CN103937764A - Mutated Enzyme For Producing Cephalosporin Antibiotics Raw Material (7-aca) - Google Patents
Mutated Enzyme For Producing Cephalosporin Antibiotics Raw Material (7-aca) Download PDFInfo
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- CN103937764A CN103937764A CN201310049908.7A CN201310049908A CN103937764A CN 103937764 A CN103937764 A CN 103937764A CN 201310049908 A CN201310049908 A CN 201310049908A CN 103937764 A CN103937764 A CN 103937764A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
- C12P35/02—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin by desacylation of the substituent in the 7 position
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
- C12P35/06—Cephalosporin C; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01093—Glutaryl-7-aminocephalosporanic-acid acylase (3.5.1.93)
Abstract
The invention relates to a mutated enzyme for producing cephalosporine antibiotics raw material (7-ACA), and more particularly, to the mutated cephalosporin C acylase in which the activity for preparing cephalosporin C increases through the point mutation and a method using the same for preparing 7-ACA. The mutated CPC acylase of the invention has an activity to the CPC matrix that is improved for 5-26 times than a wild type CPC acylase. Therefore the mutated enzyme has particularly remarkable effect for directly preparing 7-ACA by CPC through one step.
Description
Technical field
The present invention relates to cephalosporins (cephalosporin antibiotics) raw material 7-ACA production variant enzyme, relate in particular to a kind of variation cephalosporin C acylase (acylase) by the active increase of point mutation (Point mutation) manufacture cynnematin (Cephalosporin) C and utilize this to manufacture the manufacture method of 7-ACA.
Background technology
Cephalosporin (Cephalosporin C, below referred to as " CPC ") be beta-lactam (β-lactam) class antibiotics, produced some microorganisms producing such as cephalosporium chrysogenum (Acremonium chrysogenum) by thread fungus (Filamentous fungus) mould (mold), Gram-negative (Gram negative) bacterium is revealed to antibiosis activity by block cell wall synthetic table, but its degree is very faint.Therefore it is mainly used in the raw material of manufacturing semi-synthetic cephalosporins microbiotic (semi-synthetic cephalosporin antibiotics).Particularly, remove amino hexanedioyl side chain (D-amino adipoyl side chain) and the 7-amino-cephalosporanic acid (7-aminocephalosporanic acid, below referred to as " 7-ACA ") that obtains just uses as the raw material that accounts for the more than 40% most of cephalosporins (cephalosporin antibiotics) in global microbiotic market from CPC.
The existing technique of utilizing CPC to manufacture 7-ACA has chemical technology and enzymatic process.Reaction conditions complexity, the environmental treatment expense of chemical technology are high, and therefore present most of enterprise all produces 7ACA with environmental protection shape enzymatic process.
At present, the enzymatic process that enterprise is used with commercial aspect is made up of two stages conventionally.In the 1st stage, utilize D-AAO (D-amino acid oxidase; below referred to as " DAO ") enzyme reaction; CPC is changed into Glularyl-7-amino-cephalo-alkanoic acid (Glutaryl-7-aminocephalosporanic acid; below referred to as " Gl-7-ACA "); in the 2nd stage, by the enzyme reaction of Gl-7-ACA acyltransferase (acylase), Gl-7ACA is changed into 7ACA(with reference to figure 1).
But the production yield of the 2nd stage enzymatic process is low.Its reason is that the hydrogen peroxide that the enzyme reaction in the 1st stage generates attacks matrix (CPC), resultant of reaction (Gl-7-ACA) and DAO.Thereby be necessary to develop a kind of enzymatic process, in the 1st stage, by cephalosporin C acylase (Cephalosporin C acylase, below referred to as ' CPC acyltransferase (acylase) '), directly manufacture 7ACA from CPC.
Because CPC acyltransferase (acylase) is not present in nature, the improvement of therefore reaching the same goal is present in natural various Gl-7-ACA acyltransferase (also referred to as GA (Glutaryl amidase:GA)) and manufactures CPC acyltransferase (acylase).Gl-7-ACA acyltransferase (acylase) to the activity of CPC very a little less than, need to improve its activity.
Gl-7-ACA acyltransferase (acylase) for the 2nd stage enzymatic process is more common in various soil microorganismss; Suo Naweien (Sonawane) is divided into 5 (Sonawane of group in the mode of following [table 1] Gl-7-ACA; Crit.Rev.Biotech.26:95-120,2006).Belong in same group, protein size, aminoacid sequence, matrix feature, response feature etc. are closely similar, but the difference of different groups is larger.
[table 1]
The classification of Gl-7-ACA acyltransferase (acylase)
The Gl-7-ACA acyltransferase (acylase) that belongs to the III of group (Group) exists easily and is hindered shortcoming active, enzyme reaction poor stability by reaction product 7-ACA and inorganic salts, if the Gl-7ACA acyltransferase (acylase) of the improvement III of group,, can inherit shortcoming above.But there is not the shortcoming of the III of group enzyme in the Gl-7-ACA acyltransferase (acylase) of the II-B of group.Thereby, be necessary by the Gl-7ACA acyltransferase (acylase) of the improvement III-B of group, obtain 7-ACA and produce with the required variant enzyme of the 1st stage enzymatic process.
Accordingly; the pseudomonas (Pseudomonas) that the present inventor is subordinated to the II-B of group belongs to GK16 series Gl-7ACA acyltransferase (acylase); find the variation CPC acyltransferase (acylase) that the activity of CPC is increased, completed the present invention.
Summary of the invention
The problem of invention
The object of the present invention is to provide a kind of variation CPC acyltransferase (acylase); the wild-type CPC(Cephalosporin C that shown in its α subunit (α-subunit) by aminoacid sequence shown in sequence number 3 and sequence number 4, the β subunit of aminoacid sequence forms) in acyltransferase (acylase) sequence, F58 beta amino acids is replaced by α-amino-isovaleric acid (Valine).
Another object of the present invention is to provide a kind of gene that described variation CPC acyltransferase (acylase) is encrypted.
Another object of the present invention is to provide a kind of recombinant expression vector (expression vector) that comprises described gene.
Another object of the present invention is to provide a kind of host cell of being changed (transformation) by described expression vector (expression vector) by form quality.
Another object of the present invention is to provide a kind of microorganism of being changed (transformation) by described expression vector (expression vector) by form quality.
Another object of the present invention is to provide one to comprise 7-ACA (7-Aminocephalosporanic acid) the manufacture synthetics of described variation CPC acyltransferase (acylase).
Another object of the present invention is to provide one to utilize the 7-ACA manufacture method of described CPC acyltransferase (acylase).
Embodiment
In order to achieve the above object; the invention provides a kind of variation CPC acyltransferase (acylase); the wild-type CPC(Cephalosporin C that shown in its α subunit (α-subunit) by aminoacid sequence shown in sequence number 3 and sequence number 4, the β subunit of aminoacid sequence forms) in acyltransferase (acylase) sequence, F58 beta amino acids is replaced by α-amino-isovaleric acid (Valine).
The invention provides a kind of gene that described variation CPC acyltransferase (acylase) is encrypted.
The invention provides a kind of recombinant expression vector (expression vector) that comprises described gene.
The invention provides a kind of host cell of being changed (transformation) by described expression vector (expression vector) by form quality.
The invention provides a kind of microorganism of being changed (transformation) by described expression vector (expression vector) by form quality.
The invention provides the 7ACA manufacture synthetics that one comprises described variation CPC acyltransferase (acylase).
The invention provides one and utilize the 7-ACA manufacture method of described CPC acyltransferase (acylase).
The present invention is described in detail below.
The invention provides a kind of variation CPC acyltransferase (acylase); the wild-type CPC(Cephalosporin C that shown in its α subunit (α-subunit) by aminoacid sequence shown in sequence number 3 and sequence number 4, the β subunit of aminoacid sequence forms) in acyltransferase (acylase) sequence, F58 amino acid is replaced by α-amino-isovaleric acid (Valine).
Described variation CPC acyltransferase (acylase) can append and comprise the sudden change of selecting the group from being made up of Y153 β T, F177 β L and Y153 β T+F177 β L.It is described that ' Y153 β T+F177 β L ' means to have Y153 β T and F177 β L sudden change simultaneously.In addition, the explanation as an example of Y153 β T example, the coding of mark sudden change should be interpreted as the tyrosine (TyrosineY) of the 153rd position of β subunit (sequence number 4) and be replaced by Threonine (Threonine T).
Described variation CPC acyltransferase of the present invention (acylase), its to the activity of CPC matrix higher than wild-type CPC acyltransferase (acylase)." activity of CPC matrix is increased " of the present invention refers to that the specific activity (specific activity) to CPC matrix increases or refers to that the terminal thing being caused by reaction product suppresses (end-product inhibition) phenomenon and reduces.
Be α subunit (α-subunit), spacer peptide (Spacer Peptide), β subunit from the order of most of Gl-7-ACA acyltransferase (acylase) aminoacid sequence that gene translation obtains.The basi gene (basic gene:ga gene) using in the present invention through transcribing and translation process after generate by about 77kDa size nonactive (inertness) strand form polypeptide (polypeptide).Afterwards, spacer peptide drops, and forms two amount body (dimer) forms that are made up of about 18kDa size α subunit (α-subunit) (sequence number 3) and 58kDa size β subunit (sequence number 4).
Use the wild-type CPC acyltransferase (acylase) of manufacturing by ga gene; rely on point mutation (Point Mutation) and gene manipulation techniques, manufactured the variation CPC acyltransferase (acylase) that the activity of CPC acyltransferase (acylase) matrix is increased.
In order to select wild-type CPC acyltransferase (acylase) to cause the position of point mutation, suddenly change result as basis taking 3 d structure model and hypothesis, study known candidate variation residue (residue); 4 kinds of residue (F31 β are selected; F58 β, Y153 β, F177 β).F31 β means the phenylalanine (Phenylalanine, Phe, F) that is positioned at the 31st in β subunit aminoacid sequence.
Four of described selection kinds of residues (F31 β, F58 β, Y153 β, F177 β) are implemented to saturation mutation.Its result, after F31 β, Y153 β, 3 saturation mutations of F177 β residue, activity to CPC does not increase, and only has the mutant (F58V also shows as variant enzyme) by α-amino-isovaleric acid (Valine) displacement of F58 to increase active (reference example 2-2).Wild-type CPC acyltransferase (acylase) activity regards as at 1 o'clock, 5.3 times (reference examples 4) that the activity of described S17 variant enzyme is wild-type.
In order to increase the activity of F58 β V mutant to CPC, taking F58 β V mutant as object, the residues such as F31 β, Y153 β, F177 β are implemented respectively to saturation mutation (two point mutation).Result, the saturation mutation of F31 does not increase activity, and F58 β V/F177 β L (' A5 variant enzyme ') and F58 β V/Y153 β T (' B2 variant enzyme ') varient has increased respectively 9.8 times, 14.5 times (reference example 2-3 and embodiment 4) with respect to wild-type, activity.This presentation of results, Y153 β T, F177 β L sudden change does not have effect to wild-type, but improves the activity to CPC matrix in F58 β V mutant.F58 β V/Y153 β T/F177 β L(tri-point mutation) varient (' R1 variant enzyme '), with compared with wild-type, to the activity of CPC matrix high 16.8 times (reference example 4).
S17, A5, B2 and R1 varient can also append and comprise that I45 β or V382 beta amino acids are by the sudden change of other amino-acid substitutions.Preferably R1 varient can also append the sudden change comprising on I45 β or V382 β.
Describedly append the sudden change comprising, it is characterized in that: I45 β is by a certain amino-acid substitution in methionine(Met) (Methionine), α-amino-isovaleric acid (Valine), L-Ala (Alanine), halfcystine (Cysteine), leucine (Leucine), and V382 β is by leucine (Leucine) or Isoleucine (Isoleucine) displacement.
Variation CPC acyltransferase of the present invention (acylase), is characterized in that: described variation CPC acyltransferase (acylase) is made up of the α subunit (α-subunit) of aminoacid sequence shown in sequence number 6 and the β subunit of aminoacid sequence shown in sequence number 7.
In order to allow the F58 β V/Y153 β T/F177 β L(R1 variant enzyme of manufacturing in embodiment 2-4) activity of varient increases, embodiment random mutation.Pass through fallibility polymerase chain reaction (Error-prone PCR) induction random point mutation taking described 3 varient gene DNAs template.Its result, 4 varients finding 45 β M/F58 β V/Y153 β T/F177 β L (' M23 variant enzyme ') and F58 β V/Y153 β T/F177 β L/V382 β I (' M27 variant enzyme ') to the activity of CPC higher than R1 variant enzyme (embodiment 3-1).With wild-type comparative result, M23 is higher 18.8 times than wild-type, and M27 exceeds 20.3 times (reference examples 4) than wild-type.
Result by embodiment 3-1 is known, I45 β, and the variation of V382 β residue can improve CPC acyltransferase (acylase) activity.At this, 3 varients of F58 β V/Y153 β T/F177 β L are manufactured to I45 β and V382 β 2 saturation mutations (5 point mutation) storehouses (Library), the varient of active increase degree higher than M27 variant enzyme (Screening) in screening.Its result, select 55 varients (I45 β V/F58 β V/Y153 β T/F177 β L/V382 β L, I45 β A/F58 β V/Y153 β T/F177 β L/V382 β I, I45 β C/F58 β V/Y153 β T/F177 β L/V382 β L, I45 β M/F58 β V/Y153 β T/F177 β L/V382 β L, I45 β L/F58 β V/Y153 β T/F177 β L/V382 β I) (reference example 3-2).
In each enzyme above, the activity of PM2 (I45 β V/F58 β V/Y153 β T/F177 β L/V382 β L) variant enzyme is the highest.Described PM2 variant enzyme, is characterized in that: the β subunit by aminoacid sequence shown in the α subunit (α-subunit) of amino acid series shown in sequence number 6 and sequence number 7 forms.
Described embodiment 3-2 result is characterised in that: in I45 β residue, Isoleucine (Isoleucine) is by methionine(Met), α-amino-isovaleric acid (Valine), L-Ala, halfcystine or leucine displacement, and α-amino-isovaleric acid in V382 β (Valine) is replaced by leucine or Isoleucine.
Embodiment 3 has disclosed the active new a kind of residue (I45 β and V382 β) of raising CPC acyltransferase (acylase), has a very big significance.Fig. 2 is the varient selection process mode chart of embodiment 2 to 3.
The invention provides the gene that described various variation CPC acyltransferases (acylase) are encrypted.Preferably, described gene can represent by base sequence shown in sequence number 5.
It is GA (Glutaryl amidase:GA) gene (below referred to as " ga gene ") that belongs to GK16 series from the pseudomonas (Pseudomonas) that belongs to the II-B of group (Group) for the manufacture of the basi gene of variation CPC acyltransferase (acylase).Here,, to being the codon (codon) that protein synthesis efficiency was encrypted, improved to each amino acid, the codon often using taking colibacillus, as main, has been determined base sequence.Synthetic ga base sequence is with a sequence number 1(2079 base by this method) represent, aminoacid sequence is with a sequence number 2(692 amino acid) represent.
In described ga gene, except α subunit (α-subunit) and β subunit, also comprise the base sequence that spacer peptide is encrypted, it is also identical that this encrypts gene for variation CPC acyltransferase of the present invention (acylase).Because variant enzyme of the present invention only causes sudden change in wild-type beta subunit, therefore variant enzyme and wild-type enzyme have α subunit (α-subunit) and the spacer peptide of identical aminoacid sequence.
As its example, the gene base sequence that the highest varient PM2 of activity performance is encrypted is illustrated in sequence number 5.
The invention provides the recombinant expression vector (expression vector) that comprises the gene that variation CPC acyltransferase (acylase) is encrypted.
In the present invention, ' expression vector (expression vector) ' refers to that the polynucleotide (Polynucleotide) that thiolase (Thiolase) is encrypted are cloned plastid (Plasmid), virus or the other media body of (Cloning).The polynucleotide sequence being cloned in the present invention is operatively coupled on suitable expression and regulates in sequence.The described gene order being operably connected, can be present in an expression vector (expression vector) that simultaneously also comprises selective marker and replication orgin (replication origin).Described ' (operably linked) is operably connected ' refers to that described polynucleotide sequence is can allow the mode of genetic expression be connected in expression adjusting sequence.Described ' express and regulate sequence (expression control sequence) ' refers in particular host cell the DNA sequence dna that the expression of the polynucleotide sequence being operably connected is regulated.This adjusting sequence comprise by the promotor for implementing to transcribe (promoter), for regulate any operator gene (operator) sequence of transcribing, to suitable mRNA liposome (liposome) combining site is encoded the sequence of (coding), sequence that the end of transcribing and understanding is regulated etc. forms group selected a certain more than.
The carrier using as the maternal carrier of described expression vector (expression vector), has no particular limits.In the microorganism using in the technical field of the invention, as host cell, all can use in order to express used all plastids, virus or other media body etc.Such as there is the plastid (pBR322 from colibacillus as described plastid, pBR325, pUC118 and pUC119, pET-22b (+)), from the plastid (pUB110 and pTP5) of Bacillus subtilus (bacillus subtilis) and from the plastid (YEp13 of yeast, YEp24 and YCp50) etc., there are the insect viruses such as animal virus, baculovirus (baculovirus) such as retrovirus (Retrovirus), adenovirus (adenovirus) or vaccinia virus (vaccinia virus) as described virus, but are not limited to this.
Described recombinant expression vector (expression vector) preferably comprises the gene representing with base sequence shown in sequence number 5.
The variation CPC acyltransferase (acylase) of being encrypted by sequence number 5, as variant enzyme PM2, has been manufactured recombinant expression vector (expression vector) the pBC-PM2 plastid (reference example 4) that comprises gene shown in sequence number 5.
In addition, the invention provides the host cell and the microorganism that obtain form quality conversion (transformation) by described expression vector (expression vector).
The implication of described host cell comprises the unicellular organism of normally used all kinds, such as various bacteriums are (such as belonging to (Clostridia), colibacillus etc.) etc. the eukaryotic cell microorganism such as prokaryotic cell prokaryocyte microorganism and yeast, such as including, but not limited to clostridium (Clostridia) microorganism belonging to genus (such as, acetone-butanol fusobacterium (Clostridium acetobutylicum), Bai Shi clostridium (Clostridium beijerinckii), oxygen-butyl acetonitrile clostridium (Clostridium saccharoperbutylacetonicum) is crossed in saccharification, saccharification butyl acetonitrile clostridium (Clostridium saccharobutylicum)), colibacillus etc.
Described form quality conversion (transformation) can comprise variation CPC acyltransferase (acylase) by handle and encrypt the certain methods execution using when the expression vector (expression vector) of polynucleotide sequence is incorporated into host cell.Such as, the method that described expression vector (expression vector) is incorporated into host cell is including, but not limited to sodium-chlor (CaCl2) and thermal shocking (heat shock) method, particle gun ballistic method (particle gun bombardment), silicon carbide whisker (Silicon carbide whiskers), ultrasonication (sonication), electroporation (electroporation), by the precipitator method of PEG (polyethylenglycol).
Described microorganism is colon bacillus (Escherichia coli) MC1061/pBC-PM2 (registration number: KCTC12344BP) by obtain form quality conversion (transformation) containing recombinant expression vector (expression vector) pBC-PM2 of base sequence gene shown in sequence number 5.
The invention provides and comprise the make a variation 7-ACA manufacture synthetics of CPC acyltransferase (acylase) of the present invention.
By be inserted with the present invention make a variation CPC acyltransferase (acylase) encrypt gene or tool with it the recombinant expression vector of same function sequence gene (expression vector) obtain the host cell of form quality conversion (transformation); under suitable substratum and condition, cultivate, manufacture variation CPC acyltransferase (acylase).In addition, by be inserted with the present invention make a variation the α subunit (α-subunit) of CPC acyltransferase (acylase) encrypt gene or tool with it the recombinant expression vector of the gene of same function sequence (expression vector) obtain the host cell of form quality conversion (transformation), with by be inserted with the present invention make a variation CPC acyltransferase (acylase) β subunit encrypt gene or tool with it the recombinant expression vector of same function sequence gene (expression vector) obtain the host cell of form quality conversion (transformation), under suitable substratum and condition, cultivate respectively, manufacture after variation CPC acyltransferase (acylase) protein subunit, in vitro (in vitro) mixes two kinds of protein subunits, manufacture variation CPC acyltransferase (acylase).
The present invention who manufactures by described method makes a variation, and CPC acyltransferase (acylase) can directly use, manufacturing objective product, or the use of can purifying.As the separating and purifying method of variation CPC acyltransferase (acylase); can utilize known CPC acyltransferase (acylase) character; by the multiple separation method with chromatography (chromatography) isolated protein, or be slightly out of shape and use general separation method by experiment purpose.In addition; by utilizing bonding force or cellulose binding domain (the cellulose binding domain of Histidine peptide (histidine peptide) and nickel post (Column) composition; CBD), with affinity chromatography (affinity chromatography) method of the particular combination power character such as cellulosic bonding force, the variation CPC acyltransferase (acylase) of purifying also can.
In addition, variation CPC acyltransferase of the present invention (acylase) not only can use with unbound state, also can use with immobilized state.The immobilization of variation CPC acyltransferase (acylase), can be undertaken by the known usual method of the industry.Can use the natural polymers (polymer) such as Mierocrystalline cellulose, starch, dextran (dextran), agarose (agarose) as carrier; The synthetic polymers such as polyacrylamide (polyacrylamide), polyacrylic ester (polyacrylate), polymethacrylate (polymethacylate), acrylic C (Eupergit C); Or the mineral substance such as tripoli (silica), wilkinite (bentonite), metal.In addition, on these carriers, can pass through the modes such as covalent attachment (covalent bond), ionic bond, hydrophobic binding, physical adsorption, satellite capsule (microencapsulation), in conjunction with variation CPC acyltransferase (acylase).Also can allow these carrier-enzyme combinations under the effect of glutaraldehyde (glutaraldehyde), hydrogen bromide (cyanogen bromide) etc., form covalent attachment, fixing variation CPC acyltransferase (acylase).In addition, better method is the variation CPC acyltransferase (acylase) of not purifying, and directly the microorganism cells immobilization containing variation CPC acyltransferase (acylase) is used.When whole cell like that is fixed (whole cell immobilization), in order to improve the reactivity of the variation CPC acyltransferase (acylase) that is contained in microorganism, the applicable technology such as cell perforation or surface expression of giving.
More than comprising and making a variation the present invention the 7-ACA of CPC acyltransferase (acylase) manufactures and comprise and obtain selection in group that form quality conversion (transformation) body of form quality conversion (transformation) forms a certain by variation CPC acyltransferase (acylase), polynucleotide that described enzyme is encoded, the expression vector (expression vector) that comprises described polynucleotide, by described expression vector (expression vector) with synthetics.
The invention provides and utilize described variation CPC acyltransferase (acylase), with the compound or its salt of following Chemical formula 1, manufacture the required manufacture method of compound or its salt of following Chemical formula 2.
In chemical formula below, R is acetoxyl group (OCOCH
3), hydroxyl (OH), hydrogen base (H).Preferably, the R of chemical formula is below acetoxyl group (OCOCH
3), the salt form of the optimizing compound of chemical formula is basic metal (such as sodium salt, sylvite, lithium salts etc.) below.In addition, the compound of Chemical formula 1 is CPC matrix compounds, and the compound of Chemical formula 2 is that 7ACA compound is advisable.
Chemical formula 1
Chemical formula 2
Industry personnel can allow the variation CPC acyltransferase (acylase) manufactured by described method produce the culture of bacterial strain or with containing variation CPC acyltransferase (acylase) synthetics form; or allow the enzyme separating by unbound state or immobilized state, contact the compound of manufacturing described Chemical formula 2 with the compound of described Chemical formula 1.Variation CPC acyltransferase of the present invention (acylase) is embodied as suitable with contacting of described compound 1 in the aqueous solution (water or buffered soln).The concentration of compound 1 is preferably within the scope of 1 ~ 500mM; the present invention makes a variation CPC acyltransferase (acylase) addition preferably within the scope of 0.1 ~ 100U/Ml; the pH value of reaction mixture is preferably in 7 ~ 10 scopes; reaction times, temperature of reaction was preferably selected within the scope of 4 ~ 40 DEG C preferably within the scope of 0.1 ~ 24 hour.React the compound 2 generating by this kind of enzyme, can separate, purify from reaction solution by general method.
In addition, (in vivo) in vivo, by allowing the present invention's CPC acyltransferase (acylase) that makes a variation contact with described compound 1, manufactures described compound 2.In the bacterial strains such as the product cephalosporium chrysogenum with biosynthesis ability (Acremonium chrysogenum) of described compound 1, introducing is inserted with the present invention's CPC acyltransferase (acylase) that makes a variation and encrypts gene or have the recombinant expression vector of the derivative of same function sequence (expression vector) with it, under appropriate culture medium and condition, cultivate described form quality conversion (transformation) body, can change in form quality in (transformation) body, by allowing biosynthetic compound 1 and the present invention CPC acyltransferase (acylase) that makes a variation naturally contact, manufacture compound 2.
Invention effect
Variation CPC acyltransferase of the present invention (acylase), compared with wild-type CPC acyltransferase (acylase), increases by 500 to 26 times to the activity of CPC matrix, and the effect of only directly manufacturing 7-ACA by 1 stage with CPC is particularly remarkable.
Brief description of the drawings
Fig. 1 is the 1st stage enzymatic process and the 2nd stage enzymatic process schema of manufacturing 7ACA with CPC.
Fig. 2 is the present invention's CPC acyltransferase (acylase) manufacturing processed schematic diagram that makes a variation.
Embodiment
At this, the present invention is described in detail.
The following examples are example of the present invention, and interior receiving part of the present invention is limited to following embodiment.
< embodiment 1>
The manufacture of CPC acyltransferase (Cephalosporin C Acylase) and active detection
The manufacture of <1-1>pBC-GA plastid
Be to belong to GK16 series (Matsuda et.al. from pseudomonas (Pseudomonas) for the manufacture of the basi gene of variation CPC acyltransferase (acylase); J.Bacteriol.163:1222-1228,1985) GA (Glutaryl amidase:GA) gene (below referred to as " ga gene ").
PBC-GA plastid is that the DNA fragmentation of ga structure gene shown in sequence number 1 is inserted into the XbaI of pBC KS (+) carrier (Stratagene company, the U.S.) and NotI restriction enzyme (restriction enzyme) identification position manufacturing.
In order to introduce restriction enzyme (restriction enzyme) identification position and rrna (ribosome) combining site etc. to described structure gene, implement polymerase chain reaction (PCR).The composition proportion of PCR reaction is the GA-F treatment agent (primer of synthetic gaDNA, each 50pmol of 10ng; 13), Taq buffered soln (5mM KCl, 5mM Tris-HCl (pH8.3), 1.5mM MgCl2), 0.2mM dNTP mixed solution, 2.5 ExTaq of unit polysaccharases (Takara company, Japan) sequence number is 12) and GA-R treatment agent (sequence number:, final volume is adjusted to 100 μ l.In addition, PCR has used thermal cycler (Peltier Thermal Cycler PTC-200; MJ Research company, the U.S.), reaction conditions is at 95 DEG C, allows mixture carry out first sex change (pre-denaturation) reaction in 5 minutes, 95 DEG C of sex change (denaturation) reactions in 1 minute, under 58 DEG C of handling returns (annealing) in 30 seconds and 72 DEG C of conditions, carry out 90 seconds polymerization (polymerization) reaction, and repeatedly carry out after described reaction 25 times, under 72 DEG C of conditions, carry out the reaction of 10 minutes post polymerization (post-polymerization).After the approximately 2.1kb size PCR product obtaining by described PCR is cut off with restriction enzyme (restriction enzyme) XbaI and NotI, with equipment for purifying (QIAEX Gel ExtractionKit; Qiagen company, Germany) purify, this is used as inserting DNA.In addition, pBC KS (+) carrier DNA is cut off afterwards, used as carrier DNA using CIP dephosphorylation gained DNA fragmentation with restriction enzyme (restriction enzyme) XbaI and NotI.Described insertion DNA and carrier DNA, utilize ligase enzyme (ligase:Roche company, Germany) under 16 conditions, carry out after 12 ~ 16 hours ligation (ligation), utilize described ligation liquid, carried out the form quality conversion (transformation) of E.coli MC1061 bacterial strain with electroporation (electrophoration).Described bacterial strain is spread upon to concentration 25ug/mL containing the antibiotic LB agar of paraxin (chloramphenicol) (agar) substratum, in an evening of static cultivation under 30 DEG C of conditions, sorted each form quality conversion (transformation) body.From this form quality conversion (transformation) body separating plastid, determine to insert the base sequence of DNA, manufacture the pBC-GA of the ga structure gene that comprises base sequence shown in tool sequence number 1.
To utilize the production efficiency of producing CPC acyltransferase (acylase) containing pBC-GA plastid restructuring colibacillus in order detecting, to have manufactured the thick enzyme solution (crude enzyme solution) of CPC acyltransferase (acylase) with following method.Described colibacillus form quality conversion (transformation) body is inoculated into respectively to 3ml LB substratum (1% Bacto-tryptone containing 25ug/mL paraxin, 0.5%Yeast Extract, 0.5%NaCl), carry out 16 hours shaking culture with 30 DEG C, 200rpm condition.Afterwards nutrient solution 50uL is inoculated into after the new LB substratum of 50mL containing 25ug/mL paraxin, with 25 DEG C, 200rpm condition, carries out 48 hours shaking culture.This flask nutrient solution is carried out to centrifugation (4 DEG C, 8,000rpm, 10 minutes), reclaim after thalline, 0.1M potassiumphosphate (potassium phosphate; PH8.0), washing 2 times.After being suspended with the same buffered soln of 5mL with this thalline, under 4 DEG C of conditions, pulverize 10 minutes with ultrasonic grinder, with 4 DEG C, the centrifugation of 15,000rpm condition, after 20 minutes, is got upper strata liquid, has manufactured the thick enzyme solution of CPC acyltransferase (acylase).
<1-2>CPC acyltransferase (acylase) is active to be detected
In order to detect CPC acyltransferase (acylase) level of activity synthetic in described embodiment <1-1>; with the Piao's (Park) etc. of distortion method (Park etc.; Kor.J.Appl.Microbiol.Biotechnol.23:559-564; 1995), be implemented as follows.
CPC matrix (purity 90%; Ha Yao company, China), (pH8.0) after buffered soln, regulate pH as 8.0 taking 1N NaOH in 0.1M potassiumphosphate (potassium phosphate) with 20mg/mL solution, manufacture matrix solution.Add enzyme solution 20uL to this CPC matrix solution 20uL, under 37 DEG C of conditions, react after 5 minutes, add 50mM NaOH-20% Glacial acetic acid (glacial acetic acid; 1:2) solution 200uL, stops reaction.Carry out afterwards the upper strata liquid of centrifugation, recovery, be dissolved in 0.5% (w/v) PDAB (p-dimethylaminobenzaldehyde (dimethylaminobenzaldehyde) of methyl alcohol to this upper strata liquid 200uL, interpolation; Sigma, U.S.) 40uL, reaction be after 10 minutes, under 415 wavelength, detects absorbancy, with the quantitative curve comparison of reference material, carry out quantitatively.Here, 1 unit (unit) is defined as per minute and generates with CPC the enzyme amount of 1 micromole's (umole) 7-ACA.In addition, for the specific activity of CPC matrix, detect protein in enzyme solution with Bradford's (Bradford, M., Anal.Biochem.72:248-254,1976) method, afterwards to represent corresponding to the activity unit of 1mg protein.
By the thick enzyme solution of described method manufacture, detect CPC acyltransferase (acylase) activity.Its result, the production efficiency of producing CPC acyltransferase (acylase) by restructuring E.coli MC1061 (pBC-GA) bacterial strain is about 40 units/L.
< embodiment 2>
Utilize saturation mutation manufacture variation CPC acyltransferase (acylase)
<2-1> sorts GA variation residue according to known features information
In order to improve the CPC acyltransferase (acylase) (wild-type) of the described embodiment 1 very faint to the activity of CPC matrix, taking known 3 d structure model and hypothesis abrupt information as basis, to the processing that suddenlys change of GA reactive site.
For this reason; (the Fritz-Wolf et.al. such as Fritz-Wolf are preferentially examined; Protein Sci.11:92-103; 2002) 8 the each amino-acid residue of GA reactive site (Y149 β that propose; L24 β; Y33 β; Q50 β; R57 β, V70 β, Y153 β; F177 β); also examined golden Gl-7-ACA acyltransferase (acylase) the reactive site residue (Kim et.al., J.Biol.Chem.276:48376-48381,2001) from the unicellular genus of vacation CAD proposing that waits simultaneously.Its result, as the reactive site variation residue of wild-type GA, has selected 4 residues (F31 β, F58 β, Y153 β, F177 β).
The manufacture of <2-2>F58 β varient
Obtain in order to manufacture CPC acyltransferase (acylase) activity the variation CPC acyltransferase (acylase) increasing; to GA reactive site residue---F31 β; F58 β; Y153 β; F177 β residue; implement respectively to process with the saturation mutation (saturation mutagenesis) of 20 seed amino acid displacements, set up 4 kinds of mutation libraries.Concrete mutation library process of establishing is as follows.
Particularly, in order to build F31 β residue saturation mutation storehouse, taking pBC-GA plastid as template, implement to use M13-R treatment agent (sequence number: 11) and F31b-R treatment agent (sequence number: PCR 15), reclaimed the PCR product of 0.7kb size.In addition, taking pBC-GA plastid as template, implement to use F31b-F treatment agent (sequence number: 14) and M13-F treatment agent (sequence number: PCR 10), reclaimed the big or small PCR product of 1.5kb.Mix the PCR product of described 0.7kb and the PCR product of 1.5kb size, in the situation that not adding treatment agent, implement PCR with described method, reclaimed two kinds of PCR products and be connected to the PCR product of the approximately 2.1kb size of.Taking the PCR product of the 2.1kb size that obtains by described method as template, implement to use T3 treatment agent (sequence number: 8) and T7 treatment agent (sequence number: PCR 9), amplification some varient DNA fragmentations of about 2.1kb size.
To the mutant gene obtaining by described PCR, it is the PCR product of 2.1kb, after cutting off with restriction enzyme (restriction enzyme) XbaI and NotI, purify with equipment for purifying (QIAEX Gel Extraction Kit), use as inserting DNA.To pBC KS (+) carrier DNA, cut off with restriction enzyme (restriction enzyme) XbaI and NotI, the DNA fragmentation that carries out dephosphorylation using CIP is used as carrier DNA.To described insertion DNA and carrier DNA, utilize T4 DNA ligase, under 16 DEG C of conditions, carry out after 16 ligation, utilize described ligation liquid, carried out the form quality conversion (transformation) of E.coli MC1061 bacterial strain with electroporation.Affiliated bacterial strain is spread upon on the LB nutrient agar containing 25ug/mL concentration chloramphenicol antibiotics, under 30 DEG C of conditions, leave standstill and cultivate an evening, built F31 β residue mutant library.
With the same method, build respectively F58 β, Y153 β, F177 β residue saturation mutation storehouse.Here, 16) and F58b-R treatment agent (sequence number: 17) build and used F58b-F treatment agent (sequence number: when F58 β residue saturation mutation storehouse, 18) and Y153b-R treatment agent (sequence number: 19), used F177b-F treatment agent (sequence number: 20) and F177b-R treatment agent (sequence number: 21) build and used Y153b-F treatment agent (sequence number: when Y153 β residue saturation mutation storehouse when in structure the F177 β residue saturation mutation storehouse.
From described saturation mutation storehouse, screen the variation CPC acyltransferase (acylase) that the reactivity of CPC matrix is increased with following method.
The E.coli MC1061 form quality of CPC acyltransferase (acylase) gene containing undergoing mutation conversion (transformation) body; be inoculated into and move respectively after 96 orifice plates (well plate) that are connected to containing the antibiotic LB liquid nutrient medium of paraxin 200uL; with 30 DEG C; 180rpm condition, carries out 60 ~ 70 hours shaking culture.From described orifice plate, get respectively afterwards 100uL nutrient solution, it is moved and receives new 96-orifice plate.Described nutrient solution and 100uL cell decomposing solution (are contained to 0.67mg/mL lytic enzyme (lysozyme), 1.3mM EDTA, (pH8.0) buffered soln of 0.1M potassiumphosphate (potassium phosphate) of 0.13%Triton X-100) mix, under 30 DEG C of conditions, place 2 hours.Add and be dissolved in (pH8.0) 2.5% (w/v) CPC matrix of buffered soln of 0.1M potassiumphosphate (potassium phosphate) afterwards, under 28 DEG C of conditions, carry out CPC matrix hydrolyzable reaction in 14 ~ 16 hours.Carry out after the hydrolyzable reaction of CPC matrix, reaction solution with 4,200rpm centrifugation 20 minutes, obtain upper strata liquid.The upper strata liquid 50uL obtaining is moved and receives new 96-orifice plate.Here, add reaction stop solution (acetic acid (acetic acid): 250mM NaOH, 2:1) 160uL, after allowing enzyme reaction stop, add developer (being dissolved in 0.5% (w/v) PDAB solution of methyl alcohol) 40uL, at room temperature place 10 minutes.Use afterwards 96-orifice plate reader to detect absorbancy under 415nm, screened the active varient increasing of CPC matrix.
With the same method to F31 β, F58 β, Y153 β, screen in 4 saturation mutation storehouses of F177 β residue.Its result, in the saturation mutation storehouse that F31 β, Y153 β, F177 β residue, does not find that CPC acyltransferase (acylase) activity obtains the varient increasing compared with wild-type GA at 3.But in F58 residue saturation mutation storehouse, select active a kind of the varient (S17) increasing of CPC acyltransferase (acylase).By the gene base sequence that determines this varient to be encrypted, confirm that the F58 β residue of S17 variant enzyme is replaced by α-amino-isovaleric acid (Valine).The aminoacid sequence of S17 variant enzyme is expressed as sequence number 28.
<2-3>F31 β/F58 β, F58 β/Y153 β, the manufacture of 2 varients of F58 β/F177 β
In order further to increase CPC acyltransferase (acylase) activity of described F58 β V varient; taking F58 β V varient gene as object; with the method identical with embodiment <1-3>; to F31 β; Y153 β, F177 β residue has built saturation mutation storehouse.
Afterwards, with the method identical with embodiment <2-2>, 3 saturation mutation storehouses have been screened.Its result, fails to filter out CPC acyltransferase (acylase) activity compared with F58 β V varient and obtains the varient increasing in F31 residue saturation mutation storehouse.But from Y153 β residue and F177 β residue saturation mutation storehouse, respectively filter out the active varient increasing of a kind of (B2 and A5) CPC acyltransferase (acylase).By the gene base sequence that determines these varients to encrypt, confirm the Y153 β residue of B2 variant enzyme by propylhomoserin (Threonin) displacement, the F177 β residue of A5 variant enzyme is replaced as leucine (leucine).That is, B2 variant enzyme is 2 varients of F58 β V/Y153 β T, and A5 is 2 varients of F58 β V/F177 β L.
The manufacture of 3 varients of <2-4>F58 β/Y153 β/F177 β
From described embodiment <2-3>, can find out to F58 β V varient and introduce Y153 β T or F177 β L, contribute to improve CPC acyltransferase (acylase) activity.And then, manufacture 3 varients of F58 β V/Y153 β T/F177 β L, develop the variation CPC acyltransferase (acylase) based on known features information.
Particularly, introduce F177 β L variation to 2 varients of F58 β V/Y153 β T, manufactured 3 varients of F58 β V/Y153 β T/F177 β L.Here, 22) and F177bL-R treatment agent (sequence number: 23) use F177bL-F treatment agent (sequence number:, with the method identical with embodiment <2-2>, manufacture form quality conversion (transformation) body containing 3 mutant genes.From this form quality conversion (transformation) body separating plastid, determine base sequence, confirm with this whether variation is normally accessed.Variation CPC acyltransferase (acylase) unnamed gene that 3 variant enzymes of F58 β V/Y153 β T/F177 β L by this manufacture are encrypted is R1.
< experimental example 3>
The manufacture of the variation CPC acyltransferase (acylase) based on random mutation
The manufacture of <3-1>I45 β/F58 β/Y153 β/F177 β and 4 varients of F58 β/Y153 β/F177 β/V382 β
In order further to improve F58 β V/Y153 β T/F177 β 3 varient CPC acyltransferases of L (acylase) activity of described embodiment <2-4>; 3 varient DNA of F58 β V/Y153 β T/F177 β L are used as template DNA; implement fallibility polymerase chain reaction; on random site, bring out point mutation, built corresponding mutation library.Concrete mutation library construction process is as follows.
8) and T7 treatment agent (sequence number:, final volume is adjusted to 100uL 9), the rTaq polysaccharase (Takara company, Japan) of 5mM KCl, 5mM Tris-HCl (pH8.3), 3.5mM MgCl2, the dATP of 0.025mM MnCl2, each 0.2mM and the dCTP of dGTP, each 1mM and dTTP, 5 units forms fallibility polymerase chain reaction PCR reaction solution is by the T3 treatment agent of F58 β V/Y153 β T/F177 β L 3 varient DNA, each 50pmol as template DNA 5ng (sequence number:.PCR has used thermal cycler (Peltier Thermal Cycler), reaction conditions is at 95 DEG C, allows mixture carry out first sex change (pre-denaturation) reaction in 3 minutes, 95 DEG C of sex change (denaturation) reactions in 1 minute, under 58 DEG C of handling returns (annealing) in 30 seconds and 72 DEG C of conditions, carry out 90 seconds polymerization (polymerization) reaction, and repeatedly carry out after described reaction 25 times, under 72 DEG C of conditions, carry out the reaction of 10 minutes post polymerization (post-polymerization).
The mutant gene obtaining by described fallibility polymerase chain reaction, about 2.1kb size PCR product with restriction enzyme (restriction enzyme) XbaI and NotI cut off afterwards, with equipment for purifying (QIAEX Gel Extraction Kit; Qiagen company, Germany) purify, this is used as inserting DNA, and pBC-GA plastid is cut off to the big or small DNA fragmentation of the 3.4kb being reclaimed using restriction enzyme (restriction enzyme) XbaI and NotI use as carrier DNA.Described insertion DNA and carrier DNA, utilize ligase enzyme (ligase) under 16 DEG C of conditions, carry out after 16 hours ligation (ligation), utilize described ligation liquid, carried out the form quality conversion (transformation) of E.coli MC1061 bacterial strain with electroporation (electrophoration).Described bacterial strain is spread upon to concentration 25ug/mL containing the antibiotic LB agar of paraxin (chloramphenicol) (agar) substratum, in an evening of static cultivation under 30 DEG C of conditions, built random mutation storehouse.
Screen from described random mutation storehouse with the method for embodiment <2-2> the variation CPC acyltransferase (acylase) that the activity of CPC matrix is increased.Its result, has filtered out in approximately 25000 kinds of varients compared with 3 varients of F58 β V/Y153 β T/F177 β L, active varient 2 points (M23 and M27) that increase of CPC.By the gene base sequence that determines these varients to encrypt, the I45 β residue that confirms M23 variant enzyme is replaced by methionine(Met), and the V382 β residue of M27 variant enzyme is replaced by Isoleucine.
The manufacture of 5 varients of <3-2>I45 β/F58 β/Y153 β/F177 β/V382 β
The result of described <3-1> shows, while introducing the sudden change of I45 β M or V382 β I to 3 varients of F58 β V/Y153 β T/F177 β L, CPC acyltransferase (acylase) is active can be increased.In order further to improve CPC acyltransferase (acylase) activity, 2 saturation mutation storehouses taking 3 varients of F58 β V/Y153 β T/F177 β L as basic I45 β residue and V382 β residue are built.Concrete mutation library building process is as follows.
In order to build I45 β residue and 2 saturation mutation storehouses of V382 β residue, using 3 varient DNA of F58 β V/Y153 β T/F177 β L as template, 11) and I45b-R treatment agent (sequence number: PCR 25) implement to use M13-R treatment agent (sequence number:, reclaim the PCR product of 0.7kb size, and using 3 varient DNA of F58 β V/Y153 β T/F177 β L as template, 24) and V382b-R treatment agent (sequence number: PCR 27) implement to use I45b-F treatment agent (sequence number:, reclaim 1.0kb size PCR product, simultaneously using 3 varient DNA of F58 β V/Y153 β T/F177 β L as template, 26) and M13-F treatment agent (sequence number: PCR 10) implement to use V382b-F treatment agent (sequence number:, reclaim the PCR product of 0.5kb size.Mix the PCR product of the PCR product of described 0.7kb and the PCR product of 1.0kb and 0.5kb size, in the situation that not adding treatment agent, implement PCR, reclaimed three kinds of PCR products and connect into the PCR of the approximately 2.1kb size of.The 2.1kb size PCR product obtaining using this as template, implement to use T3 treatment agent (sequence number: 8) and T7 treatment agent (sequence number: PCR 9), amplification 5 varient DNA fragmentations of about 2.1kb size.The 2.1kb size PCR product obtaining with this, be inserted in pBC KS (+) carrier DNA with the method identical with embodiment <2-2>, implement the form quality conversion (transformation) of E.coli MC1061 bacterial strain, manufactured I45 β residue and 2 saturated mutant libraries of V382 β residue.
From described 2 saturation mutation storehouses; with the method for embodiment <2-2>, screen the variation CPC acyltransferase (acylase) increasing compared with M27 variant enzyme (4 varients of F58 β V/Y153 β T/F177 β L/V382 β I), to the activity of CPC matrix.Result; filter out active 5 kinds of 5 varients (the I45 β V/F58 β V/Y153 β T/F177 β L/V382 β L increasing of CPC acyltransferase (acylase) compared with M27 variant enzyme; I45 β A/F58 β V/Y153 β T/F177 β L/V382 β I; I45 β C/F58 β V/Y153 β T/F177 β L/V382 β L; I45 β M/F58 β V/Y153 β T/F177 β L/V382 β L, I45 β L/F58 β V/Y153 β T/F177 β L/V382 β I).
< embodiment 4>
The activity of variation CPC acyltransferase (acylase) detects
To variation CPC acyltransferase (acylase) level of activity of described embodiment 2 to embodiment 3, detect with the method identical with embodiment <1-2>.Shown in its result following [table 2].Five kinds of 5 varients all show close activity.Wherein the activity of PM2 varient is the highest, therefore, as representative, has detected PM2.The numerical value of wild-type enzyme regards 1 as, and the activity of variant enzyme represents with the multiple of relative wild-type enzyme activity.
[table 2]
Variation residue and the comparison of CPC acyltransferase (acylase) level of activity of variation CPC acyltransferase (acylase)
| Variant enzyme | Variation residue | Relative reactivity (doubly) to CPC matrix |
| GA | Wild-type | 1.0 |
| S17 | F58V | 5.3 |
| A5 | F58V/F177L | 9.8 |
| B2 | F58V/Y153T | 14.5 |
| R1 | F58V/Y153T/F177L | 16.8 |
| M23 | I45M/F58V/Y153T/F177L | 18.8 |
| M27 | F58V/Y153T/F177L/V382I | 20.3 |
| PM2 | I45V/F58V/Y153T/F177L/V382L | 25.3 |
Form quality conversion (transformation) becomes E.coli MC1061 bacterial strain called after intestinal bacteria (Escherichia coli) MC1061/pBC-PM2 containing the pBC-PM2 plastid of the gene (sequence number 5) that the PM2 variant enzyme in described variant enzyme is encrypted, and has carried out registering (registration number: KCTC12344BP) at 2012 12 approximately 30 days to Korea Institute of Bioengineering gene library.
Industry is utilized feasibility
Variation CPC acyltransferase of the present invention (acylase), compared with CPC acyltransferase (acylase), improves 5 times to 26 times to the activity of CPC matrix, only directly manufactures 7-ACA by 1 stage with CPC, in its industry, utilizes feasibility very high.
Claims (14)
1. a variation CPC acyltransferase (acylase); it is characterized in that: the wild-type CPC(Cephalosporin C being formed by the α subunit (α-subunit) of aminoacid sequence shown in sequence number 3 and the β subunit of aminoacid sequence shown in sequence number 4) in acyltransferase (acylase) sequence, F58 amino acid is replaced by α-amino-isovaleric acid (Valine).
2. variation CPC acyltransferase according to claim 1 (acylase), is characterized in that:
Described variation CPC acyltransferase (acylase) appends and comprises the sudden change of selecting the group from being made up of Y153T, F177L and Y153T+F177L.
3. variation CPC acyltransferase according to claim 2 (acylase), is characterized in that:
Described variation CPC acyltransferase (acylase) appends and comprises that I45 or V382 amino acid are by the sudden change of other amino-acid substitutions.
4. variation CPC acyltransferase according to claim 3 (acylase), is characterized in that:
In described sudden change, I45 is by methionine(Met) (Methionine), α-amino-isovaleric acid Valine), a certain amino-acid substitution in L-Ala (Alanine), halfcystine (Cysteine), leucine (Leucine), V382 is by leucine (Leucine) or Isoleucine (Isoleucine) displacement.
5. variation CPC acyltransferase according to claim 4 (acylase), is characterized in that:
Described variation CPC acyltransferase (acylase) is made up of the α subunit (α-subunit) of aminoacid sequence shown in sequence number 6 and the β subunit of aminoacid sequence shown in sequence number 7.
6. a gene, is characterized in that: require a certain the variation CPC acyltransferase (acylase) of selecting in 1 to 5 to be encrypted to Accessory Right.
7. gene according to claim 6, is characterized in that:
Described gene represents with base sequence shown in sequence number 5.
8. a recombinant expression vector (expression vector), is characterized in that:
Comprise the 6th gene.
9. recombinant expression vector (expression vector) according to claim 8, is characterized in that:
Comprise the 7th gene.
10. a host cell, is characterized in that:
Changed (transformation) by the expression vector of the 8th (expression vector) by form quality.
11. 1 kinds of microorganisms, is characterized in that:
Changed (transformation) by the expression vector of the 8th (expression vector) by form quality.
12. microorganisms according to claim 11, is characterized in that:
Described microorganism is colon bacillus (Escherichia coli) MC1061/pBC-PM2 (registration number: KCTC12344BP) that is changed (transformation) by the recombinant expression vector (expression vector) of the 9th by form quality.
13. 1 kinds of 7-ACA (7-Aminocephalosporanic acid) manufacture synthetics, is characterized in that:
A certain the described variation CPC acyltransferase (acylase) of selecting comprising from the 1st to the 5th.
Manufacture the method for following Chemical formula 2, it is characterized in that for 14. 1 kinds:
A certain the described variation CPC acyltransferase (acylase) of selecting allowing from the 1st to the 5th reacts with the compound or its salt of following Chemical formula 1, manufactures.
Chemical formula 1
Chemical formula 2
[in chemical formula above, R is acetoxyl group (OCOCH
3), hydroxyl (OH), hydrogen base (H).】
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Cited By (6)
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| CN108220276A (en) * | 2017-10-30 | 2018-06-29 | 南京朗恩生物科技有限公司 | A kind of Cephalosporin C acylase mutant and its application in 7-amino-cephalosporanic acid production |
| WO2018165881A1 (en) * | 2017-03-15 | 2018-09-20 | 上海星维生物技术有限公司 | Cephalosporin c acylase mutants and applications thereof |
| CN110214188A (en) * | 2016-08-26 | 2019-09-06 | 艾美科健株式会社 | The high concentration production of 7-amino-cephalosporanic acid recombinates the manufacturing method of cephalosporium acremonium bacterial strain and utilizes bacterial strain manufactured by its method |
| CN112662655A (en) * | 2020-12-29 | 2021-04-16 | 山东金城柯瑞化学有限公司 | Cephalosporin C acylase mutant and preparation method and application thereof |
| CN115175992A (en) * | 2019-09-18 | 2022-10-11 | 艾美科健株式会社 | Method for preparing recombinant Acremonium strains producing high concentrations of desethoxycephalosporin C for 7-ADCA production and strains prepared by the method |
| CN115851687A (en) * | 2021-12-24 | 2023-03-28 | 艾美科健株式会社 | Polypeptide with cephalosporin C acyltransferase activity and application thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1544620A (en) * | 2003-11-17 | 2004-11-10 | 清华大学 | Method for producing 7-amino cephalosporanic acid and its specific recombinase |
| WO2005014821A1 (en) * | 2003-08-11 | 2005-02-17 | Sandoz Ag | Cephalosporin c acylase mutant and method for preparing 7-aca using same |
| CN101240285A (en) * | 2008-03-19 | 2008-08-13 | 清华大学 | Cephalosporin C acrylase and its vector and application |
-
2013
- 2013-01-21 KR KR1020130006483A patent/KR101728906B1/en not_active Application Discontinuation
- 2013-02-07 CN CN201310049908.7A patent/CN103937764B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005014821A1 (en) * | 2003-08-11 | 2005-02-17 | Sandoz Ag | Cephalosporin c acylase mutant and method for preparing 7-aca using same |
| CN1836044A (en) * | 2003-08-11 | 2006-09-20 | 桑多斯股份公司 | Cephalosporin c(CPC) acylase mutant having improved reactivity at cpc substrate and improved resistance to 7-aca productand method for preparing 7-aca using same |
| CN1544620A (en) * | 2003-11-17 | 2004-11-10 | 清华大学 | Method for producing 7-amino cephalosporanic acid and its specific recombinase |
| CN101240285A (en) * | 2008-03-19 | 2008-08-13 | 清华大学 | Cephalosporin C acrylase and its vector and application |
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| CN112662655A (en) * | 2020-12-29 | 2021-04-16 | 山东金城柯瑞化学有限公司 | Cephalosporin C acylase mutant and preparation method and application thereof |
| CN115851687A (en) * | 2021-12-24 | 2023-03-28 | 艾美科健株式会社 | Polypeptide with cephalosporin C acyltransferase activity and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103937764B (en) | 2017-06-06 |
| KR20140094150A (en) | 2014-07-30 |
| KR101728906B1 (en) | 2017-04-20 |
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