerance of lysine decarboxylase mutant M64.
FIG. 4 shows temperature tolerance of lysine decarboxylase mutant M63.
FIG. 5 shows temperature tolerance of lysine decarboxylase mutant M64.
Detailed Description
The technical solutions of the present invention are further described in the following detailed description with reference to the drawings, and it should be understood by those skilled in the art that the following examples are only simple examples of the present invention, and do not represent or limit the scope of the present invention, which is defined by the claims.
Example 1
This example provides a method for constructing a genetically engineered strain.
A lysine decarboxylase mutant gene delta ldc (SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9) and a lysine pentanediamine antiporter gene cadB (GenBank: WP _000092909.1) are respectively inserted between restriction sites NcoI/SacI and Bgl II/Pac I of a pETDuet plasmid to construct a plasmid pETDuet-delta ldc-cadB, pelB is introduced in front of the cadB sequence and is connected through restriction sites NdeI/Bgl II, and finally, the constructed expression vector is pETDuet-delta ldc-pelB-cadB (shown in figure 1). And respectively transferring into E.coli BL21(DE3) chassis cells to construct genetic engineering strains for synthesizing the pentamethylene diamine, wherein the genetic engineering strains are still named as M61, M62, M63 and M64.
Example 2
In this example, the activities of the genetically engineered mutants were compared.
The engineered strains M61, M62, M63 and M64 obtained in example 1 were cultured overnight at 37 ℃ in 5mL of LB medium to which 100mg/L of ampicillin was added, to obtain a seed solution.
Transferring the seed solution into 50mL LB medium containing 100mg/L ampicillin in an amount of 1% by volume, culturing at 37 deg.C, and adjusting OD 600 When the concentration is 0.6, adding IPTG with the final concentration of 0.1mM for induction, continuously culturing for 20h at 20 ℃, centrifuging at 4000rpm, collecting thalli, and placing at-80 ℃ for freezing and storing for not less than 24 h.
The frozen cells were resuspended in 50mM phosphate buffer (pH 8) and used for whole cell catalysis experiments. In a whole cell catalytic system of 500. mu.L, the concentration of substrate L-lysine hydrochloride is 1.5M, the concentration of coenzyme PLP is 0.1mM, and the mutant gene engineering strain OD 600 Is 1.5, systemThe buffer used was 50mM phosphate buffer (pH 8). Reacting at 55 deg.C and 500rpm for 1h, centrifuging at 12000rpm for 2min, and collecting supernatant to detect pentanediamine content. The catalytic results of the engineered strain mutants are shown in Table 1, wherein the whole cell catalytic result of the mutant M63 is the best, and the content of the pentanediamine is up to 588.71 mM.
TABLE 1
Engineered bacterial strains Pentanediamine content (mM)
M61 256.53
M62 326.56
M63 588.71
M64 406.22
Example 3
This example provides a method for preparing free lysine decarboxylase mutants.
The engineered strains M63 and M64 obtained in example 1 were subjected to strain culture and protein-induced expression according to the method shown in example 2.
At 4 ℃ precooled buffer a (20mM potassium phosphate buffer, 0.1mM PLP, 500mM NaCl, 20mM imidazole, pH adjusted 7.4), buffer B (20mM potassium phosphate buffer, 0.1mM PLP, 500mM NaCl, 500mM imidazole, pH adjusted 7.4), stock (20mM potassium phosphate buffer, 0.1mM PLP, 150mM NaCl, 10% glycerol, pH adjusted 7.4). Re-suspending frozen thalli at-80 ℃ by using a precooled Binding buffer, crushing by using ultrasound with the power of 55 percent and the ultrasound for 2s, pausing for 3s, centrifuging the crushed liquid at 4 ℃ and 12000rpm for 20min to obtain supernatant, filtering the supernatant by using a 0.22 mu M filter membrane, purifying proteins by using a 5mL Histrap purification column AKTA protein purifier, replacing the preservation liquid by using a 5mL HiTrap desaling Desalting column to finally obtain pure enzymes dissolved in the preservation liquid, wherein the obtained enzymes are separated and purified lysine decarboxylase mutants M63 and M64, and the concentration of the purified lysine decarboxylase is determined by using a BCA protein quantitative method and is preserved at-80 ℃.
Example 4
The catalytic ability of lysine decarboxylase mutants M63 and M64 in different pH buffers was tested in this example.
The buffer solution was sodium acetate buffer (50mM, pH 5, 6, 0.9% NaCl), phosphate buffer (50mM, pH 7, 0.9% NaCl), Tris-HCl buffer (50mM, pH 8, 0.9% NaCl), sodium carbonate buffer (50mM, pH 9, 10, 11, 0.9% NaCl).
After the substrate L-lysine hydrochloride was present at a concentration of 10mM and the coenzyme PLP was present at a concentration of 0.1mM in a 500. mu.L reaction system, the lysine decarboxylase mutants M63 and M64 obtained in example 3 were diluted 150-fold, and 4.3. mu.L and 4.4. mu.L, respectively, were added to the reaction system at a concentration of 0.236. mu.g/mL, and the reaction was catalyzed at 55 ℃ and 500rpm for 1 hour, and centrifuged at 12000rpm for 2 minutes, and the solution was taken to examine the pentanediamine content. The reaction results of lysine decarboxylase mutants M63 and M64 under the condition of pH 5-11 are shown in table 2, wherein the mutant M63 is the highest when the pH of a reaction system reaches 8, and then the catalytic capacity is reduced along with the increase of the pH; mutant M64 was highest when the reaction system pH reached 7.
TABLE 2
Figure BDA0002992560700000101
Figure BDA0002992560700000111
Example 5
In this example the effect of different temperatures on the catalytic ability of lysine decarboxylase mutants M63 and M64 was examined.
The reaction system of lysine decarboxylase mutant M63 is 500 muL, wherein the concentration of substrate L-lysine hydrochloride is 10mM, the concentration of coenzyme PLP is 0.1mM, 4.3 muL is taken after the lysine decarboxylase mutant M63 obtained in example 3 is diluted by 150 times, the concentration of lysine decarboxylase mutant M63 in the system is 0.236 mug/mL, the reaction is catalyzed at pH 8 and 500rpm for 1h, and the solution is centrifuged at 12000rpm for 2min, and the pentanediamine content is detected.
The reaction system of lysine decarboxylase mutant M64 is 500 muL, wherein the concentration of substrate L-lysine hydrochloride is 10mM, the concentration of coenzyme PLP is 0.1mM, 4.4 muL is taken after the lysine decarboxylase mutant M64 obtained in example 3 is diluted by 150 times, the concentration of lysine decarboxylase mutant M63 in the system is 0.236 mug/mL, the reaction is catalyzed at pH 7 and 500rpm for 1h, and the solution is centrifuged at 12000rpm for 2min, and the pentanediamine content is detected by taking the solution.
Free enzyme catalysis results of M63 and M64 at 35-65 ℃ are shown in the following table 3, and the yield of the pentanediamine is easily found to increase and then decrease along with the increase of the temperature. The lysine decarboxylase mutant M63 has broad-spectrum characteristics in catalytic temperature, wherein the mutant M63 has the strongest capacity of synthesizing pentamethylene diamine at 55 ℃; the lysine decarboxylase mutant M64 has the strongest synthesizing ability of the pentamethylene diamine at 60 ℃.
TABLE 3
Figure BDA0002992560700000112
Figure BDA0002992560700000121
Example 6
The lysine decarboxylase mutants M63 and M64 were tested for their tolerance in different pH buffers in this example.
The lysine decarboxylase mutants M63 and M64 in example 3 were diluted 150-fold with buffers of different pH, stored at 4 ℃, sampled at 1h, 24h, 72h, 144h, 216h and 312h time points and reacted under the optimal conditions respectively. The buffer used in this example was the same as that described in example 4.
The reaction system of lysine decarboxylase mutant M63 was 500 μ L, the concentration of lysine decarboxylase mutant M63 was 0.236 μ g/mL, the concentration of substrate L-lysine hydrochloride was 10mM, the concentration of coenzyme PLP was 0.1mM, and the buffer solution was Tris-HCl buffer solution (pH 8, 50mM, 0.9% NaCl). Carrying out catalytic reaction at 55 ℃ and 500rpm for 1h, centrifuging at 12000rpm for 2min, and taking the solution to detect the content of the pentanediamine.
The reaction system of lysine decarboxylase mutant M64 was 500 μ L, the concentration of lysine decarboxylase mutant M64 was 0.236 μ g/mL, the concentration of substrate L-lysine hydrochloride was 10mM, the concentration of coenzyme PLP was 0.1mM, and the buffer was potassium phosphate buffer (pH 7, 50mM, 0.9% NaCl). Carrying out catalytic reaction at 60 ℃ and 500rpm for 1h, centrifuging at 12000rpm for 2min, and taking the solution to detect the content of the pentanediamine.
The results of pH tolerance of lysine decarboxylase mutants M63 and M64 are shown in fig. 2 and 3, respectively. Lysine decarboxylase mutants M63 and M64 can maintain the catalytic activity for a long time at pH 5, pH 6, pH 7 and pH 8, the most tolerant pH is pH 7, and the stability and the alkali resistance are obviously improved. Wherein, the lysine decarboxylase mutant M63 still maintains more than 50% of catalytic activity after being placed in Tris-HCl buffer solution (containing 0.9% NaCl) with the pH value of 8 for 312h, and the half-life period is as long as 330h, which is higher than that of the Escherichia coli CadA and the mutant reported at present.
Example 7
The lysine decarboxylase mutants M63 and M64 were tested for their tolerance at different temperatures in this example.
The lysine decarboxylase mutant M63 obtained in example 3 was diluted to 27.44 μ g/mL with potassium phosphate buffer (pH 7, 50mM, 0.9% NaCl), placed in a homomixer at a constant temperature of 50 ℃ and temperature of 40 ℃, and sampled at time points of 15min, 30min, 1h, 6h, 24h, 48h, 72h, 120h, 168h, 216h, 264h, and 312h to detect the activity. The lysine decarboxylase mutant M63 was a 500. mu.L reaction system in which the free enzyme concentration was 0.236. mu.g/mL, the lysine decarboxylase substrate L-lysine hydrochloride concentration was 10mM, the coenzyme PLP concentration was 0.1mM, and the buffer solution was Tris-HCl buffer (pH 8, 50mM, 0.9% NaCl). Carrying out catalytic reaction at 55 ℃ and 500rpm for 1h, centrifuging at 12000rpm for 2min, and taking the solution to detect the content of the pentanediamine.
The lysine decarboxylase mutant M64 obtained in example 3 was diluted to 26.82 μ g/mL with potassium phosphate buffer (pH 7, 50mM, 0.9% NaCl), placed in a homomixer at a constant temperature of 50 ℃ and temperature of 40 ℃, and sampled at time points of 15min, 30min, 1h, 6h, 24h, 48h, 72h, 120h, 168h, 216h, 264h, and 312h to detect the activity. The reaction system of lysine decarboxylase mutant M64 was a 500 μ L reaction system in which the concentration of free enzyme was 0.236 μ g/mL, the concentration of lysine decarboxylase substrate L-lysine hydrochloride was 10mM, the concentration of coenzyme PLP was 0.1mM, and the buffer was potassium phosphate buffer (pH 7, 50mM, 0.9% NaCl). Carrying out catalytic reaction at 60 ℃ and 500rpm for 1h, centrifuging at 12000rpm for 2min, and taking the solution to detect the content of the pentanediamine.
The results of temperature tolerance of lysine decarboxylase mutants M63 and M64 are shown in FIGS. 4 and 5, respectively. Obviously, the lysine decarboxylase mutants M63 and M64 can keep the catalytic activity for a long time at 40 ℃, the thermal stability is obviously improved, the half-life of the lysine decarboxylase mutant M63 at 50 ℃ is that the lysine decarboxylase mutant still keeps more than 50% of the catalytic activity after being placed in Tris-HCl buffer solution (containing 0.9% NaCl) with the pH value of 8 for 312h, the half-life is as long as 330h, and the lysine decarboxylase mutant is higher than that of Escherichia coli CadA and mutants reported at present, and has extremely high industrial application prospect.
In conclusion, the lysine decarboxylase mutants M63 and M64 provided by the invention not only have higher catalytic activity in neutral and alkaline environments, have good alkali resistance, but also have higher thermal stability. The optimum temperature of pure enzyme catalysis of lysine decarboxylase mutant M63 is 55 ℃, the optimum pH of a catalytic system is 8, the high-efficiency synthesis of the pentamethylene diamine is realized, the good alkali resistance is realized, the pentamethylene diamine can be stored for a long time under the pH of 8, and the half-life period is as long as 330 h; the optimum temperature of pure enzyme catalysis of lysine decarboxylase mutant M64 is 60 ℃, the optimum pH of the catalytic system is 7, the high-efficiency synthesis of the pentamethylene diamine is realized, the thermal stability is good, the pentamethylene diamine can be stored for a long time at 50 ℃, and the half-life period is as long as 130 h.
SEQUENCE LISTING
<110> institute of Process engineering of Chinese academy of sciences
<120> a lysine decarboxylase mutant for synthesizing pentanediamine
<130> 20210316
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 714
<212> PRT
<213> Artificial sequence
<400> 1
Met Asn Ile Ile Ala Ile Leu Asn His Met Gly Val Tyr Phe Lys Glu
1 5 10 15
Glu Pro Ile Arg Glu Leu His Lys Ala Leu Glu Ala Gln Asn Phe Gln
20 25 30
Ile Val Tyr Pro Asn Asp Arg Glu Asp Leu Leu Lys Leu Ile Asp Asn
35 40 45
Asn Ala Arg Leu Cys Gly Val Ile Phe Asp Trp Asp Thr Tyr Asn Leu
50 55 60
Asp Leu Cys Arg Asp Ile Ser Glu Met Asn Glu His Leu Pro Val Tyr
65 70 75 80
Ala Phe Ala Asn Thr His Ser Thr Leu Asp Val Ser Leu Ser Asp Leu
85 90 95
Arg Leu Asn Val Glu Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp
100 105 110
Ile Ala Leu Lys Ile Arg Gln Ser Thr Asp Ala Tyr Val Asp Glu Ile
115 120 125
Leu Pro Pro Leu Thr Lys Ala Leu Phe Asn Tyr Val Lys Glu Gly Lys
130 135 140
Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Phe Gln Lys
145 150 155 160
Ser Pro Val Gly Ser Leu Phe Tyr Asp Phe Tyr Gly Ala Asn Ala Met
165 170 175
Lys Ser Asp Ile Ser Ile Ser Val Ser Glu Leu Gly Ser Leu Leu Asp
180 185 190
His Thr Gly Pro His Lys Glu Ala Glu Glu Tyr Ile Ala Arg Thr Phe
195 200 205
Asn Ala Glu Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn
210 215 220
Lys Ile Val Gly Met Tyr Ser Ala Pro Ala Gly Ser Thr Ile Met Ile
225 230 235 240
Asp Arg Asn Cys His Lys Ser Leu Thr His Leu Met Met Met Ser Asp
245 250 255
Val Thr Pro Ile Tyr Phe Arg Pro Thr Arg Asn Ala Tyr Gly Ile Leu
260 265 270
Gly Gly Ile Pro Lys Ser Glu Phe Ala Arg Glu Thr Ile Glu Glu Arg
275 280 285
Val Lys Asn Thr Pro Asn Ala Thr Trp Pro Val His Ala Val Val Thr
290 295 300
Asn Ser Thr Tyr Asp Gly Leu Phe Tyr Asn Ala Glu Tyr Ile Lys Lys
305 310 315 320
Thr Leu Asp Val Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr
325 330 335
Thr Asn Phe Ser Pro Ile Tyr Lys Gly Leu Cys Gly Met Ser Gly Asp
340 345 350
Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser Thr His Lys Leu
355 360 365
Leu Ala Ala Phe Ser Gln Ala Ser Met Ile His Val Lys Gly Asp Ile
370 375 380
Asn Glu Glu Thr Phe Asn Glu Ala Phe Met Met His Thr Ser Thr Ser
385 390 395 400
Pro His Tyr Gly Ile Val Ala Ser Ile Glu Thr Ala Ala Ala Met Met
405 410 415
Lys Gly Asn Ala Gly Lys Arg Leu Ile Asn Gly Ser Ile Glu Arg Ala
420 425 430
Ile Arg Phe Arg Lys Glu Ile Lys Arg Leu Arg Ser Glu Ser Asp Gly
435 440 445
Trp Phe Phe Asp Val Trp Gln Pro Glu His Ile Asp Glu Ala Lys Cys
450 455 460
Trp Asn Leu Asp Pro Lys Glu Ser Trp His Gly Phe Lys Asp Ile Asp
465 470 475 480
Glu Asn His Met Phe Leu Asp Pro Ile Lys Val Thr Leu Leu Thr Pro
485 490 495
Gly Met Lys Glu Asp Gly Thr Met Ala Asp Thr Gly Ile Pro Ala Ser
500 505 510
Ile Val Ala Lys Tyr Leu Asp Glu His Gly Ile Ile Val Glu Lys Thr
515 520 525
Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr
530 535 540
Lys Ala Met Ser Leu Leu Arg Gly Leu Thr Asp Phe Lys Arg Ala Tyr
545 550 555 560
Asp Leu Asn Leu Arg Val Lys Asn Met Leu Pro Ser Leu Tyr Arg Glu
565 570 575
Asp Pro Glu Phe Tyr Glu Asn Met Arg Ile Gln Glu Leu Ala Gln Gly
580 585 590
Ile His Ala Leu Ile Gln His His Asn Leu Pro Asp Leu Met Tyr Arg
595 600 605
Ala Phe Glu Val Leu Pro Thr Met Val Met Asn Pro His Asn Ala Phe
610 615 620
Gln Met Glu Leu Arg Gly Gln Thr Glu Glu Val Tyr Leu Glu Glu Met
625 630 635 640
Ile Gly Lys Val Asn Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val
645 650 655
Pro Leu Val Met Pro Gly Glu Met Leu Thr Glu Glu Ser Arg Pro Val
660 665 670
Leu Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly
675 680 685
Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr
690 695 700
Thr Val Lys Val Leu Lys Thr Glu Gln Lys
705 710
<210> 2
<211> 714
<212> PRT
<213> Artificial sequence
<400> 2
Met Asn Ile Ile Ala Ile Leu Asn His Met Gly Val Tyr Phe Lys Glu
1 5 10 15
Glu Pro Ile Arg Glu Leu His Lys Ala Leu Glu Ala Gln Asn Phe Gln
20 25 30
Ile Val Tyr Pro Asn Asp Arg Glu Asp Leu Leu Lys Leu Ile Asp Asn
35 40 45
Asn Ala Arg Leu Cys Gly Val Ile Phe Asp Trp Asp Thr Tyr Asn Leu
50 55 60
Asp Leu Cys Arg Asp Ile Ser Glu Met Asn Glu His Leu Pro Val Tyr
65 70 75 80
Ala Phe Ala Asn Thr His Ser Thr Leu Asp Cys Ser Leu Ser Asp Leu
85 90 95
Arg Leu Asn Val Glu Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp
100 105 110
Ile Ala Leu Lys Ile Arg Gln Ser Thr Asp Ala Tyr Val Asp Glu Ile
115 120 125
Leu Pro Pro Leu Thr Lys Ala Leu Phe Asn Tyr Val Lys Glu Gly Lys
130 135 140
Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Phe Gln Lys
145 150 155 160
Ser Pro Val Gly Ser Leu Phe Tyr Asp Phe Tyr Gly Ala Asn Ala Met
165 170 175
Lys Ser Asp Ile Ser Ile Ser Val Ser Glu Leu Gly Ser Leu Leu Asp
180 185 190
His Thr Gly Pro His Lys Glu Ala Glu Glu Tyr Ile Ala Arg Thr Phe
195 200 205
Asn Ala Glu Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn
210 215 220
Lys Ile Val Gly Met Tyr Ser Ala Pro Ala Gly Ser Thr Ile Met Ile
225 230 235 240
Asp Arg Asn Cys His Lys Ser Leu Thr His Leu Met Met Met Ser Asp
245 250 255
Val Thr Pro Ile Tyr Phe Arg Pro Thr Arg Asn Ala Tyr Gly Ile Leu
260 265 270
Gly Gly Ile Pro Lys Ser Glu Phe Ala Arg Glu Thr Ile Glu Glu Arg
275 280 285
Val Lys Asn Thr Pro Asn Ala Thr Trp Pro Val His Ala Val Val Thr
290 295 300
Asn Ser Thr Tyr Asp Gly Leu Phe Tyr Asn Ala Glu Tyr Ile Lys Lys
305 310 315 320
Thr Leu Asp Val Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr
325 330 335
Thr Asn Phe Ser Pro Ile Tyr Lys Gly Leu Cys Gly Met Ser Gly Asp
340 345 350
Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser Thr His Lys Leu
355 360 365
Leu Ala Ala Phe Ser Gln Ala Ser Met Ile His Val Lys Gly Asp Ile
370 375 380
Asn Glu Glu Thr Phe Asn Glu Ala Phe Met Met His Thr Ser Thr Ser
385 390 395 400
Pro His Tyr Gly Ile Val Ala Ser Ile Glu Thr Ala Ala Ala Met Met
405 410 415
Lys Gly Asn Ala Gly Lys Arg Leu Ile Asn Gly Ser Ile Glu Arg Ala
420 425 430
Ile Arg Phe Arg Lys Glu Ile Lys Arg Leu Arg Ser Cys Ser Asp Gly
435 440 445
Trp Phe Phe Asp Val Trp Gln Pro Glu His Ile Asp Glu Ala Lys Cys
450 455 460
Trp Asn Leu Asp Pro Lys Glu Ser Trp His Gly Phe Lys Asp Ile Asp
465 470 475 480
Glu Asn His Met Phe Leu Asp Pro Ile Lys Val Thr Leu Leu Thr Pro
485 490 495
Gly Met Lys Glu Asp Gly Thr Met Ala Asp Thr Gly Ile Pro Ala Ser
500 505 510
Ile Val Ala Lys Tyr Leu Asp Glu His Gly Ile Ile Val Glu Lys Thr
515 520 525
Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr
530 535 540
Lys Ala Met Ser Leu Leu Arg Gly Leu Thr Asp Phe Lys Arg Ala Tyr
545 550 555 560
Asp Leu Asn Leu Arg Val Lys Asn Met Leu Pro Ser Leu Tyr Arg Glu
565 570 575
Asp Pro Glu Phe Tyr Glu Asn Met Arg Ile Gln Glu Leu Ala Gln Gly
580 585 590
Ile His Ala Leu Ile Gln His His Asn Leu Pro Asp Leu Met Tyr Arg
595 600 605
Ala Phe Glu Val Leu Pro Thr Met Val Met Asn Pro His Asn Ala Phe
610 615 620
Gln Met Glu Leu Arg Gly Gln Thr Glu Glu Val Tyr Leu Glu Glu Met
625 630 635 640
Ile Gly Lys Val Asn Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val
645 650 655
Pro Leu Val Met Pro Gly Glu Met Leu Thr Glu Glu Ser Arg Pro Val
660 665 670
Leu Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly
675 680 685
Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr
690 695 700
Thr Val Lys Val Leu Lys Thr Glu Gln Lys
705 710
<210> 3
<211> 714
<212> PRT
<213> Artificial sequence
<400> 3
Met Asn Ile Ile Ala Ile Leu Asn His Met Gly Val Tyr Phe Lys Glu
1 5 10 15
Glu Pro Ile Arg Glu Leu His Lys Ala Leu Glu Ala Gln Asn Phe Gln
20 25 30
Ile Val Tyr Pro Asn Asp Arg Glu Asp Leu Leu Lys Leu Ile Asp Asn
35 40 45
Asn Ala Arg Leu Cys Gly Val Ile Phe Asp Trp Asp Thr Tyr Asn Leu
50 55 60
Asp Leu Cys Arg Asp Ile Ser Glu Met Asn Glu His Leu Pro Val Tyr
65 70 75 80
Ala Phe Ala Asn Thr His Ser Thr Leu Asp Val Ser Leu Ser Asp Leu
85 90 95
Arg Leu Asn Val Glu Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp
100 105 110
Ile Ala Leu Lys Ile Arg Gln Ser Thr Asp Ala Tyr Val Asp Glu Cys
115 120 125
Leu Pro Pro Leu Thr Lys Ala Leu Phe Asn Tyr Val Lys Glu Gly Lys
130 135 140
Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Phe Gln Lys
145 150 155 160
Ser Pro Cys Gly Ser Leu Phe Tyr Asp Phe Tyr Gly Ala Asn Ala Met
165 170 175
Lys Ser Asp Ile Ser Ile Ser Val Ser Glu Leu Gly Ser Leu Leu Asp
180 185 190
His Thr Gly Pro His Lys Glu Ala Glu Glu Tyr Ile Ala Arg Thr Phe
195 200 205
Asn Ala Glu Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn
210 215 220
Lys Ile Val Gly Met Tyr Ser Ala Pro Ala Gly Ser Thr Ile Met Ile
225 230 235 240
Asp Arg Asn Cys His Lys Ser Leu Thr His Leu Met Met Met Ser Asp
245 250 255
Val Thr Pro Ile Tyr Phe Arg Pro Thr Arg Asn Ala Tyr Gly Ile Leu
260 265 270
Gly Gly Ile Pro Lys Ser Glu Phe Ala Arg Glu Thr Ile Glu Glu Arg
275 280 285
Val Lys Asn Thr Pro Asn Ala Thr Trp Pro Val His Ala Val Val Thr
290 295 300
Asn Ser Thr Tyr Asp Gly Leu Phe Tyr Asn Ala Glu Tyr Ile Lys Lys
305 310 315 320
Thr Leu Asp Val Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr
325 330 335
Thr Asn Phe Ser Pro Ile Tyr Lys Gly Leu Cys Gly Met Ser Gly Asp
340 345 350
Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser Thr His Lys Leu
355 360 365
Leu Ala Ala Phe Ser Gln Ala Ser Met Ile His Val Lys Gly Asp Ile
370 375 380
Asn Glu Glu Thr Phe Asn Glu Ala Phe Met Met His Thr Ser Thr Ser
385 390 395 400
Pro His Tyr Gly Ile Val Ala Ser Ile Glu Thr Ala Ala Ala Met Met
405 410 415
Lys Gly Asn Ala Gly Lys Arg Leu Ile Asn Gly Ser Ile Glu Arg Ala
420 425 430
Ile Arg Phe Arg Lys Glu Ile Lys Arg Leu Arg Ser Glu Ser Asp Gly
435 440 445
Trp Phe Phe Asp Val Trp Gln Pro Glu His Ile Asp Glu Ala Lys Cys
450 455 460
Trp Asn Leu Asp Pro Lys Glu Ser Trp His Gly Phe Lys Asp Ile Asp
465 470 475 480
Glu Asn His Met Phe Leu Asp Pro Ile Lys Val Thr Leu Leu Thr Pro
485 490 495
Gly Met Lys Glu Asp Gly Thr Met Ala Asp Thr Gly Ile Pro Ala Ser
500 505 510
Ile Val Ala Lys Tyr Leu Asp Glu His Gly Ile Ile Val Glu Lys Thr
515 520 525
Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr
530 535 540
Lys Ala Met Ser Leu Leu Arg Gly Leu Thr Asp Phe Lys Arg Ala Tyr
545 550 555 560
Asp Leu Asn Leu Arg Val Lys Asn Met Leu Pro Ser Leu Tyr Arg Glu
565 570 575
Asp Pro Glu Phe Tyr Glu Asn Met Arg Ile Gln Glu Leu Ala Gln Gly
580 585 590
Ile His Ala Leu Ile Gln His His Asn Leu Pro Asp Leu Met Tyr Arg
595 600 605
Ala Phe Glu Val Leu Pro Thr Met Val Met Asn Pro His Asn Ala Phe
610 615 620
Gln Met Glu Leu Arg Gly Gln Thr Glu Glu Val Tyr Leu Glu Glu Met
625 630 635 640
Ile Gly Lys Val Asn Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val
645 650 655
Pro Leu Val Met Pro Gly Glu Met Leu Thr Glu Glu Ser Arg Pro Val
660 665 670
Leu Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly
675 680 685
Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr
690 695 700
Thr Val Lys Val Leu Lys Thr Glu Gln Lys
705 710
<210> 4
<211> 714
<212> PRT
<213> Artificial sequence
<400> 4
Met Asn Ile Ile Ala Ile Leu Asn His Met Gly Val Tyr Phe Lys Glu
1 5 10 15
Glu Pro Ile Arg Glu Leu His Lys Ala Leu Glu Ala Gln Asn Phe Gln
20 25 30
Ile Val Tyr Pro Asn Asp Arg Glu Asp Leu Leu Lys Leu Ile Asp Asn
35 40 45
Asn Ala Arg Leu Cys Gly Val Ile Phe Asp Trp Asp Thr Tyr Asn Leu
50 55 60
Asp Leu Cys Arg Asp Ile Ser Glu Met Asn Glu His Leu Pro Val Tyr
65 70 75 80
Ala Phe Ala Asn Thr His Ser Thr Leu Asp Val Ser Leu Ser Asp Leu
85 90 95
Arg Leu Asn Val Glu Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp
100 105 110
Ile Ala Leu Lys Ile Arg Gln Ser Thr Asp Ala Tyr Val Asp Glu Ile
115 120 125
Leu Pro Pro Leu Thr Lys Ala Leu Phe Asn Tyr Val Lys Glu Gly Lys
130 135 140
Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Phe Gln Lys
145 150 155 160
Ser Pro Val Gly Ser Leu Phe Tyr Asp Phe Tyr Gly Ala Asn Ala Met
165 170 175
Lys Ser Asp Ile Ser Ile Ser Val Ser Glu Leu Gly Ser Leu Leu Asp
180 185 190
His Thr Gly Pro His Lys Glu Ala Glu Glu Tyr Ile Ala Arg Thr Phe
195 200 205
Asn Ala Glu Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn
210 215 220
Lys Ile Val Gly Met Tyr Ser Ala Cys Ala Gly Ser Thr Ile Met Ile
225 230 235 240
Asp Arg Asn Cys His Lys Ser Leu Thr His Leu Met Met Met Ser Asp
245 250 255
Val Thr Pro Ile Tyr Phe Arg Pro Thr Arg Asn Ala Tyr Gly Ile Leu
260 265 270
Gly Gly Ile Pro Lys Ser Glu Phe Ala Arg Glu Thr Ile Glu Glu Arg
275 280 285
Val Lys Asn Thr Pro Asn Ala Thr Trp Pro Val His Ala Val Val Thr
290 295 300
Asn Ser Thr Tyr Asp Gly Leu Phe Tyr Asn Ala Glu Tyr Ile Lys Lys
305 310 315 320
Thr Leu Asp Val Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr
325 330 335
Thr Asn Phe Ser Pro Ile Tyr Lys Gly Leu Cys Gly Met Ser Gly Asp
340 345 350
Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser Thr His Lys Leu
355 360 365
Leu Ala Ala Phe Ser Gln Ala Ser Met Ile His Val Lys Gly Asp Ile
370 375 380
Asn Glu Glu Thr Phe Asn Glu Ala Phe Met Met His Thr Ser Thr Ser
385 390 395 400
Pro His Tyr Gly Ile Val Ala Ser Ile Glu Thr Ala Ala Ala Met Met
405 410 415
Lys Gly Asn Ala Gly Lys Arg Leu Ile Asn Gly Ser Ile Glu Arg Ala
420 425 430
Ile Arg Phe Arg Lys Glu Ile Lys Arg Leu Arg Ser Glu Ser Asp Gly
435 440 445
Trp Phe Phe Asp Val Trp Gln Pro Glu His Ile Asp Glu Ala Lys Cys
450 455 460
Trp Asn Leu Asp Pro Lys Glu Ser Trp His Gly Phe Lys Asp Ile Asp
465 470 475 480
Glu Asn His Met Phe Leu Asp Pro Ile Lys Val Thr Leu Leu Thr Pro
485 490 495
Gly Met Lys Glu Asp Gly Thr Met Ala Asp Thr Gly Ile Pro Ala Ser
500 505 510
Ile Val Ala Lys Tyr Leu Asp Glu His Gly Ile Ile Val Glu Lys Thr
515 520 525
Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr
530 535 540
Lys Ala Met Ser Leu Leu Arg Gly Leu Thr Asp Phe Lys Arg Ala Tyr
545 550 555 560
Asp Leu Asn Leu Arg Val Lys Asn Met Leu Pro Ser Leu Tyr Arg Glu
565 570 575
Asp Pro Glu Phe Tyr Glu Asn Met Arg Ile Gln Glu Leu Ala Gln Gly
580 585 590
Ile His Ala Leu Ile Gln His His Asn Leu Pro Asp Leu Met Tyr Arg
595 600 605
Ala Phe Glu Val Leu Pro Thr Met Val Met Asn Pro His Asn Ala Phe
610 615 620
Gln Met Glu Cys Arg Gly Gln Thr Glu Glu Val Tyr Leu Glu Glu Met
625 630 635 640
Ile Gly Lys Val Asn Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val
645 650 655
Pro Leu Val Met Pro Gly Glu Met Leu Thr Glu Glu Ser Arg Pro Val
660 665 670
Leu Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly
675 680 685
Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr
690 695 700
Thr Val Lys Val Leu Lys Thr Glu Gln Lys
705 710
<210> 5
<211> 714
<212> PRT
<213> Artificial sequence
<400> 5
Met Asn Ile Ile Ala Ile Leu Asn His Met Gly Val Tyr Phe Lys Glu
1 5 10 15
Glu Pro Ile Arg Glu Leu His Lys Ala Leu Glu Ala Gln Asn Phe Gln
20 25 30
Ile Val Tyr Pro Asn Asp Arg Glu Asp Leu Leu Lys Leu Ile Asp Asn
35 40 45
Asn Ala Arg Leu Cys Gly Val Ile Phe Asp Trp Asp Thr Tyr Asn Leu
50 55 60
Asp Leu Cys Arg Asp Ile Ser Glu Met Asn Glu His Leu Pro Val Tyr
65 70 75 80
Ala Phe Ala Asn Thr His Ser Thr Leu Asp Val Ser Leu Ser Asp Leu
85 90 95
Arg Leu Asn Val Glu Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp
100 105 110
Ile Ala Leu Lys Ile Arg Gln Ser Thr Asp Ala Tyr Val Asp Glu Ile
115 120 125
Leu Pro Pro Leu Thr Lys Ala Leu Phe Asn Tyr Val Lys Glu Gly Lys
130 135 140
Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Phe Gln Lys
145 150 155 160
Ser Pro Val Gly Ser Leu Phe Tyr Asp Phe Tyr Gly Ala Asn Ala Met
165 170 175
Lys Ser Asp Ile Ser Ile Ser Val Ser Glu Leu Gly Ser Leu Leu Asp
180 185 190
His Thr Gly Pro His Lys Glu Ala Glu Glu Tyr Ile Ala Arg Thr Phe
195 200 205
Asn Ala Glu Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn
210 215 220
Lys Ile Val Gly Met Tyr Ser Ala Pro Ala Gly Ser Thr Ile Met Ile
225 230 235 240
Asp Arg Asn Cys His Lys Ser Leu Thr Cys Leu Met Met Met Ser Asp
245 250 255
Val Thr Pro Ile Tyr Phe Arg Pro Thr Arg Asn Ala Tyr Gly Ile Leu
260 265 270
Gly Gly Ile Pro Lys Ser Glu Phe Ala Arg Glu Thr Ile Glu Glu Arg
275 280 285
Val Lys Asn Thr Pro Asn Ala Thr Trp Pro Val His Ala Val Val Thr
290 295 300
Asn Ser Thr Tyr Asp Gly Leu Phe Tyr Asn Ala Glu Tyr Ile Lys Lys
305 310 315 320
Thr Leu Asp Val Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr
325 330 335
Thr Asn Phe Ser Pro Ile Tyr Lys Gly Leu Cys Gly Met Ser Gly Asp
340 345 350
Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser Thr His Lys Leu
355 360 365
Leu Ala Ala Phe Ser Gln Ala Ser Met Ile His Val Lys Gly Asp Ile
370 375 380
Asn Glu Glu Thr Phe Asn Glu Ala Phe Met Cys His Thr Ser Thr Ser
385 390 395 400
Pro His Tyr Gly Ile Val Ala Ser Ile Glu Thr Ala Ala Ala Met Met
405 410 415
Lys Gly Asn Ala Gly Lys Arg Leu Ile Asn Gly Ser Ile Glu Arg Ala
420 425 430
Ile Arg Phe Arg Lys Glu Ile Lys Arg Leu Arg Ser Glu Ser Asp Gly
435 440 445
Trp Phe Phe Asp Val Trp Gln Pro Glu His Ile Asp Glu Ala Lys Cys
450 455 460
Trp Asn Leu Asp Pro Lys Glu Ser Trp His Gly Phe Lys Asp Ile Asp
465 470 475 480
Glu Asn His Met Phe Leu Asp Pro Ile Lys Val Thr Leu Leu Thr Pro
485 490 495
Gly Met Lys Glu Asp Gly Thr Met Ala Asp Thr Gly Ile Pro Ala Ser
500 505 510
Ile Val Ala Lys Tyr Leu Asp Glu His Gly Ile Ile Val Glu Lys Thr
515 520 525
Gly Pro Tyr Asn Leu Leu Phe Leu Phe Ser Ile Gly Ile Asp Lys Thr
530 535 540
Lys Ala Met Ser Leu Leu Arg Gly Leu Thr Asp Phe Lys Arg Ala Tyr
545 550 555 560
Asp Leu Asn Leu Arg Val Lys Asn Met Leu Pro Ser Leu Tyr Arg Glu
565 570 575
Asp Pro Glu Phe Tyr Glu Asn Met Arg Ile Gln Glu Leu Ala Gln Gly
580 585 590
Ile His Ala Leu Ile Gln His His Asn Leu Pro Asp Leu Met Tyr Arg
595 600 605
Ala Phe Glu Val Leu Pro Thr Met Val Met Asn Pro His Asn Ala Phe
610 615 620
Gln Met Glu Leu Arg Gly Gln Thr Glu Glu Val Tyr Leu Glu Glu Met
625 630 635 640
Ile Gly Lys Val Asn Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val
645 650 655
Pro Leu Val Met Pro Gly Glu Met Leu Thr Glu Glu Ser Arg Pro Val
660 665 670
Leu Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly
675 680 685
Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr
690 695 700
Thr Val Lys Val Leu Lys Thr Glu Gln Lys
705 710
<210> 6
<211> 2163
<212> DNA
<213> Artificial sequence
<400> 6
atgaatatta ttgcaattct gaatcacatg ggcgtgtatt ttaaagaaga accgattcgc 60
gaactgcata aagcactgga agcacagaat tttcagattg tgtatccgaa tgatcgcgaa 120
gacctgctga aactgattga taataatgca cgcctgtgtg gcgtgatttt tgattgggat 180
acctataatc tggatctgtg tcgcgatatt agcgaaatga atgaacatct gccggtgtat 240
gcatttgcaa atacccatag taccctggat tgtagcctga gcgatctgcg cctgaatgtg 300
gaattttttg aatatgcact gggtgcagca gaagatattg cactgaaaat tcgccagtct 360
accgatgctt atgtggatga aattctgccg ccgctgacca aagcactgtt taattatgtt 420
aaagaaggta aatatacctt ttgtaccccg ggtcacatgg gcggcaccgc atttcagaaa 480
agcccggtgg gctcactgtt ttatgatttt tatggcgcaa atgcaatgaa atcagatatt 540
agtattagcg tgagcgaact gggtagcctg ctggatcata ccggtccgca taaagaagca 600
gaagaatata ttgcacgtac ctttaatgca gaacgtagct atatggtgac caatggtaca 660
tctaccgcaa ataaaattgt gggcatgtat agcgcaccgg caggctcaac cattatgatt 720
gatcgtaatt gtcataaatc actgacccat ctgatgatga tgtcagatgt gaccccgatt 780
tattttcgcc cgacccgtaa tgcctatggt attctgggcg gcattccgaa aagcgaattt 840
gcacgcgaaa ccattgaaga acgcgttaaa aataccccga atgcaacctg gccggttcat 900
gcagttgtga ccaatagtac ctatgatggc ctgttttata atgcagaata tattaaaaaa 960
accctggatg ttaaatcaat tcattttgat agcgcatggg ttccgtatac caattttagt 1020
ccgatttata aaggcctgtg tggtatgtca ggcgatcgtg tggaaggtaa agttatttat 1080
gaaacccagt ctacccataa actgctggca gcatttagcc aggcaagcat gattcatgtt 1140
aaaggcgata ttaatgaaga aacctttaat gaagcattta tgatgcatac ctcaacctct 1200
ccgcattatg gcattgttgc aagtattgaa accgcagcag caatgatgaa aggtaatgca 1260
ggcaaacgcc tgattaatgg ctccattgaa cgcgcaattc gctttcgtaa agaaattaaa 1320
cgtctgcgct cttgtagcga tggttggttt tttgatgttt ggcagccgga acatattgat 1380
gaagcaaaat gttggaatct ggacccgaaa gaatcttggc atggctttaa agatattgat 1440
gaaaatcaca tgtttttgga cccgattaaa gtgaccctgc tgaccccggg catgaaagaa 1500
gatggaacga tggcagatac cggtattccg gcaagtattg ttgcaaaata tctggatgaa 1560
catggtatta ttgtggaaaa aaccggcccg tataatctgc tgtttctgtt ttctattggc 1620
attgataaaa ccaaagcaat gtctctgctg cgcggcctga ccgattttaa acgcgcgtat 1680
gatctgaatc tgcgcgttaa aaatatgctg ccgtcactgt atcgcgaaga tccggaattt 1740
tatgaaaata tgcgtattca ggaactggca cagggtattc atgcactgat tcagcatcat 1800
aatctgccgg atctgatgta tcgtgcattt gaagtgctgc cgacaatggt tatgaatccg 1860
cataatgcat ttcagatgga actgcgcggc cagaccgaag aagtgtatct ggaagaaatg 1920
attggtaaag ttaatgcaaa tatgattctg ccgtatccgc cgggcgttcc gctggttatg 1980
ccgggcgaaa tgctgaccga agaatcacgt ccggtgctgg aatttctaca aatgctgtgt 2040
gaaattggcg cacattatcc gggctttgaa accgatattc atggcgcata tcgtcaggca 2100
gatggtcgct ataccgttaa agtgctgaaa accgaacaga aacatcatca ccatcaccat 2160
taa 2163
<210> 7
<211> 2163
<212> DNA
<213> Artificial sequence
<400> 7
atgaatatta ttgcaattct gaatcacatg ggcgtgtatt ttaaagaaga accgattcgc 60
gaactgcata aagcactgga agcacagaat tttcagattg tgtatccgaa tgatcgcgag 120
gatctgctga aactgattga taataatgca cgtctgtgtg gcgtgatttt tgattgggat 180
acctataatc tggatctgtg tcgcgatatt agcgaaatga atgaacatct gccggtgtat 240
gcatttgcaa atacccatag taccctggat gtgagcctga gcgatctgcg cctgaatgtg 300
gaattttttg aatatgcact gggcgcagca gaagatattg cactgaaaat tcgtcagtca 360
accgatgctt acgtggatga atgtctgccg ccgctgacca aagcactgtt taattatgtt 420
aaagaaggta aatatacctt ttgtaccccg ggtcacatgg gcggcaccgc atttcagaaa 480
tcaccgtgtg gctcactgtt ttatgatttt tatggtgcaa atgcaatgaa atcagatatt 540
tctattagcg tgagcgaact gggtagcctg ctggatcata ccggcccgca taaagaagca 600
gaagaatata ttgcacgtac ctttaatgca gaacgctctt atatggtgac caatggcaca 660
tctaccgcaa ataaaattgt gggcatgtat agcgcaccgg caggctcaac cattatgatt 720
gatcgtaatt gtcataaaag cctgacccat ctgatgatga tgagcgatgt taccccgatt 780
tattttcgtc cgacccgtaa tgcttacggt attctgggcg gcattccgaa aagtgaattt 840
gcacgcgaaa ccattgaaga acgcgttaaa aataccccga atgcaacctg gccggttcat 900
gcagttgtga ccaatagtac ctatgatggc ctgttttata atgcagaata tattaaaaaa 960
accctggatg ttaaatcaat tcattttgat agcgcatggg ttccgtatac caattttagt 1020
ccgatttata aaggcctgtg tggcatgtca ggcgatcgcg tggaaggcaa agttatttat 1080
gaaacccagt ctacccataa actgctggca gcatttagcc aggcaagtat gattcatgtt 1140
aaaggtgata ttaatgaaga aacctttaat gaagcattta tgatgcatac ctcaacctct 1200
ccgcattatg gtattgttgc aagcatagaa accgcagcag caatgatgaa aggtaatgca 1260
ggtaaacgcc tgattaatgg ctctattgaa cgtgcaattc gctttcgtaa agaaattaaa 1320
cgcctgcgta gcgaatcaga tggttggttt tttgatgttt ggcagccgga acatattgat 1380
gaagcaaaat gttggaattt agatccgaaa gaatcttggc atggctttaa agatattgat 1440
gaaaatcaca tgtttttaga cccgattaaa gtgaccctgc tgaccccggg tatgaaagaa 1500
gatggtacta tggcagatac cggtattccg gcaagtattg ttgcaaaata tctggatgaa 1560
catggcatta ttgtggaaaa aaccggcccg tataatctgc tgtttctgtt tagtattggc 1620
attgataaaa ccaaagcaat gtcactgctg cgcggtctga ccgattttaa acgcgcttat 1680
gatctgaatc tgcgcgttaa aaatatgctg ccgtcactgt atcgcgaaga tccggaattt 1740
tatgaaaata tgcgtattca ggaactggca cagggtattc atgcactgat tcagcatcat 1800
aatctgccgg atctgatgta tcgcgcattt gaagtgctgc cgacgatggt tatgaatccg 1860
cataatgcat ttcagatgga actgcgcggc cagaccgaag aagtgtatct ggaagaaatg 1920
attggtaaag ttaatgcaaa tatgattctg ccgtatccgc cgggcgttcc gctggttatg 1980
ccgggcgaaa tgctgaccga agaatcacgt ccggtgctgg aatttctcca gatgctgtgt 2040
gaaattggcg cacattatcc gggctttgaa accgatattc atggcgcata tcgccaggca 2100
gatggtcgct ataccgttaa agtgctgaaa accgaacaga aacatcatca ccatcaccat 2160
taa 2163
<210> 8
<211> 2163
<212> DNA
<213> Artificial sequence
<400> 8
atgaatatta ttgcaattct gaatcacatg ggcgtgtatt ttaaagaaga accgattcgc 60
gaactgcata aagcactgga agcacagaat tttcagattg tgtatccgaa tgatcgcgag 120
gacctgctga aactgattga taataatgca cgcctgtgtg gcgttatttt tgattgggat 180
acctataatc tggatctgtg tcgcgatatt agcgaaatga atgaacatct gccggtgtat 240
gcatttgcaa atacccatag taccctggat gtgagcctga gcgatctgcg cctgaatgtg 300
gaattttttg aatatgcact gggcgcagca gaagatattg cactgaaaat tcgtcagtct 360
accgatgcct atgtggatga aattctgccg ccgctgacca aagcactgtt taattatgtt 420
aaagaaggca aatatacctt ttgtaccccg ggtcacatgg gcggcaccgc atttcagaaa 480
tcaccggtgg gctcactgtt ttatgatttt tatggcgcaa atgcaatgaa atcagatatt 540
agtattagcg tgagcgaact gggtagcctg ctggatcata ccggtccgca taaagaagca 600
gaagaatata ttgcacgcac ctttaatgca gaacgtagct atatggtgac caatggaacg 660
tcaaccgcaa ataaaattgt gggcatgtat agtgcatgtg caggctcaac cattatgatt 720
gatcgtaatt gtcataaatc actgacccat ctgatgatga tgtcagatgt gaccccgatt 780
tattttcgtc cgacccgtaa tgcgtatggt attctgggcg gcattccgaa aagcgaattt 840
gcacgcgaaa ccattgaaga acgcgttaaa aataccccga atgcaacctg gccggttcat 900
gcagttgtga ccaatagtac ctatgatggc ctgttttata atgcagaata tattaaaaaa 960
accctggatg ttaaatcaat tcattttgat agcgcatggg ttccgtatac caattttagt 1020
ccgatttata aaggcctgtg tggtatgtca ggcgatcgcg tggaaggtaa agttatttat 1080
gaaacccagt ctacccataa actgctggca gcatttagcc aggcaagtat gattcatgtt 1140
aaaggcgata ttaatgaaga aacctttaat gaagcattta tgatgcatac ctcaacctct 1200
ccgcattatg gcattgttgc aagtattgaa accgcagcag caatgatgaa aggtaatgca 1260
ggtaaacgtc tgattaatgg ctctattgaa cgcgcaattc gctttcgtaa agaaattaaa 1320
cgcctgcgta gcgaaagcga tggttggttt tttgatgttt ggcagccgga acatattgat 1380
gaagcaaaat gttggaatct tgatccgaaa gaatcttggc atggctttaa agatattgat 1440
gaaaatcaca tgtttctcga cccgattaaa gtgaccctgc tgaccccggg catgaaagaa 1500
gatggtacta tggcagatac cggcattccg gcatcgatag ttgcaaaata tctggatgaa 1560
catggtatta ttgtggaaaa aaccggcccg tataatctgc tgtttctgtt ttctattggt 1620
attgataaaa ccaaagcaat gtcactgctg cgcggcctga ccgattttaa acgtgcatac 1680
gatctgaatc tgcgcgttaa aaatatgctg ccgtctctgt atcgtgaaga tccggaattt 1740
tatgaaaata tgcgtattca ggaactggca cagggtattc atgcactgat tcagcatcat 1800
aatctgccgg atctgatgta tcgcgcattt gaagtgctgc cgactatggt tatgaatccg 1860
cataatgcat ttcagatgga atgtcgcggc cagaccgaag aagtgtatct ggaagaaatg 1920
attggtaaag ttaatgcaaa tatgattctg ccgtatccgc cgggtgttcc gctggttatg 1980
ccgggcgaaa tgctgaccga agaatcacgc ccggtgctgg aatttcttca gatgctgtgt 2040
gaaattggcg cacattatcc gggctttgaa accgatattc atggcgcata tcgccaggca 2100
gatggtcgct ataccgttaa agtgctgaaa accgaacaga aacatcatca ccatcaccat 2160
taa 2163
<210> 9
<211> 2163
<212> DNA
<213> Artificial sequence
<400> 9
atgaatatta ttgcaattct gaatcacatg ggcgtgtatt ttaaagaaga accgattcgc 60
gaactgcata aagcactgga agcacagaat tttcagattg tgtatccgaa tgatcgcgag 120
gacctgctga aactgattga taataatgca cgcctgtgtg gcgttatttt tgattgggat 180
acctataatc tggatctgtg tcgcgatatt agcgaaatga atgaacatct gccggtgtat 240
gcatttgcaa atacccatag taccctggat gtgagtctga gcgatctgcg cctgaatgtg 300
gaattttttg aatatgcact gggcgcagca gaagatattg cactgaaaat tcgccagtct 360
accgatgctt atgtggatga aattctgccg ccgctgacca aagcactgtt taattatgtt 420
aaagaaggta aatatacctt ttgtaccccg ggtcacatgg gcggcaccgc atttcagaaa 480
tcaccggtgg gtagcctgtt ttatgatttt tatggtgcaa atgcaatgaa aagcgatatt 540
agtattagcg tgagcgaact gggctcactg ctggatcata ccggcccgca taaagaagca 600
gaagaatata ttgcacgtac ctttaatgca gaacgctctt atatggtgac caatggcaca 660
tctaccgcaa ataaaattgt gggtatgtat agcgcaccgg caggctcaac cattatgatt 720
gatcgtaatt gtcataaatc actgacctgt ctgatgatga tgtcagatgt gaccccgatt 780
tattttcgtc cgacccgtaa tgcctacggc attctgggcg gcattccgaa aagcgaattt 840
gcacgtgaaa ccattgaaga acgcgttaaa aataccccga atgcaacctg gccggttcat 900
gcagttgtga ccaatagtac ctatgatggc ctgttttata atgcagaata tattaaaaaa 960
accctggatg ttaaatcaat tcattttgat agcgcatggg ttccgtatac caattttagc 1020
ccgatttata aaggcctgtg tggcatgtca ggcgatcgcg tggaaggtaa agtgatttat 1080
gaaacccagt caacccataa actgctggca gcatttagcc aggcaagtat gattcatgtt 1140
aaaggcgata ttaatgaaga aacctttaat gaagcattta tgtgtcatac ctcaacctct 1200
ccgcattatg gtattgttgc aagtattgaa accgcagcag caatgatgaa aggtaatgca 1260
ggtaaacgcc tgattaatgg ctctattgaa cgcgcaattc gctttcgcaa agaaattaaa 1320
cgcctgcgta gcgaatcaga tggttggttt tttgatgttt ggcagccgga acatattgat 1380
gaagcaaaat gttggaattt agatccgaaa gaaagttggc atggctttaa agatattgat 1440
gaaaatcaca tgtttttaga tccgattaaa gtgaccctgc tgaccccggg catgaaagaa 1500
gatggcacta tggcagatac cggcattccg gcatcgatag ttgcaaaata tctggatgaa 1560
catggtatta ttgtggaaaa aaccggtccg tataatctgc tgtttctgtt ttctattggt 1620
attgataaaa ccaaagcaat gtctctgctg cgcggcctga ccgattttaa acgtgcatac 1680
gatctgaatc tgcgcgttaa aaatatgctg ccgtcactgt atcgcgaaga tccggaattt 1740
tatgaaaata tgcgtattca ggaactggca cagggtattc atgcactgat tcagcatcat 1800
aatctgccgg atctgatgta tcgtgcattt gaagtgctgc cgacaatggt tatgaatccg 1860
cataatgcat ttcagatgga actgcgcggc cagaccgaag aagtgtatct ggaagaaatg 1920
attggtaaag ttaatgcaaa tatgattctg ccgtatccgc cgggcgttcc gctggttatg 1980
ccgggcgaaa tgctgaccga agaatctcgt ccggtgctgg aatttttaca aatgctgtgt 2040
gaaattggcg cacattatcc gggctttgaa accgatattc atggcgcata tcgtcaggca 2100
gatggtcgct ataccgttaa agtgctgaaa accgaacaga aacatcatca ccatcaccat 2160
taa 2163

Claims (10)

1. A lysine decarboxylase mutant for synthesizing pentamethylene diamine, wherein the lysine decarboxylase has any one of the amino acid sequences shown in (I), (II) or (III):
(I) the amino acid sequence is obtained by mutating the sequence shown in SEQ ID NO.1, and one or more amino acid residues selected from the following group are mutated: positions 91, 128, 163, 233, 250, 395, 445 and 628, and the amino acid residue is mutated to Cys;
(II) the lysine decarboxylase has an amino acid sequence which is not less than 95% identical to the amino acid sequence of (I), preferably 99% identical to the amino acid sequence of (I);
(III) the lysine decarboxylase is an amino acid sequence formed by adding or deleting 1-35 amino acids, preferably 1-6 amino acid residues on the C terminal and/or N terminal of the amino acid sequence (I) and (II).
2. The lysine decarboxylase according to claim 1, characterized in that the amino acid sequence of said lysine decarboxylase is mutated at one or more groups of positions selected from the group consisting of: cys at position 91/445, and/or Cys at position 128/163, and/or Cys at position 233/628, and/or Cys at position 250/395.
Preferably, the lysine decarboxylase is mutated from 233/628 to Cys and from 250/395 to Cys.
Preferably, the lysine decarboxylase has the amino acid sequence of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.
3. A nucleotide sequence encoding a lysine decarboxylase as claimed in claim 1 or 2, preferably as shown in SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No. 5; more preferably, the nucleotide sequence shown as SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO. 9.
4. A gene expression vector for synthesizing pentamethylene diamine, which is characterized by comprising: an amino acid sequence encoding a lysine decarboxylase as claimed in claim 1 or 2 or a nucleotide sequence as claimed in claim 3;
preferably, the gene expression vector is a pET plasmid, preferably a pETDuet plasmid;
preferably, the gene expression vector further comprises a nucleotide sequence encoding lysine pentanediamine antiporter.
5. A method for constructing the gene expression vector according to claim 4, comprising the steps of:
the gene expression vector is obtained by inserting a nucleotide sequence encoding lysine decarboxylase as defined in claim 1 or 2 or a nucleotide sequence as defined in claim 3 between restriction enzyme sites of a plasmid.
6. The construction method according to claim 5, further comprising an operation of inserting a lysine pentanediamine antiporter gene;
preferably, the lysine pentanediamine antiporter gene also comprises a signal peptide;
preferably, the signal peptide comprises an E.coli periplasmic space secretion signal peptide;
preferably, the signal peptide comprises any one of dsbA, hlyA, lamB, malE, ompA, ompF, ompT, phoA or pelB, preferably pelB.
7. A recombinant engineered bacterium for synthesizing pentamethylene diamine, comprising the gene expression vector of claim 4 and/or an amino acid sequence encoding lysine decarboxylase of claim 1 or 2 or a nucleotide sequence of claim 3.
8. A method for preparing pentamethylene diamine by using the recombinant engineering bacteria as claimed in claim 7, which is characterized by comprising the following steps:
and centrifuging and resuspending the bacterial liquid obtained after the culture and induction of the recombinant engineering bacteria to obtain bacterial suspension, performing protein purification after ultrasonic crushing to obtain free lysine decarboxylase, mixing and reacting the free enzyme with buffer solution containing lysine hydrochloride and pyridoxal phosphate, and centrifuging to obtain the pentanediamine.
9. The method according to claim 8, wherein the reaction temperature is 35-65 ℃ and the reaction time is 0.5-24 h;
preferably, the mixing reaction time is 1-4 h;
preferably, the oscillation rate in the reaction is 400-800 rpm;
preferably, the rotating speed during centrifugation is 8000-12000 rpm, and the time is 1-3 min;
preferably, the molar concentration of lysine hydrochloride in the buffer solution is 0.1-3M;
preferably, the molar concentration of pyridoxal phosphate in the buffer is 0.1-0.5 mM;
preferably, the buffer comprises any one of a sodium acetate buffer, a phosphate buffer, a Tris-HCl buffer or a sodium carbonate buffer, more preferably a Tris-HCl buffer;
preferably, 0.9% of NaCl in mass fraction is added into the buffer solution;
preferably, the pH value of the buffer solution is 5-11, and preferably 6-10;
preferably, the method comprises the steps of:
(1) centrifuging a bacterial solution obtained after culturing and inducing the recombinant engineering bacteria, performing cryopreservation, then performing resuspension to obtain a bacterial suspension, performing lysis on the bacterial suspension by using an ultrasonic cell disruptor, and centrifuging at 8000-12000 rpm for 20min to obtain a crude enzyme solution;
(2) separating and purifying lysine decarboxylase in the crude enzyme solution by using an AKTA protein purifier, and quantifying the free enzyme by using a BCA protein quantification method;
(3) mixing the free enzyme with a buffer solution containing lysine hydrochloride and pyridoxal phosphate, wherein the molar concentration of the lysine hydrochloride in the buffer solution is 0.1-3M, the molar concentration of the pyridoxal phosphate is 0.1-0.5 mM, the buffer solution is a phosphate buffer solution, and the pH value is 6-10;
(4) oscillating and reacting at 35-65 ℃ at 400-800 rpm for 1-4 h, and centrifuging at 8000-12000 rpm for 1-3 min to obtain the pentanediamine.
10. Use of the lysine decarboxylase amino acid sequence as claimed in claim 1 or 2, the nucleotide sequence as claimed in claim 3, the gene expression vector as claimed in claim 5 or 6 or the recombinant engineered strain as claimed in claim 7 for the biobased synthesis of pentanediamine.
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