CN104120120B - Immobilization recombinant penicillin G acylase and application thereof - Google Patents
Immobilization recombinant penicillin G acylase and application thereof Download PDFInfo
- Publication number
- CN104120120B CN104120120B CN201410301057.5A CN201410301057A CN104120120B CN 104120120 B CN104120120 B CN 104120120B CN 201410301057 A CN201410301057 A CN 201410301057A CN 104120120 B CN104120120 B CN 104120120B
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- Prior art keywords
- acylase
- penicillin
- substrate
- enzyme
- immobilization
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- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 230000000702 anti-platelet effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000003796 beauty Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003782 beta lactam antibiotic agent Substances 0.000 description 1
- 102000006635 beta-lactamase Human genes 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000007797 corrosion Effects 0.000 description 1
- 238000005260 corrosion Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 125000003700 epoxy group Chemical group 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 229910052738 indium Inorganic materials 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000015689 metaplastic ossification Effects 0.000 description 1
- 150000004702 methyl esters Chemical class 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 230000002572 peristaltic effect Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 150000003242 quaternary ammonium salts Chemical class 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- YRWWOAFMPXPHEJ-OFBPEYICSA-K sodium L-ascorbic acid 2-phosphate Chemical compound [Na+].[Na+].[Na+].OC[C@H](O)[C@H]1OC(=O)C(OP([O-])([O-])=O)=C1[O-] YRWWOAFMPXPHEJ-OFBPEYICSA-K 0.000 description 1
- 229940048058 sodium ascorbyl phosphate Drugs 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 230000000707 stereoselective effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000002132 β-lactam antibiotic Substances 0.000 description 1
- 229940124586 β-lactam antibiotics Drugs 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a kind of immobilized penicillin G acylase and preparation (S) 2 aryl amino acid and the application of cephalosporin analog antibiotic; S type substrate can be converted into S type product within the shorter time by the immobilization recombinant penicillin G acylase that the present invention builds; substrate tolerance is high, has the strictest S selectivity to substrate and analogue thereof；By adding cobalt ions and phenylacetic acid and the glycerine protective agent as enzyme active center; in avoiding immobilization process, successfully the recombinant penicillin G acylase multiple spot covalency of the present invention is fixed on epoxy resin carrier surface in the case of enzyme molecule serious inactivation; process for fixation is simple; raw material is cheap, is suitable for large-scale operation；May be used for synthesizing cephalosporin analog antibiotic with immobilization recombinant penicillin G acylase and (S) 2 aryl amino acid is prepared in fractionation, more than 50 batches can be used before being decreased obviously does not occurs in immobilized enzyme continuously.
Description
(1) technical field
The present invention relates to a kind of immobilised enzymes and application, particularly to a kind of immobilization restructuring preparing high stability of operation
The method of penicillin G acylase, and utilize immobilization recombinant penicillin G acylated enzyme catalysis prepare (S)-2-aryl-
Amino acid and the method for cephalosporin analog antibiotic.
(2) background technology
Non-natural (S)-2-aryl-amino acid is the medicine intermediate that a class is important, and (S)-phenylglycine is used for synthesizing lactagogue
Element analog, AIDS protease inhibitors and taxol.(S)-o-chlorobenzene glycine, its chemical entitled 2-amino-(2-
Chlorphenyl) acetic acid, molecular formula is C8H8NO2Cl, its most important purposes is exactly that synthesizing new antiplatelet coagulates
Synandrium thing chlorobenzene Gray (Clopidogrel).Clopidogrel is developed in 1986 by the Sanifi company of France the earliest
Coming, generally using form clinically is its sulfate, its trade name Plavix (Plavix).
(S) preparation method of-o-chlorobenzene glycine includes: 1) chemical resolution method, with camphorsulfonic acid as resolving agent, tears open
Dividing racemic o-chlorobenzene glycine, actual recovery is about 31% (US 5204469A)；Or first with methyl alcohol by neighbour
Chlorobenzene glycine methyl ester metaplasia becomes racemic O-chlorobenzene glycine methyl ester, finally utilizes tartaric acid to prepare as resolving agent
(S)-o-chlorobenzene glycine (US 5204469A, 1991；US 6812363B,2004；WO 2006003671A,2004).
It is the highest that chemical method prepares the existence of (S)-o-chlorobenzene glycine many shortcoming, such as theoretical yield, and split process needs high temperature
Operation and reaction time are very long etc., it is most important that (S)-o-chlorobenzene glycine optical purity that chemical method produces is poor
(e.e. < 98%), needs repeatedly to recrystallize the purity requirement that just can obtain medicine after synthetic product chlorine is than Gray,
The process of recrystallization considerably increases the production cost of medicine.2) biological enzyme, utilizes immobilized penicillin acylated enzyme
The specific selectivity of substrate S type monomer is catalyzed N-(R, S)-phenylacetyl o-chlorobenzene glycine in an aqueous medium prepare
(S)-o-chlorobenzene glycine (Fadnavis, et al.Tetrahedron:Asymmetry, 2008.19 (20): 2363-2366；CN
101864464A,2010).(S)-o-chlorobenzene glycine optical purity prepared by enzyme process is generally higher than 99.9%, with fixing
Change the shortcoming that enzyme can also avoid the limited access times of traditional chemical catalyst as catalyst.
Cephalosporins is the semi-synthetic class beta-lactam antibiotic that a big class is main, and they have toxicity
Some advantages such as low, has a broad antifungal spectrum, resistance to beta-lactamase.Various have the semi-synthetic anti-of new antibacterial activity and antimicrobial spectrum
Raw element can be closed by catalysis with various D-amino acids side chains in acid condition with parent nucleus such as 7-ADCA, 6-APA
Become.With these antibiotic of the enzymatic clarification of immobilization penicillin acylated enzyme catalysis, there is reaction condition gentleness, operating procedure
Simply, need not the advantages such as specific groups protection.The semi-synthetic class antibiotic product of the enzymatic clarification being in the news has
Amoxicillin, ampicillin, Cefaclor, Cefradine, cefalexin, cefadroxil etc..
In view of the above-mentioned stereoselectivity that substrate is had of PA ase, will have stereoselective mould
Element acylase is made immobilised enzymes and is catalyzed and synthesized (S)-o-chlorobenzene glycine and have essential industry using value.Tradition
Enzyme immobilizatio method include absorption method, investment, cross-linking method and covalent coupling method, several method is respectively arranged with pluses and minuses.
In industrial applications, in order to reduce the cost of immobilised enzymes, it is desirable to it is steady that the immobilised enzymes of preparation has good operation
Qualitative, repeated multiple times can use, therefore select the immobilised enzymes that covalent coupling method is prepared as being firmly combined with than its other party
For method, tool has great advantage.Epoxylite is the synthetic resin that epoxy radicals is contained on a kind of surface, such carrier surface
The group such as epoxy radicals and the amino of enzyme molecular surface, carboxyl can open loop covalent bond under very mild conditions, from
And enzyme molecule is fixed on carrier surface.The reaction condition of epoxy radicals and amino is gentle, in conjunction with after enzyme molecule typically will not
Destroyed, the immobilised enzymes having good stability can be obtained after suitably processing.Some synthetic resin carriers are longer
Cycle in there is the strongest resistance to microorganism and acid and alkali corrosion effect and stronger mechanical performance, by control synthesis technique
It is readily synthesized and there is granularity adjustable microspheroidal porous material, meet very much the carrier as industrialization enzyme preparation.Wherein
The most representational is exactly Eupergit C and two kinds of synthetic macromolecule epoxy resin of Sepbeads EC-EP.Janssen
Make with Eupergit C Deng (Janeeen, et al.Biotechnology and Bioengineering, 2002,78 (4): 425-432)
Fixing penicillin G acylase for carrier, every gram of carrier at most can fix 1300 units of Penicillin G acylases；Slowly
Hat pearl etc. (Xu Guanzhu. microorganism journal, 2001.41 (2): 204-208) by outer for bacillus megaterium born of the same parents PA ase
Being bonded directly on this carrier, be made for Immobilized Penicillin Acylase On Polymer Beads, its apparent activity is up to 1400U/g.
Being the potassium salt of penicillin of 8% for catalytic level, after using 20 batches continuously, vigor can preserve initial vigor
80%.The immobilization technology research of China is started late, through study for many years arbitrarily develop diversified carrier and
Method, but still do not work out the carrier that can match in excellence or beauty with external first-class fixation support, really can put into industry should
Few especially, main cause be the reagent that used of immobilization and carrier cost high, immobilization efficiency is low, stable
Property poor, additionally maintain secrecy for business, a lot of technical process details the most do not disclose, and therefore study efficient immobilization blue or green
Mycin acylase is significant.
(3) summary of the invention
It is an object of the present invention to provide a kind of method being fixed on carrier by penicillin G acylase (PGA).The present invention
A further object be to utilize prepared immobilization recombinant penicillin G acylase as catalyst, be used for preparing
(S) acid of-2-aryl-amino and the method for cephalosporin analog antibiotic.
The technical solution used in the present invention is:
The present invention relates to a kind of immobilization recombinant penicillin G acylase, described immobilised enzymes is prepared as follows:
(1) fermented for the recombination engineering bacteria containing recombinant penicillin G acylase encoding gene cultivation is obtained zymotic fluid,
Centrifugal, taking supernatant is enzyme source, adds the cobalt chloride of final concentration of 0.5-5.0mM, add pH=7.0 in enzyme source
Salting liquid (phosphate buffer of preferably 3.0M or 3.0M ammonium sulfate solution), mixing, form enzyme to be fixed
Liquid；The amino acid sequence of described recombinant penicillin G acylase is shown in SEQ ID NO.1；Described salting liquid and enzyme
The volume ratio in source is 1:1；(2) enzyme liquid to be fixed is mixed with carrier, at 25 DEG C, stir 6-24h, take out carrier and use
(volume of phosphate buffer is with energy to put into the phosphate buffer of the 100mM that pH is 9.5-10.5 after distilled water flushing
Enough submergence carriers are advisable) in, add phenylacetic acid and glycerine, be incubated 1-4 days at 25 DEG C, it is thus achieved that the carrier after insulation；
The volumetric usage of enzyme liquid to be fixed is calculated as 5～10mL/g (preferably 5mL/g) with carrier quality；Described phenylacetic acid adds
Final concentration of 100mM, described glycerine add final concentration of 200g/L；Described carrier is epoxy resin；(3)
Carrier after step (2) being incubated immerses in the glycine solution of pH8.5,1.0-3.0M, and 25 DEG C preserve 24h,
Last again with distilled water flushing, obtain immobilization recombinant penicillin G acylase.
The functional group that carrier surface of the present invention contains is epoxy radicals, and being preferably but not limited to described carrier is epoxy resinC (purchased from rom (Roehm) company of Germany), epoxy resin Spebeads EC-EP are (purchased from Japan
Mitsubishi Chemical Ind) or epoxy resin LX-1000EP (purchased from Xi'an Lanxiao Sci-Tech Co., Ltd.).
Phosphate and ammonium sulfate used in the immobilized method of penicillin G acylase of the present invention are to utilize
" the saltouing " of zymoprotein molecule is acted on and reduces enzyme molecule solubility in solution system by salting liquid, promotes that it is at carrier
The rate of adsorption on surface.Some known and tradition use precipitating reagents include quaternary ammonium salt, polyethylene glycol etc. and for controlling
The phosphate of pH processed can use, and it selects to be to select according to the enzyme of effect, and precipitation can not be to enzyme
Activity and stability have a negative impact.Ammonium sulfate low price and penicillin G acylase of the present invention " is saltoutd "
Act on best, present invention preferably uses ammonium sulfate.
In the immobilized method of penicillin G acylase of the present invention, ammonium sulfate consumption selects the enzyme and specific according to effect
Immobilization process selection.The ammonium sulfate of low concentration is less to the detrimental effect of effective object, but adsorption of immobilization enters
Journey is veryer long；Relatively high concentration of ammonium sulfate has deactivation in various degree to effective object, but can be big
Shorten greatly immobilization process.The ammonium sulfate solution of final concentration of 1.5M in the preferred enzyme liquid to be fixed of the present invention.
In the immobilized method of penicillin G acylase of the present invention, in penicillin G acylase solution, add cobalt chloride
(CoCl2) time, be in order to utilize some metal ions and enzyme molecule to combine after, the electric charge that can affect enzyme molecular surface exists
Distribution situation during a certain specific pH environment.Penicillin G acylase is its activated centre when its isoelectric point (pH=6.7)
Around having the amino acid carrying positive charge in a large number, electrostatic adsorption causes the amino acid being positioned at these regions can be preferential
With the carrier generation covalent reaction with faint negative potential, thus the activated centre of enzyme is caused to be deteriorated or activated centre
Amino acid around causes substrate close to activated centre, cannot finally affect the activity of immobilised enzymes after being reacted away.Add
Cobalt ions (the Co added2+) can avoid or weaken this rough sledding of appearance after combining with enzyme molecule to final fixing
Change the impact that enzyme is lived.
The immobilized method of penicillin G acylase of the present invention, by controlling immobilization process holding stage protection liquid
PH condition lures that the amino of enzyme molecular surface and the epoxy radicals of carrier surface exceed the link work of single covalent bond into
With, two subunits of α, β making penicillin G acylase link self by hydrogen bond are all fixed on carrier by multiple spot covalency
Surface, the immobilised enzymes finally given has the operational stability of excellence.
The present invention also provides for a kind of described immobilization recombinant penicillin G acylase in preparation (S)-2-aryl-amino acid
Application, described application is: with immobilization recombinant penicillin G acylase as catalyst, with N-phenylacetyl-(R, S)-2-
Aryl-amino acid is substrate, in the ammonia spirit that pH value is 8.0-11.0 (preferably 8.0), (excellent at 25-60 DEG C
Select 30 DEG C), under the conditions of 180rpm after reaction completely, it is thus achieved that containing the mixed liquor of (S)-2-aryl-amino acid, by mixed liquor
Isolated and purified, it is thus achieved that the acid of described (S)-2-aryl-amino；Described initial substrate concentration is 0.05-0.3M (preferably 0.1M),
The consumption of described catalyst is 0.01～0.2g/mL, and (preferred catalyst consumption is that every milliliter of reaction system adds 0.02g
Immobilised enzymes).
Further, the most described substrate is N-phenylacetyl-(R, S)-phenylglycine, N-phenylacetyl-(R, S)-neighbour's sweet ammonia of chlorobenzene
Acid, N-phenylacetyl-(R, S)-chlorobenzene glycine, N-phenylacetyl-(R, S)-p-chlorophenylglycine or N-phenylacetyl-(R, S)-
D-pHPG.
The present invention also provides for the application in preparing cefalexin of a kind of described immobilization recombinant penicillin G acylase, institute
The application stated is: with immobilization recombinant penicillin G acylase as catalyst, remove acetoxyl group cephalo with 7-amino 3-
Alkanoic acid (7-ADCA) and Phenylglycine methyl ester hydrochloride (PGME) are substrate, in pH value be 6.0-8.0 (preferably
6.0) in cushioning liquid, under the conditions of 15-30 DEG C of (preferably 20 DEG C), 180rpm after reaction completely, it is thus achieved that containing head
The mixed liquor of cefalexin, mixed liquor is isolated and purified, it is thus achieved that described cefalexin；Described substrate 7-amino 3-removes acetyl
Epoxide cephalosporanic acid (7-ADCA) initial concentration is 0.01-0.3M (preferably 0.03M), described substrate phenylglycine
Methyl ester hydrochloride (PGME) initial concentration is that the interpolation of 0.05-0.3M (preferably 0.06M) described catalyst is the denseest
Degree is 0.01～0.2g/mL (preferably 0.02g/mL).
The present invention also provides for the application in preparing cefadroxil of a kind of described immobilization recombinant penicillin G acylase,
Described application is: with immobilization recombinant penicillin G acylase as catalyst, remove acetoxyl group head with 7-amino 3-
Spore alkanoic acid (7-ADCA) and p-hydroxyphenylglycine methyl ester hydrochloride (HPGME) are substrate, are 6.0-8.0 in pH value
The cushioning liquid of (preferably 7.0), under the conditions of 15-30 DEG C of (preferably 20 DEG C), 180rpm after reaction completely, obtains
The mixed liquor of cefadroxil must be contained, mixed liquor is isolated and purified, it is thus achieved that described cefadroxil；Described substrate 7-
Amino 3-desacetoxycephalosporanic acid (7-ADCA) initial concentration is 0.01-0.3M (preferably 0.03M), described
Substrate p-hydroxyphenylglycine methyl ester hydrochloride (HPGME) initial concentration is 0.05-0.3M (preferably 0.06M)
The interpolation final concentration of 0.01～0.2g/mL (preferably 0.02g/mL) of described catalyst.
The present invention also provides for the application in preparing Cefaclor of a kind of described immobilization recombinant penicillin G acylase, institute
The application stated is: with immobilization recombinant penicillin G acylase as catalyst, with 7-amino-3-chloro-3-cephem-4-acid
(7-ACCA) and Phenylglycine methyl ester hydrochloride (PGME) be substrate, be 6.0-8.0 (preferably 8.0 in pH value
Ammoniacal liquor buffer solution) cushioning liquid in, under the conditions of 15-30 DEG C of (preferably 20 DEG C), 180rpm after reaction completely,
Obtain the mixed liquor containing Cefaclor, mixed liquor is isolated and purified, it is thus achieved that described Cefaclor；Described substrate 7-amino
-3-chloro-3-cephem-4-acid (7-ACCA) initial concentration is 0.01-0.3M (preferably 0.03M), and described substrate benzene is sweet
Propylhomoserin methyl ester hydrochloride (PGME) initial concentration is 0.05-0.3M (preferably 0.06M), the interpolation of described catalyst
Final concentration of 0.01～0.2g/mL (preferably 0.02g/mL).
The present invention also provides for the application in preparing Cefradine of a kind of described immobilization recombinant penicillin G acylase, institute
The application stated is: with immobilization recombinant penicillin G acylase as catalyst, remove acetoxyl group cephalo with 7-amino 3-
Alkanoic acid (7-ADCA) and 2,5-bis-hydrogen-based Phenylglycine methyl ester hydrochloride is substrate, is that 6.0-8.0 is (excellent in pH value
Select 8.0) cushioning liquid, under the conditions of 15-30 DEG C of (preferably 20 DEG C), 180rpm after reaction completely, it is thus achieved that contain
The mixed liquor of Cefradine, mixed liquor is isolated and purified, it is thus achieved that described Cefradine；Described substrate-amino 3-goes second
Acyloxy cephalosporanic acid initial concentration is 0.01-0.3M (preferably 0.03M), described substrate 2, the sweet ammonia of 5-bis-hydrogen-based benzene
Acid methyl ester hydrochloride salt initial concentration is that the interpolation of 0.05-0.3M (preferably 0.06M) described catalyst is final concentration of
0.01～0.2g/mL (preferably 0.02g/mL).
Recombinant penicillin G acylase of the present invention is by the bacillus subtilis (B. containing plasmid pPZW103-PGA
Subtilis) WB800 expresses extracellular enzyme.Host Strains B.subtilis WB800 feature has been missing from 8 kinds of albumen
The expressive host of enzyme, when PGA expresses at B.subtilis WB800, it is less to the degraded of PGA, so that
PGA progressively accumulates in zymotic fluid, and therefore the expression of PGA is greatly improved.The weight built by described encoding gene
Group genetic engineering bacterium, this engineering bacteria need not add derivant when cultivating i.e. can be at extracellular expression recombinant penicillin
G acylase.
The nucleotides sequence of the gene that the present invention encodes recombinant penicillin G acylase is classified as shown in SEQ ID NO.2.
The encoding gene of the present invention application in preparing recombinant penicillin G acylase, described application is: build
Containing the recombinant vector of described recombinant penicillin G acylase, described recombinant vector is converted (excellent to bacillus subtilis
Select bacillus subtilis (B.subtilis) WB800) in, obtain the thalline containing recombinant penicillin G acylase thin
Born of the same parents, this somatic cells need not add derivant and i.e. can be acylated at extracellular expression recombinant penicillin G when cultivating
Enzyme.
In the present invention, the recombination engineering bacteria fermented cultivation acquisition containing recombinant penicillin G acylase encoding gene is sent out
Ferment liquid obtains as follows:
(1) inclined-plane is cultivated: be seeded to by the genetic engineering bacterium containing recombinant penicillin G acylase encoding gene containing the denseest
Degree is the slant medium of 50 μ g/mL kanamycins, cultivates 12h for 37 DEG C, it is thus achieved that inclined-plane thalline；Slant medium is eventually
Concentration consists of: peptone 10g/L, dusty yeast 5g/L, NaCl10g/L, and agar 20g/L, pH are natural, molten
Agent is water, stands and make test tube slant culture medium after medium sterilization after adding final concentration of 50 μ g/mL kanamycins；
(2) seed culture: picking slant strains accesses the seed culture medium of the kanamycins containing final concentration 50 μ g/mL,
Liquid amount is 25%, 37 DEG C, 150rpm cultivate 10-12 hour, it is thus achieved that seed liquor；Seed culture medium final concentration group
Becoming: peptone 10g/L, dusty yeast 5g/L, NaCl10g/L, pH are natural, and solvent is water, medium sterilization
After add final concentration of 50 μ g/mL kanamycins；
(3) fermented and cultured: the by volume inoculum concentration of concentration 2%, accesses seed liquor equipped with 100mL containing final concentration
In the 500mL triangular flask of the fermentation medium of the kanamycins of 50 μ g/mL, 37 DEG C, 150rpm cultivate 28h,
Zymotic fluid is centrifuged, collects supernatant, i.e. obtain enzyme source；
Or seed liquor is directly inoculated 5L fermentation tank culture: seed liquor be inoculated into volumetric concentration 2% inoculum concentration
In the 5L fermentation tank of dress liquid 3L fermentation medium.Fermentation medium final concentration consists of: soluble starch 10g/L,
Peptone 12g/L, dusty yeast 3g/L, NaCl10g/L, pH is natural, and solvent is water.In fermentation tank operating process
In, rotating speed is 150rpm, and throughput is 0.5vvm, and temperature keeps 37 DEG C, adds bubble enemy (0.03%, v/v)
Prevent foam from producing.After cultivating 26h under these conditions, zymotic fluid centrifugal (10000rpm, 10min) is collected supernatant
Liquid, is the fermented cultivation of recombination engineering bacteria containing recombinant penicillin G acylase encoding gene of the present invention
The zymotic fluid obtained ,-4 DEG C save backup.
N-phenylacetyl of the present invention-(R, S)-2-aryl-amino acid is prepared as follows:
(1) weigh respectively 0.1mol (R, S)-phenylglycine, (R, S)-o-chlorobenzene glycine, (R, S)-chlorobenzene glycine,
(R, S)-p-chlorophenylglycine, (R, S)-D-pHPG are put in the there-necked flask of 250mL, are separately added into 100
The mL4M NaOH aqueous solution, under condition of ice bath, stirring makes it be completely dissolved, and treats that temperature is cooled to about 0 DEG C,
Being slowly added dropwise 14.56mL (0.11mol) phenyllacetyl chloride, drip and finish, under room temperature, stirring reaction is overnight；
(2) for (R, S)-p-chlorophenylglycine, the reactant liquor of (R, S)-D-pHPG, suction filtration is directly carried out,
Respectively obtain white powdery solids and grey powder solid；For (R, S)-phenylglycine, (R, S)-adjacent sweet ammonia of chlorobenzene
Acid, the reactant liquor of (R, S)-chlorobenzene glycine, extract with dichloromethane, collects water layer and is placed in ice bath, uses 6M
Aqueous hydrochloric acid solution regulation pH1～2, separate out pulverulent solids under stirring, suction filtration obtains white powdery solids；
(3) suction filtration is obtained solid at 50 DEG C of dried substrate N-phenylacetyl-(R, S)-phenylglycine, N-phenylacetyls
-(R, S)-o-chlorobenzene glycine, N-phenylacetyl-(R, S)-chlorobenzene glycine, N-phenylacetyl-(R, S)-p-chlorophenylglycine,
N-phenylacetyl-(R, S)-D-pHPG.
In the present invention, N-phenylacetyl-(R, S)-2-aryl-amino acid and enzymolysis product thereof use high performance liquid chromatograph
(Dionex UltiMate3000, USA) detects.Testing conditions: chromatographic column is anti-phase chiral column Chirobiotic
R (4.6mm × 250mm, 5 μm, Sigma, USA), flowing is 0.5% acetic acidacetonitrile (20:80, V/V) mutually, stream
Speed is 1mL/min, and detection wavelength is 220nm, sample size: 3 μ L；Column temperature: 30 DEG C.
The optical purity of substrate and product is evaluated by calculating enantiomeric excess value (e.e.), formula:
E.e.=[cS-cR]/[cS+cR] × 100%, wherein, cSAnd cRFor product S type and the concentration of R type isomers.
Cefalexin in the present invention, cefadroxil, Cefaclor, Cefradine, 7-ADCA, 7-ACCA
Liquid phase detection method: chromatographic column: Hypersil ODS2-C18Post (4.6mm × 250mm, 5 μm, Elite, China)；
Flowing phase: methyl alcohol: phosphate buffer (10mM pH7.0)=30:70 (V/V)；Testing conditions: flow velocity: 0.7
mL/min；Wavelength: 214nm；Sample size: 10 μ L；Column temperature: 40 DEG C.
Compared with prior art, the beneficial effects are mainly as follows:
The main production process of current preparation (S)-2-aryl-amino acid and analogue thereof remains chemical method.Mould
Element G acylase utilizes it that High level of stereoselectivity selectivity of the acyl side-chain containing phenyl ring can be prepared high optical voidness
(S)-2-aryl-amino acid of degree.Enzymatic Resolution has catalytic efficiency height, stereoselectivity is strong, reaction condition is gentle
Feature, and PA ase wide material sources, fermentation costs is low, is to prepare the acid of high chiral purity (S)-2-aryl-amino
Ideal catalyst.The immobilization recombinant penicillin G acylase that the present invention builds can be by S within the shorter time
Type substrate is converted into S type product, and substrate tolerance is high, has the strictest S to select substrate and analogue thereof
Selecting property.Furthermore it is also possible to be used for producing the cephalo-types such as cefalexin, cefadroxil, Cefaclor, Cefradine
Antibiotic.
The present invention, by adding cobalt ions and phenylacetic acid and glycerine as the protective agent of enzyme active center, is avoiding immobilization
During successfully the recombinant penicillin G acylase multiple spot covalency of the present invention is fixed in the case of enzyme molecule serious inactivation
On epoxy resin carrier surface.Process for fixation is simple, and raw material is cheap, is suitable for large-scale operation.It is greatly improved
The enzyme access times as catalyst, can use 50 before being decreased obviously does not occurs in immobilized enzyme continuously
More than batch.
(4) accompanying drawing explanation
Fig. 1 is that recombinant bacterium B.subtilis WB800/pPZW103-PGA fermentation centrifuged supernatant SDS-PAGE is analyzed
As a result, (swimming lane 1: standard protein；Swimming lane 2:B.subtilis WB800 fermented liquid centrifuged supernatant；Swimming lane 3:
Restructuring B.subtilis WB800/pPZW103-PGA fermented liquid centrifuged supernatant；The α subunit of α-PGA albumen；
The α subunit of β-PGA albumen).
Fig. 2 difference concentration of cobalt ions affects column diagram to immobilization result.
Fig. 3 is immobilization recombinant penicillin G acylase operational stability curve map in packed bed.
(5) detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This:
The structure of embodiment 1 genetic engineering bacterium Bacillus subtilis WB800/pPZW103-PGA
1.1 recombinant penicillin G acylase encoding genes
Synthesis derive from B.megaterium CA4098 penicillin G acylase (PGA) (GenBank:
Gene order AF161313.1) (synthetic method bibliography: Nucleic.Acids.Res.2005,33,
W521-W525), total length is 2046bp (shown in SEQ ID NO.2), encodes 802 amino acid, Qi Zhongbian
Code 26 amino acid residues of signal peptide, 208 amino acid residues of coding for alpha subunit, 31 amino acid of encoded interval peptide
Residue, coding 537 amino acid residues of β subunit (shown in SEQ ID NO.1).The gene of synthesis is connected into PES
Plasmid (offer of Shanghai Xu Guan biotechnology Development Co., Ltd), it is thus achieved that recombinant plasmid PES-PGA, converts to clone
In host e. coli JM109 (offer of Beijing Tian Gen biochemical technology company), it is thus achieved that recombinant bacterium JM109/PES-PGA.
This experiment purpose is that PGA gene is connected to pPZW103 plasmid, converts to bacillus subtilis WB800, obtains
The recombinant bacterium Bacillus subtilis WB800/pPZW103-PGA of penicillin G acylase must be expressed.Concrete operations
Journey is as follows:
1.2 be digested
Cultivate and carry the recombinant bacterium of plasmid, the plasmid PES-PGA corresponding with plasmid extraction kit extracting and
pPZW103.With restriction endonuclease sma I and BamH1 double digestion PES-PGA and pPZWl03 plasmid respectively.
It is digested system composition and is shown in Table 1:
Table 1 endonuclease reaction system
37 DEG C of water-bath 6h, 65 DEG C of water-bath fire extinguishing 15min, terminate endonuclease reaction.
The connection of 1.3 genes
Use glue to reclaim kit (pursuing progress Bioisystech Co., Ltd purchased from love) and be separately recovered the base of plasmid PGA mesh
Because of fragment and the digestion products of plasmid pPZW103, it is thus achieved that glue reclaims product.Plasmid vector pPZW103 is digested product
Thing and purpose PGA genetic fragment are attached, and linked system composition is shown in Table 2.
Table 2 coupled reaction system
16 DEG C connect 16h, 65 DEG C of water-bath 15min and terminate reaction, and-20 DEG C of storages are standby.
Prepared by 1.4 host's bacillus subtilis bacterium competences
The solution that need to prepare:
MgCl2Mother liquor: 2.033g MgCl2With the MgCl that 100mL deionized water is configured to 100mM2The aqueous solution.
HEPES (4-HEPES) mother liquor: 2.383g HEPES and 100mL deionized water are configured to 100
The HEPES mother liquor of mM.
The preparation of SHMG: the MgCl of sucrose 85.575g, 100mM2The HEPES of mother liquor 5mL, 100mM
Mother liquor 10mL, glycerine 100mL, deionized water is settled to 1000mL.
(1) take out bacillus subtilis (B.subtilis) WB800 from-80 DEG C, be inoculated on LB flat board, 37 DEG C of trainings
Support 20h, it is thus achieved that thalline inclined-plane.LB flat board final concentration consists of: peptone 10g/L；Yeast extract 5g/L；
Sodium chloride 10g/L；Agar 20g/L；PH is natural, and solvent is water.
(2) from thalline inclined-plane picking list bacterium colony, being inoculated into 50mL LB fluid nutrient medium, 37 DEG C, 150rpm trains
Support overnight, it is thus achieved that seed liquor.LB fluid nutrient medium final concentration consists of: peptone 10g/L；Yeast extract 5g/L；
Sodium chloride 10g/L；Agar 20g/L；PH is natural, and solvent is water.
(3) taking 4mL seed liquor and be inoculated in 400mL LB fluid nutrient medium, 37 DEG C, 150rpm cultivates about 2-3h,
Until OD600Value reaches about 1.0, it is thus achieved that fermentation culture.
(4), in the Falcon pipe of the 400mL that fermentation culture proceeds to two 0 DEG C ice precooling 0.5h, 10 are placed on ice
Min, makes culture be cooled to 0 DEG C.
(5) 4 DEG C, 5000rpm, 10min, abandon supernatant, pipe be inverted 1min, make residual liquid flow to end.
(6) wash three times with 400mL, the SHMG freezing 0.5h in 0 DEG C of ice bath for every bottle, 5000rpm, 0 DEG C from
Heart 10min.
(7), after outwelling supernatant, add the SHMG to 2mL freezing 0.5h in 0 DEG C of ice bath, it is thus achieved that bacteria suspension, be withered
Grass bacillus WB800 competent cell suspension.
(8) take 100 μ L bacteria suspensions, transfer in precooling 0.5h in 0 DEG C of ice bath, aseptic 1.5mL centrifuge tube,
-80 DEG C long-term preservations.
1.5 electricity convert
With the electric revolving cup of 0.2mm, 2500V, 5ms are set, product pPZW103-PGA will be connected electroporated
Enter in the Bacillus subtilis WB800 competent cell prepared.The method utilizing bacterium colony PCR carries out screening sun
Sex clone, it is thus achieved that containing the Bacillus subtilis genes engineering bacteria (Bacillus of recombinant penicillin G acylase encoding gene
subtilisWB800/pPZW103-PGA).Recombined bacillus subtilis (the Bacillus that will obtain
SubtilisWB800/pPZW103-PGA) cultivating in LB fluid nutrient medium, in 37 DEG C, 180rpm trains
Support 24h.Supernatant, supernatant SDS-PAGE electrophoretic analysis is collected after nutrient solution 9000rpm is centrifuged 10min
As it is shown in figure 1, using bacillus subtilis (B.subtilis) WB800 as comparison, in figure, swimming lane 1 is protein
Mark, swimming lane 2 ferments centrifuged supernatant for bacillus subtilis (B.subtilis) WB800 host, and swimming lane 3 is
Recombinant bacterium B.subtilis WB800/pPZW103-PGA ferments centrifuged supernatant, occurs in that molecular size range is about 24
The α subunit of two protein bands of kDa and 61kDa, respectively penicillin G acylase and β subunit, meet pre-
The destination protein size of phase, it was demonstrated that recombined bacillus subtilis has been built up successfully.
The fermentation of embodiment 2 genetic engineering bacterium Bacillus subtilis WB800/pPZW103-PGA
Inclined-plane is cultivated: Bacillus subtilis genes engineering bacteria (Bacillus embodiment 1 built
SubtilisWB800/pPZW103-PGA) it is seeded to slant medium, cultivates 12h for 37 DEG C, it is thus achieved that inclined-plane thalline, 4
DEG C preserve.Slant medium final concentration consists of: peptone 10g/L；Dusty yeast 5g/L；NaCl10g/L；Fine jade
Fat 20g/L, pH are natural, and solvent is water.
Seed culture: the slant strains that picking 4 DEG C preserves accesses the seed of the kanamycins containing final concentration 50 μ g/mL
In culture medium (50mL seed liquor culture medium/250mL triangular flask), 37 DEG C, 150rpm incubated overnight (about cultivates
10-12 hour), it is thus achieved that seed liquor.Seed culture medium final concentration consists of: peptone 10g/L, dusty yeast 5g/L,
NaCl10g/L, pH are natural, and solvent is water.
5L fermentation tank culture: seed liquor is inoculated into dress liquid 3L fermentation medium with the inoculum concentration of volumetric concentration 2%
In 5L fermentation tank.Fermentation medium final concentration consists of: soluble starch 10g/L, peptone 12g/L, yeast
Powder 3g/L, NaCl10g/L, pH is natural, and solvent is water.In fermentation tank operating process, rotating speed is 150rpm,
Throughput is 0.5vvm, and temperature keeps 37 DEG C, adds bubble enemy (0.03%, v/v) and prevents foam from producing.Above-mentioned
Under the conditions of cultivate and zymotic fluid centrifugal (10000rpm, 10min) collected supernatant after 26h, be of the present invention
Fermented zymotic fluid (the thick enzyme cultivating acquisition of recombination engineering bacteria containing recombinant penicillin G acylase encoding gene
Liquid) ,-4 DEG C save backup.
During fermented and cultured, separated in time sampling and measuring biomass and pH change, then sample is centrifuged, take
Supernatant is surveyed enzyme and is lived, and enzyme is lived and is defined as: 30 DEG C, under the conditions of pH8.0, and catalysis N-phenylacetic acid-(R, S)-2-per minute
Aryl-amino acid generates the enzyme amount needed for (S)-2-aryl-amino acid of 1 μm ol, is 1 enzyme activity unit, uses U
Represent.Result shows: 5L fermentation tank culture gene engineering bacteria Bacillus subtilis WB800/pPZW103-PGA
In the crude enzyme liquid obtained, the vigor of recombinant penicillin G acylase is 15.65U/mL.
The mensuration of embodiment 3 recombinant penicillin G acylase activity
Take the crude enzyme liquid of the recombinant penicillin G acylase that 5L fermentation tank culture obtains in 0.2mL embodiment 2, with
The N-phenylacetyl of 0.1mmol-(R, S)-o-chlorobenzene glycine is substrate, with the ammonia spirit of pH=8.0 as reaction medium
Constitute the reaction system of 5mL, 30 DEG C, the shaking bath of 180rpm reacts 6min, take 1mL reactant liquor
It is placed in add and the EP pipe of the 20 μ L6M NaOH aqueous solution terminates reaction, in 12000rpm, the condition of 4 DEG C
Under be centrifuged 10min, take supernatant, detect with HPLC.
Enzyme is lived and is defined: 30 DEG C, and under the conditions of pH8.0, catalysis N-phenylacetic acid per minute-(R, S)-2-aryl-amino acid is raw
Become the enzyme amount needed for (S)-2-aryl-amino acid of 1 μm ol, be 1 enzyme activity unit, represent with U.
Under the conditions of said determination, genetic engineering bacterium Bacillus subtilis supported by 5L fermentation tank
The vigor of the recombinant penicillin G acylase crude extract that WB800/pPZW103-PGA obtains is 15.65U/mL.
Embodiment 4 recombinant penicillin G is acylated enzyme immobilizatio
First carry out preliminary experiment and filter out potential carrier, then the carrier selected is carried out detailed fixing condition
Research.
The selection of 4.1 carriers
The epoxy resin that different manufacturers produce is at water content, surface epoxide group density, carrier surface pore-size distribution
Show difference etc. parameter aspect, final result can be produced certain impact when carrying out enzyme immobilizatio.
Have selected the Sepabeads EC-EP of the Mitsubishi chemical company being widely used, Xi'an Lan Xiao scientific & technical corporation
LX-1000EP (C), tri-kinds of epoxy resin of ES-V-1 fixation support alternately of scientific & technical corporation of Nankai.
By embodiment 2 method for the fermented training of recombination engineering bacteria containing recombinant penicillin G acylase encoding gene
Supporting and obtain zymotic fluid, centrifugal, taking supernatant is enzyme source 50mL, adds the chlorine of final concentration of 0.5mM in enzyme source
Changing cobalt, add the phosphate buffer mixing of the 3.0M of 50mL, pH=7.0, forming buffer Final concentration is 1.5mM
Enzyme liquid to be fixed；Enzyme liquid to be fixed is mixed with carrier, stirs 12h at 25 DEG C, take out carrier distilled water flushing
After put in the phosphate buffer of the 100mM that pH is 10, add final concentration of 100mM phenylacetic acid and
The glycerine of 200g/L, is incubated 1 day at 25 DEG C, it is thus achieved that the carrier after insulation；Carrier immersion pH8.5 after insulation, 3.0
In the glycine solution of M, 25 DEG C preserve 24h, the most again with distilled water flushing, obtain immobilization recombinant penicillin
G acylase.Sampling and measuring immobilized enzyme.Enzyme activity determination method: use 0.1g immobilized enzyme replacement 0.2mL's is thick
Enzyme liquid, other operations are with embodiment 3.The result obtained is: with Sepabeads EC-EP, LX-1000EP (C),
ES-V-1 is immobilised enzymes enzyme respectively 27.5U/g, 9.8U/g, the 29.5U/g alive of carrier.
4.2 buffer salinities are on the restructuring immobilized impact of penicillin G acylase
The first step of enzyme immobilization is usually the process of enzyme molecule and a quick physical bond of carrier, this process
Mainly affected by hydrophobic adsorbent active force.Experiment promote enzyme at carrier surface hydrophobic adsorbent mainly by regulation
Suitable salt ionic concentration in immobilised enzymes liquid, utilizes the effect of saltouing of zymoprotein to promote the hydrophobic physics knot of enzyme and carrier
Close.
In order to select final suitably buffer salinity, the initial concentration preparing pH7.0 respectively is 0.1M, 0.2M,
The phosphate buffer of 1.0M, 2.0M, 3.0M.Take zymotic fluid that fermentation tank culture in 5mL embodiment 2 obtains from
The crude enzyme liquid that gains in depth of comprehension arrive, the buffer solution of the cobalt chloride and 5mL variable concentrations that add final concentration 0.5mM mixes system
Become buffer salt final concentration to be respectively the difference enzyme to be fixed liquid of 0.05M, 0.1M, 0.5M, 1.0M, 1.5M, put
Enter 50mL and convert in bottle,
Enzyme liquid to be fixed is mixed with 0.5g LX-1000EP (C) resin carrier, stirs 12h at 25 DEG C, take out carrier
With in the phosphate buffer putting into the 100mM that pH is 10 after distilled water flushing, add final concentration of 100mM
Phenylacetic acid and the glycerine of 200g/L, at 25 DEG C be incubated 1 day, it is thus achieved that the carrier after insulation；Carrier leaching after insulation
Entering in the glycine solution of pH8.5,3.0M, 25 DEG C preserve 24h, the most again with distilled water flushing, consolidate
Surely recombinant penicillin G acylase is changed.Take 0.1g immobilised enzymes mensuration enzyme to live, according to finally giving 0.5g immobilised enzymes
Calculate the total enzyme of immobilised enzymes to live and the enzyme rate of recovery alive.Immobilised enzymes enzyme activity determination method 0.1g immobilised enzymes replaces 0.2
ML crude enzyme liquid, other operations are with embodiment 3.Result is as shown in table 3, for carrier LX-1000EP (C), the suitableeest
Buffer salinity is the enzyme liquid to be fixed buffer salt containing final concentration of 1.5M.
Table 3 buffer salinity is on enzyme immobilized impact on carrier LX-1000EP (C)
4.3 different buffer salts are on the restructuring immobilized impact of penicillin G acylase
Prepare phosphate buffer and pH7.0,3.0M ammonium sulfate solution of pH7.0,3.0M respectively, use 0.1M
Ammoniacal liquor regulation pH value.Take the zymotic fluid that in 50mL embodiment 2, fermentation tank culture obtains and be centrifuged the crude enzyme liquid obtained,
Add final concentration 0.5mM CoCl2, water-soluble with the ammonium sulfate of the sodium phosphate buffer of 50mL and 50mL respectively
Put into 250mL tri-mouthfuls after liquid mixing to convert in bottle, be separately added into 10g LX-1000EP (C) resin, stir at 25 DEG C
12h, puts into after taking out carrier distilled water flushing in the phosphate buffer of the 100mM that pH is 10, adds end
Concentration is phenylacetic acid and the glycerine of 200g/L of 100mM, is incubated 1 day, it is thus achieved that the carrier after insulation at 25 DEG C；
Carrier after insulation immerses in the glycine solution of pH8.5,3.0M, and 25 DEG C preserve 24h, the most again with distillation
Water rinses, and obtains immobilization recombinant penicillin G acylase.Result: when salting liquid is ammonium sulfate, immobilised enzymes
Enzyme is lived and is reached 75U/g.When salting liquid is phosphate buffer, the enzyme of immobilised enzymes is lived and is reached 62U/g.
4.4 cobalt ions (Co2+) concentration impact on immobilized penicillin G acylase
Selecting the ammonium sulfate solution of 3.0M, pH7.0 as salting liquid, rotating speed is 180rpm, temperature is 25 DEG C
Under conditions of, the zymotic fluid that fermentation tank culture obtains in example 2 is centrifuged in the crude enzyme liquid 50ml obtained interpolation chlorine
Change cobalt, make cobalt ions final concentration in enzyme liquid be respectively 0mM, 0.5mM, 1.0mM, 2.5mM, 5.0mM,
10.0mM, after mixing stands 10min, adds 10g LX-1000EP (C) resin, stirs 12h, take at 25 DEG C
Put into after going out carrier distilled water flushing in the phosphate buffer of the 100mM that pH is 10, add final concentration of
The phenylacetic acid of 100mM and the glycerine of 200g/L, be incubated 1 day at 25 DEG C, it is thus achieved that the carrier after insulation；After insulation
Carrier immerses in the glycine solution of pH8.5,3.0M, and 25 DEG C preserve 24h, the most again with distilled water flushing,
Obtain immobilization recombinant penicillin G acylase.Sampling (0.1g) measures immobilized enzyme vigor, and enzyme activity determination method is with implementing
Example 3.Without CoCl2Time the enzyme of immobilised enzymes that obtains live and be defined as 100%.Result is as shown in Figure 2.Add not
After the cobalt ions of concentration, the enzyme work to immobilised enzymes typically has lifting effect, when containing final concentration of 0.5 in enzyme liquid
After the cobalt ions of mM, raising effect of living immobilised enzymes is the most obvious, and vigor improves about 13%.
The impact on immobilised enzymes stability of 4.5 temperature retention times
Selecting the ammonium sulfate solution of 3.0M, pH7.0 as salting liquid, rotating speed is 180rpm, temperature is 25 DEG C
Under conditions of, the zymotic fluid that fermentation tank culture obtains in example 2 is centrifuged in the crude enzyme liquid 50mL obtained interpolation chlorine
Change cobalt, make the final concentration of 0.5mM of cobalt ions in enzyme liquid, after mixing stands 10min, add 10g LX-1000EP (C)
Resin, stirs 12h at 25 DEG C, the phosphoric acid putting into the 100mM that pH is 10 after taking out carrier distilled water flushing delays
Rush in liquid, add the phenylacetic acid of final concentration of 100mM and the glycerine of 200g/L, be divided into 4 parts, 25 DEG C
Lower insulation 1 day respectively, 2 days, 3 days, 4 days, it is thus achieved that the carrier that temperature retention time is different；Carrier after insulation immerses
In the glycine solution of pH8.5,3.0M, 25 DEG C preserve 24h, the most again with distilled water flushing, are fixed
Change recombinant penicillin G acylase.Sampling (0.1g) measures immobilized enzyme vigor, and enzyme activity determination method is with embodiment 3.No
With temperature retention time, enzyme is lived impact not quite.Being incubated 1 day, 2 days, the enzyme of 3 days and 4 days was lived and is respectively as follows: 57.5U/g, and 60.3
U/g, 61.3U/g and 58.U/g.
The synthesis of embodiment 5 substrate N-phenylacetic acid-(R, S)-2-aryl-amino acid
The synthesis of 5.1N-phenylacetyl-(R, S)-phenylglycine
Weigh in the three neck round bottom flask that 0.1mol (R, S)-phenylglycine puts into 250mL, add 100mL4M
The NaOH aqueous solution, under 0 DEG C of condition of ice bath, stirring makes it be completely dissolved, and treats that temperature is cooled to about 0 DEG C, slow
Slow dropping phenyllacetyl chloride 14.56mL (0.11mol), drips and finishes, and under room temperature, stirring reaction is overnight.Extract with dichloromethane
Reactant liquor, collects water layer and is placed in ice bath, adjust pH1～2 with 6M aqueous hydrochloric acid solution, separate out white powder under stirring
Shape solid, suction filtration, 50 DEG C of dried product N-phenylacetyl-(R, S)-phenylglycine 26.36g.Product N-benzene second
The yield of acyl-(R, S)-phenylglycine is 94.05%, and purity is 98.35%.
The synthesis of 5.2N-phenylacetyl-(R, S)-o-chlorobenzene glycine, N-phenylacetyl-(R, S)-chlorobenzene glycine
(R, S)-phenylglycine (R, S)-o-chlorobenzene glycine or (R, S)-chlorobenzene glycine of same concentration are replaced, other
Operation is with embodiment 5 (5.1).The yield of product N-phenylacetyl-(R, S)-o-chlorobenzene glycine (28.35g) is 93.41%,
Purity is 99.36%.
The synthesis of 5.3N-phenylacetyl-(R, S)-p-chlorophenylglycine
Weigh in the three neck round bottom flask that 0.1mol (R, S)-p-chlorophenylglycine puts into 250mL, add 100mL4
The M NaOH aqueous solution, under 0 DEG C of condition of ice bath, stirring makes it be completely dissolved, and treats that temperature is cooled to about 0 DEG C,
Being slowly added dropwise phenyllacetyl chloride 14.56mL (0.11mol), drip and finish, under room temperature, stirring reaction is overnight.Reactant liquor is direct
Suction filtration, obtains white powdery solids.50 DEG C of dried product N-phenylacetyl-(R, S)-p-chlorophenylglycines 28.80
g.The yield of product N-phenylacetyl-(R, S)-p-chlorophenylglycine is 94.89%, and purity is 98.78%.
The synthesis of 5.4N-phenylacetyl-(R, S)-D-pHPG
Being replaced by (R, S)-p-chlorophenylglycine (R, S)-D-pHPG of same concentration, other operate same embodiment
5(5.3).The yield of product N-phenylacetyl-(R, S)-D-pHPG is 54.38%.
(S)-2-aryl is prepared in embodiment 6 recombinant penicillin G acylated enzyme catalysis N-phenylacetic acid-(R, S)-2-aryl-amino acid
-amino acid
Have the selectivity of height to the acyl side-chain containing phenyl ring according to recombinant penicillin G acylase, we have investigated this enzyme
To the phenylglycine of N-phenylacetyl, o-chlorobenzene glycine, p-chlorophenylglycine, a chlorobenzene glycine and to hydroxyl
The substrate specificity of phenylglycine.
Respectively with embodiment 5 prepare N-phenylacetyl-(R, S)-phenylglycine, N-phenylacetyl-(R, S)-o-chlorobenzene glycine,
N-phenylacetyl-(R, S)-chlorobenzene glycine, N-phenylacetyl-(R, S)-p-chlorophenylglycine, N-phenylacetyl-(R, S)-to hydroxyl
Phenylglycine is substrate, and in reaction system, the initial concentration of substrate is 0.06M, by the ammoniacal liquor of substrate Yu pH=10.0
Solution mixes, and is separately added into the crude enzyme liquid 0.2mL that embodiment 2 method obtains, and constitutes the reaction system of 5mL,
40 DEG C, the shaking bath of 180rpm reacts, take 1mL reactant liquor after reaction completely and be placed in and add 20 μ L6M
The EP pipe of the NaOH aqueous solution terminates reaction, at 12000rpm, centrifugal 10min under conditions of 4 DEG C, takes
Clear liquid, with HPLC detection substrate conversion ratio and product chiral purity.
Result is as shown in table 4, and N-phenylacetyl-p-chlorophenylglycine is converted needs by recombinant penicillin G acylase completely
Shortest time, by the longest for the time that N-phenylacetyl-o-chlorobenzene glycine converts needs completely.In addition recombinant penicillin
G acylated enzyme catalysis split each racemic substrate obtain product e.e. value all more than 99.9%, it was demonstrated that this enzyme is to respectively
The selection rate of substrate is all fine, has the stereocpecificity of height to five kinds of substrates, for strict S selectivity.
The table 4 recombinant penicillin G acylase split result to different substrates
N-phenylacetyl-(R, S)-o-chlorobenzene glycine system of embodiment 7 recombinant penicillin G acylated enzyme catalysis variable concentrations
Standby (S)-o-chlorobenzene glycine
Preparing N-phenylacetyl-(R, S)-o-chlorobenzene glycine for substrate with embodiment 5, in reaction system, substrate is the denseest
Degree is respectively 0.02M, 0.05M, 0.1M, 0.2M, 0.3M, 0.5M, by the ammoniacal liquor of substrate Yu pH=10.0
Solution mixes, and is separately added in embodiment 2 method the crude enzyme liquid 4mL obtained, and constitutes the reaction system of 100mL,
40 DEG C, the shaking bath of 180rpm reacts, timing takes 1mL reactant liquor as adding 20 μ L6M
The EP pipe of the NaOH aqueous solution terminates reaction, at 12000rpm, centrifugal 10min under conditions of 4 DEG C, takes
Clear liquid, detects production concentration with HPLC.
When concentration of substrate is 0.02M, 0.05M, 0.1M, 0.2M, S type substrate can completely convert and turn completely
Time required for change is respectively 15min, 30min, 60min, 360min, when concentration of substrate be 0.3M, 0.5
During M, due to high concentration substrate, to cause S type substrate to convert time of needs completely the inhibitory action that enzyme is lived longer.
Embodiment 8 immobilization recombinant penicillin G acylase hydrolyzing N-phenylacetyl-(R, S)-o-chlorobenzene glycine prepare (S)-
O-chlorobenzene glycine
The preparation of immobilised enzymes: (1) zymotic fluid that fermentation tank culture obtains in the embodiment 2 of 50mL is centrifugal to be obtained
Crude enzyme liquid in add the cobalt chloride (CoCl of final concentration of 0.5mM2), shake up, add pH value be 7.0,3.0M
Ammonium sulfate aqueous solution 50mL, make enzyme liquid 100mL to be fixed, make the end of ammonium sulfate in enzyme liquid to be fixed dense
Degree is 1.5M, obtains the enzyme mixation containing high salt concentration；(2) by the enzyme liquid to be fixed of 100mL with 10g's
LX-1000EP (C) carrier mixes, and stirs being fixed of 6-12h with electric mixer under the rotating speed of 180rpm,
The sodium ascorbyl phosphate being placed on the 100mM that 100mL, pH are 9.5-10.5 after taking out carrier distilled water flushing 3 times delays
Rush in liquid, under normal temperature (25 DEG C), be incubated 3 days, during insulation, add the phenylacetic acid and 200 of final concentration of 100mM
The glycerine of g/L is as protective agent；(3) carrier after taking-up insulation is in the glycine solution of pH8.5,3.0M,
Normal temperature (25 DEG C) preserves 24h, uses distilled water flushing 3 times the most again, obtains immobilization recombinant penicillin G and is acylated
Enzyme.
With 0.15g (0.5mmol) N-phenylacetyl-(R, S)-o-chlorobenzene glycine for substrate, it is the ammoniacal liquor of 8.0 with pH
Solution is reaction medium, is constituted 5mL reaction system with 0.1g immobilised enzymes for catalyst, at 30 DEG C, 180rpm
Water-bath in react, reaction 1h after with HPLC measure (method is with embodiment 3) S type product yield exceed
98%, e.e. value is more than 99.9%.
Embodiment 10 immobilization recombinant penicillin G acylase hydrolyzing N-phenylacetyl-(R, S)-phenylglycine prepares (S)-benzene
Glycine
Substrate is N-phenylacetyl-(R, S)-phenylglycine, and other operations are with embodiment 9.Measure S type after reaction 3h to produce
Thing conversion ratio is 80%, and e.e. value is more than 99.9%.
Prepared by embodiment 11 immobilization recombinant penicillin G acylase hydrolyzing N-phenylacetyl-(R, S)-p-chlorophenylglycine
(S)-p-chlorophenylglycine
Substrate is N-phenylacetyl-(R, S)-p-chlorophenylglycine, and other operations are with embodiment 9.S is measured after reaction 3h
Type product yield is 80%, and e.e. value is more than 99.9%.
Prepared by embodiment 12 immobilization recombinant penicillin G acylase hydrolyzing N-phenylacetyl-(R, S)-chlorobenzene glycine
(S)-chlorobenzene glycine
Substrate is N-phenylacetyl-(R, S)-chlorobenzene glycine, and other operations are with embodiment 9.S is measured after reaction 3h
Type product yield is 96%, and e.e. value is more than 99.9%.
Prepared by embodiment 13 immobilization recombinant penicillin G acylase hydrolyzing N-phenylacetyl-(R, S)-D-pHPG
(S)-D-pHPG
Substrate is N-phenylacetyl-(R, S)-D-pHPG, and other operations are with embodiment 9.S is measured after reaction 4h
Type product yield is 60%, and e.e. value is more than 99.9%.
Embodiment 14 immobilization recombinant penicillin G acylase prepares cefalexin
The immobilised enzymes (final concentration 10mg/mL) of 0.5g embodiment 8 preparation, the 7-amino 3-of 30mM remove acetyl
Epoxide cephalosporanic acid (7-ADCA), the Phenylglycine methyl ester hydrochloride (PGME) of 60mM, be placed on 50mL anti-
Answer in still and react.Controlling temperature is 20 DEG C, and the HCl utilizing pH-stat to add ammoniacal liquor or 1.0M controls pH
React 2h 6.0, under the conditions of 180rpm, it is thus achieved that the mixed liquor containing cefalexin, detected by liquid phase, it is thus achieved that 22mM
Cefalexin.
Embodiment 15 immobilization recombinant penicillin G acylase prepares cefadroxil
The immobilised enzymes (final concentration 10mg/mL) of 0.5g embodiment 8 preparation, the 7-amino 3-of 30mM remove acetyl
Epoxide cephalosporanic acid (7-ADCA), the p-hydroxyphenylglycine methyl ester hydrochloride (HPGME) of 60mM, be placed on
50mL reactor reacts.Controlling temperature is 20 DEG C, utilizes pH-stat to add the HCl of ammoniacal liquor or 1.0M
Control pH and react 110min 7.0, under the conditions of 180rpm, it is thus achieved that the mixed liquor containing cefadroxil, pass through liquid phase
Detection, it is thus achieved that 12mM cefadroxil.
Embodiment 16 immobilization recombinant penicillin G acylase prepares Cefaclor.
The immobilised enzymes (final concentration 10mg/mL) of 0.5g embodiment 8 preparation, the 7-amino-3-chloro-3-head of 30mM
Spore alkene-4-acid (7-ACCA), the Phenylglycine methyl ester hydrochloride (PGME) of 60mM, be placed on 50mL reaction
Still reacts.Controlling temperature is 20 DEG C, and the HCl utilizing pH-stat to add ammoniacal liquor or 1.0M controls pH and exists
8.0, react 80min under the conditions of 180rpm, it is thus achieved that the mixed liquor containing Cefaclor, detected by liquid phase, it is thus achieved that 27
MM Cefaclor.
Embodiment 17 immobilization recombinant penicillin G acylase prepares Cefradine.
The immobilised enzymes (final concentration 10mg/mL) of 0.5g embodiment 8 preparation, the 7-amino 3-of 30mM remove acetyl
Epoxide cephalosporanic acid (7-ADCA), the 2 of 60mM, 5-bis-hydrogen-based Phenylglycine methyl ester hydrochloride, be placed on 50mL
Reactor reacts.Controlling temperature is 20 DEG C, and the HCl utilizing pH-stat to add ammoniacal liquor or 1.0M controls pH
8.0, react 2h under the conditions of 180rpm, it is thus achieved that the mixed liquor containing Cefradine, detected by liquid phase, it is thus achieved that 27mM
Cefradine.
The operational stability of embodiment 18 immobilization recombinant penicillin G acylase
With N-phenylacetyl-(R, S)-o-chlorobenzene glycine for substrate, packed bed (internal diameter 1.0cm, high 10cm, outward
Band water-bath chuck；Accurate constant flow peristaltic pump BT300-2J: Baoding LanGe constant flow pump Co., Ltd) middle mensuration immobilization weight
The operational stability of group penicillin G acylase.Packed bed condition is: ratio of height to diameter is 8:1, adds 8g embodiment 8
In the immobilised enzymes that obtains, substrate solution flow velocity is 18mL/min (1.5 volume flow rates, SV), and substrate solution is
The N-phenylacetyl of the 100mM of 50mL, pH=8.0-(R, S)-o-chlorobenzene glycine aqueous solution.Packed bed and inflow are filled out
The substrate solution control temperature filling bed is 30 DEG C.Select 20min as a reaction batch, use after every batch reaction and steam
Packed bed is rinsed 20min by distilled water.
After successive reaction 50 batch, the conversion ratio of S type substrate stills remain in about 98%, and product (S)-neighbour's chlorobenzene is sweet
The e.e. value of propylhomoserin remains at the level (Fig. 3) more than 99%, demonstrates immobilized penicillin prepared by the present invention
The good operational stability that G acylase possesses.
The present invention is not specifically limited text, and the present invention can be in the range of claims be summarized
Spirit according to the present invention makes various change.These change the most within the scope of the present invention.
Claims (9)
1. an immobilization recombinant penicillin G acylase, it is characterised in that described immobilised enzymes is prepared as follows: (1)
Fermented for recombination engineering bacteria containing the recombinant penicillin G acylase encoding gene zymotic fluid obtained of cultivating is centrifuged, takes
Supernatant is enzyme source, adds the cobalt chloride of final concentration 0.5-5.0mM, add the salting liquid of pH=7.0 in enzyme source, mixed
Even, form enzyme liquid to be fixed；The amino acid sequence of described recombinant penicillin G acylase is shown in SEQ ID NO.1；Institute
The volume ratio stating salting liquid and enzyme source is 1:1；Described salting liquid be the phosphate buffer of 3.0M or 3.0M ammonium sulfate water-soluble
Liquid；(2) enzyme liquid to be fixed is mixed with carrier, stir 6-24h at 25 DEG C, put into after taking out carrier distilled water flushing
PH is in the phosphate buffer of the 100mM of 9.5-10.5, adds phenylacetic acid and glycerine, is incubated 1-4 days at 25 DEG C,
Obtain the carrier after insulation；The volumetric usage of described enzyme liquid to be fixed is calculated as 5～10ml/g with carrier quality；Described phenylacetic acid adds
The final concentration of 100mM entered, the final concentration of 200g/L that described glycerine adds；Described carrier is epoxy resin；(3) will
Carrier after step (2) insulation immerses in the glycine solution of pH8.5,1.0-3.0M, and 25 DEG C preserve 24h,
After again with distilled water flushing, obtain immobilization recombinant penicillin G acylase.
2. immobilization recombinant penicillin G acylase as claimed in claim 1, it is characterised in that described carrier is epoxy resinC, epoxy resin Sepabeads EC-EP or epoxy resin LX-1000EP.
3. immobilization recombinant penicillin G acylase as claimed in claim 1, it is characterised in that described enzyme source is as follows
Obtain:
(1) inclined-plane is cultivated: will be seeded to containing final concentration containing the genetic engineering bacterium of recombinant penicillin G acylase encoding gene
The slant medium of 50 μ g/mL kanamycins, cultivates 12h for 37 DEG C, it is thus achieved that inclined-plane thalline；Slant medium final concentration group
Becoming: peptone 10g/L, dusty yeast 5g/L, NaCl10g/L, agar 20g/L, pH are natural, and solvent is water；
(2) seed culture: picking slant strains accesses the seed culture medium of the kanamycins containing final concentration 50 μ g/mL, dress
Liquid measure is 25%, 37 DEG C, 150rpm cultivate 10-12 hour, it is thus achieved that seed liquor；Seed culture medium final concentration consists of:
Peptone 10g/L, dusty yeast 5g/L, NaCl10g/L, pH are natural, and solvent is water；
(3) fermented and cultured: the by volume inoculum concentration of concentration 2%, accesses seed liquor equipped with 100mL containing final concentration 50
In the 500mL triangular flask of the fermentation medium of the kanamycins of μ g/mL, 37 DEG C, 150rpm cultivate 28h, will fermentation
Liquid is centrifuged, and collects supernatant, i.e. obtains enzyme source；Fermentation medium final concentration consists of: soluble starch 10g/L, albumen
Peptone 12g/L, dusty yeast 3g/L, NaCl10g/L, pH is natural, and solvent is water.
4. the answering in preparation (S)-2-aryl-amino acid of immobilization recombinant penicillin G acylase described in a claim 1
With, it is characterised in that described application is: with immobilization recombinant penicillin G acylase as catalyst, with N-phenylacetyl
-(R, S)-2-aryl-amino acid is substrate, in the ammonia spirit that pH value is 8.0-11.0, at 25-60 DEG C, 180rpm bar
Under part after reaction completely, it is thus achieved that containing the mixed liquor of (S)-2-aryl-amino acid, mixed liquor is isolated and purified, it is thus achieved that described (S)-2-
Aryl-amino acid；Described initial substrate concentration is 0.05～0.3M, the interpolation final concentration of 0.01～0.2g/ml of described catalyst.
Apply the most as claimed in claim 4, it is characterised in that described substrate is N-phenylacetyl-(R, S)-phenylglycine, N-
Phenylacetyl-(R, S)-o-chlorobenzene glycine, N-phenylacetyl-(R, S)-chlorobenzene glycine, N-phenylacetyl-(R, S)-ammonia sweet to chlorobenzene
Acid or N-phenylacetyl-(R, S)-D-pHPG.
6. an immobilization recombinant penicillin G acylase application in preparing cefalexin described in claim 1, it is special
Levy and be that described application is: with immobilization recombinant penicillin G acylase as catalyst, remove acetoxyl group with 7-amino 3-
Cephalosporanic acid and Phenylglycine methyl ester hydrochloride are substrate, and regulation pH value is 6.0-8.0,15-30 DEG C, 180rpm condition
After lower reaction completely, it is thus achieved that the mixed liquor containing cefalexin, mixed liquor is isolated and purified, it is thus achieved that described cefalexin；Described
Substrate 7-amino 3-desacetoxycephalosporanic acid initial concentration is 0.01-0.3M, described substrate Phenylglycine methyl ester hydrochloride
Initial concentration is 0.05-0.3M, the interpolation final concentration of 0.01～0.2g/mL of described catalyst.
7. an immobilization recombinant penicillin G acylase application in preparing cefadroxil described in claim 1, its
The application being characterised by described is: with immobilization recombinant penicillin G acylase as catalyst, remove acetyl oxygen with 7-amino 3-
Base cephalosporanic acid and p-hydroxyphenylglycine methyl ester hydrochloride are substrate, and regulation pH value is 6.0-8.0,15-30 DEG C, 180
Under the conditions of rpm after reaction completely, it is thus achieved that the mixed liquor containing cefadroxil, mixed liquor is isolated and purified, it is thus achieved that described cephalo
Amoxycillin；Described substrate 7-amino 3-desacetoxycephalosporanic acid initial concentration is 0.01-0.3M, and described substrate is to hydroxyl
Phenylglycine methyl ester hydrochloride initial concentration is 0.05-0.3M, the interpolation final concentration of 0.01～0.2g/mL of described catalyst.
8. an immobilization recombinant penicillin G acylase application in preparing Cefaclor described in claim 1, it is special
Levy and be that described application is: with immobilization recombinant penicillin G acylase as catalyst, with 7-amino-3-chloro-3-cephem
-4-acid and Phenylglycine methyl ester hydrochloride are substrate, and regulation pH value is 6.0-8.0,15-30 DEG C, under the conditions of 180rpm instead
Should completely after, it is thus achieved that the mixed liquor containing Cefaclor, mixed liquor is isolated and purified, it is thus achieved that described Cefaclor；Described substrate
7-amino-3-chloro-3-cephem-4-acid initial concentration is 0.01-0.3M, described substrate Phenylglycine methyl ester hydrochloride initial concentration
For 0.05-0.3M, the interpolation final concentration of 0.01～0.2g/mL of described catalyst.
9. an immobilization recombinant penicillin G acylase application in preparing Cefradine described in claim 1, it is special
Levy and be that described application is: with immobilization recombinant penicillin G acylase as catalyst, remove acetoxyl group with 7-amino 3-
Cephalosporanic acid and 2,5-bis-hydrogen-based Phenylglycine methyl ester hydrochloride is substrate, and regulation pH value is 6.0-8.0,15-30 DEG C,
Under the conditions of 180rpm after reaction completely, it is thus achieved that the mixed liquor containing Cefradine, mixed liquor is isolated and purified, it is thus achieved that described head
Spore Latin；Described substrate 7-amino 3-desacetoxycephalosporanic acid initial concentration is 0.01-0.3M, described substrate 2,5-
Two hydrogen-based Phenylglycine methyl ester hydrochloride initial concentrations are 0.05-0.3M, the interpolation final concentration of 0.01～0.2 of described catalyst
g/mL。
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