WO2021218269A1 - 一种亲本植酸酶变体 - Google Patents

一种亲本植酸酶变体 Download PDF

Info

Publication number
WO2021218269A1
WO2021218269A1 PCT/CN2021/075123 CN2021075123W WO2021218269A1 WO 2021218269 A1 WO2021218269 A1 WO 2021218269A1 CN 2021075123 W CN2021075123 W CN 2021075123W WO 2021218269 A1 WO2021218269 A1 WO 2021218269A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
appa
variant
phytase
amino acid
Prior art date
Application number
PCT/CN2021/075123
Other languages
English (en)
French (fr)
Inventor
林洁
白挨玺
黄珂
孙艳
徐红
Original Assignee
南京百斯杰生物工程有限公司
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 南京百斯杰生物工程有限公司 filed Critical 南京百斯杰生物工程有限公司
Priority to CN202311652847.3A priority Critical patent/CN117683744A/zh
Priority to CN202311652868.5A priority patent/CN117683746A/zh
Priority to CN202311611465.6A priority patent/CN117701527A/zh
Priority to CN202311652859.6A priority patent/CN117683745A/zh
Priority to CN202311642247.9A priority patent/CN117721093A/zh
Priority to CN202311319920.5A priority patent/CN117535267A/zh
Priority to CN202311647341.3A priority patent/CN117683743A/zh
Priority to EP21797344.5A priority patent/EP4144840A1/en
Priority to US17/997,037 priority patent/US20230167422A1/en
Priority to CN202311634743.XA priority patent/CN117778350A/zh
Priority to CN202311319916.9A priority patent/CN117535266A/zh
Priority to CN202180006789.XA priority patent/CN114761549A/zh
Publication of WO2021218269A1 publication Critical patent/WO2021218269A1/zh

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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • 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)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03026—4-Phytase (3.1.3.26), i.e. 6-phytase

Definitions

  • the invention relates to the technical field of protein engineering. More specifically, it relates to a parent phytase variant.
  • Phytase also known as phytase, belongs to orthophosphate monoester phosphohydrolase. It is a general term for enzymes that catalyze the hydrolysis of phytate and phytate into inositol and phosphoric acid (phosphate).
  • Phytic acid is the most abundant in crop seeds such as grains, beans, oil crops, etc., up to 1% to 3%, accounting for 60% to 80% of the total phosphorus content of plants. However, the phosphorus in phytic acid cannot be directly absorbed and used, and must be hydrolyzed to inorganic phosphate in the digestive tract.
  • Phytase is pre-added to feed materials as a feed additive, and after high-temperature granulation and other processes (70-95 degrees, 30 seconds-120 seconds), feed is produced for animal feeding.
  • the stress resistance of phytase, especially the thermal stability, cannot meet the requirements of feed and feed processing, which has become one of the important restrictive factors restricting the popularization and application of phytase.
  • improving the heat resistance of phytase has become a new hot spot in the field of scientific research.
  • the use of coating technology can effectively reduce the activity loss of phytase during high-temperature and high-humidity treatments such as granulation, and greatly improve the utilization efficiency of phytase.
  • Research on the coating of phytase has been carried out at home and abroad and some progress has been made.
  • the phytase coating process developed by the well-known enzyme preparation manufacturers Novozymes and BASF, the coated phytase and the uncoated phytase Compared with acid enzyme powder, its thermal stability has been greatly improved.
  • Patent WO2007044968, CN1981597A, CN101168734A all disclose the coating process of phytase. Although the coating process can significantly improve the heat resistance of phytase, the coating process is complicated, and at the same time, it will prolong the production cycle of phytase and greatly increase the production cost of phytase.
  • the purpose of the present invention is to provide a phytase product with more excellent heat resistance through the method of protein engineering.
  • the inventors of the present invention found that on the basis of the parental phytase, one or more amino acid substitutions were introduced at positions 295, 349, and 374 corresponding to SEQ ID NO:1, and the resulting phytase Compared with its parent phytase, the active variant of phytase has significantly increased thermostability; wherein, the amino acid sequence of the parent phytase has at least 80% compared with the amino acid sequence shown in SEQ ID NO: 98. % Sequence identity.
  • the amino acid sequence of the parent phytase is at least 80%, 81%, 82%, 83%, 84%, 85%, compared with the amino acid sequence shown in SEQ ID NO: 98. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; further preferred, The amino acid sequence of the parent phytase is shown in SEQ ID NO: 98.
  • the amino acid sequence of the parent phytase is selected from SEQ ID NO: 2, SEQ ID NO: 98, SEQ ID NO: 102, SEQ ID NO: 107, SEQ ID NO: 112, or SEQ ID NO: 115.
  • the amino acid at position 295 of the variant is substituted with P, Y, G, K, L, or Q; preferably, it is substituted with P or Y; more preferably, it is substituted with Y.
  • the amino acid at position 349 of the variant is substituted with K, L, G, H, R or T; preferably, it is substituted with K or L; more preferably, it is substituted with K.
  • the amino acid at position 374 of the variant is substituted with R, V, N, S, T, F, K, P or Y; preferably, it is substituted with R or V; further preferred , Was replaced by R.
  • the variant contains any group of amino acid substitutions selected from the following group relative to its parent phytase:
  • T295P+Q349K T295P+E374R; T295P+Q349L;
  • the variant may only include amino acid substitutions selected from any of the above groups, or may include some other mutations in addition to amino acid substitutions selected from any of the above groups.
  • the variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to the amino acid sequence of its parent phytase, But less than 100% sequence identity.
  • the variant further includes at least one pair of introduced disulfide bonds, for example, the introduction of any one or more pairs of disulfide bonds disclosed in the patent WO 2019228441A1; preferably, the variant includes A disulfide bond is formed between the amino acid residue at the position corresponding to the 346th position of SEQ ID NO:1 and the amino acid residue at the position corresponding to the 393rd position of SEQ ID NO:1; further preferably, the The amino acid at the position corresponding to the 346th position of SEQ ID NO:1 of the variant is substituted with C, and the amino acid at the position corresponding to the 393rd position of SEQ ID NO:1 is substituted with C.
  • introducing does not limit the formation of disulfide bonds in any specific way.
  • "introducing" a disulfide bond can include replacing the amino acid residue at the corresponding position of the phytase sequence into which the disulfide bond is to be introduced with an amino acid residue capable of forming a disulfide bond (for example, a cysteine residue). Cys, homocysteine residues Hey, etc.); and/or insert amino acid residues capable of forming disulfide bonds at the corresponding positions.
  • substitution and/or insertion can be achieved, for example, by site-directed mutagenesis methods known in the art.
  • “Introduction” also includes the case where any one or two amino acid residues forming the disulfide bond are caused by natural mutation.
  • the amino acid sequence of the variant is SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105 , SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 117 or SEQ ID NO: 118.
  • the present invention also provides a nucleic acid encoding the parent phytase variant as described in any one of the above.
  • the present invention also provides a vector comprising the nucleic acid as described above.
  • the present invention also provides a host cell, which contains the vector as described above.
  • the host cell is a fungal cell, a bacterial cell or a plant cell.
  • the host cell is a fungal cell, and the fungal cell is selected from a Pichia pastoris cell or an Aspergillus niger cell.
  • the present invention also provides a method for producing the parent phytase variant as described in any one of the above, the method comprising:
  • one or more mutation sites as described in the present invention are introduced, especially multiple mutation sites are introduced at the same time, and the obtained phytase variant and the parent phytase In comparison, the residual vitality after 5 minutes of treatment at 85°C in a water bath increased by 16-27%. Therefore, the technical solution of the present invention can significantly improve the enzymatic activity of phytase, especially in terms of heat resistance stability, steam resistance stability, and granulation stability, which is significantly better than the existing wild-type or mutant phytic acid. Enzyme.
  • Sequence identity is defined as comparing sequences and introducing gaps when necessary to obtain the maximum percent sequence identity, and any conservative substitutions are not considered as part of sequence identity, and the candidate sequence is identical to a specific peptide or polypeptide The percentage of amino acid residues that have the same amino acid residues in the sequence. Sequence comparisons can be performed in a variety of ways within the skill of the art to determine percent amino acid sequence identity, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring the comparison, including any algorithm required to obtain the maximum comparison over the entire length of the sequence being compared.
  • Parent refers to the phytase that can produce the variant of the present invention after undergoing the mutation described in the present invention.
  • the parent may be a naturally occurring (wild-type) phytase or a mutant thereof prepared by a suitable method, and the parent may also be an allelic variant.
  • Host cell refers to any cell type that is susceptible to transformation, transfection, transduction, etc., with the vector containing the nucleic acid of the present invention.
  • the term "host cell” encompasses any progeny of a parent cell that is not exactly the same as the parent cell due to mutations that occur during replication.
  • Substitution refers to replacing the original amino acid occupying a certain position with a different amino acid.
  • Variant refers to an enzyme with phytase activity and contains one or more (several) amino acid residue substitutions at one or more (several) positions relative to its parent phytase.
  • the construction of the variant of the present invention can be achieved by culturing a host cell containing a nucleic acid sequence encoding the variant under conditions suitable for the production of the variant, and the variant can be subsequently recovered from the obtained culture broth.
  • Wild type refers to the phytase expressed by naturally occurring microorganisms, such as the natural phytase derived from E. coli found in nature.
  • Figure 1 shows the plasmid map of pPIC9K-APPA-Y0.
  • Figure 2 shows the thermal stability test results of the parent phytase and its variants in Example 1.
  • FIG. 3 shows the results of the thermal stability test of the variant of the site-directed saturation mutation of the 295th amino acid in Example 3.
  • FIG. 3 shows the results of the thermal stability test of the variant of the site-directed saturation mutation of the 295th amino acid in Example 3.
  • Figure 4 shows the results of the thermal stability test of the variant of the site-directed saturation mutation of the 349th amino acid in Example 3.
  • FIG. 5 shows the results of the thermal stability test of the variant of the site-directed saturation mutation of the 374th amino acid in Example 3.
  • FIG. 6 shows the results of the thermal stability test of the variant of the combination mutation in Example 4.
  • Figure 7 shows the results of the thermal stability test of the variant of the parent phytase APPA-An1 in Example 5.
  • Figure 8 shows the results of the thermal stability test of the variant of the parent phytase APPA-An5 in Example 6.
  • Figure 9 shows the results of the thermal stability test of the variant of the parental phytase APPA-An10 in Example 7.
  • Figure 10 shows the results of the thermal stability test of the variant of the parent phytase APPA-An15 in Example 8.
  • Figure 11 shows the results of the thermal stability test of the variant of the parent phytase APPA-An18 in Example 9.
  • the present invention quotes the amino acid sequence of the phytase mutant named APPA-M2-O in the patent WO2019228441. Its amino acid sequence is shown in SEQ ID NO: 2 in the present invention.
  • the present invention names the phytase mutant It is APPA-Y0.
  • APPA-Y0 is a wild-type phytase derived from Escherichia coli (its amino acid sequence is shown in SEQ ID NO: 1), which is a mutant of phytase obtained after mutation screening, glycosylation and introduction of disulfide bond sites.
  • the method is as described in WO2019228441, and the mutant has excellent thermal stability. This example is a further mutation and screening based on the APPA-Y0 sequence.
  • the nucleic acid sequence of APPA-Y0 expressed in Pichia pastoris was synthesized by GenScript Biotechnology Co., Ltd., and the gene was cloned into the Pichia pastoris vector, the expression vector was pPIC9K, and the Alpha factor of Saccharomyces cerevisiae was used as the signal peptide.
  • the acid enzyme expression plasmid pPIC9K-APPA-Y0 is shown in Figure 1.
  • APPA-Y0 was used as the parent phytase.
  • the inventors designed the following 37 mutants based on the analysis of the amino acid sequence and protein structure of APPA-Y0 ,As shown in Table 1.
  • each variant contains an amino acid substitution relative to the parent phytase APPA-Y0.
  • These variants are named APPA-Y1 to APPA-Y37, and their amino acid sequences are as shown in SEQ ID NO: 3-39. Show.
  • the variant plasmids were named pPIC9K-APPA-Y1 to pPIC9K-APPA-Y37 according to the variant names in the table above.
  • Pichia expression kit Invitrogen was used to manipulate Pichia pastoris GS115 and plasmids with reference to its instructions.
  • the Pichia pastoris GS115 strain was cultured on a YPD medium (1% yeast extract, 2% protein, 2% glucose and 1.5% agar) plate at 30°C for 48 hours, and then a single clone was picked into 4mLYPD liquid medium (1 % Yeast extract, 2% protein, 2% glucose), cultured at 30°C at 200rpm for 12h, then transferred to a 30mL YPD liquid medium in an Erlenmeyer flask.
  • YPD medium 1% yeast extract, 2% protein, 2% glucose and 1.5% agar
  • the OD600 value was detected to be 1.1- 1.3
  • the culture solution was centrifuged at 9,000 rpm at 4°C for 2 minutes, and 4 mL of bacteria were collected into a sterilized EP tube. The supernatant was gently discarded, and the remaining supernatant was blotted with sterile filter paper and then used with a pre-chilled 1 mL Resuspend the bacteria in sterilized water, centrifuge at 9,000 rpm at 4°C for 2 minutes, and discard the supernatant.
  • Pichia pastoris recombinant strains screened on MD plates were named APPA-Y0 and APPA-Y1 to APPA-Y37.
  • the clones obtained by the above screening were transferred to BMGY medium, cultured in a shaking shaker at 30°C and 250rpm for 24h, and then transferred to BMMY medium, maintained at 30°C and 250rpm, and 0.5% methanol was added every day to induce After expression for 120h; centrifugation at 9000-12000rpm for 10min to remove the bacteria, and the fermentation supernatant containing phytase APPA-Y0 and other 37 variants was obtained.
  • SDS-PAGE results showed APPA-Y1, APPA-Y7, APPA-Y8 The four variants of APPA-Y16 and APPA-Y16 were not expressed, and the remaining APPA-Y0 and 33 other variants were all expressed.
  • the determination of phytase activity follows the GBT 18634-2009 document standard.
  • the 33 variant samples in Example 1 and the parent phytase APPA-Y0 were diluted with water to 100 U/mL. Take 9mL of water in a 25mL colorimetric tube, pipette 1mL of enzyme sample, quickly add it to the test tube and mix quickly with a mixer, then place it in a constant temperature water bath at 85°C for 5 minutes. Quickly cool to room temperature, dilute with water, and measure the residual activity of each sample to calculate the residual activity of the enzyme at different treatment temperatures.
  • the enzyme activity before heat treatment is set as 100%, and the thermal stability data obtained is shown in Figure 2. .
  • phytase such as APPA-Y1, APPA-Y7, APPA-Y8 and APPA-Y16;
  • APPA-Y2 M29L
  • APPA-Y4 R50K
  • APPA-Y5 G52A
  • APPA-Y6 L58M
  • APPA-Y10 A105F
  • APPA-Y12 T118Q
  • APPA-Y25 T295P
  • APPA-Y28 L307I
  • APPA-Y30 Q349K
  • APPA-Y33 M360L
  • APPA-Y36(E374R) of which APPA-Y25 (T295P), APPA-Y30 (Q349K), APPA-Y36 (E374R) performed the best.
  • the residual activity of the variant increased by about 19-22%.
  • Example 3 Saturation mutation of the 3 mutation sites in Example 2
  • the three mutant strains APPA-Y25, APPA-Y30, and APPA-Y36 corresponding to the three mutation points T295P, Q349K, and E374R with the most improved heat resistance in Example 2 were subjected to saturation mutations, that is, the 295 positions, 349 positions,
  • the amino acid at position 374 is mutated to the other 18 amino acids, and the corresponding sequence names are shown in Table 2-4 below.
  • Pichia pastoris was used to express each variant, and then the thermal stability of each variant was measured according to the method in Example 2. The results are shown in Figures 3-5.
  • Some mutations even lead to abnormal expression such as APPA-Y25-2, APPA-Y25-17, APPA-Y30-3, APPA-Y30-5, APPA-Y30-8, APPA-Y30-12 and APPA-Y30 -18;
  • the three mutant strains APPA-Y25, APPA-Y30, and APPA-Y36 corresponding to the three mutation points T295P, Q349K, and E374R in Example 2 were superimposed, and the corresponding variants were constructed according to the method in Example 1. Expressed in red yeast, see Table 5 below for specific mutations.
  • the thermal stability measurement was performed according to the detection method in Example 2, and the thermal stability data obtained is shown in FIG. 6.
  • APPA-An1 is another mutant with excellent heat resistance obtained by mutation of E. coli wild-type phytase, and its amino acid sequence is shown in SEQ ID NO: 98:
  • this example uses APPA-An1 as the parent phytase, and The amino acid substitutions Q349K and E374R were added to the amino acid sequence of APPA-An1 alone or in combination, and the mutants were named according to APPA-An2 to APPA-An4, as shown in the following table. According to the method described in the patent application CN107353327A, the above-mentioned phytase mutant was expressed in Aspergillus niger.
  • Example 7 After obtaining the supernatant of the shake flask, the thermal stability was measured as described in Example 2, and incubated at 85° C. for 5 minutes. The experimental results are shown in FIG. 7. The experimental results show that the three variants all show a significant improvement in stability performance, showing better stability than the parent phytase APPA-An1, that is, introducing 1-2 of the present invention into the parent phytase After the mutation point, a more stable phytase variant can be obtained, and it can be predicted that it will perform well in feed pelleting.
  • APPA-An5 is another mutant with excellent heat resistance obtained by mutation of wild-type phytase, and its amino acid sequence is shown in SEQ ID NO: 102.
  • this example uses APPA-An5 as the parent phytase, and T295P, Q349K, and E374R were added to the amino acid sequence of APPA-An5 individually or in combination, and each mutant was named according to APPA-An6 to APPA-An9, as shown in Table 7 below.
  • Example 5 According to the method of Example 5, the above-mentioned phytase variant was expressed in Aspergillus niger. After obtaining the supernatant of the shake flask, the thermal stability was measured as described in Example 2, and incubated at 85°C for 5 minutes. The experimental results are shown in Figure 8. We found that the 4 mutants all showed a significant improvement in stability performance, showing better stability than the parent APPA-An5, that is, after introducing 1-2 mutation points of the present invention into the parent phytase mutant A phytase mutant with higher stability can be obtained, and it can be predicted that it will perform well in feed pelleting.
  • APPA-An10 is another mutant with excellent heat resistance obtained by mutation screening of wild-type phytase, and its amino acid sequence is shown in SEQ ID NO: 107.
  • this example uses APPA-An10 as the parent, and T295P, Q349K , E374R was added to the amino acid sequence of APPA-An10 alone or in combination, and each mutant was named according to APPA-An11 ⁇ APPA-An14, as shown in Table 8 below.
  • Example 5 According to the method of Example 5, the above-mentioned phytase variant was expressed in Aspergillus niger. After obtaining the supernatant of the shake flask, the thermal stability was measured as described in Example 2, and incubated at 85°C for 5 minutes. The experimental results are shown in Figure 9. We found that the four variants all showed a significant improvement in stability performance, and showed better stability than the parent APPA-An10, that is, after introducing the 1-3 mutation points of the present invention into the parent phytase mutant A phytase mutant with higher stability can be obtained, and it can be predicted that it will perform well in feed pelleting.
  • Mutant name Mutation site Serial number APPA-An10 Parents SEQ ID NO: 107 APPA-An11 E374R SEQ ID NO: 108 APPA-An12 T295P+Q349K SEQ ID NO: 109 APPA-An13 T295P+E374R SEQ ID NO: 110 APPA-An14 T295P+Q349K+E374R SEQ ID NO: 111
  • APPA-An15 is another mutant with excellent heat resistance obtained by mutation screening of wild-type phytase, and its amino acid sequence is shown in SEQ ID NO: 112.
  • this example uses APPA-An15 as the parent, and T295P, E374R Separately added to the amino acid sequence of APPA-An15, each mutant was named according to APPA-An16 ⁇ APPA-An17, as shown in the following table. According to the method of Example 5, the above-mentioned phytase mutant was expressed in Aspergillus niger.
  • Example 9 Aspergillus niger expresses a variant of the parent phytase APPA-An18
  • APPA-An18 is another mutant with excellent heat resistance obtained by mutation screening of wild-type phytase, and its amino acid sequence is shown in SEQ ID NO: 115.
  • this example uses APPA-An18 as the parent, and T295P, Q349K , E374R was added to the amino acid sequence of APPA-An18 alone or in combination, and each mutant was named according to APPA-An19 ⁇ APPA-An21, as shown in Table 10 below.
  • Example 5 According to the method of Example 5, the above-mentioned phytase variant was expressed in Aspergillus niger. After obtaining the supernatant of the shake flask, the thermal stability was measured as described in Example 2, and incubated at 85°C for 5 minutes. The experimental results are shown in Figure 11. We found that the three mutants all showed a significant improvement in stability performance, showing better stability than the parent APPA-An18, that is, after introducing the 1-3 mutation points of the present invention into the parent phytase mutant A phytase mutant with higher stability can be obtained, and it can be predicted that it will perform well in feed pelleting.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Mycology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

提供了一种亲本植酸酶变体，涉及蛋白质工程技术领域，所述变体相对于其亲本植酸酶，在对应于SEQ ID NO:1的第295位、第349位、第374位上具有一个或多个氨基酸的取代，与亲本植酸酶相比，所述变体具有增加的热稳定性。

Description

一种亲本植酸酶变体 技术领域
本发明涉及蛋白质工程技术领域。更具体地，涉及一种亲本植酸酶变体。
背景技术
植酸酶(Phytase)，又称为肌醇六磷酸酶，属于正磷酸单酯磷酸水解酶，是催化植酸及植酸盐水解成肌醇与磷酸(磷酸盐)的酶类总称。植酸在谷物、豆类、油料等作物种子中含量最为丰富，高达1％～3％，占植物总磷含量的60％～80％。但植酸中的磷不能被直接吸收利用，必须在消化道内先水解为无机磷酸盐。研究表明，单胃动物(猪、鸡、鸭、鹅等)因为缺乏植酸酶而对植酸中磷的利用率很低。同时，植酸的强烈电负性导致其通常与二价或三价阳离子，如Ca 2+、Zn 2+、Fe 2+等形成不溶性盐类，阻碍小肠对矿物质的吸收。还会与蛋白质，氨基酸以及脂肪酸等形成络合物，影响他们的吸收利用。植酸还会与胃蛋白酶、胰凝乳酶、胰蛋白酶等结合，降低消化酶活性。因此，在单胃动物饲料中添加植酸酶可提高动物饲料中磷的利用率，降低动物排泄物中的磷含量，同时能提高蛋白和饲料的能量利用率。
植酸酶作为饲料添加剂预先加入饲料原料中，经高温制粒等过程(70-95度，时间为30秒-120秒)后，生产出饲料用于动物饲养。植酸酶的抗逆性，尤其是热稳定性不能满足饲料及饲料加工的要求，成为制约植酸酶推广应用的重要限制性因素之一。鉴于植酸酶对提高畜牧业生产效益和降低磷对环境的污染有重要意义，提高植酸酶的耐热性成为科研领域研究的新热点。
采用包衣技术可以有效降低植酸酶在制粒等高温高湿处理过程中的活性损失，大大提高植酸酶的利用效率。国内外均已针对植酸酶的包衣进行研究且取得了一定进展，著名的酶制剂生产商Novozymes和BASF开发的植酸酶包衣工艺，包衣的植酸酶与未经包衣的植酸酶酶粉相比，其热稳定性得到大幅度提升。专利WO2007044968、CN1981597A、CN101168734A均公开了植酸酶的包衣工艺。虽然包衣工艺可以显著提升植酸酶的耐热性能，但是包衣的工艺复杂，同时还会延长植酸酶的生产周期，大大增加植酸酶的生产成本。
此外，通过蛋白质工程的方法在野生型植酸酶的氨基酸序列基础上，对特定 位置的氨基酸进行突变，也可以筛选获得耐热性能优异的植酸酶。专利WO2019228441A1、US9605245、EP2102334B1等均对植酸酶进行了特定氨基酸的定点突变，使最终获得的植酸酶产品相较于野生型植酸酶的耐热性能有所提升。但是在饲料高温制粒过程中，这些植酸酶产品的酶活仍然会大量损失。
因此，还需要在现有研究的基础上开发耐热性能更为优异的植酸酶产品。
发明内容
本发明的目的在于通过蛋白质工程的方法提供一种耐热性能更为优异的植酸酶产品。
为达到上述目的，本发明采用下述技术方案：
本发明的发明人发现，在亲本植酸酶的基础上，在对应于SEQ ID NO:1的第295位、第349位、第374位上引入一个或多个氨基酸的取代，得到的具有植酸酶活性的变体与其亲本植酸酶相比，具有显著增加的热稳定性；其中，所述亲本植酸酶的氨基酸序列与SEQ ID NO:98所示的氨基酸序列相比，具有至少80％的序列一致性。
在一些优选实施例中，所述亲本植酸酶的氨基酸序列与SEQ ID NO:98所示的氨基酸序列相比，具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列一致性；进一步优选的，所述亲本植酸酶的氨基酸序列如SEQ ID NO:98所示。
在一些具体实施例中，所述亲本植酸酶的氨基酸序列选自SEQ ID NO:2、SEQ ID NO:98、SEQ ID NO:102、SEQ ID NO:107、SEQ ID NO:112或SEQ ID NO:115。
在一些实施例中，所述变体在第295位的氨基酸被取代为P、Y、G、K、L或Q；优选的，被取代为P或Y；进一步优选的，被取代为Y。
在一些实施例中，所述变体在第349位的氨基酸被取代为K、L、G、H、R或T；优选的，被取代为K或L；进一步优选的，被取代为K。
在一些实施例中，所述变体在第374位的氨基酸被取代为R、V、N、S、T、F、K、P或Y；优选的，被取代为R或V；进一步优选的，被取代为R。
在一些优选实施例中，所述变体相对于其亲本植酸酶，包含选自下组的任一组氨基酸取代：
T295P；Q349K；E374R；T295Y；Q349L；E374V；
T295P+Q349K；T295P+E374R；T295P+Q349L；
T295P+E374V；T295Y+Q349K；T295Y+E374R；
T295Y+Q349L；T295Y+E374V；Q349L+E374R；
Q349K+E374R；Q349L+E374V；Q349K+E374V；
T295P+Q349K+E374R；
T295Y+Q349K+E374R；
T295P+Q349L+E374R；
T295Y+Q349L+E374R；
T295P+Q349K+E374V；
T295Y+Q349K+E374V；
T295P+Q349L+E374V；
T295Y+Q349L+E374V。
需要说明的是，所述变体相对于其亲本植酸酶，可以仅包括选自上述任一组的氨基酸取代，也可以在包含选自上述任一组氨基酸取代之外，还包含一些其它突变位点，例如现有技术中已经公开的可以提升植酸酶耐热性能的植酸酶突变位点。
优选地，所述变体与其亲本植酸酶的氨基酸序列相比，具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％，但小于100％的序列一致性。
在一些实施例中，所述变体还进一步包括至少一对引入的二硫键，例如，引入专利WO 2019228441A1中公开的任一对或多对二硫键；优选地，所述变体包括在与SEQ ID NO:1的第346位对应的位置上的氨基酸残基、以及与SEQ ID NO:1的第393位对应的位置上的氨基酸残基之间形成二硫键；进一步优选地，所述变体在与SEQ ID NO:1的第346位对应的位置上的氨基酸被取代为C，且所述变体在与SEQ ID NO:1的第393位对应的位置上的氨基酸被取代为C。
本领域技术人员可以理解的是，此处的“引入”不限定二硫键是以任何特定的方式生成的。例如，“引入”二硫键，可以包括将待引入二硫键的植酸酶序列的相应位置上的氨基酸残基替换成能够形成二硫键的氨基酸残基(例如，半胱氨酸残基Cys、同型半胱氨酸残基Hey，等等)；和/或在相应位置上插入能够形成二硫键的氨基酸残基。这样的替换和/或插入可以，例如，通过 本领域公知的定点诱变方法来实现。“引入”亦包括形成所述二硫键的任何一个或两个氨基酸残基是由于自然突变而产生的情况。
在一些具体实施例中，所述变体的氨基酸序列是SEQ ID NO:99、SEQ ID NO:100、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:104、SEQ ID NO:105、SEQ ID NO:106、SEQ ID NO:108、SEQ ID NO:109、SEQ ID NO:110、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:114、SEQ ID NO:116、SEQ ID NO:117或SEQ ID NO:118。
此外，本发明还提供一种核酸，其编码如上任一项所述的亲本植酸酶变体。
此外，本发明还提供一种载体，其包含如上所述的核酸。
此外，本发明还提供一种宿主细胞，其包含如上所述的载体。
在优选的实施例中，所述宿主细胞是真菌细胞、细菌细胞或植物细胞。
进一步优选地，所述宿主细胞为真菌细胞，所述真菌细胞选自毕赤酵母细胞或者黑曲霉细胞。
此外，本发明还提供一种如上任一项所述的亲本植酸酶变体的生产方法，该方法包括：
(a)在适于所述亲本植酸酶变体表达的条件下，对如上任一项所述的宿主细胞进行培养；
(b)回收该亲本植酸酶变体。
本发明的有益效果如下：
在植酸酶或植酸酶突变体的基础上引入如本发明所述的一个或多个突变位点，尤其是同时引入多个突变位点，得到的植酸酶变体与亲本植酸酶相比，在85℃水浴处理5分钟后的残余活力提高了16-27％。因此，本发明的技术方案可以显著提升植酸酶的酶活性质，特别是在耐热稳定性、耐蒸汽稳定性、制粒稳定性方面，显著优于现有的野生型或者突变型植酸酶。
命名及定义
序列一致性：即序列同一性，定义为对比序列并在必要时引入缺口以获取最大百分比序列同一性后，且不将任何保守替代视为序列同一性的一部分，候选序列中与特定肽或多肽序列中的氨基酸残基相同的氨基酸残基的百分率。可以本领域技术范围内的多种方式进行序列对比以测定百分比氨基酸序列同一性，例如使用公众可得到的计算机软件，诸如BLAST、BLAST-2,ALIGN或Megalign(DNASTAR)软件。本领域技术人员可决定测量对比的适宜参数， 包括对所比较的序列全长获得最大对比所需的任何算法。
亲本：是指进行了本发明所述的突变后可以产生本发明变体的植酸酶。所述亲本可以是天然产生(野生型)的植酸酶或由合适的方法制备的其突变体，所述亲本也可以是等位变体。
宿主细胞：是指任何对用包含本发明核酸的载体的转化、转染、转导等易感的细胞类型。术语“宿主细胞”涵盖由于在复制过程中发生的突变而与亲本细胞不完全相同的亲本细胞的任何后代。
取代：是指用不同的氨基酸替代占据某位置的原有氨基酸。
变体：是指具有植酸酶活性的酶，且相对于其亲本植酸酶包含在一个或多个(数个)位置的一个或多个(数个)氨基酸残基的取代。本发明变体的构建可通过在适于生产变体的条件下，培养含有编码所述变体之核酸序列的宿主细胞来实现，所述变体可随后从得到的培养液中回收。
野生型：是指天然产生的微生物表达的植酸酶，例如自然界中发现的大肠杆菌来源的天然的植酸酶。
在描述本发明的植酸酶变体时，使用了下述命名法以便于参考。在所有情况下，使用了公认的IUPAC单字符或三字符氨基酸缩写。
对于氨基酸的取代，使用了下列命名法：原始氨基酸、位置、取代氨基酸。例如，在位置226用丙氨酸(alanine)取代苏氨酸(threonine)被命名为“Thr226Ala”或“T226A”。通过加号(“+”)来分开多个突变，例如“Gly205Arg+Ser411Phe”或“G205R+S411F”，代表分别在位置205和411用精氨酸(R)取代甘氨酸(G)、用苯丙氨酸(F)取代丝氨酸(S)的突变。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细的说明。
图1示出pPIC9K-APPA-Y0的质粒图谱。
图2示出实施例1中亲本植酸酶及其变体的热稳定性检测结果。
图3示出实施例3中第295位氨基酸定点饱和突变的变体热稳定性检测结果。
图4示出实施例3中第349位氨基酸定点饱和突变的变体热稳定性检测结果。
图5示出实施例3中第374位氨基酸定点饱和突变的变体热稳定性检测结果。
图6示出实施例4中组合突变的变体热稳定性检测结果。
图7示出实施例5中亲本植酸酶APPA-An1的变体热稳定性检测结果。
图8示出实施例6中亲本植酸酶APPA-An5的变体热稳定性检测结果。
图9示出实施例7中亲本植酸酶APPA-An10的变体热稳定性检测结果。
图10示出实施例8中亲本植酸酶APPA-An15的变体热稳定性检测结果。
图11示出实施例9中亲本植酸酶APPA-An18的变体热稳定性检测结果。
具体实施方式
为了更清楚地说明本发明，下面结合优选实施例和附图对本发明做进一步的说明。本领域技术人员应当理解，下面所具体描述的内容是说明性的而非限制性的，不应以此限制本发明的保护范围。
实施例1构建毕赤酵母突变体表达菌株
本发明引用了专利WO2019228441中被命名为APPA-M2-O的植酸酶突变体氨基酸序列，其氨基酸序列如本发明中的SEQ ID NO：2所示，本发明将该植酸酶突变体命名为APPA-Y0。APPA-Y0是大肠杆菌来源的野生型植酸酶(其氨基酸序列如SEQ ID NO：1所示)经突变筛选、糖基化和二硫键位点引入后获得的植酸酶突变体，具体方法如WO2019228441所述，该突变体具有优良的耐热稳定性能。本实施例是在APPA-Y0序列的基础上做的进一步突变和筛选。
由金斯瑞生物科技股份有限公司合成APPA-Y0在毕赤酵母中表达的核酸序列，将该基因克隆到毕赤酵母载体中，表达载体为pPIC9K，使用酿酒酵母的Alpha因子作为信号肽，植酸酶表达质粒pPIC9K-APPA-Y0如图1所示。
在第一轮变体筛选中，使用APPA-Y0作为亲本植酸酶，为了改善亲本植酸酶的热稳定性，发明人基于APPA-Y0的氨基酸序列和蛋白质结构分析设计了如下37个突变体，如表1所示。设计中每个变体相对于亲本植酸酶APPA-Y0包含有一个氨基酸的取代，将这些变体分别命名为APPA-Y1至APPA-Y37，其氨基酸序列分别如SEQ ID NO：3-39所示。
表1第一轮构建的植酸酶变体
Figure PCTCN2021075123-appb-000001
Figure PCTCN2021075123-appb-000002
Figure PCTCN2021075123-appb-000003
变体质粒按照上表变体名称分别命名为pPIC9K-APPA-Y1至pPIC9K-APPA-Y37。为了将亲本植酸酶及其变体进行表达，使用Pichia expression kit(Invitrogen)并参考其说明书对毕赤酵母GS115和质粒进行操作。具体如下，将毕赤酵母GS115菌株使用YPD培养基(1％酵母提取物、2％蛋白、2％葡萄糖和1.5％琼脂)平板30℃培养48h后，挑取单克隆到4mLYPD液体培养基(1％酵母提取物、2％蛋白、2％葡萄糖)中，30℃200rpm培养12h，随后转接到30mL YPD液体培养基的三角瓶中，30℃、220rpm培养4-5h检测到OD600值在1.1-1.3范围后，将培养液在4℃9,000rpm离心2min,分别收集4mL菌体至灭菌EP管中，轻轻弃上清，用灭菌的滤纸吸干残留的上清后用预冷的1mL灭菌水重悬菌体，4℃9,000rpm离心2min,弃上清。重复上述步骤，将预冷的1mL山梨醇(1mol/L)重悬菌体；4℃、9,000rpm离心2min弃上清，预冷的100-150μl山梨醇(1mol/L)重悬菌体，至此感受态制备完成。将表达质粒pPIC9K-APPA-Y0和其他37个变体用BglII进行线性化，线性化片段纯化回收后通过电穿孔法转化上述毕赤酵母GS115感受态中，将混合物均匀涂布于MD平板上，30℃倒置培养2-3天，将平板上所有的菌落都用无菌水洗下来后涂布在含不同浓度遗传霉素的YPD(0.5mg/mL-8mg/mL)平板上筛选多拷贝的转化子。在MD平板上筛选得到毕赤酵母重组菌株命名为APPA-Y0和APPA-Y1至APPA-Y37。将上述筛选获得的克隆分别转接于BMGY培养基中，在30℃、250rpm振荡摇床中培养24h，再转入BMMY培养基中，维持30℃、250rpm条件，每天添加0.5％的甲醇，诱导表达120h后；9000-12000rpm离心10min以去除菌体，得到含植酸酶APPA-Y0和其他37个变体的发酵上清液，SDS-PAGE结果显示APPA-Y1，APPA-Y7,APPA-Y8和APPA-Y16四个变体未能表达，剩余的APPA-Y0和其它33个变体都有表达。
实施例2热稳定性测定
植酸酶活力测定遵循GBT 18634-2009文件标准。将实施例1中的33个变体样品及亲本植酸酶APPA-Y0用水稀释至100U/mL。取9mL水于25mL比色管中，用移液枪吸取酶样品1mL，快速加入到试管中并用混匀器快速混匀 后，在85℃恒温水浴中准确放置5min。迅速冷却至室温，用水进行稀释，测定各样品残余活力，从而计算不同处理温度下酶的残余活力，此处将热处理前的酶活定为100％，得到的热稳定性数据如图2所示。
根据上述实验结果，突变点的引入对植酸酶会产生显著影响：
一些突变点的引入会导致植酸酶不能正常表达，如APPA-Y1，APPA-Y7,APPA-Y8和APPA-Y16；
一些突变点的引入会导致植酸酶热稳定性降低，如APPA-Y22和APPA-Y29，上述两个突变体的热稳定性显著低于APPA-Y0的热稳定性；
还有一些突变点的引入显著提高了植酸酶的耐热性，如APPA-Y2(M29L)，APPA-Y4(R50K)，APPA-Y5(G52A)，APPA-Y6(L58M)，APPA-Y10(A105F)，APPA-Y12(T118Q)，APPA-Y25(T295P)，APPA-Y28(L307I)，APPA-Y30(Q349K)，APPA-Y33(M360L)，APPA-Y36(E374R)，其中APPA-Y25(T295P)，APPA-Y30(Q349K)，APPA-Y36(E374R)的表现最好，与亲本植酸酶APPA-Y0相比，变体的残余活力提高了19-22％左右。
实施例3对实施例2中的3个突变位点进行饱和突变
对实施例2中耐热性提高最多的3个突变菌株APPA-Y25，APPA-Y30，APPA-Y36对应的3个突变点T295P，Q349K，E374R进行饱和突变，即分别将295位、349位、374位的氨基酸突变成其他18个氨基酸，对应的序列命名见下表2-4所示。
表2第295位氨基酸的定点饱和突变
突变体名称 突变位点 序列编号
APPA-Y25-1 T295A SEQ ID NO：44
APPA-Y25-2 T295C SEQ ID NO：45
APPA-Y25-3 T295D SEQ ID NO：46
APPA-Y25-4 T295E SEQ ID NO：47
APPA-Y25-5 T295F SEQ ID NO：48
APPA-Y25-6 T295G SEQ ID NO：49
APPA-Y25-7 T295H SEQ ID NO：50
APPA-Y25-8 T295I SEQ ID NO：51
APPA-Y25-9 T295K SEQ ID NO：52
APPA-Y25-10 T295L SEQ ID NO：53
APPA-Y25-11 T295M SEQ ID NO：54
APPA-Y25-12 T295N SEQ ID NO：55
APPA-Y25-13 T295Q SEQ ID NO：56
APPA-Y25-14 T295R SEQ ID NO：57
APPA-Y25-15 T295S SEQ ID NO：58
APPA-Y25-16 T295V SEQ ID NO：59
APPA-Y25-17 T295W SEQ ID NO：60
APPA-Y25-18 T295Y SEQ ID NO：61
表3第349位氨基酸的定点饱和突变
突变体名称 突变位点 序列编号
APPA-Y30-1 Q349A SEQ ID NO：62
APPA-Y30-2 Q349C SEQ ID NO：63
APPA-Y30-3 Q349D SEQ ID NO：64
APPA-Y30-4 Q349E SEQ ID NO：65
APPA-Y30-5 Q349F SEQ ID NO：66
APPA-Y30-6 Q349G SEQ ID NO：67
APPA-Y30-7 Q349H SEQ ID NO：68
APPA-Y30-8 Q349I SEQ ID NO：69
APPA-Y30-9 Q349L SEQ ID NO：70
APPA-Y30-10 Q349M SEQ ID NO：71
APPA-Y30-11 Q349N SEQ ID NO：72
APPA-Y30-12 Q349P SEQ ID NO：73
APPA-Y30-13 Q349R SEQ ID NO：74
APPA-Y30-14 Q349S SEQ ID NO：75
APPA-Y30-15 Q349T SEQ ID NO：76
APPA-Y30-16 Q349V SEQ ID NO：77
APPA-Y30-17 Q349W SEQ ID NO：78
APPA-Y30-18 Q349Y SEQ ID NO：79
表4第374位氨基酸的定点饱和突变
突变体名称 突变位点 序列编号
APPA-Y36-1 E374A SEQ ID NO：80
APPA-Y36-2 E374C SEQ ID NO：81
APPA-Y36-3 E374D SEQ ID NO：82
APPA-Y36-4 E374F SEQ ID NO：83
APPA-Y36-5 E374G SEQ ID NO：84
APPA-Y36-6 E374H SEQ ID NO：85
APPA-Y36-7 E374I SEQ ID NO：86
APPA-Y36-8 E374K SEQ ID NO：87
APPA-Y36-9 E374L SEQ ID NO：88
APPA-Y36-10 E374M SEQ ID NO：89
APPA-Y36-11 E374N SEQ ID NO：90
APPA-Y36-12 E374P SEQ ID NO：91
APPA-Y36-13 E374Q SEQ ID NO：92
APPA-Y36-14 E374S SEQ ID NO：93
APPA-Y36-15 E374T SEQ ID NO：94
APPA-Y36-16 E374V SEQ ID NO：95
APPA-Y36-17 E374W SEQ ID NO：96
APPA-Y36-18 E374Y SEQ ID NO：97
按照实施例1中的方法，使用毕赤酵母表达各变体，随后按照实施例2中的方法测定各变体的热稳定性，结果如图3-5所示。
图中结果表明对3个位置的氨基酸分别进行饱和突变对变体有显著影响：
有的突变甚至导致了不能正常表达，如APPA-Y25-2，APPA-Y25-17，APPA-Y30-3，APPA-Y30-5，APPA-Y30-8，APPA-Y30-12和APPA-Y30-18；
另外一些突变点引入会导致植酸酶稳定性降低，如APPA-Y30-2，APPA-Y36-2和APPA-Y36-9，上述三个突变体的热稳定性显著低于APPA-Y0的热稳定性；
还有一些突变点的引入显著提高了植酸酶的耐热性，如APPA-Y25-6(T295G)，APPA-Y25-9(T295K)，APPA-Y25-10(T295L)，APPA-Y25-13(T295Q)，APPA-Y25-18(T295Y)，APPA-Y30-9(Q349L)，APPA-Y30-6(Q349G)，APPA-Y30-7(Q349H)，APPA-Y30-13(Q349R)，APPA-Y30-15(Q349T)，APPA-Y36-11(E374N)，APPA-Y36-14(E374S)，APPA-Y36-15(E374T)， APPA-Y36-16(E374V)，APPA-Y36-4(E374F),APPA-Y36-8(E374K)，APPA-Y36-12(E374P)，APPA-Y36-18(E374Y)。
通过实施例2和实施例3的实验数据可以看出，当在亲本植酸酶的基础上引入单个氨基酸的取代时，突变点T295P、Q349K、E374R、T295Y、Q349L或E374V的引入带来的效果更好，可以使植酸酶的热稳定性显著提升。
实施例4突变点的组合
将实施例2中的3个突变菌株APPA-Y25，APPA-Y30，APPA-Y36对应的3个突变点T295P，Q349K，E374R进行叠加，按照实施例1中的方法构建相应的变体并在毕赤酵母中表达，具体突变情况参见下表5。按照实施例2中的检测方法进行热稳定性测定，得到的热稳定性数据如图6所示。由检测结果可见，组合突变后的植酸酶变体，与亲本植酸酶APPA-Y0相比，耐热性能均有明显提升；一些突变的叠加使得变体耐热性与APPA-Y0相比提高了16％-27％，说明合适的组合可以创造出更稳定的突变体，可以预见其在饲料造粒中会有良好表现。
表5组合突变后的植酸酶变体
序列名称 突变位点 序列编号
APPA-Y38 T295P+Q349K SEQ ID NO：40
APPA-Y39 T295P+E374R SEQ ID NO：41
APPA-Y40 Q349K+E374R SEQ ID NO：42
APPA-Y41 T295P+Q349K+E374R SEQ ID NO：43
实施例5黑曲霉表达亲本植酸酶APPA-An1的变体
APPA-An1是大肠杆菌野生型植酸酶经突变获得的另一耐热优良突变体，其氨基酸序列如SEQ ID NO:98所示：
Figure PCTCN2021075123-appb-000004
Figure PCTCN2021075123-appb-000005
为了测试本发明中发现的突变位点在该已有的植酸酶突变体上是否也可以发挥功能，进一步提高植酸酶的稳定性，该实施例将APPA-An1作为亲本植酸酶，将氨基酸取代Q349K、E374R单独或组合添加到APPA-An1的氨基酸序列中，各突变体分别按照APPA-An2～APPA-An4进行命名，如下表所示。按照专利申请CN107353327A中描述的方法，在黑曲霉中表达上述植酸酶突变体。获得摇瓶上清液后按照实施例2描述进行热稳定性测定，85℃条件下孵育5分钟，实验结果如图7所示。实验结果表明，3个变体都表现出显著的稳定性性能提升，体现出比亲本植酸酶APPA-An1更为优秀的稳定性，即在亲本植酸酶中引入本发明的1-2个突变点后可以获得更高稳定性的植酸酶变体，可以预见其在饲料造粒中会有良好表现。
表6亲本APPA-An1的变体
突变体名称 突变位点 序列编号
APPA-An1 亲本 SEQ ID NO：98
APPA-An2 Q349K SEQ ID NO：99
APPA-An3 E374R SEQ ID NO：100
APPA-An4 Q349K+E374R SEQ ID NO：101
实施例6黑曲霉表达亲本植酸酶APPA-An5的变体
APPA-An5是野生型植酸酶经突变获得的另一耐热优良突变体，其氨基酸序列如SEQ ID NO：102所示。为了测试本发明中发现的突变位点在该现有的植酸酶突变体上是否也可以发挥功能，进一步提高植酸酶的稳定性，该实施例将APPA-An5作为亲本植酸酶，将T295P、Q349K、E374R单独或组合添加到APPA-An5的氨基酸序列中，各突变体分别按照APPA-An6～APPA-An9进行命名，如下表7所示。按照实施例5的方法，在黑曲霉中表达上述植酸酶变体。获得摇瓶上清液后按照实施例2的描述进行热稳定性测定，85℃条件下孵育5分钟。实验结果如图8所示。我们发现4个突变体都表现出显著的稳定性性能提升，体现出比亲本APPA-An5更为优秀的稳定性，即在亲本植酸酶突变体中引入本发明的1-2个突变点后可以获得更高稳定性的植酸酶突变体，可以预见其在饲料造粒中会有良好表现。
表7植酸酶突变体
突变体名称 突变位点 序列编号
APPA-An5 亲本 SEQ ID NO：102
APPA-An6 T295P SEQ ID NO：103
APPA-An7 Q349K SEQ ID NO：104
APPA-An8 E374R SEQ ID NO：105
APPA-An9 Q349K+E374R SEQ ID NO：106
实施例7黑曲霉表达亲本植酸酶APPA-An10的变体
APPA-An10是野生型植酸酶经突变筛选获得的另一耐热优良突变体，其氨基酸序列如SEQ ID NO:107所示。为了测试本发明中发现的突变位点在该现有的植酸酶突变体上是否也可以发挥功能，进一步提高植酸酶的稳定性，该实施例将APPA-An10作为亲本，将T295P、Q349K、E374R单独或组合添加到APPA-An10的氨基酸序列中，各突变体分别按照APPA-An11～APPA-An14进行命名，如下表8所示。按照实施例5的方法，在黑曲霉中表达上述植酸酶变体。获得摇瓶上清液后按照实施例2描述进行热稳定性测定，85℃条件下孵育5分钟。实验结果如图9所示。我们发现4个变体都表现出显著的稳定性性能提升，体现出比亲本APPA-An10更为优秀的稳定性，即在亲本植酸酶突变体中引入本发明的1-3个突变点后可以获得更高稳定性的植酸酶突变体，可以预见其在饲料造粒中会有良好表现。
表8植酸酶突变体
突变体名称 突变位点 序列编号
APPA-An10 亲本 SEQ ID NO：107
APPA-An11 E374R SEQ ID NO：108
APPA-An12 T295P+Q349K SEQ ID NO：109
APPA-An13 T295P+E374R SEQ ID NO：110
APPA-An14 T295P+Q349K+E374R SEQ ID NO：111
实施例8黑曲霉表达亲本植酸酶APPA-An15的变体
APPA-An15是野生型植酸酶经突变筛选获得的另一耐热优良突变体，其氨基酸序列如SEQ ID NO:112所示。为了测试本发明中发现的突变位点在该现有的植酸酶突变体上是否也可以发挥功能，进一步提高植酸酶的稳定性，该实施例将APPA-An15作为亲本，将T295P、E374R单独添加到APPA-An15的 氨基酸序列中，各突变体分别按照APPA-An16～APPA-An17进行命名，如下表所示。按照实施例5的方法，在黑曲霉中表达上述植酸酶突变体。获得摇瓶上清液后按照实施例2描述进行热稳定性测定，85℃条件下孵育5分钟。实验结果如图10所示。我们发现2个突变体都表现出显著的稳定性性能提升，体现出比亲本APPA-An15更为优秀的稳定性，即在亲本植酸酶突变体中引入本发明的1个突变点后可以获得更高稳定性的植酸酶突变体，可以预见其在饲料造粒中会有良好表现。
表9植酸酶突变体
突变体名称 突变位点 序列编号
APPA-An15 亲本 SEQ ID NO：112
APPA-An16 T295P SEQ ID NO：113
APPA-An17 E374R SEQ ID NO：114
实施例9黑曲霉表达亲本植酸酶APPA-An18的变体
APPA-An18是野生型植酸酶经突变筛选获得的另一耐热优良突变体，其氨基酸序列如SEQ ID NO:115所示。为了测试本发明中发现的突变位点在该现有的植酸酶突变体上是否也可以发挥功能，进一步提高植酸酶的稳定性，该实施例将APPA-An18作为亲本，将T295P、Q349K、E374R单独或组合添加到APPA-An18的氨基酸序列中，各突变体分别按照APPA-An19～APPA-An21进行命名，如下表10所示。按照实施例5的方法，在黑曲霉中表达上述植酸酶变体。获得摇瓶上清液后按照实施例2描述进行热稳定性测定，85℃条件下孵育5分钟。实验结果如图11所示。我们发现3个突变体都表现出显著的稳定性性能提升，体现出比亲本APPA-An18更为优秀的稳定性，即在亲本植酸酶突变体中引入本发明的1-3个突变点后可以获得更高稳定性的植酸酶突变体，可以预见其在饲料造粒中会有良好表现。
表10植酸酶突变体
突变体名称 突变位点 序列编号
APPA-An18 亲本 SEQ ID NO：115
APPA-An19 Q349K SEQ ID NO：116
APPA-An20 Q349K+E374R SEQ ID NO：117
APPA-An21 T295P+Q349K+E374R SEQ ID NO：118
显然，本发明的上述实施例仅仅是为清楚地说明本发明所作的举例，而并非是对本发明的实施方式的限定，对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动，这里无法对所有的实施方式予以穷举，凡是属于本发明的技术方案所引伸出的显而易见的变化或变动仍处于本发明的保护范围之列。

Claims (17)

  1. 一种亲本植酸酶变体，其特征在于，包括：
    (a)所述变体相对于其亲本植酸酶，在对应于SEQ ID NO:1的第295位、第349位、第374位上具有一个或多个氨基酸的取代，所述亲本植酸酶的氨基酸序列与SEQ ID NO:98相比，具有至少80％的序列一致性；
    (b)所述变体具有植酸酶活性；并且
    (c)与亲本植酸酶相比，所述变体具有增加的热稳定性。
  2. 权利要求1所述的亲本植酸酶变体，其特征在于，所述亲本植酸酶的氨基酸序列与SEQ ID NO:98相比，具有至少90％的序列一致性；优选的，具有至少95％的序列一致性；更优选的，具有至少98％的序列一致性。
  3. 根据权利要求2所述的亲本植酸酶变体，其特征在于，所述亲本植酸酶的氨基酸序列选自：SEQ ID NO:2、SEQ ID NO:98、SEQ ID NO:102、SEQ ID NO:107、SEQ ID NO:112或SEQ ID NO:115。
  4. 根据权利要求3所述的亲本植酸酶变体，其特征在于，所述变体在第295位的氨基酸被取代为P、Y、G、K、L或Q；优选地，被取代为P或Y。
  5. 根据权利要求3所述的亲本植酸酶变体，其特征在于，所述变体在第349位的氨基酸被取代为选自K、L、G、H、R或T；优选地，被取代为K或L。
  6. 根据权利要求3所述的亲本植酸酶变体，其特征在于，所述变体在第374位的氨基酸被取代为选自R、V、N、S、T、F、K、P或Y；优选地，被取代为R或V。
  7. 根据权利要求1-6中任一项所述的亲本植酸酶变体，其特征在于，所述变体相对于其亲本植酸酶，包含选自下组的氨基酸取代：
    T295P；Q349K；E374R；T295Y；Q349L；E374V；
    T295P+Q349K；T295P+E374R；T295P+Q349L；T295P+E374V；
    T295Y+Q349K；T295Y+E374R；T295Y+Q349L；T295Y+E374V；
    Q349L+E374R；Q349K+E374R；Q349L+E374V；Q349K+E374V；
    T295P+Q349K+E374R；T295Y+Q349K+E374R；
    T295P+Q349L+E374R；T295Y+Q349L+E374R；
    T295P+Q349K+E374V；T295Y+Q349K+E374V；
    T295P+Q349L+E374V；T295Y+Q349L+E374V。
  8. 根据权利要求7所述的亲本植酸酶变体，其特征在于，所述变体与其 亲本植酸酶的氨基酸序列相比，具有至少90％的序列一致性；优选的，具有至少95％的序列一致性；更优选的，具有至少98％的序列一致性。
  9. 根据权利要求1-8中任一项所述的亲本植酸酶变体，其特征在于，所述变体还进一步包括至少一对引入的二硫键。
  10. 根据权利要求9所述的亲本植酸酶变体，其特征在于，所述变体包括在与SEQ ID NO:1的第346位对应的位置上的氨基酸残基、以及与SEQ ID NO:1的第393位对应的位置上的氨基酸残基之间形成二硫键。
  11. 根据权利要求1-10中任一项所述的亲本植酸酶变体，其特征在于，所述变体的氨基酸序列选自SEQ ID NO:99、SEQ ID NO:100、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:104、SEQ ID NO:105、SEQ ID NO:106、SEQ ID NO:108、SEQ ID NO:109、SEQ ID NO:110、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:114、SEQ ID NO:116、SEQ ID NO:117或SEQ ID NO:118。
  12. 一种核酸，其编码如权利要求1-11中任一项所述的亲本植酸酶变体。
  13. 一种载体，其包含如权利要求12所述的核酸。
  14. 一种宿主细胞，其包含权利要求13所述的载体。
  15. 权利要求14所述的宿主细胞，其中该宿主细胞是真菌细胞、细菌细胞或植物细胞。
  16. 权利要求15所述的宿主细胞，其为真菌细胞，所述真菌细胞选自毕赤酵母细胞或者黑曲霉细胞。
  17. 一种如权利要求1-11中任一项所述的亲本植酸酶变体的生产方法，其特征在于，该方法包括：
    (a)在适于所述亲本植酸酶变体表达的条件下，对权利要求14-16中任一项所述的宿主细胞进行培养；
    (b)回收该亲本植酸酶变体。
PCT/CN2021/075123 2020-04-29 2021-02-03 一种亲本植酸酶变体 WO2021218269A1 (zh)

Priority Applications (12)

Application Number Priority Date Filing Date Title
CN202311652847.3A CN117683744A (zh) 2020-04-29 2021-02-03 一种亲本植酸酶变体
CN202311652868.5A CN117683746A (zh) 2020-04-29 2021-02-03 一种亲本植酸酶变体
CN202311611465.6A CN117701527A (zh) 2020-04-29 2021-02-03 一种亲本植酸酶变体
CN202311652859.6A CN117683745A (zh) 2020-04-29 2021-02-03 一种亲本植酸酶变体
CN202311642247.9A CN117721093A (zh) 2020-04-29 2021-02-03 一种亲本植酸酶变体
CN202311319920.5A CN117535267A (zh) 2020-04-29 2021-02-03 一种亲本植酸酶变体
CN202311647341.3A CN117683743A (zh) 2020-04-29 2021-02-03 一种亲本植酸酶变体
EP21797344.5A EP4144840A1 (en) 2020-04-29 2021-02-03 Parent phytase variant
US17/997,037 US20230167422A1 (en) 2020-04-29 2021-02-03 Parent Phytase Variant
CN202311634743.XA CN117778350A (zh) 2020-04-29 2021-02-03 一种亲本植酸酶变体
CN202311319916.9A CN117535266A (zh) 2020-04-29 2021-02-03 一种亲本植酸酶变体
CN202180006789.XA CN114761549A (zh) 2020-04-29 2021-02-03 一种亲本植酸酶变体

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010355432 2020-04-29
CN202010355432.X 2020-04-29

Publications (1)

Publication Number Publication Date
WO2021218269A1 true WO2021218269A1 (zh) 2021-11-04

Family

ID=76461795

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/075123 WO2021218269A1 (zh) 2020-04-29 2021-02-03 一种亲本植酸酶变体

Country Status (4)

Country Link
US (1) US20230167422A1 (zh)
EP (1) EP4144840A1 (zh)
CN (11) CN117701527A (zh)
WO (1) WO2021218269A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114807089B (zh) * 2022-06-29 2022-09-27 中国农业科学院北京畜牧兽医研究所 一种提高植酸酶热稳定性的方法及突变体APPAmut7和应用

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1309699A (zh) * 1998-03-23 2001-08-22 诺维信公司 植酸酶变体
WO2007044968A2 (en) 2005-10-12 2007-04-19 Genencor International, Inc. Stable, durable granules with active agents
CN1981597A (zh) 2005-12-13 2007-06-20 珠海天翼医药技术开发有限公司 一种饲用耐高温微丸酶的制备方法
CN101168734A (zh) 2007-09-29 2008-04-30 北京昕地美饲料科技有限公司 包衣微丸植酸酶及其制备方法
CN102575237A (zh) * 2009-10-22 2012-07-11 巴斯夫欧洲公司 合成的植酸酶变体
EP2102334B1 (de) 2006-11-10 2013-04-03 AB Enzymes GmbH Polypeptid mit phytaseaktivität und erhöhter temperaturstabilität der enzymaktivität sowie dieses codierende nukleotidsequenz
EP2800476A1 (en) * 2012-01-05 2014-11-12 Dupont Nutrition Biosciences ApS Method of feeding
US9528096B1 (en) * 2016-06-30 2016-12-27 Fornia Biosolutions, Inc. Phytases and uses thereof
US9605245B1 (en) 2016-06-30 2017-03-28 Fornia BioSoultions, Inc. Phytases and uses thereof
CN107353327A (zh) 2017-03-30 2017-11-17 南京百斯杰生物工程有限公司 植酸酶在黑曲霉中表达
CN108603181A (zh) * 2016-06-30 2018-09-28 福尼亚生物处理股份有限公司 新植酸酶及其用途
WO2019228441A1 (zh) 2018-05-30 2019-12-05 南京百斯杰生物工程有限公司 植酸酶突变体

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MXPA04011224A (es) * 2002-05-30 2005-01-25 Basf Ag Fitasas modificadas.
DK2933329T3 (en) * 2008-09-26 2017-07-24 Novozymes As Hafniaphytase variants
CN102943083B (zh) * 2012-11-27 2013-12-25 青岛根源生物技术集团有限公司 一种定点突变的耐高温植酸酶基因tp及其表达载体和应用
CN107236717B (zh) * 2016-03-28 2021-03-30 青岛蔚蓝生物集团有限公司 植酸酶突变体
US10351832B2 (en) * 2016-06-30 2019-07-16 Fornia Biosolutions, Inc. Phytases and uses thereof
EP3569703A4 (en) * 2017-01-15 2020-11-18 Feed Research Institute Chinese Academy of Agricultural Sciences PHYTASE YEAPPA MUTANTS WITH IMPROVED GASTRIC PROTEIN RESISTANCE AND ACID RESISTANCE AND INCREASED CATALYTIC EFFICIENCY

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1309699A (zh) * 1998-03-23 2001-08-22 诺维信公司 植酸酶变体
WO2007044968A2 (en) 2005-10-12 2007-04-19 Genencor International, Inc. Stable, durable granules with active agents
CN1981597A (zh) 2005-12-13 2007-06-20 珠海天翼医药技术开发有限公司 一种饲用耐高温微丸酶的制备方法
EP2102334B1 (de) 2006-11-10 2013-04-03 AB Enzymes GmbH Polypeptid mit phytaseaktivität und erhöhter temperaturstabilität der enzymaktivität sowie dieses codierende nukleotidsequenz
CN101168734A (zh) 2007-09-29 2008-04-30 北京昕地美饲料科技有限公司 包衣微丸植酸酶及其制备方法
CN102575237A (zh) * 2009-10-22 2012-07-11 巴斯夫欧洲公司 合成的植酸酶变体
EP2800476A1 (en) * 2012-01-05 2014-11-12 Dupont Nutrition Biosciences ApS Method of feeding
US9528096B1 (en) * 2016-06-30 2016-12-27 Fornia Biosolutions, Inc. Phytases and uses thereof
US9605245B1 (en) 2016-06-30 2017-03-28 Fornia BioSoultions, Inc. Phytases and uses thereof
CN108603181A (zh) * 2016-06-30 2018-09-28 福尼亚生物处理股份有限公司 新植酸酶及其用途
CN107353327A (zh) 2017-03-30 2017-11-17 南京百斯杰生物工程有限公司 植酸酶在黑曲霉中表达
WO2019228441A1 (zh) 2018-05-30 2019-12-05 南京百斯杰生物工程有限公司 植酸酶突变体

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SHIVANGE AMOL V; DENNIG ALEXANDER; SCHWANEBERG ULRICH: "Multi-Site Saturation by OmniChange Yields a pH- and Thermally Improved Phytase", JOURNAL OF BIOTECHNOLOGY, vol. 170, 4 December 2013 (2013-12-04), pages 68 - 72, XP028548059, ISSN: 0168-1656, DOI: 10.1016/j.jbiotec.2013.11.014 *
WANG XI: "Improving Thermostability of Escherichia coli phytase AppA by Multipoint Mutation", HIGH TECHNOLOGY LETTERS, vol. 24, no. 12, 31 December 2014 (2014-12-31), pages 1306 - 1313, XP055849164, ISSN: 1002-0470, DOI: 10.3772/j.issn.1002-0470.2014.12.012 *

Also Published As

Publication number Publication date
CN117721093A (zh) 2024-03-19
CN117683744A (zh) 2024-03-12
CN117535267A (zh) 2024-02-09
CN117778350A (zh) 2024-03-29
CN114761549A (zh) 2022-07-15
CN117683745A (zh) 2024-03-12
CN113025593A (zh) 2021-06-25
CN117683743A (zh) 2024-03-12
US20230167422A1 (en) 2023-06-01
CN117535266A (zh) 2024-02-09
CN117701527A (zh) 2024-03-15
CN117683746A (zh) 2024-03-12
EP4144840A1 (en) 2023-03-08
CN113025593B (zh) 2022-09-23

Similar Documents

Publication Publication Date Title
US20220025384A1 (en) Phytase mutants
CN112204136B (zh) 植酸酶突变体
CN107164344B (zh) 一类耐热型植酸酶突变体及其编码基因和应用
CN110042092B (zh) 一类稳定性提高的木聚糖酶突变体及其编码基因和应用
US20230242890A1 (en) Phytase mutant
US20220154154A1 (en) Phytase mutant
WO2021218269A1 (zh) 一种亲本植酸酶变体
CN109997970B (zh) 一类酶活和耐热性提高的酸性木聚糖酶突变体及其编码基因和应用
CN114457056B (zh) 一种盐乳芽孢杆菌木聚糖酶在改善面粉加工品质中的应用
CN102978146A (zh) 一株重组大肠杆菌及用其制备溶血性磷脂酶c的方法
CN100432213C (zh) 一种编码高温植酸酶基因的重组菌和该基因及其合成、克隆和表达
WO2020063267A1 (zh) 植酸酶突变体
CN114736886B (zh) 植酸酶突变体及其制备方法
CN116064479B (zh) 一种含木聚糖酶突变体和胆汁酸的复合制剂及其应用
KR101479712B1 (ko) 흑염소 반추위 미생물 유래의 엔도글루카나아제 ｃｅｌ－ＫＧ２４ 유전자 및 이의 용도
CN1616659A (zh) 一种耐高温、高比活植酸酶基因及其克隆和表达
KR101479693B1 (ko) 흑염소 반추위 미생물 유래의 엔도글루카나아제 ｃｅｌ８－ＫＧ４７ 유전자 및 이의 용도
KR101479708B1 (ko) 흑염소 반추위 미생물 유래의 엔도―1，4―베타―자일라나아제 ｃｅｌ１０－ＫＧ０４ 유전자 및 이의 용도
KR101479696B1 (ko) 흑염소 반추위 미생물 유래의 엔도글루카나아제 ｃｅｌ－ＫＧ３５ 유전자 및 이의 용도
CN115851672A (zh) 一种木聚糖酶突变体及胆汁酸复配酶制剂和应用
KR101483582B1 (ko) 흑염소 반추위 미생물 유래의 엔도자일라나아제 ｃｅｌ１０－ＫＧ４２ 유전자 및 이의 용도
KR101479698B1 (ko) 흑염소 반추위 미생물 유래의 셀룰라아제 유전자 ｃｅｌ－ＫＧ２０ 및 이의 용도
KR101483583B1 (ko) 흑염소 반추위 미생물 유래의 엔도자일라나아제 ｃｅｌ１０－ＫＧ４１ 유전자 및 이의 용도
KR20130068010A (ko) 흑염소 반추위 미생물 유래의 셀룰라아제 유전자 ｃｅｌ－ＫＧ５２ 및 이의 용도
KR20130067999A (ko) 흑염소 반추위 미생물 유래의 엔도글루카나아제 ｃｅｌ９－ＫＧ１０ 유전자 및 이의 용도

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21797344

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021797344

Country of ref document: EP

Effective date: 20221129