CN109851658A - A kind of method of one-step synthesis method N-BETA-Alanyl-L-histidine and truncated N-BETA-Alanyl-L-histidine synzyme - Google Patents
A kind of method of one-step synthesis method N-BETA-Alanyl-L-histidine and truncated N-BETA-Alanyl-L-histidine synzyme Download PDFInfo
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- CN109851658A CN109851658A CN201910041416.0A CN201910041416A CN109851658A CN 109851658 A CN109851658 A CN 109851658A CN 201910041416 A CN201910041416 A CN 201910041416A CN 109851658 A CN109851658 A CN 109851658A
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Abstract
The invention discloses a kind of method of one-step synthesis method N-BETA-Alanyl-L-histidine and truncated N-BETA-Alanyl-L-histidine synzyme, and this method is using Beta-alanine, L-Histidine, ATP and Quadrafos as raw material, MgCl2For activator, N-BETA-Alanyl-L-histidine synzyme and polyphosphate kinase is added, passes through the coupling catalysed synthesis carnosine of enzymatic reaction under the conditions of pH 6.5-8.5,30-45 DEG C of temperature；The present invention expresses truncated N-BETA-Alanyl-L-histidine synzyme (its amino acid sequence is as shown in SEQ ID No.3) and polyphosphate kinase using Escherichia coli respectively, dual-enzyme coupling system is mixed to form after purified, the N-BETA-Alanyl-L-histidine synthesis enzymatic Beta-alanine and L-Histidine for truncating expression synthesize N-BETA-Alanyl-L-histidine, simultaneous ATP dephosphorylation forms ADP, polyphosphate kinase be then catalyzed Quadrafos turn phosphate group give ADP formed ATP, to realize the circular regeneration of ATP；Using dual-enzyme coupling system, reaction obtains carnosine under the appropriate reaction conditions；Method of the invention has the advantages that low in raw material price, enzymatic conversion time are short, easy to operate and production cost is low etc..
Description
Technical field
The present invention relates to a kind of preparation method of carnosine, the method for specifically a kind of one-step synthesis method N-BETA-Alanyl-L-histidine belongs to life
Object production technical field.
Background technique
N-BETA-Alanyl-L-histidine (β-alanyl-L-histidin) and the like (such as homocarnosine and anserine), is to be widely present in the food in one's mouth
Natural activity dipeptides in the brain of newborn animal, muscle and other vital tissues.From active peptide discovery over more than 100 years,
There is a large amount of discovery or the proof N-BETA-Alanyl-L-histidine studied with significant anti-oxidant, elimination free radical intracellular, anti-aging isoreactivity, and
And it is used clinically for the auxiliary of hypertension, heart disease, cataract of old people, ulcer, antitumor, promotion wound healing etc.
Treatment.Since its oxidation resistant activity is strong, toxic side effect is low and has a variety of physiological activity, and the active peptide and its derivative are being cured
The fields such as medicine, health care, health, cosmetics have been widely used, and the market space is wide.
The production of N-BETA-Alanyl-L-histidine mainly uses chemical synthesis at present.Existing chemical synthesis process is relatively more, mainly can be with
It is divided into two major classes: (1) participates in synthesis using Beta-alanine.Its main route is Beta-alanine after amido protecting, activated carboxylic
It is condensed with the L-Histidine of protection, then takes off blocking group and obtain N-BETA-Alanyl-L-histidine.Difference of the route due to each blocking group
Cause synthetic route more.Wherein common method is to generate phthalyl-β-using phthalic anhydride and Beta-alanine
Alanine protects amino, and carboxyl is reacted with thionyl chloride generates phthalyl-β-alanyl chloride, then the L-Histidine with protection
Deprotection group obtains product after forming peptide bond.The route is more complicated, and yield is low, easy racemization in peptide bond forming process, influences
Product purity, and solvent consumption is big, easily causes environmental pollution；(2) reaction participated in without Beta-alanine: cardinal principle is L- group
Propylhomoserin first generates peptide bond from different Beta-alanine precursors, is further converted to carnosine.Common route is acted in sodium alkoxide
Under, acylation reaction occurs for L-Histidine and ethyl cyanoacetate, obtains cyano-acetamide-L-Histidine, obtains L- through catalytic hydrogen reduction
Carnosine.The route is relatively easy, saves the process to not isoplastic protection and deprotection, avoids the generation of racemization, but
It is to need waterless operation, it is desirable that stringent.Meanwhile ethyl cyanoacetate is environment harmful toxic matter, Yi Yinqi water pollution and toxicity are anti-
It answers.
Currently, having the report for replacing traditional chemical synthesis technology using the Enzyme catalyzed synthesis of mild environmental protection.Such as use ammonia
Peptidase catalytic Beta-alanine methyl esters and L-Histidine one-step synthesis N-BETA-Alanyl-L-histidine (the patent text that application publication number is CN107217048A
It offers).This method is still needed beta Alanine esterification, and the N-BETA-Alanyl-L-histidine synthesized in reaction solution can be degraded to β-by aminopeptidase
Alanine and L-Histidine, meanwhile, aminopeptidase will form tripeptides, and complicated reaction product, extraction purification difficulty, N-BETA-Alanyl-L-histidine is caused to obtain
Rate is lower.
Summary of the invention
The object of the present invention is to provide a kind of method of one-step synthesis method N-BETA-Alanyl-L-histidine, this method have low in raw material price,
The advantages such as the enzymatic conversion time is short, easy to operate and production cost is low have preferable commercial application potentiality.
In order to reach above-mentioned technical purpose, the technical scheme is that
A kind of method of one-step synthesis method N-BETA-Alanyl-L-histidine, this method are with Beta-alanine, L-Histidine, ATP and Quadrafos
Raw material, MgCl2For activator, N-BETA-Alanyl-L-histidine synzyme and polyphosphate kinase is added, in pH6.5-8.5,30-45 DEG C of temperature condition
Pass through the coupling catalysed synthesis carnosine of enzymatic reaction down.
Specifically, comprising the following steps:
(1) truncated N-BETA-Alanyl-L-histidine synthase gene recombinant expression carrier pET-CARNS1 and polyphosphate kinase are constructed respectively
Gene recombinant vectors pET-Ppk；Wherein, the truncated N-BETA-Alanyl-L-histidine synthase gene derives from Gallus gallus, will
The sequence its nucleotides sequence after codon optimization is classified as sequence shown in SEQ ID NO.1；Polyphosphate kinase gene source in
E.coli K-12 (MG1655), nucleotides sequence are classified as shown in SEQ ID NO.2；Truncated N-BETA-Alanyl-L-histidine synzyme amino acid sequence
Column are as shown in SEQ ID NO.3.
(2) recombinant expression carrier in step (1) is converted to E. coli BL21 (DE3) respectively, obtains base
Because of engineering recombinant bacterial strain E.coli pET-CARNS1 and E.coli PET-PPK.
(3) recombinant bacterial strain in incubation step (2), to obtain the truncated N-BETA-Alanyl-L-histidine synzyme of expression and Quadrafos swashs
The thallus of enzyme.
The cultural method of recombinant bacterial strain are as follows: recombinant bacterial strain is seeded to that is mould containing 50 μ g/mL cards by the 1-2% of percent by volume
In the LB liquid medium of element, in 37 DEG C, 180-220rpm is cultivated when reaching 0.6-0.8 to OD600, adds final concentration 0.2-
0.5mM IPTG collects thallus in 25-28 DEG C of Fiber differentiation 8-10h.
Preferably, every liter of the LB liquid medium includes 10g sodium chloride, 5g yeast powder, 10g peptone, pH value is
7.2-7.4。
(4) thallus in destruction step (3) obtains crude enzyme liquid, mixes after ni-sepharose purification.
(5) mixed enzyme solution obtained in step (4) is added and contains 80-150mM Beta-alanine, 80-150mM L- group ammonia
Acid, 5mM MgCl2, 3.0-6.0mM ATP and 30-50mM calgon catalytic liquid in, pH value be 6.5-8.5, temperature
Enzymatic reaction is carried out under the conditions of being 30-45 DEG C synthesizes carnosine.
The catalytic liquid be in 50mM phosphate buffer containing concentration be 100mM Beta-alanine, 100mM L-Histidine,
5mM MgCl2, 5mM ATP and 50mM calgon.
The method of the present invention expresses truncated N-BETA-Alanyl-L-histidine synzyme and polyphosphate kinase using Escherichia coli respectively, purified
After be mixed to form dual-enzyme coupling system.The N-BETA-Alanyl-L-histidine synthesis enzymatic Beta-alanine and L-Histidine for truncating expression synthesize L- flesh
Peptide, simultaneous ATP dephosphorylation form ADP, and polyphosphate kinase is then catalyzed Quadrafos and turns phosphate group to be formed to ADP
ATP, to realize the circular regeneration of ATP.
The detection method of carnosine are as follows: take appropriate volume reaction solution after 15000g is centrifuged 10min, take supernatant through appropriate dilute
After releasing, 30 μ l samples is taken to be mixed in the 270 μ l 0.2M borate buffers that pH value is 9.0,300 μ l 1.5mg/ml FMOC-Cl are added
Acetonitrile solution is placed at room temperature for 10 minutes, adds the acetonitrile solution of 300 μ l4mg/ml amantadine hydrochlorides, mixes, acetonitrile and water
Ratio is 1:1,0.22 μm of membrane filtration, and upper HPLC is measured.
Beneficial effects of the present invention:
Because from Gallus gallus N-BETA-Alanyl-L-histidine synzyme holoenzyme cannot in Escherichia coli successful expression.This hair
The truncation N-BETA-Alanyl-L-histidine synthase gene of bright middle design successfully obtains solubility expression in Escherichia coli, and can step catalysis β
Alanine and L-Histidine synthesize N-BETA-Alanyl-L-histidine.With it is other it has been reported that enzyme process or chemical method synthesis N-BETA-Alanyl-L-histidine method compared with,
Substrate needed for the present invention needs not move through esterification or radical protection；Required ATP is urged by polyphosphate kinase in catalysis reaction
It is constantly regenerating to change ADP phosphorylation, only need to consume a small amount of ADP can be realized the circular regeneration of ATP；And regenerative raw materials hexa metaphosphoric acid
Sodium is from a wealth of sources, low in cost, easy to operate, is easy to large-scale production, has preferable commercial application potentiality.
Detailed description of the invention
Fig. 1 is that dual-enzyme coupling reacts schematic diagram.
Fig. 2 is the chromatogram and carnosine standard chromatogram of double-enzyme catalysis reaction solution.
Specific embodiment
The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.
The preparation method of carnosine: using Beta-alanine, L-Histidine, ATP and Quadrafos as raw material, MgCl2For activator,
N-BETA-Alanyl-L-histidine synzyme and polyphosphate kinase is added, is coupled under the conditions of pH 6.5-8.5,30-45 DEG C of temperature by enzymatic reaction
Carnosine is catalyzed and synthesized, as shown in Figure 1.Specific steps:
One, recombination truncates N-BETA-Alanyl-L-histidine synzyme strain construction
1.1: according to the 480th of the carnosine synthase1 that gene accession number in Genbank is GU453679 coding the
Amino acid sequence to 930 extracts, and adds MERKT peptide fragment in amino acid, forms truncated N-BETA-Alanyl-L-histidine synzyme amino
Acid sequence after codon optimization, holds addition NcoI restriction enzyme site in optimization 5 ', 3 ' ends add as shown in SEQ ID NO.3
Add XhoI restriction enzyme site, forms the sequence as shown in SEQ ID NO.1.The sequence is closed by Sangon Biotech (Shanghai) Co., Ltd.
At.
1.2: using the genetic fragment and pET-28a carrier in restriction enzyme NcoI and XhoI double digestion 1.1, glue is returned
Target fragment is received, and connects two genetic fragments with T4DNA ligase, obtains recombinant expression carrier pET-CARNS1.
1.3: by the recombinant expression carrier Transformed E .coli BL21 (DE3) in 1.2, obtaining the L- flesh for producing and truncating expression
Peptide synthetase bacterial strain E.coli pET-CARNS1.
Two, polyphosphate kinase expresses strain construction
2.1: using E.coli K12 genome as template, with primer pair 5 '-
- the CGCGGATCCGGTACCTTATTCAGGTTGTTCGAG-3 ' of GGATCCATATGGGTCAGGAAAAGCTATAC-3 '/5 ' amplification
Ppk genetic fragment.The primer separately includes restriction enzyme site NdeI and BamHI, forms the sequence as shown in SEQ ID NO.2.
2.2: being carried using the target fragment and pET-15b obtained in restriction enzyme NdeI and BamHI double digestion 2.1
Body, glue recycle respective objects piece segment DNA.
2.3: using two genetic fragments in T4DNA ligase connection 2.2, obtain recombinant vector pET-PPK.
2.4: in the recombinant plasmid transformed E.coli BL21 (DE3) that 2.3 are obtained, obtaining expression polyphosphate kinase
E.coli PET-PPK。
Three, the preparation of N-BETA-Alanyl-L-histidine synzyme and polyphosphate kinase is truncated
It will be Step 1: the bacterial strain in two be inoculated into LB culture medium according to 1% or 2% (v/v) (containing 50 μ g/mL cards respectively
That mycin is used for E.coli pET-CARNS1, and 100 μ g/mL ampicillins are used for E.coli pET-PPK), in 37 DEG C,
200rpm, which is cultivated to OD600, reaches 0.8 or so, adds the IPTG of final concentration of 0.4mM, 25-28 DEG C of Fiber differentiation 10h.
By E.coli pET-CARNS1 fermentation liquid in 8000rpm, 4 DEG C are centrifuged 10 minutes, collect thallus.Use pH7.4's
It after PBS buffer solution is washed 2 times, is resuspended again with buffer, 1% (v/v) Triton-100 of addition, ice bath, ultrasonic disruption 4 seconds
Stop 2 seconds, maintains 15 minutes.4 DEG C, 12000rpm is centrifuged 20 minutes, takes supernatant.
By supernatant after the filtering of 0.45 micron membrane filter, Ni Focurose6FF (IMAC) is passed through with the flow velocity of 1ml/min
It after resin, is rinsed with 0.5M NaCl, 20mM the pH7.4PBS buffer of same flow velocity, then with 0.5M NaCl and 250mM imidazoles
Solution elutes destination protein with same flow velocity.
By the albumen of elution 4 DEG C of dialysis desaltings of pH7.4PBS buffer, purifying enzyme solution is obtained.
The fermentation liquor treatment method of E.coli pET-PPK is according to document (Artificial Cells, Blood
Substitutes,and Biotechnology,34:515–521,2006,DOI:10.1080/10731190600862886)
It is described to be handled.
Four, dual-enzyme coupling catalyzes and synthesizes N-BETA-Alanyl-L-histidine
4.1: configuration reaction system is wherein 100mM Beta-alanine containing concentration in pH7.4 50mM phosphate buffer,
100mM L-Histidine, 5mM MgCl2,5mM ADP and 50mM calgon.
4.2: to the N-BETA-Alanyl-L-histidine synzyme and polyphosphate kinase for adding purifying in 4.1, so that final concentration is respectively 0.2mg/
Ml and 0.4mg/ml.Reaction system is in 37 DEG C of water-bath oscillating reactions 6h.
The detection of N-BETA-Alanyl-L-histidine and substrate amino acid in reaction solution: L- in catalyst system obtained by above-mentioned N-BETA-Alanyl-L-histidine preparation method
The detection method of carnosine are as follows: take appropriate volume reaction solution after 15000g is centrifuged 10min, take supernatant after suitably diluting, take
30 μ l samples are mixed in 270 μ l 0.2M borate buffers (pH=9.0), and 300 μ l 1.5mg/ml FMOC-Cl acetonitrile solutions are added,
10 minutes are placed at room temperature for, the acetonitrile of 300 μ l4mg/ml amantadine hydrochlorides: water (1:1) solution is added, is mixed, 0.22 μm of filter
Film filtering, upper HPLC are measured.
Chromatographiccondition: Zorbax ODS C18 column, 20 μ l of applied sample amount；Flow phase composition: A: acetonitrile；B:50mM acetic acid
Sodium pH of buffer 4.2；Detection wavelength 263nm；Mobile phase total flow 1ml/min；30 DEG C of column temperature；Gradient elution program: 0-3min,
34%A, 66%B；3-10min, 45%A, 55%B；10-20min, 60%A, 40%B；20-30min, 100%A；30-
40min, 100%A.It is as shown in Figure 2 to analyze result.
Above-described embodiment is not limit the invention in any way, all to be obtained by the way of equivalent substitution or equivalent transformation
Technical solution fall within the scope of protection of the present invention.
Sequence table
<110>Changshu Institute of Technology
<120>a kind of method of one-step synthesis method N-BETA-Alanyl-L-histidine
<160> 3
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gccagtgcca cgctcacgct gctcaactgt ggcacgacct gcgtgctgac gctgaccatc 660
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aacacgatgt agatctggtt ctgttcgaag gccgcctgct gggcgcctgg gttagcgata 780
acggcccgac ccgcgttccg actttcctgg aaactgcggc gactctgccg tcctgcctgc 840
cggcggatcg ccaggctcag ctggttcgtg ctgctctgcg ttgctgccgt gcttgtggtc 900
tgcgtcatgg cgtatttaac gttgaactga aactgtcccc ggcgggcccg cgcctgctgg 960
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gtgcgggcga tggcccggcg gaagcgtgcg gccgtctgct gggcctgtgc caggccctgg 1320
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actatttccc gctacgactc catcgtcccc ggcggtcgtt atcataattt taaagacttt 900
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catatttggt ttgataaagc ccagttccgc aatggttttg atgccattcg cgaacgcgat 1020
gtgttgctct attatcctta tcacaccttt gagcatgtgc tggaactgct gcgtcaggct 1080
tcgttcgacc cgagcgtact ggcgattaaa attaacattt accgcgtggc gaaagattca 1140
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gagttacagg cgcgtttcga cgaagaagcc aacattcact gggcgaagcg cctgaccgaa 1260
gcaggcgtgc acgttatctt ctctgcgccg gggctgaaaa ttcacgccaa actgttcctg 1320
atttcacgta aagaaaacgg tgaagtggtg cgttatgcac acatcgggac cgggaacttt 1380
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accaacgaag tacggcgggt atttaacttt attgaaaacc cataccgtcc ggtgacattt 1500
gattatttaa tggtatcgcc gcaaaactcc cgccgcctat tgtatgaaat ggtggaccgc 1560
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gtcgataaag gcctggttga tcgtctgtat gcggcctcca gctccggcgt accggttaat 1680
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<212> PRT
<213>jungle fowl (Gallus gallus)
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Met Gly Ala Leu Thr Val Pro Ala Leu Leu Ala Gly Cys Leu Leu His
1 5 10 15
Ala Ala Gly Cys His Leu Val Gly Gly Leu Ala Ile Leu Leu Ile Gly
20 25 30
Ala Gly Gly Val Ser Leu Ser Pro Val Thr Gly Ala Ala Ala Gly Thr
35 40 45
Gly Leu Ala Ile His Leu Val Gly Ser Ala Pro Gly His Pro Ala Ala
50 55 60
Gly Leu Val Gly Thr Pro Leu Pro Thr Ala Ser Ala Gly His Ala Ala
65 70 75 80
Ala Gly Gly His Ala Gly Ala Val Leu Gly Met Leu Ala Ala Ala Gly
85 90 95
Leu Ala Pro Ala Ala Cys Leu Ser Thr Thr Ala Ala Cys Val Val Leu
100 105 110
Thr Ala Leu Leu Cys Gly Ala Leu Gly Leu Pro Gly Cys Pro Pro Ala
115 120 125
Ala Val Ala Leu Ala Leu Gly Leu Ser Ala Thr His Gly His Leu Gly
130 135 140
Ala Cys Ala Ala Gly Ala Pro Pro Pro Ala Ala Pro Ser Val Pro Cys
145 150 155 160
Ala Ala Leu Ala Ser His Gly Ala Val Gly Ala Ala Ala Gly Ala Val
165 170 175
Pro Pro Pro Ala Val Ala Leu Leu Gly Pro Gly Ala Gly Ala Val Gly
180 185 190
Val Ala Leu Val Gly Ala Ala Gly Gly Cys His Ala His Ala Ala Gly
195 200 205
Leu Thr His Ala Leu Ala Ala Ala Ala Ala His Pro Gly Ile Gly Leu
210 215 220
Gly Thr Gly Ala Ala Met Leu Leu Met Gly Thr Val Pro Gly Thr Gly
225 230 235 240
His Ala Val Ala Leu Val Leu Pro Gly Gly Ala Leu Leu Gly Ala Thr
245 250 255
Val Ser Ala Ala Gly Pro Thr Ala Val Pro Thr Pro Leu Gly Thr Ala
260 265 270
Ala Thr Leu Pro Ser Cys Leu Pro Ala Ala Ala Gly Ala Gly Leu Val
275 280 285
Ala Ala Ala Leu Ala Cys Cys Ala Ala Cys Gly Leu Ala His Gly Val
290 295 300
Pro Ala Val Gly Leu Leu Leu Ser Pro Ala Gly Pro Ala Leu Leu Gly
305 310 315 320
Ile Ala Pro Ala Met Gly Gly Pro Thr Leu Ala Ala Thr Met Ala Ala
325 330 335
Val Thr Gly Pro Ala Leu Leu Leu Ala Ala Val Leu Leu Ala Leu Gly
340 345 350
Leu Pro Pro Val Leu Pro Ser Ala Pro Ala Pro Ala Gly Gly Leu Ala
355 360 365
Gly Val Met Cys Leu Ala Ser Gly His Gly Ala Ala Leu Ala Gly Gly
370 375 380
Val Met Ala Ala Leu Gly Gly Leu Gly Ala Ala Gly Leu Val Ala Leu
385 390 395 400
Ala Pro Leu Pro Gly Gly Ala Gly Gly Ala Thr Gly Gly Pro Cys Leu
405 410 415
Ser Val Ala Cys Ala Gly Ala Gly Pro Ala Gly Ala Cys Gly Ala Leu
420 425 430
Leu Gly Leu Cys Gly Ala Leu Gly Ile Ala Ser Pro Gly Thr Pro Val
435 440 445
Gly His Pro Leu Ser His Pro Leu
450 455
Claims (6)
1. a kind of method of one-step synthesis method N-BETA-Alanyl-L-histidine, it is characterised in that: with Beta-alanine, L-Histidine, ATP and Quadrafos
For raw material, MgCl2For activator, N-BETA-Alanyl-L-histidine synzyme and polyphosphate kinase is added, in pH 6.5-8.5,30-45 DEG C of temperature
Under the conditions of pass through the coupling catalysed synthesis carnosine of enzymatic reaction.
2. a kind of method of one-step synthesis method N-BETA-Alanyl-L-histidine according to claim 1, it is characterised in that the following steps are included:
(1) truncated N-BETA-Alanyl-L-histidine synthase gene recombinant expression carrier pET-CARNS1 and polyphosphate kinase gene are constructed respectively
Recombinant expression carrier pET-Ppk；Wherein, the truncated N-BETA-Alanyl-L-histidine synthase gene derives from Gallus gallus, by the sequence
Column its nucleotides sequence after codon optimization is classified as sequence shown in SEQ ID NO.1；Polyphosphate kinase gene source in
E.coli K-12 (MG 1655), nucleotides sequence are classified as shown in SEQ ID NO.2；Truncated N-BETA-Alanyl-L-histidine synzyme amino acid sequence
Column are as shown in SEQ ID NO.3；
(2) recombinant expression carrier in step (1) is converted to E. coli BL21 (DE3) respectively, obtains gene work
Journey recombinant bacterial strain E.coli pET-CARNS1 and E.coli PET-PPK；
(3) recombinant bacterial strain in incubation step (2), to obtain the truncated N-BETA-Alanyl-L-histidine synzyme of expression and polyphosphate kinase
Thallus；
(4) thallus in destruction step (3) obtains crude enzyme liquid, mixes after ni-sepharose purification；
(5) by the mixed enzyme solution obtained in step (4) be added containing 80-150mM Beta-alanine, 80-150mM L-Histidine,
5mM MgCl2, 3.0-6.0mM ADP and 30-50mM calgon catalytic liquid in, be 6.5-8.5 in pH value, temperature is
Enzymatic reaction is carried out under the conditions of 30-45 DEG C synthesizes carnosine.
3. a kind of method of one-step synthesis method N-BETA-Alanyl-L-histidine according to claim 2, it is characterised in that: in the step (3)
The cultural method of recombinant bacterial strain are as follows: recombinant bacterial strain is seeded to the LB liquid containing 50 μ g/mL kanamycins by the 1-2% of percent by volume
In body culture medium, in 37 DEG C, 180-220rpm is cultivated when reaching 0.6-0.8 to OD600, adds final concentration 0.2-0.5mM
IPTG collects thallus in 25-28 DEG C of Fiber differentiation 8-10h.
4. a kind of method of one-step synthesis method N-BETA-Alanyl-L-histidine according to claim 3, it is characterised in that: the LB Liquid Culture
Every liter of base includes 10g sodium chloride, 5g yeast powder, 10g peptone, pH value 7.2-7.4.
5. a kind of method of one-step synthesis method N-BETA-Alanyl-L-histidine according to claim 2, it is characterised in that: the catalytic liquid is
Containing concentration in 50mM phosphate buffer is 100mM Beta-alanine, 100mM L-Histidine, 5mM MgCl2, 5mM ADP and
50mM calgon.
6. a kind of truncated N-BETA-Alanyl-L-histidine synzyme, it is characterised in that: its amino acid sequence is as shown in SEQ ID NO.3.
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