CN109402098A - Threonine aldolase, mutant and its preparing the application in substituted benzene serine derivative - Google Patents

Threonine aldolase, mutant and its preparing the application in substituted benzene serine derivative Download PDF

Info

Publication number
CN109402098A
CN109402098A CN201811310893.4A CN201811310893A CN109402098A CN 109402098 A CN109402098 A CN 109402098A CN 201811310893 A CN201811310893 A CN 201811310893A CN 109402098 A CN109402098 A CN 109402098A
Authority
CN
China
Prior art keywords
ala
leu
gly
val
glu
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201811310893.4A
Other languages
Chinese (zh)
Other versions
CN109402098B (en
Inventor
王喆明
张荣珍
谭昊
李利宏
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hangzhou Fazheluo Biotechnology Co Ltd
Original Assignee
Hangzhou Fazheluo Biotechnology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hangzhou Fazheluo Biotechnology Co Ltd filed Critical Hangzhou Fazheluo Biotechnology Co Ltd
Priority to CN201811310893.4A priority Critical patent/CN109402098B/en
Priority to CN202110925261.4A priority patent/CN113481188B/en
Publication of CN109402098A publication Critical patent/CN109402098A/en
Application granted granted Critical
Publication of CN109402098B publication Critical patent/CN109402098B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • 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
    • 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
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/02—Aldehyde-lyases (4.1.2)
    • C12Y401/02005—L-Threonine aldolase (4.1.2.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/02—Aldehyde-lyases (4.1.2)
    • C12Y401/02042—D-Threonine aldolase (4.1.2.42)

Landscapes

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

Abstract

Application of the recombination engineering bacteria and threonine aldolase and mutant of the recombinant vector, recombinant vector conversion acquisition that are constructed the invention discloses the encoding gene of threonine aldolase, mutant, threonine aldolase and mutant, encoding gene in the benzene serine derivative for preparing 2-/3-/4- substitution.The present invention is using threonine aldolase, mutant as biocatalyst, the benzaldehyde replaced using 2-/3-/4- is substrate, using pyridoxime 5-phosphate as coenzyme, the substrate supplemented by glycine/glycinate, enzymic catalytic reaction is carried out in felicity condition and medium, a series of benzene serine derivative of different substituents is prepared in separation, and this method total recovery range is in 76-99%, product ee value is greater than 99%, de value range in 70-99%.

Description

Threonine aldolase, mutant and its in preparing substituted benzene serine derivative Using
Technical field
The present invention relates to field of biotechnology, and in particular to threonine aldolase, mutant and its preparation 2-/3-/4- The application in benzene serine derivative that position replaces.
Background technique
The benzene serine derivative of 2-/3-/4- substitution is a kind of important pharmaceutical intermediate and chemical intermediate, especially It is 4- methylsulfonyl benzene serine, is widely used in mankind's antibiotic, the preparation of domestic birds and animals class antibiotic etc., it is mould to represent drug such as chlorine Element, Thiamphenicol, Florfenicol etc..
The benzene serine derivative synthetic method of 2-/3-/4- substitution mainly includes chemical synthesis and enzyme catalysis method.Its In, chemical synthesis is mainly to pass through addition reaction, esterification and chemical resolution method to obtain chiral 2-/3-/4- and take The benzene serine derivative in generation, a large amount of water, heavy metal and play have been used although chemical method is easy to operate, in reaction process The hydrogen sulfide of poison, and has a large amount of optical isomer as by-product, does not meet at present and in the future country and advocates and propulsion Atom economy and Environment Protection Policy.And enzyme catalysis method have mild condition, water consumption it is few (only the ten of chemical method/ One), without heavy metal pollution, severe poisonous chemicals are not used, chirality selects many advantages, such as high, has a good application prospect.
Threonine aldolase has very extensive application potential, it can be with the pharmaceutical key intermediate of catalytically synthesizing chiral Beta-hydroxy-alpha-amino acid.But since the stability of threonine aldolase is bad, the chirality of chiral selectivity especially beta-hydroxy Selectivity is insufficient, limits application of this enzyme in chiral synthesis.
Summary of the invention
It is an object of the present invention to provide threonine aldolase, mutant and its in the benzene serine for preparing 2-/3-/4- substitution Application in derivative, so as to solve the deficiencies in the prior art.
The invention adopts the following technical scheme:
In a first aspect, be selected from pseudomonad (Pseudomonas putida) the present invention provides threonine aldolase, Amino acid sequence is shown in SEQ ID NO:2, and gene order is shown in SEQ ID NO:1；Selected from crescent shank bacterium (Caulobacter crescentus), amino acid sequence are shown in SEQ ID NO:19, and gene order is SEQ ID NO: Shown in 88；Selected from Thermotoga maritima (Thermotoga maritima), amino acid sequence is shown in SEQ ID NO:23；Choosing From Listeria monocytogenes (Listeria monocytogenes), amino acid sequence is shown in SEQ ID NO:24；Selected from big Enterobacteria (Escherichia coli), amino acid sequence are shown in SEQ ID NO:25；Selected from aermonas jandaei (Aeromonas jandaei), amino acid sequence are shown in SEQ ID NO:26；Selected from saccharomyces cerevisiae (Saccharomyces Cerevisiae), amino acid sequence is shown in SEQ ID NO:27；Selected from ashbya gossypii bacterium (Ashbya gossypii), Its amino acid sequence is shown in SEQ ID NO:28；Selected from Bao Shewanella (Shewanella loihica), amino acid sequence It is classified as shown in SEQ ID NO:29；Selected from imperial Aeromonas (Aeromonas veronii) is tieed up, amino acid sequence is SEQ ID Shown in NO:30；Ground bacillus (Geobacter sulfurreducens) is restored selected from sulphur, amino acid sequence is SEQ ID NO: Shown in 31.
Second aspect, the present invention provides the mutant of threonine aldolase, are built upon Soviet Union's ammonia in different strains source The mutation in four sites of sour aldolase sports simple point mutation, double mutant, three point mutation or four point mutation, is mutated position Point and original amino acids are listed as follows:
Threonine aldolase First catastrophe point Second catastrophe point Third catastrophe point 4th catastrophe point
Ps_LTA N17 H94 S182 T212
Cc_LTA N14 H91 A178 T206
Ec_LTA T8 H83 A168 S196
Lm_LTA --- H98 A178 Y206
Tm_LTA T8 H83 A170 S198
Aj_LTA T10 H85 A170 S198
Sc_LTA T21 H97 A184 S212
Ag_LTA T22 H98 A185 S213
Sl_LTA T8 H83 A168 S196
Av_LTA T10 H85 A170 S198
Gs_LTA T10 H85 A170 S198
It is as follows that it corresponds to replaceable amino acid: the first catastrophe point N/T is changed to Q, and the second catastrophe point H is changed to W/P/F/ V/A/S, third catastrophe point S/A are changed to Y, and the 4th catastrophe point T/S/Y is changed to I；Wherein, the threonine aldehyde in different strains source Contracting enzyme is defined as follows: Ps_LTA- derives from the threonine aldolase of pseudomonad (Pseudomonas putida), Cc_LTA- From the threonine aldolase of crescent shank bacterium (Caulobacter crescentus), Ec_LTA- derives from Escherichia coli The threonine aldolase of (Escherichia coli), Lm_LTA- derive from Listeria monocytogenes (Listeria Monocytogenes threonine aldolase), Tm_LTA- derive from the Soviet Union of Thermotoga maritima (Thermotoga maritima) Propylhomoserin aldolase, Aj_LTA- derive from the threonine aldolase of aermonas jandaei (Aeromonas jandaei), Sc_LTA- From the threonine aldolase of saccharomyces cerevisiae (Saccharomyces cerevisiae), Ag_LTA- derives from cotton A Shu capsule The threonine aldolase of mould (Ashbya gossypii), Sl_LTA- derive from Bao Shewanella (Shewanella Loihica threonine aldolase), Av_LTA- is from the threonine aldehyde for tieing up imperial Aeromonas (Aeromonas veronii) Contracting enzyme, threonine aldolase of the Gs_LTA from sulphur reduction ground bacillus (Geobacter sulfurreducens)；Wherein, ammonia Base acid abbreviation meaning is as follows: N- asparagine, T- threonine, Q- glutamine, H- histidine, W- tryptophan, P- proline, F- Phenylalanine, V- valine, A- alanine, S- amino acids, Y- tyrosine, I- isoleucine.
Further, the mutant of threonine aldolase is selected from Soviet Union's ammonia of pseudomonad (Pseudomonas putida) The mutant of sour aldolase, amino acid sequence are SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: Shown in 10, gene order is the SEQ shown in SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 Mutant shown in ID NO:4 is by the 182nd mutant serine of amino acid sequence shown in SEQ ID NO:2 into tyrosine, institute Stating mutant shown in SEQ ID NO:6 is by the 94th hyte Histidine mutations of amino acid sequence shown in SEQ ID NO:2 into dried meat ammonia Acid, mutant shown in the SEQ ID NO:8 be by the 17th asparagine mutation of amino acid sequence shown in SEQ ID NO:2 at Glutamine, mutant shown in the SEQ ID NO:10 are by the 212nd Soviet Union's ammonia of amino acid sequence shown in SEQ ID NO:2 Acid mutation is at isoleucine；The mutation of threonine aldolase selected from crescent shank bacterium (Caulobacter crescentus) Body, amino acid sequence are SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:32, SEQ ID Shown in NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38；Choosing From the mutant of the threonine aldolase of Thermotoga maritima (Thermotoga maritima), amino acid sequence is SEQ ID Shown in NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43；Increase Liszt selected from single The mutant of the threonine aldolase of bacterium (Listeria monocytogenes), amino acid sequence are SEQ ID NO:44, Shown in SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48；Selected from Escherichia coli The mutant of the threonine aldolase of (Escherichia coli), amino acid sequence are SEQ ID NO:49, SEQ ID Shown in NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53；Selected from aermonas jandaei (Aeromonas Jandaei the mutant of threonine aldolase), amino acid sequence are SEQ ID NO:54, SEQ ID NO:55, SEQ ID Shown in NO:56, SEQ ID NO:57, SEQ ID NO:58；Selected from saccharomyces cerevisiae (Saccharomyces cerevisiae) The mutant of threonine aldolase, amino acid sequence are SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ Shown in ID NO:62, SEQ ID NO:63；Threonine aldolase selected from ashbya gossypii bacterium (Ashbya gossypii) is dashed forward Variant, amino acid sequence are SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID Shown in NO:68；The mutant of threonine aldolase selected from Bao Shewanella (Shewanella loihica), amino acid sequence It is classified as shown in SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73；It is selected from The mutant of the threonine aldolase of imperial Aeromonas (Aeromonas veronii) is tieed up, amino acid sequence is SEQ ID Shown in NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78；The thin end of the scroll is restored selected from sulphur The mutant of the threonine aldolase of bacterium (Geobacter sulfurreducens), amino acid sequence are SEQ ID NO: Shown in 79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83.
The third aspect, the present invention provides above-mentioned threonine aldolases, the encoding gene of the mutant of threonine aldolase.
Fourth aspect, the present invention provides the recombinant vector of above-mentioned encoding gene building, these recombinant vectors can pass through this The nucleotide sequence of threonine aldolase of the invention, the mutant of threonine aldolase is connected to various by field conventional method It is built-up on carrier.The carrier can be the various carriers of this field routine, such as each plasmid vector.
5th aspect, the present invention provides the recombination engineering bacterias that the conversion of above-mentioned recombinant vector obtains, can be by incite somebody to action this The recombinant expression carrier of invention is converted to be obtained into host microorganism.The host microorganism can be the various of this field routine Host microorganism, as long as self-replacation can be stablized and entrained threonine aldehyde contracting of the invention by meeting recombinant expression carrier Enzyme, threonine aldolase the gene of mutant can be with effective expression, such as Escherichia coli, bacillus subtilis, bar bacterium, yeast Deng.
6th aspect, the present invention provides above-mentioned threonine aldolase, threonine aldolase mutant preparation 2-/ Application in the benzene serine derivative of 3-/4- substitutions.
Further, it using the mutant of threonine aldolase or threonine aldolase as catalyst, is taken with 2-/3-/4- The benzaldehyde in generation is substrate, and using pyridoxime 5-phosphate as coenzyme, the substrate supplemented by glycine/glycinate controls temperature and stirs Under conditions of mixing speed, enzymatic is carried out in reaction medium, after reaction, is isolated and purified, and 2-/3-/4- substitution is obtained Benzene serine derivative.
Further, the substrate is one of following: benzaldehyde, 2- chlorobenzaldehyde, 2- fluorobenzaldehyde, 2- bromobenzaldehyde, 2- nitrobenzaldehyde, 2- methyl sulfone benzaldehyde, Benzaldehyde,2-hydroxy, 3- chlorobenzaldehyde, 3- fluorobenzaldehyde, 3- bromobenzaldehyde, 3- Nitrobenzaldehyde, 3- methyl sulfone benzaldehyde, 3- hydroxy benzaldehyde, 4- chlorobenzaldehyde, 4- fluorobenzaldehyde, 4- bromobenzaldehyde, 4- nitre Benzaldehyde, 4- methyl sulfone benzaldehyde, 4- hydroxy benzaldehyde；The auxiliary substrate is one of following: L- glycine, L- glycinate Hydrochlorate, L- glycine methyl ester hydrochloride, L- glycine ethyl ester hydrochloride, N-Boc-L- glycine, N-Boc-L- glycine ethyl ester, N-Z-L- glycine, N-Z-L- glycine ethyl ester, N-Z-L- glycine methyl ester；Reaction medium is one of following: the phosphorus of pH 8-9 Acid buffering system, water-ethanol system, Water-Methanol System, methanol system, ethanol system.
Further, the dosage of enzyme is 50-1000mg/L, concentration of substrate 1-100g/L, glycine/glycinate concentration For 5-200g/L, pyridoxime 5-phosphate concentration is 0.01-0.2g/L, and control temperature is at 25-45 DEG C, mixing speed 70-300rpm.
Beneficial effects of the present invention:
1, the present invention carries out rite-directed mutagenesis by the threonine aldolase to different strains source, to filter out stability Well, the high threonine aldolase mutant of chiral selectivity and it is used successfully to 2-/3-/4- of synthesis of chiral purity is high substitution Benzene serine derivative, shown outstanding application prospect.
2, using threonine aldolase, mutant as biocatalyst, the benzaldehyde replaced with 2-/3-/4- is the present invention Substrate, using pyridoxime 5-phosphate as coenzyme, the substrate supplemented by glycine/glycinate carries out enzyme in felicity condition and medium A series of benzene serine derivative of different substituents is prepared in catalysis reaction, separation, and this method total recovery range is in 76- 99%, product ee value is greater than 99%, de value range in 70-99%.
Detailed description of the invention
Fig. 1 is embodiment 19HPLC map.
Fig. 2 is 20 nuclear magnetic spectrum of embodiment.
Specific embodiment
The present invention is done below with reference to embodiment and attached drawing and is further explained.The following example is merely to illustrate this hair It is bright, but be not used to limit practical range of the invention.
Embodiment 1: the amplification of threonine aldolase gene PS ALD
The threonine aldolase gene from pseudomonad (Pseudomonas putida) included according to Genebank Information is that the total genomic dna of thallus is extracted with nucleic acid Rapid extraction instrument according to (gene order is as shown in SEQ ID NO:1), Using the genomic DNA as template, in primer 1 (ATGAATGGTGAAA CCAGCCG), primer 2 (TTAACGTTCCTGGGTGCGAT) PCR amplification is carried out under the action of.PCR reaction system (50 μ L of total volume): 10 × Pfu DNA Polymerase Buffer 5 μ L, 10mM dNTPmixture (each 2.5mM of dATP, dCTP, dGTP and dTTP) 1 μ L, concentration are equal For 50 μM of cloning primers 1, each 1 μ L of primer 2, genomic DNA l μ L, Pfu DNA Polymerase 1 μ L, seedless 40 μ of sour water L。
Using the PCR instrument of BioRad, PCR reaction condition: 95 DEG C of 5min of initial denaturation, 95 DEG C of denaturation 30s, 65 DEG C are returned fiery 45s, 72 DEG C of extension 1min, totally 30 recycle, last 72 DEG C of extensions 10min.PCR reaction solution is detected with 0.9% agarose gel electrophoresis And gel extraction purifies the segment, obtains threonine aldolase gene PS ALD.
Embodiment 2: the amplification of threonine aldolase gene C CALD
The threonine aldehyde contracting from crescent shank bacterium (Caulobacter crescentus) included according to Genebank Enzyme gene information is that total gene of thallus is extracted with nucleic acid Rapid extraction instrument according to (gene order is as shown in SEQ ID NO:88) Group DNA, using the genomic DNA as template, in primer 3 (ATGACCCAFACCGCGCCCCGCTA), primer 4 (CTAAGCCACTCGCTTCAGCGCC) PCR amplification is carried out under the action of.PCR reaction system (50 μ L of total volume): 10 × Pfu 5 μ L, 10mM dNTPmixture (each 2.5mM of dATP, dCTP, dGTP and dTTP) of DNA Polymerase Buffer 1 μ L is dense Degree is 50 μM of cloning primer 1, each 1 μ L of primer 2,1 μ L of genomic DNA l μ L, Pfu DNA Polymerase, free nucleic acid 40 μ L of water.
Using the PCR instrument of BioRad, PCR reaction condition: 95 DEG C of 5min of initial denaturation, 95 DEG C of denaturation 30s, 65 DEG C are returned fiery 45s, 72 DEG C of extension 1min, totally 30 recycle, last 72 DEG C of extensions 10min.PCR reaction solution is detected with 0.9% agarose gel electrophoresis And gel extraction purifies the segment, obtains threonine aldolase gene C CALD.
Embodiment 3: the building of the recombination bacillus coli of threonine aldolase gene PS ALD
The acquisition of one, recombinant plasmid pET28a-PSALD
PSALD gene order is obtained, using Nde I and Xho I restriction enzyme (TaKaRa) to amplification piece after sequencing Duan Jinhang processing, and utilize T4DNA ligase (TaKaRa) is by the segment with the business with identical restriction enzyme enzymatic treatment Change carrier pET28a to be attached, construction of expression vector pET28a-PSALD.
Two, recombinant plasmid transformed E. coli BL21 (DE3)
10 μ LPCR products are added in 100 μ L E.coli BL21 (DE3) competent cell suspensions of every pipe, gently mix It is even, 30min is stood in ice bath.It is transferred in 42 DEG C of water-baths, thermal shock 90s.Fast transfer cooling 3min into ice bath.It is added in every pipe 700 μ L LB liquid mediums, 37 DEG C of 100rpm shaking tables incubate culture 1h.Bacterium solution 3 after culture, 000rpm are centrifuged 2min, abandon supernatant 700 μ L, remaining bacterium solution are applied to after mixing containing 50 μ gmL-1On the LB plate of kanamycin sulfate, 37 DEG C of inversions were cultivated Night.
The selection of positive colony: picking 4 clones, switching contain 50 μ gmL equipped with 5mL's-1The LB of kanamycin sulfate In culture medium, 37 DEG C of culture 8h, using plasmid extraction kit Mini-Plasmid Rapid Isolation Kit, (Beijing is rich Big Tyke biological gene Technology Co., Ltd.) extract plasmid.It takes out 20 μ L plasmids and transfers to the raw work sequencing in Shanghai.Verifying is correct E.coli BL21(DE3)/pET28a-PSALD。
Embodiment 4: the building of the recombination bacillus coli of threonine aldolase gene C CALD
The acquisition of one, recombinant plasmid pET28a-CCALD
CCALD gene order is obtained, using Nde I and Xho I restriction enzyme (TaKaRa) to amplification piece after sequencing Duan Jinhang processing, and utilize T4DNA ligase (TaKaRa) is by the segment with the business with identical restriction enzyme enzymatic treatment Change carrier pET28a to be attached, construction of expression vector pET28a-CCALD.
Two, recombinant plasmid transformed E. coli BL21 (DE3)
10 μ LPCR products are added in 100 μ L E.coli BL21 (DE3) competent cell suspensions of every pipe, gently mix It is even, 30min is stood in ice bath.It is transferred in 42 DEG C of water-baths, thermal shock 90s.Fast transfer cooling 3min into ice bath.It is added in every pipe 700 μ L LB liquid mediums, 37 DEG C of 100rpm shaking tables incubate culture 1h.Bacterium solution 3 after culture, 000rpm are centrifuged 2min, abandon supernatant 700 μ L, remaining bacterium solution are applied to after mixing containing 50 μ gmL-1On the LB plate of kanamycin sulfate, 37 DEG C of inversions were cultivated Night.
The selection of positive colony: picking 4 clones, switching contain 50 μ gmL equipped with 5mL's-1The LB of kanamycin sulfate In culture medium, 37 DEG C of culture 8h, using plasmid extraction kit Mini-Plasmid Rapid Isolation Kit, (Beijing is rich Big Tyke biological gene Technology Co., Ltd.) extract plasmid.It takes out 20 μ L plasmids and transfers to the raw work sequencing in Shanghai.Verifying is correct E.coli BL21(DE3)/pET28a-CCALD。
Embodiment 5: expression mutant PSALD (S182Y), PSALD (H94P), PSALD (N17Q), PSALD (T212I) base The amplification of cause
Both ends primer is synthesized, sequence is respectively such as SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, shown in SEQ ID NO:18.:
Using the method for PCR, mutation is introduced, the specific method is as follows: PCR reaction system: ddH2O 22 μ L, Premix 25 μ L of PrimeSTAR, 1 μ L of upstream primer (20 μM), downstream primer (20 μM) 1 μ L, 1 μ L of Plasmid DNA.
PCR reaction: 98 DEG C of initial denaturation 30s；98 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 7min carry out 30 circulations；72 DEG C of extensions 10min.Using PSALD as template, PCR reaction is carried out with primer, obtains the full-length gene of mutation.Utilize 3S Spin Agarose Gel DNA Purification Kit (Shanghai Shenergy Biocolor BioScience & Technology Company) purifies DNA segment.
The Dpn I enzymic digestion template of gene PCR product after purification:
Digestion system: 30 5 μ L, Dpn I of μ L, 10 × Buffer of PCR product 1.5 μ L, ddH2System is supplied 50 μ L by O.
It (Shanghai Shen can lottery industry biology using 3S Spin Agarose Gel DNA Purification Kit after digestion 2h Science and Technology Ltd.) purifying DNA segment.
PCR reaction solution is detected with 0.9% agarose gel electrophoresis and gel extraction purifies the segment, obtains the contracting of threonine aldehyde Enzyme mutant gene PSALD (S182Y), PSALD (H94P), PSALD (N17Q), PSALD (T212I).
Embodiment 6: the amplification of expression mutant CCALD (A178Y), CCALD (H91S) gene
Both ends primer is synthesized, sequence is respectively such as SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID Shown in NO:87.
Using the method for PCR, mutation is introduced, the specific method is as follows: PCR reaction system: ddH2O 22 μ L, Premix 25 μ L of PrimeSTAR, 1 μ L of upstream primer (20 μM), downstream primer (20 μM) 1 μ L, 1 μ L of Plasmid DNA.
PCR reaction: 98 DEG C of initial denaturation 30s；98 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 7min carry out 30 circulations；72 DEG C of extensions 10min.Using CCALD as template, PCR reaction is carried out with primer, obtains the full-length gene of mutation.Utilize 3S Spin Agarose Gel DNA Purification Kit (Shanghai Shenergy Biocolor BioScience & Technology Company) purifies DNA segment.
The Dpn I enzymic digestion template of gene PCR product after purification:
Digestion system: 30 5 μ L, Dpn I of μ L, 10 × Buffer of PCR product 1.5 μ L, ddH2System is supplied 50 μ L by O.
It (Shanghai Shen can lottery industry biology using 3S Spin Agarose Gel DNA Purification Kit after digestion 2h Science and Technology Ltd.) purifying DNA segment.
PCR reaction solution is detected with 0.9% agarose gel electrophoresis and gel extraction purifies the segment, obtains the contracting of threonine aldehyde Enzyme mutant gene CCALD (A178Y), CCALD (H91S).
Embodiment 7: the building of the recombination bacillus coli of expression mutant S182Y, H94P, N17Q, T212I
One, recombinant plasmid pET28a-PSALD (S182Y), pET28a-PSALD (H94P), pET28a-PSALD (N17Q), The acquisition of pET28a-PSALD (T212I)
Mutant gene sequence is obtained, using Nde I and Xho I restriction enzyme (TaKaRa) to amplification piece after sequencing Duan Jinhang processing, and utilize T4DNA ligase (TaKaRa) by the segment with the business with identical restriction enzyme enzymatic treatment Change carrier pET28a be attached, construction of expression vector pET28a-PSALD (S182Y), pET28a-PSALD (H94P), pET28a-PSALD(N17Q)、pET28a-PSALD(T212I)。
Two, recombinant plasmid transformed E. coli BL21 (DE3)
10 μ LPCR products are added in 100 μ L E.coli BL21 (DE3) competent cell suspensions of every pipe, gently mix It is even, 30min is stood in ice bath.It is transferred in 42 DEG C of water-baths, thermal shock 90s.Fast transfer cooling 3min into ice bath.It is added in every pipe 700 μ L LB liquid mediums, 37 DEG C of 100rpm shaking tables incubate culture 1h.Bacterium solution 3 after culture, 000rpm are centrifuged 2min, abandon supernatant 700 μ L, remaining bacterium solution are applied to after mixing containing 50 μ gmL-1On the LB plate of kanamycin sulfate, 37 DEG C of inversions were cultivated Night.
The selection of positive colony: picking 4 clones, switching contain 50 μ gmL equipped with 5mL's-1The LB of kanamycin sulfate In culture medium, 37 DEG C of culture 8h, using plasmid extraction kit Mini-Plasmid Rapid Isolation Kit, (Beijing is rich Big Tyke biological gene Technology Co., Ltd.) extract plasmid.It takes out 20 μ L plasmids and transfers to the raw work sequencing in Shanghai.Verifying is correct E.coli BL21(DE3)/pET28a-PSALD(S182Y)、E.coli BL21(DE3)/pET28a-PSALD(H94P)、 E.coli BL21 (DE3)/pET28a-PSALD (N17Q), E.coli BL21 (DE3)/pET28a-PSALD (T212I) expression Threonine aldolase PSALD amino acid sequence, relative to sequence shown in SEQ ID NO:2, the 182nd serine is prominent Become tyrosine, the 94th Histidine mutagenesis into proline, the 17th asparagine mutation into glutamine, 212 threonines have been mutated into isoleucine.
Embodiment 8: the building of the recombination bacillus coli of expression mutant A178Y, H91S
The acquisition of one, recombinant plasmid pET28a-CCALD (A178Y), pET28a-CCALD (H91S)
Mutant gene sequence is obtained, using Nde I and Xho I restriction enzyme (TaKaRa) to amplification piece after sequencing Duan Jinhang processing, and utilize T4DNA ligase (TaKaRa) by the segment with the business with identical restriction enzyme enzymatic treatment Change carrier pET28a to be attached, construction of expression vector pET28a-CCALD (A178Y), pET28a-CCALD (H91S).
Two, recombinant plasmid transformed E. coli BL21 (DE3)
10 μ LPCR products are added in 100 μ L E.coli BL21 (DE3) competent cell suspensions of every pipe, gently mix It is even, 30min is stood in ice bath.It is transferred in 42 DEG C of water-baths, thermal shock 90s.Fast transfer cooling 3min into ice bath.It is added in every pipe 700 μ L LB liquid mediums, 37 DEG C of 100rpm shaking tables incubate culture 1h.Bacterium solution 3 after culture, 000rpm are centrifuged 2min, abandon supernatant 700 μ L, remaining bacterium solution are applied to after mixing containing 50 μ gmL-1On the LB plate of kanamycin sulfate, 37 DEG C of inversions were cultivated Night.
The selection of positive colony: picking 4 clones, switching contain 50 μ gmL equipped with 5mL's-1The LB of kanamycin sulfate In culture medium, 37 DEG C of culture 8h, using plasmid extraction kit Mini-Plasmid Rapid Isolation Kit, (Beijing is rich Big Tyke biological gene Technology Co., Ltd.) extract plasmid.It takes out 20 μ L plasmids and transfers to the raw work sequencing in Shanghai.Verifying is correct E.coli BL21 (DE3)/pET28a-CCALD (A178Y), E.coli BL21 (DE3)/pET28a-CCALD (H91S) expression Threonine aldolase CCALD amino acid sequence, relative to sequence shown in SEQ ID NO:19, the 178th alanine Tyrosine, the 91st Histidine mutagenesis have been mutated into serine.
Embodiment 9: the building of the recombined bacillus subtilis of expression mutant S182Y, H94P, N17Q, T212I
One, recombinant plasmid pHCMC04-PSALD (S182Y), pHCMC04-PSALD (H94P), pHCMC04-PSALD (N17Q), the acquisition of pHCMC04-PSALD (T212I)
Mutant gene sequence is obtained, using Kpn I and Sma I restriction enzyme (TaKaRa) to amplification piece after sequencing Duan Jinhang processing, and utilize T4DNA ligase (TaKaRa) is by the segment with the business with identical restriction enzyme enzymatic treatment Change carrier pHCMC04 be attached, construction of expression vector pHCMC04-PSALD (S182Y), pHCMC04-PSALD (H94P), pHCMC04-PSALD(N17Q)、pHCMC04-PSALD(T212I)。
Two, recombinant plasmid transformed bacillus subtilis Bacillus subtilis SCK6
Bacillus subtilis SCK6 is drawn into the T2 culture medium solid plate containing amicillin resistance, picking single colonie connects Kind in 5ml LB liquid medium, stay overnight by 37 DEG C of shaken cultivations, and 500 μ L cultures is taken to be forwarded to 10ml LB liquid medium In (OD600 is diluted to 1 or so), be added final concentration 1% (w/v) xylose, 37 DEG C shaken cultivation 2 hours, be added final concentration 10% Glycerol, by freezing after every 500 μ l of pipe packing in -70 DEG C, gained is competent cell.
Every 500 μ L competent cell is added 1 μ g Plasmid DNA, 37 DEG C shaken cultivation 1.5 hours, apply amicillin resistance T2 plate, 37 DEG C stand overnight after check conversion situation.
The selection of positive colony: picking single colonie is inoculated in 5ml LB liquid medium from conversion plate, 37 DEG C of vibrations Overnight incubation is swung, taking 3ml culture, thalline were collected by centrifugation, is resuspended with 150 μ L TE, small in 37 DEG C of incubations 1 after addition lysozyme When, (w/v) SDS of final concentration 0.5% and Proteinase K is added, is incubated to mixed liquor clarification for 37 DEG C after being mixed by inversion, 300 μ L is added Supernatant is collected by centrifugation after being mixed by inversion in solution TE, is adsorbed using the silicon matrix column in Omega mini-scale plasmid extracts kit pure Change, affords Plasmid DNA with 50 μ LTE.It takes out 20 μ L plasmids and transfers to the raw work sequencing in Shanghai.What verifying was correctly expressed Bacillus subtilis SCK6/pHCMC04-PSALD(S182Y)、Bacillus subtilis SCK6/pHCMC04- PSALD(H94P)、Bacillus subtilis SCK6/pHCMC04-PSALD(N17Q)、Bacillus subtilis The amino acid sequence of SCK6/pHCMC04-PSALD (T212I) threonine aldolase PSALD, relative to shown in SEQ ID NO:2 Sequence, the 182nd mutant serine is at tyrosine, the 94th Histidine mutagenesis at proline, the 17th day Winter amide has been mutated into glutamine, the 212nd threonine has been mutated into isoleucine.
Embodiment 10: the building of the recombination Corynebacterium glutamicum of expression mutant S182Y, H94P, N17Q, T212I
One, recombinant plasmid pUC19-PSALD (S182Y), pUC19-PSALD (H94P), pUC19-PSALD (N17Q), The acquisition of pUC19-PSALD (T212I)
Mutant gene sequence is obtained, using Hind I and BamH I restriction enzyme (TaKaRa) to amplification after sequencing Segment is handled, and utilizes T4DNA ligase (TaKaRa) is by the segment with the quotient with identical restriction enzyme enzymatic treatment Industry carrier pUC19 is attached, construction of expression vector pUC19-PSALD (S182Y), pUC19-PSALD (H94P), pUC19- PSALD(N17Q)、pUC19-PSALD(T212I)。
Two, recombinant plasmid transformed Corynebacterium glutamicum C.glutamicum TK260251
The C.glutamicum TK26 of the fresh plate of picking0251Single colonie accesses the 2mL liquid LB of 0.5% glucose In culture medium, 30 DEG C, revolving speed be 250r/min rotary shaker on cultivate 12-14h.By 1% or so inoculum concentration transfer into In 50mL LB liquid medium containing 3% glycine and 0.1%Tween-80, cultivated at 30 DEG C, until cell OD600Reach 0.4.First by bacterium solution ice bath 15min after cell culture, it is then centrifuged for collecting thallus (5500g, 20min, 4 DEG C).Use 250mL Buffer (10%w/v glycerol, 8mmol/L, Tris, pH 7.4) suspension thalline of ice is then centrifuged for collecting thallus (4 DEG C, 5500r/min, 20min).With glycerol washing thalline 4 times of the 10% of 100mL pre-cooling.Finally use 10% glycerol weight of 0.2mL Outstanding cell, is dispensed, every 50 μ L of pipe with 1.5mL centrifuge tube.The cell handled well can be directly used for electrotransformation or protect in -70 DEG C of refrigerators It deposits stand-by.
Electrotransformation: taking 50 μ L-Glu rod bacterium competence cell freeze thawing 1 time, adds 5 μ L Plasmid DNA thin to 50 μ L competence Born of the same parents mix, and mixed liquor is transferred in 0.2mm electricity revolving cup, and ice bath 5min opens electroporation, and parameter: 1.5-3.0kV is arranged, 200Q, 25 μ F.The condensed water outside electric revolving cup is dried, electric revolving cup is put into electric shock instrument, starts the electric pulse to cell.End-of-pulsing Afterwards, electric revolving cup is taken out as quickly as possible, and 1mL SOC culture medium is added.It is transferred to after mixing in EP pipe, in 30 DEG C of stationary culture 1h.
The selection of positive colony: 100-300 μ L bacterium solution is taken to be applied to 30 on the amicillin resistance plate containing 10 μ g/ml DEG C it is inverted culture, after growing bacterium colony, picking single colonie is inoculated in the LB culture medium of 3% glycine of 20 μ g/ml, 30 DEG C of cultures Overnight, plasmid enzyme restriction verifying is proposed.It takes out 20 μ L plasmids and transfers to the raw work sequencing in Shanghai.Verify correct C.glutamicum TK260251pUC19-PSALD(S182Y)、pUC19-PSALD(H94P)、pUC19-PSALD(N17Q)、pUC19-PSALD (T212I) amino acid sequence of the threonine aldolase PSALD expressed, relative to sequence shown in SEQ ID NO:2, the 182nd Position mutant serine at tyrosine, the 94th Histidine mutagenesis at proline, the 17th asparagine mutation at Glutamine, the 212nd threonine have been mutated into isoleucine.
Embodiment 11: the building of the recombinant Saccharomyces cerevisiae of expression mutant S182Y, H94P, N17Q, T212I
One, recombinant plasmid pESC-URA-PSALD (S182Y), pESC-URA-PSALD (H94P), pESC-URA-PSALD (N17Q), the acquisition of pESC-URA-PSALD (T212I)
Mutant gene sequence is obtained, using BamH I and Kpn I restriction enzyme (TaKaRa) to amplification after sequencing Segment is handled, and utilizes T4DNA ligase (TaKaRa) is by the segment with the quotient with identical restriction enzyme enzymatic treatment Industry carrier pESC-URA is attached, construction of expression vector pESC-URA-PSALD (S182Y), pESC-URA-PSALD (H94P)、pESC-URA-PSALD(N17Q)、pESC-URA-PSALD(T212I)。
Two, recombinant plasmid transformed S. cervisiae Saccharomyces cerevisiae YPH501
Saccharomyces cerevisiae YPH501 competent cell ice bath is thawed, is placed spare；Take the recombinant vector pESC-URA- of 1 μ g PSALD (S182Y), pESC-URA-PSALD (H94P), pESC-URA-PSALD (N17Q), pESC-URA-PSALD (T212I), Enter with the linear salmon finishing of 100 μ g into melted competent cell；The LTE/PEG-3350 (40%) of 300 μ L is added, It turns upside down and is mixed evenly；30 DEG C of environment culture 30min；42 DEG C of thermal shock 15min；The YPD Liquid Culture of 500 μ L preheating is added Base, 30 DEG C of culture 2h；Above-mentioned bacterium solution is applied on uracil-deficient SD-U plating medium by gradient, 30 DEG C of cultures 2~ 3d。
Bacterium solution is applied on the YPD culture medium containing ampicillin antibiotic after conversion and is screened, is obtained positive Transformant.Empty carrier pAUR123 is converted by method of the same race to the competent yeast cells containing empty carrier pESC-URA.To turn Beggar carries out bacterium colony PCR verifying, and it is as follows that system prepares such as 2.4.3, program: 94 DEG C, 5min；94 DEG C, 40s；58 DEG C, 50s；72 DEG C, 2min；②Rep30；72 DEG C, 10min；Hold, 4 DEG C.After PCR, with 1% agarose gel electrophoresis detection amplification knot Fruit.
The selection of positive colony: 100-300 μ L bacterium solution is taken to be applied to 30 DEG C of inversion trainings in the Cm resistant panel containing 10 μ g/ml It supports, after growing bacterium colony, picking single colonie is inoculated in the LB culture medium of 3% glycine of 20 μ g/ml, and 30 DEG C of overnight incubations mention Plasmid enzyme restriction verifying.It takes out 20 μ L plasmids and transfers to the raw work sequencing in Shanghai.Verify correct YPH501/pESC-PSALD (S182Y), YPH501/pESC-PSALD (H94P), YPH501/pESC-PSALD (N17Q), YPH501/pESC-PSALD (T212I) expression Threonine aldolase PSALD amino acid sequence, relative to sequence shown in SEQ ID NO:2, the 182nd serine is prominent Become tyrosine, the 94th Histidine mutagenesis into proline, the 17th asparagine mutation into glutamine, 212 threonines have been mutated into isoleucine.
Embodiment 12: the inducing expression culture of recombination bacillus coli
The E.coli BL21 (DE3) of acquisition/pET28a-PSALD (S182Y), E.coli BL21 (DE3)/pET28a- PSALD(H94P)、E.coli BL21(DE3)/pET28a-PSALD(N17Q)、E.coli BL21(DE3)/pET28a-PSALD (T212I)、E.coli BL21(DE3)/pET28a-CCALD(A178Y)、E.coli BL21(DE3)/pET28a-CCALD (H91S) and control group BL21 (pET-28a empty carrier transformant) is used as negative control, is inoculated in 5mL containing 50 μ gmL-1Sulfuric acid In the LB liquid medium of kanamycins, in 37 DEG C, 200rpm shaken cultivation is stayed overnight.Take the switching of 10mL culture solution in 1L containing 50 μ g·mL-1In the LB liquid medium of kanamycin sulfate, in 37 DEG C, 200rpm shaken cultivation to OD600About 0.6-0.8.To Isopropyl-Β-D- the thiogalactoside of final concentration of 0.5mM is added in culture, Fiber differentiation 12h is carried out at 25 DEG C. Thallus is collected, is dissolved in 20mM phosphate buffer (pH 7.0), ultrasonication 30min, working time 2s, intermittent time 3s. By broken thallus 12,000rpm, 40min.Supernatant is taken to precipitate respectively, SDS-PAGE detects protein expression.
Embodiment 13: the inducing expression culture of recombined bacillus subtilis
The bacillus subtilis for containing 4 kinds of genes obtained and control group SCK6 (pHCMC04 empty carrier transformant) are as yin Property control, be inoculated in the test tube equipped with 5ml LB liquid medium, 37 DEG C of shaken cultivations are stayed overnight.Next day, each culture took respectively 2ml is forwarded in the 250ml shaking flask equipped with 50ml 5YC fluid nutrient medium, and 33 DEG C sample after shaken cultivation 8 hours and are collected by centrifugation Supernatant and the isopropyl-Β-D- thiogalactoside induction that final concentration 1mM is added, continue culture to 48 hours, during which every 8 is small When sampling culture supernatant is collected by centrifugation.Culture medium in above-mentioned shake flat experiment contains 25 μ g/ml chloramphenicol.20 μ l are taken to cultivate 5 μ 4 × sample-loading buffers of l are added in object supernatant, and boiling water bath handles 5min, 20 μ l of applied sample amount after mixing, and gum concentration 5% is concentrated, point From gum concentration 17%, inside groove is cathode buffer when electrophoresis, and outer groove is positive buffer.
Embodiment 14: the inducing expression culture of Corynebacterium glutamicum is recombinated
The Corynebacterium glutamicum for containing 4 kinds of genes obtained and control group YPH501 (pUC19 empty carrier) are inoculated in 20mL kind In sub- culture medium, at 30 DEG C, cultivated on shaking table under conditions of 200r/min.As the OD of bacterium solution600When=1.0, by 5% Inoculum concentration accesses seed liquor in fermentation medium, at 30 DEG C, is cultivated on shaking table under conditions of 200r/min.Work as OD600 When=3.5, isopropyl-Β-D- thiogalactoside is added, makes the isopropyl-Β-D- thiogalactoside concentration in culture medium Reach 1mM, continues to cultivate 36h then at 28 DEG C, under conditions of 200r/min.30mL culture solution is at small-sized centrifugation, 4 DEG C 12000r/min is centrifuged 10min.Cell is resuspended in 30mL sodium-acetate buffer (50mmol/L, pH6.0).Cell suspension ultrasound Handle 30min.4 DEG C, 12000r/min is centrifuged 30min and removes cell fragment.Take 40 μ L supernatants that the 5xSDS-PAGE of 9 μ L is added Electrophoresis sample-loading buffer, and be sufficiently mixed.10min is boiled at 100 DEG C, sample is stored in -20 DEG C until carrying out albumen electricity Swimming analysis.
Embodiment 15: the inducing expression culture of recombinant Saccharomyces cerevisiae bacterium
The Yeast engineering bacteria for containing 4 kinds of genes and control group saccharomyces cerevisiae YPH501 (pESC-URA empty carrier) obtained is simultaneously It is inoculated in YPD fluid nutrient medium, it is 0.5,30 DEG C of oscillations that bacterium solution to new culture medium is added after being incubated overnight and adjusts OD600nm value 3d is cultivated, galactolipin inducing expression 1d is added；Yeast Cultivation liquid is collected, 6000rpm, 4 DEG C of centrifugation 10min abandon supernatant collection Bacterial sediment；With frozen-thawed bacterial sediment, cell pyrolysis liquid is added thereto, is uniformly mixed；It is crushed with cell pulverization instrument State bacterium solution, the work/interval condition 25KHz, 10s, time 20min；4 DEG C are collected after centrifugation supernatant, and as saccharomyces cerevisiae is thick Enzyme solution sample.
Embodiment 16: the purifying (recombination bacillus coli of recombinant protein S182Y, H94P, N17Q, T212I, A178Y, H91S E.coli BL21 (DE3) system expression)
Using His-Trap HP affinity column purification of recombinant proteins S182Y, H94P, N17Q, T212I, A178Y, H91S:
Picking positive colony single colonie is inoculated in 5mL containing 50 μ gmL-1In the LB liquid medium of kanamycin sulfate, in 37 DEG C, 200rpm shaken cultivation is stayed overnight.Take the switching of 10mL culture solution in 1L containing 50 μ gmL-1The LB liquid of kanamycin sulfate is trained It supports in base, in 37 DEG C, 200rpm shaken cultivation to OD600About 0.6-0.8.Inducer isopropyl-Β-is added into culture D- thiogalactoside 0.5mM carries out Fiber differentiation 12h at 25 DEG C of cultivation temperature.Recombinant Bacillus coli cells after culture 12,000rpm centrifugation 10min is simultaneously collected with brine afterwards three times.
2g wet thallus is weighed, appropriate 20mM Tris, 150mM NaCl (pH 8.0) buffer suspension cell, Yu Bing is added Ultrasonication (work 2s, be spaced 3s, working time 30min) is carried out in bath.12,000rpm is centrifuged 30min and collects under the conditions of 4 DEG C Supernatant is as crude enzyme liquid.Crude enzyme liquid is purified using the His-Trap affinity chromatography that GE company produces, pure enzyme solution ultrafiltration For specificity 2-/3-/4- substituted benzaldehyde of conversion after desalination.
Embodiment 17: mutation front and back specific enzyme activity measurement
The measurement of mutant enzyme vigor:
Threonine aldolase be catalyzed in the presence of co-factor (pyridoxime 5-phosphate, PLP) threonine be cracked into glycine and Acetaldehyde, acetaldehyde can be restored in the presence of NADH by alcohol dehydrogenase, NAD+Generation can cause the drop of light absorption value at 340nm It is low, therefore the work of phenylserine aldolase can be measured by the variation of the light absorption value at 340nm in measurement reaction process Power.
The standard conditions of enzyme activity determination: 250 μ L of total reaction volume is separately added into 100mM Hepes-NaOH buffer (pH8.0), 50uM PLP, 50mM L-threonine, 0.5mM NADH, 30U alcohol dehydrogenase, 30 DEG C of heat preservation 2min are added appropriate Start the variation of light absorption value at scanning 340nm after pure enzyme solution.Protein content determination uses Bradford method, with bovine serum albumin White BSA is standard items.Enzyme activity is defined as: under the above conditions, the enzyme amount for being catalyzed the NADH of 1 μm of ol per minute is defined as one Unit U.
The calculation formula of enzyme activity are as follows: enzyme activity (U)=EW × V × 103/ (6220 × 0.3)
Than calculation formula living: than (Umg-1)=enzyme activity (U)/protein content (mg) living
Wherein, in EW:1min at 340nm absorbance variation；V: the volume (mL) of reaction solution；6220: Molar Extinction system Number (L mol-1cm-1)；0.3: optical path length (cm).
The specific enzyme activity of 1 different mutants of table
By carrying out rite-directed mutagenesis to wild enzyme, obtained mutant specific enzyme activity has obviously compared to wild enzyme specific enzyme activity It improves.
Embodiment 18: catalysis 4- methylsulfonyl-benzaldehyde and glycine generate L-syn- β -4- methylsulfonyl Phenserine
Reaction system 2ml pH 8.0:KH2PO4(50mM) buffer, glycine (1M), 4- methyl sulfone benzaldehyde (100mM), PLP (240 μM), 2mg enzyme (S182Y).30 DEG C of 200rpm react 1h, and 1M HCl is added and adjusts pH=2 and terminates and reacts.
Embodiment 19:HPLC detects L-syn- β -4- methylsulfonyl Phenserine
Reaction system is derived with OPA/NAC, is detected with Agilent 1260.
Liquid-phase condition: 8.0 (50mM of Purospher STAR RP18 (250mm, 5um) C18 column buffer pH KH2PO4)/CH3CN=81:19, flow velocity 0.8ml/min, runing time 20min, 40 DEG C of temperature.Pass through the reaction to embodiment 18 Detection, obtain HPLC analysis yield 82%, ee value > 99%, de value 70%, as shown in Figure 1.
Embodiment 20:1H-NMR detects L-syn- β -4- methylsulfonyl Phenserine
The reaction solution of termination is centrifuged in 5000rmp, takes supernatant by 0.2 μm of membrane filtration, filtrate is in rotary evaporation 55 degree of set temperature on instrument, pressure 40mbar are spin-dried for, and the solid of collection send nuclear magnetic resonance chemical analyser to detect.Deuterated solvent is deuterium Water, detecting instrument are Bruker AVANCE III 400MHz nuclear magnetic resonance chemical analyser.Nuclear-magnetism verifying product structure is correct, non- Enantiomter meets the testing result of HPLC, as shown in Figure 2.
Embodiment 21: catalysis 3- bromobenzaldehyde and glycine generate L-syn- β -3- bromophenyl serine
Reaction system 2ml pH 8.0:KH2PO4(50mM) buffer, glycine (1M), 3- bromobenzaldehyde (100mM), PLP (240 μM), 2mg enzyme (S182Y)).30 DEG C of 200rpm react 1h, and 1M HCl is added and adjusts pH=2 and terminates and reacts.HPLC analysis is received Rate 81%, ee value > 99%, de value 90%.
Embodiment 22: catalysis 4- bromobenzaldehyde and glycine generate L-syn- β -4- bromophenyl serine
Reaction system 2ml pH 8.0:KH2PO4(50mM) buffer, glycine (1M), 4- bromobenzaldehyde (100mM), PLP (240 μM), 2mg enzyme (S182Y).30 DEG C of 200rpm react 1h, and 1M HCl is added and adjusts pH=2 and terminates and reacts.HPLC analysis is received Rate 79%, ee value > 99%, de value 70%.
Embodiment 23: catalysis 2- nitrobenzaldehyde and glycine generate L-syn- β -2- nitrobenzophenone serine
Reaction system 2ml pH 8.0:KH2PO4(50mM) buffer, glycine (1M), 2- nitrobenzaldehyde (100mM), PLP (240 μM), 2mg enzyme (A178Y).30 DEG C of 200rpm react 1h, and 1M HCl is added and adjusts pH=2 and terminates and reacts.HPLC analysis Yield 99%, ee value > 99%, de value 85%.
Embodiment 24: catalysis 3- nitrobenzaldehyde and glycine generate L-syn- β -3- nitrobenzophenone serine
Reaction system 2ml pH 8.0:KH2PO4(50mM) buffer, glycine (1M), 3- nitrobenzaldehyde (100mM), PLP (240 μM), 2mg enzyme (A178Y).30 DEG C of 200rpm react 1h, and 1M HCl is added and adjusts pH=2 and terminates and reacts.HPLC analysis Yield 83%, ee value > 99%, de value 77%.
Embodiment 25: catalysis 4- nitrobenzaldehyde and glycine generate L-syn- β -4- nitrobenzophenone serine
Reaction system 2ml pH 8.0:KH2PO4(50mM) buffer, glycine (1M), 4- nitrobenzaldehyde (100mM), PLP (240 μM), 2mg enzyme (A178Y).30 DEG C of 200rpm react 1h, and 1M HCl is added and adjusts pH=2 and terminates and reacts.HPLC analysis Yield 89%, ee value > 99%, de value 90%.
Embodiment 26: catalysis 4- methyl sulfone benzaldehyde and glycine generate L-syn- β -4- methylsulfonyl Phenserine
Reaction system 2ml pH 8.0:KH2PO4(50mM) buffer, glycine (1M), 4- methyl sulfone benzaldehyde (100mM), PLP (240 μM), 2mg enzyme (A178Y).30 DEG C of 200rpm react 1h, and 1M HCl is added and adjusts pH=2 and terminates and reacts. HPLC analyzes yield 91%, ee value > 99%, de value 92%.
Embodiment 27: catalysis 3- hydroxy benzaldehyde and glycine generate L-syn- β -3- hydroxy phenyl serine
Reaction system 2ml pH 8.0:KH2PO4(50mM) buffer, glycine (1M), 3- hydroxy benzaldehyde (100mM), PLP (240 μM), 2mg enzyme (H91S).30 DEG C of 200rpm react 1h, and 1M HCl is added and adjusts pH=2 and terminates and reacts.HPLC analysis Yield 76%, ee value > 99%, de value 71%.
Sequence table
<110>WangZhe Ming
Hangzhou Fa Zhe los biosynthesis Science and Technology Ltd.
<120>threonine aldolase, mutant and its application in substituted benzene serine derivative is being prepared
<130> 1
<160> 88
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1074
<212> DNA
<213>pseudomonad threonine aldolase gene (artificial sequence)
<400> 1
atgaatggtg aaaccagccg tccgccggca ctgggtttta gcagcgataa tattgcaggt 60
gcctctccgg aagtggcaca ggcactggtt aaacattctt caggtcaggc aggtccgtat 120
ggtaccgatg aactgaccgc acaggttaaa cgtaaatttt gtgaaatttt tgaacgtgat 180
gttgaagtgt ttctggttcc gaccggcacc gccgcaaatg cactgtgtct gagcgcaatg 240
accccgccgt ggggtaatat ttattgtcat ccggcaagcc atattaataa tgatgaatgt 300
ggtgcaccgg aattcttttc taatggtgca aaactgatga ccgttgatgg tccggccgca 360
aaactggata ttgttcgtct gcgcgaacgc acccgtgaaa aagttggtga tgttcatacc 420
acacagccgg catgtgttag tattacccag gcgaccgaag ttggtagcat ttataccctg 480
gatgaaattg aagcgattgg tgatgtttgt aaaagcagta gcctgggtct gcatatggat 540
ggtagccgtt ttgcgaatgc actggtgagc ctgggttgca gtccggcaga aatgacctgg 600
aaagcgggcg ttgatgcgct gtcttttggc gcaaccaaaa atggtgttct ggcagcagaa 660
gcaattgttc tgtttaatac cagcctggca accgaaatga gctatcgtcg taaacgcgca 720
ggtcatctga gcagtaaaat gcgttttctg agtgcacaga ttgatgccta tctgaccgat 780
gatctgtggc tgcgtaatgc gcgtaaagcc aatgccgcag cgcagcgtct ggcgcagggt 840
ctggaaggtc tgggtggtgt tgaagtgctg ggtggtaccg aagcaaatat tctgttttgt 900
cgcctggata gcgccatgat tgatgccctg ctgaaagccg gttttggttt ttatcatgat 960
cgttggggtc cgaatgtggt tcgttttgtt accagctttg ccaccaccgc agaagatgtg 1020
gatcatctgc tgaatcaggt tcgtctggca gcagatcgca cccaggaacg ttaa 1074
<210> 2
<211> 357
<212> PRT
<213>pseudomonad threonine aldolase amino acid (artificial sequence)
<400> 2
Met Asn Gly Glu Thr Ser Arg Pro Pro Ala Leu Gly Phe Ser Ser Asp
1 5 10 15
Asn Ile Ala Gly Ala Ser Pro Glu Val Ala Gln Ala Leu Val Lys His
20 25 30
Ser Ser Gly Gln Ala Gly Pro Tyr Gly Thr Asp Glu Leu Thr Ala Gln
35 40 45
Val Lys Arg Lys Phe Cys Glu Ile Phe Glu Arg Asp Val Glu Val Phe
50 55 60
Leu Val Pro Thr Gly Thr Ala Ala Asn Ala Leu Cys Leu Ser Ala Met
65 70 75 80
Thr Pro Pro Trp Gly Asn Ile Tyr Cys His Pro Ala Ser His Ile Asn
85 90 95
Asn Asp Glu Cys Gly Ala Pro Glu Phe Phe Ser Asn Gly Ala Lys Leu
100 105 110
Met Thr Val Asp Gly Pro Ala Ala Lys Leu Asp Ile Val Arg Leu Arg
115 120 125
Glu Arg Thr Arg Glu Lys Val Gly Asp Val His Thr Thr Gln Pro Ala
130 135 140
Cys Val Ser Ile Thr Gln Ala Thr Glu Val Gly Ser Ile Tyr Thr Leu
145 150 155 160
Asp Glu Ile Glu Ala Ile Gly Asp Val Cys Lys Ser Ser Ser Leu Gly
165 170 175
Leu His Met Asp Gly Ser Arg Phe Ala Asn Ala Leu Val Ser Leu Gly
180 185 190
Cys Ser Pro Ala Glu Met Thr Trp Lys Ala Gly Val Asp Ala Leu Ser
195 200 205
Phe Gly Ala Thr Lys Asn Gly Val Leu Ala Ala Glu Ala Ile Val Leu
210 215 220
Phe Asn Thr Ser Leu Ala Thr Glu Met Ser Tyr Arg Arg Lys Arg Ala
225 230 235 240
Gly His Leu Ser Ser Lys Met Arg Phe Leu Ser Ala Gln Ile Asp Ala
245 250 255
Tyr Leu Thr Asp Asp Leu Trp Leu Arg Asn Ala Arg Lys Ala Asn Ala
260 265 270
Ala Ala Gln Arg Leu Ala Gln Gly Leu Glu Gly Leu Gly Gly Val Glu
275 280 285
Val Leu Gly Gly Thr Glu Ala Asn Ile Leu Phe Cys Arg Leu Asp Ser
290 295 300
Ala Met Ile Asp Ala Leu Leu Lys Ala Gly Phe Gly Phe Tyr His Asp
305 310 315 320
Arg Trp Gly Pro Asn Val Val Arg Phe Val Thr Ser Phe Ala Thr Thr
325 330 335
Ala Glu Asp Val Asp His Leu Leu Asn Gln Val Arg Leu Ala Ala Asp
340 345 350
Arg Thr Gln Glu Arg
355
<210> 3
<211> 1074
<212> DNA
<213>pseudomonad threonine aldolase gene mutation (artificial sequence)
<400> 3
atgaatggtg aaaccagccg tccgccggca ctgggtttta gcagcgataa tattgcaggt 60
gcctctccgg aagtggcaca ggcactggtt aaacattctt caggtcaggc aggtccgtat 120
ggtaccgatg aactgaccgc acaggttaaa cgtaaatttt gtgaaatttt tgaacgtgat 180
gttgaagtgt ttctggttcc gaccggcacc gccgcaaatg cactgtgtct gagcgcaatg 240
accccgccgt ggggtaatat ttattgtcat ccggcaagcc atattaataa tgatgaatgt 300
ggtgcaccgg aattcttttc taatggtgca aaactgatga ccgttgatgg tccggccgca 360
aaactggata ttgttcgtct gcgcgaacgc acccgtgaaa aagttggtga tgttcatacc 420
acacagccgg catgtgttag tattacccag gcgaccgaag ttggtagcat ttataccctg 480
gatgaaattg aagcgattgg tgatgtttgt aaaagcagta gcctgggtct gcatatggat 540
ggttaccgtt ttgcgaatgc actggtgagc ctgggttgca gtccggcaga aatgacctgg 600
aaagcgggcg ttgatgcgct gtcttttggc gcaaccaaaa atggtgttct ggcagcagaa 660
gcaattgttc tgtttaatac cagcctggca accgaaatga gctatcgtcg taaacgcgca 720
ggtcatctga gcagtaaaat gcgttttctg agtgcacaga ttgatgccta tctgaccgat 780
gatctgtggc tgcgtaatgc gcgtaaagcc aatgccgcag cgcagcgtct ggcgcagggt 840
ctggaaggtc tgggtggtgt tgaagtgctg ggtggtaccg aagcaaatat tctgttttgt 900
cgcctggata gcgccatgat tgatgccctg ctgaaagccg gttttggttt ttatcatgat 960
cgttggggtc cgaatgtggt tcgttttgtt accagctttg ccaccaccgc agaagatgtg 1020
gatcatctgc tgaatcaggt tcgtctggca gcagatcgca cccaggaacg ttaa 1074
<210> 4
<211> 357
<212> PRT
<213>pseudomonad threonine aldolase amino acid mutation (artificial sequence)
<400> 4
Met Asn Gly Glu Thr Ser Arg Pro Pro Ala Leu Gly Phe Ser Ser Asp
1 5 10 15
Asn Ile Ala Gly Ala Ser Pro Glu Val Ala Gln Ala Leu Val Lys His
20 25 30
Ser Ser Gly Gln Ala Gly Pro Tyr Gly Thr Asp Glu Leu Thr Ala Gln
35 40 45
Val Lys Arg Lys Phe Cys Glu Ile Phe Glu Arg Asp Val Glu Val Phe
50 55 60
Leu Val Pro Thr Gly Thr Ala Ala Asn Ala Leu Cys Leu Ser Ala Met
65 70 75 80
Thr Pro Pro Trp Gly Asn Ile Tyr Cys His Pro Ala Ser His Ile Asn
85 90 95
Asn Asp Glu Cys Gly Ala Pro Glu Phe Phe Ser Asn Gly Ala Lys Leu
100 105 110
Met Thr Val Asp Gly Pro Ala Ala Lys Leu Asp Ile Val Arg Leu Arg
115 120 125
Glu Arg Thr Arg Glu Lys Val Gly Asp Val His Thr Thr Gln Pro Ala
130 135 140
Cys Val Ser Ile Thr Gln Ala Thr Glu Val Gly Ser Ile Tyr Thr Leu
145 150 155 160
Asp Glu Ile Glu Ala Ile Gly Asp Val Cys Lys Ser Ser Ser Leu Gly
165 170 175
Leu His Met Asp Gly Tyr Arg Phe Ala Asn Ala Leu Val Ser Leu Gly
180 185 190
Cys Ser Pro Ala Glu Met Thr Trp Lys Ala Gly Val Asp Ala Leu Ser
195 200 205
Phe Gly Ala Thr Lys Asn Gly Val Leu Ala Ala Glu Ala Ile Val Leu
210 215 220
Phe Asn Thr Ser Leu Ala Thr Glu Met Ser Tyr Arg Arg Lys Arg Ala
225 230 235 240
Gly His Leu Ser Ser Lys Met Arg Phe Leu Ser Ala Gln Ile Asp Ala
245 250 255
Tyr Leu Thr Asp Asp Leu Trp Leu Arg Asn Ala Arg Lys Ala Asn Ala
260 265 270
Ala Ala Gln Arg Leu Ala Gln Gly Leu Glu Gly Leu Gly Gly Val Glu
275 280 285
Val Leu Gly Gly Thr Glu Ala Asn Ile Leu Phe Cys Arg Leu Asp Ser
290 295 300
Ala Met Ile Asp Ala Leu Leu Lys Ala Gly Phe Gly Phe Tyr His Asp
305 310 315 320
Arg Trp Gly Pro Asn Val Val Arg Phe Val Thr Ser Phe Ala Thr Thr
325 330 335
Ala Glu Asp Val Asp His Leu Leu Asn Gln Val Arg Leu Ala Ala Asp
340 345 350
Arg Thr Gln Glu Arg
355
<210> 5
<211> 1074
<212> DNA
<213>pseudomonad threonine aldolase gene mutation (artificial sequence)
<400> 5
atgaatggtg aaaccagccg tccgccggca ctgggtttta gcagcgataa tattgcaggt 60
gcctctccgg aagtggcaca ggcactggtt aaacattctt caggtcaggc aggtccgtat 120
ggtaccgatg aactgaccgc acaggttaaa cgtaaatttt gtgaaatttt tgaacgtgat 180
gttgaagtgt ttctggttcc gaccggcacc gccgcaaatg cactgtgtct gagcgcaatg 240
accccgccgt ggggtaatat ttattgtcat ccggcaagcc ctattaataa tgatgaatgt 300
ggtgcaccgg aattcttttc taatggtgca aaactgatga ccgttgatgg tccggccgca 360
aaactggata ttgttcgtct gcgcgaacgc acccgtgaaa aagttggtga tgttcatacc 420
acacagccgg catgtgttag tattacccag gcgaccgaag ttggtagcat ttataccctg 480
gatgaaattg aagcgattgg tgatgtttgt aaaagcagta gcctgggtct gcatatggat 540
ggtagccgtt ttgcgaatgc actggtgagc ctgggttgca gtccggcaga aatgacctgg 600
aaagcgggcg ttgatgcgct gtcttttggc gcaaccaaaa atggtgttct ggcagcagaa 660
gcaattgttc tgtttaatac cagcctggca accgaaatga gctatcgtcg taaacgcgca 720
ggtcatctga gcagtaaaat gcgttttctg agtgcacaga ttgatgccta tctgaccgat 780
gatctgtggc tgcgtaatgc gcgtaaagcc aatgccgcag cgcagcgtct ggcgcagggt 840
ctggaaggtc tgggtggtgt tgaagtgctg ggtggtaccg aagcaaatat tctgttttgt 900
cgcctggata gcgccatgat tgatgccctg ctgaaagccg gttttggttt ttatcatgat 960
cgttggggtc cgaatgtggt tcgttttgtt accagctttg ccaccaccgc agaagatgtg 1020
gatcatctgc tgaatcaggt tcgtctggca gcagatcgca cccaggaacg ttaa 1074
<210> 6
<211> 357
<212> PRT
<213>pseudomonad threonine aldolase amino acid mutation (artificial sequence)
<400> 6
Met Asn Gly Glu Thr Ser Arg Pro Pro Ala Leu Gly Phe Ser Ser Asp
1 5 10 15
Asn Ile Ala Gly Ala Ser Pro Glu Val Ala Gln Ala Leu Val Lys His
20 25 30
Ser Ser Gly Gln Ala Gly Pro Tyr Gly Thr Asp Glu Leu Thr Ala Gln
35 40 45
Val Lys Arg Lys Phe Cys Glu Ile Phe Glu Arg Asp Val Glu Val Phe
50 55 60
Leu Val Pro Thr Gly Thr Ala Ala Asn Ala Leu Cys Leu Ser Ala Met
65 70 75 80
Thr Pro Pro Trp Gly Asn Ile Tyr Cys His Pro Ala Ser Pro Ile Asn
85 90 95
Asn Asp Glu Cys Gly Ala Pro Glu Phe Phe Ser Asn Gly Ala Lys Leu
100 105 110
Met Thr Val Asp Gly Pro Ala Ala Lys Leu Asp Ile Val Arg Leu Arg
115 120 125
Glu Arg Thr Arg Glu Lys Val Gly Asp Val His Thr Thr Gln Pro Ala
130 135 140
Cys Val Ser Ile Thr Gln Ala Thr Glu Val Gly Ser Ile Tyr Thr Leu
145 150 155 160
Asp Glu Ile Glu Ala Ile Gly Asp Val Cys Lys Ser Ser Ser Leu Gly
165 170 175
Leu His Met Asp Gly Ser Arg Phe Ala Asn Ala Leu Val Ser Leu Gly
180 185 190
Cys Ser Pro Ala Glu Met Thr Trp Lys Ala Gly Val Asp Ala Leu Ser
195 200 205
Phe Gly Ala Thr Lys Asn Gly Val Leu Ala Ala Glu Ala Ile Val Leu
210 215 220
Phe Asn Thr Ser Leu Ala Thr Glu Met Ser Tyr Arg Arg Lys Arg Ala
225 230 235 240
Gly His Leu Ser Ser Lys Met Arg Phe Leu Ser Ala Gln Ile Asp Ala
245 250 255
Tyr Leu Thr Asp Asp Leu Trp Leu Arg Asn Ala Arg Lys Ala Asn Ala
260 265 270
Ala Ala Gln Arg Leu Ala Gln Gly Leu Glu Gly Leu Gly Gly Val Glu
275 280 285
Val Leu Gly Gly Thr Glu Ala Asn Ile Leu Phe Cys Arg Leu Asp Ser
290 295 300
Ala Met Ile Asp Ala Leu Leu Lys Ala Gly Phe Gly Phe Tyr His Asp
305 310 315 320
Arg Trp Gly Pro Asn Val Val Arg Phe Val Thr Ser Phe Ala Thr Thr
325 330 335
Ala Glu Asp Val Asp His Leu Leu Asn Gln Val Arg Leu Ala Ala Asp
340 345 350
Arg Thr Gln Glu Arg
355
<210> 7
<211> 1074
<212> DNA
<213>pseudomonad threonine aldolase gene mutation (artificial sequence)
<400> 7
atgaatggtg aaaccagccg tccgccggca ctgggtttta gcagcgatca gattgcaggt 60
gcctctccgg aagtggcaca ggcactggtt aaacattctt caggtcaggc aggtccgtat 120
ggtaccgatg aactgaccgc acaggttaaa cgtaaatttt gtgaaatttt tgaacgtgat 180
gttgaagtgt ttctggttcc gaccggcacc gccgcaaatg cactgtgtct gagcgcaatg 240
accccgccgt ggggtaatat ttattgtcat ccggcaagcc atattaataa tgatgaatgt 300
ggtgcaccgg aattcttttc taatggtgca aaactgatga ccgttgatgg tccggccgca 360
aaactggata ttgttcgtct gcgcgaacgc acccgtgaaa aagttggtga tgttcatacc 420
acacagccgg catgtgttag tattacccag gcgaccgaag ttggtagcat ttataccctg 480
gatgaaattg aagcgattgg tgatgtttgt aaaagcagta gcctgggtct gcatatggat 540
ggtagccgtt ttgcgaatgc actggtgagc ctgggttgca gtccggcaga aatgacctgg 600
aaagcgggcg ttgatgcgct gtcttttggc gcaaccaaaa atggtgttct ggcagcagaa 660
gcaattgttc tgtttaatac cagcctggca accgaaatga gctatcgtcg taaacgcgca 720
ggtcatctga gcagtaaaat gcgttttctg agtgcacaga ttgatgccta tctgaccgat 780
gatctgtggc tgcgtaatgc gcgtaaagcc aatgccgcag cgcagcgtct ggcgcagggt 840
ctggaaggtc tgggtggtgt tgaagtgctg ggtggtaccg aagcaaatat tctgttttgt 900
cgcctggata gcgccatgat tgatgccctg ctgaaagccg gttttggttt ttatcatgat 960
cgttggggtc cgaatgtggt tcgttttgtt accagctttg ccaccaccgc agaagatgtg 1020
gatcatctgc tgaatcaggt tcgtctggca gcagatcgca cccaggaacg ttaa 1074
<210> 8
<211> 357
<212> PRT
<213>pseudomonad threonine aldolase amino acid mutation (artificial sequence)
<400> 8
Met Asn Gly Glu Thr Ser Arg Pro Pro Ala Leu Gly Phe Ser Ser Asp
1 5 10 15
Gln Ile Ala Gly Ala Ser Pro Glu Val Ala Gln Ala Leu Val Lys His
20 25 30
Ser Ser Gly Gln Ala Gly Pro Tyr Gly Thr Asp Glu Leu Thr Ala Gln
35 40 45
Val Lys Arg Lys Phe Cys Glu Ile Phe Glu Arg Asp Val Glu Val Phe
50 55 60
Leu Val Pro Thr Gly Thr Ala Ala Asn Ala Leu Cys Leu Ser Ala Met
65 70 75 80
Thr Pro Pro Trp Gly Asn Ile Tyr Cys His Pro Ala Ser His Ile Asn
85 90 95
Asn Asp Glu Cys Gly Ala Pro Glu Phe Phe Ser Asn Gly Ala Lys Leu
100 105 110
Met Thr Val Asp Gly Pro Ala Ala Lys Leu Asp Ile Val Arg Leu Arg
115 120 125
Glu Arg Thr Arg Glu Lys Val Gly Asp Val His Thr Thr Gln Pro Ala
130 135 140
Cys Val Ser Ile Thr Gln Ala Thr Glu Val Gly Ser Ile Tyr Thr Leu
145 150 155 160
Asp Glu Ile Glu Ala Ile Gly Asp Val Cys Lys Ser Ser Ser Leu Gly
165 170 175
Leu His Met Asp Gly Ser Arg Phe Ala Asn Ala Leu Val Ser Leu Gly
180 185 190
Cys Ser Pro Ala Glu Met Thr Trp Lys Ala Gly Val Asp Ala Leu Ser
195 200 205
Phe Gly Ala Thr Lys Asn Gly Val Leu Ala Ala Glu Ala Ile Val Leu
210 215 220
Phe Asn Thr Ser Leu Ala Thr Glu Met Ser Tyr Arg Arg Lys Arg Ala
225 230 235 240
Gly His Leu Ser Ser Lys Met Arg Phe Leu Ser Ala Gln Ile Asp Ala
245 250 255
Tyr Leu Thr Asp Asp Leu Trp Leu Arg Asn Ala Arg Lys Ala Asn Ala
260 265 270
Ala Ala Gln Arg Leu Ala Gln Gly Leu Glu Gly Leu Gly Gly Val Glu
275 280 285
Val Leu Gly Gly Thr Glu Ala Asn Ile Leu Phe Cys Arg Leu Asp Ser
290 295 300
Ala Met Ile Asp Ala Leu Leu Lys Ala Gly Phe Gly Phe Tyr His Asp
305 310 315 320
Arg Trp Gly Pro Asn Val Val Arg Phe Val Thr Ser Phe Ala Thr Thr
325 330 335
Ala Glu Asp Val Asp His Leu Leu Asn Gln Val Arg Leu Ala Ala Asp
340 345 350
Arg Thr Gln Glu Arg
355
<210> 9
<211> 1074
<212> DNA
<213>pseudomonad threonine aldolase gene mutation (artificial sequence)
<400> 9
atgaatggtg aaaccagccg tccgccggca ctgggtttta gcagcgataa tattgcaggt 60
gcctctccgg aagtggcaca ggcactggtt aaacattctt caggtcaggc aggtccgtat 120
ggtaccgatg aactgaccgc acaggttaaa cgtaaatttt gtgaaatttt tgaacgtgat 180
gttgaagtgt ttctggttcc gaccggcacc gccgcaaatg cactgtgtct gagcgcaatg 240
accccgccgt ggggtaatat ttattgtcat ccggcaagcc atattaataa tgatgaatgt 300
ggtgcaccgg aattcttttc taatggtgca aaactgatga ccgttgatgg tccggccgca 360
aaactggata ttgttcgtct gcgcgaacgc acccgtgaaa aagttggtga tgttcatacc 420
acacagccgg catgtgttag tattacccag gcgaccgaag ttggtagcat ttataccctg 480
gatgaaattg aagcgattgg tgatgtttgt aaaagcagta gcctgggtct gcatatggat 540
ggtagccgtt ttgcgaatgc actggtgagc ctgggttgca gtccggcaga aatgacctgg 600
aaagcgggcg ttgatgcgct gtcttttggc gcaatcaaaa atggtgttct ggcagcagaa 660
gcaattgttc tgtttaatac cagcctggca accgaaatga gctatcgtcg taaacgcgca 720
ggtcatctga gcagtaaaat gcgttttctg agtgcacaga ttgatgccta tctgaccgat 780
gatctgtggc tgcgtaatgc gcgtaaagcc aatgccgcag cgcagcgtct ggcgcagggt 840
ctggaaggtc tgggtggtgt tgaagtgctg ggtggtaccg aagcaaatat tctgttttgt 900
cgcctggata gcgccatgat tgatgccctg ctgaaagccg gttttggttt ttatcatgat 960
cgttggggtc cgaatgtggt tcgttttgtt accagctttg ccaccaccgc agaagatgtg 1020
gatcatctgc tgaatcaggt tcgtctggca gcagatcgca cccaggaacg ttaa 1074
<210> 10
<211> 357
<212> PRT
<213>pseudomonad threonine aldolase amino acid mutation (artificial sequence)
<400> 10
Met Asn Gly Glu Thr Ser Arg Pro Pro Ala Leu Gly Phe Ser Ser Asp
1 5 10 15
Asn Ile Ala Gly Ala Ser Pro Glu Val Ala Gln Ala Leu Val Lys His
20 25 30
Ser Ser Gly Gln Ala Gly Pro Tyr Gly Thr Asp Glu Leu Thr Ala Gln
35 40 45
Val Lys Arg Lys Phe Cys Glu Ile Phe Glu Arg Asp Val Glu Val Phe
50 55 60
Leu Val Pro Thr Gly Thr Ala Ala Asn Ala Leu Cys Leu Ser Ala Met
65 70 75 80
Thr Pro Pro Trp Gly Asn Ile Tyr Cys His Pro Ala Ser His Ile Asn
85 90 95
Asn Asp Glu Cys Gly Ala Pro Glu Phe Phe Ser Asn Gly Ala Lys Leu
100 105 110
Met Thr Val Asp Gly Pro Ala Ala Lys Leu Asp Ile Val Arg Leu Arg
115 120 125
Glu Arg Thr Arg Glu Lys Val Gly Asp Val His Thr Thr Gln Pro Ala
130 135 140
Cys Val Ser Ile Thr Gln Ala Thr Glu Val Gly Ser Ile Tyr Thr Leu
145 150 155 160
Asp Glu Ile Glu Ala Ile Gly Asp Val Cys Lys Ser Ser Ser Leu Gly
165 170 175
Leu His Met Asp Gly Ser Arg Phe Ala Asn Ala Leu Val Ser Leu Gly
180 185 190
Cys Ser Pro Ala Glu Met Thr Trp Lys Ala Gly Val Asp Ala Leu Ser
195 200 205
Phe Gly Ala Ile Lys Asn Gly Val Leu Ala Ala Glu Ala Ile Val Leu
210 215 220
Phe Asn Thr Ser Leu Ala Thr Glu Met Ser Tyr Arg Arg Lys Arg Ala
225 230 235 240
Gly His Leu Ser Ser Lys Met Arg Phe Leu Ser Ala Gln Ile Asp Ala
245 250 255
Tyr Leu Thr Asp Asp Leu Trp Leu Arg Asn Ala Arg Lys Ala Asn Ala
260 265 270
Ala Ala Gln Arg Leu Ala Gln Gly Leu Glu Gly Leu Gly Gly Val Glu
275 280 285
Val Leu Gly Gly Thr Glu Ala Asn Ile Leu Phe Cys Arg Leu Asp Ser
290 295 300
Ala Met Ile Asp Ala Leu Leu Lys Ala Gly Phe Gly Phe Tyr His Asp
305 310 315 320
Arg Trp Gly Pro Asn Val Val Arg Phe Val Thr Ser Phe Ala Thr Thr
325 330 335
Ala Glu Asp Val Asp His Leu Leu Asn Gln Val Arg Leu Ala Ala Asp
340 345 350
Arg Thr Gln Glu Arg
355
<210> 11
<211> 25
<212> DNA
<213>primer (artificial sequence)
<400> 11
atggatggtt accgttttgc gaatg 25
<210> 12
<211> 26
<212> DNA
<213>primer (artificial sequence)
<400> 12
gcattcgcaa aacggtaacc atccat 26
<210> 13
<211> 27
<212> DNA
<213>primer (artificial sequence)
<400> 13
atttattgtc atccggcaag ccctatt 27
<210> 14
<211> 29
<212> DNA
<213>primer (artificial sequence)
<400> 14
gcaccacatt catcattatt aatagggct 29
<210> 15
<211> 24
<212> DNA
<213>primer (artificial sequence)
<400> 15
agcagcgatc agattgcagg tgcc 24
<210> 16
<211> 23
<212> DNA
<213>primer (artificial sequence)
<400> 16
ggcacctgca atctgatcgc tgc 23
<210> 17
<211> 29
<212> DNA
<213>primer (artificial sequence)
<400> 17
tttggcgcaa tcaaaaatgg tgttctggc 29
<210> 18
<211> 27
<212> DNA
<213>primer (artificial sequence)
<400> 18
acaccatttt tgattgcgcc aaaagac 27
<210> 19
<211> 343
<212> PRT
<213>crescent shank bacterium threonine aldolase amino acid (artificial sequence)
<400> 19
Met Thr Gln Thr Ala Pro Arg Tyr Asp Phe Ala Ser Asp Asn Val Ala
1 5 10 15
Gly Ala Met Pro Glu Val Met Glu Ala Leu Ile Ala Ala Asn Ala Gly
20 25 30
Thr Ala Ser Gly Tyr Gly Thr Asp His Val Ser Arg Ala Ala Ala Asp
35 40 45
Arg Ile Arg Ala Ala Leu Asp Ala Asp Ala Gln Val Arg Phe Thr Ala
50 55 60
Ser Gly Thr Ala Ala Asn Ala Phe Ala Leu Thr Leu Leu Ala Gln Pro
65 70 75 80
His Glu Ala Val Leu Ala His Glu His Ala His Ile Cys Thr Asp Glu
85 90 95
Thr Gly Ala Pro Gly Phe Phe Gly Gln Gly Val Gly Leu Ile Gly Leu
100 105 110
Pro Gly Ala Ser Gly Lys Met Glu Leu Ala Ala Leu Glu Ala Ala Leu
115 120 125
Ala Gln Pro Asp Val Ser Tyr Arg Gln Pro Ala Ala Ala Leu Ser Leu
130 135 140
Thr Thr Ala Thr Glu Tyr Gly Thr Val Tyr Ser Glu Asp His Leu Arg
145 150 155 160
Ala Leu Ile Ala Pro Val Lys Ala Lys Gly Tyr Gly Val His Leu Asp
165 170 175
Gly Ala Arg Leu Ala Asn Ala Val Ala Gly Gly Phe Asp Leu Lys Ser
180 185 190
Ile Ala Lys Met Gly Val Asp Ile Leu Val Met Gly Gly Thr Lys Ala
195 200 205
Gly Ser Thr Pro Thr Glu Ala Val Val Phe Leu Asn Pro Asp His Ala
210 215 220
Lys Arg Leu Asp Ala Arg Leu Lys His Ala Gly Gln Leu Ile Ser Lys
225 230 235 240
Gly Arg Phe Leu Ala Ala Pro Trp Leu Gly Leu Leu Gly Glu Asn Gly
245 250 255
Gln Thr Ala Pro Trp Ala Ala Arg Ala Ala His Ala Asn Ala Met Ala
260 265 270
Gln Lys Leu Ala Ala Leu Met Pro Val Pro Ile Lys His Pro Val Glu
275 280 285
Ala Asn Gly Ile Phe Val Glu Met Asp Glu Leu Ala Leu Glu Arg Leu
290 295 300
Arg Gly Glu Gly Trp Phe Val Tyr Arg Phe Leu Asp Gly Thr Val Arg
305 310 315 320
Phe Met Cys Ser Trp Ala Thr Thr Pro Glu Met Val Glu Asp Leu Gly
325 330 335
Ala Ala Leu Lys Arg Val Ala
340
<210> 20
<211> 343
<212> PRT
<213>crescent shank bacterium threonine aldolase amino acid mutation (artificial sequence)
<400> 20
Met Thr Gln Thr Ala Pro Arg Tyr Asp Phe Ala Ser Asp Asn Val Ala
1 5 10 15
Gly Ala Met Pro Glu Val Met Glu Ala Leu Ile Ala Ala Asn Ala Gly
20 25 30
Thr Ala Ser Gly Tyr Gly Thr Asp His Val Ser Arg Ala Ala Ala Asp
35 40 45
Arg Ile Arg Ala Ala Leu Asp Ala Asp Ala Gln Val Arg Phe Thr Ala
50 55 60
Ser Gly Thr Ala Ala Asn Ala Phe Ala Leu Thr Leu Leu Ala Gln Pro
65 70 75 80
His Glu Ala Val Leu Ala His Glu His Ala His Ile Cys Thr Tyr Thr
85 90 95
Thr Gly Ala Pro Gly Phe Phe Gly Gln Gly Val Gly Leu Ile Gly Leu
100 105 110
Pro Gly Ala Ser Gly Lys Met Glu Leu Ala Ala Leu Glu Ala Ala Leu
115 120 125
Ala Gln Pro Asp Val Ser Tyr Arg Gln Pro Ala Ala Ala Leu Ser Leu
130 135 140
Thr Thr Ala Thr Glu Tyr Gly Thr Val Tyr Ser Glu Asp His Leu Arg
145 150 155 160
Ala Leu Ile Ala Pro Val Lys Ala Lys Gly Tyr Gly Val His Leu Asp
165 170 175
Gly Ala Arg Leu Ala Asn Ala Val Ala Gly Gly Phe Asp Leu Lys Ser
180 185 190
Ile Ala Lys Met Gly Val Asp Ile Leu Val Met Gly Gly Thr Lys Ala
195 200 205
Gly Ser Thr Pro Thr Glu Ala Val Val Phe Leu Asn Pro Asp His Ala
210 215 220
Lys Arg Leu Asp Ala Arg Leu Lys His Ala Gly Gln Leu Ile Ser Lys
225 230 235 240
Gly Arg Phe Leu Ala Ala Pro Trp Leu Gly Leu Leu Gly Glu Asn Gly
245 250 255
Gln Thr Ala Pro Trp Ala Ala Arg Ala Ala His Ala Asn Ala Met Ala
260 265 270
Gln Lys Leu Ala Ala Leu Met Pro Val Pro Ile Lys His Pro Val Glu
275 280 285
Ala Asn Gly Ile Phe Val Glu Met Asp Glu Leu Ala Leu Glu Arg Leu
290 295 300
Arg Gly Glu Gly Trp Phe Val Tyr Arg Phe Leu Asp Gly Thr Val Arg
305 310 315 320
Phe Met Cys Ser Trp Ala Thr Thr Pro Glu Met Val Glu Asp Leu Gly
325 330 335
Ala Ala Leu Lys Arg Val Ala
340
<210> 21
<211> 343
<212> PRT
<213>crescent shank bacterium threonine aldolase amino acid mutation (artificial sequence)
<400> 21
Met Thr Gln Thr Ala Pro Arg Tyr Asp Phe Ala Ser Asp Asn Val Ala
1 5 10 15
Gly Ala Met Pro Glu Val Met Glu Ala Leu Ile Ala Ala Asn Ala Gly
20 25 30
Thr Ala Ser Gly Tyr Gly Thr Asp His Val Ser Arg Ala Ala Ala Asp
35 40 45
Arg Ile Arg Ala Ala Leu Asp Ala Asp Ala Gln Val Arg Phe Thr Ala
50 55 60
Ser Gly Thr Ala Ala Asn Ala Phe Ala Leu Thr Leu Leu Ala Gln Pro
65 70 75 80
His Glu Ala Val Leu Ala His Glu His Ala His Ile Cys Thr Leu Glu
85 90 95
Thr Gly Ala Pro Gly Phe Phe Gly Gln Gly Val Gly Leu Ile Gly Leu
100 105 110
Pro Gly Ala Ser Gly Lys Met Glu Leu Ala Ala Leu Glu Ala Ala Leu
115 120 125
Ala Gln Pro Asp Val Ser Tyr Arg Gln Pro Ala Ala Ala Leu Ser Leu
130 135 140
Thr Thr Ala Thr Glu Tyr Gly Thr Val Tyr Ser Glu Asp His Leu Arg
145 150 155 160
Ala Leu Ile Ala Pro Val Lys Ala Lys Gly Tyr Gly Val His Leu Asp
165 170 175
Gly Ala Arg Leu Ala Asn Ala Val Ala Gly Gly Phe Asp Leu Lys Ser
180 185 190
Ile Ala Lys Met Gly Val Asp Ile Leu Val Met Gly Gly Thr Lys Ala
195 200 205
Gly Ser Thr Pro Thr Glu Ala Val Val Phe Leu Asn Pro Asp His Ala
210 215 220
Lys Arg Leu Asp Ala Arg Leu Lys His Ala Gly Gln Leu Ile Ser Lys
225 230 235 240
Gly Arg Phe Leu Ala Ala Pro Trp Leu Gly Leu Leu Gly Glu Asn Gly
245 250 255
Gln Thr Ala Pro Trp Ala Ala Arg Ala Ala His Ala Asn Ala Met Ala
260 265 270
Gln Lys Leu Ala Ala Leu Met Pro Val Pro Ile Lys His Pro Val Glu
275 280 285
Ala Asn Gly Ile Phe Val Glu Met Asp Glu Leu Ala Leu Glu Arg Leu
290 295 300
Arg Gly Glu Gly Trp Phe Val Tyr Arg Phe Leu Asp Gly Thr Val Arg
305 310 315 320
Phe Met Cys Ser Trp Ala Thr Thr Pro Glu Met Val Glu Asp Leu Gly
325 330 335
Ala Ala Leu Lys Arg Val Ala
340
<210> 22
<211> 343
<212> PRT
<213>crescent shank bacterium threonine aldolase amino acid mutation (artificial sequence)
<400> 22
Met Thr Gln Thr Ala Pro Arg Tyr Asp Phe Ala Ser Asp Asn Val Ala
1 5 10 15
Gly Ala Met Pro Glu Val Met Glu Ala Leu Ile Ala Ala Asn Ala Gly
20 25 30
Thr Ala Ser Gly Tyr Gly Thr Asp His Val Ser Arg Ala Ala Ala Asp
35 40 45
Arg Ile Arg Ala Ala Leu Asp Ala Asp Ala Gln Val Arg Phe Thr Ala
50 55 60
Ser Gly Thr Ala Ala Asn Ala Phe Ala Leu Thr Leu Leu Ala Gln Pro
65 70 75 80
His Glu Ala Val Leu Ala His Glu His Ala His Ile Cys Thr Asp Glu
85 90 95
Thr Gly Ala Pro Gly Phe Phe Gly Gln Gly Val Gly Leu Ile Gly Leu
100 105 110
Pro Gly Ala Ser Gly Lys Met Glu Leu Ala Ala Leu Glu Ala Ala Leu
115 120 125
Ala Gln Pro Asp Val Ser Tyr Arg Gln Pro Ala Ala Ala Leu Ser Leu
130 135 140
Thr Thr Ala Thr Glu Tyr Gly Thr Val Tyr Ser Glu Asp His Leu Arg
145 150 155 160
Ala Leu Ile Ala Pro Val Lys Ala Lys Gly Tyr Gly Val His Leu Glu
165 170 175
Gly Ala Arg Leu Ala Asn Ala Val Ala Gly Gly Phe Asp Leu Lys Ser
180 185 190
Ile Ala Lys Met Gly Val Asp Ile Leu Val Met Gly Gly Thr Lys Ala
195 200 205
Gly Ser Thr Pro Thr Glu Ala Val Val Phe Leu Asn Pro Asp His Ala
210 215 220
Lys Arg Leu Asp Ala Arg Leu Lys His Ala Gly Gln Leu Ile Ser Lys
225 230 235 240
Gly Arg Phe Leu Ala Ala Pro Trp Leu Gly Leu Leu Gly Glu Asn Gly
245 250 255
Gln Thr Ala Pro Trp Ala Ala Arg Ala Ala His Ala Asn Ala Met Ala
260 265 270
Gln Lys Leu Ala Ala Leu Met Pro Val Pro Ile Lys His Pro Val Glu
275 280 285
Ala Asn Gly Ile Phe Val Glu Met Asp Glu Leu Ala Leu Glu Arg Leu
290 295 300
Arg Gly Glu Gly Trp Phe Val Tyr Arg Phe Leu Asp Gly Thr Val Arg
305 310 315 320
Phe Met Cys Ser Trp Ala Thr Thr Pro Glu Met Val Glu Asp Leu Gly
325 330 335
Ala Ala Leu Lys Arg Val Ala
340
<210> 23
<211> 343
<212> PRT
<213>Thermotoga maritima threonine aldolase amino acid (artificial sequence)
<400> 23
Met Ile Asp Leu Arg Ser Asp Thr Val Thr Lys Pro Thr Glu Glu Met
1 5 10 15
Arg Lys Ala Met Ala Gln Ala Glu Val Gly Asp Asp Val Tyr Gly Glu
20 25 30
Asp Pro Thr Ile Asn Glu Leu Glu Arg Leu Ala Ala Glu Thr Phe Gly
35 40 45
Lys Glu Ala Ala Leu Phe Val Pro Ser Gly Thr Met Gly Asn Gln Val
50 55 60
Ser Ile Met Ala His Thr Gln Arg Gly Asp Glu Val Ile Leu Glu Ala
65 70 75 80
Asp Ser His Ile Phe Trp Tyr Glu Val Gly Ala Met Ala Val Leu Ser
85 90 95
Gly Val Met Pro His Pro Val Pro Gly Lys Asn Gly Ala Met Asp Pro
100 105 110
Asp Asp Val Arg Lys Ala Ile Arg Pro Arg Asn Ile His Phe Pro Arg
115 120 125
Thr Ser Leu Ile Ala Ile Glu Asn Thr His Asn Arg Ser Gly Gly Arg
130 135 140
Val Val Pro Leu Glu Asn Ile Lys Glu Ile Cys Thr Ile Ala Lys Glu
145 150 155 160
His Gly Ile Asn Val His Ile Asp Gly Ala Arg Ile Phe Asn Ala Ser
165 170 175
Ile Ala Ser Gly Val Pro Val Lys Glu Tyr Ala Gly Tyr Ala Asp Ser
180 185 190
Val Met Phe Cys Leu Ser Lys Gly Leu Cys Ala Pro Val Gly Ser Val
195 200 205
Val Val Gly Asp Arg Asp Phe Ile Glu Arg Ala Arg Lys Ala Arg Lys
210 215 220
Met Leu Gly Gly Gly Met Arg Gln Ala Gly Val Leu Ala Ala Ala Gly
225 230 235 240
Ile Ile Ala Leu Thr Lys Met Val Asp Arg Leu Lys Glu Asp His Glu
245 250 255
Asn Ala Arg Phe Leu Ala Leu Lys Leu Lys Glu Ile Gly Tyr Ser Val
260 265 270
Asn Pro Glu Asp Val Lys Thr Asn Met Val Ile Leu Arg Thr Asp Asn
275 280 285
Leu Lys Val Asn Ala His Gly Phe Ile Glu Ala Leu Arg Asn Ser Gly
290 295 300
Val Leu Ala Asn Ala Val Ser Asp Thr Glu Ile Arg Leu Val Thr His
305 310 315 320
Lys Asp Val Ser Arg Asn Asp Ile Glu Glu Ala Leu Asn Ile Phe Glu
325 330 335
Lys Leu Phe Arg Lys Phe Ser
340
<210> 24
<211> 358
<212> PRT
<213>Listeria monocytogenes threonine aldolase amino acid (artificial sequence)
<400> 24
Met Thr Asn Thr Leu Lys Thr Ser Tyr Gln Lys Thr Pro Tyr Lys Leu
1 5 10 15
Gly Gly Asn Gly Pro Arg Asn Val Gly Val Leu Thr Glu Ala Leu Gln
20 25 30
Asn Ile Asp Asp Asn Leu Glu Ser Asp Ile Tyr Gly Asn Gly Ala Val
35 40 45
Ile Glu Asp Phe Glu Thr Lys Ile Ala Lys Ile Leu Gly Lys Gln Ser
50 55 60
Ala Val Phe Phe Pro Ser Gly Thr Met Ala Gln Gln Ile Ala Leu Arg
65 70 75 80
Ile Trp Ala Asp Arg Lys Glu Asn Arg Arg Val Ala Tyr His Pro Leu
85 90 95
Ser His Leu Glu Ile His Glu Gln Asp Gly Leu Lys Glu Leu Gln Gln
100 105 110
Ile Thr Pro Leu Leu Leu Gly Thr Ala Asn Gln Leu Leu Thr Ile Asp
115 120 125
Asp Ile Lys Ser Leu Arg Glu Pro Val Ser Ser Val Leu Ile Glu Leu
130 135 140
Pro Gln Arg Glu Ile Gly Gly Gln Leu Pro Ala Phe Glu Glu Leu Glu
145 150 155 160
Lys Ile Ser Glu Tyr Cys His Glu Gln Gly Ile Ser Leu His Leu Asp
165 170 175
Gly Ala Arg Leu Trp Glu Ile Thr Pro Phe Tyr Gln Lys Ser Ala Glu
180 185 190
Glu Ile Cys Ala Leu Phe Asp Ser Val Tyr Val Ser Phe Tyr Lys Gly
195 200 205
Ile Gly Gly Ile Ala Gly Ala Ile Leu Ala Gly Asn Asp Asp Phe Val
210 215 220
Gln Glu Ala Lys Ile Trp Lys Arg Arg Tyr Gly Gly Asp Leu Ile Ser
225 230 235 240
Leu Tyr Pro Tyr Ile Leu Ser Ala Asp Tyr Tyr Phe Glu Lys Arg Ile
245 250 255
Gly Lys Met Ala Glu Tyr Phe Glu Ala Ala Lys Gly Leu Ala Glu Arg
260 265 270
Phe Asn Ser Cys Ser Gly Val Lys Thr Val Pro Glu Val Pro Val Ser
275 280 285
Asn Met Phe His Val Tyr Phe Glu Asn Ser Ala Asp Glu Ile Gly Ala
290 295 300
Ile Leu Thr Lys Ile Gln Asp Glu Thr Gly Val Gly Ile Ser Gly Tyr
305 310 315 320
Leu Gln Glu Lys Ser Ala Asp Val Cys Ala Phe Glu Val Ser Val Gly
325 330 335
Asp Ala Phe Ala Glu Ile Pro Ala Lys Asn Leu Glu Leu Val Phe Arg
340 345 350
Cys Leu Glu Lys Glu Leu
355
<210> 25
<211> 333
<212> PRT
<213>E. coli threonine aldolase amino acid (artificial sequence)
<400> 25
Met Ile Asp Leu Arg Ser Asp Thr Val Thr Arg Pro Ser Arg Ala Met
1 5 10 15
Leu Glu Ala Met Met Ala Ala Pro Val Gly Asp Asp Val Tyr Gly Asp
20 25 30
Asp Pro Thr Val Asn Ala Leu Gln Asp Tyr Ala Ala Glu Leu Ser Gly
35 40 45
Lys Glu Ala Ala Ile Phe Leu Pro Thr Gly Thr Gln Ala Asn Leu Val
50 55 60
Ala Leu Leu Ser His Cys Glu Arg Gly Glu Glu Tyr Ile Val Gly Gln
65 70 75 80
Ala Ala His Asn Tyr Leu Phe Glu Ala Gly Gly Ala Ala Val Leu Gly
85 90 95
Ser Ile Gln Pro Gln Pro Ile Asp Ala Ala Ala Asp Gly Thr Leu Pro
100 105 110
Leu Asp Lys Val Ala Met Lys Ile Lys Pro Asp Asp Ile His Phe Ala
115 120 125
Arg Thr Lys Leu Leu Ser Leu Glu Asn Thr His Asn Gly Lys Val Leu
130 135 140
Pro Arg Glu Tyr Leu Lys Glu Ala Trp Glu Phe Thr Arg Lys Arg Asn
145 150 155 160
Leu Ala Leu His Val Asp Gly Ala Arg Ile Phe Asn Ala Val Val Ala
165 170 175
Tyr Gly Cys Glu Leu Lys Glu Ile Thr Gln Tyr Cys Asp Ser Phe Thr
180 185 190
Ile Cys Leu Ser Lys Gly Leu Gly Thr Pro Val Gly Ser Leu Leu Val
195 200 205
Gly Asn Arg Asp Tyr Ile Lys Arg Ala Ile Arg Trp Arg Lys Met Thr
210 215 220
Gly Gly Gly Met Arg Gln Ser Gly Ile Leu Ala Ala Ala Gly Met Tyr
225 230 235 240
Ala Leu Lys Asn Asn Val Ala Arg Leu Gln Glu Asp His Asp Asn Thr
245 250 255
Ala Trp Met Ala Glu Gln Leu Arg Glu Ala Gly Ala Asp Val Met Arg
260 265 270
Gln Asp Thr Asn Met Leu Phe Val Arg Val Gly Glu Glu Asn Ala Ala
275 280 285
Ala Leu Gly Glu Tyr Met Lys Ala Arg Asn Val Leu Ile Asn Ala Ser
290 295 300
Pro Ile Val Arg Leu Val Thr His Leu Asp Val Ser Arg Ala Gln Leu
305 310 315 320
Ala Glu Val Ala Ala His Trp Arg Ala Phe Leu Ala Arg
325 330
<210> 26
<211> 338
<212> PRT
<213>aermonas jandaei threonine aldolase amino acid (artificial sequence)
<400> 26
Met Arg Tyr Ile Asp Leu Arg Ser Asp Thr Val Thr Gln Pro Thr Asp
1 5 10 15
Ala Met Arg Gln Cys Met Leu His Ala Glu Val Gly Asp Asp Val Tyr
20 25 30
Gly Glu Asp Pro Gly Val Asn Ala Leu Glu Ala Tyr Gly Ala Asp Leu
35 40 45
Leu Gly Lys Glu Ala Ala Leu Phe Val Pro Ser Gly Thr Met Ser Asn
50 55 60
Leu Leu Ala Val Met Ser His Cys Gln Arg Gly Glu Gly Ala Val Leu
65 70 75 80
Gly Ser Ala Ala His Ile Tyr Arg Tyr Glu Ala Gln Gly Ser Ala Val
85 90 95
Leu Gly Ser Val Ala Leu Gln Pro Val Pro Met Gln Ala Asp Gly Ser
100 105 110
Leu Ala Leu Ala Asp Val Arg Ala Ala Ile Ala Pro Asp Asp Val His
115 120 125
Phe Thr Pro Thr Arg Leu Val Cys Leu Glu Asn Thr His Asn Gly Lys
130 135 140
Val Leu Pro Leu Pro Tyr Leu Arg Glu Met Arg Glu Leu Val Asp Glu
145 150 155 160
His Gly Leu Gln Leu His Leu Asp Gly Ala Arg Leu Phe Asn Ala Val
165 170 175
Val Ala Ser Gly His Thr Val Arg Glu Leu Val Ala Pro Phe Asp Ser
180 185 190
Val Ser Ile Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu
195 200 205
Leu Val Gly Ser His Ala Phe Ile Ala Arg Ala Arg Arg Leu Arg Lys
210 215 220
Met Val Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Gln Ala Gly
225 230 235 240
Leu Phe Ala Leu Gln Gln His Val Val Arg Leu Ala Asp Asp His Arg
245 250 255
Arg Ala Arg Gln Leu Ala Glu Gly Leu Ala Ala Leu Pro Gly Ile Arg
260 265 270
Leu Asp Leu Ala Gln Val Gln Thr Asn Met Val Phe Leu Gln Leu Thr
275 280 285
Ser Gly Glu Ser Ala Pro Leu Leu Ala Phe Met Lys Ala Arg Gly Ile
290 295 300
Leu Phe Ser Gly Tyr Gly Glu Leu Arg Leu Val Thr His Leu Gln Ile
305 310 315 320
His Asp Asp Asp Ile Glu Glu Val Ile Asp Ala Phe Thr Glu Tyr Leu
325 330 335
Gly Ala
<210> 27
<211> 387
<212> PRT
<213>saccharomyces cerevisiae threonine aldolase amino acid (artificial sequence)
<400> 27
Met Thr Glu Phe Glu Leu Pro Pro Lys Tyr Ile Thr Ala Ala Asn Asp
1 5 10 15
Leu Arg Ser Asp Thr Phe Thr Thr Pro Thr Ala Glu Met Met Glu Ala
20 25 30
Ala Leu Glu Ala Ser Ile Gly Asp Ala Val Tyr Gly Glu Asp Val Asp
35 40 45
Thr Val Arg Leu Glu Gln Thr Val Ala Arg Met Ala Gly Lys Glu Ala
50 55 60
Gly Leu Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Ile Arg
65 70 75 80
Thr His Leu Met Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg Ala
85 90 95
His Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln Ala
100 105 110
Met Val Val Pro Val Val Pro Ser Asn Gly Asp Tyr Leu Thr Leu Glu
115 120 125
Asp Ile Lys Ser His Tyr Val Pro Asp Asp Gly Asp Ile His Gly Ala
130 135 140
Pro Thr Arg Leu Ile Ser Leu Glu Asn Thr Leu His Gly Ile Val Tyr
145 150 155 160
Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn Gly
165 170 175
Leu Lys Leu His Cys Asp Gly Ala Arg Ile Trp Asn Ala Ala Ala Gln
180 185 190
Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Ile Phe Asp Ser Ile Ser
195 200 205
Ile Cys Leu Ser Lys Ser Met Gly Ala Pro Ile Gly Ser Val Leu Val
210 215 220
Gly Asn Leu Lys Phe Val Lys Lys Ala Thr His Phe Arg Lys Gln Gln
225 230 235 240
Gly Gly Gly Ile Arg Gln Ser Gly Met Met Ala Arg Met Ala Leu Val
245 250 255
Asn Ile Asn Asn Asp Trp Lys Ser Gln Leu Leu Tyr Ser His Ser Leu
260 265 270
Ala His Glu Leu Ala Glu Tyr Cys Glu Ala Lys Gly Ile Pro Leu Glu
275 280 285
Ser Pro Ala Asp Thr Asn Phe Val Phe Ile Asn Leu Lys Ala Ala Arg
290 295 300
Met Asp Pro Asp Val Leu Val Lys Lys Gly Leu Lys Tyr Asn Val Lys
305 310 315 320
Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Val Thr Arg Asp Thr
325 330 335
Leu Glu Lys Val Lys Leu Ala Ile Ser Glu Ala Phe Asp Tyr Ala Lys
340 345 350
Glu His Pro Phe Asp Cys Asn Gly Pro Thr Gln Ile Tyr Arg Ser Glu
355 360 365
Ser Thr Glu Val Asp Val Asp Gly Asn Ala Ile Arg Glu Ile Lys Thr
370 375 380
Tyr Lys Tyr
385
<210> 28
<211> 382
<212> PRT
<213>ashbya gossypii bacterium threonine aldolase amino acid (artificial sequence)
<400> 28
Met Asn Gln Asp Met Glu Leu Pro Glu Ala Tyr Thr Ser Ala Ser Asn
1 5 10 15
Asp Phe Arg Ser Asp Thr Phe Thr Thr Pro Thr Arg Glu Met Ile Glu
20 25 30
Ala Ala Leu Thr Ala Thr Ile Gly Asp Ala Val Tyr Gln Glu Asp Ile
35 40 45
Asp Thr Leu Lys Leu Glu Gln His Val Ala Lys Leu Ala Gly Met Glu
50 55 60
Ala Gly Met Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Leu
65 70 75 80
Arg Thr His Leu Thr Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg
85 90 95
Ala His Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln
100 105 110
Ala Met Val Thr Pro Val Ile Pro Ser Asn Gly Asn Tyr Leu Thr Leu
115 120 125
Glu Asp Ile Lys Lys His Tyr Ile Pro Asp Asp Gly Asp Ile His Gly
130 135 140
Ala Pro Thr Lys Val Ile Ser Leu Glu Asn Thr Leu His Gly Ile Ile
145 150 155 160
His Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn
165 170 175
Asp Leu Arg Leu His Cys Asp Gly Ala Arg Ile Trp Asn Ala Ser Ala
180 185 190
Glu Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Leu Phe Asp Ser Ile
195 200 205
Ser Ile Cys Leu Ser Lys Ser Met Gly Ala Pro Met Gly Ser Ile Leu
210 215 220
Val Gly Ser His Lys Phe Ile Lys Lys Ala Asn His Phe Arg Lys Gln
225 230 235 240
Gln Gly Gly Gly Val Arg Gln Ser Gly Met Met Cys Lys Met Ala Met
245 250 255
Val Ala Ile Gln Gly Asp Trp Lys Gly Lys Met Arg Arg Ser His Arg
260 265 270
Met Ala His Glu Leu Ala Arg Phe Cys Ala Glu His Gly Ile Pro Leu
275 280 285
Glu Ser Pro Ala Asp Thr Asn Phe Val Phe Leu Asp Leu Gln Lys Ser
290 295 300
Lys Met Asn Pro Asp Val Leu Val Lys Lys Ser Leu Lys Tyr Gly Cys
305 310 315 320
Lys Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Ile Ser Glu Glu
325 330 335
Ser Leu Glu Lys Ile Lys Gln Ala Ile Leu Glu Ala Phe Glu Tyr Ser
340 345 350
Lys Lys Asn Pro Tyr Asp Glu Asn Gly Pro Thr Lys Ile Tyr Arg Ser
355 360 365
Glu Ser Ala Asp Ala Val Gly Glu Ile Lys Thr Tyr Lys Tyr
370 375 380
<210> 29
<211> 335
<212> PRT
<213>Bao Shewanella threonine aldolase amino acid (artificial sequence)
<400> 29
Met Ile Asp Phe Arg Ser Asp Thr Val Thr Gln Pro Thr Ala Ala Met
1 5 10 15
Arg Arg Ala Met Ala Asp Ala Gln Val Gly Asp Asp Val Tyr Gly Asp
20 25 30
Asp Pro Ser Val Asn Arg Leu Glu Ala Met Ala Ala Glu Arg Phe Gly
35 40 45
Phe Asp Ser Ala Leu Phe Thr Ser Ser Gly Thr Gln Ala Asn Leu Leu
50 55 60
Ala Leu Met Ser His Cys Asp Arg Gly Asp Glu Tyr Leu Cys Gly Gln
65 70 75 80
Gln Ala His Asn Tyr Lys Phe Glu Gly Gly Gly Ala Ala Val Leu Gly
85 90 95
Ser Ile Gln Pro Gln Pro Leu Thr Asn Gln Pro Asp Gly Ser Ile Leu
100 105 110
Leu Ser Asp Ile Glu Ala Ala Ile Lys Pro Asp Asp Phe His Phe Ala
115 120 125
Arg Thr Arg Leu Leu Ser Leu Glu Asn Thr Ile Gly Gly Lys Val Leu
130 135 140
Pro Gln Ser Tyr Leu Ala Glu Ala Gln Ala Leu Ala Phe Asn Lys Arg
145 150 155 160
Leu Lys Ile His Leu Asp Gly Ala Arg Ile Ala Asn Ala Ala Val Ala
165 170 175
His Asn Leu Asp Ile Ala Asp Ile Thr Gln Tyr Phe Asp Ser Val Ser
180 185 190
Ile Cys Leu Ser Lys Gly Leu Cys Ala Pro Val Gly Ser Leu Leu Leu
195 200 205
Gly Asp Glu Arg Leu Ile Asn Lys Ala Arg Arg Trp Arg Lys Met Leu
210 215 220
Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Val Ala Gly Glu Ile
225 230 235 240
Ala Leu Asn Glu Gln Val Ser Arg Leu Ala Val Asp His Glu Asn Ala
245 250 255
Arg Tyr Leu Ala Glu Gln Leu Ser Cys Leu Asp Glu Phe Glu Val Asp
260 265 270
Leu Gly Glu Val Gln Thr Asn Met Leu Phe Ala Arg Val Val Glu Gly
275 280 285
Val Ala Ile Asp Lys Leu Ala Thr Ser Leu Lys Ala Ser Gly Ile Leu
290 295 300
Ile Ser Pro Gly Lys Thr Leu Arg Met Val Thr His Ala Asp Ile Arg
305 310 315 320
Leu Glu Asp Ile Asp Leu Phe Ile Asp Lys Leu Lys Ser Leu Leu
325 330 335
<210> 30
<211> 339
<212> PRT
<213>imperial Aeromonas threonine aldolase amino acid (artificial sequence) is tieed up
<400> 30
Met Arg Tyr Ile Asp Leu Arg Ser Asp Thr Val Thr Gln Pro Thr Asp
1 5 10 15
Ala Met Arg Gln Ala Met Leu His Ala Glu Val Gly Asp Asp Val Tyr
20 25 30
Gly Glu Asp Pro Gly Val Asn Ala Leu Glu Ala His Gly Ala Arg Leu
35 40 45
Leu Gly Lys Gln Ala Ala Leu Phe Val Pro Ser Gly Thr Met Ser Asn
50 55 60
Leu Leu Ala Val Met Ser His Cys Gln Arg Gly Glu Gly Ala Ile Leu
65 70 75 80
Gly Asn Ala Ala His Ile Tyr Arg Tyr Glu Ala Gln Gly Ser Ala Val
85 90 95
Leu Gly Ser Val Ala Leu Gln Pro Leu Pro Met Gln Arg Asp Gly Thr
100 105 110
Leu Ala Phe Asp Asp Ile Lys Ala Ala Leu Ala Pro Asp Asp Ala His
115 120 125
Phe Val Gln Thr Arg Leu Ile Cys Leu Glu Asn Thr His Asn Gly Lys
130 135 140
Val Leu Pro Leu Ser Tyr Leu Gln Glu Met Gly Thr Phe Val Ala Glu
145 150 155 160
Arg Gly Leu Lys Leu His Leu Asp Gly Ala Arg Leu Phe Asn Ala Ala
165 170 175
Val Ala Ser Glu Thr Pro Val Ala Val Ile Ala Ala Pro Phe Asp Ser
180 185 190
Ile Ser Ile Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu
195 200 205
Leu Val Gly Ser His Asp Phe Ile Ala Arg Ala Arg Arg Leu Arg Lys
210 215 220
Met Leu Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Gln Ala Gly
225 230 235 240
Leu Phe Ala Leu Glu Gln His Val Thr Arg Leu Ala Asp Asp His Arg
245 250 255
Arg Ala Lys Arg Leu Ala Glu Gly Leu Ala Ala Leu Pro Gly Ile Glu
260 265 270
Leu Asp Leu Ser Leu Val Gln Ser Asn Met Val Phe Leu Arg Leu Arg
275 280 285
Glu Gly Glu Ser Ala Pro Leu Leu Ala Phe Met Lys Glu Arg Gly Ile
290 295 300
Leu Phe Ser Gly Tyr Gly Glu Leu Arg Leu Val Thr His Leu Gln Ile
305 310 315 320
Asn Asp Asp Asp Ile Glu Glu Val Ile Asp Ala Phe Thr Glu Tyr Leu
325 330 335
Gly Ala Arg
<210> 31
<211> 348
<212> PRT
<213>sulphur reduction ground bacillus threonine aldolase amino acid (artificial sequence)
<400> 31
Met Asn Ile Ile Asp Leu Arg Ser Asp Thr Val Thr Arg Pro Ser Pro
1 5 10 15
Ala Met Arg Ala Ala Met Ala Gly Ala Glu Val Gly Asp Asp Val Tyr
20 25 30
Gly Glu Asp Pro Thr Val Asn Arg Leu Glu Glu Leu Gly Ala Ala Thr
35 40 45
Leu Gly Thr Glu Ala Ala Leu Phe Thr Ala Ser Gly Thr Gln Ala Asn
50 55 60
Leu Leu Ala Leu Leu Ala His Cys Arg Arg Gly Asp Glu Tyr Ile Ala
65 70 75 80
Gly Gln Thr Ala His Cys Tyr Arg Tyr Glu Gly Gly Gly Ala Ala Ala
85 90 95
Leu Gly Gly Ile Gln Pro Gln Pro Leu Glu Val Glu Pro Asp Gly Thr
100 105 110
Leu Asp Leu Ser Ala Val Ala Ala Ala Ile Lys Pro Asp Asp Leu His
115 120 125
Phe Ala Arg Thr Arg Leu Leu Cys Leu Glu Asn Thr His Ala Gly Arg
130 135 140
Val Leu Pro Leu Asp Tyr Leu Ala Arg Ala Arg Arg Leu Cys Asn Glu
145 150 155 160
Gln Ser Leu Gly Leu His Met Asp Gly Ala Arg Ile Phe Asn Ala Ala
165 170 175
Val Lys Leu Gly Val Pro Val Arg Glu Ile Ala Gly His Val Asp Ser
180 185 190
Val Ser Val Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu
195 200 205
Leu Cys Gly Gly Arg Glu Phe Val Ala Thr Ala Arg Arg Trp Arg Lys
210 215 220
Ala Val Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Ala Ala Gly
225 230 235 240
Ile Leu Ala Leu Thr Glu Asn Val Asp Arg Leu Ala Glu Asp His Ala
245 250 255
Asn Ala Arg Arg Leu Ala Glu Gly Leu Ala Ala Ile Pro Gly Leu Gly
260 265 270
Leu Asp Pro Ser Gln Val Gln Thr Asn Met Val Phe Leu Cys Leu Pro
275 280 285
Leu Arg Thr Ala Asp Arg Leu Ala Ser Phe Leu Asn Glu Gln Gly Ile
290 295 300
Leu Ile Ser Gly Arg Glu Ser Ile Arg Leu Val Thr His Leu Asp Val
305 310 315 320
Thr Ser Ser Asp Val Glu Arg Val Ile Ala Ala Phe Gly Ala Phe Phe
325 330 335
Ala Gly His Gly Glu Met Gln Pro Gly Ala Ser Ser
340 345
<210> 32
<211> 343
<212> PRT
<213>crescent shank bacterium threonine aldolase amino acid mutation (artificial sequence)
<400> 32
Met Thr Gln Thr Ala Pro Arg Tyr Asp Phe Ala Ser Asp Thr Val Ala
1 5 10 15
Gly Ala Met Pro Glu Val Met Glu Ala Leu Ile Ala Ala Asn Ala Gly
20 25 30
Thr Ala Ser Gly Tyr Gly Thr Asp His Val Ser Arg Ala Ala Ala Asp
35 40 45
Arg Ile Arg Ala Ala Leu Asp Ala Asp Ala Gln Val Arg Phe Thr Ala
50 55 60
Ser Gly Thr Ala Ala Asn Ala Phe Ala Leu Thr Leu Leu Ala Gln Pro
65 70 75 80
His Glu Ala Val Leu Ala His Glu His Ala His Ile Cys Thr Asp Glu
85 90 95
Thr Gly Ala Pro Gly Phe Phe Gly Gln Gly Val Gly Leu Ile Gly Leu
100 105 110
Pro Gly Ala Ser Gly Lys Met Glu Leu Ala Ala Leu Glu Ala Ala Leu
115 120 125
Ala Gln Pro Asp Val Ser Tyr Arg Gln Pro Ala Ala Ala Leu Ser Leu
130 135 140
Thr Thr Ala Thr Glu Tyr Gly Thr Val Tyr Ser Glu Asp His Leu Arg
145 150 155 160
Ala Leu Ile Ala Pro Val Lys Ala Lys Gly Tyr Gly Val His Leu Asp
165 170 175
Gly Ala Arg Leu Ala Asn Ala Val Ala Gly Gly Phe Asp Leu Lys Ser
180 185 190
Ile Ala Lys Met Gly Val Asp Ile Leu Val Met Gly Gly Thr Lys Ala
195 200 205
Gly Ser Thr Pro Thr Glu Ala Val Val Phe Leu Asn Pro Asp His Ala
210 215 220
Lys Arg Leu Asp Ala Arg Leu Lys His Ala Gly Gln Leu Ile Ser Lys
225 230 235 240
Gly Arg Phe Leu Ala Ala Pro Trp Leu Gly Leu Leu Gly Glu Asn Gly
245 250 255
Gln Thr Ala Pro Trp Ala Ala Arg Ala Ala His Ala Asn Ala Met Ala
260 265 270
Gln Lys Leu Ala Ala Leu Met Pro Val Pro Ile Lys His Pro Val Glu
275 280 285
Ala Asn Gly Ile Phe Val Glu Met Asp Glu Leu Ala Leu Glu Arg Leu
290 295 300
Arg Gly Glu Gly Trp Phe Val Tyr Arg Phe Leu Asp Gly Thr Val Arg
305 310 315 320
Phe Met Cys Ser Trp Ala Thr Thr Pro Glu Met Val Glu Asp Leu Gly
325 330 335
Ala Ala Leu Lys Arg Val Ala
340
<210> 33
<211> 343
<212> PRT
<213>crescent shank bacterium threonine aldolase amino acid mutation (artificial sequence)
<400> 33
Met Thr Gln Thr Ala Pro Arg Tyr Asp Phe Ala Ser Asp Gln Val Ala
1 5 10 15
Gly Ala Met Pro Glu Val Met Glu Ala Leu Ile Ala Ala Asn Ala Gly
20 25 30
Thr Ala Ser Gly Tyr Gly Thr Asp His Val Ser Arg Ala Ala Ala Asp
35 40 45
Arg Ile Arg Ala Ala Leu Asp Ala Asp Ala Gln Val Arg Phe Thr Ala
50 55 60
Ser Gly Thr Ala Ala Asn Ala Phe Ala Leu Thr Leu Leu Ala Gln Pro
65 70 75 80
His Glu Ala Val Leu Ala His Glu His Ala His Ile Cys Thr Asp Glu
85 90 95
Thr Gly Ala Pro Gly Phe Phe Gly Gln Gly Val Gly Leu Ile Gly Leu
100 105 110
Pro Gly Ala Ser Gly Lys Met Glu Leu Ala Ala Leu Glu Ala Ala Leu
115 120 125
Ala Gln Pro Asp Val Ser Tyr Arg Gln Pro Ala Ala Ala Leu Ser Leu
130 135 140
Thr Thr Ala Thr Glu Tyr Gly Thr Val Tyr Ser Glu Asp His Leu Arg
145 150 155 160
Ala Leu Ile Ala Pro Val Lys Ala Lys Gly Tyr Gly Val His Leu Asp
165 170 175
Gly Ala Arg Leu Ala Asn Ala Val Ala Gly Gly Phe Asp Leu Lys Ser
180 185 190
Ile Ala Lys Met Gly Val Asp Ile Leu Val Met Gly Gly Thr Lys Ala
195 200 205
Gly Ser Thr Pro Thr Glu Ala Val Val Phe Leu Asn Pro Asp His Ala
210 215 220
Lys Arg Leu Asp Ala Arg Leu Lys His Ala Gly Gln Leu Ile Ser Lys
225 230 235 240
Gly Arg Phe Leu Ala Ala Pro Trp Leu Gly Leu Leu Gly Glu Asn Gly
245 250 255
Gln Thr Ala Pro Trp Ala Ala Arg Ala Ala His Ala Asn Ala Met Ala
260 265 270
Gln Lys Leu Ala Ala Leu Met Pro Val Pro Ile Lys His Pro Val Glu
275 280 285
Ala Asn Gly Ile Phe Val Glu Met Asp Glu Leu Ala Leu Glu Arg Leu
290 295 300
Arg Gly Glu Gly Trp Phe Val Tyr Arg Phe Leu Asp Gly Thr Val Arg
305 310 315 320
Phe Met Cys Ser Trp Ala Thr Thr Pro Glu Met Val Glu Asp Leu Gly
325 330 335
Ala Ala Leu Lys Arg Val Ala
340
<210> 34
<211> 343
<212> PRT
<213>crescent shank bacterium threonine aldolase amino acid mutation (artificial sequence)
<400> 34
Met Thr Gln Thr Ala Pro Arg Tyr Asp Phe Ala Ser Asp Asn Val Ala
1 5 10 15
Gly Ala Met Pro Glu Val Met Glu Ala Leu Ile Ala Ala Asn Ala Gly
20 25 30
Thr Ala Ser Gly Tyr Gly Thr Asp His Val Ser Arg Ala Ala Ala Asp
35 40 45
Arg Ile Arg Ala Ala Leu Asp Ala Asp Ala Gln Val Arg Phe Thr Ala
50 55 60
Ser Gly Thr Ala Ala Asn Ala Phe Ala Leu Thr Leu Leu Ala Gln Pro
65 70 75 80
His Glu Ala Val Leu Ala His Glu His Ala Trp Ile Cys Thr Asp Glu
85 90 95
Thr Gly Ala Pro Gly Phe Phe Gly Gln Gly Val Gly Leu Ile Gly Leu
100 105 110
Pro Gly Ala Ser Gly Lys Met Glu Leu Ala Ala Leu Glu Ala Ala Leu
115 120 125
Ala Gln Pro Asp Val Ser Tyr Arg Gln Pro Ala Ala Ala Leu Ser Leu
130 135 140
Thr Thr Ala Thr Glu Tyr Gly Thr Val Tyr Ser Glu Asp His Leu Arg
145 150 155 160
Ala Leu Ile Ala Pro Val Lys Ala Lys Gly Tyr Gly Val His Leu Asp
165 170 175
Gly Ala Arg Leu Ala Asn Ala Val Ala Gly Gly Phe Asp Leu Lys Ser
180 185 190
Ile Ala Lys Met Gly Val Asp Ile Leu Val Met Gly Gly Thr Lys Ala
195 200 205
Gly Ser Thr Pro Thr Glu Ala Val Val Phe Leu Asn Pro Asp His Ala
210 215 220
Lys Arg Leu Asp Ala Arg Leu Lys His Ala Gly Gln Leu Ile Ser Lys
225 230 235 240
Gly Arg Phe Leu Ala Ala Pro Trp Leu Gly Leu Leu Gly Glu Asn Gly
245 250 255
Gln Thr Ala Pro Trp Ala Ala Arg Ala Ala His Ala Asn Ala Met Ala
260 265 270
Gln Lys Leu Ala Ala Leu Met Pro Val Pro Ile Lys His Pro Val Glu
275 280 285
Ala Asn Gly Ile Phe Val Glu Met Asp Glu Leu Ala Leu Glu Arg Leu
290 295 300
Arg Gly Glu Gly Trp Phe Val Tyr Arg Phe Leu Asp Gly Thr Val Arg
305 310 315 320
Phe Met Cys Ser Trp Ala Thr Thr Pro Glu Met Val Glu Asp Leu Gly
325 330 335
Ala Ala Leu Lys Arg Val Ala
340
<210> 35
<211> 343
<212> PRT
<213>crescent shank bacterium threonine aldolase amino acid mutation (artificial sequence)
<400> 35
Met Thr Gln Thr Ala Pro Arg Tyr Asp Phe Ala Ser Asp Asn Val Ala
1 5 10 15
Gly Ala Met Pro Glu Val Met Glu Ala Leu Ile Ala Ala Asn Ala Gly
20 25 30
Thr Ala Ser Gly Tyr Gly Thr Asp His Val Ser Arg Ala Ala Ala Asp
35 40 45
Arg Ile Arg Ala Ala Leu Asp Ala Asp Ala Gln Val Arg Phe Thr Ala
50 55 60
Ser Gly Thr Ala Ala Asn Ala Phe Ala Leu Thr Leu Leu Ala Gln Pro
65 70 75 80
His Glu Ala Val Leu Ala His Glu His Ala Pro Ile Cys Thr Asp Glu
85 90 95
Thr Gly Ala Pro Gly Phe Phe Gly Gln Gly Val Gly Leu Ile Gly Leu
100 105 110
Pro Gly Ala Ser Gly Lys Met Glu Leu Ala Ala Leu Glu Ala Ala Leu
115 120 125
Ala Gln Pro Asp Val Ser Tyr Arg Gln Pro Ala Ala Ala Leu Ser Leu
130 135 140
Thr Thr Ala Thr Glu Tyr Gly Thr Val Tyr Ser Glu Asp His Leu Arg
145 150 155 160
Ala Leu Ile Ala Pro Val Lys Ala Lys Gly Tyr Gly Val His Leu Asp
165 170 175
Gly Ala Arg Leu Ala Asn Ala Val Ala Gly Gly Phe Asp Leu Lys Ser
180 185 190
Ile Ala Lys Met Gly Val Asp Ile Leu Val Met Gly Gly Thr Lys Ala
195 200 205
Gly Ser Thr Pro Thr Glu Ala Val Val Phe Leu Asn Pro Asp His Ala
210 215 220
Lys Arg Leu Asp Ala Arg Leu Lys His Ala Gly Gln Leu Ile Ser Lys
225 230 235 240
Gly Arg Phe Leu Ala Ala Pro Trp Leu Gly Leu Leu Gly Glu Asn Gly
245 250 255
Gln Thr Ala Pro Trp Ala Ala Arg Ala Ala His Ala Asn Ala Met Ala
260 265 270
Gln Lys Leu Ala Ala Leu Met Pro Val Pro Ile Lys His Pro Val Glu
275 280 285
Ala Asn Gly Ile Phe Val Glu Met Asp Glu Leu Ala Leu Glu Arg Leu
290 295 300
Arg Gly Glu Gly Trp Phe Val Tyr Arg Phe Leu Asp Gly Thr Val Arg
305 310 315 320
Phe Met Cys Ser Trp Ala Thr Thr Pro Glu Met Val Glu Asp Leu Gly
325 330 335
Ala Ala Leu Lys Arg Val Ala
340
<210> 36
<211> 343
<212> PRT
<213>crescent shank bacterium threonine aldolase amino acid mutation (artificial sequence)
<400> 36
Met Thr Gln Thr Ala Pro Arg Tyr Asp Phe Ala Ser Asp Asn Val Ala
1 5 10 15
Gly Ala Met Pro Glu Val Met Glu Ala Leu Ile Ala Ala Asn Ala Gly
20 25 30
Thr Ala Ser Gly Tyr Gly Thr Asp His Val Ser Arg Ala Ala Ala Asp
35 40 45
Arg Ile Arg Ala Ala Leu Asp Ala Asp Ala Gln Val Arg Phe Thr Ala
50 55 60
Ser Gly Thr Ala Ala Asn Ala Phe Ala Leu Thr Leu Leu Ala Gln Pro
65 70 75 80
His Glu Ala Val Leu Ala His Glu His Ala His Ile Cys Thr Asp Glu
85 90 95
Thr Gly Ala Pro Gly Phe Phe Gly Gln Gly Val Gly Leu Ile Gly Leu
100 105 110
Pro Gly Ala Ser Gly Lys Met Glu Leu Ala Ala Leu Glu Ala Ala Leu
115 120 125
Ala Gln Pro Asp Val Ser Tyr Arg Gln Pro Ala Ala Ala Leu Ser Leu
130 135 140
Thr Thr Ala Thr Glu Tyr Gly Thr Val Tyr Ser Glu Asp His Leu Arg
145 150 155 160
Ala Leu Ile Ala Pro Val Lys Ala Lys Gly Tyr Gly Val His Leu Asp
165 170 175
Gly Tyr Arg Leu Ala Asn Ala Val Ala Gly Gly Phe Asp Leu Lys Ser
180 185 190
Ile Ala Lys Met Gly Val Asp Ile Leu Val Met Gly Gly Thr Lys Ala
195 200 205
Gly Ser Thr Pro Thr Glu Ala Val Val Phe Leu Asn Pro Asp His Ala
210 215 220
Lys Arg Leu Asp Ala Arg Leu Lys His Ala Gly Gln Leu Ile Ser Lys
225 230 235 240
Gly Arg Phe Leu Ala Ala Pro Trp Leu Gly Leu Leu Gly Glu Asn Gly
245 250 255
Gln Thr Ala Pro Trp Ala Ala Arg Ala Ala His Ala Asn Ala Met Ala
260 265 270
Gln Lys Leu Ala Ala Leu Met Pro Val Pro Ile Lys His Pro Val Glu
275 280 285
Ala Asn Gly Ile Phe Val Glu Met Asp Glu Leu Ala Leu Glu Arg Leu
290 295 300
Arg Gly Glu Gly Trp Phe Val Tyr Arg Phe Leu Asp Gly Thr Val Arg
305 310 315 320
Phe Met Cys Ser Trp Ala Thr Thr Pro Glu Met Val Glu Asp Leu Gly
325 330 335
Ala Ala Leu Lys Arg Val Ala
340
<210> 37
<211> 343
<212> PRT
<213>crescent shank bacterium threonine aldolase amino acid mutation (artificial sequence)
<400> 37
Met Thr Gln Thr Ala Pro Arg Tyr Asp Phe Ala Ser Asp Asn Val Ala
1 5 10 15
Gly Ala Met Pro Glu Val Met Glu Ala Leu Ile Ala Ala Asn Ala Gly
20 25 30
Thr Ala Ser Gly Tyr Gly Thr Asp His Val Ser Arg Ala Ala Ala Asp
35 40 45
Arg Ile Arg Ala Ala Leu Asp Ala Asp Ala Gln Val Arg Phe Thr Ala
50 55 60
Ser Gly Thr Ala Ala Asn Ala Phe Ala Leu Thr Leu Leu Ala Gln Pro
65 70 75 80
His Glu Ala Val Leu Ala His Glu His Ala His Ile Cys Thr Asp Glu
85 90 95
Thr Gly Ala Pro Gly Phe Phe Gly Gln Gly Val Gly Leu Ile Gly Leu
100 105 110
Pro Gly Ala Ser Gly Lys Met Glu Leu Ala Ala Leu Glu Ala Ala Leu
115 120 125
Ala Gln Pro Asp Val Ser Tyr Arg Gln Pro Ala Ala Ala Leu Ser Leu
130 135 140
Thr Thr Ala Thr Glu Tyr Gly Thr Val Tyr Ser Glu Asp His Leu Arg
145 150 155 160
Ala Leu Ile Ala Pro Val Lys Ala Lys Gly Tyr Gly Val His Leu Asp
165 170 175
Gly Ala Arg Leu Ala Asn Ala Val Ala Gly Gly Phe Asp Leu Lys Ser
180 185 190
Ile Ala Lys Met Gly Val Asp Ile Leu Val Met Gly Gly Ile Lys Ala
195 200 205
Gly Ser Thr Pro Thr Glu Ala Val Val Phe Leu Asn Pro Asp His Ala
210 215 220
Lys Arg Leu Asp Ala Arg Leu Lys His Ala Gly Gln Leu Ile Ser Lys
225 230 235 240
Gly Arg Phe Leu Ala Ala Pro Trp Leu Gly Leu Leu Gly Glu Asn Gly
245 250 255
Gln Thr Ala Pro Trp Ala Ala Arg Ala Ala His Ala Asn Ala Met Ala
260 265 270
Gln Lys Leu Ala Ala Leu Met Pro Val Pro Ile Lys His Pro Val Glu
275 280 285
Ala Asn Gly Ile Phe Val Glu Met Asp Glu Leu Ala Leu Glu Arg Leu
290 295 300
Arg Gly Glu Gly Trp Phe Val Tyr Arg Phe Leu Asp Gly Thr Val Arg
305 310 315 320
Phe Met Cys Ser Trp Ala Thr Thr Pro Glu Met Val Glu Asp Leu Gly
325 330 335
Ala Ala Leu Lys Arg Val Ala
340
<210> 38
<211> 343
<212> PRT
<213>crescent shank bacterium threonine aldolase amino acid mutation (artificial sequence)
<400> 38
Met Thr Gln Thr Ala Pro Arg Tyr Asp Phe Ala Ser Asp Asn Val Ala
1 5 10 15
Gly Ala Met Pro Glu Val Met Glu Ala Leu Ile Ala Ala Asn Ala Gly
20 25 30
Thr Ala Ser Gly Tyr Gly Thr Asp His Val Ser Arg Ala Ala Ala Asp
35 40 45
Arg Ile Arg Ala Ala Leu Asp Ala Asp Ala Gln Val Arg Phe Thr Ala
50 55 60
Ser Gly Thr Ala Ala Asn Ala Phe Ala Leu Thr Leu Leu Ala Gln Pro
65 70 75 80
His Glu Ala Val Leu Ala His Glu His Ala His Ile Cys Thr Asp Glu
85 90 95
Thr Gly Ala Pro Gly Phe Phe Gly Gln Gly Val Gly Leu Ile Gly Leu
100 105 110
Pro Gly Ala Ser Gly Lys Met Glu Leu Ala Ala Leu Glu Ala Ala Leu
115 120 125
Ala Gln Pro Asp Val Ser Tyr Arg Gln Pro Ala Ala Ala Leu Ser Leu
130 135 140
Thr Thr Ala Thr Glu Tyr Gly Thr Val Tyr Ser Glu Asp His Leu Arg
145 150 155 160
Ala Leu Ile Ala Pro Val Lys Ala Lys Gly Tyr Gly Val His Leu Asp
165 170 175
Gly Ala Arg Leu Ala Asn Ala Val Ala Gly Gly Phe Asp Leu Lys Ser
180 185 190
Ile Ala Lys Met Gly Val Asp Ile Leu Val Met Gly Gly Ser Lys Ala
195 200 205
Gly Ser Thr Pro Thr Glu Ala Val Val Phe Leu Asn Pro Asp His Ala
210 215 220
Lys Arg Leu Asp Ala Arg Leu Lys His Ala Gly Gln Leu Ile Ser Lys
225 230 235 240
Gly Arg Phe Leu Ala Ala Pro Trp Leu Gly Leu Leu Gly Glu Asn Gly
245 250 255
Gln Thr Ala Pro Trp Ala Ala Arg Ala Ala His Ala Asn Ala Met Ala
260 265 270
Gln Lys Leu Ala Ala Leu Met Pro Val Pro Ile Lys His Pro Val Glu
275 280 285
Ala Asn Gly Ile Phe Val Glu Met Asp Glu Leu Ala Leu Glu Arg Leu
290 295 300
Arg Gly Glu Gly Trp Phe Val Tyr Arg Phe Leu Asp Gly Thr Val Arg
305 310 315 320
Phe Met Cys Ser Trp Ala Thr Thr Pro Glu Met Val Glu Asp Leu Gly
325 330 335
Ala Ala Leu Lys Arg Val Ala
340
<210> 39
<211> 343
<212> PRT
<213>Thermotoga maritima threonine aldolase amino acid mutation (artificial sequence)
<400> 39
Met Ile Asp Leu Arg Ser Asp Gln Val Thr Lys Pro Thr Glu Glu Met
1 5 10 15
Arg Lys Ala Met Ala Gln Ala Glu Val Gly Asp Asp Val Tyr Gly Glu
20 25 30
Asp Pro Thr Ile Asn Glu Leu Glu Arg Leu Ala Ala Glu Thr Phe Gly
35 40 45
Lys Glu Ala Ala Leu Phe Val Pro Ser Gly Thr Met Gly Asn Gln Val
50 55 60
Ser Ile Met Ala His Thr Gln Arg Gly Asp Glu Val Ile Leu Glu Ala
65 70 75 80
Asp Ser His Ile Phe Trp Tyr Glu Val Gly Ala Met Ala Val Leu Ser
85 90 95
Gly Val Met Pro His Pro Val Pro Gly Lys Asn Gly Ala Met Asp Pro
100 105 110
Asp Asp Val Arg Lys Ala Ile Arg Pro Arg Asn Ile His Phe Pro Arg
115 120 125
Thr Ser Leu Ile Ala Ile Glu Asn Thr His Asn Arg Ser Gly Gly Arg
130 135 140
Val Val Pro Leu Glu Asn Ile Lys Glu Ile Cys Thr Ile Ala Lys Glu
145 150 155 160
His Gly Ile Asn Val His Ile Asp Gly Ala Arg Ile Phe Asn Ala Ser
165 170 175
Ile Ala Ser Gly Val Pro Val Lys Glu Tyr Ala Gly Tyr Ala Asp Ser
180 185 190
Val Met Phe Cys Leu Ser Lys Gly Leu Cys Ala Pro Val Gly Ser Val
195 200 205
Val Val Gly Asp Arg Asp Phe Ile Glu Arg Ala Arg Lys Ala Arg Lys
210 215 220
Met Leu Gly Gly Gly Met Arg Gln Ala Gly Val Leu Ala Ala Ala Gly
225 230 235 240
Ile Ile Ala Leu Thr Lys Met Val Asp Arg Leu Lys Glu Asp His Glu
245 250 255
Asn Ala Arg Phe Leu Ala Leu Lys Leu Lys Glu Ile Gly Tyr Ser Val
260 265 270
Asn Pro Glu Asp Val Lys Thr Asn Met Val Ile Leu Arg Thr Asp Asn
275 280 285
Leu Lys Val Asn Ala His Gly Phe Ile Glu Ala Leu Arg Asn Ser Gly
290 295 300
Val Leu Ala Asn Ala Val Ser Asp Thr Glu Ile Arg Leu Val Thr His
305 310 315 320
Lys Asp Val Ser Arg Asn Asp Ile Glu Glu Ala Leu Asn Ile Phe Glu
325 330 335
Lys Leu Phe Arg Lys Phe Ser
340
<210> 40
<211> 343
<212> PRT
<213>Thermotoga maritima threonine aldolase amino acid mutation (artificial sequence)
<400> 40
Met Ile Asp Leu Arg Ser Asp Thr Val Thr Lys Pro Thr Glu Glu Met
1 5 10 15
Arg Lys Ala Met Ala Gln Ala Glu Val Gly Asp Asp Val Tyr Gly Glu
20 25 30
Asp Pro Thr Ile Asn Glu Leu Glu Arg Leu Ala Ala Glu Thr Phe Gly
35 40 45
Lys Glu Ala Ala Leu Phe Val Pro Ser Gly Thr Met Gly Asn Gln Val
50 55 60
Ser Ile Met Ala His Thr Gln Arg Gly Asp Glu Val Ile Leu Glu Ala
65 70 75 80
Asp Ser Trp Ile Phe Trp Tyr Glu Val Gly Ala Met Ala Val Leu Ser
85 90 95
Gly Val Met Pro His Pro Val Pro Gly Lys Asn Gly Ala Met Asp Pro
100 105 110
Asp Asp Val Arg Lys Ala Ile Arg Pro Arg Asn Ile His Phe Pro Arg
115 120 125
Thr Ser Leu Ile Ala Ile Glu Asn Thr His Asn Arg Ser Gly Gly Arg
130 135 140
Val Val Pro Leu Glu Asn Ile Lys Glu Ile Cys Thr Ile Ala Lys Glu
145 150 155 160
His Gly Ile Asn Val His Ile Asp Gly Ala Arg Ile Phe Asn Ala Ser
165 170 175
Ile Ala Ser Gly Val Pro Val Lys Glu Tyr Ala Gly Tyr Ala Asp Ser
180 185 190
Val Met Phe Cys Leu Ser Lys Gly Leu Cys Ala Pro Val Gly Ser Val
195 200 205
Val Val Gly Asp Arg Asp Phe Ile Glu Arg Ala Arg Lys Ala Arg Lys
210 215 220
Met Leu Gly Gly Gly Met Arg Gln Ala Gly Val Leu Ala Ala Ala Gly
225 230 235 240
Ile Ile Ala Leu Thr Lys Met Val Asp Arg Leu Lys Glu Asp His Glu
245 250 255
Asn Ala Arg Phe Leu Ala Leu Lys Leu Lys Glu Ile Gly Tyr Ser Val
260 265 270
Asn Pro Glu Asp Val Lys Thr Asn Met Val Ile Leu Arg Thr Asp Asn
275 280 285
Leu Lys Val Asn Ala His Gly Phe Ile Glu Ala Leu Arg Asn Ser Gly
290 295 300
Val Leu Ala Asn Ala Val Ser Asp Thr Glu Ile Arg Leu Val Thr His
305 310 315 320
Lys Asp Val Ser Arg Asn Asp Ile Glu Glu Ala Leu Asn Ile Phe Glu
325 330 335
Lys Leu Phe Arg Lys Phe Ser
340
<210> 41
<211> 343
<212> PRT
<213>Thermotoga maritima threonine aldolase amino acid mutation (artificial sequence)
<400> 41
Met Ile Asp Leu Arg Ser Asp Thr Val Thr Lys Pro Thr Glu Glu Met
1 5 10 15
Arg Lys Ala Met Ala Gln Ala Glu Val Gly Asp Asp Val Tyr Gly Glu
20 25 30
Asp Pro Thr Ile Asn Glu Leu Glu Arg Leu Ala Ala Glu Thr Phe Gly
35 40 45
Lys Glu Ala Ala Leu Phe Val Pro Ser Gly Thr Met Gly Asn Gln Val
50 55 60
Ser Ile Met Ala His Thr Gln Arg Gly Asp Glu Val Ile Leu Glu Ala
65 70 75 80
Asp Ser Pro Ile Phe Trp Tyr Glu Val Gly Ala Met Ala Val Leu Ser
85 90 95
Gly Val Met Pro His Pro Val Pro Gly Lys Asn Gly Ala Met Asp Pro
100 105 110
Asp Asp Val Arg Lys Ala Ile Arg Pro Arg Asn Ile His Phe Pro Arg
115 120 125
Thr Ser Leu Ile Ala Ile Glu Asn Thr His Asn Arg Ser Gly Gly Arg
130 135 140
Val Val Pro Leu Glu Asn Ile Lys Glu Ile Cys Thr Ile Ala Lys Glu
145 150 155 160
His Gly Ile Asn Val His Ile Asp Gly Ala Arg Ile Phe Asn Ala Ser
165 170 175
Ile Ala Ser Gly Val Pro Val Lys Glu Tyr Ala Gly Tyr Ala Asp Ser
180 185 190
Val Met Phe Cys Leu Ser Lys Gly Leu Cys Ala Pro Val Gly Ser Val
195 200 205
Val Val Gly Asp Arg Asp Phe Ile Glu Arg Ala Arg Lys Ala Arg Lys
210 215 220
Met Leu Gly Gly Gly Met Arg Gln Ala Gly Val Leu Ala Ala Ala Gly
225 230 235 240
Ile Ile Ala Leu Thr Lys Met Val Asp Arg Leu Lys Glu Asp His Glu
245 250 255
Asn Ala Arg Phe Leu Ala Leu Lys Leu Lys Glu Ile Gly Tyr Ser Val
260 265 270
Asn Pro Glu Asp Val Lys Thr Asn Met Val Ile Leu Arg Thr Asp Asn
275 280 285
Leu Lys Val Asn Ala His Gly Phe Ile Glu Ala Leu Arg Asn Ser Gly
290 295 300
Val Leu Ala Asn Ala Val Ser Asp Thr Glu Ile Arg Leu Val Thr His
305 310 315 320
Lys Asp Val Ser Arg Asn Asp Ile Glu Glu Ala Leu Asn Ile Phe Glu
325 330 335
Lys Leu Phe Arg Lys Phe Ser
340
<210> 42
<211> 343
<212> PRT
<213>Thermotoga maritima threonine aldolase amino acid mutation (artificial sequence)
<400> 42
Met Ile Asp Leu Arg Ser Asp Thr Val Thr Lys Pro Thr Glu Glu Met
1 5 10 15
Arg Lys Ala Met Ala Gln Ala Glu Val Gly Asp Asp Val Tyr Gly Glu
20 25 30
Asp Pro Thr Ile Asn Glu Leu Glu Arg Leu Ala Ala Glu Thr Phe Gly
35 40 45
Lys Glu Ala Ala Leu Phe Val Pro Ser Gly Thr Met Gly Asn Gln Val
50 55 60
Ser Ile Met Ala His Thr Gln Arg Gly Asp Glu Val Ile Leu Glu Ala
65 70 75 80
Asp Ser His Ile Phe Trp Tyr Glu Val Gly Ala Met Ala Val Leu Ser
85 90 95
Gly Val Met Pro His Pro Val Pro Gly Lys Asn Gly Ala Met Asp Pro
100 105 110
Asp Asp Val Arg Lys Ala Ile Arg Pro Arg Asn Ile His Phe Pro Arg
115 120 125
Thr Ser Leu Ile Ala Ile Glu Asn Thr His Asn Arg Ser Gly Gly Arg
130 135 140
Val Val Pro Leu Glu Asn Ile Lys Glu Ile Cys Thr Ile Ala Lys Glu
145 150 155 160
His Gly Ile Asn Val His Ile Asp Gly Tyr Arg Ile Phe Asn Ala Ser
165 170 175
Ile Ala Ser Gly Val Pro Val Lys Glu Tyr Ala Gly Tyr Ala Asp Ser
180 185 190
Val Met Phe Cys Leu Ser Lys Gly Leu Cys Ala Pro Val Gly Ser Val
195 200 205
Val Val Gly Asp Arg Asp Phe Ile Glu Arg Ala Arg Lys Ala Arg Lys
210 215 220
Met Leu Gly Gly Gly Met Arg Gln Ala Gly Val Leu Ala Ala Ala Gly
225 230 235 240
Ile Ile Ala Leu Thr Lys Met Val Asp Arg Leu Lys Glu Asp His Glu
245 250 255
Asn Ala Arg Phe Leu Ala Leu Lys Leu Lys Glu Ile Gly Tyr Ser Val
260 265 270
Asn Pro Glu Asp Val Lys Thr Asn Met Val Ile Leu Arg Thr Asp Asn
275 280 285
Leu Lys Val Asn Ala His Gly Phe Ile Glu Ala Leu Arg Asn Ser Gly
290 295 300
Val Leu Ala Asn Ala Val Ser Asp Thr Glu Ile Arg Leu Val Thr His
305 310 315 320
Lys Asp Val Ser Arg Asn Asp Ile Glu Glu Ala Leu Asn Ile Phe Glu
325 330 335
Lys Leu Phe Arg Lys Phe Ser
340
<210> 43
<211> 343
<212> PRT
<213>Thermotoga maritima threonine aldolase amino acid mutation (artificial sequence)
<400> 43
Met Ile Asp Leu Arg Ser Asp Thr Val Thr Lys Pro Thr Glu Glu Met
1 5 10 15
Arg Lys Ala Met Ala Gln Ala Glu Val Gly Asp Asp Val Tyr Gly Glu
20 25 30
Asp Pro Thr Ile Asn Glu Leu Glu Arg Leu Ala Ala Glu Thr Phe Gly
35 40 45
Lys Glu Ala Ala Leu Phe Val Pro Ser Gly Thr Met Gly Asn Gln Val
50 55 60
Ser Ile Met Ala His Thr Gln Arg Gly Asp Glu Val Ile Leu Glu Ala
65 70 75 80
Asp Ser His Ile Phe Trp Tyr Glu Val Gly Ala Met Ala Val Leu Ser
85 90 95
Gly Val Met Pro His Pro Val Pro Gly Lys Asn Gly Ala Met Asp Pro
100 105 110
Asp Asp Val Arg Lys Ala Ile Arg Pro Arg Asn Ile His Phe Pro Arg
115 120 125
Thr Ser Leu Ile Ala Ile Glu Asn Thr His Asn Arg Ser Gly Gly Arg
130 135 140
Val Val Pro Leu Glu Asn Ile Lys Glu Ile Cys Thr Ile Ala Lys Glu
145 150 155 160
His Gly Ile Asn Val His Ile Asp Gly Ala Arg Ile Phe Asn Ala Ser
165 170 175
Ile Ala Ser Gly Val Pro Val Lys Glu Tyr Ala Gly Tyr Ala Asp Ser
180 185 190
Val Met Phe Cys Leu Ile Lys Gly Leu Cys Ala Pro Val Gly Ser Val
195 200 205
Val Val Gly Asp Arg Asp Phe Ile Glu Arg Ala Arg Lys Ala Arg Lys
210 215 220
Met Leu Gly Gly Gly Met Arg Gln Ala Gly Val Leu Ala Ala Ala Gly
225 230 235 240
Ile Ile Ala Leu Thr Lys Met Val Asp Arg Leu Lys Glu Asp His Glu
245 250 255
Asn Ala Arg Phe Leu Ala Leu Lys Leu Lys Glu Ile Gly Tyr Ser Val
260 265 270
Asn Pro Glu Asp Val Lys Thr Asn Met Val Ile Leu Arg Thr Asp Asn
275 280 285
Leu Lys Val Asn Ala His Gly Phe Ile Glu Ala Leu Arg Asn Ser Gly
290 295 300
Val Leu Ala Asn Ala Val Ser Asp Thr Glu Ile Arg Leu Val Thr His
305 310 315 320
Lys Asp Val Ser Arg Asn Asp Ile Glu Glu Ala Leu Asn Ile Phe Glu
325 330 335
Lys Leu Phe Arg Lys Phe Ser
340
<210> 44
<211> 358
<212> PRT
<213>Listeria monocytogenes threonine aldolase amino acid mutation (artificial sequence)
<400> 44
Met Thr Asn Thr Leu Lys Thr Ser Tyr Gln Lys Thr Pro Tyr Lys Leu
1 5 10 15
Gly Gly Asn Gly Pro Arg Asn Val Gly Val Leu Thr Glu Ala Leu Gln
20 25 30
Asn Ile Asp Asp Asn Leu Glu Ser Asp Ile Tyr Gly Asn Gly Ala Val
35 40 45
Ile Glu Asp Phe Glu Thr Lys Ile Ala Lys Ile Leu Gly Lys Gln Ser
50 55 60
Ala Val Phe Phe Pro Ser Gly Thr Met Ala Gln Gln Ile Ala Leu Arg
65 70 75 80
Ile Trp Ala Asp Arg Lys Glu Asn Arg Arg Val Ala Tyr His Pro Leu
85 90 95
Ser Trp Leu Glu Ile His Glu Gln Asp Gly Leu Lys Glu Leu Gln Gln
100 105 110
Ile Thr Pro Leu Leu Leu Gly Thr Ala Asn Gln Leu Leu Thr Ile Asp
115 120 125
Asp Ile Lys Ser Leu Arg Glu Pro Val Ser Ser Val Leu Ile Glu Leu
130 135 140
Pro Gln Arg Glu Ile Gly Gly Gln Leu Pro Ala Phe Glu Glu Leu Glu
145 150 155 160
Lys Ile Ser Glu Tyr Cys His Glu Gln Gly Ile Ser Leu His Leu Asp
165 170 175
Gly Ala Arg Leu Trp Glu Ile Thr Pro Phe Tyr Gln Lys Ser Ala Glu
180 185 190
Glu Ile Cys Ala Leu Phe Asp Ser Val Tyr Val Ser Phe Tyr Lys Gly
195 200 205
Ile Gly Gly Ile Ala Gly Ala Ile Leu Ala Gly Asn Asp Asp Phe Val
210 215 220
Gln Glu Ala Lys Ile Trp Lys Arg Arg Tyr Gly Gly Asp Leu Ile Ser
225 230 235 240
Leu Tyr Pro Tyr Ile Leu Ser Ala Asp Tyr Tyr Phe Glu Lys Arg Ile
245 250 255
Gly Lys Met Ala Glu Tyr Phe Glu Ala Ala Lys Gly Leu Ala Glu Arg
260 265 270
Phe Asn Ser Cys Ser Gly Val Lys Thr Val Pro Glu Val Pro Val Ser
275 280 285
Asn Met Phe His Val Tyr Phe Glu Asn Ser Ala Asp Glu Ile Gly Ala
290 295 300
Ile Leu Thr Lys Ile Gln Asp Glu Thr Gly Val Gly Ile Ser Gly Tyr
305 310 315 320
Leu Gln Glu Lys Ser Ala Asp Val Cys Ala Phe Glu Val Ser Val Gly
325 330 335
Asp Ala Phe Ala Glu Ile Pro Ala Lys Asn Leu Glu Leu Val Phe Arg
340 345 350
Cys Leu Glu Lys Glu Leu
355
<210> 45
<211> 358
<212> PRT
<213>Listeria monocytogenes threonine aldolase amino acid mutation (artificial sequence)
<400> 45
Met Thr Asn Thr Leu Lys Thr Ser Tyr Gln Lys Thr Pro Tyr Lys Leu
1 5 10 15
Gly Gly Asn Gly Pro Arg Asn Val Gly Val Leu Thr Glu Ala Leu Gln
20 25 30
Asn Ile Asp Asp Asn Leu Glu Ser Asp Ile Tyr Gly Asn Gly Ala Val
35 40 45
Ile Glu Asp Phe Glu Thr Lys Ile Ala Lys Ile Leu Gly Lys Gln Ser
50 55 60
Ala Val Phe Phe Pro Ser Gly Thr Met Ala Gln Gln Ile Ala Leu Arg
65 70 75 80
Ile Trp Ala Asp Arg Lys Glu Asn Arg Arg Val Ala Tyr His Pro Leu
85 90 95
Ser Pro Leu Glu Ile His Glu Gln Asp Gly Leu Lys Glu Leu Gln Gln
100 105 110
Ile Thr Pro Leu Leu Leu Gly Thr Ala Asn Gln Leu Leu Thr Ile Asp
115 120 125
Asp Ile Lys Ser Leu Arg Glu Pro Val Ser Ser Val Leu Ile Glu Leu
130 135 140
Pro Gln Arg Glu Ile Gly Gly Gln Leu Pro Ala Phe Glu Glu Leu Glu
145 150 155 160
Lys Ile Ser Glu Tyr Cys His Glu Gln Gly Ile Ser Leu His Leu Asp
165 170 175
Gly Ala Arg Leu Trp Glu Ile Thr Pro Phe Tyr Gln Lys Ser Ala Glu
180 185 190
Glu Ile Cys Ala Leu Phe Asp Ser Val Tyr Val Ser Phe Tyr Lys Gly
195 200 205
Ile Gly Gly Ile Ala Gly Ala Ile Leu Ala Gly Asn Asp Asp Phe Val
210 215 220
Gln Glu Ala Lys Ile Trp Lys Arg Arg Tyr Gly Gly Asp Leu Ile Ser
225 230 235 240
Leu Tyr Pro Tyr Ile Leu Ser Ala Asp Tyr Tyr Phe Glu Lys Arg Ile
245 250 255
Gly Lys Met Ala Glu Tyr Phe Glu Ala Ala Lys Gly Leu Ala Glu Arg
260 265 270
Phe Asn Ser Cys Ser Gly Val Lys Thr Val Pro Glu Val Pro Val Ser
275 280 285
Asn Met Phe His Val Tyr Phe Glu Asn Ser Ala Asp Glu Ile Gly Ala
290 295 300
Ile Leu Thr Lys Ile Gln Asp Glu Thr Gly Val Gly Ile Ser Gly Tyr
305 310 315 320
Leu Gln Glu Lys Ser Ala Asp Val Cys Ala Phe Glu Val Ser Val Gly
325 330 335
Asp Ala Phe Ala Glu Ile Pro Ala Lys Asn Leu Glu Leu Val Phe Arg
340 345 350
Cys Leu Glu Lys Glu Leu
355
<210> 46
<211> 358
<212> PRT
<213>Listeria monocytogenes threonine aldolase amino acid mutation (artificial sequence)
<400> 46
Met Thr Asn Thr Leu Lys Thr Ser Tyr Gln Lys Thr Pro Tyr Lys Leu
1 5 10 15
Gly Gly Asn Gly Pro Arg Asn Val Gly Val Leu Thr Glu Ala Leu Gln
20 25 30
Asn Ile Asp Asp Asn Leu Glu Ser Asp Ile Tyr Gly Asn Gly Ala Val
35 40 45
Ile Glu Asp Phe Glu Thr Lys Ile Ala Lys Ile Leu Gly Lys Gln Ser
50 55 60
Ala Val Phe Phe Pro Ser Gly Thr Met Ala Gln Gln Ile Ala Leu Arg
65 70 75 80
Ile Trp Ala Asp Arg Lys Glu Asn Arg Arg Val Ala Tyr His Pro Leu
85 90 95
Ser His Leu Glu Ile His Glu Gln Asp Gly Leu Lys Glu Leu Gln Gln
100 105 110
Ile Thr Pro Leu Leu Leu Gly Thr Ala Asn Gln Leu Leu Thr Ile Asp
115 120 125
Asp Ile Lys Ser Leu Arg Glu Pro Val Ser Ser Val Leu Ile Glu Leu
130 135 140
Pro Gln Arg Glu Ile Gly Gly Gln Leu Pro Ala Phe Glu Glu Leu Glu
145 150 155 160
Lys Ile Ser Glu Tyr Cys His Glu Gln Gly Ile Ser Leu His Leu Asp
165 170 175
Gly Tyr Arg Leu Trp Glu Ile Thr Pro Phe Tyr Gln Lys Ser Ala Glu
180 185 190
Glu Ile Cys Ala Leu Phe Asp Ser Val Tyr Val Ser Phe Tyr Lys Gly
195 200 205
Ile Gly Gly Ile Ala Gly Ala Ile Leu Ala Gly Asn Asp Asp Phe Val
210 215 220
Gln Glu Ala Lys Ile Trp Lys Arg Arg Tyr Gly Gly Asp Leu Ile Ser
225 230 235 240
Leu Tyr Pro Tyr Ile Leu Ser Ala Asp Tyr Tyr Phe Glu Lys Arg Ile
245 250 255
Gly Lys Met Ala Glu Tyr Phe Glu Ala Ala Lys Gly Leu Ala Glu Arg
260 265 270
Phe Asn Ser Cys Ser Gly Val Lys Thr Val Pro Glu Val Pro Val Ser
275 280 285
Asn Met Phe His Val Tyr Phe Glu Asn Ser Ala Asp Glu Ile Gly Ala
290 295 300
Ile Leu Thr Lys Ile Gln Asp Glu Thr Gly Val Gly Ile Ser Gly Tyr
305 310 315 320
Leu Gln Glu Lys Ser Ala Asp Val Cys Ala Phe Glu Val Ser Val Gly
325 330 335
Asp Ala Phe Ala Glu Ile Pro Ala Lys Asn Leu Glu Leu Val Phe Arg
340 345 350
Cys Leu Glu Lys Glu Leu
355
<210> 47
<211> 358
<212> PRT
<213>Listeria monocytogenes threonine aldolase amino acid mutation (artificial sequence)
<400> 47
Met Thr Asn Thr Leu Lys Thr Ser Tyr Gln Lys Thr Pro Tyr Lys Leu
1 5 10 15
Gly Gly Asn Gly Pro Arg Asn Val Gly Val Leu Thr Glu Ala Leu Gln
20 25 30
Asn Ile Asp Asp Asn Leu Glu Ser Asp Ile Tyr Gly Asn Gly Ala Val
35 40 45
Ile Glu Asp Phe Glu Thr Lys Ile Ala Lys Ile Leu Gly Lys Gln Ser
50 55 60
Ala Val Phe Phe Pro Ser Gly Thr Met Ala Gln Gln Ile Ala Leu Arg
65 70 75 80
Ile Trp Ala Asp Arg Lys Glu Asn Arg Arg Val Ala Tyr His Pro Leu
85 90 95
Ser His Leu Glu Ile His Glu Gln Asp Gly Leu Lys Glu Leu Gln Gln
100 105 110
Ile Thr Pro Leu Leu Leu Gly Thr Ala Asn Gln Leu Leu Thr Ile Asp
115 120 125
Asp Ile Lys Ser Leu Arg Glu Pro Val Ser Ser Val Leu Ile Glu Leu
130 135 140
Pro Gln Arg Glu Ile Gly Gly Gln Leu Pro Ala Phe Glu Glu Leu Glu
145 150 155 160
Lys Ile Ser Glu Tyr Cys His Glu Gln Gly Ile Ser Leu His Leu Asp
165 170 175
Gly Ala Arg Leu Trp Glu Ile Thr Pro Phe Tyr Gln Lys Ser Ala Glu
180 185 190
Glu Ile Cys Ala Leu Phe Asp Ser Val Tyr Val Ser Phe Ile Lys Gly
195 200 205
Ile Gly Gly Ile Ala Gly Ala Ile Leu Ala Gly Asn Asp Asp Phe Val
210 215 220
Gln Glu Ala Lys Ile Trp Lys Arg Arg Tyr Gly Gly Asp Leu Ile Ser
225 230 235 240
Leu Tyr Pro Tyr Ile Leu Ser Ala Asp Tyr Tyr Phe Glu Lys Arg Ile
245 250 255
Gly Lys Met Ala Glu Tyr Phe Glu Ala Ala Lys Gly Leu Ala Glu Arg
260 265 270
Phe Asn Ser Cys Ser Gly Val Lys Thr Val Pro Glu Val Pro Val Ser
275 280 285
Asn Met Phe His Val Tyr Phe Glu Asn Ser Ala Asp Glu Ile Gly Ala
290 295 300
Ile Leu Thr Lys Ile Gln Asp Glu Thr Gly Val Gly Ile Ser Gly Tyr
305 310 315 320
Leu Gln Glu Lys Ser Ala Asp Val Cys Ala Phe Glu Val Ser Val Gly
325 330 335
Asp Ala Phe Ala Glu Ile Pro Ala Lys Asn Leu Glu Leu Val Phe Arg
340 345 350
Cys Leu Glu Lys Glu Leu
355
<210> 48
<211> 358
<212> PRT
<213>Listeria monocytogenes threonine aldolase amino acid mutation (artificial sequence)
<400> 48
Met Thr Asn Thr Leu Lys Thr Ser Tyr Gln Lys Thr Pro Tyr Lys Leu
1 5 10 15
Gly Gly Asn Gly Pro Arg Asn Val Gly Val Leu Thr Glu Ala Leu Gln
20 25 30
Asn Ile Asp Asp Asn Leu Glu Ser Asp Ile Tyr Gly Asn Gly Ala Val
35 40 45
Ile Glu Asp Phe Glu Thr Lys Ile Ala Lys Ile Leu Gly Lys Gln Ser
50 55 60
Ala Val Phe Phe Pro Ser Gly Thr Met Ala Gln Gln Ile Ala Leu Arg
65 70 75 80
Ile Trp Ala Asp Arg Lys Glu Asn Arg Arg Val Ala Tyr His Pro Leu
85 90 95
Ser His Leu Glu Ile His Glu Gln Asp Gly Leu Lys Glu Leu Gln Gln
100 105 110
Ile Thr Pro Leu Leu Leu Gly Thr Ala Asn Gln Leu Leu Thr Ile Asp
115 120 125
Asp Ile Lys Ser Leu Arg Glu Pro Val Ser Ser Val Leu Ile Glu Leu
130 135 140
Pro Gln Arg Glu Ile Gly Gly Gln Leu Pro Ala Phe Glu Glu Leu Glu
145 150 155 160
Lys Ile Ser Glu Tyr Cys His Glu Gln Gly Ile Ser Leu His Leu Asp
165 170 175
Gly Ala Arg Leu Trp Glu Ile Thr Pro Phe Tyr Gln Lys Ser Ala Glu
180 185 190
Glu Ile Cys Ala Leu Phe Asp Ser Val Tyr Val Ser Phe Ser Lys Gly
195 200 205
Ile Gly Gly Ile Ala Gly Ala Ile Leu Ala Gly Asn Asp Asp Phe Val
210 215 220
Gln Glu Ala Lys Ile Trp Lys Arg Arg Tyr Gly Gly Asp Leu Ile Ser
225 230 235 240
Leu Tyr Pro Tyr Ile Leu Ser Ala Asp Tyr Tyr Phe Glu Lys Arg Ile
245 250 255
Gly Lys Met Ala Glu Tyr Phe Glu Ala Ala Lys Gly Leu Ala Glu Arg
260 265 270
Phe Asn Ser Cys Ser Gly Val Lys Thr Val Pro Glu Val Pro Val Ser
275 280 285
Asn Met Phe His Val Tyr Phe Glu Asn Ser Ala Asp Glu Ile Gly Ala
290 295 300
Ile Leu Thr Lys Ile Gln Asp Glu Thr Gly Val Gly Ile Ser Gly Tyr
305 310 315 320
Leu Gln Glu Lys Ser Ala Asp Val Cys Ala Phe Glu Val Ser Val Gly
325 330 335
Asp Ala Phe Ala Glu Ile Pro Ala Lys Asn Leu Glu Leu Val Phe Arg
340 345 350
Cys Leu Glu Lys Glu Leu
355
<210> 49
<211> 333
<212> PRT
<213>E. coli threonine aldolase amino acid mutation (artificial sequence)
<400> 49
Met Ile Asp Leu Arg Ser Asp Gln Val Thr Arg Pro Ser Arg Ala Met
1 5 10 15
Leu Glu Ala Met Met Ala Ala Pro Val Gly Asp Asp Val Tyr Gly Asp
20 25 30
Asp Pro Thr Val Asn Ala Leu Gln Asp Tyr Ala Ala Glu Leu Ser Gly
35 40 45
Lys Glu Ala Ala Ile Phe Leu Pro Thr Gly Thr Gln Ala Asn Leu Val
50 55 60
Ala Leu Leu Ser His Cys Glu Arg Gly Glu Glu Tyr Ile Val Gly Gln
65 70 75 80
Ala Ala His Asn Tyr Leu Phe Glu Ala Gly Gly Ala Ala Val Leu Gly
85 90 95
Ser Ile Gln Pro Gln Pro Ile Asp Ala Ala Ala Asp Gly Thr Leu Pro
100 105 110
Leu Asp Lys Val Ala Met Lys Ile Lys Pro Asp Asp Ile His Phe Ala
115 120 125
Arg Thr Lys Leu Leu Ser Leu Glu Asn Thr His Asn Gly Lys Val Leu
130 135 140
Pro Arg Glu Tyr Leu Lys Glu Ala Trp Glu Phe Thr Arg Lys Arg Asn
145 150 155 160
Leu Ala Leu His Val Asp Gly Ala Arg Ile Phe Asn Ala Val Val Ala
165 170 175
Tyr Gly Cys Glu Leu Lys Glu Ile Thr Gln Tyr Cys Asp Ser Phe Thr
180 185 190
Ile Cys Leu Ser Lys Gly Leu Gly Thr Pro Val Gly Ser Leu Leu Val
195 200 205
Gly Asn Arg Asp Tyr Ile Lys Arg Ala Ile Arg Trp Arg Lys Met Thr
210 215 220
Gly Gly Gly Met Arg Gln Ser Gly Ile Leu Ala Ala Ala Gly Met Tyr
225 230 235 240
Ala Leu Lys Asn Asn Val Ala Arg Leu Gln Glu Asp His Asp Asn Thr
245 250 255
Ala Trp Met Ala Glu Gln Leu Arg Glu Ala Gly Ala Asp Val Met Arg
260 265 270
Gln Asp Thr Asn Met Leu Phe Val Arg Val Gly Glu Glu Asn Ala Ala
275 280 285
Ala Leu Gly Glu Tyr Met Lys Ala Arg Asn Val Leu Ile Asn Ala Ser
290 295 300
Pro Ile Val Arg Leu Val Thr His Leu Asp Val Ser Arg Ala Gln Leu
305 310 315 320
Ala Glu Val Ala Ala His Trp Arg Ala Phe Leu Ala Arg
325 330
<210> 50
<211> 333
<212> PRT
<213>E. coli threonine aldolase amino acid mutation (artificial sequence)
<400> 50
Met Ile Asp Leu Arg Ser Asp Thr Val Thr Arg Pro Ser Arg Ala Met
1 5 10 15
Leu Glu Ala Met Met Ala Ala Pro Val Gly Asp Asp Val Tyr Gly Asp
20 25 30
Asp Pro Thr Val Asn Ala Leu Gln Asp Tyr Ala Ala Glu Leu Ser Gly
35 40 45
Lys Glu Ala Ala Ile Phe Leu Pro Thr Gly Thr Gln Ala Asn Leu Val
50 55 60
Ala Leu Leu Ser His Cys Glu Arg Gly Glu Glu Tyr Ile Val Gly Gln
65 70 75 80
Ala Ala Trp Asn Tyr Leu Phe Glu Ala Gly Gly Ala Ala Val Leu Gly
85 90 95
Ser Ile Gln Pro Gln Pro Ile Asp Ala Ala Ala Asp Gly Thr Leu Pro
100 105 110
Leu Asp Lys Val Ala Met Lys Ile Lys Pro Asp Asp Ile His Phe Ala
115 120 125
Arg Thr Lys Leu Leu Ser Leu Glu Asn Thr His Asn Gly Lys Val Leu
130 135 140
Pro Arg Glu Tyr Leu Lys Glu Ala Trp Glu Phe Thr Arg Lys Arg Asn
145 150 155 160
Leu Ala Leu His Val Asp Gly Ala Arg Ile Phe Asn Ala Val Val Ala
165 170 175
Tyr Gly Cys Glu Leu Lys Glu Ile Thr Gln Tyr Cys Asp Ser Phe Thr
180 185 190
Ile Cys Leu Ser Lys Gly Leu Gly Thr Pro Val Gly Ser Leu Leu Val
195 200 205
Gly Asn Arg Asp Tyr Ile Lys Arg Ala Ile Arg Trp Arg Lys Met Thr
210 215 220
Gly Gly Gly Met Arg Gln Ser Gly Ile Leu Ala Ala Ala Gly Met Tyr
225 230 235 240
Ala Leu Lys Asn Asn Val Ala Arg Leu Gln Glu Asp His Asp Asn Thr
245 250 255
Ala Trp Met Ala Glu Gln Leu Arg Glu Ala Gly Ala Asp Val Met Arg
260 265 270
Gln Asp Thr Asn Met Leu Phe Val Arg Val Gly Glu Glu Asn Ala Ala
275 280 285
Ala Leu Gly Glu Tyr Met Lys Ala Arg Asn Val Leu Ile Asn Ala Ser
290 295 300
Pro Ile Val Arg Leu Val Thr His Leu Asp Val Ser Arg Ala Gln Leu
305 310 315 320
Ala Glu Val Ala Ala His Trp Arg Ala Phe Leu Ala Arg
325 330
<210> 51
<211> 333
<212> PRT
<213>E. coli threonine aldolase amino acid mutation (artificial sequence)
<400> 51
Met Ile Asp Leu Arg Ser Asp Thr Val Thr Arg Pro Ser Arg Ala Met
1 5 10 15
Leu Glu Ala Met Met Ala Ala Pro Val Gly Asp Asp Val Tyr Gly Asp
20 25 30
Asp Pro Thr Val Asn Ala Leu Gln Asp Tyr Ala Ala Glu Leu Ser Gly
35 40 45
Lys Glu Ala Ala Ile Phe Leu Pro Thr Gly Thr Gln Ala Asn Leu Val
50 55 60
Ala Leu Leu Ser His Cys Glu Arg Gly Glu Glu Tyr Ile Val Gly Gln
65 70 75 80
Ala Ala Pro Asn Tyr Leu Phe Glu Ala Gly Gly Ala Ala Val Leu Gly
85 90 95
Ser Ile Gln Pro Gln Pro Ile Asp Ala Ala Ala Asp Gly Thr Leu Pro
100 105 110
Leu Asp Lys Val Ala Met Lys Ile Lys Pro Asp Asp Ile His Phe Ala
115 120 125
Arg Thr Lys Leu Leu Ser Leu Glu Asn Thr His Asn Gly Lys Val Leu
130 135 140
Pro Arg Glu Tyr Leu Lys Glu Ala Trp Glu Phe Thr Arg Lys Arg Asn
145 150 155 160
Leu Ala Leu His Val Asp Gly Ala Arg Ile Phe Asn Ala Val Val Ala
165 170 175
Tyr Gly Cys Glu Leu Lys Glu Ile Thr Gln Tyr Cys Asp Ser Phe Thr
180 185 190
Ile Cys Leu Ser Lys Gly Leu Gly Thr Pro Val Gly Ser Leu Leu Val
195 200 205
Gly Asn Arg Asp Tyr Ile Lys Arg Ala Ile Arg Trp Arg Lys Met Thr
210 215 220
Gly Gly Gly Met Arg Gln Ser Gly Ile Leu Ala Ala Ala Gly Met Tyr
225 230 235 240
Ala Leu Lys Asn Asn Val Ala Arg Leu Gln Glu Asp His Asp Asn Thr
245 250 255
Ala Trp Met Ala Glu Gln Leu Arg Glu Ala Gly Ala Asp Val Met Arg
260 265 270
Gln Asp Thr Asn Met Leu Phe Val Arg Val Gly Glu Glu Asn Ala Ala
275 280 285
Ala Leu Gly Glu Tyr Met Lys Ala Arg Asn Val Leu Ile Asn Ala Ser
290 295 300
Pro Ile Val Arg Leu Val Thr His Leu Asp Val Ser Arg Ala Gln Leu
305 310 315 320
Ala Glu Val Ala Ala His Trp Arg Ala Phe Leu Ala Arg
325 330
<210> 52
<211> 333
<212> PRT
<213>E. coli threonine aldolase amino acid mutation (artificial sequence)
<400> 52
Met Ile Asp Leu Arg Ser Asp Thr Val Thr Arg Pro Ser Arg Ala Met
1 5 10 15
Leu Glu Ala Met Met Ala Ala Pro Val Gly Asp Asp Val Tyr Gly Asp
20 25 30
Asp Pro Thr Val Asn Ala Leu Gln Asp Tyr Ala Ala Glu Leu Ser Gly
35 40 45
Lys Glu Ala Ala Ile Phe Leu Pro Thr Gly Thr Gln Ala Asn Leu Val
50 55 60
Ala Leu Leu Ser His Cys Glu Arg Gly Glu Glu Tyr Ile Val Gly Gln
65 70 75 80
Ala Ala His Asn Tyr Leu Phe Glu Ala Gly Gly Ala Ala Val Leu Gly
85 90 95
Ser Ile Gln Pro Gln Pro Ile Asp Ala Ala Ala Asp Gly Thr Leu Pro
100 105 110
Leu Asp Lys Val Ala Met Lys Ile Lys Pro Asp Asp Ile His Phe Ala
115 120 125
Arg Thr Lys Leu Leu Ser Leu Glu Asn Thr His Asn Gly Lys Val Leu
130 135 140
Pro Arg Glu Tyr Leu Lys Glu Ala Trp Glu Phe Thr Arg Lys Arg Asn
145 150 155 160
Leu Ala Leu His Val Asp Gly Tyr Arg Ile Phe Asn Ala Val Val Ala
165 170 175
Tyr Gly Cys Glu Leu Lys Glu Ile Thr Gln Tyr Cys Asp Ser Phe Thr
180 185 190
Ile Cys Leu Ser Lys Gly Leu Gly Thr Pro Val Gly Ser Leu Leu Val
195 200 205
Gly Asn Arg Asp Tyr Ile Lys Arg Ala Ile Arg Trp Arg Lys Met Thr
210 215 220
Gly Gly Gly Met Arg Gln Ser Gly Ile Leu Ala Ala Ala Gly Met Tyr
225 230 235 240
Ala Leu Lys Asn Asn Val Ala Arg Leu Gln Glu Asp His Asp Asn Thr
245 250 255
Ala Trp Met Ala Glu Gln Leu Arg Glu Ala Gly Ala Asp Val Met Arg
260 265 270
Gln Asp Thr Asn Met Leu Phe Val Arg Val Gly Glu Glu Asn Ala Ala
275 280 285
Ala Leu Gly Glu Tyr Met Lys Ala Arg Asn Val Leu Ile Asn Ala Ser
290 295 300
Pro Ile Val Arg Leu Val Thr His Leu Asp Val Ser Arg Ala Gln Leu
305 310 315 320
Ala Glu Val Ala Ala His Trp Arg Ala Phe Leu Ala Arg
325 330
<210> 53
<211> 333
<212> PRT
<213>E. coli threonine aldolase amino acid mutation (artificial sequence)
<400> 53
Met Ile Asp Leu Arg Ser Asp Thr Val Thr Arg Pro Ser Arg Ala Met
1 5 10 15
Leu Glu Ala Met Met Ala Ala Pro Val Gly Asp Asp Val Tyr Gly Asp
20 25 30
Asp Pro Thr Val Asn Ala Leu Gln Asp Tyr Ala Ala Glu Leu Ser Gly
35 40 45
Lys Glu Ala Ala Ile Phe Leu Pro Thr Gly Thr Gln Ala Asn Leu Val
50 55 60
Ala Leu Leu Ser His Cys Glu Arg Gly Glu Glu Tyr Ile Val Gly Gln
65 70 75 80
Ala Ala His Asn Tyr Leu Phe Glu Ala Gly Gly Ala Ala Val Leu Gly
85 90 95
Ser Ile Gln Pro Gln Pro Ile Asp Ala Ala Ala Asp Gly Thr Leu Pro
100 105 110
Leu Asp Lys Val Ala Met Lys Ile Lys Pro Asp Asp Ile His Phe Ala
115 120 125
Arg Thr Lys Leu Leu Ser Leu Glu Asn Thr His Asn Gly Lys Val Leu
130 135 140
Pro Arg Glu Tyr Leu Lys Glu Ala Trp Glu Phe Thr Arg Lys Arg Asn
145 150 155 160
Leu Ala Leu His Val Asp Gly Ala Arg Ile Phe Asn Ala Val Val Ala
165 170 175
Tyr Gly Cys Glu Leu Lys Glu Ile Thr Gln Tyr Cys Asp Ser Phe Thr
180 185 190
Ile Cys Leu Ile Lys Gly Leu Gly Thr Pro Val Gly Ser Leu Leu Val
195 200 205
Gly Asn Arg Asp Tyr Ile Lys Arg Ala Ile Arg Trp Arg Lys Met Thr
210 215 220
Gly Gly Gly Met Arg Gln Ser Gly Ile Leu Ala Ala Ala Gly Met Tyr
225 230 235 240
Ala Leu Lys Asn Asn Val Ala Arg Leu Gln Glu Asp His Asp Asn Thr
245 250 255
Ala Trp Met Ala Glu Gln Leu Arg Glu Ala Gly Ala Asp Val Met Arg
260 265 270
Gln Asp Thr Asn Met Leu Phe Val Arg Val Gly Glu Glu Asn Ala Ala
275 280 285
Ala Leu Gly Glu Tyr Met Lys Ala Arg Asn Val Leu Ile Asn Ala Ser
290 295 300
Pro Ile Val Arg Leu Val Thr His Leu Asp Val Ser Arg Ala Gln Leu
305 310 315 320
Ala Glu Val Ala Ala His Trp Arg Ala Phe Leu Ala Arg
325 330
<210> 54
<211> 338
<212> PRT
<213>aermonas jandaei threonine aldolase amino acid mutation (artificial sequence)
<400> 54
Met Arg Tyr Ile Asp Leu Arg Ser Asp Gln Val Thr Gln Pro Thr Asp
1 5 10 15
Ala Met Arg Gln Cys Met Leu His Ala Glu Val Gly Asp Asp Val Tyr
20 25 30
Gly Glu Asp Pro Gly Val Asn Ala Leu Glu Ala Tyr Gly Ala Asp Leu
35 40 45
Leu Gly Lys Glu Ala Ala Leu Phe Val Pro Ser Gly Thr Met Ser Asn
50 55 60
Leu Leu Ala Val Met Ser His Cys Gln Arg Gly Glu Gly Ala Val Leu
65 70 75 80
Gly Ser Ala Ala His Ile Tyr Arg Tyr Glu Ala Gln Gly Ser Ala Val
85 90 95
Leu Gly Ser Val Ala Leu Gln Pro Val Pro Met Gln Ala Asp Gly Ser
100 105 110
Leu Ala Leu Ala Asp Val Arg Ala Ala Ile Ala Pro Asp Asp Val His
115 120 125
Phe Thr Pro Thr Arg Leu Val Cys Leu Glu Asn Thr His Asn Gly Lys
130 135 140
Val Leu Pro Leu Pro Tyr Leu Arg Glu Met Arg Glu Leu Val Asp Glu
145 150 155 160
His Gly Leu Gln Leu His Leu Asp Gly Ala Arg Leu Phe Asn Ala Val
165 170 175
Val Ala Ser Gly His Thr Val Arg Glu Leu Val Ala Pro Phe Asp Ser
180 185 190
Val Ser Ile Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu
195 200 205
Leu Val Gly Ser His Ala Phe Ile Ala Arg Ala Arg Arg Leu Arg Lys
210 215 220
Met Val Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Gln Ala Gly
225 230 235 240
Leu Phe Ala Leu Gln Gln His Val Val Arg Leu Ala Asp Asp His Arg
245 250 255
Arg Ala Arg Gln Leu Ala Glu Gly Leu Ala Ala Leu Pro Gly Ile Arg
260 265 270
Leu Asp Leu Ala Gln Val Gln Thr Asn Met Val Phe Leu Gln Leu Thr
275 280 285
Ser Gly Glu Ser Ala Pro Leu Leu Ala Phe Met Lys Ala Arg Gly Ile
290 295 300
Leu Phe Ser Gly Tyr Gly Glu Leu Arg Leu Val Thr His Leu Gln Ile
305 310 315 320
His Asp Asp Asp Ile Glu Glu Val Ile Asp Ala Phe Thr Glu Tyr Leu
325 330 335
Gly Ala
<210> 55
<211> 338
<212> PRT
<213>aermonas jandaei threonine aldolase amino acid mutation (artificial sequence)
<400> 55
Met Arg Tyr Ile Asp Leu Arg Ser Asp Thr Val Thr Gln Pro Thr Asp
1 5 10 15
Ala Met Arg Gln Cys Met Leu His Ala Glu Val Gly Asp Asp Val Tyr
20 25 30
Gly Glu Asp Pro Gly Val Asn Ala Leu Glu Ala Tyr Gly Ala Asp Leu
35 40 45
Leu Gly Lys Glu Ala Ala Leu Phe Val Pro Ser Gly Thr Met Ser Asn
50 55 60
Leu Leu Ala Val Met Ser His Cys Gln Arg Gly Glu Gly Ala Val Leu
65 70 75 80
Gly Ser Ala Ala Trp Ile Tyr Arg Tyr Glu Ala Gln Gly Ser Ala Val
85 90 95
Leu Gly Ser Val Ala Leu Gln Pro Val Pro Met Gln Ala Asp Gly Ser
100 105 110
Leu Ala Leu Ala Asp Val Arg Ala Ala Ile Ala Pro Asp Asp Val His
115 120 125
Phe Thr Pro Thr Arg Leu Val Cys Leu Glu Asn Thr His Asn Gly Lys
130 135 140
Val Leu Pro Leu Pro Tyr Leu Arg Glu Met Arg Glu Leu Val Asp Glu
145 150 155 160
His Gly Leu Gln Leu His Leu Asp Gly Ala Arg Leu Phe Asn Ala Val
165 170 175
Val Ala Ser Gly His Thr Val Arg Glu Leu Val Ala Pro Phe Asp Ser
180 185 190
Val Ser Ile Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu
195 200 205
Leu Val Gly Ser His Ala Phe Ile Ala Arg Ala Arg Arg Leu Arg Lys
210 215 220
Met Val Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Gln Ala Gly
225 230 235 240
Leu Phe Ala Leu Gln Gln His Val Val Arg Leu Ala Asp Asp His Arg
245 250 255
Arg Ala Arg Gln Leu Ala Glu Gly Leu Ala Ala Leu Pro Gly Ile Arg
260 265 270
Leu Asp Leu Ala Gln Val Gln Thr Asn Met Val Phe Leu Gln Leu Thr
275 280 285
Ser Gly Glu Ser Ala Pro Leu Leu Ala Phe Met Lys Ala Arg Gly Ile
290 295 300
Leu Phe Ser Gly Tyr Gly Glu Leu Arg Leu Val Thr His Leu Gln Ile
305 310 315 320
His Asp Asp Asp Ile Glu Glu Val Ile Asp Ala Phe Thr Glu Tyr Leu
325 330 335
Gly Ala
<210> 56
<211> 338
<212> PRT
<213>aermonas jandaei threonine aldolase amino acid mutation (artificial sequence)
<400> 56
Met Arg Tyr Ile Asp Leu Arg Ser Asp Thr Val Thr Gln Pro Thr Asp
1 5 10 15
Ala Met Arg Gln Cys Met Leu His Ala Glu Val Gly Asp Asp Val Tyr
20 25 30
Gly Glu Asp Pro Gly Val Asn Ala Leu Glu Ala Tyr Gly Ala Asp Leu
35 40 45
Leu Gly Lys Glu Ala Ala Leu Phe Val Pro Ser Gly Thr Met Ser Asn
50 55 60
Leu Leu Ala Val Met Ser His Cys Gln Arg Gly Glu Gly Ala Val Leu
65 70 75 80
Gly Ser Ala Ala Pro Ile Tyr Arg Tyr Glu Ala Gln Gly Ser Ala Val
85 90 95
Leu Gly Ser Val Ala Leu Gln Pro Val Pro Met Gln Ala Asp Gly Ser
100 105 110
Leu Ala Leu Ala Asp Val Arg Ala Ala Ile Ala Pro Asp Asp Val His
115 120 125
Phe Thr Pro Thr Arg Leu Val Cys Leu Glu Asn Thr His Asn Gly Lys
130 135 140
Val Leu Pro Leu Pro Tyr Leu Arg Glu Met Arg Glu Leu Val Asp Glu
145 150 155 160
His Gly Leu Gln Leu His Leu Asp Gly Ala Arg Leu Phe Asn Ala Val
165 170 175
Val Ala Ser Gly His Thr Val Arg Glu Leu Val Ala Pro Phe Asp Ser
180 185 190
Val Ser Ile Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu
195 200 205
Leu Val Gly Ser His Ala Phe Ile Ala Arg Ala Arg Arg Leu Arg Lys
210 215 220
Met Val Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Gln Ala Gly
225 230 235 240
Leu Phe Ala Leu Gln Gln His Val Val Arg Leu Ala Asp Asp His Arg
245 250 255
Arg Ala Arg Gln Leu Ala Glu Gly Leu Ala Ala Leu Pro Gly Ile Arg
260 265 270
Leu Asp Leu Ala Gln Val Gln Thr Asn Met Val Phe Leu Gln Leu Thr
275 280 285
Ser Gly Glu Ser Ala Pro Leu Leu Ala Phe Met Lys Ala Arg Gly Ile
290 295 300
Leu Phe Ser Gly Tyr Gly Glu Leu Arg Leu Val Thr His Leu Gln Ile
305 310 315 320
His Asp Asp Asp Ile Glu Glu Val Ile Asp Ala Phe Thr Glu Tyr Leu
325 330 335
Gly Ala
<210> 57
<211> 338
<212> PRT
<213>aermonas jandaei threonine aldolase amino acid mutation (artificial sequence)
<400> 57
Met Arg Tyr Ile Asp Leu Arg Ser Asp Thr Val Thr Gln Pro Thr Asp
1 5 10 15
Ala Met Arg Gln Cys Met Leu His Ala Glu Val Gly Asp Asp Val Tyr
20 25 30
Gly Glu Asp Pro Gly Val Asn Ala Leu Glu Ala Tyr Gly Ala Asp Leu
35 40 45
Leu Gly Lys Glu Ala Ala Leu Phe Val Pro Ser Gly Thr Met Ser Asn
50 55 60
Leu Leu Ala Val Met Ser His Cys Gln Arg Gly Glu Gly Ala Val Leu
65 70 75 80
Gly Ser Ala Ala His Ile Tyr Arg Tyr Glu Ala Gln Gly Ser Ala Val
85 90 95
Leu Gly Ser Val Ala Leu Gln Pro Val Pro Met Gln Ala Asp Gly Ser
100 105 110
Leu Ala Leu Ala Asp Val Arg Ala Ala Ile Ala Pro Asp Asp Val His
115 120 125
Phe Thr Pro Thr Arg Leu Val Cys Leu Glu Asn Thr His Asn Gly Lys
130 135 140
Val Leu Pro Leu Pro Tyr Leu Arg Glu Met Arg Glu Leu Val Asp Glu
145 150 155 160
His Gly Leu Gln Leu His Leu Asp Gly Tyr Arg Leu Phe Asn Ala Val
165 170 175
Val Ala Ser Gly His Thr Val Arg Glu Leu Val Ala Pro Phe Asp Ser
180 185 190
Val Ser Ile Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu
195 200 205
Leu Val Gly Ser His Ala Phe Ile Ala Arg Ala Arg Arg Leu Arg Lys
210 215 220
Met Val Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Gln Ala Gly
225 230 235 240
Leu Phe Ala Leu Gln Gln His Val Val Arg Leu Ala Asp Asp His Arg
245 250 255
Arg Ala Arg Gln Leu Ala Glu Gly Leu Ala Ala Leu Pro Gly Ile Arg
260 265 270
Leu Asp Leu Ala Gln Val Gln Thr Asn Met Val Phe Leu Gln Leu Thr
275 280 285
Ser Gly Glu Ser Ala Pro Leu Leu Ala Phe Met Lys Ala Arg Gly Ile
290 295 300
Leu Phe Ser Gly Tyr Gly Glu Leu Arg Leu Val Thr His Leu Gln Ile
305 310 315 320
His Asp Asp Asp Ile Glu Glu Val Ile Asp Ala Phe Thr Glu Tyr Leu
325 330 335
Gly Ala
<210> 58
<211> 338
<212> PRT
<213>aermonas jandaei threonine aldolase amino acid mutation (artificial sequence)
<400> 58
Met Arg Tyr Ile Asp Leu Arg Ser Asp Thr Val Thr Gln Pro Thr Asp
1 5 10 15
Ala Met Arg Gln Cys Met Leu His Ala Glu Val Gly Asp Asp Val Tyr
20 25 30
Gly Glu Asp Pro Gly Val Asn Ala Leu Glu Ala Tyr Gly Ala Asp Leu
35 40 45
Leu Gly Lys Glu Ala Ala Leu Phe Val Pro Ser Gly Thr Met Ser Asn
50 55 60
Leu Leu Ala Val Met Ser His Cys Gln Arg Gly Glu Gly Ala Val Leu
65 70 75 80
Gly Ser Ala Ala His Ile Tyr Arg Tyr Glu Ala Gln Gly Ser Ala Val
85 90 95
Leu Gly Ser Val Ala Leu Gln Pro Val Pro Met Gln Ala Asp Gly Ser
100 105 110
Leu Ala Leu Ala Asp Val Arg Ala Ala Ile Ala Pro Asp Asp Val His
115 120 125
Phe Thr Pro Thr Arg Leu Val Cys Leu Glu Asn Thr His Asn Gly Lys
130 135 140
Val Leu Pro Leu Pro Tyr Leu Arg Glu Met Arg Glu Leu Val Asp Glu
145 150 155 160
His Gly Leu Gln Leu His Leu Asp Gly Ala Arg Leu Phe Asn Ala Val
165 170 175
Val Ala Ser Gly His Thr Val Arg Glu Leu Val Ala Pro Phe Asp Ser
180 185 190
Val Ser Ile Cys Leu Ile Lys Gly Leu Gly Ala Pro Val Gly Ser Leu
195 200 205
Leu Val Gly Ser His Ala Phe Ile Ala Arg Ala Arg Arg Leu Arg Lys
210 215 220
Met Val Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Gln Ala Gly
225 230 235 240
Leu Phe Ala Leu Gln Gln His Val Val Arg Leu Ala Asp Asp His Arg
245 250 255
Arg Ala Arg Gln Leu Ala Glu Gly Leu Ala Ala Leu Pro Gly Ile Arg
260 265 270
Leu Asp Leu Ala Gln Val Gln Thr Asn Met Val Phe Leu Gln Leu Thr
275 280 285
Ser Gly Glu Ser Ala Pro Leu Leu Ala Phe Met Lys Ala Arg Gly Ile
290 295 300
Leu Phe Ser Gly Tyr Gly Glu Leu Arg Leu Val Thr His Leu Gln Ile
305 310 315 320
His Asp Asp Asp Ile Glu Glu Val Ile Asp Ala Phe Thr Glu Tyr Leu
325 330 335
Gly Ala
<210> 59
<211> 387
<212> PRT
<213>saccharomyces cerevisiae threonine aldolase amino acid mutation (artificial sequence)
<400> 59
Met Thr Glu Phe Glu Leu Pro Pro Lys Tyr Ile Thr Ala Ala Asn Asp
1 5 10 15
Leu Arg Ser Asp Gln Phe Thr Thr Pro Thr Ala Glu Met Met Glu Ala
20 25 30
Ala Leu Glu Ala Ser Ile Gly Asp Ala Val Tyr Gly Glu Asp Val Asp
35 40 45
Thr Val Arg Leu Glu Gln Thr Val Ala Arg Met Ala Gly Lys Glu Ala
50 55 60
Gly Leu Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Ile Arg
65 70 75 80
Thr His Leu Met Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg Ala
85 90 95
His Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln Ala
100 105 110
Met Val Val Pro Val Val Pro Ser Asn Gly Asp Tyr Leu Thr Leu Glu
115 120 125
Asp Ile Lys Ser His Tyr Val Pro Asp Asp Gly Asp Ile His Gly Ala
130 135 140
Pro Thr Arg Leu Ile Ser Leu Glu Asn Thr Leu His Gly Ile Val Tyr
145 150 155 160
Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn Gly
165 170 175
Leu Lys Leu His Cys Asp Gly Ala Arg Ile Trp Asn Ala Ala Ala Gln
180 185 190
Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Ile Phe Asp Ser Ile Ser
195 200 205
Ile Cys Leu Ser Lys Ser Met Gly Ala Pro Ile Gly Ser Val Leu Val
210 215 220
Gly Asn Leu Lys Phe Val Lys Lys Ala Thr His Phe Arg Lys Gln Gln
225 230 235 240
Gly Gly Gly Ile Arg Gln Ser Gly Met Met Ala Arg Met Ala Leu Val
245 250 255
Asn Ile Asn Asn Asp Trp Lys Ser Gln Leu Leu Tyr Ser His Ser Leu
260 265 270
Ala His Glu Leu Ala Glu Tyr Cys Glu Ala Lys Gly Ile Pro Leu Glu
275 280 285
Ser Pro Ala Asp Thr Asn Phe Val Phe Ile Asn Leu Lys Ala Ala Arg
290 295 300
Met Asp Pro Asp Val Leu Val Lys Lys Gly Leu Lys Tyr Asn Val Lys
305 310 315 320
Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Val Thr Arg Asp Thr
325 330 335
Leu Glu Lys Val Lys Leu Ala Ile Ser Glu Ala Phe Asp Tyr Ala Lys
340 345 350
Glu His Pro Phe Asp Cys Asn Gly Pro Thr Gln Ile Tyr Arg Ser Glu
355 360 365
Ser Thr Glu Val Asp Val Asp Gly Asn Ala Ile Arg Glu Ile Lys Thr
370 375 380
Tyr Lys Tyr
385
<210> 60
<211> 387
<212> PRT
<213>saccharomyces cerevisiae threonine aldolase amino acid mutation (artificial sequence)
<400> 60
Met Thr Glu Phe Glu Leu Pro Pro Lys Tyr Ile Thr Ala Ala Asn Asp
1 5 10 15
Leu Arg Ser Asp Thr Phe Thr Thr Pro Thr Ala Glu Met Met Glu Ala
20 25 30
Ala Leu Glu Ala Ser Ile Gly Asp Ala Val Tyr Gly Glu Asp Val Asp
35 40 45
Thr Val Arg Leu Glu Gln Thr Val Ala Arg Met Ala Gly Lys Glu Ala
50 55 60
Gly Leu Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Ile Arg
65 70 75 80
Thr His Leu Met Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg Ala
85 90 95
Trp Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln Ala
100 105 110
Met Val Val Pro Val Val Pro Ser Asn Gly Asp Tyr Leu Thr Leu Glu
115 120 125
Asp Ile Lys Ser His Tyr Val Pro Asp Asp Gly Asp Ile His Gly Ala
130 135 140
Pro Thr Arg Leu Ile Ser Leu Glu Asn Thr Leu His Gly Ile Val Tyr
145 150 155 160
Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn Gly
165 170 175
Leu Lys Leu His Cys Asp Gly Ala Arg Ile Trp Asn Ala Ala Ala Gln
180 185 190
Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Ile Phe Asp Ser Ile Ser
195 200 205
Ile Cys Leu Ser Lys Ser Met Gly Ala Pro Ile Gly Ser Val Leu Val
210 215 220
Gly Asn Leu Lys Phe Val Lys Lys Ala Thr His Phe Arg Lys Gln Gln
225 230 235 240
Gly Gly Gly Ile Arg Gln Ser Gly Met Met Ala Arg Met Ala Leu Val
245 250 255
Asn Ile Asn Asn Asp Trp Lys Ser Gln Leu Leu Tyr Ser His Ser Leu
260 265 270
Ala His Glu Leu Ala Glu Tyr Cys Glu Ala Lys Gly Ile Pro Leu Glu
275 280 285
Ser Pro Ala Asp Thr Asn Phe Val Phe Ile Asn Leu Lys Ala Ala Arg
290 295 300
Met Asp Pro Asp Val Leu Val Lys Lys Gly Leu Lys Tyr Asn Val Lys
305 310 315 320
Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Val Thr Arg Asp Thr
325 330 335
Leu Glu Lys Val Lys Leu Ala Ile Ser Glu Ala Phe Asp Tyr Ala Lys
340 345 350
Glu His Pro Phe Asp Cys Asn Gly Pro Thr Gln Ile Tyr Arg Ser Glu
355 360 365
Ser Thr Glu Val Asp Val Asp Gly Asn Ala Ile Arg Glu Ile Lys Thr
370 375 380
Tyr Lys Tyr
385
<210> 61
<211> 387
<212> PRT
<213>saccharomyces cerevisiae threonine aldolase amino acid mutation (artificial sequence)
<400> 61
Met Thr Glu Phe Glu Leu Pro Pro Lys Tyr Ile Thr Ala Ala Asn Asp
1 5 10 15
Leu Arg Ser Asp Thr Phe Thr Thr Pro Thr Ala Glu Met Met Glu Ala
20 25 30
Ala Leu Glu Ala Ser Ile Gly Asp Ala Val Tyr Gly Glu Asp Val Asp
35 40 45
Thr Val Arg Leu Glu Gln Thr Val Ala Arg Met Ala Gly Lys Glu Ala
50 55 60
Gly Leu Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Ile Arg
65 70 75 80
Thr His Leu Met Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg Ala
85 90 95
Pro Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln Ala
100 105 110
Met Val Val Pro Val Val Pro Ser Asn Gly Asp Tyr Leu Thr Leu Glu
115 120 125
Asp Ile Lys Ser His Tyr Val Pro Asp Asp Gly Asp Ile His Gly Ala
130 135 140
Pro Thr Arg Leu Ile Ser Leu Glu Asn Thr Leu His Gly Ile Val Tyr
145 150 155 160
Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn Gly
165 170 175
Leu Lys Leu His Cys Asp Gly Ala Arg Ile Trp Asn Ala Ala Ala Gln
180 185 190
Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Ile Phe Asp Ser Ile Ser
195 200 205
Ile Cys Leu Ser Lys Ser Met Gly Ala Pro Ile Gly Ser Val Leu Val
210 215 220
Gly Asn Leu Lys Phe Val Lys Lys Ala Thr His Phe Arg Lys Gln Gln
225 230 235 240
Gly Gly Gly Ile Arg Gln Ser Gly Met Met Ala Arg Met Ala Leu Val
245 250 255
Asn Ile Asn Asn Asp Trp Lys Ser Gln Leu Leu Tyr Ser His Ser Leu
260 265 270
Ala His Glu Leu Ala Glu Tyr Cys Glu Ala Lys Gly Ile Pro Leu Glu
275 280 285
Ser Pro Ala Asp Thr Asn Phe Val Phe Ile Asn Leu Lys Ala Ala Arg
290 295 300
Met Asp Pro Asp Val Leu Val Lys Lys Gly Leu Lys Tyr Asn Val Lys
305 310 315 320
Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Val Thr Arg Asp Thr
325 330 335
Leu Glu Lys Val Lys Leu Ala Ile Ser Glu Ala Phe Asp Tyr Ala Lys
340 345 350
Glu His Pro Phe Asp Cys Asn Gly Pro Thr Gln Ile Tyr Arg Ser Glu
355 360 365
Ser Thr Glu Val Asp Val Asp Gly Asn Ala Ile Arg Glu Ile Lys Thr
370 375 380
Tyr Lys Tyr
385
<210> 62
<211> 387
<212> PRT
<213>saccharomyces cerevisiae threonine aldolase amino acid mutation (artificial sequence)
<400> 62
Met Thr Glu Phe Glu Leu Pro Pro Lys Tyr Ile Thr Ala Ala Asn Asp
1 5 10 15
Leu Arg Ser Asp Thr Phe Thr Thr Pro Thr Ala Glu Met Met Glu Ala
20 25 30
Ala Leu Glu Ala Ser Ile Gly Asp Ala Val Tyr Gly Glu Asp Val Asp
35 40 45
Thr Val Arg Leu Glu Gln Thr Val Ala Arg Met Ala Gly Lys Glu Ala
50 55 60
Gly Leu Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Ile Arg
65 70 75 80
Thr His Leu Met Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg Ala
85 90 95
His Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln Ala
100 105 110
Met Val Val Pro Val Val Pro Ser Asn Gly Asp Tyr Leu Thr Leu Glu
115 120 125
Asp Ile Lys Ser His Tyr Val Pro Asp Asp Gly Asp Ile His Gly Ala
130 135 140
Pro Thr Arg Leu Ile Ser Leu Glu Asn Thr Leu His Gly Ile Val Tyr
145 150 155 160
Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn Gly
165 170 175
Leu Lys Leu His Cys Asp Gly Tyr Arg Ile Trp Asn Ala Ala Ala Gln
180 185 190
Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Ile Phe Asp Ser Ile Ser
195 200 205
Ile Cys Leu Ser Lys Ser Met Gly Ala Pro Ile Gly Ser Val Leu Val
210 215 220
Gly Asn Leu Lys Phe Val Lys Lys Ala Thr His Phe Arg Lys Gln Gln
225 230 235 240
Gly Gly Gly Ile Arg Gln Ser Gly Met Met Ala Arg Met Ala Leu Val
245 250 255
Asn Ile Asn Asn Asp Trp Lys Ser Gln Leu Leu Tyr Ser His Ser Leu
260 265 270
Ala His Glu Leu Ala Glu Tyr Cys Glu Ala Lys Gly Ile Pro Leu Glu
275 280 285
Ser Pro Ala Asp Thr Asn Phe Val Phe Ile Asn Leu Lys Ala Ala Arg
290 295 300
Met Asp Pro Asp Val Leu Val Lys Lys Gly Leu Lys Tyr Asn Val Lys
305 310 315 320
Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Val Thr Arg Asp Thr
325 330 335
Leu Glu Lys Val Lys Leu Ala Ile Ser Glu Ala Phe Asp Tyr Ala Lys
340 345 350
Glu His Pro Phe Asp Cys Asn Gly Pro Thr Gln Ile Tyr Arg Ser Glu
355 360 365
Ser Thr Glu Val Asp Val Asp Gly Asn Ala Ile Arg Glu Ile Lys Thr
370 375 380
Tyr Lys Tyr
385
<210> 63
<211> 387
<212> PRT
<213>saccharomyces cerevisiae threonine aldolase amino acid mutation (artificial sequence)
<400> 63
Met Thr Glu Phe Glu Leu Pro Pro Lys Tyr Ile Thr Ala Ala Asn Asp
1 5 10 15
Leu Arg Ser Asp Thr Phe Thr Thr Pro Thr Ala Glu Met Met Glu Ala
20 25 30
Ala Leu Glu Ala Ser Ile Gly Asp Ala Val Tyr Gly Glu Asp Val Asp
35 40 45
Thr Val Arg Leu Glu Gln Thr Val Ala Arg Met Ala Gly Lys Glu Ala
50 55 60
Gly Leu Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Ile Arg
65 70 75 80
Thr His Leu Met Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg Ala
85 90 95
His Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln Ala
100 105 110
Met Val Val Pro Val Val Pro Ser Asn Gly Asp Tyr Leu Thr Leu Glu
115 120 125
Asp Ile Lys Ser His Tyr Val Pro Asp Asp Gly Asp Ile His Gly Ala
130 135 140
Pro Thr Arg Leu Ile Ser Leu Glu Asn Thr Leu His Gly Ile Val Tyr
145 150 155 160
Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn Gly
165 170 175
Leu Lys Leu His Cys Asp Gly Ala Arg Ile Trp Asn Ala Ala Ala Gln
180 185 190
Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Ile Phe Asp Ser Ile Ser
195 200 205
Ile Cys Leu Ile Lys Ser Met Gly Ala Pro Ile Gly Ser Val Leu Val
210 215 220
Gly Asn Leu Lys Phe Val Lys Lys Ala Thr His Phe Arg Lys Gln Gln
225 230 235 240
Gly Gly Gly Ile Arg Gln Ser Gly Met Met Ala Arg Met Ala Leu Val
245 250 255
Asn Ile Asn Asn Asp Trp Lys Ser Gln Leu Leu Tyr Ser His Ser Leu
260 265 270
Ala His Glu Leu Ala Glu Tyr Cys Glu Ala Lys Gly Ile Pro Leu Glu
275 280 285
Ser Pro Ala Asp Thr Asn Phe Val Phe Ile Asn Leu Lys Ala Ala Arg
290 295 300
Met Asp Pro Asp Val Leu Val Lys Lys Gly Leu Lys Tyr Asn Val Lys
305 310 315 320
Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Val Thr Arg Asp Thr
325 330 335
Leu Glu Lys Val Lys Leu Ala Ile Ser Glu Ala Phe Asp Tyr Ala Lys
340 345 350
Glu His Pro Phe Asp Cys Asn Gly Pro Thr Gln Ile Tyr Arg Ser Glu
355 360 365
Ser Thr Glu Val Asp Val Asp Gly Asn Ala Ile Arg Glu Ile Lys Thr
370 375 380
Tyr Lys Tyr
385
<210> 64
<211> 382
<212> PRT
<213>ashbya gossypii bacterium threonine aldolase amino acid mutation (artificial sequence)
<400> 64
Met Asn Gln Asp Met Glu Leu Pro Glu Ala Tyr Thr Ser Ala Ser Asn
1 5 10 15
Asp Phe Arg Ser Asp Gln Phe Thr Thr Pro Thr Arg Glu Met Ile Glu
20 25 30
Ala Ala Leu Thr Ala Thr Ile Gly Asp Ala Val Tyr Gln Glu Asp Ile
35 40 45
Asp Thr Leu Lys Leu Glu Gln His Val Ala Lys Leu Ala Gly Met Glu
50 55 60
Ala Gly Met Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Leu
65 70 75 80
Arg Thr His Leu Thr Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg
85 90 95
Ala His Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln
100 105 110
Ala Met Val Thr Pro Val Ile Pro Ser Asn Gly Asn Tyr Leu Thr Leu
115 120 125
Glu Asp Ile Lys Lys His Tyr Ile Pro Asp Asp Gly Asp Ile His Gly
130 135 140
Ala Pro Thr Lys Val Ile Ser Leu Glu Asn Thr Leu His Gly Ile Ile
145 150 155 160
His Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn
165 170 175
Asp Leu Arg Leu His Cys Asp Gly Ala Arg Ile Trp Asn Ala Ser Ala
180 185 190
Glu Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Leu Phe Asp Ser Ile
195 200 205
Ser Ile Cys Leu Ser Lys Ser Met Gly Ala Pro Met Gly Ser Ile Leu
210 215 220
Val Gly Ser His Lys Phe Ile Lys Lys Ala Asn His Phe Arg Lys Gln
225 230 235 240
Gln Gly Gly Gly Val Arg Gln Ser Gly Met Met Cys Lys Met Ala Met
245 250 255
Val Ala Ile Gln Gly Asp Trp Lys Gly Lys Met Arg Arg Ser His Arg
260 265 270
Met Ala His Glu Leu Ala Arg Phe Cys Ala Glu His Gly Ile Pro Leu
275 280 285
Glu Ser Pro Ala Asp Thr Asn Phe Val Phe Leu Asp Leu Gln Lys Ser
290 295 300
Lys Met Asn Pro Asp Val Leu Val Lys Lys Ser Leu Lys Tyr Gly Cys
305 310 315 320
Lys Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Ile Ser Glu Glu
325 330 335
Ser Leu Glu Lys Ile Lys Gln Ala Ile Leu Glu Ala Phe Glu Tyr Ser
340 345 350
Lys Lys Asn Pro Tyr Asp Glu Asn Gly Pro Thr Lys Ile Tyr Arg Ser
355 360 365
Glu Ser Ala Asp Ala Val Gly Glu Ile Lys Thr Tyr Lys Tyr
370 375 380
<210> 65
<211> 382
<212> PRT
<213>ashbya gossypii bacterium threonine aldolase amino acid mutation (artificial sequence)
<400> 65
Met Asn Gln Asp Met Glu Leu Pro Glu Ala Tyr Thr Ser Ala Ser Asn
1 5 10 15
Asp Phe Arg Ser Asp Thr Phe Thr Thr Pro Thr Arg Glu Met Ile Glu
20 25 30
Ala Ala Leu Thr Ala Thr Ile Gly Asp Ala Val Tyr Gln Glu Asp Ile
35 40 45
Asp Thr Leu Lys Leu Glu Gln His Val Ala Lys Leu Ala Gly Met Glu
50 55 60
Ala Gly Met Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Leu
65 70 75 80
Arg Thr His Leu Thr Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg
85 90 95
Ala Trp Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln
100 105 110
Ala Met Val Thr Pro Val Ile Pro Ser Asn Gly Asn Tyr Leu Thr Leu
115 120 125
Glu Asp Ile Lys Lys His Tyr Ile Pro Asp Asp Gly Asp Ile His Gly
130 135 140
Ala Pro Thr Lys Val Ile Ser Leu Glu Asn Thr Leu His Gly Ile Ile
145 150 155 160
His Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn
165 170 175
Asp Leu Arg Leu His Cys Asp Gly Ala Arg Ile Trp Asn Ala Ser Ala
180 185 190
Glu Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Leu Phe Asp Ser Ile
195 200 205
Ser Ile Cys Leu Ser Lys Ser Met Gly Ala Pro Met Gly Ser Ile Leu
210 215 220
Val Gly Ser His Lys Phe Ile Lys Lys Ala Asn His Phe Arg Lys Gln
225 230 235 240
Gln Gly Gly Gly Val Arg Gln Ser Gly Met Met Cys Lys Met Ala Met
245 250 255
Val Ala Ile Gln Gly Asp Trp Lys Gly Lys Met Arg Arg Ser His Arg
260 265 270
Met Ala His Glu Leu Ala Arg Phe Cys Ala Glu His Gly Ile Pro Leu
275 280 285
Glu Ser Pro Ala Asp Thr Asn Phe Val Phe Leu Asp Leu Gln Lys Ser
290 295 300
Lys Met Asn Pro Asp Val Leu Val Lys Lys Ser Leu Lys Tyr Gly Cys
305 310 315 320
Lys Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Ile Ser Glu Glu
325 330 335
Ser Leu Glu Lys Ile Lys Gln Ala Ile Leu Glu Ala Phe Glu Tyr Ser
340 345 350
Lys Lys Asn Pro Tyr Asp Glu Asn Gly Pro Thr Lys Ile Tyr Arg Ser
355 360 365
Glu Ser Ala Asp Ala Val Gly Glu Ile Lys Thr Tyr Lys Tyr
370 375 380
<210> 66
<211> 382
<212> PRT
<213>ashbya gossypii bacterium threonine aldolase amino acid mutation (artificial sequence)
<400> 66
Met Asn Gln Asp Met Glu Leu Pro Glu Ala Tyr Thr Ser Ala Ser Asn
1 5 10 15
Asp Phe Arg Ser Asp Thr Phe Thr Thr Pro Thr Arg Glu Met Ile Glu
20 25 30
Ala Ala Leu Thr Ala Thr Ile Gly Asp Ala Val Tyr Gln Glu Asp Ile
35 40 45
Asp Thr Leu Lys Leu Glu Gln His Val Ala Lys Leu Ala Gly Met Glu
50 55 60
Ala Gly Met Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Leu
65 70 75 80
Arg Thr His Leu Thr Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg
85 90 95
Ala Pro Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln
100 105 110
Ala Met Val Thr Pro Val Ile Pro Ser Asn Gly Asn Tyr Leu Thr Leu
115 120 125
Glu Asp Ile Lys Lys His Tyr Ile Pro Asp Asp Gly Asp Ile His Gly
130 135 140
Ala Pro Thr Lys Val Ile Ser Leu Glu Asn Thr Leu His Gly Ile Ile
145 150 155 160
His Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn
165 170 175
Asp Leu Arg Leu His Cys Asp Gly Ala Arg Ile Trp Asn Ala Ser Ala
180 185 190
Glu Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Leu Phe Asp Ser Ile
195 200 205
Ser Ile Cys Leu Ser Lys Ser Met Gly Ala Pro Met Gly Ser Ile Leu
210 215 220
Val Gly Ser His Lys Phe Ile Lys Lys Ala Asn His Phe Arg Lys Gln
225 230 235 240
Gln Gly Gly Gly Val Arg Gln Ser Gly Met Met Cys Lys Met Ala Met
245 250 255
Val Ala Ile Gln Gly Asp Trp Lys Gly Lys Met Arg Arg Ser His Arg
260 265 270
Met Ala His Glu Leu Ala Arg Phe Cys Ala Glu His Gly Ile Pro Leu
275 280 285
Glu Ser Pro Ala Asp Thr Asn Phe Val Phe Leu Asp Leu Gln Lys Ser
290 295 300
Lys Met Asn Pro Asp Val Leu Val Lys Lys Ser Leu Lys Tyr Gly Cys
305 310 315 320
Lys Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Ile Ser Glu Glu
325 330 335
Ser Leu Glu Lys Ile Lys Gln Ala Ile Leu Glu Ala Phe Glu Tyr Ser
340 345 350
Lys Lys Asn Pro Tyr Asp Glu Asn Gly Pro Thr Lys Ile Tyr Arg Ser
355 360 365
Glu Ser Ala Asp Ala Val Gly Glu Ile Lys Thr Tyr Lys Tyr
370 375 380
<210> 67
<211> 382
<212> PRT
<213>ashbya gossypii bacterium threonine aldolase amino acid mutation (artificial sequence)
<400> 67
Met Asn Gln Asp Met Glu Leu Pro Glu Ala Tyr Thr Ser Ala Ser Asn
1 5 10 15
Asp Phe Arg Ser Asp Thr Phe Thr Thr Pro Thr Arg Glu Met Ile Glu
20 25 30
Ala Ala Leu Thr Ala Thr Ile Gly Asp Ala Val Tyr Gln Glu Asp Ile
35 40 45
Asp Thr Leu Lys Leu Glu Gln His Val Ala Lys Leu Ala Gly Met Glu
50 55 60
Ala Gly Met Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Leu
65 70 75 80
Arg Thr His Leu Thr Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg
85 90 95
Ala His Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln
100 105 110
Ala Met Val Thr Pro Val Ile Pro Ser Asn Gly Asn Tyr Leu Thr Leu
115 120 125
Glu Asp Ile Lys Lys His Tyr Ile Pro Asp Asp Gly Asp Ile His Gly
130 135 140
Ala Pro Thr Lys Val Ile Ser Leu Glu Asn Thr Leu His Gly Ile Ile
145 150 155 160
His Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn
165 170 175
Asp Leu Arg Leu His Cys Asp Gly Tyr Arg Ile Trp Asn Ala Ser Ala
180 185 190
Glu Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Leu Phe Asp Ser Ile
195 200 205
Ser Ile Cys Leu Ser Lys Ser Met Gly Ala Pro Met Gly Ser Ile Leu
210 215 220
Val Gly Ser His Lys Phe Ile Lys Lys Ala Asn His Phe Arg Lys Gln
225 230 235 240
Gln Gly Gly Gly Val Arg Gln Ser Gly Met Met Cys Lys Met Ala Met
245 250 255
Val Ala Ile Gln Gly Asp Trp Lys Gly Lys Met Arg Arg Ser His Arg
260 265 270
Met Ala His Glu Leu Ala Arg Phe Cys Ala Glu His Gly Ile Pro Leu
275 280 285
Glu Ser Pro Ala Asp Thr Asn Phe Val Phe Leu Asp Leu Gln Lys Ser
290 295 300
Lys Met Asn Pro Asp Val Leu Val Lys Lys Ser Leu Lys Tyr Gly Cys
305 310 315 320
Lys Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Ile Ser Glu Glu
325 330 335
Ser Leu Glu Lys Ile Lys Gln Ala Ile Leu Glu Ala Phe Glu Tyr Ser
340 345 350
Lys Lys Asn Pro Tyr Asp Glu Asn Gly Pro Thr Lys Ile Tyr Arg Ser
355 360 365
Glu Ser Ala Asp Ala Val Gly Glu Ile Lys Thr Tyr Lys Tyr
370 375 380
<210> 68
<211> 382
<212> PRT
<213>ashbya gossypii bacterium threonine aldolase amino acid mutation (artificial sequence)
<400> 68
Met Asn Gln Asp Met Glu Leu Pro Glu Ala Tyr Thr Ser Ala Ser Asn
1 5 10 15
Asp Phe Arg Ser Asp Thr Phe Thr Thr Pro Thr Arg Glu Met Ile Glu
20 25 30
Ala Ala Leu Thr Ala Thr Ile Gly Asp Ala Val Tyr Gln Glu Asp Ile
35 40 45
Asp Thr Leu Lys Leu Glu Gln His Val Ala Lys Leu Ala Gly Met Glu
50 55 60
Ala Gly Met Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Leu
65 70 75 80
Arg Thr His Leu Thr Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg
85 90 95
Ala His Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln
100 105 110
Ala Met Val Thr Pro Val Ile Pro Ser Asn Gly Asn Tyr Leu Thr Leu
115 120 125
Glu Asp Ile Lys Lys His Tyr Ile Pro Asp Asp Gly Asp Ile His Gly
130 135 140
Ala Pro Thr Lys Val Ile Ser Leu Glu Asn Thr Leu His Gly Ile Ile
145 150 155 160
His Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn
165 170 175
Asp Leu Arg Leu His Cys Asp Gly Ala Arg Ile Trp Asn Ala Ser Ala
180 185 190
Glu Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Leu Phe Asp Ser Ile
195 200 205
Ser Ile Cys Leu Ile Lys Ser Met Gly Ala Pro Met Gly Ser Ile Leu
210 215 220
Val Gly Ser His Lys Phe Ile Lys Lys Ala Asn His Phe Arg Lys Gln
225 230 235 240
Gln Gly Gly Gly Val Arg Gln Ser Gly Met Met Cys Lys Met Ala Met
245 250 255
Val Ala Ile Gln Gly Asp Trp Lys Gly Lys Met Arg Arg Ser His Arg
260 265 270
Met Ala His Glu Leu Ala Arg Phe Cys Ala Glu His Gly Ile Pro Leu
275 280 285
Glu Ser Pro Ala Asp Thr Asn Phe Val Phe Leu Asp Leu Gln Lys Ser
290 295 300
Lys Met Asn Pro Asp Val Leu Val Lys Lys Ser Leu Lys Tyr Gly Cys
305 310 315 320
Lys Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Ile Ser Glu Glu
325 330 335
Ser Leu Glu Lys Ile Lys Gln Ala Ile Leu Glu Ala Phe Glu Tyr Ser
340 345 350
Lys Lys Asn Pro Tyr Asp Glu Asn Gly Pro Thr Lys Ile Tyr Arg Ser
355 360 365
Glu Ser Ala Asp Ala Val Gly Glu Ile Lys Thr Tyr Lys Tyr
370 375 380
<210> 69
<211> 335
<212> PRT
<213>Bao Shewanella threonine aldolase amino acid mutation (artificial sequence)
<400> 69
Met Ile Asp Phe Arg Ser Asp Gln Val Thr Gln Pro Thr Ala Ala Met
1 5 10 15
Arg Arg Ala Met Ala Asp Ala Gln Val Gly Asp Asp Val Tyr Gly Asp
20 25 30
Asp Pro Ser Val Asn Arg Leu Glu Ala Met Ala Ala Glu Arg Phe Gly
35 40 45
Phe Asp Ser Ala Leu Phe Thr Ser Ser Gly Thr Gln Ala Asn Leu Leu
50 55 60
Ala Leu Met Ser His Cys Asp Arg Gly Asp Glu Tyr Leu Cys Gly Gln
65 70 75 80
Gln Ala His Asn Tyr Lys Phe Glu Gly Gly Gly Ala Ala Val Leu Gly
85 90 95
Ser Ile Gln Pro Gln Pro Leu Thr Asn Gln Pro Asp Gly Ser Ile Leu
100 105 110
Leu Ser Asp Ile Glu Ala Ala Ile Lys Pro Asp Asp Phe His Phe Ala
115 120 125
Arg Thr Arg Leu Leu Ser Leu Glu Asn Thr Ile Gly Gly Lys Val Leu
130 135 140
Pro Gln Ser Tyr Leu Ala Glu Ala Gln Ala Leu Ala Phe Asn Lys Arg
145 150 155 160
Leu Lys Ile His Leu Asp Gly Ala Arg Ile Ala Asn Ala Ala Val Ala
165 170 175
His Asn Leu Asp Ile Ala Asp Ile Thr Gln Tyr Phe Asp Ser Val Ser
180 185 190
Ile Cys Leu Ser Lys Gly Leu Cys Ala Pro Val Gly Ser Leu Leu Leu
195 200 205
Gly Asp Glu Arg Leu Ile Asn Lys Ala Arg Arg Trp Arg Lys Met Leu
210 215 220
Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Val Ala Gly Glu Ile
225 230 235 240
Ala Leu Asn Glu Gln Val Ser Arg Leu Ala Val Asp His Glu Asn Ala
245 250 255
Arg Tyr Leu Ala Glu Gln Leu Ser Cys Leu Asp Glu Phe Glu Val Asp
260 265 270
Leu Gly Glu Val Gln Thr Asn Met Leu Phe Ala Arg Val Val Glu Gly
275 280 285
Val Ala Ile Asp Lys Leu Ala Thr Ser Leu Lys Ala Ser Gly Ile Leu
290 295 300
Ile Ser Pro Gly Lys Thr Leu Arg Met Val Thr His Ala Asp Ile Arg
305 310 315 320
Leu Glu Asp Ile Asp Leu Phe Ile Asp Lys Leu Lys Ser Leu Leu
325 330 335
<210> 70
<211> 335
<212> PRT
<213>Bao Shewanella threonine aldolase amino acid mutation (artificial sequence)
<400> 70
Met Ile Asp Phe Arg Ser Asp Thr Val Thr Gln Pro Thr Ala Ala Met
1 5 10 15
Arg Arg Ala Met Ala Asp Ala Gln Val Gly Asp Asp Val Tyr Gly Asp
20 25 30
Asp Pro Ser Val Asn Arg Leu Glu Ala Met Ala Ala Glu Arg Phe Gly
35 40 45
Phe Asp Ser Ala Leu Phe Thr Ser Ser Gly Thr Gln Ala Asn Leu Leu
50 55 60
Ala Leu Met Ser His Cys Asp Arg Gly Asp Glu Tyr Leu Cys Gly Gln
65 70 75 80
Gln Ala Trp Asn Tyr Lys Phe Glu Gly Gly Gly Ala Ala Val Leu Gly
85 90 95
Ser Ile Gln Pro Gln Pro Leu Thr Asn Gln Pro Asp Gly Ser Ile Leu
100 105 110
Leu Ser Asp Ile Glu Ala Ala Ile Lys Pro Asp Asp Phe His Phe Ala
115 120 125
Arg Thr Arg Leu Leu Ser Leu Glu Asn Thr Ile Gly Gly Lys Val Leu
130 135 140
Pro Gln Ser Tyr Leu Ala Glu Ala Gln Ala Leu Ala Phe Asn Lys Arg
145 150 155 160
Leu Lys Ile His Leu Asp Gly Ala Arg Ile Ala Asn Ala Ala Val Ala
165 170 175
His Asn Leu Asp Ile Ala Asp Ile Thr Gln Tyr Phe Asp Ser Val Ser
180 185 190
Ile Cys Leu Ser Lys Gly Leu Cys Ala Pro Val Gly Ser Leu Leu Leu
195 200 205
Gly Asp Glu Arg Leu Ile Asn Lys Ala Arg Arg Trp Arg Lys Met Leu
210 215 220
Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Val Ala Gly Glu Ile
225 230 235 240
Ala Leu Asn Glu Gln Val Ser Arg Leu Ala Val Asp His Glu Asn Ala
245 250 255
Arg Tyr Leu Ala Glu Gln Leu Ser Cys Leu Asp Glu Phe Glu Val Asp
260 265 270
Leu Gly Glu Val Gln Thr Asn Met Leu Phe Ala Arg Val Val Glu Gly
275 280 285
Val Ala Ile Asp Lys Leu Ala Thr Ser Leu Lys Ala Ser Gly Ile Leu
290 295 300
Ile Ser Pro Gly Lys Thr Leu Arg Met Val Thr His Ala Asp Ile Arg
305 310 315 320
Leu Glu Asp Ile Asp Leu Phe Ile Asp Lys Leu Lys Ser Leu Leu
325 330 335
<210> 71
<211> 335
<212> PRT
<213>Bao Shewanella threonine aldolase amino acid mutation (artificial sequence)
<400> 71
Met Ile Asp Phe Arg Ser Asp Thr Val Thr Gln Pro Thr Ala Ala Met
1 5 10 15
Arg Arg Ala Met Ala Asp Ala Gln Val Gly Asp Asp Val Tyr Gly Asp
20 25 30
Asp Pro Ser Val Asn Arg Leu Glu Ala Met Ala Ala Glu Arg Phe Gly
35 40 45
Phe Asp Ser Ala Leu Phe Thr Ser Ser Gly Thr Gln Ala Asn Leu Leu
50 55 60
Ala Leu Met Ser His Cys Asp Arg Gly Asp Glu Tyr Leu Cys Gly Gln
65 70 75 80
Gln Ala Pro Asn Tyr Lys Phe Glu Gly Gly Gly Ala Ala Val Leu Gly
85 90 95
Ser Ile Gln Pro Gln Pro Leu Thr Asn Gln Pro Asp Gly Ser Ile Leu
100 105 110
Leu Ser Asp Ile Glu Ala Ala Ile Lys Pro Asp Asp Phe His Phe Ala
115 120 125
Arg Thr Arg Leu Leu Ser Leu Glu Asn Thr Ile Gly Gly Lys Val Leu
130 135 140
Pro Gln Ser Tyr Leu Ala Glu Ala Gln Ala Leu Ala Phe Asn Lys Arg
145 150 155 160
Leu Lys Ile His Leu Asp Gly Ala Arg Ile Ala Asn Ala Ala Val Ala
165 170 175
His Asn Leu Asp Ile Ala Asp Ile Thr Gln Tyr Phe Asp Ser Val Ser
180 185 190
Ile Cys Leu Ser Lys Gly Leu Cys Ala Pro Val Gly Ser Leu Leu Leu
195 200 205
Gly Asp Glu Arg Leu Ile Asn Lys Ala Arg Arg Trp Arg Lys Met Leu
210 215 220
Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Val Ala Gly Glu Ile
225 230 235 240
Ala Leu Asn Glu Gln Val Ser Arg Leu Ala Val Asp His Glu Asn Ala
245 250 255
Arg Tyr Leu Ala Glu Gln Leu Ser Cys Leu Asp Glu Phe Glu Val Asp
260 265 270
Leu Gly Glu Val Gln Thr Asn Met Leu Phe Ala Arg Val Val Glu Gly
275 280 285
Val Ala Ile Asp Lys Leu Ala Thr Ser Leu Lys Ala Ser Gly Ile Leu
290 295 300
Ile Ser Pro Gly Lys Thr Leu Arg Met Val Thr His Ala Asp Ile Arg
305 310 315 320
Leu Glu Asp Ile Asp Leu Phe Ile Asp Lys Leu Lys Ser Leu Leu
325 330 335
<210> 72
<211> 335
<212> PRT
<213>Bao Shewanella threonine aldolase amino acid mutation (artificial sequence)
<400> 72
Met Ile Asp Phe Arg Ser Asp Thr Val Thr Gln Pro Thr Ala Ala Met
1 5 10 15
Arg Arg Ala Met Ala Asp Ala Gln Val Gly Asp Asp Val Tyr Gly Asp
20 25 30
Asp Pro Ser Val Asn Arg Leu Glu Ala Met Ala Ala Glu Arg Phe Gly
35 40 45
Phe Asp Ser Ala Leu Phe Thr Ser Ser Gly Thr Gln Ala Asn Leu Leu
50 55 60
Ala Leu Met Ser His Cys Asp Arg Gly Asp Glu Tyr Leu Cys Gly Gln
65 70 75 80
Gln Ala His Asn Tyr Lys Phe Glu Gly Gly Gly Ala Ala Val Leu Gly
85 90 95
Ser Ile Gln Pro Gln Pro Leu Thr Asn Gln Pro Asp Gly Ser Ile Leu
100 105 110
Leu Ser Asp Ile Glu Ala Ala Ile Lys Pro Asp Asp Phe His Phe Ala
115 120 125
Arg Thr Arg Leu Leu Ser Leu Glu Asn Thr Ile Gly Gly Lys Val Leu
130 135 140
Pro Gln Ser Tyr Leu Ala Glu Ala Gln Ala Leu Ala Phe Asn Lys Arg
145 150 155 160
Leu Lys Ile His Leu Asp Gly Tyr Arg Ile Ala Asn Ala Ala Val Ala
165 170 175
His Asn Leu Asp Ile Ala Asp Ile Thr Gln Tyr Phe Asp Ser Val Ser
180 185 190
Ile Cys Leu Ser Lys Gly Leu Cys Ala Pro Val Gly Ser Leu Leu Leu
195 200 205
Gly Asp Glu Arg Leu Ile Asn Lys Ala Arg Arg Trp Arg Lys Met Leu
210 215 220
Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Val Ala Gly Glu Ile
225 230 235 240
Ala Leu Asn Glu Gln Val Ser Arg Leu Ala Val Asp His Glu Asn Ala
245 250 255
Arg Tyr Leu Ala Glu Gln Leu Ser Cys Leu Asp Glu Phe Glu Val Asp
260 265 270
Leu Gly Glu Val Gln Thr Asn Met Leu Phe Ala Arg Val Val Glu Gly
275 280 285
Val Ala Ile Asp Lys Leu Ala Thr Ser Leu Lys Ala Ser Gly Ile Leu
290 295 300
Ile Ser Pro Gly Lys Thr Leu Arg Met Val Thr His Ala Asp Ile Arg
305 310 315 320
Leu Glu Asp Ile Asp Leu Phe Ile Asp Lys Leu Lys Ser Leu Leu
325 330 335
<210> 73
<211> 335
<212> PRT
<213>Bao Shewanella threonine aldolase amino acid mutation (artificial sequence)
<400> 73
Met Ile Asp Phe Arg Ser Asp Thr Val Thr Gln Pro Thr Ala Ala Met
1 5 10 15
Arg Arg Ala Met Ala Asp Ala Gln Val Gly Asp Asp Val Tyr Gly Asp
20 25 30
Asp Pro Ser Val Asn Arg Leu Glu Ala Met Ala Ala Glu Arg Phe Gly
35 40 45
Phe Asp Ser Ala Leu Phe Thr Ser Ser Gly Thr Gln Ala Asn Leu Leu
50 55 60
Ala Leu Met Ser His Cys Asp Arg Gly Asp Glu Tyr Leu Cys Gly Gln
65 70 75 80
Gln Ala His Asn Tyr Lys Phe Glu Gly Gly Gly Ala Ala Val Leu Gly
85 90 95
Ser Ile Gln Pro Gln Pro Leu Thr Asn Gln Pro Asp Gly Ser Ile Leu
100 105 110
Leu Ser Asp Ile Glu Ala Ala Ile Lys Pro Asp Asp Phe His Phe Ala
115 120 125
Arg Thr Arg Leu Leu Ser Leu Glu Asn Thr Ile Gly Gly Lys Val Leu
130 135 140
Pro Gln Ser Tyr Leu Ala Glu Ala Gln Ala Leu Ala Phe Asn Lys Arg
145 150 155 160
Leu Lys Ile His Leu Asp Gly Ala Arg Ile Ala Asn Ala Ala Val Ala
165 170 175
His Asn Leu Asp Ile Ala Asp Ile Thr Gln Tyr Phe Asp Ser Val Ser
180 185 190
Ile Cys Leu Ile Lys Gly Leu Cys Ala Pro Val Gly Ser Leu Leu Leu
195 200 205
Gly Asp Glu Arg Leu Ile Asn Lys Ala Arg Arg Trp Arg Lys Met Leu
210 215 220
Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Val Ala Gly Glu Ile
225 230 235 240
Ala Leu Asn Glu Gln Val Ser Arg Leu Ala Val Asp His Glu Asn Ala
245 250 255
Arg Tyr Leu Ala Glu Gln Leu Ser Cys Leu Asp Glu Phe Glu Val Asp
260 265 270
Leu Gly Glu Val Gln Thr Asn Met Leu Phe Ala Arg Val Val Glu Gly
275 280 285
Val Ala Ile Asp Lys Leu Ala Thr Ser Leu Lys Ala Ser Gly Ile Leu
290 295 300
Ile Ser Pro Gly Lys Thr Leu Arg Met Val Thr His Ala Asp Ile Arg
305 310 315 320
Leu Glu Asp Ile Asp Leu Phe Ile Asp Lys Leu Lys Ser Leu Leu
325 330 335
<210> 74
<211> 339
<212> PRT
<213>imperial Aeromonas threonine aldolase amino acid mutation (artificial sequence) is tieed up
<400> 74
Met Arg Tyr Ile Asp Leu Arg Ser Asp Gln Val Thr Gln Pro Thr Asp
1 5 10 15
Ala Met Arg Gln Ala Met Leu His Ala Glu Val Gly Asp Asp Val Tyr
20 25 30
Gly Glu Asp Pro Gly Val Asn Ala Leu Glu Ala His Gly Ala Arg Leu
35 40 45
Leu Gly Lys Gln Ala Ala Leu Phe Val Pro Ser Gly Thr Met Ser Asn
50 55 60
Leu Leu Ala Val Met Ser His Cys Gln Arg Gly Glu Gly Ala Ile Leu
65 70 75 80
Gly Asn Ala Ala His Ile Tyr Arg Tyr Glu Ala Gln Gly Ser Ala Val
85 90 95
Leu Gly Ser Val Ala Leu Gln Pro Leu Pro Met Gln Arg Asp Gly Thr
100 105 110
Leu Ala Phe Asp Asp Ile Lys Ala Ala Leu Ala Pro Asp Asp Ala His
115 120 125
Phe Val Gln Thr Arg Leu Ile Cys Leu Glu Asn Thr His Asn Gly Lys
130 135 140
Val Leu Pro Leu Ser Tyr Leu Gln Glu Met Gly Thr Phe Val Ala Glu
145 150 155 160
Arg Gly Leu Lys Leu His Leu Asp Gly Ala Arg Leu Phe Asn Ala Ala
165 170 175
Val Ala Ser Glu Thr Pro Val Ala Val Ile Ala Ala Pro Phe Asp Ser
180 185 190
Ile Ser Ile Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu
195 200 205
Leu Val Gly Ser His Asp Phe Ile Ala Arg Ala Arg Arg Leu Arg Lys
210 215 220
Met Leu Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Gln Ala Gly
225 230 235 240
Leu Phe Ala Leu Glu Gln His Val Thr Arg Leu Ala Asp Asp His Arg
245 250 255
Arg Ala Lys Arg Leu Ala Glu Gly Leu Ala Ala Leu Pro Gly Ile Glu
260 265 270
Leu Asp Leu Ser Leu Val Gln Ser Asn Met Val Phe Leu Arg Leu Arg
275 280 285
Glu Gly Glu Ser Ala Pro Leu Leu Ala Phe Met Lys Glu Arg Gly Ile
290 295 300
Leu Phe Ser Gly Tyr Gly Glu Leu Arg Leu Val Thr His Leu Gln Ile
305 310 315 320
Asn Asp Asp Asp Ile Glu Glu Val Ile Asp Ala Phe Thr Glu Tyr Leu
325 330 335
Gly Ala Arg
<210> 75
<211> 339
<212> PRT
<213>imperial Aeromonas threonine aldolase amino acid mutation (artificial sequence) is tieed up
<400> 75
Met Arg Tyr Ile Asp Leu Arg Ser Asp Thr Val Thr Gln Pro Thr Asp
1 5 10 15
Ala Met Arg Gln Ala Met Leu His Ala Glu Val Gly Asp Asp Val Tyr
20 25 30
Gly Glu Asp Pro Gly Val Asn Ala Leu Glu Ala His Gly Ala Arg Leu
35 40 45
Leu Gly Lys Gln Ala Ala Leu Phe Val Pro Ser Gly Thr Met Ser Asn
50 55 60
Leu Leu Ala Val Met Ser His Cys Gln Arg Gly Glu Gly Ala Ile Leu
65 70 75 80
Gly Asn Ala Ala Trp Ile Tyr Arg Tyr Glu Ala Gln Gly Ser Ala Val
85 90 95
Leu Gly Ser Val Ala Leu Gln Pro Leu Pro Met Gln Arg Asp Gly Thr
100 105 110
Leu Ala Phe Asp Asp Ile Lys Ala Ala Leu Ala Pro Asp Asp Ala His
115 120 125
Phe Val Gln Thr Arg Leu Ile Cys Leu Glu Asn Thr His Asn Gly Lys
130 135 140
Val Leu Pro Leu Ser Tyr Leu Gln Glu Met Gly Thr Phe Val Ala Glu
145 150 155 160
Arg Gly Leu Lys Leu His Leu Asp Gly Ala Arg Leu Phe Asn Ala Ala
165 170 175
Val Ala Ser Glu Thr Pro Val Ala Val Ile Ala Ala Pro Phe Asp Ser
180 185 190
Ile Ser Ile Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu
195 200 205
Leu Val Gly Ser His Asp Phe Ile Ala Arg Ala Arg Arg Leu Arg Lys
210 215 220
Met Leu Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Gln Ala Gly
225 230 235 240
Leu Phe Ala Leu Glu Gln His Val Thr Arg Leu Ala Asp Asp His Arg
245 250 255
Arg Ala Lys Arg Leu Ala Glu Gly Leu Ala Ala Leu Pro Gly Ile Glu
260 265 270
Leu Asp Leu Ser Leu Val Gln Ser Asn Met Val Phe Leu Arg Leu Arg
275 280 285
Glu Gly Glu Ser Ala Pro Leu Leu Ala Phe Met Lys Glu Arg Gly Ile
290 295 300
Leu Phe Ser Gly Tyr Gly Glu Leu Arg Leu Val Thr His Leu Gln Ile
305 310 315 320
Asn Asp Asp Asp Ile Glu Glu Val Ile Asp Ala Phe Thr Glu Tyr Leu
325 330 335
Gly Ala Arg
<210> 76
<211> 339
<212> PRT
<213>imperial Aeromonas threonine aldolase amino acid mutation (artificial sequence) is tieed up
<400> 76
Met Arg Tyr Ile Asp Leu Arg Ser Asp Thr Val Thr Gln Pro Thr Asp
1 5 10 15
Ala Met Arg Gln Ala Met Leu His Ala Glu Val Gly Asp Asp Val Tyr
20 25 30
Gly Glu Asp Pro Gly Val Asn Ala Leu Glu Ala His Gly Ala Arg Leu
35 40 45
Leu Gly Lys Gln Ala Ala Leu Phe Val Pro Ser Gly Thr Met Ser Asn
50 55 60
Leu Leu Ala Val Met Ser His Cys Gln Arg Gly Glu Gly Ala Ile Leu
65 70 75 80
Gly Asn Ala Ala Pro Ile Tyr Arg Tyr Glu Ala Gln Gly Ser Ala Val
85 90 95
Leu Gly Ser Val Ala Leu Gln Pro Leu Pro Met Gln Arg Asp Gly Thr
100 105 110
Leu Ala Phe Asp Asp Ile Lys Ala Ala Leu Ala Pro Asp Asp Ala His
115 120 125
Phe Val Gln Thr Arg Leu Ile Cys Leu Glu Asn Thr His Asn Gly Lys
130 135 140
Val Leu Pro Leu Ser Tyr Leu Gln Glu Met Gly Thr Phe Val Ala Glu
145 150 155 160
Arg Gly Leu Lys Leu His Leu Asp Gly Ala Arg Leu Phe Asn Ala Ala
165 170 175
Val Ala Ser Glu Thr Pro Val Ala Val Ile Ala Ala Pro Phe Asp Ser
180 185 190
Ile Ser Ile Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu
195 200 205
Leu Val Gly Ser His Asp Phe Ile Ala Arg Ala Arg Arg Leu Arg Lys
210 215 220
Met Leu Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Gln Ala Gly
225 230 235 240
Leu Phe Ala Leu Glu Gln His Val Thr Arg Leu Ala Asp Asp His Arg
245 250 255
Arg Ala Lys Arg Leu Ala Glu Gly Leu Ala Ala Leu Pro Gly Ile Glu
260 265 270
Leu Asp Leu Ser Leu Val Gln Ser Asn Met Val Phe Leu Arg Leu Arg
275 280 285
Glu Gly Glu Ser Ala Pro Leu Leu Ala Phe Met Lys Glu Arg Gly Ile
290 295 300
Leu Phe Ser Gly Tyr Gly Glu Leu Arg Leu Val Thr His Leu Gln Ile
305 310 315 320
Asn Asp Asp Asp Ile Glu Glu Val Ile Asp Ala Phe Thr Glu Tyr Leu
325 330 335
Gly Ala Arg
<210> 77
<211> 339
<212> PRT
<213>imperial Aeromonas threonine aldolase amino acid mutation (artificial sequence) is tieed up
<400> 77
Met Arg Tyr Ile Asp Leu Arg Ser Asp Thr Val Thr Gln Pro Thr Asp
1 5 10 15
Ala Met Arg Gln Ala Met Leu His Ala Glu Val Gly Asp Asp Val Tyr
20 25 30
Gly Glu Asp Pro Gly Val Asn Ala Leu Glu Ala His Gly Ala Arg Leu
35 40 45
Leu Gly Lys Gln Ala Ala Leu Phe Val Pro Ser Gly Thr Met Ser Asn
50 55 60
Leu Leu Ala Val Met Ser His Cys Gln Arg Gly Glu Gly Ala Ile Leu
65 70 75 80
Gly Asn Ala Ala His Ile Tyr Arg Tyr Glu Ala Gln Gly Ser Ala Val
85 90 95
Leu Gly Ser Val Ala Leu Gln Pro Leu Pro Met Gln Arg Asp Gly Thr
100 105 110
Leu Ala Phe Asp Asp Ile Lys Ala Ala Leu Ala Pro Asp Asp Ala His
115 120 125
Phe Val Gln Thr Arg Leu Ile Cys Leu Glu Asn Thr His Asn Gly Lys
130 135 140
Val Leu Pro Leu Ser Tyr Leu Gln Glu Met Gly Thr Phe Val Ala Glu
145 150 155 160
Arg Gly Leu Lys Leu His Leu Asp Gly Tyr Arg Leu Phe Asn Ala Ala
165 170 175
Val Ala Ser Glu Thr Pro Val Ala Val Ile Ala Ala Pro Phe Asp Ser
180 185 190
Ile Ser Ile Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu
195 200 205
Leu Val Gly Ser His Asp Phe Ile Ala Arg Ala Arg Arg Leu Arg Lys
210 215 220
Met Leu Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Gln Ala Gly
225 230 235 240
Leu Phe Ala Leu Glu Gln His Val Thr Arg Leu Ala Asp Asp His Arg
245 250 255
Arg Ala Lys Arg Leu Ala Glu Gly Leu Ala Ala Leu Pro Gly Ile Glu
260 265 270
Leu Asp Leu Ser Leu Val Gln Ser Asn Met Val Phe Leu Arg Leu Arg
275 280 285
Glu Gly Glu Ser Ala Pro Leu Leu Ala Phe Met Lys Glu Arg Gly Ile
290 295 300
Leu Phe Ser Gly Tyr Gly Glu Leu Arg Leu Val Thr His Leu Gln Ile
305 310 315 320
Asn Asp Asp Asp Ile Glu Glu Val Ile Asp Ala Phe Thr Glu Tyr Leu
325 330 335
Gly Ala Arg
<210> 78
<211> 339
<212> PRT
<213>imperial Aeromonas threonine aldolase amino acid mutation (artificial sequence) is tieed up
<400> 78
Met Arg Tyr Ile Asp Leu Arg Ser Asp Thr Val Thr Gln Pro Thr Asp
1 5 10 15
Ala Met Arg Gln Ala Met Leu His Ala Glu Val Gly Asp Asp Val Tyr
20 25 30
Gly Glu Asp Pro Gly Val Asn Ala Leu Glu Ala His Gly Ala Arg Leu
35 40 45
Leu Gly Lys Gln Ala Ala Leu Phe Val Pro Ser Gly Thr Met Ser Asn
50 55 60
Leu Leu Ala Val Met Ser His Cys Gln Arg Gly Glu Gly Ala Ile Leu
65 70 75 80
Gly Asn Ala Ala His Ile Tyr Arg Tyr Glu Ala Gln Gly Ser Ala Val
85 90 95
Leu Gly Ser Val Ala Leu Gln Pro Leu Pro Met Gln Arg Asp Gly Thr
100 105 110
Leu Ala Phe Asp Asp Ile Lys Ala Ala Leu Ala Pro Asp Asp Ala His
115 120 125
Phe Val Gln Thr Arg Leu Ile Cys Leu Glu Asn Thr His Asn Gly Lys
130 135 140
Val Leu Pro Leu Ser Tyr Leu Gln Glu Met Gly Thr Phe Val Ala Glu
145 150 155 160
Arg Gly Leu Lys Leu His Leu Asp Gly Ala Arg Leu Phe Asn Ala Ala
165 170 175
Val Ala Ser Glu Thr Pro Val Ala Val Ile Ala Ala Pro Phe Asp Ser
180 185 190
Ile Ser Ile Cys Leu Ile Lys Gly Leu Gly Ala Pro Val Gly Ser Leu
195 200 205
Leu Val Gly Ser His Asp Phe Ile Ala Arg Ala Arg Arg Leu Arg Lys
210 215 220
Met Leu Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Gln Ala Gly
225 230 235 240
Leu Phe Ala Leu Glu Gln His Val Thr Arg Leu Ala Asp Asp His Arg
245 250 255
Arg Ala Lys Arg Leu Ala Glu Gly Leu Ala Ala Leu Pro Gly Ile Glu
260 265 270
Leu Asp Leu Ser Leu Val Gln Ser Asn Met Val Phe Leu Arg Leu Arg
275 280 285
Glu Gly Glu Ser Ala Pro Leu Leu Ala Phe Met Lys Glu Arg Gly Ile
290 295 300
Leu Phe Ser Gly Tyr Gly Glu Leu Arg Leu Val Thr His Leu Gln Ile
305 310 315 320
Asn Asp Asp Asp Ile Glu Glu Val Ile Asp Ala Phe Thr Glu Tyr Leu
325 330 335
Gly Ala Arg
<210> 79
<211> 348
<212> PRT
<213>sulphur reduction ground bacillus threonine aldolase amino acid mutation (artificial sequence)
<400> 79
Met Asn Ile Ile Asp Leu Arg Ser Asp Gln Val Thr Arg Pro Ser Pro
1 5 10 15
Ala Met Arg Ala Ala Met Ala Gly Ala Glu Val Gly Asp Asp Val Tyr
20 25 30
Gly Glu Asp Pro Thr Val Asn Arg Leu Glu Glu Leu Gly Ala Ala Thr
35 40 45
Leu Gly Thr Glu Ala Ala Leu Phe Thr Ala Ser Gly Thr Gln Ala Asn
50 55 60
Leu Leu Ala Leu Leu Ala His Cys Arg Arg Gly Asp Glu Tyr Ile Ala
65 70 75 80
Gly Gln Thr Ala His Cys Tyr Arg Tyr Glu Gly Gly Gly Ala Ala Ala
85 90 95
Leu Gly Gly Ile Gln Pro Gln Pro Leu Glu Val Glu Pro Asp Gly Thr
100 105 110
Leu Asp Leu Ser Ala Val Ala Ala Ala Ile Lys Pro Asp Asp Leu His
115 120 125
Phe Ala Arg Thr Arg Leu Leu Cys Leu Glu Asn Thr His Ala Gly Arg
130 135 140
Val Leu Pro Leu Asp Tyr Leu Ala Arg Ala Arg Arg Leu Cys Asn Glu
145 150 155 160
Gln Ser Leu Gly Leu His Met Asp Gly Ala Arg Ile Phe Asn Ala Ala
165 170 175
Val Lys Leu Gly Val Pro Val Arg Glu Ile Ala Gly His Val Asp Ser
180 185 190
Val Ser Val Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu
195 200 205
Leu Cys Gly Gly Arg Glu Phe Val Ala Thr Ala Arg Arg Trp Arg Lys
210 215 220
Ala Val Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Ala Ala Gly
225 230 235 240
Ile Leu Ala Leu Thr Glu Asn Val Asp Arg Leu Ala Glu Asp His Ala
245 250 255
Asn Ala Arg Arg Leu Ala Glu Gly Leu Ala Ala Ile Pro Gly Leu Gly
260 265 270
Leu Asp Pro Ser Gln Val Gln Thr Asn Met Val Phe Leu Cys Leu Pro
275 280 285
Leu Arg Thr Ala Asp Arg Leu Ala Ser Phe Leu Asn Glu Gln Gly Ile
290 295 300
Leu Ile Ser Gly Arg Glu Ser Ile Arg Leu Val Thr His Leu Asp Val
305 310 315 320
Thr Ser Ser Asp Val Glu Arg Val Ile Ala Ala Phe Gly Ala Phe Phe
325 330 335
Ala Gly His Gly Glu Met Gln Pro Gly Ala Ser Ser
340 345
<210> 80
<211> 348
<212> PRT
<213>sulphur reduction ground bacillus threonine aldolase amino acid mutation (artificial sequence)
<400> 80
Met Asn Ile Ile Asp Leu Arg Ser Asp Thr Val Thr Arg Pro Ser Pro
1 5 10 15
Ala Met Arg Ala Ala Met Ala Gly Ala Glu Val Gly Asp Asp Val Tyr
20 25 30
Gly Glu Asp Pro Thr Val Asn Arg Leu Glu Glu Leu Gly Ala Ala Thr
35 40 45
Leu Gly Thr Glu Ala Ala Leu Phe Thr Ala Ser Gly Thr Gln Ala Asn
50 55 60
Leu Leu Ala Leu Leu Ala His Cys Arg Arg Gly Asp Glu Tyr Ile Ala
65 70 75 80
Gly Gln Thr Ala Trp Cys Tyr Arg Tyr Glu Gly Gly Gly Ala Ala Ala
85 90 95
Leu Gly Gly Ile Gln Pro Gln Pro Leu Glu Val Glu Pro Asp Gly Thr
100 105 110
Leu Asp Leu Ser Ala Val Ala Ala Ala Ile Lys Pro Asp Asp Leu His
115 120 125
Phe Ala Arg Thr Arg Leu Leu Cys Leu Glu Asn Thr His Ala Gly Arg
130 135 140
Val Leu Pro Leu Asp Tyr Leu Ala Arg Ala Arg Arg Leu Cys Asn Glu
145 150 155 160
Gln Ser Leu Gly Leu His Met Asp Gly Ala Arg Ile Phe Asn Ala Ala
165 170 175
Val Lys Leu Gly Val Pro Val Arg Glu Ile Ala Gly His Val Asp Ser
180 185 190
Val Ser Val Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu
195 200 205
Leu Cys Gly Gly Arg Glu Phe Val Ala Thr Ala Arg Arg Trp Arg Lys
210 215 220
Ala Val Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Ala Ala Gly
225 230 235 240
Ile Leu Ala Leu Thr Glu Asn Val Asp Arg Leu Ala Glu Asp His Ala
245 250 255
Asn Ala Arg Arg Leu Ala Glu Gly Leu Ala Ala Ile Pro Gly Leu Gly
260 265 270
Leu Asp Pro Ser Gln Val Gln Thr Asn Met Val Phe Leu Cys Leu Pro
275 280 285
Leu Arg Thr Ala Asp Arg Leu Ala Ser Phe Leu Asn Glu Gln Gly Ile
290 295 300
Leu Ile Ser Gly Arg Glu Ser Ile Arg Leu Val Thr His Leu Asp Val
305 310 315 320
Thr Ser Ser Asp Val Glu Arg Val Ile Ala Ala Phe Gly Ala Phe Phe
325 330 335
Ala Gly His Gly Glu Met Gln Pro Gly Ala Ser Ser
340 345
<210> 81
<211> 348
<212> PRT
<213>sulphur reduction ground bacillus threonine aldolase amino acid mutation (artificial sequence)
<400> 81
Met Asn Ile Ile Asp Leu Arg Ser Asp Thr Val Thr Arg Pro Ser Pro
1 5 10 15
Ala Met Arg Ala Ala Met Ala Gly Ala Glu Val Gly Asp Asp Val Tyr
20 25 30
Gly Glu Asp Pro Thr Val Asn Arg Leu Glu Glu Leu Gly Ala Ala Thr
35 40 45
Leu Gly Thr Glu Ala Ala Leu Phe Thr Ala Ser Gly Thr Gln Ala Asn
50 55 60
Leu Leu Ala Leu Leu Ala His Cys Arg Arg Gly Asp Glu Tyr Ile Ala
65 70 75 80
Gly Gln Thr Ala Pro Cys Tyr Arg Tyr Glu Gly Gly Gly Ala Ala Ala
85 90 95
Leu Gly Gly Ile Gln Pro Gln Pro Leu Glu Val Glu Pro Asp Gly Thr
100 105 110
Leu Asp Leu Ser Ala Val Ala Ala Ala Ile Lys Pro Asp Asp Leu His
115 120 125
Phe Ala Arg Thr Arg Leu Leu Cys Leu Glu Asn Thr His Ala Gly Arg
130 135 140
Val Leu Pro Leu Asp Tyr Leu Ala Arg Ala Arg Arg Leu Cys Asn Glu
145 150 155 160
Gln Ser Leu Gly Leu His Met Asp Gly Ala Arg Ile Phe Asn Ala Ala
165 170 175
Val Lys Leu Gly Val Pro Val Arg Glu Ile Ala Gly His Val Asp Ser
180 185 190
Val Ser Val Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu
195 200 205
Leu Cys Gly Gly Arg Glu Phe Val Ala Thr Ala Arg Arg Trp Arg Lys
210 215 220
Ala Val Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Ala Ala Gly
225 230 235 240
Ile Leu Ala Leu Thr Glu Asn Val Asp Arg Leu Ala Glu Asp His Ala
245 250 255
Asn Ala Arg Arg Leu Ala Glu Gly Leu Ala Ala Ile Pro Gly Leu Gly
260 265 270
Leu Asp Pro Ser Gln Val Gln Thr Asn Met Val Phe Leu Cys Leu Pro
275 280 285
Leu Arg Thr Ala Asp Arg Leu Ala Ser Phe Leu Asn Glu Gln Gly Ile
290 295 300
Leu Ile Ser Gly Arg Glu Ser Ile Arg Leu Val Thr His Leu Asp Val
305 310 315 320
Thr Ser Ser Asp Val Glu Arg Val Ile Ala Ala Phe Gly Ala Phe Phe
325 330 335
Ala Gly His Gly Glu Met Gln Pro Gly Ala Ser Ser
340 345
<210> 82
<211> 348
<212> PRT
<213>sulphur reduction ground bacillus threonine aldolase amino acid mutation (artificial sequence)
<400> 82
Met Asn Ile Ile Asp Leu Arg Ser Asp Thr Val Thr Arg Pro Ser Pro
1 5 10 15
Ala Met Arg Ala Ala Met Ala Gly Ala Glu Val Gly Asp Asp Val Tyr
20 25 30
Gly Glu Asp Pro Thr Val Asn Arg Leu Glu Glu Leu Gly Ala Ala Thr
35 40 45
Leu Gly Thr Glu Ala Ala Leu Phe Thr Ala Ser Gly Thr Gln Ala Asn
50 55 60
Leu Leu Ala Leu Leu Ala His Cys Arg Arg Gly Asp Glu Tyr Ile Ala
65 70 75 80
Gly Gln Thr Ala His Cys Tyr Arg Tyr Glu Gly Gly Gly Ala Ala Ala
85 90 95
Leu Gly Gly Ile Gln Pro Gln Pro Leu Glu Val Glu Pro Asp Gly Thr
100 105 110
Leu Asp Leu Ser Ala Val Ala Ala Ala Ile Lys Pro Asp Asp Leu His
115 120 125
Phe Ala Arg Thr Arg Leu Leu Cys Leu Glu Asn Thr His Ala Gly Arg
130 135 140
Val Leu Pro Leu Asp Tyr Leu Ala Arg Ala Arg Arg Leu Cys Asn Glu
145 150 155 160
Gln Ser Leu Gly Leu His Met Asp Gly Tyr Arg Ile Phe Asn Ala Ala
165 170 175
Val Lys Leu Gly Val Pro Val Arg Glu Ile Ala Gly His Val Asp Ser
180 185 190
Val Ser Val Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu
195 200 205
Leu Cys Gly Gly Arg Glu Phe Val Ala Thr Ala Arg Arg Trp Arg Lys
210 215 220
Ala Val Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Ala Ala Gly
225 230 235 240
Ile Leu Ala Leu Thr Glu Asn Val Asp Arg Leu Ala Glu Asp His Ala
245 250 255
Asn Ala Arg Arg Leu Ala Glu Gly Leu Ala Ala Ile Pro Gly Leu Gly
260 265 270
Leu Asp Pro Ser Gln Val Gln Thr Asn Met Val Phe Leu Cys Leu Pro
275 280 285
Leu Arg Thr Ala Asp Arg Leu Ala Ser Phe Leu Asn Glu Gln Gly Ile
290 295 300
Leu Ile Ser Gly Arg Glu Ser Ile Arg Leu Val Thr His Leu Asp Val
305 310 315 320
Thr Ser Ser Asp Val Glu Arg Val Ile Ala Ala Phe Gly Ala Phe Phe
325 330 335
Ala Gly His Gly Glu Met Gln Pro Gly Ala Ser Ser
340 345
<210> 83
<211> 348
<212> PRT
<213>sulphur reduction ground bacillus threonine aldolase amino acid mutation (artificial sequence)
<400> 83
Met Asn Ile Ile Asp Leu Arg Ser Asp Thr Val Thr Arg Pro Ser Pro
1 5 10 15
Ala Met Arg Ala Ala Met Ala Gly Ala Glu Val Gly Asp Asp Val Tyr
20 25 30
Gly Glu Asp Pro Thr Val Asn Arg Leu Glu Glu Leu Gly Ala Ala Thr
35 40 45
Leu Gly Thr Glu Ala Ala Leu Phe Thr Ala Ser Gly Thr Gln Ala Asn
50 55 60
Leu Leu Ala Leu Leu Ala His Cys Arg Arg Gly Asp Glu Tyr Ile Ala
65 70 75 80
Gly Gln Thr Ala His Cys Tyr Arg Tyr Glu Gly Gly Gly Ala Ala Ala
85 90 95
Leu Gly Gly Ile Gln Pro Gln Pro Leu Glu Val Glu Pro Asp Gly Thr
100 105 110
Leu Asp Leu Ser Ala Val Ala Ala Ala Ile Lys Pro Asp Asp Leu His
115 120 125
Phe Ala Arg Thr Arg Leu Leu Cys Leu Glu Asn Thr His Ala Gly Arg
130 135 140
Val Leu Pro Leu Asp Tyr Leu Ala Arg Ala Arg Arg Leu Cys Asn Glu
145 150 155 160
Gln Ser Leu Gly Leu His Met Asp Gly Ala Arg Ile Phe Asn Ala Ala
165 170 175
Val Lys Leu Gly Val Pro Val Arg Glu Ile Ala Gly His Val Asp Ser
180 185 190
Val Ser Val Cys Leu Ile Lys Gly Leu Gly Ala Pro Val Gly Ser Leu
195 200 205
Leu Cys Gly Gly Arg Glu Phe Val Ala Thr Ala Arg Arg Trp Arg Lys
210 215 220
Ala Val Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Ala Ala Gly
225 230 235 240
Ile Leu Ala Leu Thr Glu Asn Val Asp Arg Leu Ala Glu Asp His Ala
245 250 255
Asn Ala Arg Arg Leu Ala Glu Gly Leu Ala Ala Ile Pro Gly Leu Gly
260 265 270
Leu Asp Pro Ser Gln Val Gln Thr Asn Met Val Phe Leu Cys Leu Pro
275 280 285
Leu Arg Thr Ala Asp Arg Leu Ala Ser Phe Leu Asn Glu Gln Gly Ile
290 295 300
Leu Ile Ser Gly Arg Glu Ser Ile Arg Leu Val Thr His Leu Asp Val
305 310 315 320
Thr Ser Ser Asp Val Glu Arg Val Ile Ala Ala Phe Gly Ala Phe Phe
325 330 335
Ala Gly His Gly Glu Met Gln Pro Gly Ala Ser Ser
340 345
<210> 84
<211> 24
<212> DNA
<213>primer (artificial sequence)
<400> 84
ctggacggct accggctggc caat 24
<210> 85
<211> 24
<212> DNA
<213>primer (artificial sequence)
<400> 85
attggccagc cggtagccgt ccag 24
<210> 86
<211> 22
<212> DNA
<213>primer (artificial sequence)
<400> 86
agcacgccag catctgcacg ga 22
<210> 87
<211> 22
<212> DNA
<213>primer (artificial sequence)
<400> 87
tccgtgcaga tgctggcgtg ct 22
<210> 88
<211> 1032
<212> DNA
<213>crescent shank bacterium threonine aldolase gene (artificial sequence)
<400> 88
atgacccaga ccgcgccccg ctacgatttc gcctccgaca acgtcgccgg cgccatgccc 60
gaggtgatgg aggcgttgat cgcggccaat gcggggacgg cctcgggcta cgggaccgac 120
catgtcagcc gtgccgccgc cgatcgcatc cgcgcggcgc tggacgccga cgcgcaggtg 180
cggttcacag cctcgggcac ggcggccaac gccttcgccc tgaccctgct ggcccagccg 240
cacgaggcgg tgctggccca cgagcacgcc cacatctgca cggacgagac cggcgcgccc 300
ggcttctttg gccagggcgt cggcctgatc ggcttgccgg gcgcatcggg caagatggag 360
ttggcggccc tggaggcggc gctggcccag ccggacgtct cgtaccgcca gccggcggcg 420
gccttgtcgc tgaccaccgc caccgagtac ggcacggtct attcggaaga ccacctgcgg 480
gccctgatcg cgccggtgaa ggccaagggc tatggcgtcc acctggacgg cgcgcggctg 540
gccaatgcgg tcgcgggggg ctttgacctg aaaagcatcg ccaagatggg cgtcgacatc 600
ctggtgatgg gcggcaccaa ggccggctcg acgcccaccg aggccgtggt gttcctgaac 660
cccgaccacg ccaaacgcct ggacgcgcgc ctcaagcacg ccggccagct gatctcgaag 720
ggacgcttcc tggccgcgcc gtggctgggc ctgctgggcg agaacggtca gaccgccccc 780
tgggccgccc gcgccgccca cgccaacgcc atggcgcaga agctggccgc gctgatgccc 840
gtcccgatca agcacccggt cgaggccaac ggcatcttcg tcgagatgga cgaacttgcc 900
ctggaacgcc tgcgcggcga aggctggttc gtctatcgct tcctggacgg cacggtgcgc 960
ttcatgtgct cgtgggccac gacgcccgag atggtcgagg atctgggcgc ggcgctgaag 1020
cgagtggctt ag 1032

Claims (10)

1. threonine aldolase, which is characterized in that be selected from pseudomonad (Pseudomonas putida), amino acid sequence is Shown in SEQ ID NO:2；Selected from crescent shank bacterium (Caulobacter crescentus), amino acid sequence is SEQ ID Shown in NO:19；Selected from Thermotoga maritima (Thermotoga maritima), amino acid sequence is shown in SEQ ID NO:23； Selected from Listeria monocytogenes (Listeria monocytogenes), amino acid sequence is shown in SEQ ID NO:24；It is selected from Escherichia coli (Escherichia coli), amino acid sequence are shown in SEQ ID NO:25；Selected from aermonas jandaei (Aeromonas jandaei), amino acid sequence are shown in SEQ ID NO:26；Selected from saccharomyces cerevisiae (Saccharomyces Cerevisiae), amino acid sequence is shown in SEQ ID NO:27；Selected from ashbya gossypii bacterium (Ashbya gossypii), Its amino acid sequence is shown in SEQ ID NO:28；Selected from Bao Shewanella (Shewanella loihica), amino acid sequence It is classified as shown in SEQ ID NO:29；Selected from imperial Aeromonas (Aeromonas veronii) is tieed up, amino acid sequence is SEQ ID Shown in NO:30；Ground bacillus (Geobacter sulfurreducens) is restored selected from sulphur, amino acid sequence is SEQ ID NO: Shown in 31.
2. the mutant of threonine aldolase, which is characterized in that be built upon the four of the threonine aldolase in different strains source The mutation in a site sports simple point mutation, double mutant, three point mutation or four point mutation, mutational site and raw bits ammonia Base acid is listed as follows:
Threonine aldolase First catastrophe point Second catastrophe point Third catastrophe point 4th catastrophe point Ps_LTA N17 H94 S182 T212 Cc_LTA N14 H91 A178 T206 Ec_LTA T8 H83 A168 S196 Lm_LTA --- H98 A178 Y206 Tm_LTA T8 H83 A170 S198 Aj_LTA T10 H85 A170 S198 Sc_LTA T21 H97 A184 S212 Ag_LTA T22 H98 A185 S213 Sl_LTA T8 H83 A168 S196 Av_LTA T10 H85 A170 S198 Gs_LTA T10 H85 A170 S198
It is as follows that it corresponds to replaceable amino acid: the first catastrophe point N/T is changed to Q, and the second catastrophe point H is changed to W/P/F/V/A/ S, third catastrophe point S/A are changed to Y, and the 4th catastrophe point T/S/Y is changed to I；Wherein, the threonine aldolase in different strains source Be defined as follows: Ps_LTA- derives from the threonine aldolase of pseudomonad (Pseudomonas putida), the source Cc_LTA- In the threonine aldolase of crescent shank bacterium (Caulobacter crescentus), Ec_LTA- derives from Escherichia coli The threonine aldolase of (Escherichia coli), Lm_LTA- derive from Listeria monocytogenes (Listeria Monocytogenes threonine aldolase), Tm_LTA- derive from the Soviet Union of Thermotoga maritima (Thermotoga maritima) Propylhomoserin aldolase, Aj_LTA- derive from the threonine aldolase of aermonas jandaei (Aeromonas jandaei), Sc_LTA- From the threonine aldolase of saccharomyces cerevisiae (Saccharomyces cerevisiae), Ag_LTA- derives from cotton A Shu capsule The threonine aldolase of mould (Ashbya gossypii), Sl_LTA- derive from Bao Shewanella (Shewanella Loihica threonine aldolase), Av_LTA- is from the threonine aldehyde for tieing up imperial Aeromonas (Aeromonas veronii) Contracting enzyme, threonine aldolase of the Gs_LTA from sulphur reduction ground bacillus (Geobacter sulfurreducens)；Wherein, ammonia Base acid abbreviation meaning is as follows: N- asparagine, T- threonine, Q- glutamine, H- histidine, W- tryptophan, P- proline, F- Phenylalanine, V- valine, A- alanine, S- amino acids, Y- tyrosine, I- isoleucine.
3. the mutant of threonine aldolase, which is characterized in that be selected from Soviet Union's ammonia of pseudomonad (Pseudomonas putida) The mutant of sour aldolase, amino acid sequence are SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: Shown in 10；The mutant of threonine aldolase selected from crescent shank bacterium (Caulobacter crescentus), amino acid Sequence is SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:32, SEQ ID NO:33, SEQ ID Shown in NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38；Selected from Thermotoga maritima The mutant of the threonine aldolase of (Thermotoga maritima), amino acid sequence are SEQ ID NO:39, SEQ ID Shown in NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43；Selected from Listeria monocytogenes (Listeria Monocytogenes the mutant of threonine aldolase), amino acid sequence are SEQ ID NO:44, SEQ ID NO:45, Shown in SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48；Soviet Union selected from Escherichia coli (Escherichia coli) The mutant of propylhomoserin aldolase, amino acid sequence are SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ Shown in ID NO:52, SEQ ID NO:53；Threonine aldolase selected from aermonas jandaei (Aeromonas jandaei) Mutant, amino acid sequence are SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ Shown in ID NO:58；The mutant of threonine aldolase selected from saccharomyces cerevisiae (Saccharomyces cerevisiae), Amino acid sequence is SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63 It is shown；The mutant of threonine aldolase selected from ashbya gossypii bacterium (Ashbya gossypii), amino acid sequence are Shown in SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68；Selected from Bao Xi The mutant of the threonine aldolase of watt Salmonella (Shewanella loihica), amino acid sequence are SEQ ID NO:69, Shown in SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73；Selected from the imperial Aeromonas of dimension The mutant of the threonine aldolase of (Aeromonas veronii), amino acid sequence are SEQ ID NO:74, SEQ ID Shown in NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78；Ground bacillus (Geobacter is restored selected from sulphur Sulfurreducens the mutant of threonine aldolase), amino acid sequence are SEQ ID NO:79, SEQ ID NO: Shown in 80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83.
4. the mutant of threonine aldolase described in threonine aldolase, claim 2 described in claim 1, claim 3 institute State the encoding gene of the mutant of threonine aldolase.
5. the recombinant vector of the building of encoding gene described in claim 4.
6. the recombination engineering bacteria that the conversion of recombinant vector described in claim 5 obtains.
7. the mutant of threonine aldolase described in threonine aldolase, claim 2 described in claim 1, claim 3 institute State application of the mutant of threonine aldolase in the benzene serine derivative for preparing 2-/3-/4- substitution.
8. application as claimed in claim 7, which is characterized in that with the mutant of threonine aldolase or threonine aldolase be urge Agent, the benzaldehyde replaced using 2-/3-/4-, using pyridoxime 5-phosphate as coenzyme, are with glycine/glycinate as substrate Auxiliary substrate carries out enzymatic in reaction medium, after reaction, isolates and purifies under conditions of controlling temperature and mixing speed, Obtain the benzene serine derivative of 2-/3-/4- substitution.
9. application according to any one of claims 8, which is characterized in that the substrate is one of following: benzaldehyde, 2- chlorobenzaldehyde, 2- fluorobenzaldehyde, 2- bromobenzaldehyde, 2- nitrobenzaldehyde, 2- methyl sulfone benzaldehyde, Benzaldehyde,2-hydroxy, 3- chlorobenzaldehyde, 3- Fluorobenzaldehyde, 3- bromobenzaldehyde, 3- nitrobenzaldehyde, 3- methyl sulfone benzaldehyde, 3- hydroxy benzaldehyde, 4- chlorobenzaldehyde, 4- fluorine Benzaldehyde, 4- bromobenzaldehyde, 4- nitrobenzaldehyde, 4- methyl sulfone benzaldehyde, 4- hydroxy benzaldehyde；The auxiliary substrate be it is following it One: L- glycine, L- glycine hydrochloride, L- glycine methyl ester hydrochloride, L- glycine ethyl ester hydrochloride, the sweet ammonia of N-Boc-L- Acid, N-Boc-L- glycine ethyl ester, N-Z-L- glycine, N-Z-L- glycine ethyl ester, N-Z-L- glycine methyl ester；Reaction medium It is one of following: the phosphoric acid buffer system of pH 8-9, water-ethanol system, Water-Methanol System, methanol system, ethanol system.
10. application according to any one of claims 8, which is characterized in that the dosage of enzyme is 50-1000mg/L, concentration of substrate 1- 100g/L, glycine/glycinate concentration are 5-200g/L, and pyridoxime 5-phosphate concentration is 0.01-0.2g/L, and control temperature exists 25-45 DEG C, mixing speed 70-300rpm.
CN201811310893.4A 2018-11-06 2018-11-06 Threonine aldolase, mutant and application of mutant in preparation of substituted phenylserine derivative Active CN109402098B (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201811310893.4A CN109402098B (en) 2018-11-06 2018-11-06 Threonine aldolase, mutant and application of mutant in preparation of substituted phenylserine derivative
CN202110925261.4A CN113481188B (en) 2018-11-06 2018-11-06 Threonine aldolase mutant and application thereof in preparation of substituted phenylserine derivative

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201811310893.4A CN109402098B (en) 2018-11-06 2018-11-06 Threonine aldolase, mutant and application of mutant in preparation of substituted phenylserine derivative

Related Child Applications (1)

Application Number Title Priority Date Filing Date
CN202110925261.4A Division CN113481188B (en) 2018-11-06 2018-11-06 Threonine aldolase mutant and application thereof in preparation of substituted phenylserine derivative

Publications (2)

Publication Number Publication Date
CN109402098A true CN109402098A (en) 2019-03-01
CN109402098B CN109402098B (en) 2021-09-17

Family

ID=65471763

Family Applications (2)

Application Number Title Priority Date Filing Date
CN201811310893.4A Active CN109402098B (en) 2018-11-06 2018-11-06 Threonine aldolase, mutant and application of mutant in preparation of substituted phenylserine derivative
CN202110925261.4A Active CN113481188B (en) 2018-11-06 2018-11-06 Threonine aldolase mutant and application thereof in preparation of substituted phenylserine derivative

Family Applications After (1)

Application Number Title Priority Date Filing Date
CN202110925261.4A Active CN113481188B (en) 2018-11-06 2018-11-06 Threonine aldolase mutant and application thereof in preparation of substituted phenylserine derivative

Country Status (1)

Country Link
CN (2) CN109402098B (en)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110272856A (en) * 2019-05-08 2019-09-24 江南大学 A kind of recombinant bacterium that expressing D-Thr aldolase and its construction method and application
CN110330401A (en) * 2019-08-06 2019-10-15 王喆明 The synthetic method of benzene serine derivative based on fixed bed reactors
CN110373440A (en) * 2019-07-23 2019-10-25 长兴制药股份有限公司 The method of one pot of enzyme process preparation DL-serine
CN110592058A (en) * 2019-05-30 2019-12-20 重庆大学 Threonine aldolase, coding gene thereof and application of threonine aldolase in droxidopa biosynthesis
CN111139230A (en) * 2020-01-17 2020-05-12 浙江大学 L-threonine aldolase mutant, gene and method for preparing L-syn-p-methylsulfonylphenylserine
CN111394343A (en) * 2020-04-22 2020-07-10 重庆大学 L-threonine aldolase mutant R318L and application thereof
CN111849952A (en) * 2020-04-22 2020-10-30 重庆大学 L-threonine aldolase mutant R318V and application thereof
CN111849951A (en) * 2020-04-22 2020-10-30 重庆大学 L-threonine aldolase mutant R318M and application thereof
CN112280773A (en) * 2020-11-06 2021-01-29 杭州勇诚睿生物科技有限公司 Bio-enzyme catalysis flow process for preparing 2-amino-3-substituted phenyl-3-hydroxypropionic acid
CN112301067A (en) * 2020-11-06 2021-02-02 杭州勇诚睿生物科技有限公司 Environment-friendly process for preparing 2-amino-3-substituted phenyl-3-hydroxypropionic acid
CN112387299A (en) * 2020-11-30 2021-02-23 江南大学 Method for preparing L-furan serine by biomass chemical-enzymatic method
CN115433727A (en) * 2021-06-02 2022-12-06 弈柯莱生物科技(上海)股份有限公司 L-threonine aldolase, and preparation method and application thereof

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114134134B (en) * 2021-10-29 2023-07-14 宁波泓森生物科技有限公司 L-threonine aldolase mutant and application thereof in synthesis of L-syn-p-methylsulfonyl phenylserine
CN116376989B (en) * 2022-04-11 2023-10-24 元素驱动(杭州)生物科技有限公司 Method for preparing keto acid and application of method in preparation of amino acid or amino acid derivative
CN114703169B (en) * 2022-04-29 2023-10-31 重庆大学 L-threonine aldolase mutant R318L/H128N and application thereof
CN115109769B (en) * 2022-05-20 2023-07-14 华东理工大学 L-threonine aldolase mutant and application thereof in synthesis of L-serine
CN114736939B (en) * 2022-06-13 2022-09-02 山东国邦药业有限公司 Method for promoting enzymatic preparation of (2R, 3S) -p-methylsulfonylphenylserine

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000029603A2 (en) * 1998-11-18 2000-05-25 Neose Technologies, Inc. Low cost manufacture of oligosaccharides
EP2302067B1 (en) * 2003-10-21 2014-03-05 Cargill, Incorporated Production of monatin and monatin precursors
CN103881997A (en) * 2014-03-31 2014-06-25 邦泰生物工程(深圳)有限公司 Cubilose acid aldolase mutant as well as coding gene and application thereof
CN107858340A (en) * 2017-12-22 2018-03-30 浙江大学 The phosphate aldolase A mutant of D fructose 6, recombinant expression carrier, genetic engineering bacterium and its application of high catalytic activity
CN110869383A (en) * 2017-05-27 2020-03-06 宁波酶赛生物工程有限公司 Engineered polypeptide and application thereof in synthesis of β -hydroxy- α -amino acid

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1578975A2 (en) * 2002-12-20 2005-09-28 Metanomics GmbH & Co. KGaA Method for producing aminoacids
JP2009533034A (en) * 2006-04-13 2009-09-17 ディーエスエム アイピー アセッツ ビー．ブイ． Process for the preparation of enantiomerically enriched β-amino alcohols starting from glycine and aldehydes in the presence of threonine aldolase and decarboxylase
CN102782137B (en) * 2009-12-30 2016-01-06 代谢探索者公司 The bacterial strain produced for methionine(Met) and method
BR112016027518A2 (en) * 2014-05-23 2018-01-30 Evonik Degussa Gmbh biosynthetic production of acyl amino acids ".

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000029603A2 (en) * 1998-11-18 2000-05-25 Neose Technologies, Inc. Low cost manufacture of oligosaccharides
EP2302067B1 (en) * 2003-10-21 2014-03-05 Cargill, Incorporated Production of monatin and monatin precursors
CN103881997A (en) * 2014-03-31 2014-06-25 邦泰生物工程(深圳)有限公司 Cubilose acid aldolase mutant as well as coding gene and application thereof
CN110869383A (en) * 2017-05-27 2020-03-06 宁波酶赛生物工程有限公司 Engineered polypeptide and application thereof in synthesis of β -hydroxy- α -amino acid
CN107858340A (en) * 2017-12-22 2018-03-30 浙江大学 The phosphate aldolase A mutant of D fructose 6, recombinant expression carrier, genetic engineering bacterium and its application of high catalytic activity

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GENBANK: BAD91544.1: "phenylserine aldolase [Pseudomonas putida]", 《GENBANK DATABASE》 *
余进海等: "L-苏氨酸醛缩酶催化合成L-4-硝基苯基丝氨酸", 《精细化工》 *

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110272856A (en) * 2019-05-08 2019-09-24 江南大学 A kind of recombinant bacterium that expressing D-Thr aldolase and its construction method and application
CN110592058A (en) * 2019-05-30 2019-12-20 重庆大学 Threonine aldolase, coding gene thereof and application of threonine aldolase in droxidopa biosynthesis
CN110592058B (en) * 2019-05-30 2021-11-05 重庆大学 Threonine aldolase, coding gene thereof and application of threonine aldolase in droxidopa biosynthesis
CN110373440A (en) * 2019-07-23 2019-10-25 长兴制药股份有限公司 The method of one pot of enzyme process preparation DL-serine
CN110373440B (en) * 2019-07-23 2022-03-01 长兴制药股份有限公司 Method for preparing DL-serine by one-pot enzyme method
CN110330401A (en) * 2019-08-06 2019-10-15 王喆明 The synthetic method of benzene serine derivative based on fixed bed reactors
CN110330401B (en) * 2019-08-06 2022-06-21 王喆明 Method for synthesizing phenylserine derivative based on fixed bed reactor
CN111139230B (en) * 2020-01-17 2021-05-14 浙江大学 L-threonine aldolase mutant, gene and method for preparing L-syn-p-methylsulfonylphenylserine
CN111139230A (en) * 2020-01-17 2020-05-12 浙江大学 L-threonine aldolase mutant, gene and method for preparing L-syn-p-methylsulfonylphenylserine
CN113249366B (en) * 2020-01-17 2023-12-19 浙江大学 L-threonine aldolase mutant, gene and application
CN113249366A (en) * 2020-01-17 2021-08-13 浙江大学 L-threonine aldolase mutant, gene and application
CN111394343A (en) * 2020-04-22 2020-07-10 重庆大学 L-threonine aldolase mutant R318L and application thereof
CN111849951A (en) * 2020-04-22 2020-10-30 重庆大学 L-threonine aldolase mutant R318M and application thereof
CN111849951B (en) * 2020-04-22 2022-09-20 重庆大学 L-threonine aldolase mutant R318M and application thereof
CN111394343B (en) * 2020-04-22 2022-09-20 重庆大学 L-threonine aldolase mutant R318L and application thereof
CN111849952B (en) * 2020-04-22 2022-09-20 重庆大学 L-threonine aldolase mutant R318V and application thereof
CN111849952A (en) * 2020-04-22 2020-10-30 重庆大学 L-threonine aldolase mutant R318V and application thereof
CN112301067A (en) * 2020-11-06 2021-02-02 杭州勇诚睿生物科技有限公司 Environment-friendly process for preparing 2-amino-3-substituted phenyl-3-hydroxypropionic acid
CN112280773A (en) * 2020-11-06 2021-01-29 杭州勇诚睿生物科技有限公司 Bio-enzyme catalysis flow process for preparing 2-amino-3-substituted phenyl-3-hydroxypropionic acid
CN112280773B (en) * 2020-11-06 2022-07-12 杭州濡湜生物科技有限公司 Bio-enzyme catalysis flow process for preparing 2-amino-3-substituted phenyl-3-hydroxypropionic acid
CN112387299A (en) * 2020-11-30 2021-02-23 江南大学 Method for preparing L-furan serine by biomass chemical-enzymatic method
CN115433727A (en) * 2021-06-02 2022-12-06 弈柯莱生物科技(上海)股份有限公司 L-threonine aldolase, and preparation method and application thereof
CN115433727B (en) * 2021-06-02 2023-11-17 弈柯莱生物科技(集团)股份有限公司 L-threonine aldolase and preparation method and application thereof

Also Published As

Publication number Publication date
CN109402098B (en) 2021-09-17
CN113481188B (en) 2023-03-24
CN113481188A (en) 2021-10-08

Similar Documents

Publication Publication Date Title
CN109402098A (en) Threonine aldolase, mutant and its preparing the application in substituted benzene serine derivative
CN109777763B (en) Genetically engineered bacterium for producing L-theanine and construction and application thereof
CN110791484A (en) Glufosinate-ammonium dehydrogenase mutant and application thereof in production of L-glufosinate-ammonium
CN105331642B (en) Method for catalytically producing α -ketoglutaric acid by using L-glutamic acid oxidase
CN109355266B (en) Imine reductase mutant and application thereof in synthesis of optically active 1-substituted-tetrahydroisoquinoline derivative
CN108823179A (en) A kind of transaminase from actinomyces, mutant, recombinant bacterium and application
CN102277338A (en) Diketoreductase mutant and application thereof
CN112359030B (en) Omega-aminotransferase mutant and application thereof
CN114317507B (en) Nitrile hydratase mutant and application thereof
CN112322606B (en) Nitrile hydratase mutant and application thereof
CN114134134B (en) L-threonine aldolase mutant and application thereof in synthesis of L-syn-p-methylsulfonyl phenylserine
CN111154746B (en) Amidase mutant and application thereof in catalytic synthesis of 2-chloronicotinic acid
CN108570460A (en) Short-chain dehydrogenase enzyme mutant and application thereof
CN107858364A (en) A kind of high temperature resistant height suitable for methanol yeast expression is than bacterial phytases gene living
CN110904062B (en) Strain capable of producing L-alanine at high yield
CN110358751A (en) A kind of recombinant lipase mutant, encoding gene, recombination engineering and application
CN105950595B (en) (-)-gamma-lactam enzyme, gene, mutant, carrier and its preparation and application
CN114277020A (en) Nitrilase mutant, engineering bacteria and application thereof
CN109837267A (en) A kind of phenylalanine lyases and its application in D-trp preparation
CN114196642B (en) Glutamate dehydrogenase variants and their use in the preparation of L-amino acids
CN111518789B (en) Recombinant lipase mutant, gene, vector and application thereof
CN116254253B (en) Glutamic acid decarboxylase mutant obtained through DNA synthesis shuffling combination mutation and application
CN116286910A (en) Recombinant gene and recombinant enzyme for synthesizing L-aspartic acid by maleic acid one-pot biosynthesis and application thereof
CN116555214A (en) Acyl-coa synthetase mutant and efficient synthesis of malonyl-coa thereof
WO2024124605A1 (en) 2,3-butanediol dehydrogenase mutant and use thereof

Legal Events

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

Denomination of invention: Threonine aldolase, mutants and their application in the preparation of substituted phenylserine derivatives

Effective date of registration: 20230215

Granted publication date: 20210917

Pledgee: Mount Huangshan Jiangnan Financing Guarantee Co.,Ltd.

Pledgor: HANGZHOU FAZHELUO BIOTECHNOLOGY Co.,Ltd.|Wang Zheming

Registration number: Y2023980032566