CN109468303A

CN109468303A - A kind of carnosine hydrolase, gene, mutant and its application

Info

Publication number: CN109468303A
Application number: CN201811437512.9A
Authority: CN
Inventors: 潘江; 殷东亚; 许建和; 钱小龙
Original assignee: Fuan Suzhou Hundred Zymotechnic Co Ltd; East China University of Science and Technology
Current assignee: Fuan Suzhou Hundred Zymotechnic Co Ltd; East China University of Science and Technology
Priority date: 2018-11-28
Filing date: 2018-11-28
Publication date: 2019-03-15

Abstract

The invention belongs to technical field of bioengineering, it is related to a kind of carnosine hydrolase and its mutant, recombinant expression carrier and recombinant expression transformants containing the enzyme and mutant gene, the preparation method of the recombinase, the process for fixation of recombinase, and the method that levo form N-BETA-Alanyl-L-histidine is prepared by inverse hydrolysis using the recombinase.Compared with prior art; carnosine hydrolytic enzyme activities disclosed by the invention are high, thermal stability is good; N-BETA-Alanyl-L-histidine is prepared by direct polycondensation using the enzymatic Beta-alanine and L-Histidine; avoid the step of protecting and being deprotected in the method for conventional chemistry synthetic peptide; it is simple process, mild condition, environmentally protective, therefore had a good application prospect in the industrialized production of N-BETA-Alanyl-L-histidine.

Description

A kind of carnosine hydrolase, gene, mutant and its application

Technical field

The invention belongs to technical field of bioengineering, and in particular to a kind of carnosine hydrolase and its mutant compile carnosine water The nucleic acid of solution enzyme, recombinant expression carrier and recombinant expression transformants containing nucleic acid, the preparation method of the recombination carnosine hydrolase, And its it is used for N-BETA-Alanyl-L-histidine synthetic method.

Background technique

L-Carnosine [L-carnosine, N- β-Alanyl-L-histidine] also known as β-alanyl-L-histidin, point Minor: C₉H₁₄N₄O₃, molecular weight 226.23, No. CAS is 305-84-0, is one kind by two kinds of ammonia of Beta-alanine and L-Histidine The dipeptides that base acid is condensed to yield is crystalline solid, is existing a kind of natural in organism.

Auto-oxidation reaction can occur for the lipid in human body, and this chain reaction belongs to radical reaction, and reaction produces The unsaturated fatty acid that the further catalytic attack of raw free radical easily aoxidizes, causes oxidative damage, this kinds of oxidation reaction Termination is realized through the combination between free radical molecule or in conjunction with antioxidant.Carnosine has good pH resiliency Can, it is a kind of good antioxidant, wound healing accelerator and ion chelating agent (especially to Cu²⁺And Zn²⁺), antioxygen Change mechanism and specifically includes that buffered physiologic pH, chelated metal ions and capture free radical etc..The carboxyl of N-BETA-Alanyl-L-histidine is capable of providing hydrogen Ion and amino can receive hydrionic characteristic, make it have pH buffering ability, when to strenuous exercise muscle generate it is big Measuring lactic acid has certain neutralization；Carnosine can also chelate transhipment metal ion, to inhibit the rouge of metal ion catalysis Matter oxidation；In addition, carnosine can also connect into carnosine albumen with oxidative carbonyl protein, oxidative carbonyl protein is prevented further to be crosslinked, To inhibit the oxidative modification of protein.Based on the above reasons, carnosine can be used as copper and rely on toxic neuroprotective agent inhibition copper The toxicant and antioxidant of dependence.These copper and zinc mediate neurotoxin related with a variety of diseases, such as amyotrophic lateral sclerosis side Rope sclerosis, Alzheimer disease, Parkinson's disease can be prevented by endogenous metal-chelator (such as carnosine), same with this When, carnosine can be reached by mechanism the effects of scavenging capacity oxygen radical, inhibition glycosylation, stimulation collagen synthesis Anti-aging function.Therefore, before carnosine and its derived product have wide application in fields such as drug, health care product, cosmetics Scape.

Currently, the production method of N-BETA-Alanyl-L-histidine includes two major classes: chemical synthesis and biological synthesis process.Using chemical synthesis N-BETA-Alanyl-L-histidine is prepared, the active group to substrate Beta-alanine and L-Histidine is needed to be protected or activated, can be divided into following several Class: 1918, Baumann etc. under the catalysis of β-propidium jodiole chlorine, was prepared using L-Histidine and β-alanyl chloride as substrate The higher N-BETA-Alanyl-L-histidine of optical purity, the method reaction step is less, without carry out Beta-alanine amido protecting (J Biol Chem, 1918,35:263).2005, Yamaoka etc. was improved on the basis of this method, with L-Histidine hydrochloride and β-alanyl Villaumite hydrochlorate is raw material, obtains N-BETA-Alanyl-L-histidine, yield 68% by two-step reaction, but logical nitrogen protection is needed in reaction process, And the reaction time is up to 40h, and part racemization (JP2005306782A) can occur for final products.2007, Hildbrand etc. Using ethyl cyanoacetate and L-Histidine as substrate, N-BETA-Alanyl-L-histidine is synthesized, without being protected and being deprotected accordingly, yield can reach 70%, but need to lead under the high pressure of 45bar hydrogen 1h in reaction process, reaction condition is more harsh (US7164028B2). Nineteen fifty-nine, Schwarz etc. carry out the amido protecting of Beta-alanine with tertbutyloxycarbonyl (Boc), and with n-hydroxysuccinimide As the carboxyl activator of Beta-alanine, preparation synthesis N-BETA-Alanyl-L-histidine, yield be only 42% (J Am Chem Soc, 1959,81 (21):5691-5695).1964, the Beta-alanine methyl esters or acyl chlorides and L- that Rinderknecht etc. is protected with phthalyl Histidine is that substrate is synthesized, and reaction product passes through hydrazinolysis, obtains N-BETA-Alanyl-L-histidine, product yield be 43% (J Org Chem, 1964,29(7):1968-1970).The method and its subsequent improved document report are more, are in the industrial production of current N-BETA-Alanyl-L-histidine The main method of use, domestic Shanghai Institute of Organic Chemistry improve on the basis of the method, product yield are increased to exceed 53% (CN103193712A).But this method reaction step is more, and reaction condition is harsher, needs the items such as ice salt bath reaction Part, and the deprotection process of amino needs to use reagent hydrazine, since hydrazine is a kind of highly toxic compound, in product N-BETA-Alanyl-L-histidine Do not allow to remain, therefore has very high requirement to the extraction of product, purification.

Although generally needing complicated protection-deprotection step in conclusion chemical synthesis development is more early, existing Synthesis step it is cumbersome, the problems such as severe reaction conditions, product yield is low, and pollution is big, and there are toxic reagent residuals.Phase therewith Than biological synthesis process has many advantages, such as that synthetic route is short, reaction condition is mild, environmental-friendly, product optical purity is high, is more suitable for In industrial application, traditional chemical synthesis process will be gradually replaced.

According to the difference of the enzyme and enzyme process carnosine synthetic reaction type that use, biological enzyme is synthetically prepared N-BETA-Alanyl-L-histidine can be with It is divided into following two categories: (1) β-aminopeptidase catalysis β-alanimamides/Beta-alanine ester and L-Histidine condensation Synthesis L- flesh Peptide；(2) carnosine hydrolyzes enzymatic Beta-alanine and synthesizes N-BETA-Alanyl-L-histidine with the inverse hydrolysis of L-Histidine.Swiss Confederation's aquatic science N-BETA-Alanyl-L-histidine can be synthesized by reporting three kinds of β-aminopeptidases with technical research institute Heck etc., and reaction condition is more mild, but yield is very It is low, highest only (Chem Biodivers, 2007,4 (9): 2016-2030) 81 μ g/mL.For this purpose, the seminar is with β-alanyl Amine and L-Histidine are substrate, are transformed to DmpA gene, are reacted using the intact cell catalysis containing recombinase, by output increased To 3.7g/L (Microb Biotechnol, 2010,3 (1): 74-83).Nevertheless, reaction in substrate L-Histidine addition Amount needs to reach 16 times of β-alanimamides or more, and another substrate β-alanimamides price is 40 times of Beta-alanine, with The price of N-BETA-Alanyl-L-histidine remains basically stable, therefore the synthesis technology is at high cost, it is difficult to realize industrial application.2011, Japan Takeshita et al. is the study found that the aminopeptidase RhDmpA3 from RhodotoruLa minuta IFO0879 can be catalyzed β- Alanine ester and L-Histidine condensation generate N-BETA-Alanyl-L-histidine (US2011/0081678 A1), but this report does not announce enzymatic Synthesize the yield data of N-BETA-Alanyl-L-histidine.

The N-BETA-Alanyl-L-histidine synthetic reaction of aminopeptidase catalysis, compared with chemical synthesis, although having reaction condition mild, chemical The high advantage of selectivity it is not necessary that substrate is protected and is deprotected comprehensively, but still is needed using chemical method to Beta-alanine Carboxyl or the amino of L-Histidine activated, increase additional step, and bring environmental pollution and cost increase Problem.

In vivo, carnosine hydrolase or two peptidohydrolases can hydrolyze carnosine in water phase, generate Beta-alanine and L-Histidine, this reaction is a reversible reaction.Ueda seminar, Kyoto Univ Japan using human brain cDNA as template, gram The carnosine hydrolase hCN1 of grand acquisition, carries out surface display in yeast, and use in a two-phase system the whole cell of yeast as Catalyst is catalyzed Beta-alanine with the inverse hydrolysis of L-Histidine and synthesizes N-BETA-Alanyl-L-histidine, and L-Histidine concentration is in reaction system 100mM, Beta-alanine concentration are 500mM, and the concentration of product N-BETA-Alanyl-L-histidine is only 4.5mM (Appl Microbiol Biotechnol,2010,86(6):1895-1902)。

Compared with the N-BETA-Alanyl-L-histidine synthetic reaction of aminopeptidase catalysis, the N-BETA-Alanyl-L-histidine of carnosine hydrolysis enzymatic is not necessarily to against hydrolysis Any additional chemical activation and protection are carried out to substrate, step is few, more environmentally protective；Pass through the original of product in reaction process Displacement removes, and can be easily carried out the serialization and automation of production；Simple process, it is high-efficient, there is good industry to answer Use prospect.Nevertheless, in the prior art, the carnosine hydrolase less varieties of open report, catalytic activity is low, the reaction time Long, production concentration is low, cannot reach industrialization production requirements.Therefore, it is necessary to screen active higher, the better enzyme of stability, with Just the target product that higher concentration is obtained within the shorter reaction time, to meet the technical requirements of industrialized production N-BETA-Alanyl-L-histidine.

Summary of the invention

The present invention is directed to the enzyme process of carnosine hydrolysis enzymatic against the deficiency in the prior art in hydrolysis N-BETA-Alanyl-L-histidine, provides The carnosine hydrolase and its mutant that a kind of catalytic activity is high, substrate tolerance is good, contain the carnosine hydrolase and mutant The recombinant expression carrier and recombinant expression transformants of gene, the preparation method of the recombination carnosine hydrolase, and use recombination flesh The method of peptidohydrolase synthesis N-BETA-Alanyl-L-histidine.

The purpose of the present invention can be achieved through the following technical solutions:

One of the technical solution adopted by the present invention:

A kind of carnosine hydrolase is following (a) or protein (b):

(a) protein that the amino acid sequence shown in SEQ ID No.2 forms；

(b) amino acid sequence as shown in SEQ ID No.2 by replacement, missing or adds one or more amino acid and obtains The derived protein with N-BETA-Alanyl-L-histidine synthesizing activity arrived.

The preparation method of the protein (a) is preferably: dividing from serratia marcescens Serratia marcescens From acquisition, or separates and obtain from the transformant for recombinantly expressing the carnosine hydrolase, it can also be artificial synthesized.

The present invention has carried out the screening of carnosine hydrolysing activity to the bacterial strain of laboratory preservation, by training to different strain It supports, using N-BETA-Alanyl-L-histidine as substrate, carries out the measurement of whole cell effect and carnosine hydrolysing activity, the cement that discovery number is ECU1010 Serratieae carnosine hydrolysing activity with higher.The serratia marcescens Serratia marcescens ECU1010 is What inventor separated from soil in Previous work, it has been stored in China General Microbiological culture presevation administrative center at present, The deposit date is on September 14th, 2004, number was CGMCC No.1219 (see the China of authorization Publication No. CN100334198C Patent of invention).

The high activity carnosine hydrolase in Serratieae CGMCC No.1219 is cloned using shotgun.Using limit Property restriction endonuclease Sau3AI processed is partially digested to the Serratieae total DNA progress of extraction, collects the segment of about 2~4kb, is connected to On plasmid pUC118, and it is transformed into bacillus coli DH 5 alpha competent cell.Monoclonal in picking solid medium tablets, point Be not inoculated into deep-well plates cultivated, the identification of inducing expression and carnosine hydrolysing activity.N-BETA-Alanyl-L-histidine hydrolysate L- group ammonia Acid can react the Schiff for generating and having hyperfluorescence signal, λ with o-phthalaldehyde (OPA)_Exc=355nm, λ_Em=460nm, Rapid identification can be carried out by fluorescence microplate reader.Using the strategy, the positive clone molecule for generating obvious product is screened. Positive clone molecule commission Shanghai Sai Yin Bioisystech Co., Ltd is subjected to sequencing, is obtained as shown in SEQ ID NO.1 Nucleic acid sequence, the amino acid sequence speculated according to the nucleic acid sequence is as shown in SEQ ID NO.2, carnosine which is expressed Hydrolase is named as SmPepD.The amino acid sequence as shown in SEQ ID NO.2 is scanned for comparing in ncbi database, is sent out Existing its has highest similar to β-alanyl-histidine dipeptidase amino acid sequence that accession number is WP_033634091.1 Property, the two homology is higher than 99%, but there are still the difference of two amino acid, the amino acid sequence of SmPepD of the present invention 92nd is threonine, and the 271st is aspartic acid；And β-alanyl-histidine dipeptidase WP_033634091.1 amino The 92nd of acid sequence is alanine, and the 271st is glutamic acid.

On this basis, evolution transformation is oriented to SmPepD using fallibility PCR strategy, using OPA derivatization method The high flux screening of mutation library is carried out, screening obtains the carnosine hydrolysis enzyme mutant that a collection of activity significantly improves, specifically, its sequence Column are as follows:

(1) the 18th glutamic acid of the amino acid sequence as shown in SEQ ID No.2 is replaced with into threonine, the 22nd bad ammonia Acid replaces with alanine, and the 67th phenylalanine replaces with alanine；

(2) the 17th threonine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into alanine, the 39th sweet ammonia Acid replaces with aspartic acid；

(3) the 18th glutamic acid of the amino acid sequence as shown in SEQ ID No.2 is replaced with into threonine；

(4) the 22nd lysine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into aspartic acid, the 64th essence Propylhomoserin replaces with glutamic acid；

(5) the 22nd lysine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into aspartic acid, the 64th essence Propylhomoserin replaces with glutamic acid, and the 93rd cysteine replaces with threonine；

(6) the 39th glycine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into alanine, the 114th figured silk fabrics Propylhomoserin replaces with asparagine；

(7) the 39th glycine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into alanine, the 93rd half Guang Propylhomoserin replaces with threonine, and the 114th valine replaces with asparagine, and 243 asparagines replace with serine；

(8) the 44th lysine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into glycine, the 93rd half Guang Propylhomoserin replaces with threonine, and 243 asparagines replace with serine；

(9) the 44th lysine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into glycine, the 64th smart ammonia Acid replaces with glutamic acid, and the 67th phenylalanine replaces with alanine；243rd asparagine replaces with leucine；

(10) the 64th arginine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into glutamic acid, the 67th benzene Alanine replaces with alanine；

(11) the 64th arginine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into glutamic acid, the 67th benzene Alanine replaces with alanine；243rd asparagine replaces with leucine；

(12) the 67th phenylalanine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into alanine, the 93rd Cysteine replaces with isoleucine, and the 114th valine replaces with asparagine, and the 243rd asparagine replaces with bright ammonia Acid；

(13) the 93rd cysteine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into isoleucine, the 114th Position valine replaces with tyrosine；

(14) the 93rd cysteine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into isoleucine, the 114th Position valine replaces with tyrosine, and the 243rd asparagine replaces with leucine；

(15) the 93rd cysteine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into isoleucine, the 243rd Position asparagine replaces with leucine.

The two of the technical solution adopted by the present invention:

A kind of nucleic acid of coding carnosine hydrolase as described in technical solution one.

The preparation method of nucleic acid of the present invention is this field customary preparation methods, and the preparation method is preferably comprised:

The nucleic acid point of naturally occurring coding carnosine hydrolase SmPepD is extracted from wild type serratia marcescens thallus Son；Or the genomic nucleic acid molecule of coding carnosine hydrolase SmPepD and its mutant is obtained by gene clone technology, or pass through Artificial complete sequence synthetic method obtains the nucleic acid molecules of coding carnosine hydrolase SmPepD and its mutant.

The gene core of the present invention that coding carnosine hydrolase SmPepD and its mutant are obtained by gene clone technology The method of acid molecule are as follows: with

Forward primer 5'-CCGGAATTCGTGTCTGAATTGTCTCAGCTTT-3',

Reverse primer 5'-CCGCTCGAGTTACGCGCGCTCAGGGATCGCTTT-3',

Using polymerase chain reaction technology to the DNA sequence of the coding SmPepD and its mutant that are obtained in technical solution one Column carry out gene magnification:

PCR system (50 μ L): 25 5 μ L, dNTP mix of μ L, 10 × Buffer of rTaq 4 μ L, template plasmid about 100ng, Upstream and downstream primer (10 μM) each 2 μ L, diH₂O complements to 50 μ L.

PCR response procedures: (1) 98 DEG C of denaturation 3min；(2) 98 DEG C of denaturation 30s, (3) 55 DEG C of annealing 30s, (4) 72 DEG C extend 1.5min；(5) step (2)-(4) carry out 30 circulations, last 72 DEG C of extensions 10min, 4 DEG C of preservations altogether.

The three of the technical solution adopted by the present invention:

A kind of recombinant expression carrier comprising carnosine hydrolase of the present invention and mutant nucleic acid sequence.It can pass through this The nucleic acid sequence of carnosine hydrolase gene or the nucleic acid sequence of mutant gene of the invention are connected to respectively by field conventional method It is built-up on the suitable carrier of kind.The carrier can be the various conventional carriers of this field, preferred plasmid, more preferable plasmid pET28a.The carnosine hydrolase gene can be operatively connected to be suitble under the regulating and controlling sequence of expression in selected carrier Trip, to realize the composing type or inducible expression of the carnosine hydrolase.

Preferably, as an example, recombinant expression carrier of the present invention can be made by following methods: PCR will be passed through Gene order DNA fragmentation restriction enzyme EcoRI and the XhoI double digestion of resulting carnosine hydrolase SmPepD is expanded, together When gene DNA fragment by empty plasmid pET28a restriction enzyme EcoRI and XhoI double digestion, after recycling above-mentioned digestion And pET28a plasmid, it is connected using T4DNA ligase, building obtains the recombination comprising the carnosine hydrolase SmPepD gene Expression vector pET28a-SmPepD.

The four of the technical solution adopted by the present invention:

A kind of recombinant expression transformants comprising carnosine hydrolase gene of the present invention or its recombinant expression carrier.It can The recombinant expression transformants are made by converting recombinant expression carrier of the present invention into host cell.The place Chief cell can be the various conventional host cells of this field, on condition that the recombinant expression carrier can be made steadily voluntarily multiple System, and the carnosine hydrolase gene entrained by it can be by effective expression.Preferred host cell of the present invention is Escherichia coli, more preferably E. coli DH5 α or E. coli BL21 (DE3).

The five of the technical solution adopted by the present invention:

A kind of preparation method recombinating carnosine hydrolase includes the following steps: that cultivating recombinant expression of the present invention turns Change body, obtains recombination carnosine hydrolase.Wherein, cultivating culture medium used in the recombinant expression transformants can be selected from this field Conventional medium, on condition that transformants grew can be made and generate carnosine hydrolase of the invention.Cultivate the concrete operations of transformant It can be carried out by this field routine operation.

The recombination bacillus coli that will be constructed by above-mentioned technical proposal is seeded to the LB culture medium containing 50 μ g/mL kanamycins In (peptone 10g/L, yeast extract 5g/L, NaCl 10g/L, pH 7.0), 37 DEG C of shaken cultivations are stayed overnight, by connecing for 1% (v/v) The access of kind amount is placed in 37 DEG C, 180rpm shaking table shaken cultivation, works as training equipped in the 500mL conical flask of 100mL LB culture medium The OD of nutrient solution₆₀₀When reaching 0.6, isopropyl-β-D-thiogalactoside (IPTG) conduct of final concentration of 0.1mmol/L is added Inducer, by medium centrifugal, collects cell after 16 DEG C of inductions for 24 hours, and twice with brine, obtains resting cell. Resulting resting cell is suspended in the buffer of 10mL pH 8.0, supernatant is collected by centrifugation in the ultrasonication in ice-water bath Liquid, the as crude enzyme liquid of recombinase, for crude enzyme liquid through Polyacrylamide Gel Electrophoresis, recombinant protein can with part in cell Molten form exists, and in addition has part albumen to be present in cell fragment.

Protein in the present invention contains histidine tag in N-terminal, therefore can carry out protein purification using nickel column.The following are Zymoprotein purification buffer formula: A liquid: Tris-HCl buffer (50mM, pH 8.0), containing 0.5M NaCl, 20mM imidazoles and The glycerol of 10% (w/v)；B liquid: Tris-HCl buffer (50mM, pH 8.0) contains 0.5M NaCl, 500mM imidazoles and 10% (w/v) glycerol.The recombinant protein of expression is loaded in nickel column, elutes foreign protein with A liquid first, then with gradient imidazoles B liquid elutes target protein, according to the protein of the collection purifying of SDS-PAGE detection, is concentrated by ultrafiltration, is added final concentration of The glycerol of 20% (w/v) is saved in -80 DEG C.

The six of the technical solution adopted by the present invention:

Carnosine hydrolase of the present invention prepares the application in N-BETA-Alanyl-L-histidine in catalysis Beta-alanine and L-Histidine condensation. The application is that carnosine hydrolase of the present invention is added in the buffer solution containing Beta-alanine and L-Histidine, The inverse hydrolysis for being catalyzed Beta-alanine and L-Histidine synthesizes N-BETA-Alanyl-L-histidine.

Wherein the buffer salt system of the buffer solution is unlimited, as long as its pH range is 6.0-9.0；It is preferred slow Rushing salt system is Tris-HCl, and pH range is 7.0~8.5.Reaction temperature is 25~60 DEG C, preferably 30-40 DEG C.The buffering is molten Appropriate water-soluble solvent or water insoluble solvent can be added in liquid.The water-soluble solvent can be methanol, ethyl alcohol, isopropyl One of alcohol, dimethyl sulfoxide, dimethyl imide or dioxane or any combination thereof, adding proportion are 0-20% (v/ v)；The water insoluble solvent can be isooctane, toluene, isopropyl ether, methyl tertiary butyl ether(MTBE) or ethyl butyrate, slow with water phase The ratio of fliud flushing is 5:1-1:5 (v/v).Other reaction conditions such as concentration of substrate, enzyme dosage etc. can be by the such reaction in this field Normal condition is selected.

Carnosine hydrolase of the present invention is used as catalyst, can also be thin by the tranquillization as described in technical solution five Born of the same parents, crude enzyme liquid, pure enzyme, or zymoprotein is fixed to the immobilised enzymes obtained on suitable carrier as the carnosine hydrolase Catalyst.

Intermittent sampling in reaction process carries out conversion ratio analysis using liquid chromatography.The chromatographic column used is chiral hat Ether column (CR (+), φ 40mm × 150mm) makes a concrete analysis of condition are as follows: using the perchloric acid solution of 0.1M as mobile phase, mobile phase stream Fast 0.3mL/min, 25 DEG C of column temperature, Detection wavelength 220nm.

After reaction, reaction solution, concentration are separated, Beta-alanine preferential crystallization is separated off the beta-amino third of crystallization Acid, mother liquor continue to be concentrated, and N-BETA-Alanyl-L-histidine is added as crystal seed, can be obtained the N-BETA-Alanyl-L-histidine with higher degree.To product repeatedly into Row recrystallization, can be obtained the N-BETA-Alanyl-L-histidine of high-purity.

The positive effect of the present invention is that: carnosine hydrolytic enzyme activities of the present invention are high, thermal stability is good, this hair The bright carnosine hydrolase synthesizes N-BETA-Alanyl-L-histidine, avoids conventional chemistry directly using Beta-alanine and L-Histidine as substrate The step of protecting and be deprotected in the method for synthetic peptide.Relative to other reported N-BETA-Alanyl-L-histidine preparation methods, the present invention is used Method prepare N-BETA-Alanyl-L-histidine, have reaction system simple, raw material is easy without protection or activation, synthesis technology, is easy to industry and puts It is big to wait significant advantages；And production concentration is high, and reaction condition is mild, and it is environmentally friendly, before there is the exploitation of good industrial application Scape.

Specific embodiment

The present invention is described in detail combined with specific embodiments below.

The screening of 1. carnosine hydrolase producing strains of embodiment

500 multi-strain bacteria strains of laboratory preservation are cultivated, medium centrifugal respectively, collect cell, carries out carnosine water Solve screening active ingredients.Preservation strain is inoculated into the test tube of the culture medium containing 4mL, 30 DEG C, 180rpm shaking culture 48h, culture knot Shu Hou, 4 DEG C, 8000rpm cell is collected by centrifugation, be resuspended in Tris-HCl (50mM, pH containing 100mmol/L N-BETA-Alanyl-L-histidine 8.0) it in buffer, reacts in 30 DEG C, 1000rpm shaking for 24 hours, with the hydrolysis conversion of efficient liquid phase chromatographic analysis carnosine.Make Chromatographic column is chiral crown ether column (CR (+), φ 40mm × 150mm) concrete analysis condition are as follows: molten with the perchloric acid of pH 1.0 Liquid is mobile phase, flow rate of mobile phase 0.3mL/min, Detection wavelength 220nm, 25 DEG C of column temperature.It the results are shown in Table 1, in bacterium, 9 plants of bacterium have apparent carnosine hydrolysing activity, and the serratia marcescens that wherein laboratory number is ECU1010 has highest flesh Hydrolase polypeptide activity, the bacterial strain have been stored in China General Microbiological culture presevation administrative center at present, and the deposit date is 2004 9 The moon 14, number CGMCCNo.1219.

The carnosine hydrolysing activity of 1 different strains of table compares

Note: "+" indicates hydrolysis conversion in 0-10%；" ++ " indicates hydrolysis conversion in 10-30%；

" +++ " indicates that hydrolysis conversion is higher than 50%.

The clone of 2. carnosine hydrolase SmPepD gene of embodiment

Bacterial strain serratia marcescens Serratia marcescens ECU1010 is inoculated into LB culture medium and is trained It supports, high-purity, the genome DNA of large fragment is extracted using cetyl trimethylammonium bromide (CTAB) method.By appropriate cement Liquid nitrogen frozen is added in Serratieae thallus, pulverizes, and appropriate 2 × CTAB Extraction buffer (100mmol/L Tris- is added HCl, pH 8.0, EDTA containing 20mmol/L, 1.4moI/L NaCl, 20g/L CTAB, 40mmol/L mercaptoethanol), 65 DEG C of guarantors Temperature 10 minutes, interval are shaken.Isometric chloroform/isoamyl alcohol is then added, light and slow reverse centrifuge tube mixes, at room temperature, 12000rpm is centrifuged 10min, and supernatant is transferred in another centrifuge tube, isometric chloroform/isoamyl alcohol is added, and overturns centrifuge tube It mixes, room temperature, 12000rpm is centrifuged 10 minutes.Upper strata aqueous phase is transferred in new centrifuge tube, it is mixed that isometric isopropanol is added It is even, it is placed at room temperature for 30 minutes.4000rpm is centrifuged 10 minutes, removes supernatant, is rinsed with 70% ethyl alcohol, and it is small to be added 20 after air-drying When TE buffer (100mM Tris-HCl, 10mM EDTA, pH 8.0) dissolving DNA, -20 DEG C save backup.

Using shotgun to the high activity carnosine hydrolase in serratia marcescens CGMCC No.1219 described in embodiment 1 Carry out gene cloning.It is partially digested using Serratieae total DNA progress of the restriction enzyme Sau3AI to extraction, after digestion DNA fragmentation is purified by electrophoresis, and the segment of about 2~4kb is recycled using glue recovery purifying kit, and the DNA of recycling is molten Solution is placed in -20 DEG C and saves backup in Tris-HCl (10mmol/L, pH 8.0).

DNA fragmentation will be recycled by following reaction system to be attached with vector plasmid pUC118:

16 DEG C incubate 12 hours, take 10 μ L enzyme-linked products convert 200 μ L bacillus coli DH 5 alpha competent cells (TaKaRa, Code:D9057), picking monoclonal is inoculated into the deep hole of the LB culture medium (containing 100 μ g/mL card that penicillin) added with 300 μ L In plate, 37 DEG C of shaken cultivations are stayed overnight, and the 50 μ L that then transfer are to the LB culture medium added with 600 μ L (containing 100 μ g/mL card that mould Element) second level deep-well plates, the IPTG of final concentration of 0.2mmol/L is added after 37 DEG C of shaken cultivation 3h, 16 DEG C of inductions are for 24 hours.Take 10 μ L bacterium solution is added in Tris-HCl (50mM, pH 8.0) buffer that 300 μ L contain 100mmol/L N-BETA-Alanyl-L-histidine, 37 DEG C of oscillations 1h is reacted, the o-phthalaldehyde (OPA) of final concentration of 50mg/mL is added, continues oscillating reactions 30min.If there is high activity Carnosine hydrolase, can be catalyzed N-BETA-Alanyl-L-histidine hydrolysis generate L-Histidine, the latter can with O-phthalic aldehyde reaction generate have The Schiff of hyperfluorescence signal, λ_Exc=355nm, λ_Em=460nm can carry out Rapid identification by fluorescence microplate reader.Using This strategy screens the positive clone molecule generated with obvious product.By positive clone molecule commission Shanghai match sound biology Technology Co., Ltd. carries out sequencing, obtains the nucleic acid sequence as shown in SEQ ID NO.1, is speculated according to the nucleic acid sequence Amino acid sequence as shown in SEQ ID NO.2, the carnosine hydrolase which expresses is named as SmPepD.

The preparation of the recombination of embodiment 3. carnosine hydrolase SmPepD

With forward primer 5'-CCGGAATTCGTGTCTGAATTGTCTCAGCTTT-3', reverse primer 5'-CCGCTCGAG TTACGCGCGCTCAGGGATCGCTTT-3', using polymerase chain reaction technology to the carnosine hydrolase obtained in embodiment 2 The nucleotide sequence of SmPepD is expanded, by the amplification of DNA fragments restriction enzyme containing SmPepD sequence of acquisition EcoRI and XhoI double digestion is then carried out with the plasmid pET28a for also passing through restriction enzyme EcoRI and XhoI double digestion Connection obtains recombinant plasmid pET28a-SmPepD.

The recombinant plasmid pET28a-SmPepD of acquisition is transformed into E. coli BL21, by the recombination of building Escherichia coli be seeded to containing 50 μ g/ml kanamycins LB culture medium (peptone l0g/L, yeast extract 5g/L, NaC1 10g/L, PH 7.0) in, 37 DEG C of shaken cultivations are stayed overnight, and are burnt by the inoculum concentration access of 1% (v/v) equipped with the 2L triangle of 600mL LB culture medium In bottle, 37 DEG C, 220rpm shaking table shaken cultivation are set, as the 0D of culture solution₆₀₀When reaching 1.2, final concentration of 0.2mmol/L is added IPTG after 16 DEG C of inductions for 24 hours, by medium centrifugal, collect cell as inducer, and twice with brine, obtain To resting cell.Obtained resting cell is suspended in Tris-HCl buffer (50mM, pH 8.0), high pressure homogenizer is broken It is broken, the crude enzyme liquid of recombination carnosine hydrolase SmPepD is obtained, crude enzyme liquid is freeze-dried and obtains freeze-drying recombination carnosine water Enzyme SmPepD is solved, the vigor that enzyme powder is lyophilized is 6.1U/g (dry powder).

The crude enzyme liquid of above-mentioned SmPepD recombinase is uploaded in nickel column, elutes foreign protein with A liquid first, then uses B liquid Target protein SmPepD is eluted, according to the testing result of polyacrylamide gel electrophoresis, SmPepD solubility expression is fine.It collects Target protein after purification is added the glycerol that concentration is 20% (w/v), saves backup in -20 DEG C, the A liquid are as follows: Tris-HCl Buffer (20mM, pH 8.0), the glycerol containing 0.5M NaCl, 20mM imidazoles and 10% (w/v)；The B liquid are as follows: Tris- HCl buffer (20mM, pH 8.0), the glycerol containing 0.5M NaCl, 500mM imidazoles and 10% (w/v).

The Determination of Kinetic Parameters of compound direction, the V of enzyme are carried out to the SmPepD of purifying_maxFor

V_max=110 μm of ol/min/mg albumen.

The molecular modification of the recombination of embodiment 4. carnosine hydrolase SmPepD

The random mutant library that carnosine hydrolase SmPepD is established using fallibility round pcr, is dashed forward using OPA derivatization Become the high flux screening in library.

Both ends primer is designed first:

Forward primer 5'-CCGGAATTCGTGTCTGAATTGTCTCAGCTTT-3',

Reverse primer 5

'-CTCGAGTGCGGCCGCAAGCTTACCGAGGCTCGAGATGAA-3'

It is expanded using the recombinant plasmid pET28a-SmPepD obtained of embodiment 3 as template.Polymerase chain reaction PCR system (50 μ L): 0.25 5 μ L, dNTP Mix of μ L, 10 × Buffer of rTaq 4 μ L, template plasmid about 100ng, Primer 2 μ L, Primer R of F 2 μ L, MnCl₂(10mM) 0.5 μ L, diH₂O complements to 50 μ L.PCR program are as follows: 98 DEG C of 3min, 98 DEG C 30s, 55 DEG C of 30s, 72 DEG C of 10min are recycled 30 times, 72 DEG C of 10min.PCR product is saved backup at 4 DEG C.

PCR product segment containing random mutation site is after EcoR1 and XhoI digestion and with identical restriction enzyme site PET28a (+) plasmid is ligated and transformed into E.coli BL21 competent cell, and be spread evenly across containing 50 μ g/ml cards that On the LB agar plate of mycin.After 37 DEG C are incubated overnight, picking monoclonal is inoculated into the LB culture medium added with 300 μ L and (contains 100 μ g/mL card that penicillin) deep-well plates in, 37 DEG C of shaken cultivations are stayed overnight, and the 50 μ L that then transfer are to the LB culture medium added with 600 μ L The second level deep-well plates of (containing 100 μ g/mL card that penicillin) are added final concentration of 0.2mmol/L's after 37 DEG C of shaken cultivation 3h IPTG, 16 DEG C of inductions are for 24 hours.It takes 40 μ L bacterium solutions to be added to 160 μ L and contains 180mmol/L Beta-alanine and 20mmol/L N-BETA-Alanyl-L-histidine Tris-HCl (50mM, pH 8.0) buffer in, the O-phthalic of final concentration of 50mg/mL is added in 37 DEG C of oscillating reactions 1h Aldehyde (OPA) continues oscillating reactions 30min, carries out fluorescence detection, excitation wavelength 355nm, launch wavelength by fluorescence microplate reader 460nm judges enzymatic activity height according to fluorescence power, and so screening obtains the mutant of activity raising, send match sound biotechnology (Shanghai) Co., Ltd. carries out sequencing.Sequencing result is carried out with ApE software and carnosine hydrolase gene (SmPepD) sequence It compares, the mutant for screening some high activities of acquisition is listed in Table 2 below.In table 2, sequential labeling is corresponded respectively to behind table 2 A series of sequences.In active column, compared with maternal SmPepD, a plus sige "+" indicates that the vigor of mutant protein is promoted 1.2-5 again；Two plus siges " ++ " indicate that mutant protein vigor promotes 5-10 times；Three plus siges " +++ " indicate mutant protein Synthesis of dynamic be lifted beyond 10 times.

2. carnosine hydrolase mutant sequence of table and corresponding activity improve list

The amino acid sequence difference that 7 β-HSDH mutant of label are corresponded in table is as follows:

5. carnosine of embodiment hydrolyzes enzyme mutant SmPepD_M13Preparation

Extract the recombinant plasmid pET28a-SmPepD as obtained in embodiment 4_M13, it is transformed into Escherichia coli In E.coli BL21, it is seeded to LB culture medium (the peptone 10g/L, yeast extract 5g/L, NaC1 of the kanamycins containing 50mg/ml 10g/L, pH 7.0) in, 37 DEG C of shaken cultivations are stayed overnight, and are transferred to by the inoculum concentration of 1% (v/v) equipped with 600mL LB culture medium In 2L conical flask, it is placed in 37 DEG C, 180rpm shaking table shaken cultivation, as the OD of culture solution₆₀₀When reaching 1.2, it is added final concentration of The IPTG of 0.1mmol/L by medium centrifugal, collects cell, and washed with physiology salt after 16 DEG C of inductions for 24 hours as inducer It washs twice, obtains resting cell.Resulting resting cell is suspended in Tris-HCl buffer (20mM, pH8.0), height is used Pressure homogenate is broken, is crushed liquid freeze-drying to get to carnosine and hydrolyzes enzyme mutant SmPepD_M13Freeze-drying enzyme powder, freeze-drying enzyme powder is living Power is that enzyme powder is lyophilized in 11.8U/g.

The different immobilization materials of embodiment 6. are to recombination SmPepD_M13The influence of synthesizing activity

With different immobilization materials (as listed in table 3), to recombination carnosine hydrolase SmPepD_M13It immobilizes.Institute It states Immobilized Resin material to be pre-processed by product description, then adding 1ml enzyme solution by 0.2g fixation support, (10mg is lyophilized Enzyme powder/mL) ratio mixed, 30 DEG C of shakings for 24 hours, filter and remove solvent, carry out vitality test to immobilised enzymes, calculate solid Surely change the Activity recovery of enzyme.

With Tris-HCl (50mM, pH 8.0) for buffer, the N-BETA-Alanyl-L-histidine of final concentration of 100mM is substrate, and measurement is fixed Change the activity of enzyme, react at 30 DEG C, carried out in 1000rpm, measures reaction conversion ratio with efficient liquid phase chromatographic analysis.Immobilised enzymes Activity recovery be defined as immobilised enzymes vigor free enzyme activity corresponding to before immobilization ratio, the results are shown in Table 3.

The vigour of 3. immobilized enzyme catalysis of table synthesis N-BETA-Alanyl-L-histidine

7. temperature of embodiment is on the active influence of carnosine hydrolase SmPepD

Under different temperatures (20~60 DEG C), takes the 20 pure enzymes of μ L (zymoprotein concentration 5mg/mL) to be added to 180 μ L and contain In Tris-HCl (50mM, pH 8.0) buffer of 20mmol/L N-BETA-Alanyl-L-histidine, 37 DEG C of oscillating reactions 5-20min use efficient liquid phase Chromatography measures conversion ratio.The results are shown in Table 4, and the enzyme is in 50 DEG C of performance most highly actives, by activity definition at this temperature It is 100%, when more than 50 DEG C, the active rapid decrease of enzyme.

The activity of 4. carnosine hydrolase SmpepD of table at different temperatures

Embodiment 8.pH is on the active influence of carnosine hydrolase SmPepD

When survey temperature living is 50 DEG C, takes the pure enzyme (enzyme concentration 5mg/mL) of 20 μ L to be added to 180 μ L and contain 20mmol/L L- flesh In Tris-HCl (50mM, pH 8.0) buffer of peptide, 37 DEG C of oscillating reactions 5-20min investigate carnosine hydrolase SmPepD and exist Activity in different pH buffers.Buffer solution system used is respectively sodium phosphate buffer (pH 6.0-7.5) and Tris-HCl (pH 7.5-9.0).The results are shown in Table 5, in the Tris-HCl buffer that pH is 8.0, the relative activity highest of enzyme, and definition It is 100%.

Activity of the 5. carnosine hydrolase SmPepD of table in different pH buffers

Influence of 9. organic solvent of embodiment to carnosine synthesizing activity

With Tris-HCl (50mM, pH 8.0) for buffer, a certain proportion of organic solvent is added, including water-soluble organic Solvent and water-insoluble organic solvents, type and adding proportion are distinguished as shown in table 6 and table 7, with the β-the third of final concentration of 2M The L-Histidine of propylhomoserin and 100mM are substrate, add the SmPepD of 10mg/mL such as embodiment 5_M13Freeze-drying freeze-drying enzyme powder, reaction are total 200 μ L of system, is reacted at 30 DEG C, converts 30min, analyzes measurement L-Histidine with high performance liquid chromatography (chiral crown ether column) Conversion ratio, be as a result shown in Table 6 and table 7 respectively.

6. water-miscible organic solvent type of table and adding proportion are to SmPepD_M13Active influence

Note: institute's column data is carnosine hydrolase SmPepD in the case of different solvents addition in table_M13It is catalyzed the initial velocity of reaction Rate, unit are μm ol/min/g enzyme powder.

The influence of 7. water-insoluble organic solvents type of table and adding proportion to SmPepD synthesis of dynamic

Note: in the case that institute's column data is different organic solvents and buffer ratio in table, carnosine hydrolase SmPepD_M13 It is catalyzed the first rate of reaction, unit is μm ol/min/g enzyme powder.

Influence of 10. concentration of substrate of embodiment to carnosine Synthesis conversion

100mg is taken to recombinate carnosine hydrolase SmPepD as described in Example 5_M13Thick enzyme powder, be dissolved in 10mL Tris- In HCl buffer (50mM, pH8.0), substrate Beta-alanine and L-Histidine are separately added into final concentration as listed by table 8,30 DEG C, Magnetic agitation reaction.The conversion ratio that measurement L-Histidine is analyzed with high performance liquid chromatography (CR (+), φ 40mm × 150mm), until anti- Reaction is terminated when conversion ratio being answered to stop rising, and the results are shown in Table 8.

The different concentration of substrate of table 8. and the corresponding production concentration of ratio

It is prepared by the gram-grade scale of embodiment 11L- carnosine

Reaction carries out in 2L three-necked flask, sequentially adds 50mM Tris-HCl buffer (pH 8.0) 500mL, β-the third Propylhomoserin 690g and L-Histidine 75.5g, isooctane 200mL, the recombination carnosine hydrolase SmPepD prepared such as embodiment 5_M13It is thick Enzyme powder 10g, carnosine concentration reaches 78mM in 30 DEG C, 200rpm mechanic whirl-nett reaction 60h, water phase.After reaction, separation is anti- Liquid, rotary evaporation concentration are answered, Beta-alanine preferential crystallization comes out, and is separated off the Beta-alanine of crystallization, be concentrated repeatedly, N-BETA-Alanyl-L-histidine is added as crystal seed in mother liquor concentrations to final volume about 50mL by crystallization, and 0 DEG C stands for 24 hours, filters, acquisition has higher The N-BETA-Alanyl-L-histidine of purity.The N-BETA-Alanyl-L-histidine crude product of acquisition is dissolved in 50mL deionized water, magnetic agitation is heated to 80 DEG C, then Be slowly added to ethyl alcohol, until solution occur it is muddy, add it is several drip, clarify solution, slowly cool to 0 DEG C, filter, filter cake dries It is dry, obtain N-BETA-Alanyl-L-histidine 5.7g, purity 98%.

12. packed bed enzyme reactor of embodiment catalyzes and synthesizes N-BETA-Alanyl-L-histidine

With immobilised enzymes SmPepD described in embodiment 6_M13@ESR-103 is inserted in column reactor, reactor inside diameter 3cm, bed height about 15cm.With Tris-HCl (50mM, pH 8.0) for buffer, the Beta-alanine and 100mM of final concentration 2M L-Histidine be substrate, substrate solution total volume 1L, in the liquid storage bottle.It is using peristaltic pump, substrate solution is anti-by pillar Device bottom is answered to be pumped into, liquid storage bottle, flow velocity 1mL/min are flowed back in top outflow.30 DEG C of reactor constant temperature are reacted, and are reacted 2 days Afterwards, carnosine concentration is 73mM.After reaction, reaction solution, rotary evaporation concentration are separated, Beta-alanine preferential crystallization, separation removes The Beta-alanine to decrystallize is concentrated repeatedly, crystallizes, and by mother liquor concentrations to final volume about 50mL, N-BETA-Alanyl-L-histidine conduct is added Crystal seed, 0 DEG C stands for 24 hours, filters, obtains the N-BETA-Alanyl-L-histidine with higher degree.The N-BETA-Alanyl-L-histidine crude product of acquisition is dissolved in 50mL In ionized water, magnetic agitation is heated to 80 DEG C, is then slowly added into ethyl alcohol, until solution occur it is muddy, add it is several drip, make Solution clarification, slowly cools to 0 DEG C, filters, and filter cake drying obtains N-BETA-Alanyl-L-histidine 5.1g, purity 98%.

1. β of comparative example-alanyl-histidine dipeptidase WP_033634091.1 clone, expression and vitality test

Homologous comparison is carried out in ncbi database with amino acid sequence shown in SEQ ID No.2, with its homology highest Amino acid sequence be protein that accession number is WP_033634091.1, which is predicted as β-alanyl-histidine dipeptides Enzyme, for the two only there are two amino acid of differences, homology is higher than 99%.Its difference is: carnosine hydrolase of the present invention The 92nd of the amino acid sequence of SmPepD is threonine, and the 271st is aspartic acid, and β-alanyl-histidine dipeptidase The 92nd of the amino acid sequence of WP_033634091.1 is alanine, and the 271st is glutamic acid.According to β-alanyl-group ammonia The nucleic acid sequence of sour dipeptidase WP_033634091.1 has carried out gene chemical synthesis, recombinantly expresses in Escherichia coli, and to expression Protein purified (with the operating procedure of embodiment 3), through synthesizing directional dynamics parametric measurement, the V of pure enzyme_max For 8.3 μm of ol/min/mg_Albumen, far below SmPepD (V of the invention_max=110 μm of ol/min/mg_Albumen)。

The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention. Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general Principle is applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be of the invention Within protection scope.

Sequence table

<110>East China University of Science, hundred Fuan zymotechnic Co., Ltd of Suzhou

<120>a kind of carnosine hydrolase, gene, mutant and its application

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1461

<212> DNA

<213> Serratia marcescens

<400> 1

gtgtctgaat tgtctcagct ttcgccacag ccgctgtggg atattttcgc caagatctgt 60

tctatcccgc acccttctta tcatgaagaa gcgctggcgc agcacatcct cacctgggcc 120

aaagaaaaga acctgcacgc cgagcgcgat caggtcggca atatcctgct gcgcaagccg 180

gccaccaaag gcatggaaaa ccgcaagccg gtcgcgctgc aggcgcacct ggacatggtg 240

ccgcaaaaga ataacgacac cgtgcacgac ttcaccaagg atccgatcca gccttatatc 300

gccggtgagt gggtgaaagc gcgcggcacc acgctgggcg ccgacaacgg catcggcatg 360

gcctccgccc tggcggtgct ggccgacgac agcgttgaac acggcccgct ggaagtgctg 420

ctgaccatga ccgaagaagc cggcatggac ggcgccttcg ggctgcagcc gaactggctg 480

caggcggaca tcctgatcaa taccgattcc gaagaagaag gcgaaatcta catgggttgc 540

gccggcggca tcgacttcat caccaccctg ccgctgcagc gcgaagcggt gccggccggt 600

taccaaaccc tgaagctgac cctcaagggc ctgaaaggcg gccactccgg cgccgagatc 660

cacgtcgggc tgggcaacgc caacaaactg ctggcgcgct tcctgttcgc ccatgcggcg 720

gcgctgaacc tgcgcgtgct ggacctgaac ggcggcaccc tgcgcaatgc catcccgcgt 780

gaagcctccg cggtggtagc ggtgccggcg gacaaagccg acgcgctgaa agcgctaagc 840

caggagttcc tggccgtgct gcagaacgaa ctctctgcca aagagaagaa catcaccgtg 900

ctgctggagc cggccaccag cgcttcgcag gcgctgagcg ccgacagcca gcaacgcttc 960

ctggcgctgc tgaacggcac gccgaacggc gtgatccgca tgagcgacgc ggtgaaaggc 1020

gtggtggaaa cctcgctgaa cgtcggggtg gtcaccacca gcgaaaacga agcggaaatc 1080

atctgcctga tccgctccct gatcgacagc ggcaaagact acgtagtcga gatgctgacc 1140

gcgctgggcc agttggccgg cgccaaggtg gcgccgaagg gcggctaccc gggctggcag 1200

ccggacgccg actcgccggt gatgcacctg gtgcgagagc tgtatcagga tctgttcaac 1260

aagacgccga acatcatggt gatccacgcc ggcctggaat gcggtctgtt caaaaagccg 1320

tatccaaaca tggacatggt atcgatcggg ccaaccatca ccggcccaca ctcgccggat 1380

gagcaggtgc atatcgaaag cgtcggtctg tactggaagc tgctgacctc gttgctgaaa 1440

gcgatccctg agcgcgcgta a 1461

<210> 2

<211> 486

<212> PRT

<213> Serratia marcescens

<400> 2

Val Ser Glu Leu Ser Gln Leu Ser Pro Gln Pro Leu Trp Asp Ile Phe

1 5 10 15

Ala Lys Ile Cys Ser Ile Pro His Pro Ser Tyr His Glu Glu Ala Leu

20 25 30

Ala Gln His Ile Leu Thr Trp Ala Lys Glu Lys Asn Leu His Ala Glu

35 40 45

Arg Asp Gln Val Gly Asn Ile Leu Leu Arg Lys Pro Ala Thr Lys Gly

50 55 60

Met Glu Asn Arg Lys Pro Val Ala Leu Gln Ala His Leu Asp Met Val

65 70 75 80

Pro Gln Lys Asn Asn Asp Thr Val His Asp Phe Thr Lys Asp Pro Ile

85 90 95

Gln Pro Tyr Ile Ala Gly Glu Trp Val Lys Ala Arg Gly Thr Thr Leu

100 105 110

Gly Ala Asp Asn Gly Ile Gly Met Ala Ser Ala Leu Ala Val Leu Ala

115 120 125

Asp Asp Ser Val Glu His Gly Pro Leu Glu Val Leu Leu Thr Met Thr

130 135 140

Glu Glu Ala Gly Met Asp Gly Ala Phe Gly Leu Gln Pro Asn Trp Leu

145 150 155 160

Gln Ala Asp Ile Leu Ile Asn Thr Asp Ser Glu Glu Glu Gly Glu Ile

165 170 175

Tyr Met Gly Cys Ala Gly Gly Ile Asp Phe Ile Thr Thr Leu Pro Leu

180 185 190

Gln Arg Glu Ala Val Pro Ala Gly Tyr Gln Thr Leu Lys Leu Thr Leu

195 200 205

Lys Gly Leu Lys Gly Gly His Ser Gly Ala Glu Ile His Val Gly Leu

210 215 220

Gly Asn Ala Asn Lys Leu Leu Ala Arg Phe Leu Phe Ala His Ala Ala

225 230 235 240

Ala Leu Asn Leu Arg Val Leu Asp Leu Asn Gly Gly Thr Leu Arg Asn

245 250 255

Ala Ile Pro Arg Glu Ala Ser Ala Val Val Ala Val Pro Ala Asp Lys

260 265 270

Ala Asp Ala Leu Lys Ala Leu Ser Gln Glu Phe Leu Ala Val Leu Gln

275 280 285

Asn Glu Leu Ser Ala Lys Glu Lys Asn Ile Thr Val Leu Leu Glu Pro

290 295 300

Ala Thr Ser Ala Ser Gln Ala Leu Ser Ala Asp Ser Gln Gln Arg Phe

305 310 315 320

Leu Ala Leu Leu Asn Gly Thr Pro Asn Gly Val Ile Arg Met Ser Asp

325 330 335

Ala Val Lys Gly Val Val Glu Thr Ser Leu Asn Val Gly Val Val Thr

340 345 350

Thr Ser Glu Asn Glu Ala Glu Ile Ile Cys Leu Ile Arg Ser Leu Ile

355 360 365

Asp Ser Gly Lys Asp Tyr Val Val Glu Met Leu Thr Ala Leu Gly Gln

370 375 380

Leu Ala Gly Ala Lys Val Ala Pro Lys Gly Gly Tyr Pro Gly Trp Gln

385 390 395 400

Pro Asp Ala Asp Ser Pro Val Met His Leu Val Arg Glu Leu Tyr Gln

405 410 415

Asp Leu Phe Asn Lys Thr Pro Asn Ile Met Val Ile His Ala Gly Leu

420 425 430

Glu Cys Gly Leu Phe Lys Lys Pro Tyr Pro Asn Met Asp Met Val Ser

435 440 445

Ile Gly Pro Thr Ile Thr Gly Pro His Ser Pro Asp Glu Gln Val His

450 455 460

Ile Glu Ser Val Gly Leu Tyr Trp Lys Leu Leu Thr Ser Leu Leu Lys

465 470 475 480

Ala Ile Pro Glu Arg Ala

485

Claims

1. a kind of carnosine hydrolase, which is characterized in that it is following (a) or protein (b):

Protein (a): the protein that the amino acid sequence shown in SEQ ID No.2 forms；

Protein (b): the amino acid sequence as shown in SEQ ID No.2 is by replacing, missing or adding one or more amino acid Obtained from carnosine hydrolytic enzyme activities the protein as derived from (a).

2. a kind of carnosine hydrolase according to claim 1, which is characterized in that the protein (b) has following sequence:

(1) the 18th glutamic acid of the amino acid sequence as shown in SEQ ID No.2 is replaced with into threonine, the 22nd lysine replaces It is changed to alanine, the 67th phenylalanine replaces with alanine；

(2) the 17th threonine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into alanine, the 39th glycine replaces It is changed to aspartic acid；

(4) the 22nd lysine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into aspartic acid, the 64th arginine Replace with glutamic acid；

(5) the 22nd lysine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into aspartic acid, the 64th arginine Glutamic acid is replaced with, the 93rd cysteine replaces with threonine；

(6) the 39th glycine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into alanine, the 114th valine Replace with asparagine；

(7) the 39th glycine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into alanine, the 93rd cysteine Threonine is replaced with, the 114th valine replaces with asparagine, and 243 asparagines replace with serine；

(8) the 44th lysine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into glycine, the 93rd cysteine Threonine is replaced with, 243 asparagines replace with serine；

(9) the 44th lysine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into glycine, the 64th arginine replaces It is changed to glutamic acid, the 67th phenylalanine replaces with alanine；243rd asparagine replaces with leucine；

(10) the 64th arginine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into glutamic acid, the 67th phenylpropyl alcohol ammonia Acid replaces with alanine；

(11) the 64th arginine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into glutamic acid, the 67th phenylpropyl alcohol ammonia Acid replaces with alanine；243rd asparagine replaces with leucine；

(12) the 67th phenylalanine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into alanine, the 93rd half Guang Propylhomoserin replaces with isoleucine, and the 114th valine replaces with asparagine, and the 243rd asparagine replaces with leucine；

(13) the 93rd cysteine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into isoleucine, the 114th figured silk fabrics Propylhomoserin replaces with tyrosine；

(14) the 93rd cysteine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into isoleucine, the 114th figured silk fabrics Propylhomoserin replaces with tyrosine, and the 243rd asparagine replaces with leucine；

(15) the 93rd cysteine of the amino acid sequence as shown in SEQ ID No.2 is replaced with into isoleucine, the 243rd day Winter amide replaces with leucine.

3. a kind of isolated nucleic acid, which is characterized in that the nucleic acid encode carnosine hydrolase as claimed in claim 1 or 2.

4. a kind of recombinant expression carrier, which is characterized in that include nucleic acid as claimed in claim 3.

5. a kind of recombinant expression transformants, which is characterized in that include recombinant expression carrier as claimed in claim 4.

6. a kind of preparation method of carnosine hydrolase as claimed in claim 1 or 2, which is characterized in that comprise the following steps: culture Recombinant expression transformants as claimed in claim 5, separation obtain the carnosine hydrolase of recombinant expression.

7. a kind of carnosine hydrolase as claimed in claim 1 or 2 synthesis N-BETA-Alanyl-L-histidine in application, which is characterized in that including with Lower step: using carnosine hydrolase as claimed in claim 1 or 2, and the inverse hydrolysis for being catalyzed Beta-alanine and L-Histidine is raw At N-BETA-Alanyl-L-histidine, then from reaction mixture separation and Extraction product N-BETA-Alanyl-L-histidine.

8. the use as claimed in claim 7, which is characterized in that the carnosine hydrolase can also be such as claims 1 or 2 The carnosine hydrolase is fixed on immobilised enzymes obtained on carrier；

Preferably, the carrier is macromolecule resin, contains epoxy group or amino group.

9. the use as claimed in claim 7, which is characterized in that the reaction medium system is one of following system: The single_phase system or water buffer and water-insoluble that pure water, water buffer, water buffer are constituted with water-miscible organic solvent have The two-phase system that solvent is constituted；

Optionally, the water-miscible organic solvent be selected from methanol, ethyl alcohol, isopropanol, dimethyl sulfoxide, dimethyl imide or One of dioxane or their any combination, the adding proportion of water-miscible organic solvent are 0~20% (v/v)；

Optionally, the water-insoluble organic solvents are selected from isooctane, toluene, isopropyl ether, methyl tertiary butyl ether(MTBE) or butyric acid second Ester, the ratio with water phase buffer solution are 5:1~1:5 (v/v).

10. the use as claimed in claim 7, which is characterized in that reaction temperature is 20~60 DEG C, and reaction pH is 6.0~9.0, The concentration of substrate Beta-alanine is 200mM~saturated concentration, and the concentration of substrate L-Histidine is 100mM~saturated concentration.