CN113584008B - Penicillin G acylase mutant and application thereof - Google Patents

Penicillin G acylase mutant and application thereof Download PDF

Info

Publication number
CN113584008B
CN113584008B CN202110868353.3A CN202110868353A CN113584008B CN 113584008 B CN113584008 B CN 113584008B CN 202110868353 A CN202110868353 A CN 202110868353A CN 113584008 B CN113584008 B CN 113584008B
Authority
CN
China
Prior art keywords
ala
gly
leu
thr
val
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202110868353.3A
Other languages
Chinese (zh)
Other versions
CN113584008A (en
Inventor
周晶辉
赵强
刘亚
赵士敏
刘洋
许岗
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hunan Flag Biotechnology Co ltd
Original Assignee
Hunan Flag Biotechnology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hunan Flag Biotechnology Co ltd filed Critical Hunan Flag Biotechnology Co ltd
Priority to CN202110868353.3A priority Critical patent/CN113584008B/en
Publication of CN113584008A publication Critical patent/CN113584008A/en
Application granted granted Critical
Publication of CN113584008B publication Critical patent/CN113584008B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C12N9/84—Penicillin amidase (3.5.1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01011—Penicillin amidase (3.5.1.11), i.e. penicillin-amidohydrolase

Abstract

The invention belongs to the technical field of enzyme engineering, and relates to a penicillin G acylase mutant and application thereof. The penicillin G acylase mutant has a plurality of mutated amino acid sites in an amino acid sequence shown in SEQ ID NO. 1. The penicillin G acylase mutant of the invention is used for preparing 7-amino-3-desacetoxy cephalosporanic acid (7-ADCA) by catalyzing and hydrolyzing 7-phenylacetylaminoacetoxy cephalosporanic acid (G-7-ADCA), and compared with the enzyme before mutation, the penicillin G acylase mutant has higher specific activity, higher conversion efficiency, lower substrate residue and better organic solvent tolerance, and is more suitable for industrial large-scale production and application.

Description

Penicillin G acylase mutant and application thereof
Technical Field
The invention belongs to the technical field of enzyme engineering, and relates to a penicillin G acylase mutant and a method for preparing 7-ADCA by using the same.
Background
The beta-lactam antibiotics are the antibiotics which are most used clinically and have the widest application, and are mainly classified into penicillin antibiotics and cephalosporin antibiotics. Penicillin antibiotics have the defects of relatively narrow antibacterial spectrum, instability to acid and the like; the cephalosporin antibiotics have strong inhibition effect on gram-positive bacteria and gram-negative bacteria and good broad spectrum. The key intermediates for the synthesis of cephalosporin antibiotics are 7-aminocephalosporanic acid (7-ACA) and 7-amino-3-desacetoxycephalosporanic acid (7-ADCA, structural formula shown in figure 1). The 7-ACA is mainly prepared by taking cephalosporin C as a raw material and hydrolyzing a side chain by an enzyme method, and the prior art is very mature; 7-ADCA is an important medical intermediate for preparing semi-synthetic cephalosporins such as cefadroxil, cefradine, cefaclor and the like, and has large market consumption. The processes for the preparation of 7-ADCA are mainly based on the following two routes: the chemical method and semi-synthetic enzyme method, the chemical method mainly uses penicillin G potassium as raw material, and uses chemical oxidation to obtain penicillin G sulfoxide, then uses silicon esterification and makes chemical ring-expanding rearrangement reaction to obtain expanded cyclic acid (G-7-ADCA). Hydrolyzing the 7-phenylacetyl side chain of the G-7-ADCA through chemical cracking to finally obtain 7-ADCA, wherein the whole process is gradually replaced by using a large amount of organic solvents due to complex process and multiple working procedures; the semisynthetic enzyme method uses penicillin G potassium as substrate, and produces G-7-ADCA by chemical or enzymatic ring expansion, and then produces 7-ADCA by hydrolyzing side chain with penicillin acylase (FIG. 2). As can be seen from the above process routes, the preparation of 7-ADCA, whether by chemical or semi-synthetic enzymatic methods, requires removal of the side chain by G-7-ADCA, and the processes used in the prior art are all prepared by hydrolysis of the side chain by immobilized enzymes.
However, the industrial application shows that: the following problems remain in the preparation of 7-ADCA by the hydrolysis of the side chain by penicillin acylase: the enzyme has poor tolerance to organic solvents (organic solvent residue exists in G-7-ADCA generated by chemical ring expansion), so that the enzyme catalysis performance is reduced, and the reaction of multiple batches is easy to inactivate; the existing enzyme catalysis efficiency and conversion rate are low, a large amount of G-7-ADCA residues exist in the reaction process, and the problems can affect the quality of 7-ADCA and subsequent series antibiotic products taking 7-ADCA as an intermediate, on the basis, the G-7-ADCA is taken as a substrate on the basis of the existing enzyme, the directional screening of the mutant enzyme with better substrate specificity, higher organic solvent tolerance and higher conversion rate and excellent performance is very important when applied to the semi-synthetic antibiotic industry, and the method has very important significance for improving the product quality and quality level of beta-lactam antibiotics in China.
In the previous work, it was found that the catalytic hydrolysis of side chains to form 7-ADCA with mutant penicillin G acylase (patent No. ZL 201510638013.6) as catalyst and G-7-ADCA as substrate has better effect, so as to further improve the catalytic efficiency and reduce the substrate residue. On the basis of the patent, the mutant penicillin G acylase PGA-6 is used as the starting enzyme, and further superposition mutation and directional screening are carried out on the basis, so that the mutated enzyme has higher conversion rate, lower substrate residue and better organic solvent tolerance compared with the PGA-6, and is more suitable for industrial large-scale application.
Disclosure of Invention
The primary object of the present invention is to provide a penicillin G acylase mutant, which can make the mutant have higher specific activity than PGA-6, and can have higher conversion rate, lower substrate residue and better organic solvent tolerance when G-7-ADCA is used as a substrate for catalyzing and preparing 7-ADCA.
To achieve this object, in a basic embodiment, the present invention provides a penicillin G acylase mutant, said mutant having a plurality of amino acid sites mutated in penicillin G acylase PGA-6 of the amino acid sequence shown in SEQ ID No.1, the plurality of amino acid sites mutated including at least one of G477D, L567M and Q683R.
In a preferred embodiment, the present invention provides a mutant of penicillin G acylase, the mutation pattern comprising any of the following 7:
G477D; L567M; Q683R; G477D and L567M; G477D and Q683R; L567M and Q683R; G477D and L567M and Q683R.
Furthermore, the amino acid sequences of the seven mutants are shown in SEQ ID NO.2-8 in sequence.
It is a second object of the present invention to provide a polynucleotide encoding the aforementioned penicillin G acylase mutant, so as to enable the encoded penicillin G acylase mutant to have higher catalytic efficiency, lower substrate residue and better organic solvent resistance than the penicillin G acylase mutant PGA-6. To achieve this, in a basic embodiment, the present invention provides polynucleotides encoding the aforementioned mutants.
The third purpose of the invention is to provide the application of the penicillin G acylase mutant, mainly the method for preparing 7-ADCA by using the penicillin G acylase mutant, so as to better prepare 7-ADCA.
To achieve this object, in a basic embodiment, the present invention provides the use of a penicillin G acylase mutant for preparing 7-ADCA by catalyzing the hydrolysis of G-7-ADCA with the penicillin G acylase mutant as described above in a reaction system.
In a preferred embodiment, the present invention provides the use of a penicillin G acylase mutant with a substrate G-7-ADCA concentration of 6% to 8% and an enzyme activity of 3000 to 6000U/L.
Furthermore, the reaction temperature is 25-28 ℃, and the reaction time is 0.5-3.5h.
Further, the substrate solvent is 0.05-0.2mol/L boric acid buffer solution, and the reaction pH is 7.5-8.5.
In a preferred embodiment the present invention provides a process for the preparation of 7-ADCA wherein said penicillin G acylase mutant is an immobilized penicillin G acylase mutant.
The invention has the advantages that the penicillin G acylase mutant has higher specific activity than PGA-6, higher conversion rate, lower substrate residue and better organic solvent tolerance when catalyzing and preparing 7-ADCA, and is more suitable for industrial large-scale application.
The invention takes the mutant penicillin G acylase PGA-6 with the patent number ZL201510638013.6 as a starting point, and has the following advantages compared with penicillin G acylases from other or wild type sources: higher reaction activity, higher speed, higher substrate concentration, high conversion rate and less residual amount of the substrate. On the basis of PGA-6, the invention mutates PGA-6 by means of genetic engineering and enzyme engineering technology, and the obtained penicillin G acylase mutant has the characteristics and advantages of higher enzyme specific activity, higher catalytic rate and conversion yield, less substrate residue, stronger organic solvent tolerance and the like compared with PGA-6, thereby being more suitable for large-scale application and being used for producing 7-ADCA.
Drawings
FIG. 1 is a schematic diagram of the catalytic synthesis of 7-ADCA by penicillin G acylase.
FIG. 2 is a schematic diagram of the semi-synthetic enzymatic route to 7-ADCA.
Detailed Description
The following description will further describe embodiments of the present invention with reference to the accompanying drawings.
The enzyme activity detection method of the penicillin G acylase and the mutant thereof comprises the following steps:
substrate concentration: 6 percent, a proper amount of a substrate of the ring-expanding acid (G-7-ADCA) is weighed and dissolved in 150mL of 0.1mol/L borate buffer solution with the pH value of 8.0, the volume of the buffer solution is determined to be 200mL, and the solution needs to be prepared for use.
Collecting the fermented thalli, performing ball milling or ultrasonic disruption, centrifuging (12000g 5 min), accurately measuring 0.5mL of the supernatant obtained after the thalli disruption, placing the supernatant into a jacketed glass reactor with 20mL of substrate solution at 28 ℃, stirring, keeping the temperature of a water bath at 28 ℃, adjusting the pH to 8.00 with 0.1mol/L of sodium hydroxide titration solution, reacting for 3min, and recording the consumption of the sodium hydroxide titration solution. (0.1 mol/L sodium hydroxide titration solution is prepared by preparing standard titration solution)
And (3) detecting the activity of the immobilized enzyme, namely accurately weighing 0.20g of immobilized enzyme sample, adding the immobilized enzyme sample into a jacketed glass reactor filled with 80mL of substrate solution preheated to 28 ℃, stirring, keeping the temperature of a water bath at 28 ℃, adjusting the pH to 8.00 by using 0.1mol/L sodium hydroxide titration solution, reacting for 10min, and recording the consumption of the sodium hydroxide titration solution.
Unit of enzyme activity: under certain reaction conditions, the amount of enzyme consuming 1. Mu. MoL NaOH per minute is one unit.
Liquid phase method for detection of product 7-ADCA: a chromatographic column: spherisorb ODS 1 5 μm 4.6X 200mm. Mobile phase: ammonium acetate 1.542g is weighed and added into 965mL of ultrapure water for dissolving, then the pH is adjusted to 7.0 by using ammonia water of 3moL/L, a 0.45 mu m water system membrane is used for filtering, 50mL of chromatographic pure acetonitrile is added, and the mixture is evenly mixed and degassed for 30min for standby. Flow rate: 1.0mL/min, detection: UV225 nm, sample injection: the sample solution and the standard solution were injected in 20. Mu.L each.
Example 1: activation of PGA-6 prokaryotic expression strain E.coli BL21 (DE 3)/pET 30a (+) -PGA-6 and construction of error-prone mutation library
According to the strain information stored in the mutant penicillin G acylase PGA-6 in the patent ZL201510638013.6, a glycerol strain stored at minus 80 ℃ is taken out, streaked on a kanamycin LB solid culture medium containing 50 mu G/mL, cultured overnight at 37 ℃, a single colony is picked the next day, inoculated in a kanamycin LB liquid culture medium containing 50 mu G/mL, cultured overnight at 220rpm and 37 ℃, a plasmid kit (OMEGA) is used for extracting a plasmid, and the plasmid pET30a (+) -PGA-6 is used as a template to construct an error-prone mutation library, wherein the specific process of constructing the error-prone mutation library is as follows:
selecting a conventional universal primer for PCR reaction, wherein the name and the sequence of the primer are as follows: (name of Universal primer: T7F:5'-TAATACGACTCACTATAGGG-3' SEQ ID NO.9, T7R 2+ 、Mn 2+ dCTP and dTTP oligonucleotide concentration, so that the base mismatching rate of the mutant library is only two thousandth, namely ensuring that only 1 to 2 amino acids of one mutant are mutated.
Figure BDA0003188098140000051
/>
Figure BDA0003188098140000061
Error-prone PCR reaction procedure: pre-denaturation at 95 ℃ for 5min; then carrying out denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 1min, and extending at 72 ℃ for 1.5min for 30-35 cycles; finally, extension is carried out for 10min at 72 ℃. And (3) sampling 2 mu L of the error-prone PCR product, carrying out agarose gel electrophoresis detection, and purifying by using a PCR product purification kit after the detection is error-free. Carrying out double digestion on the PCR purified product and a prokaryotic expression vector pET30a (+) by Nde I restriction enzymes and Xho I restriction enzymes respectively at 37 ℃, carrying out gel cutting on the digestion product, recovering (wherein the size of the fragment of the recovered PCR purified product is about 2700bp, and the size of the fragment of the recovered vector pET30a (+) is about 5400 bp) and then according to error-prone PCR products: prokaryotic expression vector pET30a (+) is 3:1, and T4 DNA ligase was added thereto and ligated overnight at 16 ℃. And (3) performing column-loading purification on the ligation product by using a DNA product purification and recovery kit the next day, and then performing an electric shock transformation process. And (3) coating the products after electric shock transformation on a solid culture medium containing 50 mu g/mL kanamycin LB, and growing a bacterial colony on a flat plate to obtain the error-prone random mutant library based on the PGA-6.
Example 2: screening of error-prone mutation library based on PGA-6
Using the sterilized toothpicks, single colonies of the mutant library (1 single colony per toothpick) were carefully picked and inoculated into different wells of a 96-well cell culture plate (LB liquid medium containing 50. Mu.g/mL kanamycin had been added to each well). The 96-well cell culture plate is placed in a constant temperature shaking table to be cultured for 6 hours at 37 ℃ and 700rpm, 50 mu L of cell culture solution is taken out from the culture plate and added into the 96-well enzyme label plate which is treated by aseptic treatment to be used as seed solution for storage, then lactose with the final concentration of 1% (m/v) is added into the 96-well culture plate by an 8-channel pipette, and the induction culture is carried out for 8 hours at 25 ℃ and 250 rpm. After induction culture is finished, the 96-hole cell culture plate is placed into an ultralow temperature refrigerator with the temperature of minus 80 ℃ for freezing for 2 hours, the cell culture plate is taken out and placed at room temperature for half an hour, then 4000r/min is carried out, the cell culture plate is centrifuged for 20 minutes at the temperature of 4 ℃,100 mu L of supernatant is taken from each hole and placed into a 96-hole enzyme label plate for subsequent high-flux screening reaction. Preparing reagents related to the high-throughput screening reaction:
substrate solution: 2G/L of G-7-ADCA ring-expanding acid solution, and is prepared by 0.05M pH 8.0 borate buffer solution;
reaction termination solution: 100mM NaOH,40% acetic acid, in a ratio of 1
Color development liquid: 0.5% (W/V) PDAB (p-dimethylaminobenzaldehyde solution) is prepared with methanol,
high-throughput screening of color reaction:
in a 96-well microplate containing 100. Mu.L of the disrupted cell supernatant, the ratio of 1:1 was added to the reaction substrate solution at 30 ℃. Incubating for 15-30min;
adding 50 μ L of stop solution, mixing, and centrifuging at 4000rpm for 10min;
and adding 50 mu L of supernatant into 50 mu L of developing solution, detecting and analyzing by using an enzyme-linked immunosorbent assay, and selecting the supernatant with high OD405nm light absorption value for subsequent sequencing and conversion experiment verification.
Through several rounds of mutation library construction and screening, repeated screening and verification (about 20000 clones), strains with high light absorption values are selected for sequencing analysis, enzyme activity determination and the like, and 4 strains with higher activity to the expanded ring acid than PGA-6 are obtained, wherein the specific conditions are as shown in Table 1:
table 1: mutant obtained by error-prone mutation screening on the basis of PGA-6
Figure BDA0003188098140000071
Figure BDA0003188098140000081
As can be seen from Table 1, compared with the starting template PGA-6, the fermentation activities of the bacteria of the mutants are obviously improved, the specific activities of the proteins are higher than that of the starting template PGA-6, wherein the activities of the PGA-7 mutants and the PGA-8 mutants are obviously improved, so that the superposition mutation and screening can be considered on the basis of the PGA-7 mutants and the PGA-8 mutants, the catalytic performance of the enzyme on G-7-ADCA is further improved, and the industrial scale application requirements are met.
Example 3: construction and verification of superposition mutants based on PGA-7 and PGA-8
Taking PGA-7 and PGA-8 strains as starting strains, selecting a single colony, inoculating the single colony in an LB liquid culture medium containing 50 mu g/mL of kanamycin, culturing at 220rpm at 37 ℃, collecting thalli the next day, extracting plasmids by using a plasmid kit (OMEGA), taking plasmids pET30a (+) -PGA-7 and pET30a (+) -PGA-8 as templates, carrying out overlapping mutation on obtained mutation sites, and then carrying out favorable mutation site screening, wherein the finally obtained favorable mutation sites and strains are shown in the following table 2:
table 2: construction of additive mutants based on PGA-7 and PGA-8
Figure BDA0003188098140000082
Figure BDA0003188098140000091
As can be seen from Table 2, the activities of the stacked mutants, such as PGA-10, PGA-11, and PGA-13, were all significantly improved, while the activities of the mutant PGA-12 were reduced. In order to further verify the catalytic reaction capability of each enzyme, expression strains of PGA-6 (SEQ ID NO. 1), PGA-7 (SEQ ID NO. 2), PGA-8 (SEQ ID NO. 3), PGA-9 (SEQ ID NO. 4), PGA-10 (SEQ ID NO. 5), PGA-11 (SEQ ID NO. 6), PGA-12 (SEQ ID NO. 7) and PGA-13 (SEQ ID NO. 8) are selected for fermentation culture, and target proteins are respectively separated, purified and immobilized for catalyzing the hydrolysis of G-7-ADCA to generate 7-ADCA.
Example 4: PGA-6 and mutant protein purification and immobilization
The supernatants obtained in examples 2 and 3 were purified using His-tag carried by PGA-6 and mutant recombinant proteins using activated IDA Resin (purchased from Andron, beijing Biotechnology Ltd., specific model: his. Bind Resin, ni-charged) by the following method: centrifuging the fermentation liquid for 10min at 4 ℃ and 10000r/min, discarding the supernatant, collecting the thallus, repeatedly washing the thallus twice with phosphate buffer solution (pH 8.0 and 0.1 mol/L), centrifuging, and concentrating the thallus by 5 times and suspending in 20mL of phosphate buffer solution (pH 8.0 and 0.1 mol/L). And (3) placing the treated bacterial liquid in ice water for ultrasonic crushing until the bacterial liquid is clarified, wherein the ultrasonic crushing conditions are as follows: work 2s, interval 5s, ultrasonic power 500W. And (3) placing the crushed lysate into a low-temperature high-speed centrifuge for centrifugation (12000 rpm, 4 ℃ and 20 min), and collecting supernatant to obtain crude protein. Loading the crude protein onto the activated IDA resin, performing gradient elution by using imidazole solution (2 mM-20 mM), performing real-time monitoring by using a protein chromatography system (Bio-Rad), and collecting the stable protein peak, namely the MATI recombinant protein purified protein, for preparing immobilized enzyme.
The purified PGA-6 and the recombinant protein mutant are used for preparing immobilized enzyme, and the specific method comprises the following steps:
(1) Activating an immobilized carrier: accurately measuring 30mL of 60% (m/v) glutaraldehyde and dipotassium hydrogen phosphate (K) 2 HPO 4 ·3H 2 O) 4.76g is added into 600mL deionized water, dissolved and then the volume is adjusted to 1000mL by deionized water, and the pH value is adjusted to 8.0 by phosphoric acid solution. Adding epoxy carrier ECEP (Italy research S.r.l) 250g into the above solution, activating at 25 deg.C with low speed stirring for 2 hr, filtering to collect carrier, washing with sterile deionized water for 2-3 timesAnd (5) filtering the mixture to dryness in vacuum for later use.
(2) And (3) immobilizing the PGA-6 and the recombinant protein mutant: diluting a certain amount of the purified MATI recombinant protein with a phosphate buffer solution (pH 8.0 and 0.1 mol/L), adding 50g of activated carrier, immobilizing for 48h at 25 ℃ and 120rpm, washing the obtained immobilized enzyme with deionized water for 3-5 times, and vacuum filtering to obtain the final immobilized enzyme product.
Example 5: analysis of reaction performance of PGA-6 and mutant immobilized enzyme catalyzed 7-ADCA
The mutant PGA-7, PGA-8, PGA-9, PGA-10, PGA-11, PGA-12, PGA-13 and the starting protein PGA-6 obtained in example 2 and example 3 were prepared as immobilized enzymes for catalyzing the reaction of producing 7-ADCA under the same conditions, and the activities of the prepared immobilized enzymes were as follows:
table 3: comparison of activities of PGA-6 and mutant immobilized enzymes
Figure BDA0003188098140000101
Figure BDA0003188098140000111
As can be seen from Table 3, the immobilized enzymes of the mutants prepared on the basis of PGA-6 are all higher than that of the original protein PGA-6, and except that the activity of the immobilized enzyme of the PGA-12 mutant is not obviously improved, the activities of the immobilized enzymes prepared by other mutants are obviously improved. In order to further verify the catalytic performance of the immobilized enzyme, the following reaction parameters were set for the evaluation of the catalytic performance of each immobilized enzyme.
Basic reaction conditions: the immobilized enzyme (activity 300-600U) with the same mass was added, the concentration of the substrate cyclizing acid G-7-ADCA was 6% (dissolved in 0.05mol/L boric acid buffer), the pH was kept constant at about 8.0 with 3mol/L ammonia water, the reaction temperature was 28 ℃ and the reaction system was 100mL.
(1) Sampling and determining the residual amount of the substrate cyclized acid G-7-ADCA in the reaction system at different reaction time periods, and performing comparative analysis by taking the residual amount as a measurement standard, wherein the results are as follows:
table 4: PGA-6 and analysis of substrate residues in different time periods of each mutant reaction
Figure BDA0003188098140000112
As can be seen from Table 4, the original enzyme PGA-6 has a significantly greater substrate residue than the mutant enzyme with the reaction time being longer, and the reaction time is 3.5 hours, which shows a significant tailing phenomenon, while the mutant PGA-11 and PGA-13 have a complete substrate reaction with 3 hours, which is more valuable for industrial application than PGA-6.
(2) Influence of organic solvents on the enzymatic performance. In the above reaction system, 0.5% toluene +0.5% pyridine was added to examine the effect on the catalytic reaction process of PGA-6 and mutants, as shown in Table 5:
table 5: influence of organic solvent on catalytic performance of PGA-6 and mutants
Figure BDA0003188098140000121
As is apparent from Table 5, after the organic solvent is added, the catalytic performance of PGA-6 is obviously inhibited, which results in the slowing of the catalytic reaction rate and the large substrate residue, 15.47mg/mL of substrate residue still exists in 3.5h, while the mutants PGA-10, PGA-11 and PGA-13 have smaller tolerance to the organic solvent, the catalytic efficiency is obviously superior to that of PGA-6, especially the PGA-13 mutant, the substrate is completely reacted when the reaction is carried out for 3h, the catalytic performance is hardly affected by the organic solvent, and the method has industrial application value compared with PGA-6 and other mutants.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is intended to include such modifications and variations. The foregoing examples or embodiments are merely illustrative of the present invention, which may be embodied in other specific forms or in other specific forms without departing from the spirit or essential characteristics thereof. The described embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. The scope of the invention should be indicated by the appended claims, and any changes that are equivalent to the intent and scope of the claims should be construed to be included therein.
Sequence listing
<110> Hunan Fulaige Biotechnology Ltd
<120> penicillin G acylase mutant and application thereof
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 823
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Glu Gln Ser Ser Ser Glu Ile Lys Ile Val Arg Asp Glu Tyr Gly
1 5 10 15
Met Pro His Ile Tyr Ala Asn Asp Thr Trp His Leu Phe Tyr Gly Tyr
20 25 30
Gly Tyr Val Val Ala Gln Asp Arg Leu Phe Gln Met Glu Met Ala Arg
35 40 45
Arg Ser Thr Gln Gly Thr Val Ala Glu Val Leu Gly Lys Asp Phe Val
50 55 60
Lys Phe Asp Lys Asp Ile Arg Arg Asn Tyr Trp Pro Asp Ala Ile Arg
65 70 75 80
Ala Gln Ile Ala Ala Leu Ser Pro Glu Asp Met Ser Ile Leu Gln Gly
85 90 95
Tyr Ala Asp Gly Met Asn Ala Trp Ile Asp Lys Val Asn Thr Asn Pro
100 105 110
Glu Thr Leu Leu Pro Lys Gln Phe Asn Thr Phe Gly Phe Thr Pro Lys
115 120 125
Arg Trp Glu Pro Phe Asp Val Ala Met Ile Phe Val Gly Thr Met Ala
130 135 140
Asn Arg Phe Ser Asp Ser Thr Ser Glu Ile Asp Asn Leu Ala Leu Leu
145 150 155 160
Thr Ala Leu Lys Asp Lys Tyr Gly Val Ser Gln Gly Met Ala Val Phe
165 170 175
Asn Gln Phe Lys Trp Leu Val Asn Pro Ser Ala Pro Thr Thr Ile Ala
180 185 190
Val Gln Glu Ser Asn Tyr Pro Leu Lys Phe Asn Gln Gln Asp Ser Gln
195 200 205
Ser Ala Ala Leu Leu Pro Arg Tyr Asp Leu Pro Ala Pro Met Leu Asp
210 215 220
Arg Pro Ala Lys Gly Ala Asp Gly Ala Leu Leu Ala Leu Thr Ala Gly
225 230 235 240
Lys Asn Arg Glu Thr Ile Val Ala Gln Phe Ala Gln Gly Gly Ala Asn
245 250 255
Gly Leu Ala Gly Tyr Pro Thr Thr Ser Asn Met Trp Val Ile Gly Lys
260 265 270
Ser Lys Ala Gln Asp Ala Lys Ala Ile Met Val Asn Gly Pro Gln Phe
275 280 285
Gly Trp Tyr Ala Pro Ala Tyr Thr Tyr Gly Ile Gly Leu His Gly Ala
290 295 300
Gly Tyr Asp Val Thr Gly Asn Thr Pro Phe Ala Tyr Pro Gly Leu Val
305 310 315 320
Phe Gly His Asn Gly Val Ile Ser Trp Gly Ser Thr Ala Gly Phe Gly
325 330 335
Asp Asp Val Asp Ile Phe Ala Glu Arg Leu Ser Ala Glu Lys Pro Gly
340 345 350
Tyr Tyr Leu His Asn Gly Lys Trp Val Lys Met Leu Ser Arg Glu Glu
355 360 365
Thr Ile Thr Val Lys Asn Gly Gln Ala Glu Thr Phe Thr Val Trp Arg
370 375 380
Thr Val His Gly Asn Ile Leu Gln Thr Asp Gln Thr Thr Gln Thr Ala
385 390 395 400
Tyr Ala Lys Ser Arg Ala Trp Asp Gly Lys Glu Val Ala Ser Leu Leu
405 410 415
Ala Trp Thr His Gln Met Lys Ala Lys Asn Trp Gln Glu Trp Thr Gln
420 425 430
Gln Ala Ala Lys Gln Ala Leu Thr Ile Asn Trp Tyr Tyr Ala Asp Val
435 440 445
Asn Gly Asn Ile Gly Tyr Val His Thr Gly Ala Tyr Pro Asp Arg Gln
450 455 460
Ser Gly His Asp Pro Arg Leu Pro Val Pro Gly Thr Gly Lys Trp Asp
465 470 475 480
Trp Lys Gly Leu Leu Pro Phe Glu Met Asn Pro Lys Val Tyr Asn Pro
485 490 495
Gln Ser Gly Tyr Ile Ala Asn Trp Asn Asp Ser Pro Gln Lys Asp Tyr
500 505 510
Pro Ala Ser Asp Leu Phe Ala Phe Leu Trp Gly Gly Ala Asp Arg Val
515 520 525
Thr Glu Ile Asp Arg Leu Leu Glu Gln Lys Pro Arg Leu Thr Ala Asp
530 535 540
Gln Ala Trp Asp Val Ile Arg Gln Thr Ser Arg Gln Asp Leu Asn Leu
545 550 555 560
Arg Leu Phe Leu Pro Thr Leu Gln Ala Ala Thr Ser Gly Leu Thr Gln
565 570 575
Ser Asp Pro Arg Arg Gln Leu Val Glu Thr Leu Thr Arg Trp Asp Gly
580 585 590
Ile Asn Leu Leu Asn Asp Asp Gly Lys Gln Trp Gln Gln Pro Gly Ser
595 600 605
Ala Ile Leu Asn Val Trp Leu Thr Ser Met Leu Lys Arg Thr Val Val
610 615 620
Ala Ala Val Pro Met Pro Phe Asp Lys Trp Tyr Ser Ala Ser Gly Tyr
625 630 635 640
Glu Thr Thr Gln Asp Gly Pro Thr Gly Ser Leu Asn Ile Ser Val Gly
645 650 655
Ala Lys Ile Leu Tyr Glu Ala Val Gln Gly Asp Lys Ser Pro Ile Pro
660 665 670
Gln Ala Val Asp Leu Phe Ala Gly Lys Pro Gln Gln Glu Val Val Leu
675 680 685
Ala Ala Leu Glu Asp Thr Trp Glu Thr Leu Ser Lys Arg Tyr Gly Asn
690 695 700
Asn Val Ser Asn Trp Lys Thr Pro Ala Met Ala Leu Thr Phe Arg Ala
705 710 715 720
Asn Asn Phe Phe Gly Val Pro Gln Ala Ala Ala Glu Glu Thr Arg His
725 730 735
Gln Ala Glu Tyr Gln Asn Arg Gly Thr Glu Asn Val Met Ile Val Phe
740 745 750
Ser Pro Thr Thr Ser Asp Arg Pro Val Leu Ala Trp Asp Val Val Ala
755 760 765
Pro Gly Gln Ser Gly Phe Ile Ala Pro Asp Gly Thr Val Asp Lys His
770 775 780
Tyr Glu Asp Gln Leu Lys Met Tyr Glu Asn Phe Gly Arg Lys Ser Leu
785 790 795 800
Trp Leu Thr Lys Gln Asp Val Glu Ala His Lys Glu Ser Gln Glu Val
805 810 815
Leu His Val Gln Arg Leu Glu
820
<210> 2
<211> 823
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Glu Gln Ser Ser Ser Glu Ile Lys Ile Val Arg Asp Glu Tyr Gly
1 5 10 15
Met Pro His Ile Tyr Ala Asn Asp Thr Trp His Leu Phe Tyr Gly Tyr
20 25 30
Gly Tyr Val Val Ala Gln Asp Arg Leu Phe Gln Met Glu Met Ala Arg
35 40 45
Arg Ser Thr Gln Gly Thr Val Ala Glu Val Leu Gly Lys Asp Phe Val
50 55 60
Lys Phe Asp Lys Asp Ile Arg Arg Asn Tyr Trp Pro Asp Ala Ile Arg
65 70 75 80
Ala Gln Ile Ala Ala Leu Ser Pro Glu Asp Met Ser Ile Leu Gln Gly
85 90 95
Tyr Ala Asp Gly Met Asn Ala Trp Ile Asp Lys Val Asn Thr Asn Pro
100 105 110
Glu Thr Leu Leu Pro Lys Gln Phe Asn Thr Phe Gly Phe Thr Pro Lys
115 120 125
Arg Trp Glu Pro Phe Asp Val Ala Met Ile Phe Val Gly Thr Met Ala
130 135 140
Asn Arg Phe Ser Asp Ser Thr Ser Glu Ile Asp Asn Leu Ala Leu Leu
145 150 155 160
Thr Ala Leu Lys Asp Lys Tyr Gly Val Ser Gln Gly Met Ala Val Phe
165 170 175
Asn Gln Phe Lys Trp Leu Val Asn Pro Ser Ala Pro Thr Thr Ile Ala
180 185 190
Val Gln Glu Ser Asn Tyr Pro Leu Lys Phe Asn Gln Gln Asp Ser Gln
195 200 205
Ser Ala Ala Leu Leu Pro Arg Tyr Asp Leu Pro Ala Pro Met Leu Asp
210 215 220
Arg Pro Ala Lys Gly Ala Asp Gly Ala Leu Leu Ala Leu Thr Ala Gly
225 230 235 240
Lys Asn Arg Glu Thr Ile Val Ala Gln Phe Ala Gln Gly Gly Ala Asn
245 250 255
Gly Leu Ala Gly Tyr Pro Thr Thr Ser Asn Met Trp Val Ile Gly Lys
260 265 270
Ser Lys Ala Gln Asp Ala Lys Ala Ile Met Val Asn Gly Pro Gln Phe
275 280 285
Gly Trp Tyr Ala Pro Ala Tyr Thr Tyr Gly Ile Gly Leu His Gly Ala
290 295 300
Gly Tyr Asp Val Thr Gly Asn Thr Pro Phe Ala Tyr Pro Gly Leu Val
305 310 315 320
Phe Gly His Asn Gly Val Ile Ser Trp Gly Ser Thr Ala Gly Phe Gly
325 330 335
Asp Asp Val Asp Ile Phe Ala Glu Arg Leu Ser Ala Glu Lys Pro Gly
340 345 350
Tyr Tyr Leu His Asn Gly Lys Trp Val Lys Met Leu Ser Arg Glu Glu
355 360 365
Thr Ile Thr Val Lys Asn Gly Gln Ala Glu Thr Phe Thr Val Trp Arg
370 375 380
Thr Val His Gly Asn Ile Leu Gln Thr Asp Gln Thr Thr Gln Thr Ala
385 390 395 400
Tyr Ala Lys Ser Arg Ala Trp Asp Gly Lys Glu Val Ala Ser Leu Leu
405 410 415
Ala Trp Thr His Gln Met Lys Ala Lys Asn Trp Gln Glu Trp Thr Gln
420 425 430
Gln Ala Ala Lys Gln Ala Leu Thr Ile Asn Trp Tyr Tyr Ala Asp Val
435 440 445
Asn Gly Asn Ile Gly Tyr Val His Thr Gly Ala Tyr Pro Asp Arg Gln
450 455 460
Ser Gly His Asp Pro Arg Leu Pro Val Pro Gly Thr Asp Lys Trp Asp
465 470 475 480
Trp Lys Gly Leu Leu Pro Phe Glu Met Asn Pro Lys Val Tyr Asn Pro
485 490 495
Gln Ser Gly Tyr Ile Ala Asn Trp Asn Asp Ser Pro Gln Lys Asp Tyr
500 505 510
Pro Ala Ser Asp Leu Phe Ala Phe Leu Trp Gly Gly Ala Asp Arg Val
515 520 525
Thr Glu Ile Asp Arg Leu Leu Glu Gln Lys Pro Arg Leu Thr Ala Asp
530 535 540
Gln Ala Trp Asp Val Ile Arg Gln Thr Ser Arg Gln Asp Leu Asn Leu
545 550 555 560
Arg Leu Phe Leu Pro Thr Leu Gln Ala Ala Thr Ser Gly Leu Thr Gln
565 570 575
Ser Asp Pro Arg Arg Gln Leu Val Glu Thr Leu Thr Arg Trp Asp Gly
580 585 590
Ile Asn Leu Leu Asn Asp Asp Gly Lys Gln Trp Gln Gln Pro Gly Ser
595 600 605
Ala Ile Leu Asn Val Trp Leu Thr Ser Met Leu Lys Arg Thr Val Val
610 615 620
Ala Ala Val Pro Met Pro Phe Asp Lys Trp Tyr Ser Ala Ser Gly Tyr
625 630 635 640
Glu Thr Thr Gln Asp Gly Pro Thr Gly Ser Leu Asn Ile Ser Val Gly
645 650 655
Ala Lys Ile Leu Tyr Glu Ala Val Gln Gly Asp Lys Ser Pro Ile Pro
660 665 670
Gln Ala Val Asp Leu Phe Ala Gly Lys Pro Gln Gln Glu Val Val Leu
675 680 685
Ala Ala Leu Glu Asp Thr Trp Glu Thr Leu Ser Lys Arg Tyr Gly Asn
690 695 700
Asn Val Ser Asn Trp Lys Thr Pro Ala Met Ala Leu Thr Phe Arg Ala
705 710 715 720
Asn Asn Phe Phe Gly Val Pro Gln Ala Ala Ala Glu Glu Thr Arg His
725 730 735
Gln Ala Glu Tyr Gln Asn Arg Gly Thr Glu Asn Val Met Ile Val Phe
740 745 750
Ser Pro Thr Thr Ser Asp Arg Pro Val Leu Ala Trp Asp Val Val Ala
755 760 765
Pro Gly Gln Ser Gly Phe Ile Ala Pro Asp Gly Thr Val Asp Lys His
770 775 780
Tyr Glu Asp Gln Leu Lys Met Tyr Glu Asn Phe Gly Arg Lys Ser Leu
785 790 795 800
Trp Leu Thr Lys Gln Asp Val Glu Ala His Lys Glu Ser Gln Glu Val
805 810 815
Leu His Val Gln Arg Leu Glu
820
<210> 3
<211> 823
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Met Glu Gln Ser Ser Ser Glu Ile Lys Ile Val Arg Asp Glu Tyr Gly
1 5 10 15
Met Pro His Ile Tyr Ala Asn Asp Thr Trp His Leu Phe Tyr Gly Tyr
20 25 30
Gly Tyr Val Val Ala Gln Asp Arg Leu Phe Gln Met Glu Met Ala Arg
35 40 45
Arg Ser Thr Gln Gly Thr Val Ala Glu Val Leu Gly Lys Asp Phe Val
50 55 60
Lys Phe Asp Lys Asp Ile Arg Arg Asn Tyr Trp Pro Asp Ala Ile Arg
65 70 75 80
Ala Gln Ile Ala Ala Leu Ser Pro Glu Asp Met Ser Ile Leu Gln Gly
85 90 95
Tyr Ala Asp Gly Met Asn Ala Trp Ile Asp Lys Val Asn Thr Asn Pro
100 105 110
Glu Thr Leu Leu Pro Lys Gln Phe Asn Thr Phe Gly Phe Thr Pro Lys
115 120 125
Arg Trp Glu Pro Phe Asp Val Ala Met Ile Phe Val Gly Thr Met Ala
130 135 140
Asn Arg Phe Ser Asp Ser Thr Ser Glu Ile Asp Asn Leu Ala Leu Leu
145 150 155 160
Thr Ala Leu Lys Asp Lys Tyr Gly Val Ser Gln Gly Met Ala Val Phe
165 170 175
Asn Gln Phe Lys Trp Leu Val Asn Pro Ser Ala Pro Thr Thr Ile Ala
180 185 190
Val Gln Glu Ser Asn Tyr Pro Leu Lys Phe Asn Gln Gln Asp Ser Gln
195 200 205
Ser Ala Ala Leu Leu Pro Arg Tyr Asp Leu Pro Ala Pro Met Leu Asp
210 215 220
Arg Pro Ala Lys Gly Ala Asp Gly Ala Leu Leu Ala Leu Thr Ala Gly
225 230 235 240
Lys Asn Arg Glu Thr Ile Val Ala Gln Phe Ala Gln Gly Gly Ala Asn
245 250 255
Gly Leu Ala Gly Tyr Pro Thr Thr Ser Asn Met Trp Val Ile Gly Lys
260 265 270
Ser Lys Ala Gln Asp Ala Lys Ala Ile Met Val Asn Gly Pro Gln Phe
275 280 285
Gly Trp Tyr Ala Pro Ala Tyr Thr Tyr Gly Ile Gly Leu His Gly Ala
290 295 300
Gly Tyr Asp Val Thr Gly Asn Thr Pro Phe Ala Tyr Pro Gly Leu Val
305 310 315 320
Phe Gly His Asn Gly Val Ile Ser Trp Gly Ser Thr Ala Gly Phe Gly
325 330 335
Asp Asp Val Asp Ile Phe Ala Glu Arg Leu Ser Ala Glu Lys Pro Gly
340 345 350
Tyr Tyr Leu His Asn Gly Lys Trp Val Lys Met Leu Ser Arg Glu Glu
355 360 365
Thr Ile Thr Val Lys Asn Gly Gln Ala Glu Thr Phe Thr Val Trp Arg
370 375 380
Thr Val His Gly Asn Ile Leu Gln Thr Asp Gln Thr Thr Gln Thr Ala
385 390 395 400
Tyr Ala Lys Ser Arg Ala Trp Asp Gly Lys Glu Val Ala Ser Leu Leu
405 410 415
Ala Trp Thr His Gln Met Lys Ala Lys Asn Trp Gln Glu Trp Thr Gln
420 425 430
Gln Ala Ala Lys Gln Ala Leu Thr Ile Asn Trp Tyr Tyr Ala Asp Val
435 440 445
Asn Gly Asn Ile Gly Tyr Val His Thr Gly Ala Tyr Pro Asp Arg Gln
450 455 460
Ser Gly His Asp Pro Arg Leu Pro Val Pro Gly Thr Gly Lys Trp Asp
465 470 475 480
Trp Lys Gly Leu Leu Pro Phe Glu Met Asn Pro Lys Val Tyr Asn Pro
485 490 495
Gln Ser Gly Tyr Ile Ala Asn Trp Asn Asp Ser Pro Gln Lys Asp Tyr
500 505 510
Pro Ala Ser Asp Leu Phe Ala Phe Leu Trp Gly Gly Ala Asp Arg Val
515 520 525
Thr Glu Ile Asp Arg Leu Leu Glu Gln Lys Pro Arg Leu Thr Ala Asp
530 535 540
Gln Ala Trp Asp Val Ile Arg Gln Thr Ser Arg Gln Asp Leu Asn Leu
545 550 555 560
Arg Leu Phe Leu Pro Thr Met Gln Ala Ala Thr Ser Gly Leu Thr Gln
565 570 575
Ser Asp Pro Arg Arg Gln Leu Val Glu Thr Leu Thr Arg Trp Asp Gly
580 585 590
Ile Asn Leu Leu Asn Asp Asp Gly Lys Gln Trp Gln Gln Pro Gly Ser
595 600 605
Ala Ile Leu Asn Val Trp Leu Thr Ser Met Leu Lys Arg Thr Val Val
610 615 620
Ala Ala Val Pro Met Pro Phe Asp Lys Trp Tyr Ser Ala Ser Gly Tyr
625 630 635 640
Glu Thr Thr Gln Asp Gly Pro Thr Gly Ser Leu Asn Ile Ser Val Gly
645 650 655
Ala Lys Ile Leu Tyr Glu Ala Val Gln Gly Asp Lys Ser Pro Ile Pro
660 665 670
Gln Ala Val Asp Leu Phe Ala Gly Lys Pro Gln Gln Glu Val Val Leu
675 680 685
Ala Ala Leu Glu Asp Thr Trp Glu Thr Leu Ser Lys Arg Tyr Gly Asn
690 695 700
Asn Val Ser Asn Trp Lys Thr Pro Ala Met Ala Leu Thr Phe Arg Ala
705 710 715 720
Asn Asn Phe Phe Gly Val Pro Gln Ala Ala Ala Glu Glu Thr Arg His
725 730 735
Gln Ala Glu Tyr Gln Asn Arg Gly Thr Glu Asn Val Met Ile Val Phe
740 745 750
Ser Pro Thr Thr Ser Asp Arg Pro Val Leu Ala Trp Asp Val Val Ala
755 760 765
Pro Gly Gln Ser Gly Phe Ile Ala Pro Asp Gly Thr Val Asp Lys His
770 775 780
Tyr Glu Asp Gln Leu Lys Met Tyr Glu Asn Phe Gly Arg Lys Ser Leu
785 790 795 800
Trp Leu Thr Lys Gln Asp Val Glu Ala His Lys Glu Ser Gln Glu Val
805 810 815
Leu His Val Gln Arg Leu Glu
820
<210> 4
<211> 823
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Met Glu Gln Ser Ser Ser Glu Ile Lys Ile Val Arg Asp Glu Tyr Gly
1 5 10 15
Met Pro His Ile Tyr Ala Asn Asp Thr Trp His Leu Phe Tyr Gly Tyr
20 25 30
Gly Tyr Val Val Ala Gln Asp Arg Leu Phe Gln Met Glu Met Ala Arg
35 40 45
Arg Ser Thr Gln Gly Thr Val Ala Glu Val Leu Gly Lys Asp Phe Val
50 55 60
Lys Phe Asp Lys Asp Ile Arg Arg Asn Tyr Trp Pro Asp Ala Ile Arg
65 70 75 80
Ala Gln Ile Ala Ala Leu Ser Pro Glu Asp Met Ser Ile Leu Gln Gly
85 90 95
Tyr Ala Asp Gly Met Asn Ala Trp Ile Asp Lys Val Asn Thr Asn Pro
100 105 110
Glu Thr Leu Leu Pro Lys Gln Phe Asn Thr Phe Gly Phe Thr Pro Lys
115 120 125
Arg Trp Glu Pro Phe Asp Val Ala Met Ile Phe Val Gly Thr Met Ala
130 135 140
Asn Arg Phe Ser Asp Ser Thr Ser Glu Ile Asp Asn Leu Ala Leu Leu
145 150 155 160
Thr Ala Leu Lys Asp Lys Tyr Gly Val Ser Gln Gly Met Ala Val Phe
165 170 175
Asn Gln Phe Lys Trp Leu Val Asn Pro Ser Ala Pro Thr Thr Ile Ala
180 185 190
Val Gln Glu Ser Asn Tyr Pro Leu Lys Phe Asn Gln Gln Asp Ser Gln
195 200 205
Ser Ala Ala Leu Leu Pro Arg Tyr Asp Leu Pro Ala Pro Met Leu Asp
210 215 220
Arg Pro Ala Lys Gly Ala Asp Gly Ala Leu Leu Ala Leu Thr Ala Gly
225 230 235 240
Lys Asn Arg Glu Thr Ile Val Ala Gln Phe Ala Gln Gly Gly Ala Asn
245 250 255
Gly Leu Ala Gly Tyr Pro Thr Thr Ser Asn Met Trp Val Ile Gly Lys
260 265 270
Ser Lys Ala Gln Asp Ala Lys Ala Ile Met Val Asn Gly Pro Gln Phe
275 280 285
Gly Trp Tyr Ala Pro Ala Tyr Thr Tyr Gly Ile Gly Leu His Gly Ala
290 295 300
Gly Tyr Asp Val Thr Gly Asn Thr Pro Phe Ala Tyr Pro Gly Leu Val
305 310 315 320
Phe Gly His Asn Gly Val Ile Ser Trp Gly Ser Thr Ala Gly Phe Gly
325 330 335
Asp Asp Val Asp Ile Phe Ala Glu Arg Leu Ser Ala Glu Lys Pro Gly
340 345 350
Tyr Tyr Leu His Asn Gly Lys Trp Val Lys Met Leu Ser Arg Glu Glu
355 360 365
Thr Ile Thr Val Lys Asn Gly Gln Ala Glu Thr Phe Thr Val Trp Arg
370 375 380
Thr Val His Gly Asn Ile Leu Gln Thr Asp Gln Thr Thr Gln Thr Ala
385 390 395 400
Tyr Ala Lys Ser Arg Ala Trp Asp Gly Lys Glu Val Ala Ser Leu Leu
405 410 415
Ala Trp Thr His Gln Met Lys Ala Lys Asn Trp Gln Glu Trp Thr Gln
420 425 430
Gln Ala Ala Lys Gln Ala Leu Thr Ile Asn Trp Tyr Tyr Ala Asp Val
435 440 445
Asn Gly Asn Ile Gly Tyr Val His Thr Gly Ala Tyr Pro Asp Arg Gln
450 455 460
Ser Gly His Asp Pro Arg Leu Pro Val Pro Gly Thr Gly Lys Trp Asp
465 470 475 480
Trp Lys Gly Leu Leu Pro Phe Glu Met Asn Pro Lys Val Tyr Asn Pro
485 490 495
Gln Ser Gly Tyr Ile Ala Asn Trp Asn Asp Ser Pro Gln Lys Asp Tyr
500 505 510
Pro Ala Ser Asp Leu Phe Ala Phe Leu Trp Gly Gly Ala Asp Arg Val
515 520 525
Thr Glu Ile Asp Arg Leu Leu Glu Gln Lys Pro Arg Leu Thr Ala Asp
530 535 540
Gln Ala Trp Asp Val Ile Arg Gln Thr Ser Arg Gln Asp Leu Asn Leu
545 550 555 560
Arg Leu Phe Leu Pro Thr Leu Gln Ala Ala Thr Ser Gly Leu Thr Gln
565 570 575
Ser Asp Pro Arg Arg Gln Leu Val Glu Thr Leu Thr Arg Trp Asp Gly
580 585 590
Ile Asn Leu Leu Asn Asp Asp Gly Lys Gln Trp Gln Gln Pro Gly Ser
595 600 605
Ala Ile Leu Asn Val Trp Leu Thr Ser Met Leu Lys Arg Thr Val Val
610 615 620
Ala Ala Val Pro Met Pro Phe Asp Lys Trp Tyr Ser Ala Ser Gly Tyr
625 630 635 640
Glu Thr Thr Gln Asp Gly Pro Thr Gly Ser Leu Asn Ile Ser Val Gly
645 650 655
Ala Lys Ile Leu Tyr Glu Ala Val Gln Gly Asp Lys Ser Pro Ile Pro
660 665 670
Gln Ala Val Asp Leu Phe Ala Gly Lys Pro Arg Gln Glu Val Val Leu
675 680 685
Ala Ala Leu Glu Asp Thr Trp Glu Thr Leu Ser Lys Arg Tyr Gly Asn
690 695 700
Asn Val Ser Asn Trp Lys Thr Pro Ala Met Ala Leu Thr Phe Arg Ala
705 710 715 720
Asn Asn Phe Phe Gly Val Pro Gln Ala Ala Ala Glu Glu Thr Arg His
725 730 735
Gln Ala Glu Tyr Gln Asn Arg Gly Thr Glu Asn Val Met Ile Val Phe
740 745 750
Ser Pro Thr Thr Ser Asp Arg Pro Val Leu Ala Trp Asp Val Val Ala
755 760 765
Pro Gly Gln Ser Gly Phe Ile Ala Pro Asp Gly Thr Val Asp Lys His
770 775 780
Tyr Glu Asp Gln Leu Lys Met Tyr Glu Asn Phe Gly Arg Lys Ser Leu
785 790 795 800
Trp Leu Thr Lys Gln Asp Val Glu Ala His Lys Glu Ser Gln Glu Val
805 810 815
Leu His Val Gln Arg Leu Glu
820
<210> 5
<211> 823
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Met Glu Gln Ser Ser Ser Glu Ile Lys Ile Val Arg Asp Glu Tyr Gly
1 5 10 15
Met Pro His Ile Tyr Ala Asn Asp Thr Trp His Leu Phe Tyr Gly Tyr
20 25 30
Gly Tyr Val Val Ala Gln Asp Arg Leu Phe Gln Met Glu Met Ala Arg
35 40 45
Arg Ser Thr Gln Gly Thr Val Ala Glu Val Leu Gly Lys Asp Phe Val
50 55 60
Lys Phe Asp Lys Asp Ile Arg Arg Asn Tyr Trp Pro Asp Ala Ile Arg
65 70 75 80
Ala Gln Ile Ala Ala Leu Ser Pro Glu Asp Met Ser Ile Leu Gln Gly
85 90 95
Tyr Ala Asp Gly Met Asn Ala Trp Ile Asp Lys Val Asn Thr Asn Pro
100 105 110
Glu Thr Leu Leu Pro Lys Gln Phe Asn Thr Phe Gly Phe Thr Pro Lys
115 120 125
Arg Trp Glu Pro Phe Asp Val Ala Met Ile Phe Val Gly Thr Met Ala
130 135 140
Asn Arg Phe Ser Asp Ser Thr Ser Glu Ile Asp Asn Leu Ala Leu Leu
145 150 155 160
Thr Ala Leu Lys Asp Lys Tyr Gly Val Ser Gln Gly Met Ala Val Phe
165 170 175
Asn Gln Phe Lys Trp Leu Val Asn Pro Ser Ala Pro Thr Thr Ile Ala
180 185 190
Val Gln Glu Ser Asn Tyr Pro Leu Lys Phe Asn Gln Gln Asp Ser Gln
195 200 205
Ser Ala Ala Leu Leu Pro Arg Tyr Asp Leu Pro Ala Pro Met Leu Asp
210 215 220
Arg Pro Ala Lys Gly Ala Asp Gly Ala Leu Leu Ala Leu Thr Ala Gly
225 230 235 240
Lys Asn Arg Glu Thr Ile Val Ala Gln Phe Ala Gln Gly Gly Ala Asn
245 250 255
Gly Leu Ala Gly Tyr Pro Thr Thr Ser Asn Met Trp Val Ile Gly Lys
260 265 270
Ser Lys Ala Gln Asp Ala Lys Ala Ile Met Val Asn Gly Pro Gln Phe
275 280 285
Gly Trp Tyr Ala Pro Ala Tyr Thr Tyr Gly Ile Gly Leu His Gly Ala
290 295 300
Gly Tyr Asp Val Thr Gly Asn Thr Pro Phe Ala Tyr Pro Gly Leu Val
305 310 315 320
Phe Gly His Asn Gly Val Ile Ser Trp Gly Ser Thr Ala Gly Phe Gly
325 330 335
Asp Asp Val Asp Ile Phe Ala Glu Arg Leu Ser Ala Glu Lys Pro Gly
340 345 350
Tyr Tyr Leu His Asn Gly Lys Trp Val Lys Met Leu Ser Arg Glu Glu
355 360 365
Thr Ile Thr Val Lys Asn Gly Gln Ala Glu Thr Phe Thr Val Trp Arg
370 375 380
Thr Val His Gly Asn Ile Leu Gln Thr Asp Gln Thr Thr Gln Thr Ala
385 390 395 400
Tyr Ala Lys Ser Arg Ala Trp Asp Gly Lys Glu Val Ala Ser Leu Leu
405 410 415
Ala Trp Thr His Gln Met Lys Ala Lys Asn Trp Gln Glu Trp Thr Gln
420 425 430
Gln Ala Ala Lys Gln Ala Leu Thr Ile Asn Trp Tyr Tyr Ala Asp Val
435 440 445
Asn Gly Asn Ile Gly Tyr Val His Thr Gly Ala Tyr Pro Asp Arg Gln
450 455 460
Ser Gly His Asp Pro Arg Leu Pro Val Pro Gly Thr Asp Lys Trp Asp
465 470 475 480
Trp Lys Gly Leu Leu Pro Phe Glu Met Asn Pro Lys Val Tyr Asn Pro
485 490 495
Gln Ser Gly Tyr Ile Ala Asn Trp Asn Asp Ser Pro Gln Lys Asp Tyr
500 505 510
Pro Ala Ser Asp Leu Phe Ala Phe Leu Trp Gly Gly Ala Asp Arg Val
515 520 525
Thr Glu Ile Asp Arg Leu Leu Glu Gln Lys Pro Arg Leu Thr Ala Asp
530 535 540
Gln Ala Trp Asp Val Ile Arg Gln Thr Ser Arg Gln Asp Leu Asn Leu
545 550 555 560
Arg Leu Phe Leu Pro Thr Met Gln Ala Ala Thr Ser Gly Leu Thr Gln
565 570 575
Ser Asp Pro Arg Arg Gln Leu Val Glu Thr Leu Thr Arg Trp Asp Gly
580 585 590
Ile Asn Leu Leu Asn Asp Asp Gly Lys Gln Trp Gln Gln Pro Gly Ser
595 600 605
Ala Ile Leu Asn Val Trp Leu Thr Ser Met Leu Lys Arg Thr Val Val
610 615 620
Ala Ala Val Pro Met Pro Phe Asp Lys Trp Tyr Ser Ala Ser Gly Tyr
625 630 635 640
Glu Thr Thr Gln Asp Gly Pro Thr Gly Ser Leu Asn Ile Ser Val Gly
645 650 655
Ala Lys Ile Leu Tyr Glu Ala Val Gln Gly Asp Lys Ser Pro Ile Pro
660 665 670
Gln Ala Val Asp Leu Phe Ala Gly Lys Pro Gln Gln Glu Val Val Leu
675 680 685
Ala Ala Leu Glu Asp Thr Trp Glu Thr Leu Ser Lys Arg Tyr Gly Asn
690 695 700
Asn Val Ser Asn Trp Lys Thr Pro Ala Met Ala Leu Thr Phe Arg Ala
705 710 715 720
Asn Asn Phe Phe Gly Val Pro Gln Ala Ala Ala Glu Glu Thr Arg His
725 730 735
Gln Ala Glu Tyr Gln Asn Arg Gly Thr Glu Asn Val Met Ile Val Phe
740 745 750
Ser Pro Thr Thr Ser Asp Arg Pro Val Leu Ala Trp Asp Val Val Ala
755 760 765
Pro Gly Gln Ser Gly Phe Ile Ala Pro Asp Gly Thr Val Asp Lys His
770 775 780
Tyr Glu Asp Gln Leu Lys Met Tyr Glu Asn Phe Gly Arg Lys Ser Leu
785 790 795 800
Trp Leu Thr Lys Gln Asp Val Glu Ala His Lys Glu Ser Gln Glu Val
805 810 815
Leu His Val Gln Arg Leu Glu
820
<210> 6
<211> 823
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Met Glu Gln Ser Ser Ser Glu Ile Lys Ile Val Arg Asp Glu Tyr Gly
1 5 10 15
Met Pro His Ile Tyr Ala Asn Asp Thr Trp His Leu Phe Tyr Gly Tyr
20 25 30
Gly Tyr Val Val Ala Gln Asp Arg Leu Phe Gln Met Glu Met Ala Arg
35 40 45
Arg Ser Thr Gln Gly Thr Val Ala Glu Val Leu Gly Lys Asp Phe Val
50 55 60
Lys Phe Asp Lys Asp Ile Arg Arg Asn Tyr Trp Pro Asp Ala Ile Arg
65 70 75 80
Ala Gln Ile Ala Ala Leu Ser Pro Glu Asp Met Ser Ile Leu Gln Gly
85 90 95
Tyr Ala Asp Gly Met Asn Ala Trp Ile Asp Lys Val Asn Thr Asn Pro
100 105 110
Glu Thr Leu Leu Pro Lys Gln Phe Asn Thr Phe Gly Phe Thr Pro Lys
115 120 125
Arg Trp Glu Pro Phe Asp Val Ala Met Ile Phe Val Gly Thr Met Ala
130 135 140
Asn Arg Phe Ser Asp Ser Thr Ser Glu Ile Asp Asn Leu Ala Leu Leu
145 150 155 160
Thr Ala Leu Lys Asp Lys Tyr Gly Val Ser Gln Gly Met Ala Val Phe
165 170 175
Asn Gln Phe Lys Trp Leu Val Asn Pro Ser Ala Pro Thr Thr Ile Ala
180 185 190
Val Gln Glu Ser Asn Tyr Pro Leu Lys Phe Asn Gln Gln Asp Ser Gln
195 200 205
Ser Ala Ala Leu Leu Pro Arg Tyr Asp Leu Pro Ala Pro Met Leu Asp
210 215 220
Arg Pro Ala Lys Gly Ala Asp Gly Ala Leu Leu Ala Leu Thr Ala Gly
225 230 235 240
Lys Asn Arg Glu Thr Ile Val Ala Gln Phe Ala Gln Gly Gly Ala Asn
245 250 255
Gly Leu Ala Gly Tyr Pro Thr Thr Ser Asn Met Trp Val Ile Gly Lys
260 265 270
Ser Lys Ala Gln Asp Ala Lys Ala Ile Met Val Asn Gly Pro Gln Phe
275 280 285
Gly Trp Tyr Ala Pro Ala Tyr Thr Tyr Gly Ile Gly Leu His Gly Ala
290 295 300
Gly Tyr Asp Val Thr Gly Asn Thr Pro Phe Ala Tyr Pro Gly Leu Val
305 310 315 320
Phe Gly His Asn Gly Val Ile Ser Trp Gly Ser Thr Ala Gly Phe Gly
325 330 335
Asp Asp Val Asp Ile Phe Ala Glu Arg Leu Ser Ala Glu Lys Pro Gly
340 345 350
Tyr Tyr Leu His Asn Gly Lys Trp Val Lys Met Leu Ser Arg Glu Glu
355 360 365
Thr Ile Thr Val Lys Asn Gly Gln Ala Glu Thr Phe Thr Val Trp Arg
370 375 380
Thr Val His Gly Asn Ile Leu Gln Thr Asp Gln Thr Thr Gln Thr Ala
385 390 395 400
Tyr Ala Lys Ser Arg Ala Trp Asp Gly Lys Glu Val Ala Ser Leu Leu
405 410 415
Ala Trp Thr His Gln Met Lys Ala Lys Asn Trp Gln Glu Trp Thr Gln
420 425 430
Gln Ala Ala Lys Gln Ala Leu Thr Ile Asn Trp Tyr Tyr Ala Asp Val
435 440 445
Asn Gly Asn Ile Gly Tyr Val His Thr Gly Ala Tyr Pro Asp Arg Gln
450 455 460
Ser Gly His Asp Pro Arg Leu Pro Val Pro Gly Thr Asp Lys Trp Asp
465 470 475 480
Trp Lys Gly Leu Leu Pro Phe Glu Met Asn Pro Lys Val Tyr Asn Pro
485 490 495
Gln Ser Gly Tyr Ile Ala Asn Trp Asn Asp Ser Pro Gln Lys Asp Tyr
500 505 510
Pro Ala Ser Asp Leu Phe Ala Phe Leu Trp Gly Gly Ala Asp Arg Val
515 520 525
Thr Glu Ile Asp Arg Leu Leu Glu Gln Lys Pro Arg Leu Thr Ala Asp
530 535 540
Gln Ala Trp Asp Val Ile Arg Gln Thr Ser Arg Gln Asp Leu Asn Leu
545 550 555 560
Arg Leu Phe Leu Pro Thr Leu Gln Ala Ala Thr Ser Gly Leu Thr Gln
565 570 575
Ser Asp Pro Arg Arg Gln Leu Val Glu Thr Leu Thr Arg Trp Asp Gly
580 585 590
Ile Asn Leu Leu Asn Asp Asp Gly Lys Gln Trp Gln Gln Pro Gly Ser
595 600 605
Ala Ile Leu Asn Val Trp Leu Thr Ser Met Leu Lys Arg Thr Val Val
610 615 620
Ala Ala Val Pro Met Pro Phe Asp Lys Trp Tyr Ser Ala Ser Gly Tyr
625 630 635 640
Glu Thr Thr Gln Asp Gly Pro Thr Gly Ser Leu Asn Ile Ser Val Gly
645 650 655
Ala Lys Ile Leu Tyr Glu Ala Val Gln Gly Asp Lys Ser Pro Ile Pro
660 665 670
Gln Ala Val Asp Leu Phe Ala Gly Lys Pro Arg Gln Glu Val Val Leu
675 680 685
Ala Ala Leu Glu Asp Thr Trp Glu Thr Leu Ser Lys Arg Tyr Gly Asn
690 695 700
Asn Val Ser Asn Trp Lys Thr Pro Ala Met Ala Leu Thr Phe Arg Ala
705 710 715 720
Asn Asn Phe Phe Gly Val Pro Gln Ala Ala Ala Glu Glu Thr Arg His
725 730 735
Gln Ala Glu Tyr Gln Asn Arg Gly Thr Glu Asn Val Met Ile Val Phe
740 745 750
Ser Pro Thr Thr Ser Asp Arg Pro Val Leu Ala Trp Asp Val Val Ala
755 760 765
Pro Gly Gln Ser Gly Phe Ile Ala Pro Asp Gly Thr Val Asp Lys His
770 775 780
Tyr Glu Asp Gln Leu Lys Met Tyr Glu Asn Phe Gly Arg Lys Ser Leu
785 790 795 800
Trp Leu Thr Lys Gln Asp Val Glu Ala His Lys Glu Ser Gln Glu Val
805 810 815
Leu His Val Gln Arg Leu Glu
820
<210> 7
<211> 823
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Met Glu Gln Ser Ser Ser Glu Ile Lys Ile Val Arg Asp Glu Tyr Gly
1 5 10 15
Met Pro His Ile Tyr Ala Asn Asp Thr Trp His Leu Phe Tyr Gly Tyr
20 25 30
Gly Tyr Val Val Ala Gln Asp Arg Leu Phe Gln Met Glu Met Ala Arg
35 40 45
Arg Ser Thr Gln Gly Thr Val Ala Glu Val Leu Gly Lys Asp Phe Val
50 55 60
Lys Phe Asp Lys Asp Ile Arg Arg Asn Tyr Trp Pro Asp Ala Ile Arg
65 70 75 80
Ala Gln Ile Ala Ala Leu Ser Pro Glu Asp Met Ser Ile Leu Gln Gly
85 90 95
Tyr Ala Asp Gly Met Asn Ala Trp Ile Asp Lys Val Asn Thr Asn Pro
100 105 110
Glu Thr Leu Leu Pro Lys Gln Phe Asn Thr Phe Gly Phe Thr Pro Lys
115 120 125
Arg Trp Glu Pro Phe Asp Val Ala Met Ile Phe Val Gly Thr Met Ala
130 135 140
Asn Arg Phe Ser Asp Ser Thr Ser Glu Ile Asp Asn Leu Ala Leu Leu
145 150 155 160
Thr Ala Leu Lys Asp Lys Tyr Gly Val Ser Gln Gly Met Ala Val Phe
165 170 175
Asn Gln Phe Lys Trp Leu Val Asn Pro Ser Ala Pro Thr Thr Ile Ala
180 185 190
Val Gln Glu Ser Asn Tyr Pro Leu Lys Phe Asn Gln Gln Asp Ser Gln
195 200 205
Ser Ala Ala Leu Leu Pro Arg Tyr Asp Leu Pro Ala Pro Met Leu Asp
210 215 220
Arg Pro Ala Lys Gly Ala Asp Gly Ala Leu Leu Ala Leu Thr Ala Gly
225 230 235 240
Lys Asn Arg Glu Thr Ile Val Ala Gln Phe Ala Gln Gly Gly Ala Asn
245 250 255
Gly Leu Ala Gly Tyr Pro Thr Thr Ser Asn Met Trp Val Ile Gly Lys
260 265 270
Ser Lys Ala Gln Asp Ala Lys Ala Ile Met Val Asn Gly Pro Gln Phe
275 280 285
Gly Trp Tyr Ala Pro Ala Tyr Thr Tyr Gly Ile Gly Leu His Gly Ala
290 295 300
Gly Tyr Asp Val Thr Gly Asn Thr Pro Phe Ala Tyr Pro Gly Leu Val
305 310 315 320
Phe Gly His Asn Gly Val Ile Ser Trp Gly Ser Thr Ala Gly Phe Gly
325 330 335
Asp Asp Val Asp Ile Phe Ala Glu Arg Leu Ser Ala Glu Lys Pro Gly
340 345 350
Tyr Tyr Leu His Asn Gly Lys Trp Val Lys Met Leu Ser Arg Glu Glu
355 360 365
Thr Ile Thr Val Lys Asn Gly Gln Ala Glu Thr Phe Thr Val Trp Arg
370 375 380
Thr Val His Gly Asn Ile Leu Gln Thr Asp Gln Thr Thr Gln Thr Ala
385 390 395 400
Tyr Ala Lys Ser Arg Ala Trp Asp Gly Lys Glu Val Ala Ser Leu Leu
405 410 415
Ala Trp Thr His Gln Met Lys Ala Lys Asn Trp Gln Glu Trp Thr Gln
420 425 430
Gln Ala Ala Lys Gln Ala Leu Thr Ile Asn Trp Tyr Tyr Ala Asp Val
435 440 445
Asn Gly Asn Ile Gly Tyr Val His Thr Gly Ala Tyr Pro Asp Arg Gln
450 455 460
Ser Gly His Asp Pro Arg Leu Pro Val Pro Gly Thr Gly Lys Trp Asp
465 470 475 480
Trp Lys Gly Leu Leu Pro Phe Glu Met Asn Pro Lys Val Tyr Asn Pro
485 490 495
Gln Ser Gly Tyr Ile Ala Asn Trp Asn Asp Ser Pro Gln Lys Asp Tyr
500 505 510
Pro Ala Ser Asp Leu Phe Ala Phe Leu Trp Gly Gly Ala Asp Arg Val
515 520 525
Thr Glu Ile Asp Arg Leu Leu Glu Gln Lys Pro Arg Leu Thr Ala Asp
530 535 540
Gln Ala Trp Asp Val Ile Arg Gln Thr Ser Arg Gln Asp Leu Asn Leu
545 550 555 560
Arg Leu Phe Leu Pro Thr Met Gln Ala Ala Thr Ser Gly Leu Thr Gln
565 570 575
Ser Asp Pro Arg Arg Gln Leu Val Glu Thr Leu Thr Arg Trp Asp Gly
580 585 590
Ile Asn Leu Leu Asn Asp Asp Gly Lys Gln Trp Gln Gln Pro Gly Ser
595 600 605
Ala Ile Leu Asn Val Trp Leu Thr Ser Met Leu Lys Arg Thr Val Val
610 615 620
Ala Ala Val Pro Met Pro Phe Asp Lys Trp Tyr Ser Ala Ser Gly Tyr
625 630 635 640
Glu Thr Thr Gln Asp Gly Pro Thr Gly Ser Leu Asn Ile Ser Val Gly
645 650 655
Ala Lys Ile Leu Tyr Glu Ala Val Gln Gly Asp Lys Ser Pro Ile Pro
660 665 670
Gln Ala Val Asp Leu Phe Ala Gly Lys Pro Arg Gln Glu Val Val Leu
675 680 685
Ala Ala Leu Glu Asp Thr Trp Glu Thr Leu Ser Lys Arg Tyr Gly Asn
690 695 700
Asn Val Ser Asn Trp Lys Thr Pro Ala Met Ala Leu Thr Phe Arg Ala
705 710 715 720
Asn Asn Phe Phe Gly Val Pro Gln Ala Ala Ala Glu Glu Thr Arg His
725 730 735
Gln Ala Glu Tyr Gln Asn Arg Gly Thr Glu Asn Val Met Ile Val Phe
740 745 750
Ser Pro Thr Thr Ser Asp Arg Pro Val Leu Ala Trp Asp Val Val Ala
755 760 765
Pro Gly Gln Ser Gly Phe Ile Ala Pro Asp Gly Thr Val Asp Lys His
770 775 780
Tyr Glu Asp Gln Leu Lys Met Tyr Glu Asn Phe Gly Arg Lys Ser Leu
785 790 795 800
Trp Leu Thr Lys Gln Asp Val Glu Ala His Lys Glu Ser Gln Glu Val
805 810 815
Leu His Val Gln Arg Leu Glu
820
<210> 8
<211> 823
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Met Glu Gln Ser Ser Ser Glu Ile Lys Ile Val Arg Asp Glu Tyr Gly
1 5 10 15
Met Pro His Ile Tyr Ala Asn Asp Thr Trp His Leu Phe Tyr Gly Tyr
20 25 30
Gly Tyr Val Val Ala Gln Asp Arg Leu Phe Gln Met Glu Met Ala Arg
35 40 45
Arg Ser Thr Gln Gly Thr Val Ala Glu Val Leu Gly Lys Asp Phe Val
50 55 60
Lys Phe Asp Lys Asp Ile Arg Arg Asn Tyr Trp Pro Asp Ala Ile Arg
65 70 75 80
Ala Gln Ile Ala Ala Leu Ser Pro Glu Asp Met Ser Ile Leu Gln Gly
85 90 95
Tyr Ala Asp Gly Met Asn Ala Trp Ile Asp Lys Val Asn Thr Asn Pro
100 105 110
Glu Thr Leu Leu Pro Lys Gln Phe Asn Thr Phe Gly Phe Thr Pro Lys
115 120 125
Arg Trp Glu Pro Phe Asp Val Ala Met Ile Phe Val Gly Thr Met Ala
130 135 140
Asn Arg Phe Ser Asp Ser Thr Ser Glu Ile Asp Asn Leu Ala Leu Leu
145 150 155 160
Thr Ala Leu Lys Asp Lys Tyr Gly Val Ser Gln Gly Met Ala Val Phe
165 170 175
Asn Gln Phe Lys Trp Leu Val Asn Pro Ser Ala Pro Thr Thr Ile Ala
180 185 190
Val Gln Glu Ser Asn Tyr Pro Leu Lys Phe Asn Gln Gln Asp Ser Gln
195 200 205
Ser Ala Ala Leu Leu Pro Arg Tyr Asp Leu Pro Ala Pro Met Leu Asp
210 215 220
Arg Pro Ala Lys Gly Ala Asp Gly Ala Leu Leu Ala Leu Thr Ala Gly
225 230 235 240
Lys Asn Arg Glu Thr Ile Val Ala Gln Phe Ala Gln Gly Gly Ala Asn
245 250 255
Gly Leu Ala Gly Tyr Pro Thr Thr Ser Asn Met Trp Val Ile Gly Lys
260 265 270
Ser Lys Ala Gln Asp Ala Lys Ala Ile Met Val Asn Gly Pro Gln Phe
275 280 285
Gly Trp Tyr Ala Pro Ala Tyr Thr Tyr Gly Ile Gly Leu His Gly Ala
290 295 300
Gly Tyr Asp Val Thr Gly Asn Thr Pro Phe Ala Tyr Pro Gly Leu Val
305 310 315 320
Phe Gly His Asn Gly Val Ile Ser Trp Gly Ser Thr Ala Gly Phe Gly
325 330 335
Asp Asp Val Asp Ile Phe Ala Glu Arg Leu Ser Ala Glu Lys Pro Gly
340 345 350
Tyr Tyr Leu His Asn Gly Lys Trp Val Lys Met Leu Ser Arg Glu Glu
355 360 365
Thr Ile Thr Val Lys Asn Gly Gln Ala Glu Thr Phe Thr Val Trp Arg
370 375 380
Thr Val His Gly Asn Ile Leu Gln Thr Asp Gln Thr Thr Gln Thr Ala
385 390 395 400
Tyr Ala Lys Ser Arg Ala Trp Asp Gly Lys Glu Val Ala Ser Leu Leu
405 410 415
Ala Trp Thr His Gln Met Lys Ala Lys Asn Trp Gln Glu Trp Thr Gln
420 425 430
Gln Ala Ala Lys Gln Ala Leu Thr Ile Asn Trp Tyr Tyr Ala Asp Val
435 440 445
Asn Gly Asn Ile Gly Tyr Val His Thr Gly Ala Tyr Pro Asp Arg Gln
450 455 460
Ser Gly His Asp Pro Arg Leu Pro Val Pro Gly Thr Asp Lys Trp Asp
465 470 475 480
Trp Lys Gly Leu Leu Pro Phe Glu Met Asn Pro Lys Val Tyr Asn Pro
485 490 495
Gln Ser Gly Tyr Ile Ala Asn Trp Asn Asp Ser Pro Gln Lys Asp Tyr
500 505 510
Pro Ala Ser Asp Leu Phe Ala Phe Leu Trp Gly Gly Ala Asp Arg Val
515 520 525
Thr Glu Ile Asp Arg Leu Leu Glu Gln Lys Pro Arg Leu Thr Ala Asp
530 535 540
Gln Ala Trp Asp Val Ile Arg Gln Thr Ser Arg Gln Asp Leu Asn Leu
545 550 555 560
Arg Leu Phe Leu Pro Thr Met Gln Ala Ala Thr Ser Gly Leu Thr Gln
565 570 575
Ser Asp Pro Arg Arg Gln Leu Val Glu Thr Leu Thr Arg Trp Asp Gly
580 585 590
Ile Asn Leu Leu Asn Asp Asp Gly Lys Gln Trp Gln Gln Pro Gly Ser
595 600 605
Ala Ile Leu Asn Val Trp Leu Thr Ser Met Leu Lys Arg Thr Val Val
610 615 620
Ala Ala Val Pro Met Pro Phe Asp Lys Trp Tyr Ser Ala Ser Gly Tyr
625 630 635 640
Glu Thr Thr Gln Asp Gly Pro Thr Gly Ser Leu Asn Ile Ser Val Gly
645 650 655
Ala Lys Ile Leu Tyr Glu Ala Val Gln Gly Asp Lys Ser Pro Ile Pro
660 665 670
Gln Ala Val Asp Leu Phe Ala Gly Lys Pro Arg Gln Glu Val Val Leu
675 680 685
Ala Ala Leu Glu Asp Thr Trp Glu Thr Leu Ser Lys Arg Tyr Gly Asn
690 695 700
Asn Val Ser Asn Trp Lys Thr Pro Ala Met Ala Leu Thr Phe Arg Ala
705 710 715 720
Asn Asn Phe Phe Gly Val Pro Gln Ala Ala Ala Glu Glu Thr Arg His
725 730 735
Gln Ala Glu Tyr Gln Asn Arg Gly Thr Glu Asn Val Met Ile Val Phe
740 745 750
Ser Pro Thr Thr Ser Asp Arg Pro Val Leu Ala Trp Asp Val Val Ala
755 760 765
Pro Gly Gln Ser Gly Phe Ile Ala Pro Asp Gly Thr Val Asp Lys His
770 775 780
Tyr Glu Asp Gln Leu Lys Met Tyr Glu Asn Phe Gly Arg Lys Ser Leu
785 790 795 800
Trp Leu Thr Lys Gln Asp Val Glu Ala His Lys Glu Ser Gln Glu Val
805 810 815
Leu His Val Gln Arg Leu Glu
820
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
taatacgact cactataggg 20
<210> 10
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gctagttatt gctcagcgg 19

Claims (7)

1. A penicillin G acylase mutant characterized by: is to mutate the amino acid sequence shown in SEQ ID NO.1,
the mutation mode is any one of the following modes:
G477D; G477D and L567M; G477D and Q683R; G477D and L567M and Q683R.
2. A nucleotide encoding the penicillin G acylase mutant of claim 1.
3. Use of a penicillin G acylase mutant according to claim 1 characterized in that: catalyzing and synthesizing 7-ADCA by taking G-7-ADCA as a substrate.
4. Use according to claim 3, characterized in that the substrate G-7-ADCA concentration is 6% -8% and the enzyme activity is 3000-6000U/L.
5. Use according to claim 3, wherein the reaction temperature is 25-28 ℃ and the reaction time is 0.5-3.5h.
6. The use according to claim 3, wherein the substrate solvent is 0.05-0.2mol/L boric acid buffer, and the reaction pH is 7.5-8.5.
7. Use according to claim 3, characterized in that: the penicillin G acylase mutant is an immobilized penicillin G acylase mutant.
CN202110868353.3A 2021-07-30 2021-07-30 Penicillin G acylase mutant and application thereof Active CN113584008B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110868353.3A CN113584008B (en) 2021-07-30 2021-07-30 Penicillin G acylase mutant and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110868353.3A CN113584008B (en) 2021-07-30 2021-07-30 Penicillin G acylase mutant and application thereof

Publications (2)

Publication Number Publication Date
CN113584008A CN113584008A (en) 2021-11-02
CN113584008B true CN113584008B (en) 2023-04-11

Family

ID=78252349

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110868353.3A Active CN113584008B (en) 2021-07-30 2021-07-30 Penicillin G acylase mutant and application thereof

Country Status (1)

Country Link
CN (1) CN113584008B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114958877B (en) * 2022-06-14 2024-02-20 河南省健康元生物医药研究院有限公司 Deacetylated oxo cephalosporin C synthetase mutant, encoding gene and application thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108660127A (en) * 2017-03-27 2018-10-16 珠海联邦制药股份有限公司 The penicillin G acylation zymogen and its coded sequence of a kind of engineer and application

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8541199B2 (en) * 2008-12-23 2013-09-24 Dsm Sinochem Pharmaceuticals Netherlands B.V. Mutant penicillin G acylases
CN103834631B (en) * 2014-02-20 2015-12-30 浙江普洛得邦制药有限公司 A kind of penicillin G acylase mutant and encoding gene thereof and application
CN105087533B (en) * 2015-09-30 2018-03-27 湖南福来格生物技术有限公司 A kind of mutant of penicillin G acylase and its preparation method and application
KR101985911B1 (en) * 2017-12-28 2019-06-04 아미코젠주식회사 Mutants of penicillin G acylase from Achromobacter sp. CCM 4824, and uses thereof
WO2021140526A1 (en) * 2020-01-08 2021-07-15 Fermenta Biotech Limited Mutant penicillin g acylases of achromobacter ccm4824
CN113373123B (en) * 2021-07-30 2022-05-24 湖南福来格生物技术有限公司 Tyrosinase mutant and application thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108660127A (en) * 2017-03-27 2018-10-16 珠海联邦制药股份有限公司 The penicillin G acylation zymogen and its coded sequence of a kind of engineer and application

Also Published As

Publication number Publication date
CN113584008A (en) 2021-11-02

Similar Documents

Publication Publication Date Title
CN109402098B (en) Threonine aldolase, mutant and application of mutant in preparation of substituted phenylserine derivative
JP3072101B2 (en) 7-A new method for biochemical production of ADCA
CN111235126B (en) S-adenosylmethionine synthetase mutant and preparation method using same
CN105274082B (en) A kind of synthesis penicillin G acylase mutant and its application in preparing Amoxicillin
CN113584008B (en) Penicillin G acylase mutant and application thereof
CN105087533B (en) A kind of mutant of penicillin G acylase and its preparation method and application
CN113481189B (en) Sucrose isomerase mutant and application thereof
CN103695405B (en) A kind of production method of novel ss-lactam class antibiotic synthetic enzymes
CN110358751B (en) Recombinant lipase mutant, encoding gene, recombinant engineering bacterium and application
CN114736884B (en) Cytidine monophosphate kinase mutant and gene and application thereof
RU2388826C2 (en) RECOMBINANT DNA, WHICH CODES FUNCTIONALLY ACTIVE HYBRID PROTEIN GL7ACA-ACYLASE WITH CHITIN-BINDING DOMAIN (BrdGL7ACA-cbd) AND RECOMBINANT PLASMID pSVH0108, PROVIDING FOR ITS SYNTHESIS IN CELLS Escherichia coli, RECOMBINANT STRAIN Escherichia coli BL21(DE3)/pSVH0108-PRODUCER BrdGL7ACA-cbd
CN109161540B (en) Penicillin V acylase mutant, coding sequence, recombinant expression vector, genetic engineering bacterium and application
CN111826372A (en) Engineering strain for producing butanol by using xylose and construction method and application thereof
CN111500564B (en) Penicillin G acylase mutant and application thereof in enzymatic synthesis of cefamandole
CN117247928A (en) Cephalosporin C acylase catalyzed acylation reaction and application thereof
CN117247927A (en) Artificial synthesis of cephalosporin C acylase and mutant application thereof
CN112852913B (en) Deacetoxycephalosporin C synthetase mutant and application thereof in synthesis of beta-lactam antibiotic parent nucleus
CN110904087B (en) L-arabinose epimerase mutant and application thereof
CN117947137A (en) Penicillin amidase for synthesizing cefaclor
CN117247926A (en) Cephalosporin C acylase mutant and application thereof
Illanes et al. Evaluation of penicillin acylase production by two strains of Bacillus megaterium
CN117247925A (en) Cephalosporin C acylase mutant and application thereof in cephalosporin mother nucleus synthesis
CN116497010A (en) G-7-ADCA synthetase and application thereof in preparation of cephalosporanic acid compounds
CN113943721A (en) Lipase mutant and application thereof
CN114317631A (en) Application of monoamine oxidase in preparation of tropinone

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
PE01 Entry into force of the registration of the contract for pledge of patent right

Denomination of invention: A Penicillin G Acylase Mutant and Its Application

Granted publication date: 20230411

Pledgee: Changsha Bank city branch of Limited by Share Ltd.

Pledgor: HUNAN FLAG BIOTECHNOLOGY Co.,Ltd.

Registration number: Y2024980012003

PE01 Entry into force of the registration of the contract for pledge of patent right