WO2021233193A1 - Mutant de phytase - Google Patents

Mutant de phytase Download PDF

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Publication number
WO2021233193A1
WO2021233193A1 PCT/CN2021/093532 CN2021093532W WO2021233193A1 WO 2021233193 A1 WO2021233193 A1 WO 2021233193A1 CN 2021093532 W CN2021093532 W CN 2021093532W WO 2021233193 A1 WO2021233193 A1 WO 2021233193A1
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Prior art keywords
mutant
phytase
seq
amino acid
phy
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PCT/CN2021/093532
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English (en)
Chinese (zh)
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李馨培
吴秀秀
黄亦钧
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青岛蔚蓝生物集团有限公司
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Publication of WO2021233193A1 publication Critical patent/WO2021233193A1/fr

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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (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)
    • C12Y301/02—Thioester hydrolases (3.1.2)
    • C12Y301/02006—Hydroxyacylglutathione hydrolase (3.1.2.6)
    • 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)
    • C12Y301/03008—3-Phytase (3.1.3.8)

Definitions

  • the present invention relates to the field of biotechnology, in particular to a phytase mutant, its preparation method and application, and DNA molecules, vectors and host cells encoding the phytase mutant.
  • Phytase is a phosphatase that can hydrolyze phytate. It can degrade phytate phosphorus (inositol hexaphosphate) into inositol and inorganic phosphoric acid. This enzyme is divided into two categories: 3-phytase (EC.3.1.3.8) and 6-phytase (EC.3.1.2.6).
  • Phytase is widely present in plants, animals and microorganisms, such as higher plants such as corn and wheat, prokaryotic microorganisms such as Bacillus subtilis, Pseudomonas, Lactobacillus, Escherichia coli, and eukaryotic microorganisms such as yeast, Rhizopus, Aspergillus, etc. .
  • the basic storage form of phosphorus is phytate phosphorus, and its content is as high as 1% to 3%, and it accounts for 60% to 80% of the total phosphorus in plants.
  • phosphorus in the form of phytate phosphorus is difficult to use due to the lack of enzymes that can decompose phytate in monogastric animals, and its utilization rate is only 0%-40%, which causes many problems: firstly, waste of phosphorus sources. On the one hand, the phosphorus source in the feed cannot be effectively utilized.
  • inorganic phosphorus must be added to the feed, which increases the cost of the feed; secondly, the formation of high-phosphorus manure pollutes the environment. About 85% of the phytate phosphorus in the feed will be directly excreted by the animals, and the large amount of phytate phosphorus in the feces will seriously pollute the water and soil.
  • phytate phosphorus is also an anti-nutritional factor, it will chelate with a variety of metal ions such as Zn 2+ , Ca 2+ , Cu 2+ , Fe 2+, etc. and protein during the digestion and absorption process of the animal’s gastrointestinal tract. The corresponding insoluble compounds reduce the effective utilization of these nutrients by animals.
  • Phytase can be used as a feed additive for monogastric animals, and its feeding effect has been confirmed worldwide. It can increase the utilization rate of phosphorus in plant feeds by 60% and reduce the excretion of phosphorus by 40% in feces. At the same time, it can also reduce the anti-nutritional effect of phytic acid. Therefore, adding phytase to feed is of great significance for improving the production efficiency of livestock and poultry industry and reducing the pollution of phytate phosphorus to the environment.
  • the industrial phytases are mainly fungal phytases derived from Aspergillus niger and bacterial phytases derived from Escherichia coli.
  • the phytase APPA from E. coli has the characteristics of high specific activity and good digestive tract stability. At present, it is mainly applied in the feed industry by directly adding powder feed or spraying after pellet feed.
  • Bacterial phytase APPA has poor thermal stability. Its aqueous solution is kept at 70°C for 5 minutes and the remaining enzyme activity is less than 30%. It is directly added to animal feed for pelleting and the remaining enzyme activity is generally less than 20%, so that APPA plant
  • the application of acid enzymes in pellet feed is restricted.
  • the method of spraying the phytase solution onto the feed after pelleting not only increases the equipment investment, but also cannot guarantee the stability of the enzyme preparation and the uniformity of the distribution in the feed. Therefore, improving the thermal stability has important practical significance for the current phytase used in feed.
  • the present invention provides a mutant of phytase to obtain mutant protein and improve its heat resistance, thereby facilitating the wide application of phytase in the field of feed.
  • the present invention relates to a phytase mutant, which comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 3, and comprises an amino acid in at least one position selected from the following group compared with SEQ ID NO: 3 Replaced by: 36,111,202.
  • the amino acid sequence of the mutant is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% compared with SEQ ID NO: 3, Or at least 99% identity.
  • the amino acid sequence of the mutant has at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% compared with SEQ ID NO: 3, Or at least 99.9% identity.
  • the mutant comprises a substitution of at least one amino acid in the following group: A36P, T111P, A202P.
  • the substitution or combination of substitutions contained in the mutant is selected from the following substitutions and combinations of substitutions: A36P, T111P, A202P, A36P/T111P, A36P/A202P, T111P/A202P, A36P/ T111P/A202P.
  • the present invention also relates to DNA molecules encoding the aforementioned phytase mutants.
  • the present invention also relates to a recombinant expression vector containing the above-mentioned DNA molecule.
  • the present invention also relates to a host cell comprising the above-mentioned recombinant expression vector.
  • the host cell is Pichia pastoris.
  • the host cell is Trichoderma reesei.
  • the present invention also provides a method for preparing the above-mentioned phytase mutant, which includes:
  • Step 1 Obtain a DNA molecule encoding a phytase mutant, the phytase mutant comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 3 and is selected from SEQ ID NO: 1 At least one position in the following group contains at least one amino acid substitution: 36, 111, 202;
  • Step 2 Fusion the DNA molecule obtained in step 1 with an expression vector, construct a recombinant expression vector, and transform the host cell;
  • Step 3 Induce the host cell containing the recombinant expression vector to express the fusion protein, and separate and purify the expressed fusion protein.
  • the phytase mutant described in step 1 contains at least one amino acid substitution in the following group: A36P, T111P, A202P.
  • the host cell described in step 2 is Pichia pastoris.
  • the host cell described in step 2 is Trichoderma reesei.
  • the invention also provides the application of the above-mentioned phytase mutant in feed.
  • the present invention provides a mutant containing at least one mutation site among A36P, T111P, and A202P. Compared with APPA-N0, the heat resistance of the mutant is significantly improved.
  • mutants PHY-N1, PHY-N2, and PHY-N3 containing single point mutations of A36P, T111P, and A202P increased by 21.2%, 7.4%, and 14.3%, respectively;
  • the residual rate of N7 enzyme activity was increased by 28.9%, 36.9%, 8.0%, 45.1%, respectively; after being treated at 85°C for 5 minutes, the mutant PHY-N3 containing a single point mutation of A202P, containing two A36P/T111P and A36P/A202P respectively
  • the present invention discloses a phytase mutant, its preparation method and application, and DNA molecules, vectors, and host cells encoding the phytase mutant. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters.
  • the method and application of the present invention have been described through the preferred embodiments. It is obvious that relevant persons can make changes or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.
  • the nomenclature used to define amino acid positions is based on the amino acid sequence of E. coli phytase deposited in Genbank under the number ABF60232, which is given in the sequence table as SEQ ID NO:1 (SEQ ID NO:1 Amino acids 1-410). Therefore, in this context, the base SEQ ID NO: 1 for position numbering starts at Q1 (Gln1) and ends at L410 (Leu410). SEQ ID NO: 1 serves as the standard for position numbering, and therefore serves as the basis for naming.
  • the present invention uses conventional techniques and methods used in the fields of genetic engineering and molecular biology, such as MOLECULAR CLONING: A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001) and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003) .
  • These general references provide definitions and methods known to those skilled in the art.
  • those skilled in the art can use other conventional methods, experimental schemes, and reagents in the field on the basis of the technical solutions described in the present invention, and are not limited to the specific embodiments of the present invention.
  • the present invention can choose the following experimental materials and reagents:
  • Escherichia coli DH5 ⁇ , Pichia pastoris GS115, vectors pPIC9k, Amp, and G418 were purchased from Invitrogen.
  • Enzymes and kits PCR enzymes and ligases were purchased from Takara, restriction endonucleases were purchased from Fermentas, plasmid extraction kits and gel purification recovery kits were purchased from Omega, GeneMorph II random mutagenesis kits were purchased from Beijing Bomaisi Biological Technology Co., Ltd.
  • E. coli medium (LB medium): 0.5% yeast extract, 1% peptone, 1% NaCL, pH 7.0);
  • Yeast medium 1% yeast extract, 2% peptone, 2% glucose;
  • Yeast selection medium 2% peptone, 2% agarose;
  • BMGY medium 2% peptone, 1% yeast extract, 100 mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 4 ⁇ 10-5 biotin, 1% glycerol;
  • BMMY medium 2% peptone, 1% yeast extract, 100 mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 4 ⁇ 10-5 biotin, 0.5% methanol;
  • LB-AMP medium 0.5% yeast extract, 1% peptone, 1% NaCL, 100 ⁇ g/mL ampicillin, pH 7.0;
  • LB-AMP plate 0.5% yeast extract, 1% peptone, 1% NaCL, 1.5% agar, 100 ⁇ g/mL ampicillin, pH7.0;
  • Lower medium plate 2% glucose, 0.5% (NH 4 ) 2 SO 4 , 1.5% KH 2 PO 4 , 0.06% MgSO 4 , 0.06% CaCl 2 , 1.5% agar.
  • the applicant has made mutations (W46E, Q62W, G70E, A73P, T114H, A73P, T114H, N137V, D142R, S146E, R159Y, Y255D) to obtain the phytase mutant APPA-N0, the amino acid sequence of which is SEQ ID NO: 3, and a coding nucleotide sequence of SEQ ID NO: 4 was synthesized by referring to this sequence.
  • the heat resistance of mutant APPA-N0 is significantly improved. After being treated at 75°C for 5 minutes, the residual enzyme activity of phytase APPA is less than 10%, while the residual enzyme activity of mutant APPA-N0 is high. At 85%.
  • the applicant conducted a protein structure analysis of its gene.
  • the protein has two domains: 134 amino acid residues at the N-terminal and 152 amino acid residues at the C-terminal. It constitutes domain 1, and the remaining 124 amino acid residues in the middle constitute domain 2.
  • the conserved sequence and active center are both located in domain 1.
  • the gene is further mutated without destroying the secondary structure and active center of the protein.
  • N0-F1 GGC GAATTC CAGTCAGAACCAGAGTTGAAGTT (underlined is restriction enzyme EcoRI recognition site);
  • N0-R1 ATA GCGGCCGC TTACAAGGAACAAGCAGGGAT (underlined is the restriction enzyme NotI recognition site).
  • APPA-N0 gene (SEQ ID NO: 4) as a template, using the above primers to perform PCR amplification with GeneMorph II Random Mutation PCR Kit (Stratagene), the PCR product is recovered by gel, EcoRI and NotI are digested with the same enzyme treatment Connect the digested pET21a vector, transform it into E. coli BL21(DE3), spread it on LB+Amp plate, and invert it at 37°C.
  • the present invention provides single point mutants containing single mutation sites of A36P, T111P, and A202P, respectively named PHY-N1, PHY-N2, PHY-N3, and their amino acid sequences They are SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9, and their coding nucleotide sequences are SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10.
  • the present invention further provides mutants containing a combination of two mutation sites A36P/T111P, A36P/A202P, and T111P/A202P, respectively named PHY-N4, PHY-N5, and PHY-N6, and their amino acid sequences are respectively SEQ ID NO :11, SEQ ID NO: 13, SEQ ID NO: 15, and their coding nucleotide sequences are SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16.
  • the present invention also provides a mutant containing a combination of three mutation sites of A36P/T111P/A202P, named PHY-N7, whose amino acid sequence is SEQ ID NO: 17, and its coding nucleotide sequence is SEQ ID NO: 18 .
  • the gene sequence SEQ ID NO: 4 of APPA-N0 and the gene sequence of the mutant were optimized and synthesized, and EcoRI and NotI were added to the 5'and 3'ends of the synthetic sequence, respectively.
  • a restriction site According to the coding preference of Pichia pastoris, the gene sequence SEQ ID NO: 4 of APPA-N0 and the gene sequence of the mutant were optimized and synthesized, and EcoRI and NotI were added to the 5'and 3'ends of the synthetic sequence, respectively. A restriction site.
  • the synthesized APPA-N0 and mutant gene sequences were digested with EcoRI and NotI respectively, and then ligated with the pPIC-9K vector after the same digestion at 16°C overnight, and transformed into E. coli DH5a, and spread on LB+Amp Plate, inverted culture at 37°C, after transformant appeared, colony PCR (reaction system: template picked monoclonal, rTaqDNA polymerase 0.5ul, 10 ⁇ Buffer 2.0 ⁇ L, dNTPs (2.5mM) 2.0 ⁇ L, 5'AOX primer (10M): 0.5 ⁇ L, 3'AOX primer: 0.5 ⁇ L, ddH 2 O 14.5 ⁇ L, reaction program: 95°C pre-denaturation 5min, 30 cycles: 94°C30sec, 55°C30sec, 72°C2min, 72°C10min) . The positive clones were verified, and the correct recombinant expression plasmid was obtained after sequencing verification.
  • the Pichia pastoris GS115 strain was activated on a YPD plate. After culturing at 30°C for 48 hours, the activated GS115 monoclonal was inoculated in 6mL YPD liquid medium at 30°C and 220 rpm for about 12 hours. Then the bacterial solution was transferred to 30mL YPD liquid culture. Incubate at 30°C and 220rpm for about 5 hours in a basic Erlenmeyer flask, and measure the cell density with an ultraviolet spectrophotometer.
  • centrifuge at 4°C at 9000rpm for 2min to collect 4mL cells respectively to sterilize EP In the tube, gently discard the supernatant, absorb the remaining supernatant with sterile filter paper, resuspend the bacteria in pre-chilled 1mL sterile water, centrifuge at 4°C, 9000rpm for 2min, gently discard the supernatant, and repeat 1mL
  • centrifuge at 4°C, 9000rpm for 2min gently discard the supernatant, and resuspend the bacteria in pre-cooled 1mL sorbitol (1mol/L); centrifuge at 4°C, 9000rpm for 2min, gently discard the supernatant, and pre-cool 100-150 ⁇ l of sorbitol (1mol/L) to gently resuspend the bacteria.
  • the expression plasmids constructed in 2.1 were linearized with Sac I. After the linearized fragments were purified and recovered, the Pichia pastoris GS115 was transformed by electroporation, and the Pichia pastoris recombinant strains were screened on MD plates, and then genetically modified at different concentrations. Multiple copies of transformants were screened on YPD plates (0.5 mg/mL-8 mg/mL) with mycin.
  • the obtained transformants were transferred to BMGY medium and cultured with shaking at 30°C and 250rpm for 1d; then transferred to BMMY medium and cultured with shaking at 30°C and 250rpm; 0.5% methanol was added every day to induce expression for 4d; centrifuged at 9000rpm After removing the bacteria in 10 minutes, fermentation supernatants containing phytase APPA-N0 and phytase mutants were obtained.
  • X enzyme activity unit, U/g(mL);
  • m the mass or volume of the sample, g/mL
  • the phytase activity was measured on the fermentation supernatant of the constructed Pichia pastoris recombinant strain using the above methods.
  • the gene sequence SEQ ID NO: 4 of APPA-N0 and the gene sequence of the mutant were optimized and synthesized, and KpnI and MluI were added to the 5'and 3'ends of the synthetic sequence, respectively. Two restriction sites.
  • the synthesized phytase gene fragment and pSC1G vector were digested with restriction enzymes KpnI and MluI (Fermentas), respectively, and the digested product was purified using a gel purification kit, and T4 DNA ligase (Fermentas) was used respectively.
  • the phytase gene and the digested product of the pSC1G vector were ligated and transformed into E. coli Trans5 ⁇ (Transgen), selected with ampicillin, and the clone was sequenced (Invitrogen) for verification. After the sequencing is correct, a recombinant plasmid containing the phytase gene is obtained.
  • Trichoderma reesei UE spore suspension Take the host bacterium Trichoderma reesei UE spore suspension, inoculate it on a PDA plate, culture at 30°C for 6 days; after the spores are abundant, cut a colony of about 1cm ⁇ 1cm and place it with 120mL YEG+U( In a liquid medium containing 0.5% yeast powder, 1% glucose, and 0.1% uridine), shake culture at 30°C and 220 rpm for 14 to 16 hours;
  • the applicant separately constructed and obtained the engineered strains of Trichoderma reesei that recombinantly express APPA-N0 and the above-mentioned phytase mutant.
  • the engineered strains of Trichoderma reesei obtained by the above construction were respectively inoculated into PDA solid plates, and incubated in a constant temperature incubator at 30°C for 6-7 days. After the spores were enriched, two hypha blocks with a diameter of 1 cm were respectively inoculated with 50 mL Fermentation medium (1.5% glucose, 1.7% lactose, 2.5% corn steep liquor, 0.44% (NH 4 ) 2 SO 4 , 0.09% MgSO 4 , 2% KH 2 PO 4 , 0.04% CaCl 2 , 0.018% Tween-80 , 0.018% trace elements) in a 250mL Erlenmeyer flask, incubated at 30°C for 48 hours, and then at 25°C for 48 hours. Centrifuge the fermentation broth to obtain fermentation supernatants containing phytase APPA-N0 and the above-mentioned phytase mutant respectively.
  • Example 2 The method described in Example 2 was used to determine the phytase activity of the constructed fermentation supernatant of the recombinant Trichoderma reesei strain.
  • Enzyme activity residual rate (%) enzyme activity of untreated sample/enzyme activity of heat-treated sample ⁇ 100%.
  • the phytase mutant PHY-N3 containing a single point mutation of A202P
  • the phytase mutant PHY-N4 and PHY-N5 containing a combination of two mutation sites of A36P/T111P and A36P/A202P respectively
  • the phytase mutant PHY-N7 which contains a combination of three mutation sites of A36P/T111P/A202P, has an enzyme activity residue rate that is 18.4%, 13.1%, 25.0%, 30.8% higher than that of phytase APPA-N0, respectively.
  • the mutation sites A36P, T111P, and A202P provided by the present invention can significantly improve the heat resistance of phytase, thereby facilitating the wide application of phytase in feed.

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Abstract

L'invention concerne un mutant de phytase, son procédé de préparation et son utilisation, une molécule d'ADN codant pour le mutant de phytase, un vecteur et une cellule hôte. Le mutant contient le substituant d'un acide aminé à un minimum d'une position choisie dans le groupe suivant : 36, 111 et 202. La résistance à la chaleur du mutant est considérablement améliorée, ce qui facilite l'application large de la phytase dans l'alimentation.
PCT/CN2021/093532 2020-05-22 2021-05-13 Mutant de phytase WO2021233193A1 (fr)

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CN202110496409.7A CN113717959B (zh) 2020-05-22 2021-05-07 植酸酶突变体
CN202110496409.7 2021-05-07

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