CN103275960B - Manually designed penicillin G acylation zymogen, coding sequence and fermentation method - Google Patents

Manually designed penicillin G acylation zymogen, coding sequence and fermentation method Download PDF

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CN103275960B
CN103275960B CN201310256792.4A CN201310256792A CN103275960B CN 103275960 B CN103275960 B CN 103275960B CN 201310256792 A CN201310256792 A CN 201310256792A CN 103275960 B CN103275960 B CN 103275960B
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penicillin
acylase
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赖红星
肖拥军
曹春来
陈康月
张秋华
马文柱
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ZHUHAI LIANBANG PHARMACEUTICAL CO Ltd
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Abstract

The invention discloses a manually designed penicillin G acylation zymogen, a coding sequence and a fermentation method. The penicillin G acylation zymogen comprises an alpha subunit with an amino acid sequence shown as SEQ ID No. 1 (Sequence Identifier Number 1) and a beta subunit with an amino acid sequence shown as SEQ ID No. 2. The zymogen can be processed into a mature penicillin G acylation zymogen and can be used for synthesis of beta-lactam antibiotics such as amoxicillin. A nucleotide sequence for coding the zymogen is shown as SEQ ID No. 4, is obtained through optimization, and is suitable for expression in escherichia coli. The nucleotide sequence is recombined on an escherichia coli expression vector and then converted into an escherichia coli expression strain, and fermentation is performed by using a fermentation medium consisting of an organic nitrogen source, a carbon source and inorganic salt and by matching with a supplemented medium. The enzyme activity of penicillin G acylase obtained by the fermentation method can reach 28000U/L for a constitutive expression strain, and 35000U/L for an inducible expression strain.

Description

A kind of penicillin G acylase of artificial design former and encoding sequence and fermentation process
Technical field
The invention belongs to biological technical field, particularly former and encoding sequence and fermentation process of a kind of penicillin G acylase of artificial design.
Background technology
Penicillin acylase (E.C.3.5.1.11) is a kind of important industrial enzyme, is widely used in microbiotic industrial production.Due to the substrate specificity difference of these enzymes, can be divided three classes: penicillin G acylase (PGA), GDA (PVA) and penbritin acylase (AEH).Penicillin G acylase energy catalytic hydrolysis penicillin G generates 6-amino-penicillanic acid (6-APA) or hydrolysis cephamycin G generates 7-aminodeacetoxycephalosporanic acid (7-ADCA).In addition, this enzyme can also catalysis said hydrolyzed the reversed reaction of reaction, be connected different side chains at 6-APA with on 7-ADCA parent nucleus, generate dissimilar semisynthetic antibiotics, as amoxycilline Trihydrate bp etc.But the penicillin G acylase different in kind of different sources, some enzymes tend to cartalytic decomposition effect, and other tends to catalytic synthesis.
The source of penicillin G acylase is very extensive, comprises bacterium, actinomycetes, yeast and fungi etc.From the Protein Data Bank (http://www.ncbi.nlm.nih.gov/protein/) of state-run biotechnology information center of the U.S. (NCBI); retrieval penicillin G acylase has obtained 2595 sequences altogether; wherein 162 of intestinal bacteria, 125 of Pseudomonas aeruginosas.In addition, Bacillus foecalis alkaligenes (United States Patent (USP) 5695978), bacillus megaterium (United States Patent (USP) 3145395), achromobacter (United States Patent (USP) 2010/0112673A1) are also the important sources of penicillin G acylase.
Achromobacter penicillin G acylase has some special character, has therefore been subject to part Study person's attention.(the Gang Cai et al such as the Gang Cai of Chinese Academy of Sciences's Shanghai school of life and health sciences; Appl.Environ.Microbiol.; 2004; 70 (5): 2764-2770) be separated to a kind of new penicillin G acylase from achromobacter (Achromobacter xylosoxidans); the thermostability of this kind of enzyme is than thermotolerance alcaligenes faecalis penicillin G acylase (the Verhaert et al reporting before; Appl.Environ.Microbiol; 1997,63:3412-3418) thermostability taller.This achromobacter penicillin G acylase, is 55min the transformation period of 55 DEG C, is the highest more thermally-stabilised penicillin G acylase of reporting up to now.2003; (the Enzyme and Microbial Technology such as Czech scientist Skrob; 2003,32:738-744) be separated to a kind of brand-new penicillin G acylase from achromobacter (Achromobacter sp.CCM4824), research finds that this kind of enzyme has unique substrate specificity.The discoveries such as Skrob, with penicillin G acylase (Kutzbach et al, the Hoppe-Seylers Z Physiol Chem1974 of known report; 354:45 – 53; Baker et al, J Appl Bacteriol1992; 73:14 – 22; Robak et al, Acta Biochim Pol1981; 28:275 – 84.) compare; achromobacter penicillin G acylase not only can hydrolyzing penicillin G; and can also be hydrolyzed Ampicillin Trihydrate (ampicillin), amoxycilline Trihydrate bp (amoxicillin) and Cephalexin Monohydrate Micro/Compacted (cephalexin), the more important thing is that this enzyme is taller to the substrate specificity comparison penicillin G of Ampicillin Trihydrate, amoxycilline Trihydrate bp and Cephalexin Monohydrate Micro/Compacted.This is indicating that achromobacter penicillin G acylase has important using value in microbiotic compound probability.India scientist Kyslik etc. (United States Patent (USP) 2010112673A1); isolate from achromobacter (Achromobacter sp.CCM4824) nucleotide sequence that a segment length is 2646bp; by recombinant DNA technology, this section of nucleotide sequence imported to e. coli bl21, obtained the recombinant microorganism that can produce penicillin acylase.Find through fermenting experiment, Penicillin Acylase Activity is the highest can reach 18000U/L fermented liquid.
Summary of the invention
Primary and foremost purpose of the present invention is that the shortcoming that overcomes prior art, with not enough, provides a kind of penicillin G acylase of artificial design former.This penicillin G acylase is former under suitable condition, can be processed as ripe penicillin G acylase, is made up of α subunit and two subunits of β subunit.
Another object of the present invention is to provide the former nucleotide sequence of penicillin G acylase of the above-mentioned artificial design of coding.
A further object of the present invention is the former fermentation process of penicillin G acylase of the artificial design that provides described.
Object of the present invention is achieved through the following technical solutions: a kind of penicillin G acylase of artificial design is former, comprises α subunit and the aminoacid sequence β subunit as SEQ ID NO.2 as shown in of aminoacid sequence as shown in SEQ ID NO.1;
The penicillin G acylase of described artificial design is former, and preferably by aminoacid sequence, the α subunit as shown in SEQ ID NO.1 is connected and obtains successively with the β subunit of aminoacid sequence as shown in SEQ ID NO.2;
The former aminoacid sequence of the penicillin G acylase of described artificial design is preferably as shown in SEQ ID NO.3;
The former nucleotide sequence of the penicillin G acylase of above-mentioned artificial design of encoding is as shown in SEQ ID NO.4;
Express the former recombinant vectors of penicillin G acylase of above-mentioned artificial design, for the nucleotide sequence as shown in SEQ ID NO.4 is reconstituted on expression vector and is obtained;
Described expression vector is preferably pET9a, pET28a, pET28a-c (+), pET30a-c (+) or pET33b (+);
In the time that expression vector is preferably pET9a, the former recombinant vectors of the penicillin G acylase of the above-mentioned artificial design of expression obtaining is constitutive expression plasmid;
In the time that expression vector is preferably pET28a, the former recombinant vectors of the penicillin G acylase of the above-mentioned artificial design of expression obtaining is inducible expression plasmid;
The former recombinant vectors of penicillin G acylase of the described above-mentioned artificial design of expression, preferably obtains by following steps:
(1) add NdeI restriction enzyme site at the 5' of the nucleotide sequence as shown in SEQ ID NO.4 end, add BamHI restriction enzyme site at 3' end, obtain the sequence that contains NdeI and BamHI double enzyme site;
(2) utilize NdeI and BamHI restriction enzyme double digestion step (1) finally obtains respectively sequence and expression vector; Sequence after double digestion is connected with the expression vector after double digestion, obtains expressing the former recombinant vectors of penicillin G acylase of artificial design;
The former bacterial strain of penicillin G acylase of expressing artificial design, obtains for the recombinant vectors former penicillin G acylase of above-mentioned artificial design is transformed into host strain;
Described host strain is preferably intestinal bacteria (Escherichia coli) BL21 or e. coli bl21 (DE3);
The fermentation process of the former bacterial strain of the penicillin G acylase of the artificial design of described expression, comprises the steps: the inoculation former penicillin G acylase of expressing artificial design, in fermention medium, to ferment, specific as follows:
1. in the time that the recombinant vectors in the former bacterial strain of the penicillin G acylase of the artificial design of expression is inducible expression plasmid, fermentation condition is as follows: temperature is controlled between 28 DEG C～32 DEG C, pH value is controlled between 6.5～7.0, mixing speed is controlled between 150rpm～700rpm, air velocity is controlled between 200L/h～1600L/h, dissolved oxygen (DO) is controlled at 5～50% maximum oxygen saturation ratio, and the addition of inductor IPTG is between 0.3mM～0.6mM; In the time that carbon source exhausts, start feed supplement, adopt at the uniform velocity feed supplement, feed rate is controlled at 0.5mLmin -1l -1～1mLmin -1l -1between;
2. in the time that the recombinant vectors in the former bacterial strain of the penicillin G acylase of the artificial design of expression is constitutive expression plasmid, fermentation condition is as follows: temperature is controlled between 28 DEG C～32 DEG C, pH is controlled between 6.5～7.0, mixing speed is controlled between 150rpm～700rpm, air velocity is controlled between 200L/h～1600L/h, and dissolved oxygen (DO) is controlled between 20～50% maximum oxygen saturation ratio; In the time that exhausting, carbon source starts feed supplement, initial feed rate 0.2mLmin -1l -1, progressively increase, but should control the maximum oxygen saturation ratio that DO is greater than 20%, to prevent the too fast plasmid loss that causes of feed rate;
Described fermention medium composed as follows: every liter contains yeast powder 2～5g, peptone 3～8g, sodium-chlor 1～2g, potassium primary phosphate 2～5g, Sodium phosphate dibasic 2～5g, CALCIUM CHLORIDE DIHYDRATE 0.01～0.02g, magnesium sulfate 1～2g, glycerine 4～7g, ammonium sulfate 5～7g, micro-0.875mL; Water is settled to 1L, pH6.5～7.0;
The composition of described fermention medium is preferably as follows: every liter contains yeast powder 3g, peptone 5g, sodium-chlor 1g, potassium primary phosphate 3g, Sodium phosphate dibasic 3.25g, CALCIUM CHLORIDE DIHYDRATE 0.014g, magnesium sulfate 1g, glycerine 4.125g, ammonium sulfate 6g, micro-0.875mL; Water is settled to 1L, pH6.5～7.0;
Described micro-composed as follows: every liter contain Iron dichloride tetrahydrate 20～30g, zinc chloride 1～3g, cobalt chloride hexahydrate 2～4g, two molybdic acid hydrate sodium 2～4g, CALCIUM CHLORIDE DIHYDRATE 1～2g, Copper dichloride dihydrate 1～2g, boric acid 0.4～0.6g, Manganous sulfate monohydrate 2～3g, concentration is the concentrated hydrochloric acid 100mL of mass percent 37%, water is settled to 1L;
Described micro-composition is preferably as follows: every liter contain Iron dichloride tetrahydrate 22.87g, zinc chloride 1.31g, cobalt chloride hexahydrate 2g, two molybdic acid hydrate sodium 2g, CALCIUM CHLORIDE DIHYDRATE 1g, Copper dichloride dihydrate 1.25g, boric acid 0.5g, Manganous sulfate monohydrate 2.17g, concentration is the concentrated hydrochloric acid 100mL of mass percent 37%, water is settled to 1L;
Described feed supplement composed as follows: every liter contains 250～500g glycerine, 25～50g yeast powder, 40～60g peptone, surplus is water;
The composition of described feed supplement is preferably as follows: every liter contains 500g glycerine, 25g yeast powder, and 40g peptone, surplus is water.
The present invention has following advantage and effect with respect to prior art:
(1) the invention provides a kind of penicillin G acylase of artificial design former, this proenzyme can be processed as ripe penicillin G acylase, can be used for the synthetic of the β-lactam antibiticss such as amoxycilline Trihydrate bp.
(2) the invention provides a kind of artificial former nucleotide sequence of penicillin G acylase designing of encoding.This nucleotides sequence is classified as through optimizing and is obtained, and is suitable for expressing in intestinal bacteria.
(3) the invention provides the fermentation process of expressing the former bacterial strain of the artificial penicillin G acylase designing, fermention medium wherein, is made up of organic nitrogen source, carbon source and inorganic salt, and this substratum is applicable to producing the expression of Penicillin G Acylase Gene engineering bacteria; A kind of feed supplement is also provided simultaneously, and this feed supplement can be avoided the loss of expression vector.The enzyme work of the penicillin G acylase obtaining by this fermentation process has reached 28000U/L(composing type) or 35000U/L(induction type) expression vector.
Brief description of the drawings
Fig. 1 is the aminoacid sequence comparing result figure of penicillin G acylase proenzyme and achromobacter penicillin G acylase proenzyme in the present invention.
Fig. 2 is the building process schematic diagram of recombinant expression vector pET28a-PGA.
Fig. 3 is the building process schematic diagram of recombinant expression vector pET9a-PGA.
Fig. 4 is engineering bacteria BL21 (DE3)/pET28a-PGA and the engineering bacteria BL21/pET9a-PGA growth curve chart in fermentation culture process.
Fig. 5 is the product enzyme graphic representation of induction type bacterial classification BL21 (DE3)/pET28a-PGA and composing type bacterial classification BL21/pET9a-PGA.
Fig. 6 is engineering bacteria BL21 (DE3)/pET28a-PGA figure of the SDS-PAGE before induction and after induction respectively in the present invention.
Fig. 7 is the HPLC collection of illustrative plates of amoxycilline Trihydrate bp standard substance.
Fig. 8 is that reaction system adds testing sample HPLC collection of illustrative plates before.
Fig. 9 is the HPLC collection of illustrative plates that reaction system adds testing sample reaction 30min.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
Experimentation general introduction of the present invention:
One, the artificial design of penicillin G acylase zymogen protein matter
Penicillin G acylase is a kind of heterozygosis protein dimer, comprises a less α subunit and a larger β subunit.In the wild microorganism of product penicillin G acylase, first it is to be synthesized with a kind of precursor protein matter of non-activity.This precursor protein matter is called again penicillin G acylase proenzyme, has comprised α subunit and β subunit, and connects the spacer peptide of the two, and proenzyme just becomes activated penicillin G acylase through a series of processing and modification.
Penicillin G acylase belongs to N-end nucleophilic hydrolase family (N-terminal nucleophile; Ntn); this is enzyme (the Gang Cai et al that a class has an active centre and has a nucleophilic catalysis body at N-terminal; Appl.Environ.Microbiol.; 2004,70 (5): 2764-2770).The catalytic active center of penicillin G acylase is the Serine (Ser) that is positioned at β subunit N-terminal.The N-terminal nucleophilic catalysis body (Ntn_PGA) of penicillin G acylase is positioned at β subunit; its conservative property is very high, does is the accession number in the conserved sequence database (CDD) on NCBI cd03748(http: //www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi? uid=73358).
Achromobacter penicillin G acylase has thermostability and to beta-lactam class microbiotic substrate specificity advantages of higher (Enzyme and Microbial Technology, 2003,32:738-744; Gang Cai et al; Appl.Environ.Microbiol.; 2004; 70 (5): 2764-2770); researchist of the present invention is through further investigation; in conjunction with the conserved sequence of penicillin G acylase and the aminoacid sequence of part achromobacter penicillin G acylase, considering the aspects such as catalytic activity, protein structure stability, manually design α subunit (24KD) and the β subunit (58KD) of penicillin G acylase.The aminoacid sequence of α subunit is as shown in SEQ ID NO:1, and the aminoacid sequence of β subunit is as shown in SEQ ID NO:2.α subunit is connected with β subunit, just becomes proenzyme, and its aminoacid sequence is as shown in SEQ ID NO:3.
Penicillin G acylase proenzyme aminoacid sequence of the present invention is taking achromobacter penicillin G acylase proenzyme as parent, in the situation that considering the factors such as catalytic active center, conserved sequence and protein result stability, designs.It and achromobacter penicillin G acylase proenzyme have 96.2% homology (using software Vector NTI Suite9 to analyze), and Fig. 1 is shown in its aminoacid sequence contrast.
Two, the design and optimization of penicillin G acylase proenzyme encoding gene
The aminoacid sequence of penicillin G acylase proenzyme is as shown in SEQ ID NO:3.According to password sublist, these aminoacid sequences are converted to nucleotide sequence, in switching process, according to the codon usage frequency database of Host Strains (http://www.kazusa.or.jp/codon/), the codon that choice for use frequency is the highest.The present invention, according to colibacillary codon usage data storehouse, has obtained corresponding nucleotide sequence, submits the nucleotide sequence obtaining to Invitrogen company, through sequence optimisation, as shown in SEQ ID NO:4.In order to facilitate being connected of gene and carrier, add restriction enzyme site NdeI(CATATG at 5 ' of this nucleotide sequence), 3 ' end adds restriction enzyme site BamHI(GGATCC), called after PGA, this sequence is submitted to Nanjing Genscript Biotechnology Co., Ltd. synthesizes, synthetic PGA sequence is connected to carrier pUC57 by Nanjing Genscript Biotechnology Co., Ltd., the recombinant vectors pUC57-PGA of acquisition.
Three, the structure of recombinant expression vector
The pET serial carrier of Novagen company exploitation, uses the T7 promotor with strong startup ability, and expression that can high efficiency drive goal gene, has now become one of the most frequently used prokaryotic expression carrier.The carrier pET28a(+ that the present invention selects) use T7lac combined promoter, the expression that can freely close and open gene, before induction, gene is not expressed substantially, greatly reduces the load of Host Strains, after induction, can give expression to rapidly a large amount of target proteins.Designer of the present invention considers; the purification step that adds characteristic label (as His Tag, T7Tag) to simplify in the N of enzyme end, C end or inside; again because the catalytic center of penicillin G acylase at the N-terminal of β subunit; for fear of the impact on catalytic center structure, therefore added histidine-tagged (6His) at the N-terminal of α subunit.Use restriction enzyme site NdeI and BamHI that PGA is cloned into carrier pET28a(+), can realize and add histidine-tagged object, the recombinant expression vector name pET28a-PGA of acquisition.
The present invention also selects pET9a as expression vector, and it has independent T7 promotor, can, in the situation that there is no inductor, start the expression of goal gene.First Penicillin G Acylase Gene is expressed as penicillin G acylase proenzyme in Host Strains, is then become and is had bioactive maturing enzyme by processing and modification in a series of born of the same parents.As can be seen here, the too fast expression of exogenous protein must cause forming inclusion body, and can not process more maturing enzyme.Constitutive expression can regulate the resultant current of protein, improves the enzymatic productivity of Host Strains.Equally, use restriction enzyme site NdeI and BamHI that PGA is cloned into carrier pET9a, can realize above-mentioned purpose, the recombinant expression vector called after pET9a-PGA of acquisition.
Four, the foundation of penicillin G acylase expression library and screening
Recombinant expression vector is imported in escherichia coli expression host by Calcium Chloride Method, according to the various combination of carrier and Host Strains, by obtained genetic engineering bacterium called after BL21/pET28a-PGA, BL21 (DE3)/pET28a-PGA, DH5 α/pET28a-PGA and TOP10/pET28a-PGA respectively.Because these four kinds of genetic engineering bacteriums must, in the situation that inductor exists, could be expressed, therefore be referred to as inducible genes engineering bacteria., will not import in escherichia coli expression host containing the carrier pET28a of goal gene, the recombinant bacterium obtaining as a control group, is distinguished called after BL21/pET28a, BL21 (DE3)/pET28a, DH5 α/pET28a and TOP10/pET28a meanwhile.In host cell, can oneself be processed as activated penicillin G acylase because penicillin G acylase is former, therefore, the former final product of expressing of bacterial strain of penicillin G acylase of expressing artificial design is activated penicillin G acylase.
6-nitro-3-phenylacetylamino phenylformic acid (NIPAB) is joined in LB solid medium, and the penicillin G acylase that genetic engineering bacterium produces just can hydrolyzing N IPAB(white), generate material 6-nitro-3-benzaminic acid of yellow color.Therefore, can judge by the size of the yellow circle of periphery of bacterial colonies on flat board and the depth of color the height that enzyme is lived.
Adopt identical method for transformation, recombinant expression vector pET9a-PGA is imported in four kinds of escherichia coli expression hosts, and the genetic engineering bacterium obtaining is called after BL21/pET9a-PGA, BL21 (DE3)/pE9a-PGA, DH5 α/pET9a-PGA and TOP10/pET9a-PGA respectively.Because these four kinds of genetic engineering bacteriums need to just can not be expressed in the situation that inductor exists, therefore be referred to as constitutive gene engineering bacteria.Do not import in four kinds of escherichia coli expression hosts containing the carrier pET9a of goal gene, the recombinant bacterium of acquisition is as expression control group, respectively called after BL21/pET9a, BL21 (DE3)/pET9a, DH5 α/pET9a and TOP10/pET9a.
Five, the fermentation culture of engineering bacteria
The invention discloses a kind of fermention medium, be made up of organic nitrogen source, carbon source and inorganic salt, this substratum is applicable to producing the expression of Penicillin G Acylase Gene engineering bacteria.During the fermentation, must avoid the loss of expression vector as far as possible, use the strategy of restriction feed supplement to have certain mitigation to the loss of expression vector.The present invention is through experiment showed, that the feed rate of induction type bacterial classification is controlled at 0.5mLmin -1l -1～1mLmin -1l -1be advisable, and the initial feed rate of composing type bacterial classification is 0.2mLmin -1l -1, progressively increase, guarantee the maximum oxygen saturation ratio that DO is greater than 20%.
Embodiment 1
Recombinant DNA technology
Use restriction enzyme NdeI and the BamHI of TaKaRa company to carry out double digestion to recombinant vectors pUC57-PGA, getting 5 μ L enzymes cuts product mass volume ratio 1% agarose gel electrophoresis and analyzes, after confirming that enzyme cuts entirely, whole enzymes are cut to product and carry out mass volume ratio 0.8% agarose electrophoresis, cut off the gel containing the DNA fragmentation of 2.6kb, use the sepharose recovery test kit of TIANGEN company that the DNA fragmentation in gel is purified in 30 μ L deionized waters, just obtain the gene fragment with sticky end.Equally, plasmid pET28a is carried out to double digestion with NdeI and BamHI, the plasmid fragment purification after enzyme is cut is in 20 μ L deionized waters.Use the DNA Ligation Kit2.0 of TaKaRa company that gene fragment is connected with plasmid fragment, condition of contact is 16 DEG C, spends the night.Get 10 μ L spend the night connect product, join 80 μ L CaCl 2in bacillus coli DH 5 alpha (purchased from invitrogen company) competent cell prepared by method (" molecular cloning experiment guide " third edition that Cold Spring Harbor Laboratory is published), process 90s for 42 DEG C, add rapidly 300 μ L through the LB liquid nutrient medium of 37 DEG C of preheatings (wherein, peptone 10g/L, yeast powder 5g/L, sodium-chlor 5g/L, pH7.0～7.5), in 37 DEG C of shaking tables, low-speed oscillation is cultivated 45min, then get 100 μ L culture coatings and added kantlex (Kanamycin, final concentration is 50 μ g/ml) solid LB substratum (wherein, peptone 10g/L, yeast powder 5g/L, sodium-chlor 5g/L, agar powder 15g/L, pH7.0～7.5).In 37 DEG C of incubators, be inverted and cultivate the about 18h of this flat board, until grow single bacterium colony, random picking part list bacterium colony carries out bacterium liquid PCR qualification, and the condition of PCR reaction is: 94 DEG C of 5min; 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 30 circulations; 72 DEG C of 5min; Primer is: T7 upstream primer 5 '-TAATACGACTCACTATAGGG-3 ', T7 downstream primer 5 '-GCTAGTTATTGCTCAGCGG-3 '.The positive colony extracting plasmid that preliminary screening is obtained, carries out enzyme by NdeI and BamHI and cuts qualification, obtains the clone of the gene fragment of 2.6kb and the carrier segments of 5.3kb, is the clone who contains PGA fragment.The clone that qualification is contained to PGA fragment delivers to Invitrogen company and checks order.Order-checking is proved not have to the mono-clonal of base mutation and reading frame displacement, be seeded to 50ml and added kantlex (Kanamycin, 50 μ g/ml) LB liquid nutrient medium, 37 DEG C, 250rpm, cultivate 18h, the little middle amount test kit extracting plasmid (by specification operates) of carrying of plasmid of TIANGEN company for the culture of acquisition, the recombinant expression plasmid called after pET28a-PGA(of acquisition is shown in Fig. 2).
By above-mentioned identical method by PGA gene clone to plasmid pET9a, obtain recombinant expression plasmid pET9a-PGA(and see Fig. 3).
Embodiment 2
The structure of penicillin G acylase expression library and screening
The Calcium Chloride Method that " molecular cloning experiment guide " third edition of publishing according to Cold Spring Harbor Laboratory provides, prepares respectively e. coli bl21 (purchased from invitrogen company), BL21 (DE3) (purchased from invitrogen company), DH5 α and TOP10(purchased from invitrogen company) competent cell.The recombinant expression plasmid pET28a-PGA getting in 1 μ L embodiment 3 transforms respectively above-mentioned four kinds of competent escherichia coli cells, and method for transformation carries out according to the Calcium Chloride Method of " molecular cloning experiment guide " third edition equally.Conversion fluid is applied to respectively to the solid LB substratum that has added kantlex (Kanamycin, final concentration is 50 μ g/ml), cultivates until there is single bacterium colony, obtained penicillin G acylase inducible expression strain library for 37 DEG C.
With the aseptic toothpick equivalent thalline of the above-mentioned four kinds of engineering bacterias of picking respectively, be seeded to and added 6-nitro-3-phenylacetylamino phenylformic acid (NIPAB, final concentration is 0.9mg/ml) and sec.-propyl-β-D-sulfo-galactopyranoside (IPTG, final concentration is 1mmol/L) solid LB substratum, in the incubator of 28 DEG C, cultivate after 5h, examine periphery of bacterial colonies and have or not yellow color to occur.Experimental result shows, the yellow circle maximum of genetic engineering bacterium BL21 (DE3)/pET28a-PGA periphery of bacterial colonies, and color is the darkest, illustrates that its enzymatic productivity is the strongest.
Recombinant expression plasmid pET9a is transformed in e. coli bl21, BL21 (DE3), DH5 α and tetra-kinds of hosts of TOP10 by above-mentioned same method, has obtained penicillin G acylase constitutive expression strain library.With the aseptic toothpick equivalent thalline of the above-mentioned four kinds of engineering bacterias of picking respectively, be seeded to the solid LB substratum that has added 0.9mg/ml NIPAB, in the incubator of 28 DEG C, cultivate after 5h, examine periphery of bacterial colonies and have or not yellow color to occur.Result shows, the yellow circle maximum that genetic engineering bacterium BL21/pET9a-PGA periphery of bacterial colonies forms, and color is the darkest, illustrates that its enzymatic productivity is the strongest.
By two kinds of genetic engineering bacterium BL21 (DE3) of above-mentioned enzymatic productivity the best/pET28a-PGA and BL21/pET9a-PGA, at fresh LB solid medium (Kanamycin, final concentration is 50 μ g/ml) upper line, carry out pure culture, picking list bacterium colony is cultured to OD with LB liquid nutrient medium (Kanamycin, final concentration is 50 μ g/ml) 600=0.6～0.8, make the bacterium liquid containing volume percent 15% glycerine, deposit to-70 DEG C of refrigerators for subsequent use.
Embodiment 3
(1) fermentation of induction type Penicillin G Acylase Gene engineering bacteria
1. prepare inoculum
Get 20 μ L at the bacterial classification BL21 of-70 DEG C of freezing preservations (DE3)/pET28a-PGA, be seeded to the LB liquid nutrient medium that 50ml has added kantlex (Kanamycin, final concentration is 50 μ g/ml), at 28 DEG C, in the shaking table of 250rpm, cultivate 16 hours, thus activated spawn.The bacterial classification again 50ml being activated is inoculated in the LB liquid nutrient medium that 400ml added 50 μ g/ml kantlex, at 28 DEG C, continues to cultivate 3h under the condition of 250rpm, obtains inoculum, controls its cell concentration OD 600between 0.8～1.2.
2. the fermentation culture in 20L fermentor tank
Use 20L stirred-tank fermenter (Nanjing Hua Long company), feed intake according to the fermentative medium formula shown in table 1, the volume that feeds intake is 8L.Strict controlled fermentation condition: temperature is controlled between 28 DEG C～32 DEG C, pH is controlled between 6.5～7.0, fermentation rotating speed is controlled at 150rpm～700rpm(according to the variation regulation and control of DO) between, air velocity is controlled between 200L/h～1600L/h (according to the variation regulation and control of DO), and dissolved oxygen (DO) is controlled between 5～50% maximum oxygen saturation ratio.Be cultured to when carbon source exhausts and start feed supplement (wherein every L contains 500g glycerine, 25g yeast powder, 40g peptone, and water is settled to 1L), (feed rate is controlled at 0.6mLmin to adopt at the uniform velocity feed supplement -1l -1), the consumption of feed supplement is 4L.When being cultured to cell concentration OD 600when ≈ 30, start to add IPTG to final concentration 0.3mM, start induction.Induction time is 10h, and fermentation period is that 24h(is shown in Fig. 4).
Table 1
Table 2
(2) mensuration of the degrading activity of penicillin G acylase
Get the fermented liquid that 1mL step (1) obtains, through centrifugation thalline, with 1mL distilled water wash twice, then centrifugation, thalline is kept to-20 DEG C.Thalline after thawing is resuspended in 1mL0.05mol/L potassium phosphate buffer (pH8.0), is testing sample.
Get 0.05mol/L potassium phosphate buffer (pH8.0) 100mL, 37 DEG C of preheating numbers minute, add 1g potassium penicillin G, stir, then add 1mL testing sample, in the time of pH to 8.00, start timing, maintain pH=8.00 by dripping 0.1mol/L standard NaOH solution, react 8～10 minutes, accurate recording consumes volume and the reaction times of NaOH solution.
Enzyme is lived and is defined as, and, is hydrolyzed the amount of the required penicillin G acylase of 1 μ mol potassium penicillin G in 1 minute under certain condition.The calculation formula that enzyme is lived is:
Enzyme=100 × (t) U/L of V ÷ alive
Wherein, V represents to consume the number of minutes of NaOH solution, and t represents the number of minutes in reaction times.
According to this method, the degrading activity of measuring induction type bacterial classification expression penicillin G acylase of the present invention is up to 35000U/L(Fig. 5).
Embodiment 4
(1) fermentation of composing type Penicillin G Acylase Gene engineering bacteria
1. prepare inoculum
Use constitutive expression bacterial classification BL21/pET9a-PGA, method is with embodiment 5.
2. the fermentation culture in 20L fermentor tank
Feed intake according to the fermentative medium formula shown in table 1, the volume that feeds intake is 8L.Strict controlled fermentation condition: temperature is controlled between 28 DEG C～32 DEG C, pH is controlled between 6.5～7.0, fermentation rotating speed is controlled between 150rpm～700rpm, and air velocity is controlled between 200L/h～1600L/h, and dissolved oxygen (DO) is controlled between 20%～50% maximum oxygen saturation ratio.Be cultured to and when carbon source exhausts, start feed supplement (formula of feed supplement is with implementing 3).Due to constitutive expression bacterial classification, all larger at the load of whole fermentation period, in order to reduce the loss of plasmid, must the strict flow velocity of controlling feed supplement.Initial feed rate control is 0.2mLmin -1l -1, progressively increasing, every 6h increases once, increases 0.1mLmin at every turn -1l -1, increase to 0.6mLmin -1l -1shi Ze no longer increases.The generalized case feed supplement time is 26h, and feed supplement consumption is in about 4.6L, but should control the maximum oxygen saturation ratio that DO is greater than 20%, to prevent the too fast plasmid loss that causes of feed rate.Induction type bacterial classification does not need induction, generally cultivates 35h, and carrier is lost more serious, stops the (see figure 4) of fermenting.
Utilize the method for embodiment 3, the highest decomposition enzyme of measuring constitutive expression bacterial classification in the present invention is lived and is seen Fig. 5 for 28000U/L().
Embodiment 5
The electrophoretic analysis of penicillin G acylase and amoxycilline Trihydrate bp synthesis capability detect
(1) protein electrophoresis
Get 2g fermentation thalline, resuspended with 10mL50mM sodium phosphate buffer (pH8.0), ultrasonication 10 minutes.After centrifugation, get supernatant, use Polyacrylamide Gel Electrophoresis (SDS-PAGE, resolving gel concentration is 12%, concentrated gum concentration is 5%), the results are shown in Figure 6.As can be seen here, penicillin G acylase of the present invention is made up of α, two subunits of β, and size is about respectively 28KD and 55KD.
(2) detection of amoxycilline Trihydrate bp synthesis capability
Get 1mL fermented liquid, through centrifugation thalline, with 1mL distilled water wash thalline twice, then centrifugation, thalline is kept to-20 DEG C.Thalline after thawing is resuspended in 1mL0.05mol/L potassium phosphate buffer (pH8.0), is testing sample.
In 100mL water, add 0.5g6-APA(6-aminopenicillanic acid), with the NaOH solution adjust pH to 6.3 of mass volume ratio 40%, then add 0.5g HPGMe.HCl(D-p-hydroxyphenylglycine methyl ester hydrochloride), with the NaOH solution adjust pH to 6.3 of mass volume ratio 40%, add 1ml sample, under the water bath condition of 37 DEG C, react 30 minutes.Use high-efficient liquid phase chromatogram technique analysis reaction product, chromatographic column: Luna, 5 μ C18,100A, 150 × 4.60mm(Phenomenex); Moving phase: A phase: 0.05M sodium phosphate buffer, pH=3.1
B phase: methyl alcohol; Column temperature: 30 DEG C; Flow velocity: 1.0mLmin; Detect wavelength: 215nm.
Detected result is shown in Fig. 7～9, and wherein Fig. 7 is the color atlas of amoxycilline Trihydrate bp standard substance (concentration 0.946mg/mL), and retention time is 6.3min.Fig. 8 is that reaction system adds testing sample color atlas before, and 6-APA concentration is 5mg/mL, retention time 2.4min, and HPGMe.HCl concentration is 5mg/mL, retention time 4.8min.Figure 10 adds the reaction product color atlas of example reaction after 30 minutes, has 4 kinds of materials, HPG(D-D-pHPG), 6-APA, HPGMe.HCl and amoxycilline Trihydrate bp, retention time is respectively 1.7min, 2.4min, 4.8min, 6.3min.Wherein HPG and amoxycilline Trihydrate bp are the products generating after reaction.
By above analysis, illustrate that penicillin G acylase of the present invention has the ability that catalysis 6-APA and HPGMe.HCl have synthesized amoxycilline Trihydrate bp.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.

Claims (9)

1. the penicillin G acylase of an artificial design is former; it is characterized in that: the α subunit by aminoacid sequence as shown in SEQ ID NO.1 is connected and obtains successively with the β subunit of aminoacid sequence as shown in SEQ ID NO.2, and aminoacid sequence is as shown in SEQ ID NO.3.
2. the former nucleotide sequence of penicillin G acylase of coding artificial design claimed in claim 1, is characterized in that: as shown in SEQ ID NO.4.
3. the former recombinant vectors of penicillin G acylase of expressing artificial design, is characterized in that: nucleotide sequence claimed in claim 2 is reconstituted on expression vector and is obtained.
4. recombinant vectors according to claim 3, is characterized in that: described expression vector is pET9a, pET28a, pET28a-c (+), pET30a-c (+) or pET33b (+).
5. recombinant vectors according to claim 3, is characterized in that: obtain by following steps:
(1) add NdeI restriction enzyme site at the 5' of nucleotide sequence claimed in claim 2 end, add BamHI restriction enzyme site at 3' end, obtain the sequence that contains NdeI and BamHI double enzyme site;
(2) by NdeI and BamHI double digestion step (1) finally obtains respectively sequence and expression vector; Sequence after double digestion is connected with the expression vector after double digestion, obtains expressing the former recombinant vectors of penicillin G acylase of artificial design.
6. the former bacterial strain of penicillin G acylase of expressing artificial design, is characterized in that: recombinant vectors claimed in claim 3 is transformed into host strain and obtains.
7. the former bacterial strain of penicillin G acylase of the artificial design of expression according to claim 6, is characterized in that: described host strain is intestinal bacteria (Escherichia coli) BL21 or e. coli bl21 (DE3).
8. the fermentation process of the former bacterial strain of the penicillin G acylase of the artificial design of expression claimed in claim 7; it is characterized in that comprising the steps: by the inoculation former penicillin G acylase of expressing artificial design in fermention medium; ferment, specific as follows:
1. in the time that the recombinant vectors in the former bacterial strain of the penicillin G acylase of the artificial design of expression is inducible expression plasmid, fermentation condition is as follows: temperature is controlled between 28 DEG C～32 DEG C, pH value is controlled between 6.5～7.0, mixing speed is controlled between 150rpm～700rpm, air velocity is controlled between 200L/h～1600L/h, it is 5～50% that dissolved oxygen is controlled at oxygen saturation, and the addition of inductor IPTG is at 0.3mM～0.6mM; In the time that carbon source exhausts, start feed supplement, adopt at the uniform velocity feed supplement, feed rate is controlled at 0.5mLmin -1l -1～1mLmin -1l -1between;
2. in the time that the recombinant vectors in the former bacterial strain of the penicillin G acylase of the artificial design of expression is constitutive expression plasmid, fermentation condition is as follows: temperature is controlled between 28 DEG C～32 DEG C, pH is controlled between 6.5～7.0, mixing speed is controlled between 150rpm～700rpm, air velocity is controlled between 200L/h～1600L/h, and it is 20～50% that dissolved oxygen is controlled at oxygen saturation; In the time that exhausting, carbon source starts feed supplement, initial feed rate 0.2mLmin -1l -1, progressively increasing, oxygen saturation can not be lower than being 20%;
Described fermention medium composed as follows: every liter contains yeast powder 2～5g, peptone 3～8g, sodium-chlor 1～2g, potassium primary phosphate 2～5g, Sodium phosphate dibasic 2～5g, CALCIUM CHLORIDE DIHYDRATE 0.01～0.02g, magnesium sulfate 1～2g, glycerine 4～7g, ammonium sulfate 5～7g, micro-0.875mL; Water is settled to 1L, pH6.5～7.0;
Described micro-composed as follows: every liter contain Iron dichloride tetrahydrate 20～30g, zinc chloride 1～3g, cobalt chloride hexahydrate 2～4g, two molybdic acid hydrate sodium 2～4g, CALCIUM CHLORIDE DIHYDRATE 1～2g, Copper dichloride dihydrate 1～2g, boric acid 0.4～0.6g, Manganous sulfate monohydrate 2～3g, concentration is the concentrated hydrochloric acid 100mL of mass percent 37%, water is settled to 1L;
Described feed supplement composed as follows: every liter contains 250～500g glycerine, 25～50g yeast powder, 40～60g peptone, surplus is water.
9. the fermentation process of the former bacterial strain of the penicillin G acylase of the artificial design of expression according to claim 8, is characterized in that:
Described fermention medium composed as follows: every liter contains yeast powder 3g, peptone 5g, sodium-chlor 1g, potassium primary phosphate 3g, Sodium phosphate dibasic 3.25g, CALCIUM CHLORIDE DIHYDRATE 0.014g, magnesium sulfate 1g, glycerine 4.125g, ammonium sulfate 6g, micro-0.875mL; Water is settled to 1L, pH6.5～7.0;
Described micro-composed as follows: every liter contain Iron dichloride tetrahydrate 22.87g, zinc chloride 1.31g, cobalt chloride hexahydrate 2g, two molybdic acid hydrate sodium 2g, CALCIUM CHLORIDE DIHYDRATE 1g, Copper dichloride dihydrate 1.25g, boric acid 0.5g, Manganous sulfate monohydrate 2.17g, concentration is the concentrated hydrochloric acid 100mL of mass percent 37%, water is settled to 1L;
Described feed supplement composed as follows: every liter contains 500g glycerine, 25g yeast powder, 40g peptone, surplus is water.
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CN105647807A (en) * 2016-04-11 2016-06-08 浙江拓普药业股份有限公司 Culture method of producing bacteria of penicillin acylase
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