WO2024032012A1 - 苯丙氨酸解氨酶突变体及其应用 - Google Patents

苯丙氨酸解氨酶突变体及其应用 Download PDF

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WO2024032012A1
WO2024032012A1 PCT/CN2023/087182 CN2023087182W WO2024032012A1 WO 2024032012 A1 WO2024032012 A1 WO 2024032012A1 CN 2023087182 W CN2023087182 W CN 2023087182W WO 2024032012 A1 WO2024032012 A1 WO 2024032012A1
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mutated
alanine
threonine
amino acid
histidine
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PCT/CN2023/087182
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French (fr)
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柴成程
王晶
李华珍
章家泉
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百葵锐(深圳)生物科技有限公司
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Priority claimed from CN202210955082.XA external-priority patent/CN117586997A/zh
Priority claimed from CN202211738582.4A external-priority patent/CN118272358A/zh
Priority claimed from CN202310055051.3A external-priority patent/CN118440926A/zh
Application filed by 百葵锐(深圳)生物科技有限公司 filed Critical 百葵锐(深圳)生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/14—Plant cells
    • 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
    • C12N9/88—Lyases (4.)
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention belongs to the field of protein engineering and biotechnology, and specifically relates to a phenylalanine ammonia lyase mutant derived from Rhodotorula glutinosa and its application.
  • Phenylalanine ammonialyase belongs to the aromatic amino acid lyase family (EC4.3.1.23-1.25 and 4.3.1.3). Phenylalanine ammonia lyase (PAL) is a therapeutic enzyme with biomedical applications discovered in recent years. It is a non-hydrolytic enzyme that can catalyze the non-oxidative deamination of L-phenylalanine to generate trans-cinnamon. acid and ammonia. PAL is a non-mammalian enzyme that is widely present in higher plants and is also found in some microorganisms such as algae, ferns and bacteria, but does not exist in animals and humans.
  • PAL enzyme can be used as a protein to treat the human metabolic disorder phenylketonuria (PKU).
  • PKU is a rare autosomal recessive disorder caused by mutations in the gene encoding phenylalanine hydroxylase (PAH) or an enzyme involved in the synthesis or recycling of the cofactor tetrahydrobiopterin, resulting in partial loss of enzyme function. Or caused by loss of all functions.
  • PAH phenylalanine hydroxylase
  • the PAH enzyme is responsible for regulating L-phenylalanine levels in plasma. Lack of PAH causes the accumulation and increase of phenylalanine content in the human body, and phenylalanine is converted into phenylpyruvic acid and other derivatives.
  • PKU patients Depending on the type of mutation, PKU patients usually have >360 ⁇ M phenylalanine in their blood. If PKU patients are not treated early, high levels of phenylalanine and some of its breakdown products may cause major medical problems, affect people's cognitive functions, and lead to diseases such as tremors, epilepsy, autism, and chronic mental abnormalities. Due to the instability of PAH, it is difficult to obtain it in vitro. Therefore, PAL provides an alternative for PKU patients by converting toxic phenylalanine into non-toxic, excreted metabolic compounds, namely trans-cinnamic acid and ammonia.
  • Palynziq TM is obtained by PEG (polyethylene glycol) oxidation of PAL.
  • the drug is obtained by using enzyme immobilization technology.
  • PEGylation has been shown to improve enzyme half-life and reduce subject antigen responses (see WO 2008/153776; Sarkissian et al., PNAS 105:20894-20899 [2008]).
  • Oral PAL drugs are currently in the development and application stage.
  • Oral PAL preparations are developed to reduce the content of phenylalanine in PKU subjects (Babich et al., Pharmaceuticals 13, 63 [2020] and Hoskins et al., Lancet1 (8165): 392-394[1980]), by developing engineered probiotics capable of expressing PAL enzyme to consume phenylalanine in the human gastrointestinal tract (see WO 2021/188819). In addition, in animal experiments, it was reduced through the intestinal circulation pathway. Phenylalanine levels in the blood have been demonstrated (Isabella et al., Nature Biotechnology 39:857-867 [2018]). However, the above methods all have shortcomings.
  • PEG-PAL injection drugs for example, they are expensive, have numerous adverse reactions, immunogenicity issues (efficacy decreases over time), and long-term injections cause pain, etc.; in PAL probiotic drugs Aspects: It is dose-dependent and has many adverse reactions. The oral method is difficult to accept. The amount of phenylalanine consumed per unit of enzyme production by probiotics is small. The enzyme activity of the selected phenylalanine ammonia lyase is not high. Material specificity is not strong, etc.
  • PAL enzymes derived from different organisms vary greatly, among which PAL derived from fungi has higher advantages and has higher activity than PAL enzymes derived from other microorganisms (Kawatra et al., Biochimie 177: 142-152 [2020] and Zhu et al., Biotechnol Lett5:751-756[2013]).
  • the enzyme activity of RgPAL derived from Rhodotorulaglutinis under optimal conditions is 4.2U/mg, and its enzyme activity is higher than that of R.toruloides, R.aurantiaca KM-1, P.crispum, S.maritimus, A. variabilis, and PAL enzyme activity derived from N.
  • PALs exert their highest enzymatic activity in an alkaline environment, with an optimal pH range of 8.5 to 9.5.
  • PAL obtained in vitro has certain limitations, such as reduced specific activity, short half-life, and loss of activity due to protein degradation at pH 7 due to long contact time with phenylalanine in the body.
  • the present invention improves the activity performance of phenylalanine ammonia lyase RgPAL derived from Rhodotorula glutinis through random evolution and directed transformation. For example, it is optimized to enhance the resistance to proteases and improve the enzyme's ability to operate under acidic pH conditions. catalytic activity under high temperature conditions, or improve stability under high temperature conditions.
  • the present invention conducts mutation screening for the wild-type phenylalanine ammonia lyase RgPAL disclosed in the prior art.
  • This enzyme is screened from Rhodotorulaglutinis.
  • the nucleic acid coding sequence is shown in SEQ ID NO.1 and the amino acid sequence is shown in SEQ ID NO.2.
  • Its optimal pH is 8 to 9, and it is sensitive to acidic pH, proteolytic enzymes and high temperature environments. Therefore, dominant mutants were screened through random mutation of the wild-type enzyme in order to obtain phenylalanine ammonia lyase mutants with improved phenylalanine conversion rate in simulated intestinal environment.
  • the object of the present invention is to provide a phenylalanine ammonia lyase mutant with improved catalytic activity and its application.
  • the present invention conducts a large number of mutant screenings through random mutation to obtain phenylalanine ammonia-lyase mutants with stronger catalytic activity, and constructs a genetic engineering strain that recombinantly expresses phenylalanine ammonia-lyase, in order to realize its application Foundation.
  • the present invention provides a phenylalanine ammonia lyase mutant, the mutant has the activity of catalyzing phenylalanine, and the catalytic activity of the mutant is higher than that of wild-type phenylalanine in a specific environment. Catalytic activity of acid ammonia lyase.
  • the amino acid sequence of the mutant is based on the amino acid sequence shown in SEQ ID NO. 2, and the amino acid at at least one position among K92, Q488, and Q576 is mutated; or the benzene
  • the amino acid sequence of the alanine ammonia lyase mutant has the mutation site in the mutated amino acid sequence.
  • the phenylalanine ammonia lyase mutant includes a mutation corresponding to SEQ ID NO. 2, with the following sites: one or two or three or more than four of K92E, Q488E, Q576E The combination.
  • lysine at position 92 is mutated to glutamic acid; glutamine at position 488 is mutated into glutamic acid; glutamine at position 576 is mutated is glutamic acid; glutamine at position 488 is mutated to glutamic acid, and glutamine at position 576 is mutated to glutamic acid; lysine at position 92 is mutated to glutamic acid, and glutamine at position 488 is mutated to Glutamic acid, and glutamine at position 576 is mutated to glutamic acid.
  • the present invention in order to further improve the catalytic activity of engineered phenylalanine ammonia-lyase, provides phenylalanine ammonia-lyase mutants with continuously improved enzyme activity, which are better mutants obtained above.
  • Variant#5 the amino acid sequence is shown in SEQ ID NO. 4
  • further mutation transformation was carried out.
  • the amino acid sequence of the mutant is based on SEQ ID NO.4, with A13, N18, T28, S29, R77, I89, I127, S145, L151, T169, I184, K231, Q237, M239, T275, T279, T342, H376, S379, N399, N444, D513, E542, E544, A557, T560, S592, E606, A623, I624, A636, I654 in At least one amino acid position is mutated; or the amino acid sequence of the phenylalanine ammonia lyase mutant has the mutation site in the mutated amino acid sequence.
  • the phenylalanine ammonia lyase mutant includes mutations corresponding to SEQ ID NO.4, with the following sites: A13T, N18D, T28I, S29G, R77G, I89V, I127T, S145N, L151Q, T169A , I184V, K231H, K231Y, Q237R, Q237E, M239T, T275P, T279S, T342I, H376R, S379G, N399S, N444S, E488A, D513E, E542G, E544G, A557V, T560S, E576A, S5 92G, E606V, A623G, I624V, A636V , one or a combination of two or three or more than four of I654M.
  • the isoleucine at position 89 is mutated to valine; the isoleucine at position 89 is mutated to valine, and the isoleucine at position 444 is mutated to valine;
  • Paragine is mutated to serine; aspartic acid at position 513 is mutated to glutamic acid; aspartic acid at position 513 is mutated to glutamic acid, and isoleucine at position 654 is mutated to methionine; position 13 Alanine is mutated to threonine, isoleucine at position 127 is mutated into threonine, and aspartic acid at position 513 is mutated into glutamic acid; asparagine at position 18 is mutated into aspartic acid;
  • the leucine at position 151 is mutated to glutamine; the asparagine at position 18 is mutated to aspartic acid, and the leucine at position
  • Thionine is mutated to threonine, and alanine at position 557 is mutated to valine; glutamine at position 237 is mutated to glutamic acid, and alanine at position 636 is mutated to valine; position 237 Glutamine is mutated to arginine, glutamic acid at position 542 is mutated to glycine, and glutamic acid at position 606 is mutated to valine; serine at position 29 is mutated into glycine, and serine at position 145 is mutated into asparagine.
  • threonine at position 279 was mutated to serine; arginine at position 77 was mutated to glycine, isoleucine at position 89 was mutated to valine, leucine at position 151 was mutated into glutamine, and glutamine at position 237 Aminoamide is mutated to glutamic acid, methionine at position 239 is mutated to threonine, and aspartic acid at position 513 is mutated to glutamic acid; threonine at position 342 is mutated to isoleucine; No.
  • the isoleucine at position 184 is mutated into valine, and the threonine at position 342 is mutated into isoleucine; the isoleucine at position 184 is mutated into valine, and the threonine at position 342 is mutated into isoleucine , and serine at position 592 is mutated to glycine; isoleucine at position 89 is mutated to valine, leucine at position 151 is mutated to glutamine, glutamine at position 237 is mutated to glutamic acid, and glutamine at position 342
  • the threonine at position 342 is mutated to isoleucine; the lysine at position 231 is mutated to tyrosine, and the threonine at position 342 is mutated to isoleucine; the threonine at position 342 is mutated to isoleucine, And glutamic acid at position 544 is mutated to
  • threonine at position 342 is mutated to isoleucine
  • threonine at position 275 is mutated to proline
  • methionine at position 239 is mutated to threonine
  • threonine at position 342 is mutated is isoleucine
  • threonine at position 169 is mutated to alanine
  • threonine at position 275 is mutated to proline
  • methionine at position 239 is mutated to threonine
  • threonine at position 342 Mutation to isoleucine
  • arginine at position 77 to glycine, threonine at position 169 to alanine, methionine at position 239 to threonine, and threonine at position 342 is isoleucine
  • isoleucine at position 89 is mutated to valine
  • leucine at position 151 is mutated to glutamine, methi
  • the invention provides a phenylalanine ammonia-lyase mutant whose catalytic activity is further improved under the action of trypsin, that is, in the wild-type phenylalanine ammonia-lyase RgPAL (corresponding to SEQ ID NO. 2), further mutation transformation is carried out.
  • the amino acid sequence of the mutant is based on SEQ ID NO. 2, and the amino acid at at least one position among K26, Y64, R177, R445, and K676 is mutated; or the phenylalanine is decomposed
  • the amino acid sequence of the ammonia enzyme mutant has the mutation site in the mutated amino acid sequence.
  • the phenylalanine ammonia lyase mutant includes a mutation corresponding to SEQ ID NO. 2 with the following sites: one or both of K26A, K26P, Y64S, Y64H, R177M, R445A, and K676S. species or a combination of three or more.
  • Lysine at position 26 is mutated to alanine; Lysine at position 26 is mutated to proline; Tyrosine at position 64 is mutated is serine; tyrosine at position 64 is mutated into histidine; arginine at position 177 is mutated into methionine; arginine at position 445 is mutated into alanine; lysine at position 676 is mutated into serine; The tyrosine at position 64 is mutated to serine, and the arginine at position 445 is mutated to alanine; the lysine at position 26 is mutated to proline, and the arginine at position 177 is mutated to methionine; Lysine at position 26 is mutated to proline, and tyrosine at position 64 is mutated to serine; tyrosine at position 64 is mutated to histidine,
  • the amino acid is mutated to histidine, and arginine at position 445 is mutated to alanine; lysine at position 26 is mutated into alanine, tyrosine at position 64 is mutated into histidine, and arginine at position 177
  • the acid is mutated to methionine, and arginine at position 445 is mutated into alanine; lysine at position 26 is mutated into proline, tyrosine at position 64 is mutated into serine, and arginine at position 177 is mutated is methionine, and arginine at position 445 is mutated to alanine.
  • the invention provides a phenylalanine ammonia-lyase mutant with enhanced temperature stability, that is, based on the wild-type phenylalanine ammonia-lyase RgPAL (corresponding to SEQ ID NO. 2) Mutational transformation.
  • the amino acid sequence of the mutant is based on SEQ ID NO. 2, and the amino acid at at least one position among R101, E124, H340, E341, and V344 is mutated; or the phenylalanine is decomposed
  • the amino acid sequence of the ammonia enzyme mutant has the mutation site in the mutated amino acid sequence.
  • the phenylalanine ammonia lyase mutant includes a mutation corresponding to SEQ ID NO. 2, with the following sites: one or both of R101Q, E124A, E124Q, H340D, H340V, E341A, and V344A. species or a combination of three or more.
  • the present invention gradually improves the trypsin resistance, catalytic activity and temperature stability of the engineered phenylalanine ammonia lyase through combined mutations of two or more sites. That is, combinatorial mutation transformation is carried out on the basis of wild-type phenylalanine ammonia lyase.
  • the amino acid sequence of the mutant is based on SEQ ID NO. 2, at least two of the K26, Y64, E124, T169, R177, M239, H340, H341, V344, R445, Q488, and Q576 positions.
  • An amino acid mutation occurs at an amino acid position; or the amino acid sequence of the phenylalanine ammonia lyase mutant has the mutation site in the mutated amino acid sequence.
  • the phenylalanine ammonia lyase mutant includes mutations corresponding to SEQ ID NO. 2, with the following sites: K26A, K26P, Y64S, Y64H, E124A, T169A, R177M, M239T, H340D, H340V , one or two or a combination of three or more of H341A, V344A, R445A, Q488E and Q576E.
  • lysine at position 26 is mutated to alanine, and valine at position 344 is mutated to alanine; lysine at position 26 Mutation is proline, and valine at position 344 is mutated into alanine; tyrosine at position 64 is mutated into serine, and histidine at position 340 is mutated into aspartic acid; tyrosine at position 64 is mutated is histidine, and histidine at position 340 is mutated to aspartic acid; tyrosine at position 64 is mutated to serine, and histidine at position 340 is mutated to valine; tyrosine at position 64 is mutated to Histidine, and histidine at position 340 is mutated to valine; lysine at position 26 is mutated to alanine, tyrosine at position 64 is mutated to histidine, and histidine at position 340 is mutated to valine; lysine at position
  • Arginine at position 340 is mutated to methionine, histidine at position 340 is mutated to valine, and valine at position 344 is mutated to alanine;
  • tyrosine at position 64 is mutated to histidine, and tyrosine at position 340 Histidine is mutated to aspartate, glutamine at position 488 is mutated to glutamic acid, and glutamine at position 576 is mutated to glutamic acid;
  • tyrosine at position 64 is mutated to histidine, and glutamine at position 340 Histidine is mutated to valine, glutamine at position 488 is mutated to glutamic acid, and glutamine at position 576 is mutated to glutamic acid;
  • tyrosine at position 64 is mutated into histidine, and glutamine at position 169 Threonine was mutated to alanine, methionine
  • Lysine at position 64 was mutated to alanine, tyrosine at position 64 was mutated to histidine, threonine at position 169 was mutated to alanine, arginine at position 177 was mutated into methionine, and arginine at position 239 Methionine is mutated to threonine, and histidine at position 340 is mutated to aspartic acid; lysine at position 26 is mutated to alanine, tyrosine at position 64 is mutated to histidine, and tyrosine at position 169 The threonine at position 239 was mutated to alanine, the methionine at position 239 was mutated into threonine, the histidine at position 340 was mutated into aspartic acid, and the arginine at position 445 was mutated into alanine.
  • the present invention also provides a coding gene encoding the above-mentioned phenylalanine ammonia lyase mutant.
  • the present invention also provides a genetically engineered bacterium expressing the above-mentioned phenylalanine ammonia lyase mutant, which contains a polynucleotide encoding the phenylalanine ammonia lyase mutant.
  • the genetically engineered bacterium is a recombinant strain obtained by ligating the nucleic acid vector to obtain a recombinant expression vector and then introducing it into a protein-expressing host bacterium.
  • the host is any one of Escherichia coli, Bacillus subtilis, lactic acid bacteria or yeast.
  • the protein expression host bacterium is E. coli, more preferably it is E. coli BL21 (DE3).
  • the expression vector is pET-30a(+).
  • the nucleic acid and the expression vector are connected by DNA ligase or by seamless cloning PCR recombination technology to form a recombinant expression vector.
  • the present invention also provides a method for constructing the above-mentioned genetically engineered bacteria, which includes the steps of ligating the nucleic acid vector to obtain a recombinant vector, and then introducing the expression host strain to obtain a recombinant strain.
  • the present invention provides the use of the genetically engineered bacteria in preparing phenylalanine ammonia lyase mutants.
  • the present invention further provides a method for preparing the phenylalanine ammonia lyase mutant, including the step of cultivating the genetically engineered bacterium to express the gene encoding the phenylalanine ammonia lyase mutant.
  • the LB medium contains 10g/L peptone, 5g/L yeast extract and 10g/L NaCl.
  • the preparation method further includes the step of purifying the phenylalanine ammonia lyase mutant from the cultured recombinant expression strain. That is, the cultured recombinant bacterial cells are further subjected to ultrasonic disruption, and the expression supernatant after cell disruption is collected. The expression supernatant contains the phenylalanine ammonia lyase mutant target protein, and the target protein is purified with a Ni column. The high-purity phenylalanine ammonia lyase mutant target protein was obtained by elution with high-concentration imidazole buffer.
  • the present invention also provides the use of the phenylalanine ammonia lyase mutant in catalytic degradation of L-phenylalanine.
  • the product generated in the catalytic reaction in which the mutant participates is trans-cinnamon. acid and ammonia.
  • the present invention also provides a method for degrading phenylalanine, which includes incubating the phenylalanine ammonia lyase mutant with phenylalanine to perform a catalytic degradation reaction.
  • the temperature of the catalytic reaction is 25-40°C, preferably 25°C; preferably, the concentration of phenylalanine in the catalytic reaction system is 20mM.
  • the pH of the catalytic reaction system is 6-9, preferably pH 7-8.8.
  • the present invention also provides a method for screening mutants with high activity of phenylalanine ammonia lyase, including the following steps: establishing a mutant library: constructing, isolating and high-throughput culturing strains; expressing phenylalanine ammonia lyase. High-throughput fragmentation and cleavage of host strains of ammonia enzyme mutant proteins; catalytic reaction of lysates containing phenylalanine ammonia lyase mutants under specific conditions; measuring the absorbance of the catalytic reaction solution at 290 nm with a microplate reader changes to select mutants with high catalytic activity. The more obvious the absorbance rising trend is, the more trans-cinnamic acid content generated by the reaction is, which means the higher the catalytic activity of the mutant.
  • the specific condition is the catalytic reaction of the phenylalanine ammonia lyase mutant at pH 6 and/or 8.8; in one embodiment, the specific condition is the catalytic reaction of the phenylalanine ammonia lyase mutant in pancreatic The catalytic reaction that protease takes part in after action; in another embodiment, the specific condition is the catalytic reaction that phenylalanine ammonia-lyase takes part in after being stored at 37°C or 50°C for a certain period of time.
  • the present invention further provides the use of the phenylalanine ammonia lyase mutant in the preparation of oral medicine for treating phenylketonuria.
  • the engineered phenylalanine ammonia lyase is a mutant enzyme.
  • the mutant enzyme has higher catalytic activity, stronger protease tolerance and temperature stability than the wild-type enzyme, and can consume more phenylalanine.
  • the present invention screens out a batch of phenylalanine ammonia-lyase mutants by constructing a high-throughput screening method for mutants.
  • the trypsin resistance of the mutants is Specific activity under pH 6 conditions, stable temperature
  • the qualitative performance is significantly improved compared to wild-type phenylalanine ammonia lyase, and it can be expressed efficiently in Escherichia coli.
  • through rational design of phenylalanine ammonia lyase and analysis of its amino acid sequence and protein structure and function its catalytic activity was gradually improved.
  • phenylalanine ammonia-lyase has broad application value. Therefore, the mutant is more advantageous for its application in oral drug treatment of phenylketonuria disease process.
  • Figure 1 Schematic diagram of the high-throughput screening process for mutants.
  • FIG. 2 SDS-PAGE results of crude enzyme solution after cell lysis. SDS-PAGE results of the crude enzyme solution after cell lysis of wild-type phenylalanine ammonia lyase RgPAL and mutant Variant#1 after the expression strain was cultured in 96-well plate high-throughput cells.
  • FIG. 3 SDS-PAGE results after purification of RgPAL and some of its mutant proteins. SDS-PAGE results of protein expression and post-purification of wild-type phenylalanine ammonia lyase RgPAL and some mutants (Variant#5, 23, 41, 60, 74 and 92).
  • Figure 4 Relative enzyme activities of RgPAL and some of its mutants under pH 8.8 conditions.
  • Figure 5 Relative enzyme activities of RgPAL and some of its mutants under pH6 conditions. Comparison of relative enzyme activities of wild-type phenylalanine ammonia lyase RgPAL and some mutants (Variant#4, 5, 12, 15, 27, 35, 41, 44, 45 and 48) after catalytic reactions under pH 6 conditions result.
  • Figure 6 Relative enzyme activities of RgPAL and some of its mutants in simulated intestinal fluid. Wild-type phenylalanine ammonia lyase RgPAL and some mutants (Variant#4, 45, 48, 54, 55, 59, 60, 61, 62, 63, 64, 81 and 96) in simulated intestinal fluid containing trypsin Comparative results of relative enzyme activity after catalytic reaction in .
  • Figure 7 Dynamic changes in the production of cinnamic acid by RgPAL and some of its mutants in simulated intestinal fluid. Wild-type phenylalanine ammonia lyase RgPAL and some mutants (Variant#54, 55, 59, 60, 61, 62, 63 and 64) degrade phenylalanine in simulated intestinal fluid containing trypsin, and the reaction takes 15 minutes. The production amount of internal product cinnamic acid changes with time.
  • Figure 8 Relative enzyme activities of RgPAL and some of its mutants after incubation at 37°C for 4 hours.
  • Figure 9 Relative enzyme activities of RgPAL and some of its mutants after incubation at 50°C for 4 hours. Relative residues of wild-type phenylalanine ammonia lyase RgPAL and some mutants (Variant#68, 69, 70, 71, 76, 81, 82, 83, 84, 85, 86 and 96) after incubation at 50°C for 4 hours Enzyme activity comparison results.
  • amino acids are represented by single-letter or three-letter codes, which have the following meanings: G (Gly-glycine), A (Ala-alanine), V (Val-valine), L (Leu-leucine) , I(Ile-isoleucine), P(Pro-proline), F(Phe-phenylalanine), Y(Tyr-tyrosine), W(Trp-tryptophan), S( Ser-serine), T(Thr-threonine), C(Cys-cysteine), M(Met-methionine), N(Asn-asparagine), Q(Gln-glutamine) ), D (Asp-aspartic acid), E (Glu-glutamic acid), K (Lys-lysine), R (Arg-arginine), H (His-histidine).
  • primer refers to an initial nucleic acid fragment, usually an RNA oligonucleotide, DNA oligonucleotide or chimeric sequence that is complementary to the primer binding site of all or part of the target nucleic acid molecule.
  • Primer strands may contain natural, synthetic or modified nucleotides.
  • the lower limit of the primer length is the minimum length required to form a stable double strand under the nucleic acid amplification reaction conditions.
  • mutant refers to a specific amino acid sequence, such as a wild-type sequence.
  • SEQ ID NO.2 is derived from the phenylalanine ammonia lyase of Rhodotorula glutinosa, or is derived from such an enzyme, and contains changes in one or more amino acids, that is, amino acid substitutions, insertions and/or deletions, and Still retains phenylalanine ammonia lyase activity.
  • Mutants can be obtained by various techniques known in the art. Exemplary techniques for modifying coding DNA sequences include, but are not limited to, directed mutagenesis, random mutagenesis, and the construction of synthetic oligonucleotides to produce mutants with altered amino acid sequences.
  • the homology or sequence identity may be more than 90%, preferably more than 95%, more preferably 98% homology.
  • the mutant site is expressed by the position number of the mutation site and the amino acid type of the site.
  • K92E indicates that compared with SEQ ID NO.2, the lysine at position 92 corresponding to SEQ ID NO.2 is mutated to glutamic acid.
  • "/" is used to indicate a combination of mutation sites.
  • “Q488E/Q576E” indicates that both glutamine at position 488 and glutamic acid at position 576 are mutated, including two mutation sites, namely position 488.
  • Glutamine is mutated to glutamic acid and glutamic acid at position 576 is mutated to glutamic acid, which is a double mutant.
  • K92E/Q488E/Q576E means that the corresponding mutations occur at the three corresponding sites at the same time, which is a triple mutant.
  • Example 1 Obtaining wild-type phenylalanine ammonia lyase RgPAL gene and construction of expression vector
  • the present invention is based on the phenylalanine ammonia lyase RgPAL disclosed in the prior art. It screens the self-adhesive red yeast Rhodotorulaglutinis JN-1. Its optimal pH for catalyzing the phenylalanine reaction is 8-9, and the nucleic acid coding sequence As shown in SEQ ID NO.1, the amino acid sequence is as shown in SEQ ID NO.2, and it was expressed and prepared in E. coli BL21 (DE3). Genscript Biotechnology Co., Ltd. was entrusted to artificially synthesize the gene after codon optimization, and then connected it into the pET-30a(+) vector between the NdeI and XhoI restriction sites, and transformed it into the E.
  • coli cloning host Top10 and the expression host.
  • E.coliBL21 (DE3), screen on LB plates containing 50 ⁇ g/mL kanamycin, and culture overnight at 37°C.
  • the expression vector contained in the screened positive clone transformants is named pET30a-RgPAL.
  • the plasmid is extracted and sequenced. Verified correctly.
  • the present invention establishes a strategy for efficient screening of RgPAL mutants by enzyme-linked reaction, achieving high-throughput screening of mutants.
  • the schematic diagram of the process is shown in Figure 1.
  • the specific method is as follows:
  • telomere sequence is connected to the expression plasmid to form a recombinant vector.
  • RgPAL as an intracellular enzyme in E. coli
  • the cells need to be broken, and the broken supernatant is used as crude enzyme solution, and the substrate is added to carry out the enzymatic reaction.
  • 400 ⁇ L/well lysis buffer 50mM Tris-HCl, 150mM NaCl, pH8.0; 2mM EDTA; 0.5% Triton X-100; 1mM DTT; 5mg/mL lysozyme
  • 400 ⁇ L/well lysis buffer 50mM Tris-HCl, 150mM NaCl, pH8.0; 2mM EDTA; 0.5% Triton X-100; 1mM DTT; 5mg/mL lysozyme
  • the mixture is at room temperature Shake and stir for 2-3 hours, then centrifuge (4000 rpm, 20 min).
  • the activity of RgPAL mutants was determined and compared by assessing the production of trans-cinnamic acid as a function of absorbance at 290 nm.
  • 250 ⁇ L/well enzyme catalyzed reaction solution (20mM phenylalanine, 100mM sodium borate buffer, pH8.8/50mM MES buffer, pH6, 20 ⁇ L crude enzyme lysis solution) is added to a 96-well enzyme plate (Costar #3635, Corning), and the enzyme activity was determined by tracking the changes in absorbance at 290 nm over time (15 min, 1 min/time reading) using a microplate reader.
  • mutants in Example 2 Through the high-throughput analysis of mutants in Example 2, mutants whose relative enzyme activity is higher than that of wild-type phenylalanine ammonia lyase are screened out, and their amino acid mutation sites are analyzed through sequencing. Combined mutation of two or more sites is carried out, the corresponding mutant expression strain is cultured, the expressed target protein is purified, and then the enzymatic properties of the mutant are quantitatively analyzed.
  • the amino acid sequence of the dominant mutant Variant#5 obtained by screening is shown in SQEIDNO.4, and the screening results are shown in Table 1.
  • Random mutations were carried out on the basis of mutant Variant#5, efficient mutants were screened according to the screening strategy shown in Example 2, and combined mutations of two or more sites were performed at the same time. The screening results are shown in Table 2.
  • RgPAL phenylalanine ammonia lyase derived from Rhodotorula torhodopsin and phenylalanine ammonia lyase reported in the Genbank database was performed; at the same time, its protein structure was predicted and used Websites and software such as Swiss-Model and PyMOL perform homology modeling on wild-type RgPAL and use phenylalanine molecules as substrates for molecular docking to predict the catalytic active sites and substrate binding sites of RgPAL and analyze these. The intermolecular interactions of amino acid residues near the site are used to design the amino acid mutation site of RgPAL.
  • RgPAL phenylalanine ammonia lyase
  • the protease hydrolysis site of RgPAL was also analyzed using a protease hydrolysis site prediction website. It was determined that K26, Y64, F115, R177, K258, K345, R445, and K676 corresponding to the wild-type phenylalanine ammonia lyase amino acid sequence (SQEIDNO.2) were selected as mutation sites for saturation mutation, and a single-site saturation mutation was established. library. Then, the high-throughput screening strategy in Example 2 was used to screen the transformants in the mutant library for enzyme activity. After further combined mutations, mutants with significantly improved enzymatic properties were obtained. The mutant screening results are shown in Table 3.
  • Example 5 Improving the temperature stability of wild-type RgPAL through site-directed mutation and combination mutation
  • the protein thermal stability analysis website (HotSpot Wizard and FireProt: Design stable proteins) was used to analyze the amino acid sequence of wild-type phenylalanine ammonia lyase RgPAL and predict the mutation site.
  • the mutation sites are R101D, R101E, R101Q, T123L, T123V, E124A, E124Q, H340D, H340V, E341A, E343A, V344A, L353D, L353H and R354M.
  • Site-directed mutation primer sequences are designed respectively, mutants are constructed, the corresponding mutant expression strains are cultured, the expressed target protein is purified, and then the enzymatic properties of the mutants are quantitatively analyzed.
  • the enzyme activity of the mutants was gradually improved through combined mutations of two or more sites.
  • the mutant screening results are shown in Table 4.
  • Example 6 Combined mutations to further improve the enzyme activity of phenylalanine ammonia lyase
  • the method of transforming competent cells of E. coli using plasmids well known in the art The E. coli BL21 (DE3) competent cells used were purchased from Beijing Qingke Biotechnology Co., Ltd., and the wild-type synthesized in the above examples was transformed by heat shock method.
  • the expression vector pET30a-RgPAL and the mutant expression vector were transformed into competent cells respectively, and positive clones were selected for PCR verification and sequencing verification before protein fermentation and expression.
  • Example 7 Collect the bacterial cells after fermentation and expression of the protein in Example 7 by centrifugation (8000rpm, 4°C, 10min), resuspend the cells in 20mL Tris-HCl, 150mM NaCl buffer (pH7.5), and disrupt with ultrasonic waves in an ice bath; centrifuge at 12000rpm for 30min to collect the supernatant , which is the crude enzyme extract expressed in Escherichia coli cells, filtered with a water-based filter membrane with a pore size of 0.22 ⁇ m.
  • the above target protein was purified by affinity chromatography using AKTA affinity chromatography system and Ni 2+ chromatography column.
  • Enzyme activity definition (Active Unit, U): The amount of enzyme consumed to generate 1 ⁇ m product cinnamic acid per minute.
  • Specific Activity definition The enzyme activity per mg of enzyme (hereinafter referred to as specific activity).
  • Enzyme activity detection method Measure in 100mM sodium borate buffer at pH 8.8 and 50mM MES buffer at pH 6, using 20mM L-phenylalanine as the substrate, and add the purified enzyme solution with a final concentration of 50 ⁇ g/mL; Continuously react at room temperature for 5 minutes, measure the cinnamic acid content generated by the reaction based on the change in absorbance at 290 nm, determine the enzyme activity and specific activity of the mutant, and calculate the ratio of the wild type and mutant specific enzyme activities to obtain the relative enzyme activity (vs. wild type comparison).
  • the relative enzyme activity results of RgPAL wild-type enzyme and some of its mutants under pH8.8 and pH6 conditions are shown in Figure 4 and Figure 5 respectively.
  • Protein concentration determination Protein concentration was determined according to the Lowry method, using bovine serum albumin as the standard.
  • the catalytic activity of phenylalanine ammonia lyase mutants was determined in simulated intestinal fluid containing trypsin.
  • Example 9 According to the relative enzyme activity determination method in Example 9 1), detect the residual enzyme activity of the phenylalanine ammonia lyase mutant after incubation for 4 hours at 37°C and 50°C respectively, and calculate and compare the relative enzyme activity of the RgPAL wild type and its mutant. Live, the results are shown in Figure 8 and Figure 9.
  • Example 2 The high-throughput screening strategy in Example 2 was used to screen transformants from the wild-type enzyme mutant library. From more than 1,000 transformants, 3 mutants with overall improved enzyme activity were screened and sequenced. After combining the mutations, the mutant protein was purified and verified for enzymatic properties analysis under different conditions. The relative enzyme activity was obtained by comparing it with the enzyme activity of wild-type phenylalanine ammonia lyase. The specific results are shown in Table 1.
  • Example 2 high-throughput screening of mutant Variant#5 was performed to construct a large number of mutant libraries. Through combined mutation, protein expression purification, and enzymatic property analysis, the specific results were compared with the enzyme activity of mutant Variant#5, as shown in the table. 2 shown.
  • Example 4 was used to conduct site-specific saturation of some amino acid sites of wild-type phenylalanine ammonia-lyase RgPAL (SEQ ID NO. 2). Mutations to construct mutant libraries High-throughput screening was performed, and the enzyme activity was obtained through further combination of mutations, protein expression and purification, and enzymatic property analysis, and compared with the enzyme activity of wild-type RgPAL. The specific results are shown in Table 3.
  • Example 5 In order to improve the thermal stability of the wild-type phenylalanine ammonia lyase RgPAL, Example 5 was used to carry out site-directed mutation and combined mutation of a single site on RgPAL (SEQ ID NO. 2) to construct a mutant, and through protein expression and purification, The enzyme activity was obtained through enzymatic property analysis and compared with the enzyme activity of wild-type RgPAL. The specific results are shown in Table 4.

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Abstract

提供了一种苯丙氨酸解氨酶突变体、其编码基因、基因工程菌以及该苯丙氨酸解氨酶突变体的制备方法。还提供了该苯丙氨酸解氨酶突变体在催化降解L-苯丙氨酸和制备治疗苯丙酮尿症疾病的口服药物中的应用。

Description

苯丙氨酸解氨酶突变体及其应用 技术领域
本发明属于蛋白质工程领域和生物技术领域，具体涉及粘红酵母来源的苯丙氨酸解氨酶突变体及其应用。
背景技术
苯丙氨酸解氨酶(Phenylalanineammonialyase,PAL)属芳香族氨基酸裂解酶家族(EC4.3.1.23-1.25和4.3.1.3)。苯丙氨酸解氨酶(PAL)是近年来研究发现的一种具有生物医学应用的治疗酶，它是一种非水解酶能够催化L-苯丙氨酸通过非氧化脱氨生成反式肉桂酸和氨。PAL是一种非哺乳动物酶，广泛存在于高等植物中，在一些藻类、蕨类和细菌等微生物中也被发现，但在动物和人体中不存在。此外，许多上述来源的苯丙氨酸解氨酶已经在大肠杆菌中获得了重组表达和性质研究(Moffitt等，Biochemistry 46：1004-1012[2007]；Sarkissian等，Proc.Natl.Acad.Sci.USA 96：2339-2344[1999]；Williams等，Microbiology 151：2543–2550[2005]和Xiang等，277：32505–32509[2002])。
PAL酶可用作治疗人体代谢紊乱病——苯丙酮尿症(PKU)的蛋白质。PKU是一种罕见的常染色体隐性遗传性疾病，由编码苯丙氨酸羟化酶(PAH)或参与辅因子四氢生物喋呤合成或循环的酶的基因突变而导致酶的部分功能缺失或全部功能丧失而引起。PAH酶负责调节血浆中L-苯丙氨酸的水平。缺乏PAH导致苯丙氨酸含量在人体内堆积和增加，苯丙氨酸转化为苯丙酮酸和其他衍生物。根据突变类型不同，PKU患者血液中的苯丙氨酸含量通常>360μM。PKU患者如果不及早治疗，高水平的苯丙氨酸及其一些分解产物可能会导致重大的医疗问题，影响人的认知功能，导致震颤、癫痫、自闭症和慢性精神畸形等疾病。由于PAH具有不稳定性，在体外难以获得。因此，PAL为PKU患者提供了另一种选择，PAL将有毒的苯丙氨酸转化为无毒的、可排泄的代谢化合物，即反式肉桂酸和氨。
许多研究都集中在PAL在酶替代治疗PKU中的应用(Ambrus等，Science 201：837-839[1978]；Hendrikse等，ScientificReports10：1315-1337[2020]；Kim等，Molecular Therapy 10：220-224[2004]和Sarkissian等，Proc.Natl.Acad.Sci.USA 96：2339-2344[1999])。目前，PAL用于治疗PKU疾病的注射药剂已被批准生产，以及PAL口服药物正处于研发和临床应用阶段。PAL注射制剂(药物名：PalynziqTM)最近已批准用于治疗成人PKU患者，PalynziqTM是PAL经过PEG(聚乙二醇)化得到的，该药物利用酶的固定化技术得 以开发应用，聚乙二醇化已被证明改进酶的半衰期并降低受试者抗原反应(参见WO 2008/153776；Sarkissian等，PNAS 105：20894-20899[2008])。针对PAL口服药物，目前已经处于开发和应用阶段，通过研制口服PAL制剂来降低PKU受试者体内苯丙氨酸的含量(Babich等，Pharmaceuticals13，63[2020]和Hoskins等，Lancet1(8165)：392-394[1980])，通过开发能够表达PAL酶的工程益生菌来消耗人体胃肠道内的苯丙氨酸(参见WO 2021/188819)，此外，在动物实验中，通过肠循环途径来降低血液中苯丙氨酸水平已经被证实(Isabella等，Nature Biotechnology39：857-867[2018])。但以上方式都存在缺点，在PEG-PAL注射药剂方面：如，价格昂贵，不良反应众多，免疫原性问题(疗效随时间下降)，长时间注射使人产生痛苦感等；在PAL益生菌药物方面：具有剂量依赖性，不良反应众多，口服方法比较难以接受，单位益生菌产酶数消耗的苯丙氨酸的量较少，所选苯丙氨酸解氨酶的酶活性不高，底物特异性不强等。
目前，对来源于藻类植物Anabaena variabilis；真菌类Rhodosporidiumtoruloides，Rhodotorulaglutinis和Pseudozyma antarctica；以及细菌类Streptomyces maritimus，Photorhabdusluminescens和Rubrobacterxylanophilus等的PAL研究较多。不同生物体来源的PAL酶的活性相差较大，其中真菌来源的PAL具有较高优势，比其他微生物来源的PAL酶活性更高(Kawatra等，Biochimie177：142-152[2020]和Zhu等，Biotechnol Lett5：751-756[2013])。其中粘红酵母(Rhodotorulaglutinis)来源的RgPAL在最适条件下的酶活力为4.2U/mg，其酶活力高于R.toruloides，R.aurantiaca KM-1，P.crispum，S.maritimus，A.variabilis，和N.punctiforme来源的PAL酶活力(Zhu等，Biotechnol Lett 5：751-756[2013])。大多数PAL在碱性环境中发挥其最高的酶催化活性，最适pH范围为8.5至9.5。体外获得的PAL具有一定的局限性，如比活性降低、半衰期短，以及由于在体内与苯丙氨酸接触时间较长而在pH 7时发生蛋白质降解而失去活性等等。因此，一些研究集中在改善PAL性能方面(Gamez等，Molecular Therapy9：124-129[2004]；Babich等，Heliyon 6：e03096[2020]；Zhu等，Biotechnology Reports 3：21-26[2014]；WO 2014/172541和US 2021/0222145)，即对胃酸的耐受性，对蛋白酶的抵抗力，酶的稳定性以及耐受时间，目的是能够成功的实现口服PAL酶类药物。
为了通过口服PAL途径治疗PKU疾病，实现PAL在人体胃肠道环境中的应用价值，有必要通过一些技术方法改善PAL的活性，以期提高工程苯丙氨酸解氨酶对蛋白酶的耐受性，在酸性条件下的催化活性，以及对高温储存的稳定性。已知微生物来源的酶的天然催化活性可以通过各种蛋白质工程方法来提高，包括诱变、定向进化、聚乙二醇化和包埋。这些工程变异体、突变体或其多肽在生化上毒性较低、作用高效，并且免疫原性刺激较低。
通过蛋白质工程技术改善酶的性质的方法有很多，包括随机突变，定向进化，理性设计以及固定化等等。通过定向改造野生酶，从而获得具有优良性能的突变体以满足人们对该酶的工业化需求。
发明内容
本发明通过随机进化与定向改造的方法改善粘红酵母(Rhodotorulaglutinis)来源的苯丙氨酸解氨酶RgPAL的活性性能，例如被优化以增强对蛋白酶的耐受性能，提高该酶在酸性pH条件下的催化活性，或提高在高温条件下的稳定性。
本发明针对现有技术公开的野生型苯丙氨酸解氨酶RgPAL进行突变筛选。该酶筛选自粘红酵母(Rhodotorulaglutinis)，核酸编码序列如SEQ ID NO.1所示，氨基酸序列为SEQ ID NO.2所示。其最适pH为8～9，对酸性pH，蛋白水解酶及高温环境敏感。因此，通过对该野生型酶进行随机突变筛选优势突变体，以期获得在模拟肠道环境中苯丙氨酸转化率提高的苯丙氨酸解氨酶突变体。
本发明的目的是提供一种催化活性提高的苯丙氨酸解氨酶突变体及其应用。本发明通过随机突变进行大量的突变体筛选，获得催化活性更强的苯丙氨酸解氨酶突变体，并构建得到了重组表达苯丙氨酸解氨酶的基因工程菌株，为实现其应用奠定了基础。
第一方面，本发明提供一种苯丙氨酸解氨酶突变体，所述突变体具有催化苯丙氨酸的活性，且在特定环境中所述突变体的催化活性高于野生型苯丙氨酸解氨酶的催化活性。
根据本发明的实施方案，所述突变体的氨基酸序列是在SEQ ID NO.2所示的氨基酸序列基础上，在K92，Q488，Q576位中的至少一个位置的氨基酸发生突变；或者所述苯丙氨酸解氨酶突变体的氨基酸序列具有所述发生突变的氨基酸序列中的所述突变位点。
更优选地，所述苯丙氨酸解氨酶突变体包括对应于SEQ ID NO.2，存在如下位点的突变：K92E，Q488E，Q576E中的一种或两种或三种或四种以上的组合。
更具体地，对应于SEQ ID NO.2，存在如下位点的突变：第92位赖氨酸突变为谷氨酸；第488位谷氨酰胺突变为谷氨酸；第576位谷氨酰胺突变为谷氨酸；第488位谷氨酰胺突变为谷氨酸，且第576位谷氨酰胺突变为谷氨酸；第92位赖氨酸突变为谷氨酸，第488位谷氨酰胺突变为谷氨酸，且第576位谷氨酰胺突变为谷氨酸。
根据本发明的实施方案，为进一步提高工程苯丙氨酸解氨酶的催化活性，本发明提供酶活性继续提高的苯丙氨酸解氨酶突变体，其在上述获得的较优的突变体Variant#5(氨基酸序列如SEQ ID NO.4所示)的基础上，进一步进行突变改造。具体来说，所述突变体的氨基酸序列是在SEQ ID NO.4的基础上，在A13，N18，T28，S29，R77，I89，I127，S145， L151，T169，I184，K231，Q237，M239，T275，T279，T342，H376，S379，N399，N444，D513，E542，E544，A557，T560，S592，E606，A623，I624，A636，I654位中的至少一个位置的氨基酸发生突变；或者所述苯丙氨酸解氨酶突变体的氨基酸序列具有所述发生突变的氨基酸序列中的所述突变位点。
更优选地，所述苯丙氨酸解氨酶突变体包括对应于SEQ ID NO.4，存在如下位点的突变：A13T，N18D，T28I，S29G，R77G，I89V，I127T，S145N，L151Q，T169A，I184V，K231H，K231Y，Q237R，Q237E，M239T，T275P，T279S，T342I，H376R，S379G，N399S，N444S，E488A，D513E，E542G，E544G，A557V，T560S，E576A，S592G，E606V，A623G，I624V，A636V，I654M中的一种或两种或三种或四种以上的组合。
更具体地，对应于SEQ ID NO.4，存在如下位点的突变：第89位异亮氨酸突变为缬氨酸；第89位异亮氨酸突变为缬氨酸，且第444位天冬酰胺突变为丝氨酸；第513位天冬氨酸突变为谷氨酸；第513位天冬氨酸突变为谷氨酸，且第654位异亮氨酸突变为甲硫氨酸；第13位丙氨酸突变为苏氨酸，第127位异亮氨酸突变为苏氨酸，且第513位天冬氨酸突变为谷氨酸；第18位天冬酰胺突变为天冬氨酸；第151位亮氨酸突变为谷氨酰胺；第18位天冬酰胺突变为天冬氨酸，且第151位亮氨酸突变为谷氨酰胺；第151位亮氨酸突变为谷氨酰胺，且第231位赖氨酸突变为组氨酸；第151位亮氨酸突变为谷氨酰胺，且第231位赖氨酸突变为酪氨酸；第151位亮氨酸突变为谷氨酰胺，第231位赖氨酸突变为酪氨酸，且第623位丙氨酸突变为甘氨酸；第151位亮氨酸突变为谷氨酰胺，第376位组氨酸突变为精氨酸，且第557位丙氨酸突变为缬氨酸；第151位亮氨酸突变为谷氨酰胺，第231位赖氨酸突变为酪氨酸，第376位组氨酸突变为精氨酸，且第557位丙氨酸突变为缬氨酸；第18位天冬酰胺突变为天冬氨酸，第89位异亮氨酸突变为缬氨酸，第127位异亮氨酸突变为苏氨酸，第151位亮氨酸突变为谷氨酰胺，且第513位天冬氨酸突变为谷氨酸；第77位精氨酸突变为甘氨酸；第77位精氨酸突变为甘氨酸，且第654位异亮氨酸突变为甲硫氨酸；第77位精氨酸突变为甘氨酸，第279位苏氨酸突变为丝氨酸，且第488位谷氨酸突变为丙氨酸；第28位苏氨酸突变为异亮氨酸，第77位精氨酸突变为甘氨酸，第237位谷氨酰胺突变为谷氨酸，且第399位天冬酰胺突变为丝氨酸；第77位精氨酸突变为甘氨酸，第89位异亮氨酸突变为缬氨酸，第151位亮氨酸突变为谷氨酰胺，第231位赖氨酸突变为酪氨酸，第279位苏氨酸突变为丝氨酸，且第513位天冬氨酸突变为谷氨酸；第239位甲硫氨酸突变为苏氨酸；第239位甲硫氨酸突变为苏氨酸，且第560位苏氨酸突变为丝氨酸；第77位精氨酸突变为甘氨酸，第89位异亮氨酸突变为缬氨酸，且第239位甲硫氨酸突变为苏氨酸；第77位精氨酸突变为甘氨酸，第89位异亮氨酸突变为缬氨酸，第239位甲硫氨酸突变为苏氨酸，且第576 位谷氨酸突变为丙氨酸；第239位甲硫氨酸突变为苏氨酸，且第542位谷氨酸突变为甘氨酸；第151位亮氨酸突变为谷氨酰胺，第239位甲硫氨酸突变为苏氨酸，且第557位丙氨酸突变为缬氨酸；第237位谷氨酰胺突变为谷氨酸，且第636位丙氨酸突变为缬氨酸；第237位谷氨酰胺突变为精氨酸，第542位谷氨酸突变为甘氨酸，且第606位谷氨酸突变为缬氨酸；第29位丝氨酸突变为甘氨酸，第145位丝氨酸突变为天冬酰胺，且第279位苏氨酸突变为丝氨酸；第77位精氨酸突变为甘氨酸，第89位异亮氨酸突变为缬氨酸，第151位亮氨酸突变为谷氨酰胺，第237位谷氨酰胺突变为谷氨酸，第239位甲硫氨酸突变为苏氨酸，且第513位天冬氨酸突变为谷氨酸；第342位苏氨酸突变为异亮氨酸；第184位异亮氨酸突变为缬氨酸，且第342位苏氨酸突变为异亮氨酸；第184位异亮氨酸突变为缬氨酸，第342位苏氨酸突变为异亮氨酸，且第592位丝氨酸突变为甘氨酸；第89位异亮氨酸突变为缬氨酸，第151位亮氨酸突变为谷氨酰胺，第237位谷氨酰胺突变为谷氨酸，且第342位苏氨酸突变为异亮氨酸；第231位赖氨酸突变为酪氨酸，且第342位苏氨酸突变为异亮氨酸；第342位苏氨酸突变为异亮氨酸，且第544位谷氨酸突变为甘氨酸；第239位甲硫氨酸突变为苏氨酸，且第342位苏氨酸突变为异亮氨酸；第89位异亮氨酸突变为缬氨酸，第239位甲硫氨酸突变为苏氨酸，且第342位苏氨酸突变为异亮氨酸；第169位苏氨酸突变为丙氨酸，第239位甲硫氨酸突变为苏氨酸，且第342位苏氨酸突变为异亮氨酸；第275位苏氨酸突变为脯氨酸，第239位甲硫氨酸突变为苏氨酸，且第342位苏氨酸突变为异亮氨酸；第169位苏氨酸突变为丙氨酸，第275位苏氨酸突变为脯氨酸，第239位甲硫氨酸突变为苏氨酸，且第342位苏氨酸突变为异亮氨酸；第77位精氨酸突变为甘氨酸，第169位苏氨酸突变为丙氨酸，第239位甲硫氨酸突变为苏氨酸，且第342位苏氨酸突变为异亮氨酸；第89位异亮氨酸突变为缬氨酸，第151位亮氨酸突变为谷氨酰胺，第239位甲硫氨酸突变为苏氨酸，且第342位苏氨酸突变为异亮氨酸；第169位苏氨酸突变为丙氨酸，第275位苏氨酸突变为脯氨酸，第239位甲硫氨酸突变为苏氨酸，第342位苏氨酸突变为异亮氨酸，且第513位天冬氨酸突变为谷氨酸；第77位精氨酸突变为甘氨酸，第169位苏氨酸突变为丙氨酸，第239位甲硫氨酸突变为苏氨酸，第342位苏氨酸突变为异亮氨酸，且第513位天冬氨酸突变为谷氨酸；第77位精氨酸突变为甘氨酸，第151位亮氨酸突变为谷氨酰胺，第169位苏氨酸突变为丙氨酸，第239位甲硫氨酸突变为苏氨酸，第342位苏氨酸突变为异亮氨酸，且第513位天冬氨酸突变为谷氨酸；第77位精氨酸突变为甘氨酸，第89位异亮氨酸突变为缬氨酸，第151位亮氨酸突变为谷氨酰胺，第169位苏氨酸突变为丙氨酸，第237位谷氨酰胺突变为精氨酸，第239位甲硫氨酸突变为苏氨酸，第275位苏氨酸突变为脯氨酸，第342位苏氨酸突变为异亮氨酸，且第513位天冬氨酸突变为谷氨酸。
根据本发明的实施方案，本发明提供了在胰蛋白酶作用下催化活性进一步提高的苯丙氨酸解氨酶突变体，即在野生型苯丙氨酸解氨酶RgPAL(对应于SEQ ID NO.2)的基础上，进一步进行突变改造。具体地，所述突变体的氨基酸序列是在SEQ ID NO.2的基础上，在K26，Y64，R177，R445，K676位中的至少一个位置的氨基酸发生突变；或者所述苯丙氨酸解氨酶突变体的氨基酸序列具有所述发生突变的氨基酸序列中的所述突变位点。
更优选地，所述苯丙氨酸解氨酶突变体包括对应于SEQ ID NO.2，存在如下位点的突变：K26A，K26P，Y64S，Y64H，R177M，R445A，K676S中的一种或两种或三种或四种以上的组合。
更具体地，对应于SEQ ID NO.2，存在如下位点的突变：第26位赖氨酸突变为丙氨酸；第26位赖氨酸突变为脯氨酸；第64位酪氨酸突变为丝氨酸；第64位酪氨酸突变为组氨酸；第177位精氨酸突变为甲硫氨酸；第445位精氨酸突变为丙氨酸；第676位赖氨酸突变为丝氨酸；第64位酪氨酸突变为丝氨酸，且第445位精氨酸突变为丙氨酸；第26位赖氨酸突变为脯氨酸，且第177位精氨酸突变为甲硫氨酸；第26位赖氨酸突变为脯氨酸，且第64位酪氨酸突变为丝氨酸；第64位酪氨酸突变为组氨酸，且第177位精氨酸突变为甲硫氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为丝氨酸，且第177位精氨酸突变为甲硫氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为组氨酸，且第445位精氨酸突变为丙氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为组氨酸，第177位精氨酸突变为甲硫氨酸，且第445位精氨酸突变为丙氨酸；第26位赖氨酸突变为脯氨酸，第64位酪氨酸突变为丝氨酸，第177位精氨酸突变为甲硫氨酸，且第445位精氨酸突变为丙氨酸。
根据本发明的实施方案，本发明提供温度稳定性增强的苯丙氨酸解氨酶突变体，即在野生型苯丙氨酸解氨酶RgPAL(对应于SEQ ID NO.2)的基础上进行突变改造。具体地，所述突变体的氨基酸序列是在SEQ ID NO.2的基础上，在R101，E124，H340，E341，V344位中的至少一个位置的氨基酸发生突变；或者所述苯丙氨酸解氨酶突变体的氨基酸序列具有所述发生突变的氨基酸序列中的所述突变位点。
更优选地，所述苯丙氨酸解氨酶突变体包括对应于SEQ ID NO.2，存在如下位点的突变：R101Q，E124A，E124Q，H340D，H340V，E341A，V344A中的一种或两种或三种或四种以上的组合。
更具体地，对应于SEQ ID NO.2，存在如下位点的突变：第101位精氨酸突变为谷氨酰胺；第124位谷氨酸突变为丙氨酸；第124位谷氨酸突变为谷氨酰胺；第340位组氨酸突变为天冬氨酸；第340位组氨酸突变为缬氨酸；第341位谷氨酸突变为丙氨酸；第344位缬氨酸突变为丙氨酸；第340位组氨酸突变为缬氨酸，第341位谷氨酸突变为丙氨酸，且第344 位缬氨酸突变为丙氨酸；第124位谷氨酸突变为丙氨酸，第340位组氨酸突变为天冬氨酸，第341位谷氨酸突变为丙氨酸，且第344位缬氨酸突变为丙氨酸；第124位谷氨酸突变为丙氨酸，第340位组氨酸突变为缬氨酸，第341位谷氨酸突变为丙氨酸，且第344位缬氨酸突变为丙氨酸；第124位谷氨酸突变为谷氨酰胺，第340位组氨酸突变为天冬氨酸，第341位谷氨酸突变为丙氨酸，且第344位缬氨酸突变为丙氨酸；第124位谷氨酸突变为谷氨酰胺，第340位组氨酸突变为缬氨酸，第341位谷氨酸突变为丙氨酸，且第344位缬氨酸突变为丙氨酸。
根据本发明的实施方案，本发明通过两个或两个以上位点的组合突变逐步提高工程苯丙氨酸解氨酶的胰蛋白酶耐受性，pH6条件下的催化活性和温度稳定性。即在野生型苯丙氨酸解氨酶的基础上进行组合突变改造。具体地，所述突变体的氨基酸序列是在SEQ ID NO.2的基础上，在K26，Y64，E124，T169，R177，M239，H340，H341，V344，R445，Q488，Q576位中的至少两个位置发生氨基酸突变；或者所述苯丙氨酸解氨酶突变体的氨基酸序列具有所述发生突变的氨基酸序列中的所述突变位点。
更优选地，所述苯丙氨酸解氨酶突变体包括对应于SEQ ID NO.2，存在如下位点的突变：K26A，K26P，Y64S，Y64H，E124A，T169A，R177M，M239T，H340D，H340V，H341A，V344A，R445A，Q488E和Q576E中的一种或两种或三种或四种以上的组合。
更具体地，对应于SEQ ID NO.2，存在如下位点的突变：第26位赖氨酸突变为丙氨酸，且第344位缬氨酸突变为丙氨酸；第26位赖氨酸突变为脯氨酸，且第344位缬氨酸突变为丙氨酸；第64位酪氨酸突变为丝氨酸，且第340位组氨酸突变为天冬氨酸；第64位酪氨酸突变为组氨酸，且第340位组氨酸突变为天冬氨酸；第64位酪氨酸突变为丝氨酸，且第340位组氨酸突变为缬氨酸；第64位酪氨酸突变为组氨酸，且第340位组氨酸突变为缬氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为组氨酸，且第340位组氨酸突变为天冬氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为组氨酸，且第340位组氨酸突变为缬氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为组氨酸，且第341位组氨酸突变为丙氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为组氨酸，且第344位缬氨酸突变为丙氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为组氨酸，第177位精氨酸突变为甲硫氨酸，且第340位组氨酸突变为天冬氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为组氨酸，第177位精氨酸突变为甲硫氨酸，且第340位组氨酸突变为缬氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为组氨酸，第124位谷氨酸突变为丙氨酸，第177位精氨酸突变为甲硫氨酸，且第340位组氨酸突变为缬氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为组氨酸，第124位谷氨酸突变为丙氨酸，第177 位精氨酸突变为甲硫氨酸，第340位组氨酸突变为缬氨酸，且第344位缬氨酸突变为丙氨酸；第64位酪氨酸突变为组氨酸，第340位组氨酸突变为天冬氨酸，第488位谷氨酰胺突变为谷氨酸，且第576位谷氨酰胺突变为谷氨酸；第64位酪氨酸突变为组氨酸，第340位组氨酸突变为缬氨酸，第488位谷氨酰胺突变为谷氨酸，且第576位谷氨酰胺突变为谷氨酸；第64位酪氨酸突变为组氨酸，第169位苏氨酸突变为丙氨酸，第239位甲硫氨酸突变为苏氨酸，且第340位组氨酸突变为天冬氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为组氨酸，第169位苏氨酸突变为丙氨酸，第239位甲硫氨酸突变为苏氨酸，且第445位精氨酸突变为丙氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为组氨酸，第169位苏氨酸突变为丙氨酸，第177位精氨酸突变为甲硫氨酸，第239位甲硫氨酸突变为苏氨酸，且第340位组氨酸突变为天冬氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为组氨酸，第169位苏氨酸突变为丙氨酸，第239位甲硫氨酸突变为苏氨酸，第340位组氨酸突变为天冬氨酸，且445位精氨酸突变为丙氨酸。
根据本发明的实施方案，还提供了含有与SEQ ID NO.2和4至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的氨基酸序列的工程苯丙氨酸解氨酶突变体或其功能片段。
第二方面，本发明还提供编码上述苯丙氨酸解氨酶突变体的编码基因。
第三方面，本发明还提供表达上述苯丙氨酸解氨酶突变体的基因工程菌，其包含编码所述苯丙氨酸解氨酶突变体的多核苷酸。具体地，所述基因工程菌是将所述核酸载体连接得到重组表达载体，再导入蛋白表达宿主菌中得到的重组菌株。
根据本发明的实施方案，所述宿主为大肠杆菌、枯草芽孢杆菌、乳酸菌或者酵母菌中的任一种。优选地，所述蛋白表达宿主菌为大肠杆菌，更优选地其是E.coli BL21(DE3)。
根据本发明的实施方案，所述表达载体为pET-30a(+)。
根据本发明的实施方案，所述核酸与表达载体通过DNA连接酶或者通过无缝克隆PCR重组技术连接形成重组表达载体。
第四方面，本发明还提供上述基因工程菌的构建方法，包括将所述核酸载体连接得到重组载体，再导入表达宿主菌种得到重组菌株的步骤。
第五方面，本发明提供该基因工程菌在制备苯丙氨酸解氨酶突变体中的应用。
第六方面，本发明进一步提供所述苯丙氨酸解氨酶突变体的制备方法，包括培养所述基因工程菌，使其表达编码所述苯丙氨酸解氨酶突变体基因的步骤。
根据本发明的实施方案，所述的培养条件是将所述重组菌株接种于含50μg/mL卡那霉素抗性的LB培养基中，37℃、220rpm振荡培养至OD600＝0.6-0.8时，加入诱导剂IPTG至终浓度为0.5mM，转至16℃、150rpm培养16-18h使苯丙氨酸解氨酶突变体蛋白表达。
根据本发明的实施方案，所述LB培养基含有10g/L蛋白胨，5g/L酵母提取物和10g/L NaCl。
根据本发明的实施方案，所述制备方法还包括从培养的重组表达菌株中提纯得到苯丙氨酸解氨酶突变体的步骤。即进一步将所述培养后的重组菌体进行超声波破碎，收集细胞破碎后的表达上清，所述表达上清含有苯丙氨酸解氨酶突变体目的蛋白，用Ni柱纯化目的蛋白，通过高浓度咪唑缓冲液洗脱得到纯度较高的苯丙氨酸解氨酶突变体目的蛋白。
第七方面，本发明还提供所述的苯丙氨酸解氨酶突变体在催化降解L-苯丙氨酸中的应用，所述突变体参与的催化反应中所生成的产物为反式肉桂酸和氨。
第八方面，本发明还提供了一种苯丙氨酸的降解方法，包括使所述苯丙氨酸解氨酶突变体与苯丙氨酸孵育，进行催化降解反应。
根据本发明筛选突变体的实施方案，所述催化反应的温度为25-40℃，优选25℃；优选地，所述催化反应体系中苯丙氨酸的浓度为20mM。优选地，所述催化反应体系的pH为6-9，优选pH7-8.8。
第九方面，本发明还提供一种苯丙氨酸解氨酶高活性突变体的筛选方法，包括如下步骤：建立突变体文库：构建、分离和高通量培养菌株；表达苯丙氨酸解氨酶突变体蛋白的宿主菌株的高通量破碎裂解；含苯丙氨酸解氨酶突变体的裂解物的在特定条件下进行催化反应；用酶标仪测定催化反应液在290nm下的吸光度变化，选择催化活性高的突变体。所述吸光度上升趋势越明显，则反应生成的反式肉桂酸含量越多，表示突变体的催化活性越高。
具体地，所述特定条件是苯丙氨酸解氨酶突变体在pH6和/或8.8下的催化反应；在一个实施例中，所述特定条件是苯丙氨酸解氨酶突变体在胰蛋白酶作用后参与的催化反应；在又一实施例中，所述特定条件是苯丙氨酸解氨酶在37℃或50℃保存一定时间后的参与的催化反应。
第十方面，本发明进一步提供所述的苯丙氨酸解氨酶突变体在制备治疗苯丙酮尿症疾病的口服药物中的应用。在一些实施例中，该工程苯丙氨酸解氨酶是突变体酶。所述的突变酶在胃肠道环境中，与野生型酶相比具有更高的催化活性，更强的蛋白酶耐受性和温度稳定性，能够消耗更多的苯丙氨酸。
本发明根据苯丙氨酸解氨酶的催化性质，通过构建突变体的高通量筛选方法，筛选出一批苯丙氨酸解氨酶的突变体，该突变体的胰蛋白酶耐受性，在pH6条件下的比活，温度稳 定性较野生型苯丙氨酸解氨酶显著提升，并可以在大肠杆菌种高效表达。此外，通过对苯丙氨酸解氨酶进行理性设计，分析其氨基酸序列及蛋白质结构功能，逐步提高其催化活性。通过在含有胰蛋白酶的模拟肠液中进行酶活性分析，对苯丙氨酸解氨酶具有广泛的应用价值。因此，所述突变体更加有利于其在口服药物治疗苯丙酮尿症疾病过程的应用。
附图说明
图1：突变体高通量筛选流程示意图。
图2：细胞裂解后粗酶液的SDS-PAGE结果。表达菌株经96孔板高通量细胞培养后，野生型苯丙氨酸解氨酶RgPAL和突变体Variant#1细胞裂解后粗酶液的SDS-PAGE结果。
图3：RgPAL及其部分突变体蛋白纯化后的SDS-PAGE结果。野生型苯丙氨酸解氨酶RgPAL和部分突变体(Variant#5，23，41，60，74和92)蛋白表达并后纯化的SDS-PAGE结果。
图4：RgPAL及其部分突变体在pH8.8条件下的相对酶活力。野生型苯丙氨酸解氨酶RgPAL及部分突变体(Variant#4，5，12，15，27，35，41，44，45和48)在pH8.8条件下进行催化反应后的相对酶活力对比结果。
图5：RgPAL及其部分突变体在pH6条件下的相对酶活力。野生型苯丙氨酸解氨酶RgPAL及部分突变体(Variant#4，5，12，15，27，35，41，44，45和48)在pH6条件下进行催化反应后的相对酶活力对比结果。
图6：RgPAL及其部分突变体在模拟肠液中的相对酶活力。野生型苯丙氨酸解氨酶RgPAL及部分突变体(Variant#4，45，48，54，55，59，60，61，62，63，64，81和96)在含有胰蛋白酶的模拟肠液中进行催化反应后的相对酶活力对比结果。
图7：RgPAL及其部分突变体在模拟肠液中生成肉桂酸的动态变化。野生型苯丙氨酸解氨酶RgPAL及部分突变体(Variant#54，55，59，60，61，62，63和64)在含有胰蛋白酶的模拟肠液中降解苯丙氨酸，在反应15min内产物肉桂酸的生成量随时间的变化。
图8：RgPAL及其部分突变体在37℃孵育4h后的相对酶活力。野生型苯丙氨酸解氨酶RgPAL及部分突变体(Variant#70，71，73，74，76，78，81，82，83，84，85，86和96)在37℃孵育4h后的相对残余酶活力对比结果。
图9：RgPAL及其部分突变体在50℃孵育4h后的相对酶活力。野生型苯丙氨酸解氨酶RgPAL及部分突变体(Variant#68，69，70，71，76，81，82，83，84，85，86和96)在50℃孵育4h后的相对残余酶活力对比结果。
具体实施方式
本发明中氨基酸由单字母或三字母代码表示，具有如下含义：G(Gly-甘氨酸)，A(Ala-丙氨酸)，V(Val-缬氨酸)，L(Leu-亮氨酸)，I(Ile-异亮氨酸)，P(Pro-脯氨酸)，F(Phe-苯丙氨酸)，Y(Tyr-酪氨酸)，W(Trp-色氨酸)，S(Ser-丝氨酸)，T(Thr-苏氨酸)，C(Cys-半胱氨酸)，M(Met-甲硫氨酸)，N(Asn-天冬酰胺)，Q(Gln-谷氨酰胺)，D(Asp-天冬氨酸)，E(Glu-谷氨酸)，K(Lys-赖氨酸)，R(Arg-精氨酸)，H(His-组氨酸)。
本发明中，“同源性”具有本领域常规的含义，是指两个核苷酸或者氨基酸序列之间的同一性。
在本发明中，术语“引物”是指初始的核酸片段，通常是与由目标核酸分子全部或部分的引物结合位点互补的RNA寡核苷酸、DNA寡核苷酸或嵌合序列。引物链可包含天然的、合成的或修饰的核苷酸。引物长度的下限为在核酸扩增反应条件下可以形成稳定双链所需的最小长度。
在本发明中，术语“突变体”、“突变体蛋白”、“突变体酶”和“变异体”可以互换使用，这些表达是指相对于某一特定的氨基酸序列，例如野生型的序列SEQ ID NO.2来源于粘红酵母的苯丙氨酸解氨酶，或来源于此类酶的基础上，包含一个或更多位置氨基酸的改变，即氨基酸取代、插入和/或缺失，并仍保留苯丙氨酸解氨酶的活性。突变体可以通过本领域已知的各种技术方法获得。用于修饰编码DNA序列的示例性技术包括但不限于：定向诱变、随机突变和合成寡核苷酸的构建，进而得到氨基酸序列发生改变的突变体。
本文所用的术语“对应于”具有本领域普通技术人员通常理解的意义。具体地说，“对应于”表示两条序列经过比对后，一条序列与另一条序列中的指定位置相对应的位置。
具体实施方案中，所述同源性或序列相同性可以是90％以上，优选95％以上，更优选98％的同源性。本文中通过突变位点的位置编号和该位点的氨基酸种类表达突变体位点。例如K92E表示与SEQ ID NO.2比对，在对应于SEQ ID NO.2第92位置的赖氨酸突变为谷氨酸。本发明中，采用“/”表示突变位点的组合，例如“Q488E/Q576E”表示第488位谷氨酰胺和第576位谷氨酸均发生突变，包含两个突变位点，即第488位谷氨酰胺突变为谷氨酸和第576位谷氨酸突变为谷氨酸，为双突变体。依此类推，“K92E/Q488E/Q576E”表示相应的三个位点同时发生相应的突变，为三突变体。
下面将结合实施例对本发明的方法做进一步说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。在实施例中未注明具体条件的实验方法，通常可按照分子生物学领域的常规实验中的条件，或按照质粒、菌株等商品化生产厂商的说明书进行操作。突变PCR为本领域技术人员所熟悉的易错PCR。除非特别说明，以下实施例中使用的试剂和仪器均为市售可得产品，或者可以通过已知方法制备所得。
实施例一：野生型苯丙氨酸解氨酶RgPAL基因的获得及表达载体的构建
本发明以现有技术公开的苯丙氨酸解氨酶RgPAL为基础，其筛选自粘红酵母Rhodotorulaglutinis JN-1，其催化苯丙氨酸反应的最适作用pH为8-9，核酸编码序列如SEQ ID NO.1所示，氨基酸序列为SEQ ID NO.2所示，在大肠杆菌E.coli BL21(DE3)中表达制备。委托金斯瑞生物技术有限公司经过密码子优化后人工合成该基因，然后连接入pET-30a(+)载体的NdeI和XhoI的酶切位点之间，转化入大肠杆菌克隆宿主Top10和表达宿主E.coliBL21(DE3)中，于含有50μg/mL卡那霉素的LB平板上筛选，37℃过夜培养，筛选阳性克隆转化子中包含的表达载体被命名为pET30a-RgPAL，抽提质粒，测序验证正确。
实施例二：RgPAL突变体高通量筛选方法
本发明建立了酶联反应高效筛选RgPAL突变体的策略，实现突变体的高通量筛选，该过程示意图如图1所示。具体方法如下：
1)RgPAL突变体的高通量培养
以重组质粒pET30a-RgPAL为模板，分别设计随机突变引物，进行易错PCR，对目的基因进行单点或多点随机突变以及两个或两个以上位点的组合突变，通过同源重组技术将突变后的目的基因片段与表达质粒连接形成重组载体。将重组质粒转化入表达宿主E.coliBL21(DE3)中，在含有10g/L蛋白胨，5g/L酵母提取物，10g/L NaCl，15g/L琼脂粉和50μg/mL卡那霉素的LB琼脂平板上培养，筛选出阳性克隆的大肠杆菌细胞。在37℃孵育过夜后，将单克隆菌落接种于含有50μg/mL卡那霉素，400μL/孔LB培养基的96深孔板中，在孔板摇床(800rpm，37℃)过夜培养12-16h作为种子液。
取50μL过夜生长的细胞种子液转接至含有50μg/mL卡那霉素，450μL/孔LB培养基的96深孔板中，放置孔板摇床(800rpm，37℃)中培养120min。然后用终浓度为0.5mMIPTG诱导细胞表达蛋白，在16℃摇床中培养16-18h。将含有种子液的96孔板放置-80℃保存。蛋白表达后的96孔板在离心机中离心(4000rpm，20min)，去除培养基上清液，留细胞沉淀进行下一步分析。
以上操作过程均在无菌超净工作台中进行，且96孔筛选板中设置野生型苯丙氨酸解氨酶表达宿主作为对照。
2)RgPAL突变体蛋白的高通量裂解
大肠杆菌表达RgPAL为胞内酶，需要对菌体进行破碎，以破碎上清为粗酶液，加入底物进行酶催化反应。首先，在含有细胞沉淀的96深孔板中加入400μL/孔裂解缓冲液(50mM Tris-HCl，150mM NaCl，pH8.0；2mM EDTA；0.5％Triton X-100；1mM DTT；5mg/mL溶菌酶和0.5mg/mL多粘菌素B)，用孔板振荡器将菌体沉淀重悬。混合物在室温 下震荡搅拌2-3h后离心(4000rpm，20min)，收集细胞裂解后上清液即含有苯丙氨酸解氨酶的粗酶液，用来进行下一步酶活力高通量分析。取野生型和Variant#1细胞裂解后的粗酶液进行SDS-PAGE分析表明(如图2所示)，在表观相对分子质量为～75kDa处存在一个过表达的蛋白，这与RgPAL的预期相对分子质量一致，且目的蛋白在大肠杆菌中实现高效可溶性表达。
3)RgPAL突变体粗酶裂解液酶活的高通量分析
通过在290nm处的吸光度随时间的变化来评估反式肉桂酸的生成，从而确定与比较RgPAL突变体的活性。在本发明中，将250μL/孔酶催化反应液(20mM苯丙氨酸，100mM硼酸钠缓冲液，pH8.8/50mM MES缓冲液，pH6，20μL粗酶裂解液)加入到96孔酶标板中(Costar#3635，Corning)混合反应，并通过使用酶标仪跟踪290nm处吸光度随时间的变化(15min，1min/次读数)来确定酶活力。
4)RgPAL突变体粗酶液酶活在胰蛋白酶作用下的高通量分析
模拟含有胰蛋白酶的肠道环境，测定RgPAL突变体在胰蛋白酶作用后的残余酶活力。首先，在96孔酶标板中加入100μL混合液(100μg/mL胰蛋白酶，0.68％的KH2PO4，pH6.8，80μL粗酶裂解液)，将孔板放置37℃，400rpm孵育30min后进行分析。根据实施例二第3)点的高通量酶活力分析方法，检测苯丙氨酸解氨酶突变体粗酶液在胰蛋白酶作用后的残余酶活力。
5)高温贮藏后RgPAL突变体粗酶液酶活的高通量分析
各取100μL突变体的裂解液粗酶液分别放置37℃和50℃条件下孵育4h，孵育过程中析出的不溶物质通过离心(4000rpm，20min)被去除。取20μL粗酶裂解液上清，根据实施例二中第3)点的高通量酶活力分析方法，检测苯丙氨酸解氨酶突变体粗酶液高温孵育后的残余酶活力。
实施例三：通过组合突变和叠代突变提高RgPAL酶活力
通过实施例二中对突变体的高通量分析，筛选出相对酶活力高于野生型苯丙氨酸解氨酶的突变体，通过测序分析其氨基酸突变位点。进行两个或两个以上位点的组合突变，并对相应的突变体表达菌株进行培养，对表达的目的蛋白进行纯化，进而对突变体酶学性质进行定量分析。所筛选得到的优势突变体Variant#5，其氨基酸序列如SQEIDNO.4所示，筛选结果如表1所示。
在突变体Variant#5的基础上进行随机突变，根据实施例二所示的筛选策略筛选高效突变体，同时进行两个或两个以上位点的组合突变，筛选结果如表2所示。
实施例四：通过定点饱和突变及组合突变提高野生型RgPAL的胰蛋白酶耐受性
将粘红酵母来源的苯丙氨酸解氨酶(RgPAL)与Genbank数据库中已报道的苯丙氨酸解氨酶的氨基酸序列进行同源性比对分析；同时对其蛋白结构进行预测，利用Swiss-Model和PyMOL等网站和软件对野生型RgPAL进行同源建模，利用苯丙氨酸分子为底物进行分子对接，从而预测RgPAL的催化活性位点及底物结合位点，并分析这些位点附近氨基酸残基的分子间相互作用，从而设计RgPAL的氨基酸突变位点。
在上述蛋白质结构分析的基础上，还利用蛋白酶水解位点预测网站分析了RgPAL的蛋白酶水解位点。共确定选择对应于野生型苯丙氨酸解氨酶氨基酸序列(SQEIDNO.2)的K26，Y64，F115，R177，K258，K345，R445，K676为突变位点进行饱和突变，建立单位点饱和突变文库。然后利用实施例二中的高通量筛选策略对突变体文库中的转化子进行酶活力筛选，经过进一步组合突变得到酶学性质显著提高的突变体，突变体筛选结果如表3所示。
实施例五：通过定点突变及组合突变提高野生型RgPAL的温度稳定性
为提高RgPAL的热稳定性，利用蛋白质热稳定性分析网站(HotSpot Wizard和FireProt:Design stable proteins)分析野生型苯丙氨酸解氨酶RgPAL的氨基酸序列并预测突变位点。选择对应于野生型苯丙氨酸解氨酶(SEQIDNO.2)的氨基酸序列进行定点突变，突变位点为R101D，R101E，R101Q，T123L，T123V，E124A，E124Q，H340D，H340V，E341A，E343A，V344A，L353D，L353H和R354M。分别设计定点突变引物序列，构建突变体，并对相应的突变体表达菌株进行培养，对表达的目的蛋白进行纯化，进而对突变体酶学性质进行定量分析。
通过两个或两个以上位点的组合突变逐步提高突变体的酶活力，突变体筛选结果如表4所示。
实施例六：组合突变进一步提高苯丙氨酸解氨酶的酶活力
通过上述实施例二-实施例五过程中筛选得到的优势突变位点，进行两个或两个以上位点的组合突变，构建突变体，对相应的突变体表达菌株进行发酵表达得到突变体蛋白，进行目的蛋白纯化，并进一步分析突变体酶活力。筛选结果如表5所示。
实施例七：RgPAL及其突变体蛋白的发酵表达
利用本领域公知的质粒转化感受态细胞大肠杆菌的方法，所用大肠杆菌E.coliBL21(DE3)感受态细胞购买于北京擎科生物科技有限公司，并用热激法将上述实施例中合成的野生型表达载体pET30a-RgPAL和突变体表达载体分别转化入感受态细胞，筛选阳性克隆PCR验证及测序验证正确后进行蛋白发酵表达。
接种针挑取野生型与突变体阳性单克隆菌株接种于5mL LB培养基中，以37℃、220rpm过夜培养16-18h，然后以按2％(V/V)接种量接种于250mL LB培养基中，以37℃、 220rpm培养2-3h。当细菌密度OD600达到0.6-0.8时，加入终浓为0.5mM的IPTG诱导蛋白表达，为防止包涵体的形成，表达条件为150rpm，16℃低温过夜诱导蛋白表达。
实施例八：RgPAL及其突变体蛋白的纯化
离心收集实施例七中蛋白发酵表达后的菌体(8000rpm、4℃、10min)，20mLTris-HCl，150mMNaCl缓冲液(pH7.5)重悬细胞，冰浴超声波破碎；12000rpm，30min离心收集上清，即为大肠杆菌胞内表达的粗酶提取液，用孔径0.22μm水系滤膜过滤。采用AKTA亲和层析系统，利用Ni2+层析柱对上述目的蛋白进行亲和层析纯化，咪唑洗脱后经过脱盐柱脱盐，蛋白保存在Tris-HCl/NaCl(pH7.5)缓冲液中备用，野生型蛋白命名为RgPAL，突变型蛋白分别命名为Variant#加编号。野生型酶和部分突变体蛋白纯化后的SDS-PAGE结果如图3所示。
实施例九：纯化的RgPAL及其突变体蛋白的酶学性质分析
通过上述实施例中的高通量初步筛选，蛋白结构分析，定点饱和突变，定点突变和组合突变得到酶活力提高的突变体共96个，对这些突变体蛋白的表达菌株进行培养，加入0.5mM IPTG进行诱导，然后利用Ni柱纯化突变体蛋白，对纯化后的蛋白进行酶学性质分析。
1)苯丙氨酸解氨酶RgPAL及其突变体的相对酶活力
酶活力定义(Active Unit，U)：每分钟生成1μm产物肉桂酸消耗的酶量。
比酶活定义(Specific Activity，U/mg)：每mg酶具有的酶活力(以下简称比活)。
酶活力检测方法：测定在pH8.8的100mM硼酸钠缓冲液和pH6的50mM MES缓冲液中，以20mM L-苯丙氨酸为底物，加入终浓为50μg/mL纯化后的酶液；在室温下连续反应5min，根据在290nm的吸光度的变化测定反应生成的肉桂酸含量，确定突变体的酶活力和比活，并计算野生型与突变体比酶活的比值得到相对酶活力(与野生型比较)。RgPAL野生型酶和其部分突变体在pH8.8和pH6条件下的相对酶活力结果分别如图4和图5所示。
蛋白浓度测定：蛋白质浓度的测定依照Lowry方法，以牛血清白蛋白作为标准。
2)苯丙氨酸解氨酶RgPAL及突变体在含有胰蛋白酶的模拟肠液中的催化反应
测定苯丙氨酸解氨酶突变体在含有胰蛋白酶的模拟肠液中的催化活性。
模拟含有胰蛋白酶的肠液环境，取浓度为0.5mg/mL的RgPAL野生型酶或突变体酶在含有20mM L-苯丙氨酸，100μg/mL胰蛋白酶的0.68％KH2PO4缓冲液(pH6.8)中进行催化反应，在37℃条件下连续反应15min，根据在290nm的吸光度的变化测定反应生成的肉桂酸含量，确定酶的酶活力以及比活，计算比较RgPAL野生型及其突变体的相对酶活力，结果如图6所示。反应15min内，RgPAL野生型及部分突变体在胰蛋白酶作用下生成肉桂酸的动力学结果如图7所示。
3)苯丙氨酸解氨酶RgPAL及其突变体的温度稳定性
取0.5mg/mLRgPAL野生型酶和突变体酶分别放置于37℃和50℃水浴锅中静置孵育4h，离心(12000rpm，5min)去除不溶物质，取上清进行酶活力测定。
根据实施例九1)中相对酶活测定方法，检测苯丙氨酸解氨酶突变体分别在37℃和50℃孵育4h后的残余酶活力，计算比较RgPAL野生型及其突变体的相对酶活，结果如图8和图9所示。
筛选结果
1)野生型苯丙氨酸解氨酶RgPAL随机突变高通量筛选结果：
利用实施例二中的高通量筛选策略对野生型酶突变体文库转化子进行筛选，从1000余个转化子中筛选获得了3个酶活力总体提高的突变体，对其进行测序，并进行组合突变，突变体蛋白纯化后验证不同条件下进行酶学性质分析，与野生型苯丙氨酸解氨酶的酶活力进行比较得到相对酶活力，具体结果如表1所示。
表1
注：以野生型苯丙氨酸解氨酶RgPAL(SEQ ID NO.2)的酶活力为100％，将突变体的酶活力与野生型进行比较得到其他突变体的相对酶活力(Relative activity，RA)。
2)突变体Variant#5随机突变高通量筛选结果：
利用实施例二对突变体Variant#5进行高通量筛选构建大量的突变体文库，通过组合突变，蛋白表达纯化，酶学性质分析，与突变体Variant#5酶活力相比，具体结果如表2所示。
表2

注：以野生型苯丙氨酸解氨酶RgPAL(SEQ ID NO.2)的酶活力为100％，将突变体的酶活力与野生型进行比较得到其他突变体的相对酶活力(Relative activity，RA)。
3)RgPAL定点饱和突变及组合突变筛选结果：
为了提高野生型苯丙氨酸解氨酶RgPAL对胰蛋白酶的耐受性，利用实施例四对野生型苯丙氨酸解氨酶RgPAL(SEQ ID NO.2)的部分氨基酸位点进行定点饱和突变构建突变体文库 并进行高通量筛选，通过进一步组合突变，蛋白表达与纯化，酶学性质分析得到酶活力，并与野生型RgPAL的酶活力进行比较，具体结果如表3所示。
表3
注：以野生型苯丙氨酸解氨酶RgPAL(SEQ ID NO.2)的酶活力为100％，将突变体的酶活力与野生型进行比较得到其他突变体的相对酶活力(Relative activity，RA)。
4)RgPAL定点突变及组合突变筛选结果：
为了提高野生型苯丙氨酸解氨酶RgPAL的热稳定性，利用实施例五对RgPAL(SEQ ID NO.2)进行单个位点的定点突变及组合突变构建突变体，通过蛋白表达与纯化，酶学性质分析得到酶活力，并与野生型RgPAL的酶活力进行比较，具体结果如表4所示。
表4

注：以野生型苯丙氨酸解氨酶RgPAL(SEQ ID NO.2)的酶活力为100％，将突变体的酶活力与野生型进行比较得到其他突变体的相对酶活力(Relative activity，RA)。
5)组合突变筛选结果：
表5
注：以野生型苯丙氨酸解氨酶RgPAL(SEQ ID NO.2)的酶活力为100％，将突变体的酶活力与野生型进行比较得到其他突变体的相对酶活力(Relative activity，RA)。

Claims (12)

  1. 一种苯丙氨酸解氨酶突变体，所述突变体具有催化苯丙氨酸的活性，且在特定环境中所述突变体的催化活性高于野生型苯丙氨酸解氨酶的催化活性，具体地其降低了对蛋白酶的敏感性以增强对蛋白酶水解的耐受性，在酸性pH条件下具有更高的催化活性，提高了对高温储存的耐受性。
  2. 如权利要求1所述的苯丙氨酸解氨酶突变体，其特征在于，所述突变体的氨基酸序列是在SEQ ID NO.2所示的氨基酸序列基础上，在K92，Q488，Q576位中的至少一个位置的氨基酸发生突变；或者所述苯丙氨酸解氨酶突变体的氨基酸序列具有所述发生突变的氨基酸序列中的所述突变位点，且与SEQ ID NO.2有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的氨基酸序列的苯丙氨酸解氨酶突变体或其功能片段；
    更优选地，所述苯丙氨酸解氨酶突变体包括对应于SEQ ID NO.2所示氨基酸序列，存在如下位点的突变：K92E，Q488E，Q576E中的一种或两种或三种的组合；
    更具体地，对应于SEQ ID NO.2所示氨基酸序列，存在如下位点的突变：第92位赖氨酸突变为谷氨酸；第488位谷氨酰胺突变为谷氨酸；第576位谷氨酰胺突变为谷氨酸；第488位谷氨酰胺突变为谷氨酸，且第576位谷氨酰胺突变为谷氨酸；第92位赖氨酸突变为谷氨酸，第488位谷氨酰胺突变为谷氨酸，且第576位谷氨酰胺突变为谷氨酸。
  3. 如权利要求1所述的苯丙氨酸解氨酶突变体，其特征在于，所述突变体的氨基酸序列是在SEQ ID NO.4的基础上，在A13，N18，T28，S29，R77，I89，I127，S145，L151，T169，I184，K231，Q237，M239，T275，T279，T342，H376，S379，N399，N444，D513，E542，E544，A557，T560，S592，E606，A623，I624，A636，I654位中的至少一个位置的氨基酸发生突变；或者所述苯丙氨酸解氨酶突变体的氨基酸序列具有所述发生突变的氨基酸序列中的所述突变位点，且与SEQ ID NO.4有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的氨基酸序列的苯丙氨酸解氨酶突变体或其功能片段；
    更优选地，所述苯丙氨酸解氨酶突变体包括对应于SEQ ID NO.4所示氨基酸序列，存在如下位点的突变：A13T，N18D，T28I，S29G，R77G，I89V，I127T，S145N，L151Q，T169A，I184V，K231H，K231Y，Q237R，Q237E，M239T，T275P，T279S，T342I，H376R，S379G，N399S，N444S，E488A，D513E，E542G，E544G，A557V，T560S，E576A，S592G，E606V，A623G，I624V，A636V，I654M中的一种或两种或三种或四种以上的组合；
    更优选地，对应于SEQ ID NO.4，存在如下位点的突变：第89位异亮氨酸突变为缬氨酸；第89位异亮氨酸突变为缬氨酸，且第444位天冬酰胺突变为丝氨酸；第513位天冬氨酸突变为谷氨酸；第513位天冬氨酸突变为谷氨酸，且第654位异亮氨酸突变为甲硫氨酸；第13位丙氨酸突变为苏氨酸，第127位异亮氨酸突变为苏氨酸，且第513位天冬氨酸突变为谷氨酸；第18位天冬酰胺突变为天冬氨酸；第151位亮氨酸突变为谷氨酰胺；第18位天冬酰胺突变为天冬氨酸，且第151位亮氨酸突变为谷氨酰胺；第151位亮氨酸突变为谷氨酰胺，且第231位赖氨酸突变为组氨酸；第151位亮氨酸突变为谷氨酰胺，且第231位赖氨酸突变为酪氨酸；第151位亮氨酸突变为谷氨酰胺，第231位赖氨酸突变为酪氨酸，且第623位丙氨酸突变为甘氨酸；第151位亮氨酸突变为谷氨酰胺，第376位组氨酸突变为精氨酸，且第557位丙氨酸突变为缬氨酸；第151位亮氨酸突变为谷氨酰胺，第231位赖氨酸突变为酪氨酸，第376位组氨酸突变为精氨酸，且第557位丙氨酸突变为缬氨酸；第18位天冬酰胺突变为天冬氨酸，第89位异亮氨酸突变为缬氨酸，第127位异亮氨酸突变为苏氨酸，第151位亮氨酸突变为谷氨酰胺，且第513位天冬氨酸突变为谷氨酸；第77位精氨酸突变为甘氨酸；第77位精氨酸突变为甘氨酸，且第654位异亮氨酸突变为甲硫氨酸；第77位精氨酸突变为甘氨酸，第279位苏氨酸突变为丝氨酸，且第488位谷氨酸突变为丙氨酸；第28位苏氨酸突变为异亮氨酸，第77位精氨酸突变为甘氨酸，第237位谷氨酰胺突变为谷氨酸，且第399位天冬酰胺突变为丝氨酸；第77位精氨酸突变为甘氨酸，第89位异亮氨酸突变为缬氨酸，第151位亮氨酸突变为谷氨酰胺，第231位赖氨酸突变为酪氨酸，第279位苏氨酸突变为丝氨酸，且第513位天冬氨酸突变为谷氨酸；第239位甲硫氨酸突变为苏氨酸；第239位甲硫氨酸突变为苏氨酸，且第560位苏氨酸突变为丝氨酸；第77位精氨酸突变为甘氨酸，第89位异亮氨酸突变为缬氨酸，且第239位甲硫氨酸突变为苏氨酸；第77位精氨酸突变为甘氨酸，第89位异亮氨酸突变为缬氨酸，第239位甲硫氨酸突变为苏氨酸，且第576位谷氨酸突变为丙氨酸；第239位甲硫氨酸突变为苏氨酸，且第542位谷氨酸突变为甘氨酸；第151位亮氨酸突变为谷氨酰胺，第239位甲硫氨酸突变为苏氨酸，且第557位丙氨酸突变为缬氨酸；第237位谷氨酰胺突变为谷氨酸，且第636位丙氨酸突变为缬氨酸；第237位谷氨酰胺突变为精氨酸，第542位谷氨酸突变为甘氨酸，且第606位谷氨酸突变为缬氨酸；第29位丝氨酸突变为甘氨酸，第145位丝氨酸突变为天冬酰胺，且第279位苏氨酸突变为丝氨酸；第77位精氨酸突变为甘氨酸，第89位异亮氨酸突变为缬氨酸，第151位亮氨酸突变为谷氨酰胺，第237位谷氨酰胺突变为谷氨酸，第239位甲硫氨酸突变为苏氨酸，且第513位天冬氨酸突变为谷氨酸；第342位苏氨酸突变为异亮氨酸；第184位异亮氨酸突变为缬氨酸，且第342位苏氨酸突变为异亮氨酸；第184位异亮氨酸突变为缬氨酸，第342位苏氨酸突变 为异亮氨酸，且第592位丝氨酸突变为甘氨酸；第89位异亮氨酸突变为缬氨酸，第151位亮氨酸突变为谷氨酰胺，第237位谷氨酰胺突变为谷氨酸，且第342位苏氨酸突变为异亮氨酸；第231位赖氨酸突变为酪氨酸，且第342位苏氨酸突变为异亮氨酸；第342位苏氨酸突变为异亮氨酸，且第544位谷氨酸突变为甘氨酸；第239位甲硫氨酸突变为苏氨酸，且第342位苏氨酸突变为异亮氨酸；第89位异亮氨酸突变为缬氨酸，第239位甲硫氨酸突变为苏氨酸，且第342位苏氨酸突变为异亮氨酸；第169位苏氨酸突变为丙氨酸，第239位甲硫氨酸突变为苏氨酸，且第342位苏氨酸突变为异亮氨酸；第275位苏氨酸突变为脯氨酸，第239位甲硫氨酸突变为苏氨酸，且第342位苏氨酸突变为异亮氨酸；第169位苏氨酸突变为丙氨酸，第275位苏氨酸突变为脯氨酸，第239位甲硫氨酸突变为苏氨酸，且第342位苏氨酸突变为异亮氨酸；第77位精氨酸突变为甘氨酸，第169位苏氨酸突变为丙氨酸，第239位甲硫氨酸突变为苏氨酸，且第342位苏氨酸突变为异亮氨酸；第89位异亮氨酸突变为缬氨酸，第151位亮氨酸突变为谷氨酰胺，第239位甲硫氨酸突变为苏氨酸，且第342位苏氨酸突变为异亮氨酸；第169位苏氨酸突变为丙氨酸，第275位苏氨酸突变为脯氨酸，第239位甲硫氨酸突变为苏氨酸，第342位苏氨酸突变为异亮氨酸，且第513位天冬氨酸突变为谷氨酸；第77位精氨酸突变为甘氨酸，第169位苏氨酸突变为丙氨酸，第239位甲硫氨酸突变为苏氨酸，第342位苏氨酸突变为异亮氨酸，且第513位天冬氨酸突变为谷氨酸；第77位精氨酸突变为甘氨酸，第151位亮氨酸突变为谷氨酰胺，第169位苏氨酸突变为丙氨酸，第239位甲硫氨酸突变为苏氨酸，第342位苏氨酸突变为异亮氨酸，且第513位天冬氨酸突变为谷氨酸；第77位精氨酸突变为甘氨酸，第89位异亮氨酸突变为缬氨酸，第151位亮氨酸突变为谷氨酰胺，第169位苏氨酸突变为丙氨酸，第237位谷氨酰胺突变为精氨酸，第239位甲硫氨酸突变为苏氨酸，第275位苏氨酸突变为脯氨酸，第342位苏氨酸突变为异亮氨酸，且第513位天冬氨酸突变为谷氨酸。
  4. 如权利要求1所述的苯丙氨酸解氨酶突变体，其特征在于，所述突变体的氨基酸序列是在SEQ ID NO.2所示氨基酸序列的基础上，在K26，Y64，R177，R445，K676位中的至少一个位置的氨基酸发生突变；或者所述苯丙氨酸解氨酶突变体的氨基酸序列具有所述发生突变的氨基酸序列中的所述突变位点，且与SEQ ID NO.2有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的氨基酸序列的苯丙氨酸解氨酶突变体或其功能片段；
    更优选地，所述苯丙氨酸解氨酶突变体包括对应于SEQ ID NO.2所示氨基酸序列，存在如下位点的突变：K26A，K26P，Y64S，Y64H，R177M，R445A，K676S中的一种或两种或三种或四种以上的组合；
    更优选地，对应于SEQ ID NO.2，存在如下位点的突变：第26位赖氨酸突变为丙氨酸；第26位赖氨酸突变为脯氨酸；第64位酪氨酸突变为丝氨酸；第64位酪氨酸突变为组氨酸；第177位精氨酸突变为甲硫氨酸；第445位精氨酸突变为丙氨酸；第676位赖氨酸突变为丝氨酸；第64位酪氨酸突变为丝氨酸，且第445位精氨酸突变为丙氨酸；第26位赖氨酸突变为脯氨酸，且第177位精氨酸突变为甲硫氨酸；第26位赖氨酸突变为脯氨酸，且第64位酪氨酸突变为丝氨酸；第64位酪氨酸突变为组氨酸，且第177位精氨酸突变为甲硫氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为丝氨酸，且第177位精氨酸突变为甲硫氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为组氨酸，且第445位精氨酸突变为丙氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为组氨酸，第177位精氨酸突变为甲硫氨酸，且第445位精氨酸突变为丙氨酸；第26位赖氨酸突变为脯氨酸，第64位酪氨酸突变为丝氨酸，第177位精氨酸突变为甲硫氨酸，且第445位精氨酸突变为丙氨酸。
  5. 如权利要求1所述的苯丙氨酸解氨酶突变体，其特征在于，所述突变体的氨基酸序列是在SEQ ID NO.2所示氨基酸序列的基础上，在R101，E124，H340，E341，V344位中的至少一个位置的氨基酸发生突变；或者所述苯丙氨酸解氨酶突变体的氨基酸序列具有所述发生突变的氨基酸序列中的所述突变位点，且与SEQ ID NO.2有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的氨基酸序列的苯丙氨酸解氨酶突变体或其功能片段；
    更优选地，所述苯丙氨酸解氨酶突变体包括对应于SEQ ID NO.2所示氨基酸序列，存在如下位点的突变：R101Q，E124A，E124Q，H340D，H340V，E341A，V344A中的一种或两种或三种或四种以上的组合；
    更具体地，对应于SEQ ID NO.2所示氨基酸序列，存在如下位点的突变：第101位精氨酸突变为谷氨酰胺；第124位谷氨酸突变为丙氨酸；第124位谷氨酸突变为谷氨酰胺；第340位组氨酸突变为天冬氨酸；第340位组氨酸突变为缬氨酸；第341位谷氨酸突变为丙氨酸；第344位缬氨酸突变为丙氨酸；第340位组氨酸突变为缬氨酸，第341位谷氨酸突变为丙氨酸，且第344位缬氨酸突变为丙氨酸；第124位谷氨酸突变为丙氨酸，第340位组氨酸突变为天冬氨酸，第341位谷氨酸突变为丙氨酸，且第344位缬氨酸突变为丙氨酸；第124位谷氨酸突变为丙氨酸，第340位组氨酸突变为缬氨酸，第341位谷氨酸突变为丙氨酸，且第344位缬氨酸突变为丙氨酸；第124位谷氨酸突变为谷氨酰胺，第340位组氨酸突变为天冬氨酸，第341位谷氨酸突变为丙氨酸，且第344位缬氨酸突变为丙氨酸；第124位谷氨酸突变为谷氨酰胺，第340位组氨酸突变为缬氨酸，第341位谷氨酸突变为丙氨酸，且第344位缬氨酸突变为丙氨酸。
  6. 如权利要求1所述的苯丙氨酸解氨酶突变体，其特征在于，所述突变体的氨基酸序列是在SEQ ID NO.2所示氨基酸序列的基础上，在K26，Y64，E124，T169，R177，M239，H340，H341，V344，R445，Q488，Q576位中的至少两个位置发生氨基酸突变；或者所述苯丙氨酸解氨酶突变体的氨基酸序列具有所述发生突变的氨基酸序列中的所述突变位点，且与SEQ ID NO.2有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的氨基酸序列的苯丙氨酸解氨酶突变体或其功能片段；
    更优选地，所述苯丙氨酸解氨酶突变体包括对应于SEQ ID NO.2，存在如下位点的突变：K26A，K26P，Y64S，Y64H，E124A，T169A，R177M，M239T，H340D，H340V，H341A，V344A，R445A，Q488E和Q576E中的一种或两种或三种或四种以上的组合；
    更具体地，对应于SEQ ID NO.2，存在如下位点的突变：第26位赖氨酸突变为丙氨酸，且第344位缬氨酸突变为丙氨酸；第26位赖氨酸突变为脯氨酸，且第344位缬氨酸突变为丙氨酸；第64位酪氨酸突变为丝氨酸，且第340位组氨酸突变为天冬氨酸；第64位酪氨酸突变为组氨酸，且第340位组氨酸突变为天冬氨酸；第64位酪氨酸突变为丝氨酸，且第340位组氨酸突变为缬氨酸；第64位酪氨酸突变为组氨酸，且第340位组氨酸突变为缬氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为组氨酸，且第340位组氨酸突变为天冬氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为组氨酸，且第340位组氨酸突变为缬氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为组氨酸，且第341位组氨酸突变为丙氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为组氨酸，且第344位缬氨酸突变为丙氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为组氨酸，第177位精氨酸突变为甲硫氨酸，且第340位组氨酸突变为天冬氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为组氨酸，第177位精氨酸突变为甲硫氨酸，且第340位组氨酸突变为缬氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为组氨酸，第124位谷氨酸突变为丙氨酸，第177位精氨酸突变为甲硫氨酸，且第340位组氨酸突变为缬氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为组氨酸，第124位谷氨酸突变为丙氨酸，第177位精氨酸突变为甲硫氨酸，第340位组氨酸突变为缬氨酸，且第344位缬氨酸突变为丙氨酸；第64位酪氨酸突变为组氨酸，第340位组氨酸突变为天冬氨酸，第488位谷氨酰胺突变为谷氨酸，且第576位谷氨酰胺突变为谷氨酸；第64位酪氨酸突变为组氨酸，第340位组氨酸突变为缬氨酸，第488位谷氨酰胺突变为谷氨酸，且第576位谷氨酰胺突变为谷氨酸；第64位酪氨酸突变为组氨酸，第169位苏氨酸突变为丙氨酸，第239位甲硫氨酸突变为苏氨酸，且第340位组氨酸突变为天冬氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为组 氨酸，第169位苏氨酸突变为丙氨酸，第239位甲硫氨酸突变为苏氨酸，且第445位精氨酸突变为丙氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为组氨酸，第169位苏氨酸突变为丙氨酸，第177位精氨酸突变为甲硫氨酸，第239位甲硫氨酸突变为苏氨酸，且第340位组氨酸突变为天冬氨酸；第26位赖氨酸突变为丙氨酸，第64位酪氨酸突变为组氨酸，第169位苏氨酸突变为丙氨酸，第239位甲硫氨酸突变为苏氨酸，第340位组氨酸突变为天冬氨酸，且445位精氨酸突变为丙氨酸。
  7. 如权利要求1至6任一项所述的苯丙氨酸解氨酶突变体的编码基因。
  8. 表达如权利要求1至6任一项所述的苯丙氨酸解氨酶突变体的基因工程菌，其包含所述苯丙氨酸解氨酶突变体的编码基因；具体地，所述基因工程菌是将所述编码基因载体连接得到重组表达载体，再导入蛋白表达宿主菌中得到的重组菌株；优选地，所述蛋白表达宿主菌为大肠杆菌，更优选地其是E.coli BL21(DE3)；所述表达载体为pET-30a(+)。
  9. 如权利要求1至6任一项所述苯丙氨酸解氨酶突变体的制备方法，包括培养如权利要求8所述的基因工程菌，使其表达编码所述苯丙氨酸解氨酶突变体基因的步骤；
    任选地，所述制备方法还包括从培养的重组表达菌株中提纯得到苯丙氨酸解氨酶突变体的步骤；具体地，将所述培养后的重组菌体进行超声波破碎，收集细胞破碎后的表达上清，所述表达上清含有苯丙氨酸解氨酶突变体目的蛋白，用Ni柱纯化目的蛋白，通过高浓度咪唑缓冲液洗脱得到纯度较高的苯丙氨酸解氨酶突变体目的蛋白。
  10. 如权利要求1至6任一项所述的苯丙氨酸解氨酶突变体在催化降解L-苯丙氨酸中的应用，具体地，其参与的催化反应中所生成的产物为反式肉桂酸和氨。
  11. 一种苯丙氨酸的降解方法，包括使如权利要求1至6任一项所述苯丙氨酸解氨酶突变体与苯丙氨酸孵育，进行催化降解反应；具体地，所述催化反应的温度为25-40℃，优选25℃；优选地，所述催化反应体系中苯丙氨酸的浓度为20mM。优选地，所述催化反应体系的pH为6-9，优选为pH7-8.8；更优选地，所述催化体系在人体肠道pH环境下具有催化活性。
  12. 如权利要求1至6任一项所述的苯丙氨酸解氨酶突变体在制备治疗苯丙酮尿症疾病的口服药物中的应用。
PCT/CN2023/087182 2022-08-10 2023-04-08 苯丙氨酸解氨酶突变体及其应用 WO2024032012A1 (zh)

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