CN1544620A - Method for producing 7-amino cephalosporanic acid and its specific recombinase - Google Patents

Abstract

The invention discloses a method of producing 7-amido-cephalosporanic acid and its special recombinant enzyme, providing a recombinant enzyme with the activity of glutaryl-7-amido- cephalosporanic acid acylase and D-amino acid oxidase and its coding gene, and a method of using the recombinant enzyme to catalyze cephalosporin C so as to directly produce 7-amido- cephalosporanic acid. The recombinant enzyme is a protein with aminophenol residual group order of SEQID No:6 in the order table a protein obtained by making an or several aminophenol residual group substitutions, deletion or addition on the aminophenol residual group order of SEQID No:6, derived from SEQID No:6 and having the same activity as the aminophenol residual group order of SEQID No:6. The method of producing 7-amido-cephalosporanic acid uses the recombinant enzyme to catalyze cephalosporin C so as to produce 7-amido- cephalosporanic acid. The recombinant enzyme can be widely applied to production of 7-amido- cephalosporanic acid. It provides a new way for the recombinant enzyme catalyzing cephalosporin C to produce 7-amido- cephalosporanic acid.

Description

A kind of method and special-purpose recombinase thereof of producing 7-amino-cephalosporanic acid

Technical field

The present invention relates to particularly a kind of method and special-purpose recombinase thereof of producing 7-amino-cephalosporanic acid in the bioengineering field of biotechnology.

Background technology

7-amino-cephalosporanic acid (7-aminocephalosporanic acid, 7-ACA) be the important source material of present semi-synthetic cynnematin, mainly (Cephalosporin C CPC) sloughs by chemical method or enzymatic cleavage that the D-alpha-amino group hexanedioyl side chain of 7-position obtains by cephalosporin.

Use enzyme process to substitute traditional chemical method cracking cephalosporin and produce 7-ACA, have advantages such as simple, efficient, safe, the pollution-free and constant product quality of technology, on economical and protection environment, bigger competitive edge is arranged all.In recent years, people have dropped into a large amount of man power and materials to the research and development of Production by Enzymes semisynthetic antibiotics.Produce 7-ACA with enzymatic cleavage, be divided into a step enzyme method and two step enzyme methods.Wherein a step enzyme method utilizes directly catalysis cephalosporin of cephalosporin C acrylase (Cephalosporin C acylase), sloughs the D-alpha-amino group hexanedioyl side chain of 7-position, generates 7-ACA.Though this method step is simple, the CPC acylase vigor of having found at present is all very low, far can not satisfy the needs of Industrial Catalysis.Two step enzyme methods are to utilize two different enzymes of source to carry out two-step catalysis, and at first, cephalosporin is at D-amino-acid oxidase (D-Amino Acid Oxidase, DAAO) under the effect, produce the intermediate with ketone group, this intermediate is unstable, is easy to the H that is generated simultaneously ₂O ₂The chemical oxidation decarboxylation, be transformed into glutaryl--7-amino-cephalosporanic acid (Glutaryl-7-amidocephalosporanicAcid, GL-7-ACA); Under the effect of GL-7-ACA acylase, slough its side chain then, generate 7-ACA (as shown in Figure 1).

The DAAO that uses in two step enzyme methods is a kind of typical flavoprotein (coenzyme is a flavin adenine dinucleotide), extensively is present in pluck and the microorganism, as pig kidney, trigonopsis variabilis, rhodotorula, fusarium solanae is mould and aspergillus etc.The Substratspezifitaet of DAAO is lower, can act on the compound (as cephalosporin) of nearly all D-amino acid and the various D-of having amino.

Most of bacterial strain that produces the GL-7-ACA acylase is pseudomonas (Pseudomonas sp.).Can be having catalysis GL-7-ACA activity, the enzyme that generates 7-ACA falls into 5 types, and same zymoid gene all has the homology more than 95%, and zymologic property is almost completely identical.

At present, utilize two step enzyme method catalytic pyrolysis cephalosporins to make the technology industrialization abroad of 7-ACA, domestic also introduction of foreign technology carry out 7-ACA and produce, but carry out the production cost height from the foreign procurement enzyme, lack competitive power economically with chemical method.Along with China's entry into the WTO, the external cynnematin of producing pours in China, and domestic microbiotic industry will face severe situation.Therefore, step up to develop the proprietary technology of the Production by Enzymes 7-ACA with advanced level, satisfy domestic needs and strengthen competitiveness in the international market extremely urgent.

Along with development of biology, people more and more pay attention to producing enzyme and transforming enzyme with engineered method.Laboratory, the present inventor place is cloned from trigonopsis variabilis and has been obtained the DAAO gene, and it has successfully been made up genetic engineering bacterium BL21 (DE3)/pET-DAAO, realized DAAO efficiently expressing in intestinal bacteria, the work of DAAO enzyme can reach 175U/mL, has substantially exceeded the bibliographical information value.(cloning and expression of daao gene in intestinal bacteria, the microorganism journal is employed; Its physical map as shown in Figure 4)

For the GL-7-ACA acylase, people generally are by a large amount of screenings, obtain having the active wild strain of GL-7-ACA acylase, and then through complicated molecular biology operation, the GL-7-ACA acylase gene are cloned and expressed.But the bacterial screening workload is big, length consuming time, and also difficulty is also big, often needs the time in several years just can obtain certain progress.The present inventor has invented a kind of method of obtaining the GL-7-ACA acylase gene fast; the method of promptly utilizing microbiology and molecular biology to combine; obtain goal gene through the method that microbial selective is cultivated and specific PCR increases fast from occurring in nature; significantly reduce the workload of screening wild strain and clone's goal gene, applied for Chinese patent (application number: 03143198.4).And (the GL-7-ACA acylase gene is at the cloning and expression of intestinal bacteria, and the microbiology circular is employed the gained gene to be used for making up genetic engineering bacterium BL21 (DE3)/pET-ACY; Its physical map is as shown in Figure 4), realized the quick expression of GL-7-ACA acylase.

Producing in the research of 7-ACA with D-amino-acid oxidase and GL-7-ACA acylated enzyme catalysis; people cultivate respectively two bacterial strains earlier and express; carry out the separation and purification of enzyme then respectively; then carry out the immobilization of enzyme more respectively; at last, immobilized enzyme being mixed one goes on foot catalysis CPC or carries out catalysis in two steps.In decades, people's research emphasis all concentrates on the vigor and stability that improves enzyme, and is then less relatively to the improved research of cephalosporin Catalytic processes, therefore, in existing Catalytic processes, also has many can the simplification and improved place.

In actual multistep enzyme catalysis was produced, in order to simplify technology, people often carried out co-immobilization with two or more enzymes, and polystep reaction can be carried out simultaneously.But still need cultivate respectively each enzyme, separation and purification, and the optimal fixation condition of each enzyme may have nothing in common with each other, and need optimize repeatedly the co-immobilization condition.

In recent years, the development of structure biology and genetic engineering technique makes people effectively to transform the enzyme molecule, even can design the original non-existent new enzyme of occurring in nature targetedly, make its performance better or give its new function, fusion gene promptly is one of them important research direction.So-called fusion gene is meant that the coding region with two or more genes joins end to end, and places the mosaic gene that constitutes under same set of regulating and controlling sequence (comprising promotor, enhanser, ribosome binding sequence, the terminator etc.) control.The development of modern DNA recombinant technology makes the fusion of different genes or gene fragment to carry out easily, after fusion gene is expressed via appropriate expression system, can obtain the Multifunction albumen that together formed by the amalgamation of difference in functionality albumen, we are called fusion rotein (Fusion protein).

In utilizing the constructed fusion protein molecule of gene fusion, if the complete encoding sequence of each enzyme molecule is retained in the new fusion protein molecule, and fusion rotein can correctly fold in the expression process, and fusion rotein generally can keep the enzyme molecule enzymic activity separately that is constituted.Compare with one enzyme, the enzyme of fusion rotein may decrease than work, but for the catalytic reaction of multienzyme order, because " proximity effect " (proximityeffect), might improve total transformation efficiency of multienzymatic reaction.Also promptly Catalytic processes is improved, can make it to become more simple, efficient by engineered means.Therefore, construction of fusion protein is the another novel method that has using value after methods such as several enzyme co-immobilizations (co-immobilization) and chemically crosslinked.

In a word, no matter be or aspect the industrial application, fusion rotein all has very high researching value in basic theory, be the big promising research direction of biological technical field.

The innovation and creation content

The purpose of this invention is to provide a kind of have glutaryl--7-amino-cephalosporanic acid acylase and active recombinase of D-amino-acid oxidase and encoding gene thereof.

Recombinase provided by the present invention, be to have SEQ ID № in the sequence table: the protein of six amino acid residue sequence, or with SEQ ID №: 6 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino-acid residue and have the № with SEQ ID: 6 amino acid residue sequence is identical active by SEQ ID №: 6 deutero-protein.

SEQ ID № in the sequence table: the 6th, the protein of forming by 1060 amino-acid residues.

A kind of encoding gene of above-mentioned recombinase is one of following nucleotide sequences:

1) SEQ ID № in the sequence table: 5 dna sequence dna;

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 6 protein sequences;

3) with sequence table in SEQ ID №: 5 dna sequence dnas that limit have 90% above homology, and the identical function protein DNA sequence of encoding.

The dna sequence dna of sequence 5 is by 3183 based compositions in the sequence table, and the reading frame of this gene is to hold the 1st to the 3183rd bit base from 5 '.

The expression vector and the clone that contain this recombinase encoding gene also belong to protection scope of the present invention; utilize existing molecular biological method can obtain different expression vectors and engineering bacteria, as recombinant expression vector pET-ALD (collection of illustrative plates as shown in Figure 2) and engineering bacteria E.coli BL21 (DE3)/pET-ALD.

Second purpose of the present invention provides a kind of method of producing 7-amino-cephalosporanic acid.

The method of production 7-amino-cephalosporanic acid provided by the present invention is to produce 7-amino-cephalosporanic acid by above-mentioned recombinase catalysis cephalosporin.

Recombinase of the present invention has kept the script vigor of GL-7-ACA acylase and DAAO, and can be used for the direct catalytic production 7-ACA of cephalosporin.Method of the present invention is expressed respectively with the method for two-step catalysis with two traditional genes and is compared, can be so that the Catalytic processes of the separation and purification of the culture expression of genetic engineering bacterium, enzyme and cephalosporin all obtains simplifying, that is to say by engineered means downstream catalytic technology is improved, for the catalysis of 7-ACA provides new approaches, and accelerated the industrialized paces of cephalosporin catalytic production 7-ACA, have important significance for theories and practical significance.

Description of drawings

Fig. 1 is the synoptic diagram that two step enzyme methods are produced 7-ACA

Fig. 2 is the physical map that contains the recombinant plasmid pET-ALD of recombinase encoding gene

Fig. 3 is the full bacterium SDS-PAGE electrophorogram behind the recombinase genetic engineering bacterium abduction delivering

Fig. 4 is the building process synoptic diagram that contains the recombinant plasmid pET-ALD of recombinase encoding gene

Fig. 5 is the HPLC color atlas of the reaction product of reorganization enzyme catalysis cephalosporin 60min and 120min

Embodiment

Embodiment 1, contain the structure of the recombinant plasmid pET-ALD of recombinase encoding gene (ACY-linker-DAAO, be called for short ALD)

The building process of recombinant plasmid pET-ALD as shown in Figure 4, concrete steps are as follows:

1, the clone of daao gene

(1) (its correlated character is Kan with the recombinant plasmid pET-DAAO that has the DAAO gene ^r, T7 lacpromotor, DAAO; Its physical map is as shown in Figure 4) be template, be that primer carries out pcr amplification with sequence in the sequence table 1 and sequence 2, sequence 1 is made up of 25 nucleotide residues in the sequence table, and sequence 2 is made up of 29 nucleotide residues.In a sterilized 0.2mL PCR thin-walled tube, add 5 μ L sterilized waters, 10 μ L amplification buffers, four kinds of deoxynucleotide (2.5mM of 1 μ L successively, TAKARA company), 1 μ L primer 1 (final concentration 50pmol), 1 μ L primer 2 (final concentration 50pmol), 0.5 μ L plasmid pET-DAAO solution (concentration is 50ng/ μ L), 0.2 μ Lpfu archaeal dna polymerase (5U/ μ L, Shanghai Sangon Biological Engineering Technology And Service Co., Ltd), complementing to cumulative volume with sterilized water is 20 μ L, and centrifuge tube is placed temperature varying system.In the PCR reaction, the temperature changing process of PCR reaction is: be warming up to 94 ℃ earlier, kept 1 minute, then by following temperature variation program loop 30 times: be warming up to 94 ℃, kept 1 minute, be cooled to 56 ℃, kept 1.5 minutes, and be warming up to 72 ℃, kept 2.5 minutes, kept the end amplified reaction 10 minutes in 72 ℃ at last.

(2) through pcr amplification, obtain having the DAAO gene of BamH I and Hind III restriction enzyme site, and the DAAO gene that obtains is carried out enzyme with BamH I and Hind III cut, and the colibacillus expression plasmid pET-28 that cuts with same BamH I and Hind III enzyme (available from Novagen company, its correlated character is Kan ^r, T7 lacpromotor; Its physical map is as shown in Figure 4) connect with the T4 dna ligase, (available from Invitrogen company, correlated character has F-mcrA Δ (mrr-hsdRMS-mcrBC) φ 80lacZ Δ M15 Δ lacX74deoRrecAlaraD139 (ara-leu) 7697galU galK rpsL (Str to connect sample transformed into escherichia coli TOP-10F ' ^R) endAl nupG) competent cell, after cultivating 16hr on the LB solid medium that contains 100 μ g/mL kantlex, the some single bacterium colonies of picking overnight incubation in liquid LB substratum, collect thalline and extract plasmid, 0.7% agarose gel electrophoresis detects, and screening obtains having the recombinant plasmid pET-DAAO fusion of new DAAO gene, and (its correlated character is Kan ^r, T7 lac promotor, DAAOfusion).

2, the clone who has the GL-7-ACA acylase gene of connection peptides sequence

(1) (its correlated character is Kan with the recombinant plasmid pET-ACY that has the GL-7-ACA acylase gene ^r, T7lac promotor, ACY; Its physical map is as shown in Figure 4) be template, be that primer carries out pcr amplification with sequence in the sequence table 3 and sequence 4, sequence 3 is made up of 31 nucleotide residues, and sequence 4 is made up of 58 nucleotide residues.In a sterilized 0.2mL PCR thin-walled tube, add 5 μ L sterilized waters, 10 μ L amplification buffers, four kinds of deoxynucleotides of 1 μ L (2.5mM), 1 μ L primer 1 (final concentration 50pmol), 1 μ L primer 2 (final concentration 50pmol), 0.5 μ L plasmid pET-ACY solution (concentration is 50ng/ μ L), 0.2 μ LLA Taq archaeal dna polymerase (5U/ μ L successively, TAKARA company), complement to cumulative volume 20 μ L with sterilized water, centrifuge tube is placed temperature varying system.In the PCR reaction, the temperature changing process of PCR reaction is: be warming up to 94 ℃ earlier, kept 1 minute, then by following temperature variation program loop 30 times: be warming up to 94 ℃, kept 1 minute, be cooled to 58 ℃, kept 1.5 minutes, and be warming up to 72 ℃, kept 2.5 minutes, kept the end amplified reaction 10 minutes in 72 ℃ at last.

(2) the GL-7-ACA acylase gene that has Nde I and BamHI restriction enzyme site that obtains through pcr amplification has been introduced one section correspondence (Gly-Ser) simultaneously in the PCR downstream primer ₆The connection peptides encoding sequence: GGATCAGGCTCTGGGTCTGGCTCTGGATCAGGATCC.

The insertion of this connection peptides is for increasing by two distances between the enzyme molecule, reduce the sterically hindered of them, making fusion rotein keep its original biologic activity to greatest extent.And amplification obtains the GL-7-ACA acylase gene and has been removed terminator codon TGA; fusion gene can not stop when translating GL-7-ACA acylase C end; and can continue to translate connection peptides and daao gene, thereby be expressed as a complete fusion rotein.

The GL-7-ACA acylase gene that PCR is obtained carries out enzyme with Nde I and BamH I and cuts; and the GL-7-ACA acylase gene that obtains is carried out enzyme with BamH I and Nde I cut; and be connected with the T4 dna ligase with the DAAO recombinant plasmid pET-DAAO fusion that Nde I enzyme is cut with same BamH I; connect sample transformed into escherichia coli TOP-10F ' competent cell; after cultivating 16hr on the LB solid medium that contains 100 μ g/mL kantlex; the some single bacterium colonies of picking overnight incubation in liquid LB substratum; collect thalline and extract plasmid; 0.7% agarose gel electrophoresis detects; screening obtains having the recombinant plasmid pET-ALD of DAAO and GL-7-ACA acylase antigen-4 fusion protein gene; its physical map as shown in Figure 2; it is a double-stranded cyclic DNA; length is 8.5kb, and its correlated character is Kan ^r, T7 lac promotor, ALD.Make up the gene order of the recombinase (fusion rotein) that obtains and corresponding amino acid sequence shown in sequence in the sequence table 5 and sequence 6 through this method.

The expression of embodiment 2, recombinase (fusion rotein)

To recombinate, (available from Promega company, correlated character has M15 Tn10 (ter to pET-ALD transformed host cell E.coli BL21 (DE3) ^r) HsdS gal (λ cIts857 indl Sam7 nin5 lacUV5-T7 Genel)), obtain genetic engineering bacterium E.coli BL21 (the DE3)/pET-ALD of recombinase (fusion rotein).

Genetic engineering bacterium is inoculated in (the LB substratum consists of: yeast powder 0.5%, Tryptones 1%, NaCl 1% in the LB liquid nutrient medium, pH7.0), 37 ℃ of shaking table overnight incubation, being transferred to the 300mL that 50mL LB substratum is housed with 5% inoculum size again shakes in the bottle, continue 37 ℃ and cultivate 2hr, add 0.5mM IPTG, induce in 28 ℃, behind the 20hr, centrifugal collection thalline.Measure the activity of DAAO and GL-7-ACA acylase in the genetic engineering bacterium respectively, record the DAAO enzyme and live and be that 3.4U/mL, GL-7-ACA acidated enzyme live and be 54U/L.

The thalline that collection is obtained carries out full bacterium SDS-PAGE electrophoresis, as shown in Figure 3, show that genetic engineering bacterium has given expression to recombinase (fusion rotein), among the figure standard protein molecular weight be respectively 94,67,43,31,21,14kD, swimming lane 1 is induced preceding thalline sample for IPTG, swimming lane 2 is the thalline sample of IPTG after inducing, and swimming lane 3 is the standard protein sample.Two tangible protein bands about 18kD and 94kD, have been given expression to; the α subunit of the corresponding GL-7-ACA acylase of 18kD protein band wherein, the fusion fragment of β subunit, connection peptides and the D-amino-acid oxidase of the corresponding GL-7-ACA acylase of the protein band about 94kD.α subunit and β subunit that GL-7-ACA acylase in the recombinase (fusion rotein) is described are correctly processed, and recombinase (fusion rotein) is successful expression.

Embodiment 3, recombinase (fusion rotein) directly catalysis cephalosporin are produced 7-ACA

The genetic engineering bacterium liquid of abduction delivering among the embodiment 2 is centrifugal, collect thalline, (pH8.0) is resuspended in the 50mL triangular flask with the 0.1mol phosphate buffered saline buffer, adds with same phosphate buffered saline buffer dissolved cephalosporin solution (final concentration is 5g/L), and 28 ℃ of shaking tables react 60min and 120min.Adopt the HPLC method detection reaction product of Shimadzu C18 post, moving phase is 7.5% acetonitrile-15% methyl alcohol-1% acetate, flow velocity is 1mL/min, chromatographic separation result as shown in Figure 5, when showing example reaction 60min, CPC still unreacted is intact, and existing 7-ACA generates, but has the GL-7-ACA of suitable volume simultaneously.This explanation in recombinase (fusion rotein), DAAO and GL-7-ACA acylase active unbalanced, the excessive part of intermediate product GL-7-ACA that caused of DAAO accumulates.During example reaction 120min, CPC primitive reaction is complete, and GL-7-ACA has also substantially changed into 7-ACA, and its production rate is about 78%.

Sequence table

<160>6

<210>1

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>1

cgggatccat?ggctaaaatc?gttgt 25

<210>2

<211>29

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>2

gctcaagctt?ctaaaggttt?ggacgagta 29

<210>3

<211>31

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>

ggaattccat?atggagccga?cctcgacgcc?g 31

<210>4

<211>58

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>4

atggatcctg?atccagagcc?agacccagag?cctgatcctg?gcttgaagtt?gaagggcg 58

<210>5

<211>3183

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>5

atggagccga?cctcgacgcc?gcaggcgccg?attgcggcct?ataaaccgag?aagcaatgag 60

atcctgtggg?acggctacgg?cgtcccgcac?atctacgggg?tcgacgcgcc?ctccgccttc 120

tacggctacg?gctgggccca?ggcgcgaagc?cacggcgaca?atatcctgcg?cctgtatgga 180

gaagcgcggg?gcaagggggc?cgaatactgg?ggcccggatt?acgaacagac?gaccgtctgg 240

ctgctgacca?acggcgtgcc?ggagcgcgcc?cagcagtggt?atgcgcagca?gtcgctggat 300

ttccgcgcca?acctcgacgc?cttcgcggcg?ggcatcaacg?cctatgccga?acagaaccca 360

gacgacatct?cgcccgaggt?gcggcaggtg?ctgccggttt?ccggcgccga?cgtggtggcg 420

cacgcccatc?gcctgatgaa?cttcctctat?gtcgcgtcgc?ccggccgcac?cctgggcgag 480

ggcgatccgc?cggacctggc?cgatcagggg?tccaactcct?gggccgtggc?gccgggcaag 540

acggcgaacg?ggaacgccct?gctgctgcag?aacccgcacc?tgtcctggac?gacggactac 600

ttcacctact?acgaggcgca?tctcgtcacg?ccggacttcg?aggtctatgg?cgcgacccag 660

atcggcctgc?cggtcatccg?cttcgccttc?aatcagcgga?tgggcatcac?caataccgtc 720

aacggcatgg?tgggggccac?caactatcgg?ctgacgcttc?aggacggcgg?ctatctgtac 780

gacggtcagg?tgcggccgtt?cgagcggcgt?caggcctcgt?atcgcctgcg?tcaggcggac 840

gggtcgacgg?tcgacaagcc?attggagatt?cgctccagcg?tccatggccc?ggtcttcgag 900

cgcgcggacg?gcacggccgt?cgccgttcgg?gtcgccggtt?tggatcggcc?gggcatgctc 960

gagcagtatt?tcgacatgat?cacggccgac?agcttcgacg?actacgaagc?cgccatggcg 1020

cggatgcagg?tgccgacctt?caacatcgtc?tacgccgacc?gcgaagggac?catcaactac 1080

agcttcaacg?gcgtggcgcc?caaacgggcc?gagggcgaca?tcgccttctg?gcaggggctc 1140

gtgcctggcg?attcctcgcg?ttacctgtgg?accgagaccc?acccgctgga?cgatctgccg 1200

cgcgtcacca?atccgccggg?cggcttcgtg?cagaactcca?atgatccgcc?gtggacgccg 1260

acctggcccg?tcacctacac?gcccaaggac?ttcccctcct?atctggcgcc?ccagacgccg 1320

cattccctgc?gcgcgcaaca?aagcgtgcgt?ctgatgtccg?agaacgacga?cctgacgctg 1380

gagcgcttca?tggcgctgca?gttgagccac?cgcgccgtca?tggccgaccg?cacgttgccg 1440

gatctgattc?cggccgccct?gatcgacccc?gatcccgagg?tccaggcggc?ggcgcgcctg 1500

ctggcggcgt?gggatcgcga?gttcgccagc?gacagccggg?ccgccctgct?gttcgaggaa 1560

tgggcgcgtc?tgttcgccgg?ccagaatttc?gccggccagg?cgggtttcgc?cacgccctgg 1620

tcgctggata?agccggtcag?cacgccctac?ggcgtccgcg?acaccaaggc?cgctgtcgat 1680

caactgcgga?ccgccatcgc?caacaccaag?cgcaagtacg?gcgcgatcga?ccggccgttc 1740

ggcgacgcct?cgcgcatgat?cctgaacaat?gtgaatgttc?cgggcgccgc?cggctacggc 1800

aacctgggtt?ccttccgggt?cttcacctgg?tccgatcctg?acgaaaacgg?ggttcgcacg 1860

cccgtccacg?gcgagacgtg?ggtggcgatg?atcgagttct?ccaccccggt?gcgggcctat 1920

ggcctgatga?gctacggcaa?ttctcgccag?ccgggcacga?cgcactacag?cgatcagatc 1980

gaacgcgtgt?cgcgcgccga?cttccgcgag?ctgttgttgc?ggcgagagca?ggtcgaggcc 2040

gccgtccagg?aacgcacgcc?cttcaacttc?aagccaggat?caggctctgg?gtctggctct 2100

ggatcaggat?ccatggctaa?aatcgttgtt?attggtgccg?gtgttgccgg?tttaactaca 2160

gctcttcaac?ttcttcgtaa?aggacatgag?gttacaattg?tgtccgagtt?tacgcccggt 2220

gatcttagta?tcggatatac?c?tcgccttgg?gcaggtgcca?actggctcac?attttacgat 2280

ggaggcaagt?tagccgacta?cgatgccgtc?tcttatccta?tcttgcgaga?gctggctcga 2340

agcagccccg?aggctggaat?t?cgactcatc?aaccaacgct?cccatgttct?caagcgtgat 2400

cttcctaaac?tggaaggtgc?catgtcggcc?atctgtcaac?gcaacccctg?gttcaaaaac 2460

acagtcgatt?ctttcgagat?tatcgaggac?aggtccagga?ttgtccacga?tgatgtggct 2520

tatctagtcg?aatttgcttc?cgtttgtatc?cacaccggag?tctacttgaa?ctggctgatg 2580

tcccaatgct?tatcgctcgg?cgccacggtg?gttaaacgtc?gagtgaacca?tatcaaggat 2640

gccaatttac?tacactcctc?aggatcacgc?cccgacgtga?ttgtcaactg?tagtggtctc 2700

tttgcccggt?tcttgggagg?cgtcgaggac?aagaagatgt?accctattcg?aggacaagtc 2760

gtccttgttc?gaaactctct?tccttttatg?gcctcctttt?ccagcactcc?tgaaaaagaa 2820

aatgaagacg?aagctctata?tatcatgacc?cgattcgatg?gtacttctat?cattggcggt 2880

tgtttccaac?ccaacaactg?gtcatccgaa?cccgatcctt?ctctcaccca?tcgaatcctg 2940

tctagagccc?tcgaccgatt?cccggaactg?accaaagatg?gccctcttga?cattgtgcgc 3000

gaatgcgttg?gccaccgtcc?tggtagagag?ggcggtcccc?gagtagaatt?agagaagatc 3060

cccggcgttg?gctttgttgt?ccataactat?ggtgccgccg?gtgctggtta?ccagtcctct 3120

tacggcatgg?ctgatgaagc?tatttcttac?gtcgaaagag?ctcttactcg?tccaaacctt 3180

tag 3183

<210>6

<211>1060

<212>PRT

＜213〉artificial sequence

<400>6

Met?Glu?Pro?Thr?Ser?Thr?Pro?Gln?Ala?Pro?Ile?Ala?Ala?Tyr?Lys?Pro

1 5 10 15

Arg?Ser?Asn?Glu?Ile?Leu?Trp?Asp?Gly?Tyr?Gly?Val?Pro?His?Ile?Tyr

20 25 30

Gly?Val?Asp?Ala?Pro?Ser?Ala?Phe?Tyr?Gly?Tyr?Gly?Trp?Ala?Gln?Ala

35 40 45

Arg?Ser?His?Gly?Asp?Asn?Ile?Leu?Arg?Leu?Tyr?Gly?Glu?Ala?Arg?Gly

50 55 60

Lys?Gly?Ala?Glu?Tyr?Trp?Gly?Pro?Asp?Tyr?Glu?Gln?Thr?Thr?Val?Trp

65 70 75 80

Leu?Leu?Thr?Asn?Gly?Val?Pro?Glu?Arg?Ala?Gln?Gln?Trp?Tyr?Ala?Gln

85 90 95

Gln?Ser?Leu?Asp?Phe?Arg?Ala?Asn?Leu?Asp?Ala?Phe?Ala?Ala?Gly?Ile

100 105 110

Asn?Ala?Tyr?Ala?Glu?Gln?Asn?Pro?Asp?Asp?Ile?Ser?Pro?Glu?Val?Arg

115 120 125

Gln?Val?Leu?Pro?Val?Ser?Gly?Ala?Asp?Val?Val?Ala?His?Ala?His?Arg

130 135 140

Leu?Met?Asn?Phe?Leu?Tyr?Val?Ala?Ser?Pro?Gly?Arg?Thr?Leu?Gly?Glu

145 150 155 160

Gly?Asp?Pro?Pro?Asp?Leu?Ala?Asp?Gln?Gly?Ser?Asn?Ser?Trp?Ala?Val

165 170 175

Ala?Pro?Gly?Lys?Thr?Ala?Asn?Gly?Asn?Ala?Leu?Leu?Leu?Gln?Asn?Pro

180 185 190

His?Leu?Ser?Trp?Thr?Thr?Asp?Tyr?Phe?Thr?Tyr?Tyr?Glu?Ala?His?Leu

195 200 205

Val?Thr?Pro?Asp?Phe?Glu?Val?Tyr?Gly?Ala?Thr?Gln?Ile?Gly?Leu?Pro

210 215 220

Val?Ile?Arg?Phe?Ala?Phe?Asn?Gln?Arg?Met?Gly?Ile?Thr?Asn?Thr?Val

225 230 235 240

Asn?Gly?Met?Val?Gly?Ala?Thr?Asn?Tyr?Arg?Leu?Thr?Leu?Gln?Asp?Gly

245 250 255

Gly?Tyr?Leu?Tyr?Asp?Gly?Gln?Val?Arg?Pro?Phe?Glu?Arg?Arg?Gln?Ala

260 265 270

Ser?Tyr?Arg?Leu?Arg?Gln?Ala?Asp?Gly?Ser?Thr?Val?Asp?Lys?Pro?Leu

275 280 285

Glu?Ile?Arg?Ser?Ser?Val?His?Gly?Pro?Val?Phe?Glu?Arg?Ala?Asp?Gly

290 295 300

Thr?Ala?Val?Ala?Val?Arg?Val?Ala?Gly?Leu?Asp?Arg?Pro?Gly?Met?Leu

305 310 315 320

Glu?Gln?Tyr?Phe?Asp?Met?Ile?Thr?Ala?Asp?Ser?Phe?Asp?Asp?Tyr?Glu

325 330 335

Ala?Ala?Met?Ala?Arg?Met?Gln?Val?Pro?Thr?Phe?Asn?Ile?Val?Tyr?Ala

340 345 350

Asp?Arg?Glu?Gly?Thr?Ile?Asn?Tyr?Ser?Phe?Asn?Gly?Val?Ala?Pro?Lys

355 360 365

Arg?Ala?Glu?Gly?Asp?Ile?Ala?Phe?Trp?Gln?Gly?Leu?Val?Pro?Gly?Asp

370 375 380

Ser?Ser?Arg?Tyr?Leu?Trp?Thr?Glu?Thr?His?Pro?Leu?Asp?Asp?Leu?Pro

385 390 395 400

Arg?Val?Thr?Asn?Pro?Pro?Gly?Gly?Phe?Val?Gln?Asn?Ser?Asn?Asp?Pro

405 410 415

Pro?Trp?Thr?Pro?Thr?Trp?Pro?Val?Thr?Tyr?Thr?Pro?Lys?Asp?Phe?Pro

420 425 430

Ser?Tyr?Leu?Ala?Pro?Gln?Thr?Pro?His?Ser?Leu?Arg?Ala?Gln?Gln?Ser

435 440 445

Val?Arg?Leu?Met?Ser?Glu?Asn?Asp?Asp?Leu?Thr?Leu?Glu?Arg?Phe?Met

450 455 460

Ala?Leu?Gln?Leu?Ser?His?Arg?Ala?Val?Met?Ala?Asp?Arg?Thr?Leu?Pro

465 470 475 480

Asp?Leu?Ile?Pro?Ala?Ala?Leu?Ile?Asp?Pro?Asp?Pro?Glu?Val?Gln?Ala

485 490 495

Ala?Ala?Arg?Leu?Leu?Ala?Ala?Trp?Asp?Arg?Glu?Phe?Ala?Ser?Asp?Ser

500 505 510

Arg?Ala?Ala?Leu?Leu?Phe?Glu?Glu?Trp?Ala?Arg?Leu?Phe?Ala?Gly?Gln

515 520 525

Asn?Phe?Ala?Gly?Gln?Ala?Gly?Phe?Ala?Thr?Pro?Trp?Ser?Leu?Asp?Lys

530 535 540

Pro?Val?Ser?Thr?Pro?Tyr?Gly?Val?Arg?Asp?Thr?Lys?Ala?Ala?Val?Asp

545 550 555 560

Gln?Leu?Arg?Thr?Ala?Ile?Ala?Asn?Thr?Lys?Arg?Lys?Tyr?Gly?Ala?Ile

565 570 575

Asp?Arg?Pro?Phe?Gly?Asp?Ala?Ser?Arg?Met?Ile?Leu?Asn?Asn?Val?Asn

580 585 590

Val?Pro?Gly?Ala?Ala?Gly?Tyr?Gly?Asn?Leu?Gly?Ser?Phe?Arg?Val?Phe

595 600 605

Thr?Trp?Ser?Asp?Pro?Asp?Glu?Asn?Gly?Val?Arg?Thr?Pro?Val?His?Gly

610 615 620

Glu?Thr?Trp?Val?Ala?Met?Ile?Glu?Phe?Ser?Thr?Pro?Val?Arg?Ala?Tyr

625 630 635 640

Gly?Leu?Met?Ser?Tyr?Gly?AsnSer?Arg?Gln?Pro?Gly?Thr?Thr?His?Tyr

645 650 655

Ser?Asp?Gln?Ile?Glu?Arg?Val?Ser?Arg?Ala?Asp?Phe?Arg?Glu?Leu?Leu

660 665 670

Leu?Arg?Arg?Glu?Gln?Val?Glu?Ala?Ala?Val?Gln?Glu?Arg?Thr?Pro?Phe

675 680 685

Asn?Phe?Lys?Pro?Gly?Ser?Gly?Ser?Gly?Ser?Gly?Ser?Gly?Ser?Gly?Ser

690 695 700

Met?Ala?Lys?Ile?Val?Val?Ile?Gly?Ala?Gly?Val?Ala?Gly?Leu?Thr?Thr

705 710 715 720

Ala?Leu?Gln?Leu?Leu?Arg?Lys?Gly?His?Glu?Val?Thr?Ile?Val?Ser?Glu

725 730 735

Phe?Thr?Pro?Gly?Asp?Leu?Ser?Ile?Gly?Tyr?Thr?Ser?Pro?Trp?Ala?Gly

740 745 750

Ala?Asn?Trp?Leu?Thr?Phe?Tyr?Asp?Gly?Gly?Lys?Leu?Ala?Asp?Tyr?Asp

755 760 765

Ala?Val?Ser?Tyr?Pro?Ile?Leu?Arg?Glu?Leu?Ala?Arg?Ser?Ser?Pro?Glu

770 775 780

Ala?Gly?Ile?Arg?Leu?Ile?Asn?Gln?Arg?Ser?His?Val?Leu?Lys?Arg?Asp

785 790 795 800

Leu?Pro?Lys?Leu?Glu?Gly?Ala?Met?Ser?Ala?Ile?Cys?Gln?Arg?Asn?Pro

805 810 815

Trp?Phe?Lys?Asn?Thr?Val?Asp?Ser?Phe?Glu?Ile?Ile?Glu?Asp?Arg?Ser

820 825 830

Arg?Ile?Val?His?Asp?Asp?Val?Ala?Tyr?Leu?Val?Glu?Phe?Ala?Ser?Val

835 840 845

Cys?Ile?His?Thr?Gly?Val?Tyr?Leu?Asn?Trp?Leu?Met?Ser?Gln?Cys?Leu

850 855 860

Ser?Leu?Gly?Ala?Thr?Val?Val?Lys?Arg?Arg?Val?Asn?His?Ile?Lys?Asp

865 870 875 880

Ala?Asn?Leu?Leu?His?Ser?Ser?Gly?Ser?Arg?Pro?Asp?Val?Ile?Val?Asn

885 890 895

Cys?Ser?Gly?Leu?Phe?Ala?Arg?Phe?Leu?Gly?Gly?Val?Glu?Asp?Lys?Lys

900 905 910

Met?Tyr?Pro?Ile?Arg?Gly?Gln?Val?Val?Leu?Val?Arg?Asn?Ser?Leu?Pro

915 920 925

Phe?Met?Ala?Ser?Phe?Ser?Ser?Thr?Pro?Glu?Lys?Glu?Asn?Glu?Asp?Glu

930 935 940

Ala?Leu?Tyr?Ile?Met?Thr?Arg?Phe?Asp?Gly?Thr?Ser?Ile?Ile?Gly?Gly

945 950 955 960

Cys?Phe?Gln?Pro?Asn?Asn?Trp?Ser?Ser?Glu?Pro?Asp?Pro?Ser?Leu?Thr

965 970 975

His?Arg?Ile?Leu?Ser?Arg?Ala?Leu?Asp?Arg?Phe?Pro?Glu?Leu?Thr?Lys

980 985 990

Asp?Gly?Pro?Leu?Asp?Ile?Val?Arg Glu?Cys?Val?Gly?His Arg?Pro?Gly

995 1000 1005

Arg?Glu Gly?Gly?Pro?Arg?Val Glu?Leu?Glu?Lys?Ile Pro?Gly?Val

1010 1015 1020

Gly?Phe Val?Val?His?Asn?Tyr Gly?Ala?Ala?Gly?Ala Gly?Tyr?Gln

1025 1030 1035

Ser?Ser Tyr?Gly?Met?Ala?Asp Glu?Ala?Ile?Ser?Tyr Val?Glu?Arg

1040 1045 1050

Ala?Leu Thr?Arg?Pro?Asn?Leu

1055 1060

Claims (10)

1, a kind of recombinase, be to have SEQ ID № in the sequence table: the protein of six amino acid residue sequence, or with the amino acid residue sequence of SEQ ID NQ:6 through replacement, disappearance or the interpolation of one or several amino-acid residue and have № with SEQ ID: 6 amino acid residue sequence is identical active by SEQ ID №: 6 deutero-protein.

2, recombinase according to claim 1 is characterized in that: described recombinase is the SEQ ID № in the sequence table: 6.

3, a kind of encoding gene of recombinase is one of following nucleotide sequences:

1) SEQ ID № in the sequence table: 5 dna sequence dna;

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 6 protein sequences;

3) with sequence table in SEQ ID №: 5 dna sequence dnas that limit have 90% above homology, and the identical function protein DNA sequence of encoding.

4, gene according to claim 1 is characterized in that: the encoding gene of described recombinase is the SEQ ID № in the sequence table: 5.

5, contain the described expression carrier of claim 3.

6, expression vector according to claim 5 is characterized in that: described expression vector is pET-ALD.

7, the clone that contains the described gene of claim 3.

8, clone according to claim 7 is characterized in that: described cell is E.coli BL21 (DE3)/pET-ALD.

9, a kind of method of producing 7-amino-cephalosporanic acid is to produce 7-amino-cephalosporanic acid by recombinase catalysis cephalosporin.

10, the application of the described recombinase of claim 1 in producing 7-amino-cephalosporanic acid.

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