Page 1

(19) State Intellectual Property Office of the People's Republic of China http://epub.cnipa.gov.cn/patent/CN109161540A

φ
(21 ) Application number 201811135223.3

(22 ) Application date 2018.09.28

(12) Invention patent application
(10 ) Application publication number CN 109161540 A
(43 ) Application Announcement Date 2019. 01.08

(71) Applicant Hunan Fulaige Biotechnology Co., Ltd.

Address 410000 Changsha Economic Technology, Changsha City, Hunan Province
No. 7 Lixiang West Road , Development Zone

(72) Inventor Huang Bin Zhao Qiang Zhou Jinghui Xu Gang

(74 ) Patent agency Changsha Rongzhi Patent Office

43114
Agent Yuan Jing

(51)lnt.CI.
C12N ?/ 54(2006.01)
C12N / 5 / 55 (2006.01)
C12N /// ¢72(2006.01)
C12P 37 / 06 ( 2006 . 01 )
Claims 1 page specification 12 pages
Sequence Listing 8 Pages Attached Page 1

(54 ) Title of invention

Penicillin V acylase mutant, coding sequence, recombination
Expression vector, genetic engineering bacteria and application
(57 ) Summary
The invention provides a mutant of penicillin V acylase,
Coding sequence, recombinant expression vector, genetic engineering bacteria and application. Correct
Penicillin from Bacillus sphaericus
Line mutation, the specific activity of the obtained mutant PVA-3 increased by 9 times,
Immobilization activity increased from 92U / g to 820U / g , at a concentration of

30 % penicillin V potassium salt incubation for 1 hour, residual activity

89.1 % ; immobilized mutant PVA-3 is applied to pH 6 . 0 , 25 [deg.] C
Cleaved under 25 % concentration of penicillin V Preparation of 6-APA anti
The response time was shortened from 180 minutes to 60 minutes, and the substrate conversion rate increased
Up to 99.5 % or more, and continuous use of more than 600 batches, vitality
There is no obvious loss, and it has good operation stability. After mutation

< PVA-3 greatly reduces production costs and improves production

^ Production efficiency is more suitable for industrial applications.
LO
TH
CD
TH

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Claims
CN 109161540 A
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1. A penicillin V acylase mutant characterized by having at least the amino acid sequence shown in SEQ ID N 0.2
The threonine at position 91 is mutated to alanine, preferably penicillin V acylase having the amino acid sequence shown in SEQ ID N 0.2
The threonine at position 91 is mutated to alanine, and the preferred mutant is named PVA-I . Its amino acid sequence is shown as SEQ ID N 0.4
Shown.
2. A mutant of penicillin V acylase, characterized in that it has at least the amino acid number shown in SEQ ID N 0.2
 The threonine at position 91 is mutated to alanine, and the isoleucine at position 280 is mutated to phenylalanine, preferably with SEQ ID

The amino acid shown at N 0.2 has the threonine at position 91 mutated to alanine, and the isoleucine at position 280 has been mutated to phenylalanine.
The selected mutant was named PVA -2, and its amino acid sequence is shown in SEQ ID N 0.6.
3. A penicillin V acylase mutant, characterized in that it has at least the amino acid number shown in SEQ ID N 0.2
The threonine at position 91 is mutated to alanine, the isoleucine at position 280 is mutated to phenylalanine, and the serine at position 110 is mutated
Into cysteine, it is preferred to mutate the threonine having the amino acid position 91 shown in SEQ ID N 0.2 to alanine, the 280th
The isoleucine at position is mutated to phenylalanine, the serine at position 110 is mutated to cysteine, and the preferred mutant is named

The amino acid sequence of PVA -3 is shown in SEQ ID N 0.8.

4. A gene sequence encoding a penicillin V acylase mutant, characterized in that it encodes any one of claims 1-3
The gene sequence of any one of the penicillin V acylase mutants described in the item .
5. A recombinant expression vector containing one or more of the coding genes of claim 4.
6. A genetically engineered bacterium containing one or more of the recombinant expression vectors of claim 5.
7. An immobilized penicillin V acylase mutant, characterized in that the right is protected by an epoxy carrier or an amino carrier
The penicillin V acylase mutant according to any one of claims 1 to 3 is required for immobilization.
8. Use of a penicillin V acylase mutant, characterized in that the penicillin V according to any one of claims 1 to 4 is used
Immobilized penicillin acylase mutant of claim 7, or claims V catalyzed hydrolysis of penicillin acylase mutant V Preparation
6 -APAo
9. The use of the penicillin V acylase mutant according to claim 8 , wherein the immobilized penicillium
When V- acylase mutant hydrolyzes penicillin V to prepare 6- APA , the conversion conditions are: substrate penicillin V concentration ( W / V ) is 15%
~30%, preferably 20%~30%, more preferably 25%, conversion temperature 20~25° C , pH 6.0~6.5.

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Penicillin V acylase mutant, coding sequence, recombinant expression vector, genetic engineering
Cheng Bacteria and its application

Technical field
[0001] The present invention belongs to the field of genetic engineering and enzyme engineering, in particular to a penicillin V acylase mutant and coding sequence
List, recombinant expression vectors, genetically engineered bacteria and their applications.

Background technique
[0002] Penicillin V is an early-used penicillin antibiotic, which is effective against most Gram-positive bacteria and Gram-negative spheres.
Bacteria, Gram-negative bacilli individual (e.g., Haemophilus), spirochetes, and actinomycetes have antibacterial activity, but producing β within lactamase _
This type of strain has no antibacterial effect. At present, most of the penicillin V ( PenV ) produced by fermentation is used to produce β -lactam antibiotics
Firing the intermediate 6-aminopenicillanic acid (6- Amino-penicillanic acid , 6_ APA ) starting material. 6- APA is a synthetic amoxi
Lin and ampicillin are an important intermediate.
[0003] Currently, β -lactam antibiotics industrial production of 6- APA mainly uses penicillin G acylase (penicillin

G acylase , PGA, EC 3.5.1.11) obtained by hydrolyzing the potassium salt of penicillin G , while some manufacturers have passed chemical methods
Or use penicillin V acylase (penicillin V acylase , PVA , EC 3.5.1.11) to hydrolyze the potassium salt of penicillin V
Formula 6- APA , the reaction principle of penicillin V acylase is shown in the figure below.

PVA
[0004] Penicillin V + water = 6-aminopenicillanic acid + phenoxyacetic acid

[0005] Compared with the use of PGA enzymatic production of 6- APA , the use of PVA enzymatic production of 6- APA has the following advantages: (1) can be catalyzed
 Higher substrate concentration. The maximum substrate concentration of penicillin G potassium salt is 8%, and its conversion rate is 97.5%, while penicillin V potassium salt

The substrate concentration can reach 12%, the conversion rate is about 99%, 6- APA yield is higher; ⑵ has a better reaction pHJGA
The reaction pH is around 8.0, more by-products are produced under this pH condition, and the optimal reaction pH of PVA is between 5.5-8.5
In the meantime, there are fewer acidic neutral pH by -products and 6- APA is more stable; (3) Product 6- APA has better quality. 6- APA produced by PGA
The by-product phenylacetic acid is difficult to separate and is easily brought into downstream synthetic antibiotics, causing allergic reactions, and 6- APA produced by PVA
The by-product is phenoxyacetic acid, which can be easily separated from 6- APA , and the resulting 6- APA has higher purity and quality
better. Therefore, the development of an excellent penicillin V acylase in the antibiotic industry has very good market prospects.
[0006] Wild-type penicillin V acylase is widely distributed in various types of microorganisms. Many wild-type PVA are used by domestic and foreign
Researchers screen, clone and express the genes they encode. For example, Chinese patent CN 1132251 discloses a Fusarium oxysporum
Gene encoding a novel penicillin V acylase and expressed in a heterologous host, its activity is 130 U / ml ; Feng Ying, Dong Zhiyang
The fermentation, purification and immobilization of penicillin V acylase derived from Fusarium oxysporum FP 941 were studied.
The force is 210 U / g . After 25 batches are used, the enzyme activity remains 90%; Patent W 02005/066336 discloses a source

The Bacillus sphaericus penicillin V acylase gene was recombinantly expressed in E. coli and its fermentation activity reached
203.68 U / g dry cell weight; Demin Zhang et al. disclosed a new type derived from Streptomyces mobaraensis
Gene encoding penicillin V acylase and characterization of the enzyme; Jesus Torres - Bacete et al.

Streptomyces Iavendulae- derived PVA has been immobilized and has been used continuously for 50 batches.
Weakened; V · S · Avinash et al . performed large intestine on penicillin V acylase derived from Pectobacterium atrosepticum
Recombinant expression of Bacillus, its specific activity can reach 430 U / g .

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[0007] With the development of genetic engineering and protein engineering technology, the cloning and expression of wild-type penicillin V acylase has
There are many reports, but there are few reports on the mutation and property modification of penicillin V acylase. Currently, Penicillium is used in API companies
During the pilot production of 6- APA by the enzymatic method of V- acylase , it was found that the wild-type penicillin V acylase has low catalytic activity and reaction.
The shortcomings of long time and few batches of use seriously restrict the promotion and application of the enzyme, so a high catalytic activity and reaction
The new penicillin V acylase with fast rate and good operational stability has a good market prospect.

Summary of the invention

[0008] The primary object of the present invention is to provide a highly active penicillin V acylase mutant. Mutants are passed randomly
Mutation, site-directed mutagenesis and other non-rational and rational enzyme engineering technology designed to BaciIlus sphaericus green sources
Amycin V acylase is obtained by mutation, its reaction activity is higher, the reaction rate is faster, the stability is better, it can be more effective and fast
Quickly convert penicillin V to 6- APA .

[0009] Specifically, the invention

[0010] The mutants having at least SEQ ID N0.2 amino acid sequence of the 91 threonine at position mutated to alanine.

[0011] preferably having SEQ ID N0.2 penicillin amino acid sequence of V acylase of 91 threonine mutation at position
To form alanine, the preferred mutant is named PVA-I , and its amino acid sequence is shown in SEQ ID N0.4 . Activity of this mutant
Higher than wild-type penicillin V acylase.

[0012] Further, at least the threonine having amino acid position 91 of SEQ ID N0.2 is mutated to alanine,
The isoleucine at position 280 was mutated to phenylalanine.

[0013] preferably having N0.2 SEQ ID amino acid of 91 threonine to alanine mutation at position, the first 280 bits
Isoleucine is mutated to phenylalanine. The preferred mutant is named PVA-2, and its amino acid sequence is shown in SEQ ID N0.6 .
The mutant meets the requirements of high activity and also has tolerance to high concentration penicillin V.

[0014] Further, at least the amino acid shown in SEQ ID N0.2 threonine mutation at 91 to alanine
Acid, the isoleucine at position 280 is mutated to phenylalanine, and the serine at position 110 is mutated to cysteine, preferably having

The amino acid at position 91 of SEQ ID N0.2 is mutated to alanine, and the isoleucine at position 280 is mutated to phenylalanine
Amino acid, the serine at position 110 is mutated to cysteine, the preferred mutant is named PVA-3 , and its amino acid sequence is as SEQ

ID N0.8 . This mutant meets the requirements of high activity, has better tolerance to high concentration penicillin V , and is stable
It has better sex and its hydrolysis activity is much higher than that of mutant PVA-2 .

[0015] The second object of the present invention is to provide the above-mentioned gene sequence encoding a penicillin V acylase mutant.

[0016] The sequence is the gene sequence of any one of the penicillin V acylase mutants.

[0017] Further, the gene sequence of the penicillin V acylase mutant is any one of a ~ d ,

[0018] a) having the nucleotide sequence of SEQ ID N0.3 in the sequence listing ;

[0019] b) having the nucleotide sequence of SEQ ID N0.5 in the sequence listing ;

[0020] c) having the nucleotide sequence of SEQ ID N0.7 in the sequence listing ;
[0021] d ) under stringent conditions can be hybridized with a nucleotide sequence defined in any one of a , b or c and encodes a penicillin V acyl
The nucleotide sequence of the protein with enzyme activity;
[0022] The third object of the present invention is to provide a recombination table of the above-mentioned gene sequences encoding penicillin V acylase mutants
 Up to vector.
[0023] Specifically, it is a recombinant expression vector containing one or more of the coding genes.

[0024] The fourth object of the present invention is to provide recombinant expression of the coding gene sequence of the above-mentioned penicillin V acylase mutant

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Vector of genetically engineered bacteria.

[0025] Specifically, it is a genetically engineered bacterium containing one or more of the above recombinant expression vectors.
[0026] The fifth object of the present invention is to provide an immobilized penicillin V acylase mutant. Specifically using epoxy
The base carrier or amino carrier immobilizes the above-mentioned penicillin V acylase mutant.
[0027] The sixth object of the present invention is to provide a penicillin V acylase mutant application, using the above-mentioned penicillium
Su V acylase mutant, or the above-described immobilized penicillin V acylase mutant catalyzed hydrolysis of penicillin V Preparation of 6- APA .
[0028] Specifically, when the immobilized penicillin V acylase mutant hydrolyzes penicillin V to prepare 6- APA , the conversion strip
The pieces are: the concentration of the substrate penicillin V ( ff / ν ) is 15% to 30% , preferably 20% to 30%, more preferably 25%. Conversion temperature
It is 20 ~ 25° C , pH is 6.0~6.5.
[0029] The preparation method of the aforementioned mutant penicillin V acylase of the present invention includes the following steps:
[0030] a ) random mutation, saturation mutation, iterative saturation of the nucleotide sequence having SEQ ID NO . 1 in the sequence table
Mutation and other steps to obtain a mutated gene encoding SEQ ID NO. 4 , SEQ ID N 0.6 or SEQ ID N 0.8 amino acid sequence;
[0031] b ) inserting the mutant gene into a plasmid to obtain an expression vector;
[0032] c ) transforming the expression vector into the strain to obtain recombinant genetically engineered bacteria;
[0033] d ) Penicillin V acylase is obtained by fermentation of recombinant genetically engineered bacteria with 1% lactose .
[0034] Further, it also includes a purification step, the purification step is to break down the recombinant genetically engineered bacteria producing penicillin V acylase

After crushing, centrifuging, and collecting, it was purified by immobilized metal chelating affinity chromatography to obtain pure penicillin V acylase enzyme.
[0035] Further, it also includes an enzyme immobilization step, the immobilization step is the above purified enzyme solution, using phosphate
Buffer (the pH 6.0,0. I mole / L ) were dissolved, then added to 50 g at 25 ° after the treatment the activated support, C , 120 RPM under conditions
Stirring and immobilizing at low speed for 48 h until the adsorption is saturated, the obtained immobilized enzyme is washed repeatedly with deionized water 3 to 5 times, vacuum filtered
After drying, the final immobilized enzyme product is obtained.
[0036] The activation process of the carrier: accurately measure 60% glutaraldehyde 30 ml , dipotassium hydrogen phosphate ( K 2 HPO 4 · 3 H 2 O )
4.76 g was dissolved in 600 ml of deionized water, and finally made up to 1000 ml with deionized water , while adjusting its pH with phosphoric acid solution

6.0, after sterilization standby; epoxy-based support ECEP or amino vector the ECHA / S (Italy Resindion S . R & lt . Company. 1)
250 g was put into the above solution, and activated at 25 °C with low speed stirring for 2 h , the carrier was collected by filtration, and repeated with sterile deionized water
After rinsing for 2~3 times, vacuum dry.
[0037] Further, the immobilized carrier is an epoxy carrier ECEP or an amino carrier ECHA / S .
[0038] Further, the plasmid preferably the pET 30 A (+), preferably a strain of E . Coli BL 21 is ( DE . 3).
[0039] The present invention uses random mutations, site-directed mutations and other enzyme engineering techniques to source Bacillus sphaericus
The penicillin V acylase was mutated to obtain improved catalytic activity, reduced inhibition of high concentration substrates and enhanced stability

PVA mutants can more efficiently and quickly catalyze the hydrolysis of penicillin V to prepare 6- APA .
[0040] First, a preferred mutant named PVA - I was obtained , and the threonine at position 91 of the penicillin V acylase was mutated to propionate
Lysine. The activity of this mutant is higher than that of wild-type penicillin V acylase. It is further preferred to obtain penicillin V acylase No. 91
The threonine at position 280 was mutated to alanine, and the isoleucine at position 280 at phenylalanine was named PVA -2. The
The mutant meets the requirements of high activity and also has tolerance to high concentrations of penicillin V. Especially the mutations obtained by the present invention
Body the PVA -3 (penicillin V acylase threonine at position 91 mutated to alanine, at position 280 mutated to isoleucine benzenepropanoic
Amino acid, the serine at position 110 was mutated to cysteine), the specific activity was increased by 9 times, and the immobilization activity was increased from 92 U / g to
820 U / g , incubation for 1 hour under the condition of 30% penicillin V potassium salt, the residual activity is 89.1%; immobilized mutant

The PVA -3 applied to the pH 6.0,25 [deg.] C cleaved 25% strength under conditions of penicillin V Preparation of 6- APA reaction time of 180 minutes

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Shortened to 60 minutes, substrate conversion rate increased to more than 99.5%, and continuous use of more than 600 batches, no significant loss of vitality
Loss, with good operational stability. The mutant PVA -3 greatly reduces the production cost and improves the production efficiency,
More suitable for industrial applications.

[0041] The drawings that form a part of this application are used to provide a further understanding of the present invention, but do not constitute a
 Improperly specified.

BRIEF DESCRIPTION

[0042] FIG. 1 is a flowchart of construction of a recombinant expression vector pET-pva-wt according to an embodiment of the present invention ;

detailed description

[0043] The following examples and their descriptions are used to explain the present invention, but do not constitute an undue limitation of the present invention.

[0044] 1. The methods used in the following examples are conventional methods unless otherwise specified, such as "Molecular Cloning Experiment Guide"
South "( J . Sambrook, D . W . Russell with, Huang Peitang, Wang Jia Xi, Zhu thick foundation, translated 3rd edition, Beijing: Science Publishing
Co., 2002). Mutant primers and sequence sequencing are both Taihe Yongchang (Changsha) Biotechnology Co., Ltd.
Division is completed. The E. coli host strain was purchased from Merck , E. coli BL 21 ( DE 3) . The E. coli host strain can also be

E.coli BL 21 ( DE 3) plys , purchased from Tiangen Company, prokaryotic expression vector pET 30 a (+) was purchased from Merck company ANA Endo
The enzyme Dpn I was purchased from Fermentas . The automatic potentiometric titrator was purchased from Metrohm, Switzerland, the model 8 series was purchased from METT
Le Toledo, the rest of the materials are commercially available. Meanwhile, the amino acids in the present invention are abbreviated or used unless otherwise specified.
The code number is indicated (see the English and Chinese amino acid names and their abbreviations and code numbers in Table 1).

[0045] Table 1 Chinese and English names of amino acids and their abbreviations and codes
[0046]
Chinese name English name abbreviation
Code Chinese name English name abbreviation
Code
Alanine Alanine Ala A Proline Proline Pro P
Arginine Arginine Arg R Leucine Leucine Leu L
Asparagine Asparagine Asn N Isoleucine Isoleucine lie I
Aspartic acid Asp D Glycine Glycine Gly G
Cysteine Cysteine Cys C Phenylalanine Phenylalanine Phe F
Glutamine Glutamine Gln Q Methionine Methionine Met M
Glutamate Glutamicacid Glu E Lysine Lysine Lys K
Threonine Threonine Thr T Histidine Histidine His H
Tryptophan Tryptophan Trp W Valine Valine Val V
Serine Serine Ser S Tyrosine Tyrosine Tyr Y
[0047] 2 , penicillin V enzyme activity detected acylating

[0048] ⑴ Titration
[0049] Principle: Penicillin V is converted into 6-APA and phenoxyacetic acid under the action of penicillin V acylase , neutralized with NaOH solution
Phenoxyacetic acid, calculated penicillin V acylase activity according to the consumption of NaOH .

[0050] DETAILED OPERATION: . 1 . . 5 m. 1 centrifuge tube previously added glass beads, glass beads reached when the scale centrifuge tube
Stop at 1.0 ml , accurately measure 0.5 ml of the penicillin V acylase in Examples 1 to 3 in a centrifuge tube at an oscillation frequency of
1500 / min to MS3 on oscillator IOmin , the sample together with glass beads, is added to a [deg.] With preheated to 25 C in

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In a reactor of 30 ml of a 1% penicillin V potassium salt substrate solution, adjust the temperature of the water bath to 25° C and use 0. lmol / L sodium hydroxide
When the titrant adjusts the pH to 6.00, start timing. Keep the pH at 6.00 and stir for 5 minutes to react. Use a fully automatic potentiometric titrator
Detect the consumption of sodium hydroxide titrant. The calculation formula of enzyme activity in the titration method is:

X ( X 10()0
[0051] Enzymatic activity =... tons (iv)%) (Tao or special

[0052] where V n- 3OH : sodium hydroxide solution consumption, ml .

Lmol / L 0 [0053] C _ h : the molar concentration of sodium hydroxide titration solution is 0. lmol / L 0
[0054] T : reaction time, min .

[0055] V sample: sample volume of enzyme solution, ml .

[°°56] Sample W : weight of immobilized enzyme sample, g .

[0057] 1000 : from mmol to ymol .

[0058] The enzyme activity unit is defined as ⑹: at 25 [deg.] C , pH 6.0 under conditions, Imin the catalyst substrate was penicillin V generating 1μ
The amount of enzyme required for mol phenoxyacetic acid is 1U .
[0059] (2) High performance liquid chromatography (HPLC) method
[0060] Accurately take 20 μl of the penicillin V acylase in Examples 1 to 5 and perform centrifugal filtration to take the supernatant. Supernatant
The solution was mixed with 0.01 mol / L , pH 6.00 phosphate buffer containing 5% ( W / V ) penicillin V to a final volume of 200 μ1 to obtain
The system at the beginning of the reaction. After reacting the system at 25° C for 10 minutes at the beginning of the reaction , 200 μ 1 of 40% glacial acetic acid was added to terminate
Reaction, the system at the end of the reaction. The system at the beginning of the reaction and the system at the end of the reaction were analyzed by HPLC .
[0061] Dissolve sample preparation: Weigh 1.542 g of ammonium acetate and 950 ml of ultrapure water to dissolve, adjust the pH to 3 mol / L ammonia
 7.0, 0.45 μηι water-based membrane filter, mix evenly and set aside.
[0062] Mobile phase preparation: Weigh 1.542g ammonium acetate and 965ml ultrapure water to dissolve, adjust the pH to 3mol / L ammonia water

7.0, 0.45μηι water-based membrane filter, add 50ml of chromatographically pure acetonitrile, mix and degas for 30min .

[0063] Detection wavelength: 254 nm .

[0064] Column type: BDS C 18 200 mmX 4.6 mm .

[0065] Total flow rate: 1.0 ml / min .
[0066] Injection volume: 20 μl .

[0067] The enzyme activity unit is defined as ⑹: at 25 [deg.] C , pH 6.0 under conditions, Imin the catalytic substrate penicillin V generating 1μ
mol product was 6-ΑΡΑ amount of enzyme is needed. 1 the U- .
[0068] Example 1 : Construction and expression of wild-type PVA recombinant genetically engineered bacteria and purification and immobilization of recombinant proteins

[0069] 1-1 Construction of wild-type PVA prokaryotic expression vector and construction of recombinant genetically engineered bacteria
[°07°] The wild-type PVA gene and amino acid sequence used in the present invention are derived from Bacillus sphaericus

(GeneBank accession number: P12256.1) , the coding gene is optimized and carried out by E. coli codon preference
Whole gene synthesis, and the wild-type penicillin V acylase was named PVA-WT , and its coding gene was named pva-wt , nucleoside
The acid sequence and amino acid sequence are shown in SEQ ID NO : 1 and SEQ ID NO : 2 .

[0071] Referring to FIG. 1, the wild-type PVA coding gene pva - wt synthesized with the whole gene and the prokaryotic expression vector pET 30 a (+)
Were EcoR I and Xho I double digestion, respectively, were digested Gel Extraction 3 hours, the product is recovered according to the product: containing
The molar ratio of 3:1 was mixed, and T 4 DNA Iigase was added at 16° C for overnight ligation. Transfer 5 μ 1 of ligation product to 50 μ
1 DH . 5 A competent E. coli, the coating containing 50 YG / ml kanamycin LB solid medium plates [deg.] To 37 [ C underway
Incubate overnight. Select single colonies for colony PCR verification, inoculate positive clones in LB culture containing 50 yg / ml kanamycin

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Culture overnight, extract plasmids, verify with EcoR I and Xho I double digestion, and send clones of correct size to sequencing company
After DNA sequencing verification, after sequence alignment is correct, the recombinant expression vector was named pET-pva-wt , so that we got
There is a His-tag plasmid at both the N- terminus and C- terminus , and the wild-type PVA protein expressed with two histidine tags can be
Protein purification was performed by immobilized metal chelate affinity chromatography (IMC) .

[0072] The above-mentioned recombinant expression vector pET-pva-wt is transformed into E. coli BL21 (DE3) or by chemical transformation
By E. coli BL21 (DE3) plys competent cells, transformed cells were plated containing 50yg / ml kanamycin LB solid culture
Recombinant wild-type PVA genetically engineered bacteria were obtained by culturing the base plate at 37°C overnight .

1-2 Expression of recombinant wild-type PVA protein

[0074 ] The above recombinant genetically engineered bacteria were inoculated in a shake flask containing 50 μg / ml kanamycin 100 ml LB medium (connected
Species . 3 th flasks) within 37 ° C shaker in culture fermentation, until 0 D 600 value of 0 .8 _ 1.2 , the addition of O . 5 mM of

IPTG induced expression for 8 hours. A certain bacterial solution was taken to determine the activity of recombinant genetically engineered PVA enzyme. The results showed that: wild type

PVA enzyme activity is 5U / ml .

1-3 Isolation, purification and immobilization of recombinant wild-type PVA protein

[0076] (1) Isolation and purification of recombinant wild-type PVA protein

[0077] Since the introduction of the expression vector in prokaryotic expression vector was constructed during the pET 30 A (+) in the N and C terminal 2
His - tag , therefore, the inventors used the histidine tag to perform immobilized metal chelate affinity chromatography ( IMAC ) to purify heavy
The specific method of histone is as follows.

[0078] Take 100 mL of wild-type PVA fermentation broth after overnight induction , centrifuge and discard the supernatant to collect bacterial cells (10 OOOrpm , 4
° C , IOmin ), wash the cells twice with phosphate buffer (pH 6.0, 0.1 Imo 1/ L ), collect the cells by centrifugation, and concentrate 5
Resuspended fold 20 is ml phosphate buffer (the pH 6.0,0. Lmol / L ) of. Place the bacterial solution after the above treatment in ice water
Ultrasonic crushing until clarification, the ultrasonic crushing conditions are: working 2 s , interval 5 s . Place the broken lysate above
Centrifuge in a low-temperature high-speed centrifuge (12,000 rpm , 4° C , 20 min ), collect the supernatant, and obtain recombinant wild-type PVA protein.
The crude recombinant protein was injected onto IDA resin that had been activated and bound to Ni + , and gradient elution was performed with imidazole at different concentrations.
The protein chromatography system ( Bio - Rad ) monitors in real time. When a stable protein peak appears in the computer, the collection starts until the
Until the peak disappears. The recombinant enzyme protein was separated and purified, sealed in a sterile bag, and placed in a refrigerator at 4° C for subsequent experiments.
The specific activity of the wild-type PVA was determined to be 6U / mg .

[0079] (2) Immobilization of recombinant wild-type PVA protein

[0080] A , immobilization activated

[0081] Accurate measurement of 60 % glutaraldehyde 30ml , dipotassium hydrogen phosphate (K2HPO4·3H20) 4.76g dissolved in 600ml to dissociate
In deionized water, finally make up to 1000ml with deionized water , and adjust its pH to 6.0 with phosphoric acid solution , after sterilization;
Oxygen carrier ECEP or amino carrier ECHA / S (Italian ResindionS.r. 1 company) 250g was put into the above solution, and

Stir and activate at 25°C for 2h at low speed , collect the carrier by filtration, and rinse it repeatedly with sterile deionized water for 2 to 3 times, then vacuum filter to dry
use.

[0082] B. Enzyme immobilization

[0083] Take a certain amount of the above purified enzyme solution, dissolve with phosphate buffer (pH 6.0, 0. lmol / L) , and then add
 50g of the activated carrier was stirred and immobilized at 25°C and 120rpm at low speed for 48h , and the resulting immobilized enzyme was used.
Ionized water was repeatedly washed 3 to 5 times, and the final product of immobilized enzyme was obtained after vacuum filtration. Accurately weigh Ig for the above immobilized enzyme
Viability assay to ECEP and the ECHA / S as the carrier were immobilized enzyme activity of 92U / G and 71U / G , an epoxy group ECEP solid
The activity of the stabilizing enzyme is significantly higher than that of the amino carrier ECHA / S immobilizing enzyme. Therefore, ECEP was selected as the immobilization of penicillin V acylating enzyme

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化carrier.
[0084] Example 2: Preparation of high activity PVA mutants
[0085] 2-1 Construction of PVA mutant library

[0086] In order to improve the vitality of wild-type PVA , the inventors used recombinant expression vector pET - pva - wt as a DNA template, which
The middle primer is T 7 universal primer ( SEQ ID N0 : 9 and 10) , a random mutant library is constructed by error-prone PCR method,
And by adjusting the concentration of Mg 2+ and Mn 2+ in the error-prone PCR reaction system and the concentration of dCTP and dTTP oligonucleotides, the mutant

The base mismatch rate of the library is five thousandths, that is to ensure that 1 to 3 amino acids of a mutant are mutated to construct a mutant
The specific process of the library is as follows.

[0087] Error-prone PCR reaction system:

IOX Buffer 5 μίν

2 mmol / L dNTPS 5 |xL

100 mmol / L dCTP 0.5 μ 1.

1 OOmmoH dTTP 0>5 (iL

10 mmol / L MnCb 5 |xL

50 mmol / L MgSOj 5 pL

Primer T 7 promoter 2 pL

Bow丨T 7 Terminator 2 ^iL

Template pET - pw-wt IgL

TaqDNA polymerase 1.5 tuL

ddH 20 22.5 'uL

[0090] The error-prone PCR products obtained above are subjected to electrophoresis and gel extraction to recover and purify the purified products and the prokaryotic table
The vector pET 30 a (+) was digested with EcoR I and Xho I , and digested for 3 hours, and then cut and recovered separately.
The materials were mixed in a 3:1 molar ratio of product to carrier, and T 4 DNA 1 igase was added and ligated at 16° C overnight. second
One day, constructing engineering bacteria according to the method of Example 1-1, a random mutant library with a large library capacity can be obtained.
[0091] 2-2 High -throughput screening of high-activity PVA mutant libraries and preparation of mutants
[0092] Using sterile toothpicks, inoculate a single colony in the PVA mutant library into 96-well cell culture with LB medium
In the mother board, the volume of LB medium is 200 yL /well, and contains 50 yg / ml of kanamycin at 37° C , constant 250 rmp
After incubating for 8 hours in a warm shaker, take 50 μL of bacterial solution in another new 96-well cell culture plate as a daughter plate and place it at 4 °C
Refrigerator for conservation. After conservation, was added 0.5 mM of IPTG to a motherboard, [deg.] To 25 C , 250 RMP constant temperature shaker in the induction medium and then
Keep 8 hours. After induction, place the 96-well cell culture plate in an ultra-low temperature refrigerator at -86° C for 2 hours for freezing and thawing, remove and place
Place at room temperature for half an hour, then place in a 96-well cell culture plate centrifuge at 4,000 rpm and centrifuge at 4° C for 20 minutes. above
Said enzyme solution, a 1: 2 volume ratio was added 0.05 mol / L , the pH phosphate buffer, 6.0 [deg.] To 25 C and incubated for 1 hour.
[0093] Take 10 μL of supernatant and add it to a 96-well microtiter plate containing 100 μL concentration ( W / V ) of 10% penicillin V potassium salt and place
[Deg.] At 25 C the reaction temperature and humidity incubator two hours, the solution was added to terminate 160. yL after, plated in 96 well cell culture plates were centrifuged

Page 10

Instructions
CN 109161540 A
8 / 12 Ye

Centrifuge at 4, OOOrmp , 4° C for 20 minutes in the machine .

[0094] taken 50yL supernatant solution containing IOOyL color development solution coloration microtiter plates 3 Minutes later, placed on a microplate reader
In, the absorbance is measured at a wavelength of 405 nm .
 [0095] Termination solution: first dispose 100mmol / L NaOH and 40% acetic acid, and mix according to the ratio of 1:2.
[0096] color liquid : 0 . . 5 % (W is / V) of dimethylaminobenzaldehyde (PDAB) was added.

[0097] Approximately 20,000 clones were screened from the above mutant library, and 10 color changes were obtained with significant values
- growing PVA high mutants. Then rescreen these 10 mutants, the specific process is: inoculate these 10 mutants in
Ferment and induce in a 500 ml shake flask containing 100 ml LB medium , and measure the viability by titration to obtain a comparison
The clone with a 2-fold higher activity was named PVA - I . The sequencing results showed that the amino acid of PVA - I was changed at position 91
Change from T mutation became A .

[0098] Example 3: Preparation of high substrate concentration tolerant PVA mutants

[0099] Since the wild-type PVA activity by penicillin V concentration inhibiting, in order to obtain Penicillin V higher concentrations tolerated mutant
To improve its catalytic efficiency, the inventors conducted directed evolution mutation based on the above mutant PVA - I again, according to
The method in the above example was used to construct a mutant library and conduct screening (the concentration of penicillin V potassium salt ( W / V ) was increased from 10% to
20%). Approximately 20,000 clones were selected from the above mutant library, and 5 color changes were obtained and the value was significantly higher than that of PVA-I
High mutant. After repeated screening in a shake flask, a clone with twice the activity of PVA - I was obtained and named as PVA -2.
The results show that PVA -2 has an amino acid change at position 91 relative to the wild-type penicillin V acylase, which is mutated from T to

A , the 280th site amino acids changed from I mutation would be F. . And the mutant has high tolerance to penicillin V in high concentration
To improve.
Example 4: Preparation of PVA mutants with improved stability
[0101] Many studies have shown that the formation of disulfide bonds has the function of stabilizing the spatial structure of proteins and maintaining their active functions.
Extremely important influence. Therefore, we use the disulfide bond prediction analysis software ( DiANNAl . 1) to analyze the whole protein of PVA ,
It was found to have 3 Cs , of which 2 Cs at positions 4 and 286 formed a pair of disulfide bonds, and the C at position 116 was free
The free C will affect the stability of PVA protein structure. In order for the C at position 116 to form a disulfide bond, we will
The S at position 110 is mutated into C at a certain point , and analyzed by DiANNAl.1 software, these two Cs form a stable disulfide bond, and the analysis result
As shown in Table 2 below.
Table 2

10

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Instructions
CN 109161540 A
9 / 12 Ye

DhsJ on ted sccir<i»

.1 ^ vx ^^ cv ·

[0103]

B: m light d ship g

[0104] The specific experimental process is as follows:

[0105] Mutant primer:

[0106] SllOCf : TATGTGATTTGCCAGGTGCTGGGCAACTGCGTGACCGTG

[0107] SllOCr : CAGCACCTGGCAAATCACATACACCGGATTAATGCCGGTG
[0108] Using PVA-2 as a template and Sl IOCf / SI IOCr as a mutant primer, full- length amplification is performed using whole plasmid PCR technology.
The amplified full-length gene was digested with DpnI to remove the template, purified, and transformed into BL 21 ( DE 3) at 37° C .
 After incubation at night, after sequencing is correct, a mutant that mutates Ser at the 110th position into Cys is obtained, and we name it as
The PVA -3, wherein PCR used of KOD - Plus - Neo fidelity DNA polymerase and the appropriate PCR buffer, of Mg 2+, dNTPs solutions were
Purchased from Τ 0 Υ 0 Β 0 company, and refer to its manual to configure the PCR reaction system and set the PCR reaction conditions.

[0109] PVA -3 was subjected to shake flask fermentation, purification and immobilization according to the method in Example 1 . At the same time, different PVA in
The thermal stability at 50° C was measured and analyzed.
[0110] It was found that the mutants PVA - UPVA -2 and PVA -3 have a half-life of 50° C relative to the wild-type PVA
There is an improvement, of which the effect of PVA -3 is the most significant, and the half-life is increased to 8 times of the original. The specific results are shown in Table 3. This is because

The Sl IOC mutation introduced in PVA -3 forms a disulfide bond with C at position 116 , which makes the enzyme protein structure more stable,
Thereby significantly improving the thermal stability of the enzyme.
[0111] Table 3 The thermal stability of each PVA mutant and wild-type PVA at 50 ° C

[ 0112 ]
Numbering Amino acid mutation point
Ti /2 (min) Numbering Amino acid mutation point Ti /2 (min)

PVA-WT Wild type 11 PVA-2 T91A / I280F 15.8

PVA-I T91A 15.6 PVA-3 T91A / I280F / S110C 88.2

[0113] Example 5 mutant PVA-3 immobilized enzyme properties and preparation of 6-APA application
[0114] 5-1 mutant PVA-3 enzymatic properties

[0115] According to the method of Example 1 , the above-mentioned wild-type and mutant PVA pure enzymes and immobilized enzymes were subjected to activity detection,
The enzyme properties of mutant PVA-3 and other mutants are shown in Table 4 .

[0116] Table 4 wild type and mutant PVA enzymatic properties comparison
[0117]
Enzyme Mutation site Specific activity ( U / mg ) Immobilized enzyme activity ( U / g )

11

Page 12

CN 109161540 A Instructions 10 / 12 Ye

PVA-WT no 6 92

PVA-I T 9 IA 11.3 177

PVA -2 T 91 A / I 280 F 23.2 328

PVA -3 T 91 A / I 280 F / S 110 C 55.3 820

[0118] It can be seen from Table 3 that the mutant PVA has a 9-fold increase in specific activity relative to the wild-type PVA , and the immobilization activity is from 92 U / g
Increased to 820 U / g , indicating that the mutant immobilized enzyme has a better application prospect than the wild-type immobilized enzyme.
[0119] 5-2 mutant PVA -3 immobilized enzyme substrate tolerance experiment
[0120] The above-mentioned wild-type and mutant PVA immobilized enzyme, with the same enzyme amount (1000 U activity) were placed in 5%,
10%, 15%, 20%, 25% and 30% penicillin V potassium salt substrate solution, incubate at 25° C for 1 hour, then remove and fix
The enzyme is washed repeatedly with sterile deionized water 4 to 5 times to ensure that there is no buffer residue in the immobilized enzyme.
The residual activity of the immobilized enzyme was measured. The specific results are shown in Table 5.
[0121] Table 5 wild type and mutant PVA immobilized enzyme substrate tolerance comparison
[0122]

Kyrgyzstan ADM V concentration

Numbering Residual vitality Remnant Penicillin V concentrationNumbering Residual vitality ( U) Residual rate

Degree (W/V) CU.) mm.

PVA-WT 966 96.6% IiVA-WT 486 48.6%

5% PVA--I '972 97.2% 20% PVA-I 658 65.8%

PVA-2 99.9 99.9% PVA-2 966 96.6%

[0123]

PVA-3 : 1000 100% PVA-3 996 99 . 6 %

FVA-W 1 813 8:1,3% PVA-W 1 334 33,4 : %·

10% PVA-1 92§: 92,8% 25% PVA-I 524 52.4%

PVA-2 995 99.5% PVA-2 9V) 93.9%

PVA-3 9^9 99.9% PVA-3 981 98.1%

PVA-WT 635 6.3.:5:% PVA-WT 185 18.5%

15% PVA-I 788 78,8% 30% PVA-I 369 36.9%

PVA-2 982 _9.8 , 2% PVA-2 827 8.17%

()VA-3 998 99.8% PVV\-3 H9I 89.1%

[0124] According to the results in Table 5, it can be seen that PVA-3 immobilized enzyme maintains about 98 % of the activity under the condition of 25 % substrate concentration ,
 Under the condition of 30% substrate concentration, it still maintains about 90% vitality, and the activity is basically unchanged under the condition of 5%-20%, indicating that
The enzyme remains stable under conditions of high substrate concentration.

[0125] 5-3 wild type PVA and PVA-3 immobilized enzyme hydrolyzed penicillin V to prepare 6-APA experiment
[0126] (1) Hydrolysis

[0127] The above wild-type PVA and PVA-3 immobilized enzyme, the same amount of enzyme (5000U) were placed at a certain concentration

12

Page 13

Instructions
CN 109161540 A
11 / 12 Ye

(10 % , 15 % , 20 % , 25 % and 30 %) in penicillin V solution at 25 °C , pH 6.0 , during the reaction

Continuously add 3 mol / L concentrated ammonia water to neutralize the acid generated during the reaction to keep the pH constant at 6.00, and record the amount of ammonia water added,
When the reaction reaches the end point (the method for determining the end point is: ammonia water automatically stops adding, the pH value remains unchanged for more than 3 min ), remember
Record the total reaction time. The above hydrolysate is filtered, and the immobilized enzyme is repeatedly washed 4 to 5 times with sterile deionized water for use.
[0128] (2). 6 _ APA crystallization reaction
[0129] The above-mentioned filtered hydrolysate was placed in a water bath with a temperature of 0 to 8 °C , and pre-cooled
Methanol in water in the hydrolysis solution, the opening and slowly added dropwise with stirring. 6 mol / L concentration the HC . 1; in 30~45 min dropwise addition of the HCl to

PH 4.20; continue to crystallize in a water bath at 0~4 °C for 1.5 h ; filter the crystallization solution, wash with pre-cooled deionized water first, and then use
Wash three times with acetone, vacuum dry and place in a desiccator to dry overnight. After weighing and bagging, check the 6- APA moisture and content to
And the penicillin V residue and calculate the conversion rate of penicillin V , the specific experimental comparison data is shown in Table 6.
[0130] Table 6 wild type PVA and PVA -3 immobilized enzyme conversion application experiment comparison

Penicillin V concentration Numbering Conversion time Conversion rate ( %Substrate

) residue ( % )

(W/V) (min)

10% PVA-WT 60 99.5 0.5

Ρ\ν\-3 3Q 10() 0

15% PVA-WT 75. 94.36 5.64

PVA-3 40 : 99.9 (U

20% PVA-WT 120 9 !.2 §.:8

PVA-3 50 99.H 0.2

25% PVA--WT 18() 90.5 9.5

PVA-3 60 99.6 0.4

PVA-WT 360 mi !0. 8

PVA-3 80 98.2 IS

[0132] Batch experiment of 5-4 mutant PVA -3 immobilized enzyme operation stability
[0133] It can be seen from Table 5 that the optimal substrate concentration for the PVA -3 immobilized enzyme reaction is 25%. Therefore, in order to better reflect this
For the stability of the mutant, the inventors set the reaction conditions as follows during the experiment: the concentration of penicillin V is 25%, PVA -6
The total amount of immobilized enzyme (820 U / g ) is 5000 U , the reaction pH is 6.0, the reaction temperature is 25° C , and the reaction volume is 1000 ml .
The experimental results are shown in Table 7.
[0134] Table 7 Mutant PVA -3 immobilized enzyme operation stability batch experiment

[0135]
batch Reaction time ( min ) Residual vitality (u/g) batch Reaction time ( min ) Residual vitality (u/g)
1 60 820 300 70 753

50 60 812 400 75 712

100 63 798 500 80 668

13

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Instructions
CN 109161540 A
12 / 12 Ye

200 65 775 600 90 603

[0136] It can be known from the PVA -3 immobilized enzyme operation stability batch experiment that the PVA -3 immobilized enzyme prepared by the present invention undergoes
 In 600 consecutive batches of conversion experiments, the reaction time was not significantly prolonged, and the activity of immobilized enzymes did not decrease significantly.
The PVA -3 immobilized enzyme prepared by the invention has good operational stability.
[0137] The above description of the embodiments is for the convenience of those skilled in the art to understand and apply the present invention
Bright. It is obvious that those skilled in the art can easily make various modifications to these embodiments, and describe the
The general principles are applied to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiment,
According to the disclosure of the present invention, those skilled in the art should make improvements and modifications without departing from the scope of the present invention.
Within the scope of protection.

14

Page 15
Sequence listing
CN 109161540 A 1 / 8 Ye

Sequence listing

< 110 > Hunan Fulaige Biotechnology Co., Ltd.

< 120 > — A penicillin V acylase mutant, coding sequence, recombinant expression vector, genetically engineered bacteria and application

< 130 > 201809

< 160 > 12

< 170 > SlPOSequenceListing 1.0

< 210 > 1

< 211 > 1014

< 212 > DNA
< 213 > Bacillus sphaericus wild-type penicillin V acylase amino acid sequence ( )

< 400 > 1

atgctgggtt gtagtagcct gagcattcgc acgacggacg ataaaagtct gtttgcccgc 60

acgatggatt tcacgatgga accggatagc aaagtgatta ttgtgccgcg taactatggc 120

attcgcctgc tggaaaaaga aaacgtggtg BCBBBBBBCB gctatgcgtt tgtgggcatg 180

ggcagcaccg atattaccag CCCggtgCtg tatgatggcg tgaatgaaaa aggcctgatg 240

ggcgcgatgc tgtattatgc gacctttgcg acctatgcgg atgaaccgaa gaaaggcacc 300

accggcatta atccggtgta tgtgattagc caggtgctgg gcaactgcgt gaccgtggat 360
 gatgtgattg aaaaactgac cagctatacc ctgctgaacg aagcgaatat tattctgggc 420

tttgcgccgc cgctgcatta tacctttacc gatgcgagcg gcgaaagcat tgtgattgaa 480

ccggataaaa ccggcattac cattcatcgt aaaaccattg gcgtgatgac caacagcccg 540

ggctatgaat ggcatcagac caatctgcgc gcgtatattg gcgtgacccc gaacccgccg 600

caggatatta tgatgggcga tctggatctg accccgtttg gtcagggtgc gggcggcctg 660

ggtctgccgg gtgattttac cccgagcgcc cgttttctgc gtgtggccta ctggaaaaaa 720

tacaccgaaa aagcgaaaaa cgaaaccgaa ggcgtgacca acctgtttca tattctgagc 780

agcgtgaaca ttccgaaagg cgtggtgctg accaatgaag gcaaaaccga ttataccatt 840

tataccagcg cgatgtgcgc gcagagcaaa aactactact tcaaactgta cgataatagc 900

cgtattagcg ccgtgagcct gatggcggaa aacctgaata gccaggatct gattaccttt 960

gaatgggacc gtaaacagga ctgaaccaag tgaatgtgat gage 1014

< 210 > 2

< 211 > 338

< 212 > PRT

< 213 > Bacillus sphaericus wild-type penicillin V acylase amino acid sequence ( )

< 400 > 2

Met Leu Gly Cys Ser Ser Leu Ser lie Arg Thr Thr Asp Asp Lys Ser

1 5 10 15

Leu Phe Ala Arg Thr Met Asp Phe Thr Met Glu Pro Asp Ser Lys Val

20 25 30

lie lie Val Pro Arg Asn Tyr Gly lie Arg Leu Leu Glu Lys Glu Asn

15

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CN 109161540 A Sequence listing

2 / 8 Ye

35 40 45

Val Val lie Asn Asn Ser Tyr Ala Phe Val Gly Met Gly Ser Thr Asp
50 55 60

lie Thr Ser Pro Val Leu Tyr Asp Gly Val Asn Glu Lys Gly Leu Met
65 70 75 80

Gly Ala Met Leu Tyr Tyr Ala Thr Phe Ala Thr Tyr Ala Asp Glu Pro
85 90 95

Lys Lys Gly Thr Thr Gly lie Asn Pro Val Tyr Val lie Ser Gln Val
100 105 110

Leu Gly Asn Cys Val Thr Val Asp Asp Val lie Glu Lys Leu Thr Ser
115 120 125

Tyr Thr Leu Leu Asn Glu Ala Asn lie lie Leu Gly Phe Ala Pro Pro
130 135 140

Leu His Tyr Thr Phe Thr Asp Ala Ser Gly Glu Ser lie Val lie Glu
145 150 155 160

Pro Asp Lys Thr Gly lie Thr lie His Arg Lys Thr lie Gly Val Met
165 170 175

Thr Asn Ser Pro Gly Tyr Glu Trp His Gln Thr Asn Leu Arg Ala Tyr
180 185 190

lie Gly Val Thr Pro Asn Pro Pro Gln Asp lie Met Met Gly Asp Leu
195 200 205

Asp Leu Thr Pro Phe Gly Gln Gly Ala Gly Gly Leu Gly Leu Pro Gly
210 215 220

Asp Phe Thr Pro Ser Ala Arg Phe Leu Arg Val Ala Tyr Trp Lys Lys
225 230 235 240

Tyr Thr Glu Lys Ala Lys Asn Glu Thr Glu Gly Val Thr Asn Leu Phe
245 250 255

His lie Leu Ser Ser Val Asn lie Pro Lys Gly Val Val Leu Thr Asn
260 265 270

Glu Gly Lys Thr Asp Tyr Thr lie Tyr Thr Ser Ala Met Cys Ala Gln
275 280 285

Ser Lys Asn Tyr Tyr Phe Lys Leu Tyr Asp Asn Ser Arg lie Ser Ala
290 295 300
 Val Ser Leu Met Ala Glu Asn Leu Asn Ser Gln Asp Leu lie Thr Phe
305 310 315 320

Glu Trp Asp Arg Lys Gln Asp lie Lys Gln Leu Asn Gln Val Asn Val
325 330 335

Met Ser
<210> 3

16

Page 17
Sequence listing
CN 109161540 A 3 / 8 Ye

< 211 > 1014

< 212 > DNA

< 213 > PVA-I mutant (T91A coding gene)

< 400 > 3

atgctgggtt gtagtagcct gagcattcgc acgacggacg ataaaagtct gtttgcccgc 60
acgatggatt tcacgatgga accggatagc aaagtgatta ttgtgccgcg taactatggc 120

attcgcctgc tggaaaaaga aaacgtggtg atcaacaaca gctatgcgtt tgtgggcatg 180

ggcagcaccg atattaccag cccggtgctg tatgatggcg tgaatgaaaa aggcctgatg 240

ggcgcgatgc tgtattatgc gacctttgcg gcctatgcgg atgaaccgaa gaaaggcacc 300

accggcatta atccggtgta tgtgattagc caggtgctgg gcaactgcgt gaccgtggat 360

gatgtgattg aaaaactgac cagctatacc ctgctgaacg aagcgaatat tattctgggc 420

tttgcgccgc cgctgcatta tacctttacc gatgcgagcg gcgaaagcat tgtgattgaa 480

ccggataaaa ccggcattac cattcatcgt aaaaccattg gcgtgatgac caacagcccg 540

ggctatgaat ggcatcagac caatctgcgc gcgtatattg gcgtgacccc gaacccgccg 600

caggatatta tgatgggcga tctggatctg accccgtttg gtcagggtgc gggcggcctg 660

ggtctgccgg gtgattttac cccgagcgcc cgttttctgc gtgtggccta ctggaaaaaa 720

tacaccgaaa aagcgaaaaa cgaaaccgaa ggcgtgacca acctgtttca tattctgagc 780

agcgtgaaca ttccgaaagg cgtggtgctg accaatgaag gcaaaaccga ttataccatt 840

tataccagcg cgatgtgcgc gcagagcaaa aactactact tcaaactgta cgataatagc 900

cgtattagcg ccgtgagcct gatggcggaa aacctgaata gccaggatct gattaccttt 960

gaatgggacc gtaaacagga tattaaacaa ctgaaccaag tgaatgtgat gage 1014

< 210 > 4

< 211 > 338

< 212 > PRT

< 213 > PVA-I mutant (T91A amino acid sequence)

< 400 > 4

Met Leu Gly Cys Ser Ser Leu Ser lie Arg Thr Thr Asp Asp Lys Ser
15 10 15

Leu Phe Ala Arg Thr Met Asp Phe Thr Met Glu Pro Asp Ser Lys Val

20 25 30
lie lie Val Pro Arg Asn Tyr Gly lie Arg Leu Leu Glu Lys Glu Asn

35 40 45
Val Val lie Asn Asn Ser Tyr Ala Phe Val Gly Met Gly Ser Thr Asp

50 55 60

lie Thr Ser Pro Val Leu Tyr Asp Gly Val Asn Glu Lys Gly Leu Met

65 70 75 80

Gly Ala Met Leu Tyr Tyr Ala Thr Phe Ala Ala Tyr Ala Asp Glu Pro

85 90 95
Lys Lys Gly Thr Thr Gly lie Asn Pro Val Tyr Val lie Ser Gln Val

17

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CN 109161540 A Sequence listing 4/8 pages

100 105 110

 Leu Gly Asn Cys Val Thr Val Asp Asp Val lie Glu Lys Leu Thr Ser
115 120 125

Tyr Thr Leu Leu Asn Glu Ala Asn lie lie Leu Gly Phe Ala Pro Pro

130 135 140

Leu His Tyr Thr Phe Thr Asp Ala Ser Gly Glu Ser lie Val lie Glu

145 150 155 160

Pro Asp Lys Thr Gly lie Thr lie His Arg Lys Thr lie Gly Val Met

165 170 175

Thr Asn Ser Pro Gly Tyr Glu Trp His Gln Thr Asn Leu Arg Ala Tyr

180 185 190

lie Gly Val Thr Pro Asn Pro Pro Gln Asp lie Met Met Gly Asp Leu

195 200 205

Asp Leu Thr Pro Phe Gly Gln Gly Ala Gly Gly Leu Gly Leu Pro Gly

210 215 220

Asp Phe Thr Pro Ser Ala Arg Phe Leu Arg Val Ala Tyr Trp Lys Lys
225 230 235 240

Tyr Thr Glu Lys Ala Lys Asn Glu Thr Glu Gly Val Thr Asn Leu Phe

245 250 255

His lie Leu Ser Ser Val Asn lie Pro Lys Gly Val Val Leu Thr Asn

260 265 270

Glu Gly Lys Thr Asp Tyr Thr lie Tyr Thr Ser Ala Met Cys Ala Gln

275 280 285

Ser Lys Asn Tyr Tyr Phe Lys Leu Tyr Asp Asn Ser Arg lie Ser Ala
290 295 300

Val Ser Leu Met Ala Glu Asn Leu Asn Ser Gln Asp Leu lie Thr Phe

305 310 315 320

Glu Trp Asp Arg Lys Gln Asp lie Lys Gln Leu Asn Gln Val Asn Val

325 330 335

Met Ser
< 210 > 5
< 211 > 1014

< 212 > DNA

< 213 > PVA-2 mutant (T91A / I280F coding gene)

< 400 > 5

atgctgggtt gtagtagcct gagcattcgc acgacggacg ataaaagtct gtttgcccgc 60

acgatggatt tcacgatgga accggatagc aaagtgatta ttgtgccgcg taactatggc 120

attcgcctgc tggaaaaaga aaacgtggtg atcaacaaca gctatgcgtt tgtgggcatg 180

ggcagcaccg atattaccag cccggtgctg tatgatggcg tgaatgaaaa aggcctgatg 240

18

Page 19
Sequence listing
CN 109161540 A 5 / 8 Ye

ggcgcgatgc tgtattatgc gacctttgcg gcctatgcgg atgaaccgaa gaaaggcacc 300

accggcatta atccggtgta tgtgattagc caggtgctgg gcaactgcgt gaccgtggat 360

gatgtgattg aaaaactgac cagctatacc ctgctgaacg aagcgaatat tattctgggc 420

tttgcgccgc cgctgcatta tacctttacc gatgcgagcg gcgaaagcat tgtgattgaa 480

ccggataaaa ccggcattac cattcatcgt aaaaccattg gcgtgatgac caacagcccg 540

ggctatgaat ggcatcagac caatctgcgc gcgtatattg gcgtgacccc gaacccgccg 600

caggatatta tgatgggcga tctggatctg accccgtttg gtcagggtgc gggcggcctg 660

ggtctgccgg gtgattttac cccgagcgcc cgttttctgc gtgtggccta ctggaaaaaa 720

tacaccgaaa aagcgaaaaa cgaaaccgaa ggcgtgacca acctgtttca tattctgagc 780

agcgtgaaca ttccgaaagg cgtggtgctg accaatgaag gcaaaaccga ttataccttt 840

tataccagcg cgatgtgcgc gcagagcaaa aactactact tcaaactgta cgataatagc 900

cgtattagcg ccgtgagcct gatggcggaa aacctgaata gccaggatct gattaccttt 960

gaatgggacc gtaaacagga tattaaacaa ctgaaccaag tgaatgtgat gage 1014

< 210 > 6

< 211 > 338

< 212 > PRT

 < 213 > PVA-2 mutant (T91A / I280F amino acid sequence)
< 400 > 6
Met Leu Gly Cys Ser Ser Leu Ser lie Arg Thr Thr Asp Asp Lys Ser

15 10 15

Leu Phe Ala Arg Thr Met Asp Phe Thr Met Glu Pro Asp Ser Lys Val

20 25 30
lie lie Val Pro Arg Asn Tyr Gly lie Arg Leu Leu Glu Lys Glu Asn

35 40 45
Val Val lie Asn Asn Ser Tyr Ala Phe Val Gly Met Gly Ser Thr Asp

50 55 60

lie Thr Ser Pro Val Leu Tyr Asp Gly Val Asn Glu Lys Gly Leu Met
65 70 75 80

Gly Ala Met Leu Tyr Tyr Ala Thr Phe Ala Ala Tyr Ala Asp Glu Pro

85 90 95
Lys Lys Gly Thr Thr Gly lie Asn Pro Val Tyr Val lie Ser Gln Val

100 105 110

Leu Gly Asn Cys Val Thr Val Asp Asp Val lie Glu Lys Leu Thr Ser

115 120 125

Tyr Thr Leu Leu Asn Glu Ala Asn lie lie Leu Gly Phe Ala Pro Pro

130 135 140

Leu His Tyr Thr Phe Thr Asp Ala Ser Gly Glu Ser lie Val lie Glu

145 150 155 160

Pro Asp Lys Thr Gly lie Thr lie His Arg Lys Thr lie Gly Val Met

19

Page 20

CN 109161540 A Sequence listing 6/8 pages

165 170 175

Thr Asn Ser Pro Gly Tyr Glu Trp His Gln Thr Asn Leu Arg Ala Tyr
180 185 190

lie Gly Val Thr Pro Asn Pro Pro Gln Asp lie Met Met Gly Asp Leu

195 200 205

Asp Leu Thr Pro Phe Gly Gln Gly Ala Gly Gly Leu Gly Leu Pro Gly

210 215 220

Asp Phe Thr Pro Ser Ala Arg Phe Leu Arg Val Ala Tyr Trp Lys Lys

225 230 235 240

Tyr Thr Glu Lys Ala Lys Asn Glu Thr Glu Gly Val Thr Asn Leu Phe

245 250 255

His lie Leu Ser Ser Val Asn lie Pro Lys Gly Val Val Leu Thr Asn

260 265 270

Glu Gly Lys Thr Asp Tyr Thr Phe Tyr Thr Ser Ala Met Cys Ala Gln

275 280 285

Ser Lys Asn Tyr Tyr Phe Lys Leu Tyr Asp Asn Ser Arg lie Ser Ala
290 295 300

Val Ser Leu Met Ala Glu Asn Leu Asn Ser Gln Asp Leu lie Thr Phe

305 310 315 320

Glu Trp Asp Arg Lys Gln Asp lie Lys Gln Leu Asn Gln Val Asn Val

325 330 335

Met Ser
< 210 > 7
< 211 > 1014

< 212 > DNA

< 213 > PVA-3 mutant (T91A / I280F / S110C coding gene)

< 400 > 7

atgctgggtt gtagtagcct gagcattcgc acgacggacg ataaaagtct gtttgcccgc 60

acgatggatt tcacgatgga accggatagc aaagtgatta ttgtgccgcg taactatggc 120

attcgcctgc tggaaaaaga aaacgtggtg atcaacaaca gctatgcgtt tgtgggcatg 180

ggcagcaccg atattaccag cccggtgctg tatgatggcg tgaatgaaaa aggcctgatg 240

ggcgcgatgc tgtattatgc gacctttgcg gcctatgcgg atgaaccgaa gaaaggcacc 300

 accggcatta atccggtgta tgtgatttgc caggtgctgg gcaactgcgt gaccgtggat 360
gatgtgattg aaaaactgac cagctatacc ctgctgaacg aagcgaatat tattctgggc 420

tttgcgccgc cgctgcatta tacctttacc gatgcgagcg gcgaaagcat tgtgattgaa 480

ccggataaaa ccggcattac cattcatcgt aaaaccattg gcgtgatgac caacagcccg 540

ggctatgaat ggcatcagac caatctgcgc gcgtatattg gcgtgacccc gaacccgccg 600

caggatatta tgatgggcga tctggatctg accccgtttg gtcagggtgc gggcggcctg 660

ggtctgccgg gtgattttac cccgagcgcc cgttttctgc gtgtggccta ctggaaaaaa 720

20

Page 21
Sequence listing
CN 109161540 A 7 / 8 Ye

tacaccgaaa aagcgaaaaa cgaaaccgaa ggcgtgacca acctgtttca tattctgagc 780

agcgtgaaca ttccgaaagg cgtggtgctg accaatgaag gcaaaaccga ttataccttt 840

tataccagcg cgatgtgcgc gcagagcaaa aactactact tcaaactgta cgataatagc 900

cgtattagcg ccgtgagcct gatggcggaa aacctgaata gccaggatct gattaccttt 960

gaatgggacc gtaaacagga tattaaacaa ctgaaccaag tgaatgtgat gage 1014

< 210 > 8

< 211 > 338

< 212 > PRT

< 213 > PVA-3 mutant (T91A / I280F / S110C amino acid sequence)

< 400 > 8

Met Leu Gly Cys Ser Ser Leu Ser lie Arg Thr Thr Asp Asp Lys Ser

15 10 15

Leu Phe Ala Arg Thr Met Asp Phe Thr Met Glu Pro Asp Ser Lys Val
20 25 30

lie lie Val Pro Arg Asn Tyr Gly lie Arg Leu Leu Glu Lys Glu Asn

35 40 45
Val Val lie Asn Asn Ser Tyr Ala Phe Val Gly Met Gly Ser Thr Asp

50 55 60

lie Thr Ser Pro Val Leu Tyr Asp Gly Val Asn Glu Lys Gly Leu Met

65 70 75 80

Gly Ala Met Leu Tyr Tyr Ala Thr Phe Ala Ala Tyr Ala Asp Glu Pro

85 90 95
Lys Lys Gly Thr Thr Gly lie Asn Pro Val Tyr Val lie Cys Gln Val

100 105 110

Leu Gly Asn Cys Val Thr Val Asp Asp Val lie Glu Lys Leu Thr Ser

115 120 125

Tyr Thr Leu Leu Asn Glu Ala Asn lie lie Leu Gly Phe Ala Pro Pro
130 135 140

Leu His Tyr Thr Phe Thr Asp Ala Ser Gly Glu Ser lie Val lie Glu

145 150 155 160

Pro Asp Lys Thr Gly lie Thr lie His Arg Lys Thr lie Gly Val Met

165 170 175

Thr Asn Ser Pro Gly Tyr Glu Trp His Gln Thr Asn Leu Arg Ala Tyr

180 185 190

lie Gly Val Thr Pro Asn Pro Pro Gln Asp lie Met Met Gly Asp Leu

195 200 205

Asp Leu Thr Pro Phe Gly Gln Gly Ala Gly Gly Leu Gly Leu Pro Gly

210 215 220

Asp Phe Thr Pro Ser Ala Arg Phe Leu Arg Val Ala Tyr Trp Lys Lys

twenty one

Page 22
Sequence listing
CN 109161540 A
8 / 8 Ye
 225 230 235 240
Tyr Thr Glu Lys Ala Lys Asn Glu Thr Glu Gly Val Thr Asn Leu Phe
245 250 255

His lie Leu Ser Ser Val Asn lie Pro Lys Gly Val Val Leu Thr Asn

260 265 270

Glu Gly Lys Thr Asp Tyr Thr Phe Tyr Thr Ser Ala Met Cys Ala Gln

275 280 285

Ser Lys Asn Tyr Tyr Phe Lys Leu Tyr Asp Asn Ser Arg lie Ser Ala

290 295 300

Val Ser Leu Met Ala Glu Asn Leu Asn Ser Gln Asp Leu lie Thr Phe

305 310 315 320

Glu Trp Asp Arg Lys Gln Asp lie Lys Gln Leu Asn Gln Val Asn Val

325 330 335

Met Ser
< 210 > 9

< 211 > 20

< 212 > DNA

< 213 > T7 promoter primer

〈400〉9

taatacgact cactataggg 20

< 210 > 10

< 211 > 19

< 212 > DNA

< 213 > T7 Terminator primer

〈400〉10

gctagttatt gctcagcgg 19
<210> 11

< 211 > 39

< 212 > DNA
< 213 > SllOCf primer ()

〈400〉11

tatgtgattt gccaggtgct gggcaactgc gtgaccgtg 39

< 210 > 12

< 211 > 40

< 212 > DNA

< 213 > SllOCr primer ()

< 400 > 12

cagcacctgg caaatcacat acaccggatt aatgccggtg 40

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Page 23

Specification drawings
CN 109161540 A 1 / 1 Ye

pETmrnm
mmm

Digest

T4 0NAigas€
figure 1

twenty three