Method for synthesizing (R) -3-amino-1-butanol by double-enzyme cascade catalysis
阅读说明：本技术 一种双酶级联催化合成(r)-3-氨基-1-丁醇的方法 (Method for synthesizing (R) -3-amino-1-butanol by double-enzyme cascade catalysis ) 是由 孙周通 王红月 曲戈 于 2020-12-01 设计创作，主要内容包括：本发明公开了一种双酶级联催化合成(R)-3-氨基-1-丁醇的方法。该方法包括如下步骤：以1,3-丁二醇为底物,经醇脱氢酶催化反应生成4-羟基-2-丁酮；以4-羟基-2-丁酮为底物,经胺脱氢酶或其突变体催化反应生成手性(R)-3-氨基-1-丁醇。本发明提供了一条全新的绿色生物合成路线,以廉价的1,3-丁二醇为原料,通过双酶细胞共表达催化合成手性(R)-3-氨基-1-丁醇。同时本发明提供的方法存在辅因子自循环系统,具有很好的经济效益。本发明具有重要的应用价值。(The invention discloses a method for synthesizing (R) -3-amino-1-butanol by double-enzyme cascade catalysis. The method comprises the following steps: 1, 3-butanediol is used as a substrate, and is catalyzed by alcohol dehydrogenase to generate 4-hydroxy-2-butanone; the chiral (R) -3-amino-1-butanol is generated by the catalytic reaction of amine dehydrogenase or mutants thereof by taking 4-hydroxy-2-butanone as a substrate. The invention provides a brand-new green biosynthesis route, which takes cheap 1, 3-butanediol as a raw material to catalytically synthesize chiral (R) -3-amino-1-butanol by double-enzyme cell co-expression. Meanwhile, the method provided by the invention has a cofactor self-circulation system and has good economic benefit. The invention has important application value.)
1. A method for synthesizing chiral (R) -3-amino-1-butanol, comprising the steps of:
(A) 1, 3-butanediol is used as a substrate, and 4-hydroxy-2-butanone is generated through the catalytic reaction of enzyme A;
(B) taking the 4-hydroxy-2-butanone generated in the step (A) as a substrate, and carrying out catalytic reaction by using an enzyme B to generate chiral (R) -3-amino-1-butanol;
the enzyme A is selected from any one of the following: alcohol dehydrogenase, alcohol dehydrogenase mutant;
the enzyme B is selected from any one of the following: amine dehydrogenase and amine dehydrogenase mutants.
2. A method for synthesizing chiral (R) -3-amino-1-butanol, comprising the steps of: 1, 3-butanediol is taken as a substrate,
carrying out catalytic reaction on the mixture by enzyme A and enzyme B to generate chiral (R) -3-amino-1-butanol;
the enzyme A is selected from any one of the following: alcohol dehydrogenase, alcohol dehydrogenase mutant;
the enzyme B is selected from any one of the following: amine dehydrogenase and amine dehydrogenase mutants.
3. The method of claim 1 or 2, wherein:
the alcohol dehydrogenase is any one of the following (a1) - (a 42):
(a1) alcohol dehydrogenase ADH1 derived from Lactobacillus brevis (Lactobacillus brevis KB290), the access Number of the amino acid sequence is BAN 05992.1;
(a2) alcohol dehydrogenase ADH2 derived from Thermoanaerobacter braskii (Thermoanaerobacter braskii), the Access Number of the amino acid sequence being CAA 46053.1;
(a3) alcohol dehydrogenase ADH3 derived from Aromatous aromaticum EbN1, the Accession Number of the amino acid sequence is CAI 07428.1;
(a4) alcohol dehydrogenase ADH4 derived from Streptomyces coelicolor, wherein the Access Number of the amino acid sequence is Q9KYM 4;
(a5) alcohol dehydrogenase ADH5 derived from Bacillus subtilis, and the Accession Number of the amino acid sequence is WP-003234015.1;
(a6) an alcohol dehydrogenase ADH6 derived from Klebsiella pneumoniae (Klebsiella pneumoniae), the Access Number of the amino acid sequence being WP _ 015958558.1;
(a7) an alcohol dehydrogenase ADH7 derived from Gluconobacter oxydans (Gluconobacter oxydans), the Access Number of the amino acid sequence being WP _ 011253549.1;
(a8) an alcohol dehydrogenase ADH8 derived from the genus Lysidium (Leifsonia sp.S749), the Access Number of the amino acid sequence being BAD 99642.1;
(a9) alcohol dehydrogenase ADH9 derived from Rhodococcus ruber (Rhodococcus ruber), the Access Number of the amino acid sequence being Q8KLT 9;
(a10) alcohol dehydrogenase ADH10 derived from Lactobacillus kefir (Lactobacillus kefir DSM20587), the Access Number of the amino acid sequence being AAP 94029.1;
(a11) an alcohol dehydrogenase ADH11 derived from Thermoanaerobacter virginelii Rt8.B1, the Access Number of the amino acid sequence being AEM 77517.1;
(a12) an alcohol dehydrogenase ADH12 derived from Lactobacillus (Lactobacillus zymae DSM 19395), the access Number of the amino acid sequence being KRL 08675.1;
(a13) alcohol dehydrogenase ADH13 derived from enterococcus alcoholifera (Oenococcus alcoholifera), the access Number of the amino acid sequence is KGO 31568.1;
(a14) alcohol dehydrogenase ADH14 derived from Thermococcus guayaensis (Thermococcus guayaensis), the Access Number of the amino acid sequence being ADV 18977.1;
(a15) alcohol dehydrogenase ADH15 derived from Clostridium botulinum (Clostridium botulinum), the Access Number of the amino acid sequence is WP _ 003399463.1;
(a16) alcohol dehydrogenase ADH16 derived from Desulfotomanum nigricans (Desulfotomanum nigricans), the Access Number of the amino acid sequence is WP _ 003542410.1;
(a17) alcohol dehydrogenase ADH17 derived from Thermoascus carbox cardiovorans (Thermosinus carboxdivorans), the Access Number of the amino acid sequence being WP _ 007290608.1;
(a18) alcohol dehydrogenase ADH18 derived from Thermoanaerobacter alkannati (Thermoanaerobacter mathranii), the Access Number of the amino acid sequence is WP _ 013150923.1;
(a19) an alcohol dehydrogenase ADH19 derived from Clostridium (Clostridium), the Access Number of the amino acid sequence being WP _ 013239134.1;
(a20) alcohol dehydrogenase ADH20 derived from a bacterium of the phylum Firmicutes (Firmicutes bacteria CAG:137), with an access Number of the amino acid sequence CDB 31037.1;
(a21) alcohol dehydrogenase ADH21 derived from Clostridium beijerinckii (Clostridium beijerinckii), the Access Number of the amino acid sequence is WP _ 026889046.1;
(a22) an alcohol dehydrogenase ADH22 derived from Clostridium (Clostridium), the Access Number of the amino acid sequence being WP _ 039771361.1;
(a23) alcohol dehydrogenase ADH23 derived from Methanobacterium acetobacter (Methanosarcina acetovorans), the access Number of the amino acid sequence is WP _ 048065256.1;
(a24) alcohol dehydrogenase ADH24 derived from Methanobacterium laochraceus (Methanosarcina lacustris), the Access Number of the amino acid sequence is WP _ 048125376.1;
(a25) alcohol dehydrogenase ADH25 derived from Methanosarcina thermophila (Methanosarcina thermophila), the access Number of the amino acid sequence is WP _ 048166386.1;
(a26) alcohol dehydrogenase ADH26 derived from Methanobacterium occidentalis (Methanosarcina siciae), the Access Number of the amino acid sequence is WP _ 048172170.1;
(a27) alcohol dehydrogenase ADH27 derived from Staphylococcus texuelis (Desnuesella massilisis), the Access Number of the amino acid sequence is WP _ 055668884.1;
(a28) an alcohol dehydrogenase ADH28 derived from Bacillus caldaniella (Caldanobacter subterraneus), the Access Number of the amino acid sequence being KUK 09008.1;
(a29) an alcohol dehydrogenase ADH29 derived from Clostridium sticklandii (Clostridium ljungdahliii), the access Number of the amino acid sequence being WP _ 063556461.1;
(a30) alcohol dehydrogenase ADH30 derived from Clostridium beijerinckii (Clostridium beijerinckii), the Access Number of the amino acid sequence is WP _ 065417405.1;
(a31) an alcohol dehydrogenase ADH31 derived from uncultured Clostridium (uncultured Clostridium sp.), the access Number of the amino acid sequence being SCI 81347.1;
(a32) an alcohol dehydrogenase ADH32 derived from Clostridium sporogenes (Clostridium taeniosporum), the access Number of the amino acid sequence being WP _ 069679756.1;
(a33) alcohol dehydrogenase ADH33 derived from Vibrio desulfovii (Desulfovibrio litoralis), the Access Number of the amino acid sequence being WP _ 072695399.1;
(a34) an alcohol dehydrogenase ADH34 derived from a Desulfuromicrobium species (Desutomaculum putei), the Access Number of the amino acid sequence being WP _ 073236284.1;
(a35) alcohol dehydrogenase ADH35 derived from Clostridium beijerinckii (Clostridium beijerinckii), the Access Number of the amino acid sequence is WP _ 077844196.1;
(a36) alcohol dehydrogenase ADH36 derived from Clostridium (Clostridium punicium), the Accession Number of the amino acid sequence is WP _ 077846831.1;
(a37) alcohol dehydrogenase ADH37 derived from Clostridium difficile (Clostridium diolis), the Access Number of the amino acid sequence is WP _ 087701616.1;
(a38) an alcohol dehydrogenase ADH38 derived from Clostridium spongium (Clostridium cochleariae), the access Number of the amino acid sequence being WP _ 089865926.1;
(a39) alcohol dehydrogenase ADH39 derived from Clostridium vaccinium (Clostridium uliginosum), the Accession Number of the amino acid sequence is WP _ 090093236.1;
(a40) an alcohol dehydrogenase ADH40 derived from Clostridium bacteria (Clostridium bacteria SK-Y3), the Access Number of the amino acid sequence being WP _ 094550774.1;
(a41) alcohol dehydrogenase ADH41 derived from Candida parapsilosis (Clostridium botulinum), the Accession Number of the amino acid sequence being WP _ 096043277.1;
(a42) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of a protein defined in any one of (a1) to (a 41);
the amine dehydrogenase is any one of the following (b1) - (b 10):
(b1) amine dehydrogenase AmDH1 derived from Geobacillus stearothermophilus (Geobacillus sterothermophilus) and having an amino acid sequence of SEQ ID No. 1;
(b2) amine dehydrogenase AmDH2 derived from Bacillus stearothermophilus (Bacillus sterothermophilus) and having an amino acid sequence of SEQ ID No. 2;
(b3) amine dehydrogenase AmDH3 derived from Sporosarcina psychrophilila (Sporosarcina psendophila) and having the amino acid sequence of SEQ ID No. 3;
(b4) amine dehydrogenase AmDH4 derived from lysine bacillus sphaericus (Lysinibacillus sphaericus), and having an amino acid sequence of SEQ ID No. 4;
(b5) amine dehydrogenase AmDH5 derived from an amine dehydrogenase of Microbacterium siberia (Exiguobacterium sibiricum), having an amino acid sequence of SEQ ID No. 5;
(b6) amine dehydrogenase AmDH6 derived from common Thermoactinomyces intermedia (Thermoactinomyces intermedia), and the amino acid sequence is SEQ ID No. 6;
(b7) amine dehydrogenase AmDH7 derived from Bacillus thermokali thermonatum (Caldalkalibacillus thermomarum) and having an amino acid sequence of SEQ ID No. 7;
(b8) amine dehydrogenase AmDH8 derived from Laceella saccharophila (Laceylla saccharophila) and having an amino acid sequence of SEQ ID No. 8;
(b9) amine dehydrogenase AmDH9 derived from Bacillus badius, and the amino acid sequence is SEQ ID No. 9;
(b10) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of a protein defined in any one of (b1) to (b 9);
the mutant of the amine dehydrogenase is (c1) or (c 2):
(c1) sequentially mutating 114 th and 291 th positions of amine dehydrogenase AmDH3 shown in SEQ ID No.3 into valine residues and cysteine residues;
(c2) the 111 th, 114 th and 294 th positions of the amine dehydrogenase AmDH3 shown in SEQ ID No.3 are mutated into a phenylalanine residue, a valine residue and a cysteine residue in sequence.
4. A method according to any of claims 1 to 3, characterized by: the enzyme A and the enzyme B are in the form of crude enzyme liquid, crude enzyme powder, pure enzyme or whole cells to perform catalysis.
5. A method according to claim 2 or 3, characterized by: the catalytic reaction by the enzyme A and the enzyme B is a total cell catalytic reaction co-expressed by the enzyme A and the enzyme B.
6. The method of claim 4 or 5, wherein:
the crude enzyme liquid, the crude enzyme powder and the pure enzyme are prepared according to the method comprising the following steps: expressing the enzyme A and/or the enzyme B in a host cell to obtain a recombinant cell; cracking the recombinant cells to obtain the crude enzyme solution, crude enzyme powder or pure enzyme;
further, the whole cells are prepared according to a method comprising the following steps: expressing the enzyme A and/or the enzyme B in a host cell to obtain a recombinant cell, namely the whole cell;
still further, the recombinant cell is prepared according to a method comprising the following steps: introducing a nucleic acid molecule capable of expressing the enzyme A and/or the enzyme B into the host cell, and obtaining the recombinant cell expressing the enzyme A and/or the enzyme B after induction culture;
further, said "nucleic acid molecule capable of expressing said enzyme A and/or said enzyme B" is introduced into said host cell in the form of a recombinant vector; the recombinant vector is a bacterial plasmid, a bacteriophage, a yeast plasmid or a retrovirus packaging plasmid carrying the coding gene of the enzyme A and/or the enzyme B;
and/or the host cell is a prokaryotic cell or a lower eukaryotic cell;
specifically, the prokaryotic cell is a bacterium; the lower eukaryotic cell is a yeast cell;
more specifically, the bacterium is escherichia coli.
7. The method of any of claims 1 to 6, wherein:
the temperature of the catalytic reaction is 25-40 ℃;
the time of the catalytic reaction is 4-48 h.
8. The method of any of claims 1 to 7, wherein:
when the enzyme A is subjected to the step (A) in the form of whole cells, crude enzyme solution, crude enzyme powder or pure enzyme, the catalytic reaction is carried out in a buffer as shown below (d 1);
when the enzyme B is subjected to the step (B) in the form of whole cells, crude enzyme solution, crude enzyme powder or pure enzyme, the catalytic reaction is carried out in a buffer as shown below (d 2);
when the enzyme A and the enzyme B co-express whole cell catalytic reaction, the catalytic reaction is performed in a buffer as shown below (d 2);
(d1) a phosphate buffer solution having a concentration of 50 to 100mM and a pH of 6.5 to 8.5;
(d2) ammonium chloride/ammonia buffer solution with the concentration of 50 mM-4M and the pH value of 7.0-9.5.
9. An enzyme system or a related product thereof, characterized in that: the enzyme system comprising the enzyme A and the enzyme B of any one of claims 1 to 8;
the related products are nucleic acid molecules capable of expressing each enzyme in the enzyme system, or expression cassettes, recombinant vectors, recombinant bacteria or transgenic cell lines containing the nucleic acid molecules.
10. Use of the enzyme system of claim 9 or a related product thereof in the synthesis of chiral (R) -3-amino-1-butanol.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for catalytically synthesizing (R) -3-amino-1-butanol by double enzyme cascade, in particular to a method for catalytically synthesizing chiral (R) -3-amino-1-butanol by alcohol dehydrogenase and amine dehydrogenase in a cascade manner.
Background
Chiral amino alcohol compounds are structural units of a plurality of bioactive molecules, are important medical and fine chemical intermediates, wherein (R) -3-amino-1-butanol is an important precursor for synthesizing anti-AIDS integrase inhibitor Dolutegravir (Dolutegravir), and can also be derived into beta-lactam for synthesizing penem antibiotics.
(R) -3-amino-1-butanol is mainly synthesized by a chemical method, and enzymatic synthesis has been reported less frequently. Gertzmann et al, 1995, used chiral (R) -alanine as a raw material, protected with amino groups, increased the carbon chain with diazomethane to become beta-amino acid ester, deprotected and reduced to obtain the target product. In 1977 Kinas et al reacted crotonate with (R) -phenethylamine to generate a group of epimers with two chiral centers, separated by silica gel column chromatography to obtain a single isomer, and then subjected to ester reduction and debenzylation to obtain (R) -3-amino-1-butanol; the method has fewer steps and easily obtained raw materials, is a method hopefully realizing industrial production, but has the following problems that the first step has poor reaction selectivity, two enantiomers with almost equal quantity are obtained, the separation and purification are difficult, the chromatographic column method is often adopted for separation, the eluent dosage is large, the loss is large, the efficiency is low, and the expensive LiAlH is used4As a reducing agent, the cost of raw materials is also obviously increased, and the method is not suitable for large-scale industrial production. In 1998, Tatsuya et al reported a method for synthesizing (R) -3-amino-1-butanol, but the method had expensive raw materials and severe reaction conditions.
In the existing synthesis method of (R) -3-amino-1-butanol, a chemical method needs high temperature, high pressure and a metal catalyst, the reaction conditions are harsh, the pollution is large, the safety coefficient is low, and the yield is 60-70%. The biological method has mild reaction conditions but low conversion rate. Therefore, a new green method is searched for synthesizing the (R) -3-amino-1-butanol, and the method has important scientific value and social significance for promoting green upgrade of the existing chemical process and strengthening environmental protection.
The 1, 3-butanediol is an important chemical raw material, has the characteristics of easy acquisition, low price and the like, is used as a synthetic raw material of (R) -3-amino-1-butanol, and has good economic benefit.
Disclosure of Invention
The object of the present invention is how to synthesize chiral (R) -3-amino-1-butanol.
The invention firstly protects a method for synthesizing chiral (R) -3-amino-1-butanol.
The method for synthesizing chiral (R) -3-amino-1-butanol, which is provided by the invention, can be specifically a method I, and can comprise the following steps:
(A) 1, 3-butanediol is used as a substrate, and 4-hydroxy-2-butanone is generated through the catalytic reaction of enzyme A;
(B) taking the 4-hydroxy-2-butanone generated in the step (A) as a substrate, and carrying out catalytic reaction by using an enzyme B to generate chiral (R) -3-amino-1-butanol;
the enzyme A is selected from any one of the following: alcohol dehydrogenase, alcohol dehydrogenase mutant;
the enzyme B is selected from any one of the following: amine dehydrogenase and amine dehydrogenase mutants.
The method for synthesizing chiral (R) -3-amino-1-butanol, which is provided by the invention, can be specifically a method II, and can comprise the following steps: 1, 3-butanediol is used as a substrate, and chiral (R) -3-amino-1-butanol is generated through the catalytic reaction of enzyme A and enzyme B;
the enzyme A is selected from any one of the following: alcohol dehydrogenase, alcohol dehydrogenase mutant;
the enzyme B is selected from any one of the following: amine dehydrogenase and amine dehydrogenase mutants.
Any one of the alcohol dehydrogenases may be any one of the following (a1) to (a 42).
(a1) An alcohol dehydrogenase ADH1 derived from Lactobacillus brevis (Lactobacillus brevis KB290), the access Number of the amino acid sequence is BAN 05992.1.
(a2) An alcohol dehydrogenase ADH2 derived from Thermoanaerobacter braskii (Thermoanaerobacter braskii), the Access Number of the amino acid sequence being CAA 46053.1.
(a3) An alcohol dehydrogenase ADH3 derived from Aromatous aromaticum EbN1, the Accession Number of the amino acid sequence is CAI 07428.1.
(a4) An alcohol dehydrogenase ADH4 derived from Streptomyces coelicolor, the access Number of the amino acid sequence is Q9KYM 4.
(a5) An alcohol dehydrogenase ADH5 derived from Bacillus subtilis, and the Accession Number of the amino acid sequence is WP _ 003234015.1.
(a6) An alcohol dehydrogenase ADH6 derived from Klebsiella pneumoniae (Klebsiella pneumoniae), the Access Number of the amino acid sequence being WP _ 015958558.1.
(a7) An alcohol dehydrogenase ADH7 derived from Gluconobacter oxydans (Gluconobacter oxydans), the Access Number of the amino acid sequence being WP _ 011253549.1.
(a8) An alcohol dehydrogenase ADH8 derived from the genus Lysidium (Leifsonia sp.S749), the Access Number of the amino acid sequence being BAD 99642.1.
(a9) Alcohol dehydrogenase ADH9 derived from Rhodococcus ruber (Rhodococcus ruber), the Access Number of the amino acid sequence being Q8KLT 9.
(a10) An alcohol dehydrogenase ADH10 derived from Lactobacillus kefir (Lactobacillus kefir DSM20587), the access Number of the amino acid sequence being AAP 94029.1.
(a11) An alcohol dehydrogenase ADH11 derived from Thermoanaerobacter virginelii Rt8.B1, the access Number of the amino acid sequence being AEM 77517.1.
(a12) An alcohol dehydrogenase ADH12 derived from Lactobacillus (Lactobacillus zymae DSM 19395) with an access Number of KRL08675.1 as amino acid sequence.
(a13) Alcohol dehydrogenase ADH13 derived from Oenococcus alcoholifera (Oenococcus alcoholifera), the Access Number of the amino acid sequence is KGO 31568.1.
(a14) An alcohol dehydrogenase ADH14 derived from Thermococcus guayaensis (Thermococcus guayaensis), the access Number of the amino acid sequence being ADV 18977.1.
(a15) An alcohol dehydrogenase ADH15 derived from Clostridium botulinum (Clostridium botulinum), the access Number of the amino acid sequence is WP _ 003399463.1.
(a16) An alcohol dehydrogenase ADH16 derived from Desulfotomanum nigricans (Desulfotomanum nigricans), the access Number of the amino acid sequence being WP _ 003542410.1.
(a17) An alcohol dehydrogenase ADH17 derived from Thermoascus carbooxydivorans (Thermosinus carboxdivorans), with an access Number of amino acid sequence WP _ 007290608.1.
(a18) An alcohol dehydrogenase ADH18 derived from Thermoanaerobacter alkannati (Thermoanaerobacter mathranii), the Access Number of the amino acid sequence being WP _ 013150923.1.
(a19) An alcohol dehydrogenase ADH19 derived from Clostridium (Clostridium), and the Access Number of the amino acid sequence is WP _ 013239134.1.
(a20) An alcohol dehydrogenase ADH20 derived from a bacterium of the phylum Firmicutes (Firmicutes bacteria CAG:137), the access Number of the amino acid sequence being CDB 31037.1.
(a21) An alcohol dehydrogenase ADH21 derived from Clostridium beijerinckii (Clostridium beijerinckii), the access Number of the amino acid sequence being WP _ 026889046.1.
(a22) An alcohol dehydrogenase ADH22 derived from Clostridium (Clostridium), and the Access Number of the amino acid sequence is WP _ 039771361.1.
(a23) An alcohol dehydrogenase ADH23 derived from Methanobacterium acetobacter (Methanosarcina acetovorans), the access Number of the amino acid sequence is WP _ 048065256.1.
(a24) An alcohol dehydrogenase ADH24 derived from Methanobacterium laochromogenes (Methanosarcina lacustris), the access Number of the amino acid sequence being WP _ 048125376.1.
(a25) An alcohol dehydrogenase ADH25 derived from Methanosarcina thermophila, the access Number of the amino acid sequence being WP _ 048166386.1.
(a26) An alcohol dehydrogenase ADH26 derived from Methanobacterium occidentalis (Methanosarcina siciae), the access Number of the amino acid sequence being WP _ 048172170.1.
(a27) An alcohol dehydrogenase ADH27 derived from Staphylococcus texuelis (Desnuesella massilisis), the Access Number of the amino acid sequence being WP _ 055668884.1.
(a28) An alcohol dehydrogenase ADH28 derived from Bacillus caldaniella (Caldanobacter subterraneus), the Access Number of the amino acid sequence being KUK 09008.1.
(a29) An alcohol dehydrogenase ADH29 derived from Clostridium closterium ljungdahliii, the access Number of the amino acid sequence being WP _ 063556461.1.
(a30) An alcohol dehydrogenase ADH30 derived from Clostridium beijerinckii (Clostridium beijerinckii), the access Number of the amino acid sequence being WP _ 065417405.1.
(a31) An alcohol dehydrogenase ADH31 derived from uncultured Clostridium (uncultured Clostridium sp.) and having an access Number of the amino acid sequence SCI 81347.1.
(a32) An alcohol dehydrogenase ADH32 derived from Clostridium sporogenes (Clostridium taeniosporum), the access Number of the amino acid sequence being WP _ 069679756.1.
(a33) An alcohol dehydrogenase ADH33 derived from Vibrio desulfovii (Desulfuricus), the Access Number of the amino acid sequence being WP _ 072695399.1.
(a34) An alcohol dehydrogenase ADH34 derived from a Desulfotomaculum (Desulfotomaculum putei) and having an Access Number of amino acid sequence WP _ 073236284.1.
(a35) An alcohol dehydrogenase ADH35 derived from Clostridium beijerinckii (Clostridium beijerinckii), the access Number of the amino acid sequence being WP _ 077844196.1.
(a36) An alcohol dehydrogenase ADH36 derived from Clostridium (Clostridium punicium) and having an access Number of WP _077846831.1 as the amino acid sequence.
(a37) An alcohol dehydrogenase ADH37 derived from Clostridium difficile (Clostridium diolis), the access Number of the amino acid sequence being WP _ 087701616.1.
(a38) An alcohol dehydrogenase ADH38 derived from Clostridium spongium (Clostridium cochleariae), the access Number of the amino acid sequence being WP _ 089865926.1.
(a39) Alcohol dehydrogenase ADH39 derived from Clostridium vaccinium (Clostridium uliginosum), the Access Number of the amino acid sequence is WP _ 090093236.1.
(a40) An alcohol dehydrogenase ADH40 derived from Clostridium bacteria (Clostridium bacteria SK-Y3) having an access Number of WP-094550774.1.
(a41) An alcohol dehydrogenase ADH41 derived from Candida parapsilosis (Clostridium botulinum), the Accession Number of the amino acid sequence being WP _ 096043277.1.
(a42) A fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (a1) to (a 41).
Any one of the above amine dehydrogenases may be any one of the following (b1) to (b 10).
(b1) Amine dehydrogenase AmDH1 derived from Geobacillus stearothermophilus (Geobacillus sterothermophilus) and having an amino acid sequence of SEQ ID No. 1.
(b2) Amine dehydrogenase AmDH2 derived from Bacillus stearothermophilus (Bacillus sterothermophilus) and having an amino acid sequence of SEQ ID No. 2.
(b3) Amine dehydrogenase AmDH3 derived from Sporosarcina psychrophilila (Sporosarcina) and having the amino acid sequence of SEQ ID No. 3.
(b4) Amine dehydrogenase AmDH4 derived from Lysinibacillus sphaericus (Lysinibacillus sphaericus) and having an amino acid sequence of SEQ ID No. 4.
(b5) Amine dehydrogenase AmDH5 derived from an amine dehydrogenase of Microbacterium siberia (Exiguobacterium sibiricum), having an amino acid sequence of SEQ ID No. 5.
(b6) Amine dehydrogenase AmDH6 derived from Thermoactinomyces intermedia (Thermoactinomyces intermedia) has an amino acid sequence of SEQ ID No. 6.
(b7) Amine dehydrogenase AmDH7 derived from Bacillus thermokali thermonatum (Caldalkalibacillus thermomarum) has an amino acid sequence of SEQ ID No. 7.
(b8) Amine dehydrogenase AmDH8 derived from Laceella saccharophila (Laceylla saccharophila) and having an amino acid sequence of SEQ ID No. 8.
(b9) Amine dehydrogenase AmDH9 derived from Bacillus badius, and the amino acid sequence is SEQ ID No. 9.
(b10) A fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (b1) to (b 9).
The mutant of any of the above amine dehydrogenases may be (c1) or (c 2).
(c1) The 114 th site and the 291 th site of the amine dehydrogenase AmDH3 shown in SEQ ID No.3 are mutated into a valine residue and a cysteine residue in turn.
(c2) The 111 th, 114 th and 294 th positions of the amine dehydrogenase AmDH3 shown in SEQ ID No.3 are mutated into a phenylalanine residue, a valine residue and a cysteine residue in sequence.
In any of the above methods, the enzyme A and the enzyme B are each catalyzed by a crude enzyme solution, a crude enzyme powder, a pure enzyme or whole cells.
In any of the above methods, the reaction catalyzed by enzyme a and enzyme B may be a reaction catalyzed by a whole cell co-expressing enzyme a and enzyme B.
In any of the above methods, the crude enzyme solution, crude enzyme powder and/or pure enzyme are prepared by a method comprising the following steps: expressing the enzyme A and/or the enzyme B in a host cell to obtain a recombinant cell; and cracking the recombinant cells to obtain the crude enzyme solution, the crude enzyme powder or the pure enzyme.
In any of the above methods, the whole cells are prepared according to a method comprising the steps of: expressing the enzyme A and/or the enzyme B in host cells, and obtaining recombinant cells, namely the whole cells.
The recombinant cell is prepared according to a method comprising the following steps: introducing a nucleic acid molecule capable of expressing the enzyme A and/or the enzyme B into the host cell, and obtaining the recombinant cell expressing the enzyme A and/or the enzyme B after induction culture.
The "nucleic acid molecule capable of expressing the enzyme A and/or the enzyme B" is introduced into the host cell in the form of a recombinant vector.
The recombinant vector may be a bacterial plasmid, a bacteriophage, a yeast plasmid or a retroviral packaging plasmid carrying the gene encoding the enzyme a and/or the enzyme B.
The host cell may be a prokaryotic cell or a lower eukaryotic cell.
The prokaryotic cell may be a bacterium. The lower eukaryotic cell may be a yeast cell.
The bacterium may be escherichia coli.
The recombinant vector is specifically a recombinant plasmid obtained by replacing a small fragment between enzyme cutting sites Nco I and Xho I of pET28a vector by the encoding gene of the alcohol dehydrogenase and/or the amine dehydrogenase or the mutant thereof.
In any of the above methods, the temperature of the catalytic reaction may be 25 to 40 ℃ (specifically, 30 ℃). The time of the catalytic reaction can be 4-48 h (specifically 24 h).
In any of the above-mentioned methods, when the enzyme A is used in the step (A) in the form of whole cells, crude enzyme solution, crude enzyme powder or pure enzyme, the catalytic reaction is carried out in a buffer as shown in the following (d 1). When the enzyme B is subjected to the step (B) in the form of whole cells, crude enzyme solution, crude enzyme powder or pure enzyme, the catalytic reaction is carried out in a buffer as shown below (d 2). When enzyme A and enzyme B co-express whole cell catalytic reactions, the catalytic reactions were performed in the buffer shown below (d 2).
(d1) A phosphate buffer solution having a concentration of 50 to 100mM and a pH of 6.5 to 8.5.
(d2) Ammonium chloride/ammonia buffer solution with the concentration of 50 mM-4M and the pH value of 7.0-9.5.
(d1) Specifically, the concentration of the buffer solution may be 100mM and the pH may be 8.0.
(d2) Specifically, the buffer solution may be an ammonium chloride/ammonia buffer solution having a concentration of 1M and a pH of 8.0.
In one embodiment of the invention, the host cell is specifically e.coli, more specifically e.coli BL21(DE 3). Correspondingly, the induction culture is to add IPTG to the culture system to a final concentration of 0.1-0.5mM (specifically 0.1mM), 20-37 deg.C (specifically 20 deg.C) for 12-24h (specifically 16 h).
In the present invention, the coenzyme of the enzyme A may be specifically oxidized coenzyme I (i.e., NAD +) or oxidized coenzyme II (i.e., NADP +). The coenzyme of the enzyme B is in particular NADH or NADPH.
The concentration of the 1, 3-butanediol in the reaction system may be 1 to 100mM (e.g., 20 mM). The concentration of the recombinant cells in the reaction system may be 50-500g/L (e.g., 100 g/L). The concentration of the crude enzyme solution or crude enzyme powder in the reaction system may be 10-50g/L (e.g., 20 g/L). The concentration of the pure enzyme in the reaction system may be 0.1 to 2g/L (e.g., 0.5 g/L). The NAD+The concentration in the reaction system may be 0.1 to 2.0mM (e.g., 1.0 mM).
The alcohol dehydrogenase is subjected to the catalytic reaction of step (A)The system comprises the following components: phosphate buffer (pH 8.0) at a concentration of 100mM, 1, 3-butanediol at a final concentration of 1-10mM, NAD at a final concentration of 1mM+Or NADP+Acetone (v/v) with the final concentration of 2 percent and crude enzyme liquid or crude enzyme powder of the alcohol dehydrogenase with the final concentration of 10-40 g/L.
The catalytic reaction system for the amine dehydrogenase or the mutant thereof to be subjected to the step (B) consists of: ammonium chloride/ammonia buffer (pH 8.5) at 1M concentration, 4-hydroxy-2-butanone at 1-20mM final concentration, GDH crude enzyme powder at 2g/L final concentration, glucose at 100mM final concentration, NAD at 1mM final concentration+And a final concentration of 100g/L of the whole cell of the amine dehydrogenase or the mutant thereof.
When the alcohol dehydrogenase (or alcohol dehydrogenase mutant) and amine dehydrogenase (or amine dehydrogenase mutant) catalyze the reaction in whole cells, the composition is as follows: ammonium chloride/ammonia buffer (pH 8.0) at a concentration of 1M, 1, 3-butanediol at a final concentration of 20mM, i.e., NAD at a final concentration of 1mM+The total cells were co-expressed with lysozyme at a final concentration of 1g/L, DNase I (deoxyribonuclease) at a final concentration of 6U/mL, alcohol dehydrogenase (or alcohol dehydrogenase mutant) and amine dehydrogenase (or amine dehydrogenase mutant) at a final concentration of 200 g/L.
The invention also protects the enzyme system or its related products.
The enzyme system comprises any one of the enzyme A and the enzyme B.
The related product can be a nucleic acid molecule capable of expressing each enzyme in the enzyme system, or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule.
The application of the enzyme system or the related products in the synthesis of chiral (R) -3-amino-1-butanol also belongs to the protection scope of the invention.
In the method for synthesizing chiral (R) -3-amino-1-butanol, a cofactor self-circulation system exists. The cofactor regeneration system is used for catalyzing the oxidation of 1, 3-butanediol into 4-hydroxy-2-butanone and NAD by the enzyme A+Is reduced to NADH; the enzyme B catalyzes the production of (R) -3-amino-1-butanol from 4-hydroxy-2-butanone and NADH is oxidized to NAD+Is living in natureFormed NAD+The oxidation of 1, 3-butanediol into 4-hydroxy-2-butanone is carried out again.
The invention provides a brand-new green biosynthesis route, which takes cheap 1, 3-butanediol as a raw material to catalytically synthesize chiral (R) -3-amino-1-butanol by double-enzyme coexpression. The method has the advantages of convenient operation, high optical purity of the product and the like, and has better industrial application prospect in the preparation of chiral (R) -3-amino-1-butanol by biocatalysis.
Drawings
FIG. 1 is a schematic diagram of a reaction for preparing (R) -3-amino-1-butanol by coupling an amine dehydrogenase with an alcohol dehydrogenase.
FIG. 2 is a GC detection spectrum of a reaction of crude enzyme powder of alcohol dehydrogenase catalyzing 1, 3-butanediol to produce 4-hydroxy-2-butanone.
FIG. 3 shows the HPLC detection results of (R) -3-amino-1-butanol prepared by amine dehydrogenase. E: liquid chromatography results of a racemic 3-amino-1-butanol standard substance; f: the sample (R) -3-amino-1-butanol was subjected to liquid chromatography.
FIG. 4 shows the HPLC detection results of (R) -3-amino-1-butanol prepared by coupling amine dehydrogenase with alcohol dehydrogenase. G: liquid chromatography results of a racemic 3-amino-1-butanol standard substance; h: the sample (R) -3-amino-1-butanol was subjected to liquid chromatography.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The quantitative tests in the following examples, all set up three replicates and the results averaged.
In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.
In the following examples, ADH1 is an alcohol dehydrogenase derived from Lactobacillus brevis (Lactobacillus brevis KB290), and the Access Number of the amino acid sequence is BAN 05992.1; ADH2 is derived from thermophilic anaerobic bacillus (Thermoanaerobacter brockii) alcohol dehydrogenase, the amino acid sequence of Access Number is CAA 46053.1; ADH3 is derived from nitrogen fixing vibrio (aromatic extracellular ebN1) alcohol dehydrogenase, amino acid sequence of the access Number is CAI 07428.1; ADH4 is derived from Streptomyces coelicolor alcohol dehydrogenase, the amino acid sequence of Access Number is Q9KYM 4; ADH5 is alcohol dehydrogenase derived from Bacillus subtilis, and the Accession Number of the amino acid sequence is WP-003234015.1; ADH6 is derived from Klebsiella pneumoniae (Klebsiella pneumoniae) alcohol dehydrogenase, amino acid sequence of Access Number WP _ 015958558.1; ADH7 is an alcohol dehydrogenase derived from Gluconobacter oxydans (Gluconobacter oxydans), and the Access Number of the amino acid sequence is WP _ 011253549.1; ADH8 is derived from lysine (Leifsonia sp.S749) alcohol dehydrogenase, amino acid sequence of Access Number BAD 99642.1; ADH9 is derived from red blood coccus (Rhodococcus ruber) alcohol dehydrogenase, amino acid sequence of Access Number for Q8KLT 9; ADH10 is derived from Lactobacillus kefir (Lactobacillus kefir DSM20587) alcohol dehydrogenase, amino acid sequence of the access Number AAP 94029.1; ADH11 is derived from Thermoanaerobacter virginiae Rt8.B1 alcohol dehydrogenase, amino acid sequence of Access Number AEM 77517.1; ADH12 is derived from Lactobacillus (Lactobacillus zymae DSM 19395) alcohol dehydrogenase, amino acid sequence of Access Number KRL 08675.1; ADH13 is derived from wine coccus (Oenococcus alcoholic dehydrogenase), the amino acid sequence of Access Number KGO 31568.1; ADH14 is derived from Thermococcus guayaensis (Thermococcus guayaensis) alcohol dehydrogenase, the amino acid sequence of Access Number for ADV 18977.1; ADH15 is derived from Clostridium botulinum (Clostridium botulinum) alcohol dehydrogenase, amino acid sequence of the Accession Number is WP _ 003399463.1; ADH16 is derived from black desulfurization intestinal bacteria (Desutomaculum nigricans) alcohol dehydrogenase, amino acid sequence of Accession Number is WP _ 003542410.1; ADH17 is derived from carbon monoxide thermophilic antrodia (Thermosinus carboxdiovarans) alcohol dehydrogenase, the amino acid sequence of Access Number is WP _ 007290608.1; ADH18 is derived from thermophilic anaerobic bacillus alkyl (Thermoanaerobacter mathranii) alcohol dehydrogenase, amino acid sequence access Number is WP _ 013150923.1; ADH19 is derived from Clostridium (Clostridium) alcohol dehydrogenase, amino acid sequence of the access Number is WP _ 013239134.1; ADH20 is derived from Firmicutes bacteria CAG (137) alcohol dehydrogenase, amino acid sequence of the access Number of CDB 31037.1; ADH21 is derived from Clostridium beijerinckii (Clostridium beijerinckii) alcohol dehydrogenase, the amino acid sequence of Access Number is WP _ 026889046.1; ADH22 is derived from Clostridium (Clostridium) alcohol dehydrogenase, amino acid sequence of the access Number is WP _ 039771361.1; ADH23 is alcohol dehydrogenase derived from Methanobacterium aceticum, and the Access Number of the amino acid sequence is WP-048065256.1; ADH24 is derived from methanogens (Methanosarcina lacustris) alcohol dehydrogenase, amino acid sequence of access Number is WP _ 048125376.1; ADH25 is alcohol dehydrogenase derived from Methanosarcina thermophila (Methanosarcina thermophila), and the Access Number of the amino acid sequence is WP-048166386.1; ADH26 is alcohol dehydrogenase derived from Methanobacterium occidentalis (Methanosarcina siciae), and the Access Number of the amino acid sequence is WP-048172170.1; ADH27 is derived from Staphylococcus aureus (Desnuesella massilisis) alcohol dehydrogenase, amino acid sequence of Access Number is WP _ 055668884.1; ADH28 is derived from Karda terrestris (Caldanaaerobacter subterraneus) alcohol dehydrogenase, amino acid sequence of Access Number KUK 09008.1; ADH29 is derived from Clostridium closterium ljungdahlii alcohol dehydrogenase, amino acid sequence of Accession Number WP _ 063556461.1; ADH30 is derived from Clostridium beijerinckii (Clostridium beijerinckii) alcohol dehydrogenase, the amino acid sequence of Access Number is WP _ 065417405.1; ADH31 is derived from uncultured Clostridium (uncultred Clostridium sp.) alcohol dehydrogenase, amino acid sequence of the access Number is SCI 81347.1; ADH32 is derived from filamentous spore Clostridium (Clostridium taeniiospora) alcohol dehydrogenase, amino acid sequence of the access Number is WP _ 069679756.1; ADH33 is an alcohol dehydrogenase derived from Vibrio desulfovii (Desulfovibrio litoralis), and the Access Number of the amino acid sequence is WP _ 072695399.1; ADH34 is derived from the desulfurization of intestinal bacteria (Desutomaculum putei) alcohol dehydrogenase, amino acid sequence of Access Number is WP _ 073236284.1; ADH35 is derived from Clostridium beijerinckii (Clostridium beijerinckii) alcohol dehydrogenase, the amino acid sequence of Access Number is WP _ 077844196.1; ADH36 is derived from Clostridium (Clostridium punicium) alcohol dehydrogenase, amino acid sequence of the access Number is WP _ 077846831.1; ADH37 is derived from Clostridium difficile (Clostridium diolis) alcohol dehydrogenase, the amino acid sequence of Access Number is WP _ 087701616.1; ADH38 is derived from Clostridium sponginum (Clostridium cochleariae) alcohol dehydrogenase, amino acid sequence of the access Number is WP _ 089865926.1; ADH39 is alcohol dehydrogenase derived from Clostridium vaccinium (Clostridium uliginosum), and the Access Number of the amino acid sequence is WP _ 090093236.1; ADH40 is derived from Clostridium bacteria (Clostridium bacteria SK-Y3) alcohol dehydrogenase, amino acid sequence of the access Number is WP _ 094550774.1; ADH41 is derived from Candida parapsilosis (Clostridium botulinum) alcohol dehydrogenase, the amino acid sequence of the Accession Number is WP _ 096043277.1; AmDH1 is amine dehydrogenase derived from Geobacillus stearothermophilus, and has an amino acid sequence of SEQ ID No. 1; AmDH2 is amine dehydrogenase derived from Bacillus stearothermophilus (Bacillus stearothermophilus), and has an amino acid sequence of SEQ ID No. 2; AmDH3 is amine dehydrogenase derived from Sporosarcina psychrophilia (Sporosarcina psychrophila), and has amino acid sequence of SEQ ID No. 3; AmDH4 is amine dehydrogenase derived from lysine bacillus sphaericus (Lysinibacillus sphaericus), and has an amino acid sequence of SEQ ID No. 4; AmDH5 is amine dehydrogenase derived from amine dehydrogenase of Microbacterium siberia (Exiguobacterium sibiricum), and has amino acid sequence of SEQ ID No. 5; AmDH6 is amine dehydrogenase derived from Thermoactinomyces intermedius (Thermoactinomyces intermedius), and has an amino acid sequence of SEQ ID No. 6; AmDH7 is amine dehydrogenase derived from Bacillus thermokali thermokalii (Caldalkalibacillus thermomarum), and has amino acid sequence of SEQ ID No. 7; AmDH8 is amine dehydrogenase derived from Laceella saccharina (Laceyella saccharai), and has amino acid sequence of SEQ ID No. 8; AmDH9 is amine dehydrogenase derived from Bacillus badius, and has an amino acid sequence of SEQ ID No. 9; AmDH3-M1 is an amine dehydrogenase mutant obtained by mutating 114 th and 291 th positions of amine dehydrogenase AmDH3 shown in SEQ ID No.3 into valine residues and cysteine residues in sequence; AmDH3-M2 is an amine dehydrogenase mutant obtained by mutating the 111 th, 114 th and 294 th positions of the amine dehydrogenase AmDH3 shown in SEQ ID No.3 into a phenylalanine residue, a valine residue and a cysteine residue in sequence.
2 x High-fidelity Master Mix is a product of Scustraceae Biotechnology Limited, Cat # TP 001.
1, 3-butanediol and 4-hydroxy-2-butanone are products of Shanghai Aladdin Biotechnology GmbH, with the serial numbers B111018 and H106330.
The racemic 3-amino-1-butanol is a Shanghai Shao Yuan reagent with the product number of SY 030187.
Through a large number of experiments, the inventor establishes a method for synthesizing chiral (R) -3-amino-1-butanol by alcohol dehydrogenase and amine dehydrogenase cascade catalysis. The reaction scheme for coupling amine dehydrogenase to produce (R) -3-amino-1-butanol is shown in FIG. 1.
Example 1 preparation of an engineered bacterium expressing alcohol dehydrogenase and an engineered bacterium expressing amine dehydrogenase
The coding genes of the alcohol dehydrogenase and the amine dehydrogenase are respectively sent to the Cherey biotechnology and Limited company for synthesis (codon optimization is carried out by taking escherichia coli as a host according to requirements), and the synthesized genes are connected to various expression vectors to construct the gene chip. The expression vector is any vector conventionally used in the art. The vector is specifically pET28a, and a small DNA segment between enzyme cutting recognition sites Nco I and Xho I of pET28a is replaced by a coding gene of related enzyme after whole-gene synthesis, so that a recombinant expression vector is obtained.
The recombinant expression vector is transformed into a suitable microbial host. The host microorganism is a variety of host microorganisms conventionally used in the art as long as stable self-replication of the above recombinant expression vector and efficient expression of the alcohol dehydrogenase or amine dehydrogenase gene are satisfied. Among them, the preferred host microorganism is Escherichia coli (Escherichia coli), preferably Escherichia coli BL21(DE3), and the above recombinant expression vector is transformed into e.coli BL21(DE3) to obtain the genetically engineered strain of the present invention.
Obtaining the mutant of the alcohol dehydrogenase gene by using a site-directed mutagenesis method, and then obtaining the corresponding genetic engineering strain for expressing the alcohol dehydrogenase mutant according to the steps.
Obtaining the mutant of the amine dehydrogenase gene by using a site-directed mutagenesis method, and then obtaining the corresponding genetic engineering strain for expressing the amine dehydrogenase mutant according to the steps.
Example 2 preparation of an engineered bacterium co-expressed with alcohol dehydrogenase and amine dehydrogenase
The gene coding for alcohol dehydrogenase (or alcohol dehydrogenase mutant) and amine dehydrogenase (or amine dehydrogenase mutant) are each synthesized as a whole gene (codon optimization is carried out using E.coli as a host as required), and the synthesized genes are ligated to various expression vectors. The expression vector is any vector conventionally used in the art. The vector of the invention is specifically pET28 a. Firstly, replacing a small DNA segment between restriction enzyme cutting recognition sites Nco I and Xho I of pET28a with a coding gene of alcohol dehydrogenase after whole-gene synthesis to obtain an intermediate vector; then, the coding gene of the amine dehydrogenase after the whole gene synthesis was linked to the coding gene of the alcohol dehydrogenase by using a recombinant cloning kit (Nanjing Nodezam Biotechnology Co., Ltd., product No. C112-02) to obtain a recombinant expression plasmid.
The recombinant expression plasmid is transformed into a suitable microbial host. The host microorganism is a variety of host microorganisms conventionally used in the art as long as stable self-replication of the above recombinant expression plasmid and efficient expression of the alcohol dehydrogenase gene and the amine dehydrogenase gene can be satisfied. Among them, the preferred host microorganism is Escherichia coli (Escherichia coli), preferably Escherichia coli BL21(DE3), and the genetically engineered strain of the present invention can be obtained by transforming the recombinant expression plasmid into e.coli BL21(DE 3).
Example 3 expression or Co-expression of alcohol dehydrogenase and amine dehydrogenase and preparation of Whole cell, crude enzyme
1. The recombinant expression vector constructed in example 1 or the recombinant expression plasmid constructed in example 2 was transferred into competent cells of Escherichia coli BL21(DE3) to obtain recombinant cells.
2. The transformants were picked up in 5mL LB liquid medium containing 50. mu.g/mL kanamycin, shaken at 37 ℃ and 220rpm overnight for 12 hours, inoculated in 1% volume percentage into TB liquid medium containing 50. mu.g/mL kanamycin, and cultured at 37 ℃ to OD600nmWhen the concentration is 0.7, adding IPTG with the final concentration of 0.1mmol/L, inducing expression for 12h at 20 ℃ and 220rpm, centrifuging for 10min at 4 ℃ and 4000rpm, collecting precipitated thalli (namely whole cells), and suspending the collected thalli by using phosphate buffer solution (50mmol/L and pH 7.4) to obtain thalli suspension; then, the bacterial cells are broken by ultrasonic under the ice-bath condition, and a broken sample (namely, crude enzyme liquid) is obtained.
3. Centrifuging the crude enzyme solution at 4 deg.C and 8000rpm for 30min, collecting supernatant, freezing the supernatant at-80 deg.C, and freeze-drying with vacuum drier to obtain crude enzyme powder.
Example 4 catalysis of 1, 3-butanediol by alcohol dehydrogenase or alcohol dehydrogenase mutant to 4-hydroxy-2-butanone
The crude enzyme powder of the alcohol dehydrogenase or the alcohol dehydrogenase mutant prepared in example 3 was used to catalyze the production of 4-hydroxy-2-butanone from 1, 3-butanediol.
To the reaction system were added phosphate buffer (pH 8.0) at a concentration of 100mM, 1, 3-butanediol at a final concentration of 5mM, and NAD at a final concentration of 1mM in this order+Or NADP+Acetone (v/v) with a final concentration of 2 percent and alcohol dehydrogenase or alcohol dehydrogenase mutant crude enzyme powder with a final concentration of 10-40 g/L. After the reaction system was reacted at 30 ℃ for 24 hours, the product was subjected to Gas Chromatography (GC).
The detection conditions for Gas Chromatography (GC) were as follows: a chromatographic column: rtx-1 (25X 0.32mm ID); temperature rising procedure: keeping the temperature at 80 ℃ for 3 minutes; heating to 120 ℃ at the speed of 5 ℃/min, and keeping for 4 minutes; the split ratio is as follows: 20: 1; flow rate of the chromatographic column: 1 mL/min; sample introduction amount: 2 mu L of the solution; operating time: for 15 minutes.
The GC detection result spectrum of the reaction for catalyzing 1, 3-butanediol to generate 4-hydroxy-2-butanone by the crude enzyme powder of the alcohol dehydrogenase is shown in figure 2(A is the gas chromatography result of a racemic 1, 3-butanediol standard substance, B is the gas chromatography result of a 4-hydroxy-2-butanone standard substance, C is the gas chromatography result of the racemic 1, 3-butanediol and 4-hydroxy-2-butanone standard substance, and D is the gas chromatography result of a sample substrate 1, 3-butanediol and an intermediate product 4-hydroxy-2-butanone).
The conversion was calculated and the results are shown in table 1. The results show that the alcohol dehydrogenase and the alcohol dehydrogenase mutant can catalyze 1, 3-butanediol to generate 4-hydroxy-2-butanone, and the conversion rate is 8-99%.
TABLE 1 results of conversion of 1, 3-butanediol to 4-hydroxy-2-butanone catalyzed by alcohol dehydrogenase and alcohol dehydrogenase mutants
Name (R)
Conversion (%)
Name (R)
Conversion (%)
ADH1
43
ADH22
46
ADH2
99
ADH23
9
ADH3
52
ADH24
12
ADH4
60
ADH25
87
ADH5
55
ADH26
13
ADH6
54
ADH27
60
ADH7
59
ADH28
11
ADH8
60
ADH29
54
ADH9
51
ADH30
15
ADH10
57
ADH31
12
ADH11
31
ADH32
20
ADH12
16
ADH33
25
ADH13
40
ADH34
40
ADH14
99
ADH35
75
ADH15
12
ADH36
51
ADH16
92
ADH37
69
ADH17
14
ADH38
11
ADH18
99
ADH39
46
ADH19
8
ADH40
9
ADH20
15
ADH41
12
ADH21
15
Note: conversion rate ═ A1/A2×100％；A1: analyzing the peak area value of the obtained 1, 3-butanediol or 4-hydroxy-2-butanone by using gas chromatography; a. the2: and (3) analyzing the peak area value of the obtained standard 1, 3-butanediol or 4-hydroxy-2-butanone by liquid chromatography.
Example 5 Whole-cell catalysis of amine dehydrogenase or amine dehydrogenase mutant for production of (R) -3-amino-1-butanol from 4-hydroxy-2-butanone
Whole-cell catalysis of 4-hydroxy-2-butanone to (R) -3-amino-1-butanol by the amine dehydrogenase or the amine dehydrogenase mutant prepared in example 3 was performed.
1. To the reaction system were added ammonium chloride/ammonia buffer solution (pH 8.5) at a concentration of 1M, 4-hydroxy-2-butanone at a final concentration of 20mM, GDH crude enzyme powder at a final concentration of 2g/L, glucose at a final concentration of 100mM, and GDH crude enzyme powder at a final concentration of 100mM in this orderNAD at a concentration of 1mM+And amine dehydrogenase or an amine dehydrogenase mutant at a final concentration of 100 g/L. The reaction system is reacted for 24 hours at the temperature of 30 ℃ to obtain reaction liquid.
2. mu.L of 0.4mol/L boric acid buffer (pH 9.5), 150. mu.L ultrapure water, 200. mu.L derivatizing agent and 100. mu.L reaction solution were thoroughly mixed and left to stand for 2 minutes, and then centrifuged at 12000rpm for 10 minutes, and the supernatant was collected and filtered with a filter.
A derivatizing agent: 0.343g of o-phthalaldehyde, 5mL of absolute ethyl alcohol, 0.147g N-acetyl-L cysteine and a proper amount of 0.4mol/L boric acid buffer solution (pH 9.5) are mixed, and then the volume is adjusted to 25mL by 0.4mol/L boric acid buffer solution (pH 9.5), and the mixture is kept in the dark for standby.
3. The filtrate was subjected to HPLC detection.
The HPLC detection conditions are as follows: an Agilent SB-Aq C18 column (4.6 x 250mm, 5 μm); the detection wavelength is 334 nm; column temperature: 35 ℃, flow rate: 1mL/min, loading amount: 10 mu L of the solution; the gradient elution procedure is shown in table 2.
TABLE 2 gradient elution procedure for HPLC
Time (min)
Mobile phase (methanol)%
Mobile phase (0.05mol/L sodium acetate)%
0-6 (excluding 6)
30
70
6-7 (including 6, excluding 7)
30
70
7-15 (including 7, not including 15)
45
55
15-15.5 (including 15, not including 15.5)
45
55
15.5-20 (including 15.5 and 20)
30
70
Note: the% in the table indicates the volume percentage.
The HPLC detection result spectrum of the reaction for catalyzing and reducing 4-hydroxy-2-butanone to generate (R) -3-amino-1-butanol by the amine dehydrogenase is shown in FIG. 3 (E is the liquid chromatography result of the racemic 3-amino-1-butanol standard, and F is the liquid chromatography result of the sample (R) -3-amino-1-butanol).
The conversion was calculated and the results of partial HPLC measurements are shown in Table 3. The result shows that the amine dehydrogenase or the mutant thereof can asymmetrically reduce and catalyze 4-hydroxy-2-butanone to generate (R) -3-amino-1-butanol, the conversion rate is 5-36 percent, and the stereoselectivity is more than 99 percent (R).
TABLE 3 results of the whole-cell catalysis of amine dehydrogenase and its mutants to (R) -3-amino-1-butanol from 4-hydroxy-2-butanone
Name (R)
Conversion (%)
ee(％)
AmDH1
36
>99(R)
AmDH2
20
>99(R)
AmDH3
23
>99(R)
AmDH3-M1
20
>99(R)
AmDH3-M2
29
>99(R)
AmDH4
26
>99(R)
AmDH5
23
>99(R)
AmDH6
＜5
>99(R)
AmDH7
＜5
>99(R)
AmDH8
＜5
>99(R)
AmDH9
＜5
>99(R)
Note: conversion rate ═ A1/A2×100％；A1: (R) -3-amino-1-butanol peak area value obtained by liquid chromatography; a. the2: the peak area value of the standard substance (R) -3-amino-1-butanol obtained by liquid chromatography analysis; ee (stereoselectivity) ═ aS-AR)/(AS+AR)×100％；AS: peak area values of (S) -3-amino-1-butanol obtained by liquid chromatography; a. theR: the peak area value of the obtained (R) -3-amino-1-butanol was analyzed by liquid chromatography.
Example 6 Co-expression of alcohol dehydrogenase (or alcohol dehydrogenase mutant) and amine dehydrogenase (or amine dehydrogenase mutant) Whole cell catalysis of production of (R) -3-amino-1-butanol from 1, 3-butanediol
Whole-cell catalysis of 1, 3-butanediol to produce (R) -3-amino-1-butanol by the alcohol dehydrogenase (or alcohol dehydrogenase mutant) and amine dehydrogenase (or amine dehydrogenase mutant) prepared in example 3.
1. To the reaction system were added ammonium chloride/ammonia buffer (pH 8.0) at a concentration of 1M, 1, 3-butanediol at a final concentration of 20mM, NAD at a final concentration of 1mM+The total cells were co-expressed with lysozyme at a final concentration of 1g/L, DNase I (deoxyribonuclease) at a final concentration of 6U/mL, alcohol dehydrogenase (or alcohol dehydrogenase mutant) and amine dehydrogenase (or amine dehydrogenase mutant) at a final concentration of 200 g/L. The reaction system is reacted for 24 hours at the temperature of 30 ℃ to obtain reaction liquid.
2. mu.L of the reaction solution, 50. mu.L of 1mol/L ammonium chloride/aqueous ammonia buffer (pH 8.5) and 500. mu.L of acetonitrile were thoroughly mixed and then filtered.
3. mu.L of the filtrate from step 2, 20. mu.L of Marfey's reagent (concentration 14mmol/L,acetonitrile as solvent), 36. mu.L NaHCO3(concentration: 1mol/L) and 100. mu.L of DMSO were mixed, reacted at 40 ℃ and 1000rpm for 2 hours, and then 40. mu.L of HCl (concentration: 1mol/L) was added to terminate the reaction, thereby obtaining a reaction solution.
4. And (4) carrying out HPLC detection on the reaction liquid obtained in the step (3).
The HPLC detection conditions are as follows: zorbax SB-C18 column (4.6X 150mm, 5 μm); the detection wavelength is 340 nm; column temperature: 25 ℃; flow rate: 1 mL/min; sample loading amount: 10 mu L of the solution; mobile phase A: ultrapure water (0.1% trifluoroacetic acid), mobile phase B: methanol (0.1% trifluoroacetic acid). Elution procedure: 60% A/40% B; 40% B for 6 min; b was increased to 60% in 9 minutes, left for 3 minutes; b was reduced to 40% in 2 minutes and left for 5 minutes.
The HPLC detection result map of the alcohol dehydrogenase (or alcohol dehydrogenase mutant) and the amine dehydrogenase (or amine dehydrogenase mutant) co-expressing whole cells catalyzing the production of (R) -3-amino-1-butanol from 1, 3-butanediol is shown in FIG. 4.
The conversion was calculated and the results of partial HPLC measurements are shown in Table 4. The results show that the alcohol dehydrogenase (or the alcohol dehydrogenase mutant) and the amine dehydrogenase (or the amine dehydrogenase mutant) can be co-expressed in a whole-cell form to catalyze 1, 3-butanediol to generate (R) -3-amino-1-butanol, the conversion rate is 9-38%, and the stereoselectivity is more than 99% (R).
TABLE 4 results of the conversion of 1, 3-butanediol to (R) -3-amino-1-butanol catalyzed by the co-expression of alcohol dehydrogenase (or alcohol dehydrogenase mutant) and amine dehydrogenase (or amine dehydrogenase mutant) in whole cells
Name (R)
Conversion (%)
ee(％)
ADH4-AmDH1
18
>99(R)
ADH4-AmDH2
9
>99(R)
ADH4-AmDH3
15
>99(R)
ADH4-AmDH4
20
>99(R)
ADH14-AmDH1
32
>99(R)
ADH14-AmDH2
16
>99(R)
ADH14-AmDH3
28
>99(R)
ADH14-AmDH4
35
>99(R)
ADH18-AmDH1
35
>99(R)
ADH18-AmDH2
18
>99(R)
ADH18-AmDH3
26
>99(R)
ADH18-AmDH4
38
>99(R)
Note: conversion rate ═ A1/A2×100％；A1: (R) -3-amino-1-butanol peak area value obtained by liquid chromatography; a. the2: the peak area value of the standard substance (R) -3-amino-1-butanol obtained by liquid chromatography analysis; ee (stereoselectivity) ═ aS-AR)/(AS+AR)×100％；AS: peak area values of (S) -3-amino-1-butanol obtained by liquid chromatography; a. theR: the peak area value of the obtained (R) -3-amino-1-butanol was analyzed by liquid chromatography.
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> method for synthesizing (R) -3-amino-1-butanol by double-enzyme cascade catalysis
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 429
<212> PRT
<213> Artificial sequence
<400> 1
Met Glu Leu Phe Lys Tyr Met Glu Thr Tyr Asp Tyr Glu Gln Val Leu
1 5 10 15
Phe Cys Gln Asp Lys Glu Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Met Tyr
35 40 45
Asn Ser Glu Glu Glu Ala Leu Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Ser Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Ala Met Phe
85 90 95
Arg Ala Phe Gly Arg Phe Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Ala Asp Met Asp Ile Ile Tyr Gln
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Glu Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Ala Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Glu Ala Phe Gly Ser Asp Ser Leu Glu Gly Lys Val Val
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr His Leu Cys Arg His Leu
180 185 190
His Glu Glu Gly Ala Lys Leu Ile Val Thr Asp Ile Asn Lys Glu Val
195 200 205
Val Ala Arg Ala Val Glu Glu Phe Gly Ala Lys Ala Val Asp Pro Asn
210 215 220
Asp Ile Tyr Gly Val Glu Cys Asp Ile Phe Ala Pro Cys Ala Leu Gly
225 230 235 240
Gly Ile Ile Asn Asp Gln Thr Ile Pro Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ser Ala Leu Asn Gln Leu Lys Glu Pro Arg His Gly Asp Ile
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Arg Glu
290 295 300
Arg Ala Met Lys Lys Ile Glu Gln Ile Tyr Asp Asn Ile Glu Lys Val
305 310 315 320
Phe Ala Ile Ala Lys Arg Asp Asn Ile Pro Thr Tyr Val Ala Ala Asp
325 330 335
Arg Met Ala Glu Glu Arg Ile Glu Thr Met Arg Lys Ala Ala Ser Gln
340 345 350
Phe Leu Gln Asn Gly His His Ile Leu Ser Arg Arg Pro Arg Pro Leu
355 360 365
Thr Ala Ala Arg Ala Gly Leu Arg Arg Ala Asp Asp Gly Gly Thr Thr
370 375 380
Thr Met Gln Glu Gln Lys Phe Arg Ile Leu Thr Ile Asn Pro Gly Ser
385 390 395 400
Thr Ser Thr Lys Ile Gly Val Phe Glu Asn Glu Arg Ala Ile Ala Ser
405 410 415
Lys Lys Arg Ser Ala Thr Arg Ala Gly Ala Ser Ala Ile
420 425
<210> 2
<211> 366
<212> PRT
<213> Artificial sequence
<400> 2
Met Thr Leu Glu Ile Phe Glu Tyr Leu Glu Lys Tyr Asp Tyr Glu Gln
1 5 10 15
Val Val Phe Cys Gln Asp Lys Glu Ser Gly Leu Lys Ala Ile Ile Ala
20 25 30
Ile His Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp
35 40 45
Thr Tyr Asp Ser Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala
50 55 60
Lys Gly Met Thr Tyr Ser Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly
65 70 75 80
Ala Lys Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Ser Glu Ala
85 90 95
Met Phe Arg Ala Leu Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr
100 105 110
Ile Thr Ala Glu Asp Val Gly Thr Thr Val Asp Asp Met Asp Ile Ile
115 120 125
His Glu Glu Thr Asp Phe Val Thr Gly Ile Ser Pro Ser Phe Gly Ser
130 135 140
Ser Gly Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met
145 150 155 160
Lys Ala Ala Ala Lys Glu Ala Phe Gly Thr Asp Asn Leu Glu Gly Lys
165 170 175
Val Ile Ala Val Gln Gly Val Gly Asn Val Ala Tyr His Leu Cys Lys
180 185 190
His Leu His Ala Glu Gly Ala Lys Leu Ile Val Thr Asp Ile Asn Lys
195 200 205
Glu Ala Val Gln Arg Ala Val Glu Glu Phe Gly Ala Ser Ala Val Glu
210 215 220
Pro Asn Glu Ile Tyr Gly Val Glu Cys Asp Ile Tyr Ala Pro Cys Ala
225 230 235 240
Leu Gly Ala Thr Val Asn Asp Glu Thr Ile Pro Gln Leu Lys Ala Lys
245 250 255
Val Ile Ala Gly Ser Ala Leu Asn Gln Leu Lys Glu Asp Arg His Gly
260 265 270
Asp Ile Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile
275 280 285
Asn Ala Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn
290 295 300
Arg Glu Arg Ala Leu Lys Arg Val Glu Ser Ile Tyr Asp Thr Ile Ala
305 310 315 320
Lys Val Ile Glu Ile Ser Lys Arg Asp Gly Ile Ala Thr Tyr Val Ala
325 330 335
Ala Asp Arg Leu Ala Glu Glu Arg Ile Ala Ser Leu Lys Asn Ser Arg
340 345 350
Ser Thr Tyr Leu Arg Asn Gly His Asp Ile Ile Ser Arg Arg
355 360 365
<210> 3
<211> 364
<212> PRT
<213> Artificial sequence
<400> 3
Met Glu Ile Phe Lys Tyr Met Glu His Gln Asp Tyr Glu Gln Leu Val
1 5 10 15
Ile Cys Gln Asp Lys Ala Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Ala Ser Glu Glu Glu Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Ser Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asn Pro Lys Thr Asp Lys Asn Asp Glu Met Phe
85 90 95
Arg Ala Phe Gly Arg Tyr Ile Glu Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Glu Ala Asp Met Asp Leu Ile Asn Leu
115 120 125
Glu Thr Asp Tyr Val Thr Gly Thr Ser Ala Gly Ala Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Ile Tyr Tyr Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Glu Ala Phe Gly Asp Asp Ser Leu Ala Gly Lys Thr Val
165 170 175
Ala Val Gln Gly Val Gly Asn Val Ala Tyr Ala Leu Cys Glu Tyr Leu
180 185 190
His Glu Glu Gly Ala Lys Leu Ile Ile Thr Asp Ile Asn Glu Glu Ala
195 200 205
Val Gln Arg Ala Val Asp Ala Phe Gly Ala Thr Ala Val Gly Ile Asn
210 215 220
Glu Ile Tyr Ser Gln Glu Ala Asp Ile Phe Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Ile Ile Asn Asp Glu Thr Ile Pro Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ser Ala Leu Asn Gln Leu Lys Glu Thr Arg His Gly Asp Leu
260 265 270
Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ser
275 280 285
Gly Gly Val Ile Asn Val Ala Asp Glu Leu Asp Gly Tyr Asn Arg Glu
290 295 300
Arg Ala Leu Lys Arg Val Glu Gly Ile Tyr Asp Val Ile Gly Lys Ile
305 310 315 320
Phe Ala Ile Ser Lys Arg Asp Asn Ile Pro Thr Tyr Val Ala Ala Asp
325 330 335
Arg Met Ala Glu Glu Arg Ile Ala Arg Val Ala Asn Thr Arg Ser Thr
340 345 350
Phe Leu Gln Asn Glu Lys Ser Val Leu Ser Arg Arg
355 360
<210> 4
<211> 362
<212> PRT
<213> Artificial sequence
<400> 4
Met Glu Ile Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Ala Ser Gly Leu Lys Ala Val Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Ala Arg Met Trp Thr Tyr
35 40 45
Ala Ser Glu Glu Asn Ala Val Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Ser Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Phe Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Leu Gly Arg Phe Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Thr Asp Met Asp Leu Ile His Glu
115 120 125
Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Glu Ala Phe Gly Ser Glu Ser Leu Glu Gly Leu Lys Ile
165 170 175
Ser Val Gln Gly Leu Gly Asn Val Ala Tyr Lys Leu Cys Glu Tyr Leu
180 185 190
His Asn Glu Gly Ala Lys Leu Val Val Thr Asp Ile Asn Gln Ala Ala
195 200 205
Ile Asp Arg Val Val Asn Asp Phe Asp Ala Ile Ala Val Ala Pro Asp
210 215 220
Glu Ile Tyr Ala Gln Glu Val Asp Ile Phe Ser Pro Cys Ala Leu Gly
225 230 235 240
Ala Ile Leu Asn Asp Glu Thr Ile Pro Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ser Ala Leu Asn Gln Leu Lys Asp Ser Arg His Gly Asp Phe
260 265 270
Leu His Glu Leu Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Arg Glu
290 295 300
Arg Ala Leu Lys Arg Val Asp Gly Ile Tyr Asp Ser Ile Glu Lys Ile
305 310 315 320
Phe Ala Ile Ser Lys Arg Asp Gly Ile Pro Thr Tyr Val Ala Ala Asn
325 330 335
Arg Leu Ala Glu Glu Arg Ile Ala Arg Val Ala Lys Ser Arg Ser Gln
340 345 350
Phe Leu Lys Asn Glu Lys Asn Ile Leu His
355 360
<210> 5
<211> 374
<212> PRT
<213> Artificial sequence
<400> 5
Met Val Glu Thr Asn Val Glu Ala Arg Phe Ser Ile Phe Glu Thr Met
1 5 10 15
Ala Met Glu Asp Tyr Glu Gln Val Val Phe Cys His Asp Lys Val Ser
20 25 30
Gly Leu Lys Ala Ile Ile Ala Ile His Asp Thr Thr Leu Gly Pro Ala
35 40 45
Leu Gly Gly Leu Arg Met Trp Asn Tyr Ala Ser Asp Glu Glu Ala Leu
50 55 60
Ile Asp Ala Leu Arg Leu Ala Lys Gly Met Thr Tyr Ser Asn Ala Ala
65 70 75 80
Ala Gly Leu Asn Leu Gly Gly Gly Lys Ala Val Ile Ile Gly Asp Ala
85 90 95
Lys Thr Gln Lys Ser Glu Ala Leu Phe Arg Ala Phe Gly Arg Tyr Val
100 105 110
Gln Ser Leu Asn Gly Arg Tyr Ile Thr Ala Glu Asp Val Asn Thr Thr
115 120 125
Val Ala Asp Met Asp Tyr Ile His Met Glu Thr Asp Phe Val Thr Gly
130 135 140
Val Ser Pro Ala Phe Gly Ser Ser Gly Asn Pro Ser Pro Val Thr Ala
145 150 155 160
Tyr Gly Val Tyr Arg Gly Met Lys Ala Ala Ala Lys Glu Val Tyr Gly
165 170 175
Thr Asp Ser Leu Gly Gly Lys Thr Val Ala Ile Gln Gly Val Gly Asn
180 185 190
Val Ala Phe Asn Leu Cys Arg His Leu His Glu Glu Gly Ala Lys Leu
195 200 205
Ile Val Thr Asp Ile Asn Gln Asp Ala Leu Arg Arg Ala Glu Glu Ala
210 215 220
Phe Gly Ala Leu Val Val Gly Pro Asp Glu Ile Tyr Ser Val Asp Ala
225 230 235 240
Asp Ile Phe Ala Pro Cys Ala Leu Gly Ala Thr Leu Asn Asp Glu Thr
245 250 255
Ile Pro Gln Leu Lys Val Lys Ile Ile Ala Gly Ala Ala Leu Asn Gln
260 265 270
Leu Lys Glu Asp Arg His Gly Asp Met Leu Gln Glu Arg Gly Ile Leu
275 280 285
Tyr Thr Pro Asp Phe Val Ile Asn Ala Gly Gly Val Ile Asn Val Ala
290 295 300
Asp Glu Leu Asp Gly Tyr Asn Arg Glu Arg Ala Met Lys Lys Val Glu
305 310 315 320
Leu Val Tyr Asp Ala Val Ala Lys Val Ile Glu Ile Ala Lys Arg Asp
325 330 335
His Leu Pro Thr Tyr Arg Ala Ala Glu Lys Met Ala Glu Glu Arg Ile
340 345 350
Ala Thr Met Gly Ser Ala Arg Ser Gln Phe Leu Arg Arg Asp Lys Asn
355 360 365
Ile Leu Gly Ser Arg Gly
370
<210> 6
<211> 366
<212> PRT
<213> Artificial sequence
<400> 6
Met Lys Ile Phe Asp Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Met Cys Gln Asp Lys Glu Ser Gly Leu Lys Ala Ile Ile Cys Ile His
20 25 30
Val Thr Thr Leu Gly Pro Ala Leu Gly Gly Met Arg Met Trp Thr Tyr
35 40 45
Ala Ser Glu Glu Glu Ala Ile Glu Asp Ala Leu Arg Leu Gly Arg Gly
50 55 60
Met Thr Tyr Ser Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Ala Met Phe
85 90 95
Arg Ala Leu Gly Arg Phe Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Glu
115 120 125
Glu Thr Arg Tyr Val Thr Gly Val Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Glu Ala Phe Gly Asp Asp Ser Leu Glu Gly Lys Val Val
165 170 175
Ala Val Gln Gly Val Gly His Val Ala Tyr Glu Leu Cys Lys His Leu
180 185 190
His Asn Glu Gly Ala Lys Leu Ile Val Thr Asp Ile Asn Lys Glu Asn
195 200 205
Ala Asp Arg Ala Val Gln Glu Phe Gly Ala Glu Phe Val His Pro Asp
210 215 220
Lys Ile Tyr Asp Val Glu Cys Asp Ile Phe Ala Pro Cys Ala Leu Gly
225 230 235 240
Ala Ile Ile Asn Asp Glu Thr Ile Glu Arg Leu Lys Cys Lys Val Val
245 250 255
Ala Gly Ser Ala Leu Asn Gln Leu Lys Glu Glu Arg His Gly Lys Met
260 265 270
Leu Glu Glu Lys Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Val Ile Asn Val Ala Asp Glu Leu Leu Gly Tyr Asn Arg Glu
290 295 300
Arg Ala Met Lys Lys Val Glu Gly Ile Tyr Asp Lys Ile Leu Lys Val
305 310 315 320
Phe Glu Ile Ala Lys Arg Asp Gly Ile Pro Ser Tyr Leu Ala Ala Asp
325 330 335
Arg Met Ala Glu Glu Arg Ile Glu Met Met Arg Lys Thr Arg Ser Thr
340 345 350
Phe Leu Gln Asp Gln Arg Asn Leu Ile Asn Phe Asn Asn Lys
355 360 365
<210> 7
<211> 370
<212> PRT
<213> Artificial sequence
<400> 7
Met Ser Thr Val Thr Phe Asp Gln Ile Ser Glu His Glu Gln Val Met
1 5 10 15
Phe Cys Asn Asp Pro His Thr Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asn Thr Thr Leu Gly Pro Ala Leu Gly Gly Cys Arg Met Leu Pro Tyr
35 40 45
Lys Ser Glu Glu Glu Ala Leu Thr Asp Val Leu Arg Leu Ser Lys Gly
50 55 60
Met Thr Tyr Ser Cys Val Ala Ala Asp Val Asp Phe Gly Gly Gly Lys
65 70 75 80
Ala Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Thr Pro Glu Leu Phe
85 90 95
Arg Ala Phe Gly Gln Phe Val Gln Ser Leu Asn Gly Arg Phe Tyr Thr
100 105 110
Gly Thr Asp Met Gly Thr Thr Pro Glu Asp Phe Val Gln Ala Tyr Lys
115 120 125
Glu Thr Ser Phe Ile Val Gly Leu Pro Glu Glu Tyr Gly Gly Asn Gly
130 135 140
Asp Ser Ser Val Thr Thr Ala Phe Gly Val Met Gln Gly Leu Arg Ala
145 150 155 160
Val Ser Gln Phe Leu Trp Gly Thr Asp Val Leu Thr Glu Arg Val Phe
165 170 175
Ala Val Gln Gly Leu Gly Lys Val Gly Phe Lys Val Ala Glu Gly Leu
180 185 190
Leu Lys Glu Gly Ala Asn Val Tyr Val Thr Asp Val Asp Pro Glu Thr
195 200 205
Ile Ala Lys Leu Glu Glu Lys Ala Tyr Gln Tyr Pro Gly His Val Gln
210 215 220
Ala Val Thr Ala Asp Asp Ile Tyr Gly Val Gly Ala Asp Val Phe Val
225 230 235 240
Pro Cys Ala Ile Gly Gly Ile Ile Asn Asp Glu Thr Ile Glu Arg Leu
245 250 255
Lys Val Lys Ala Val Cys Gly Ala Ala Leu Asn Gln Leu Leu Glu Asp
260 265 270
Arg His Gly Lys Val Leu Gln Ala Lys Asn Ile Leu Tyr Ala Pro Asp
275 280 285
Tyr Ile Val Asn Ala Gly Gly Leu Ile Gln Val Ser Asp Glu Leu Tyr
290 295 300
Gly Pro Asn Lys Ala Arg Val Leu Lys Lys Thr Arg Ala Leu Tyr Asp
305 310 315 320
Thr Leu Phe Glu Ile Phe Gln Ser Ala Glu Lys Lys Ala Val Ser Thr
325 330 335
Val Glu Ala Ala Asn Gln Phe Val Glu Glu Arg Leu Gln Lys Arg Ala
340 345 350
Arg Leu Asn Ser Phe Phe Ser Pro Asp Asn Pro Pro Lys Trp Arg Val
355 360 365
Arg Arg
370
<210> 8
<211> 366
<212> PRT
<213> Artificial sequence
<400> 8
Met Lys Ile Phe Glu Tyr Met Gly Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Leu Cys His Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Cys Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr
35 40 45
Asp Ser Glu Asp Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Ser Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Val Ile Gly Asp Pro Lys Lys Asp Lys Ser Glu Ala Leu Phe
85 90 95
Arg Ala Leu Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile Arg Glu
115 120 125
Glu Thr Lys Tyr Val Thr Gly Val Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Lys Gly Met Lys Ala
145 150 155 160
Ala Ser Lys Val Ala Phe Gly Glu Asp Ser Leu Lys Gly Lys Val Val
165 170 175
Ala Val Gln Gly Val Gly His Val Ala Tyr Asn Leu Cys Lys His Leu
180 185 190
His Ala Glu Gly Ala Lys Leu Ile Val Thr Asp Ile Asn Gln Ala Asn
195 200 205
Gly Asp Arg Ala Val Gln Glu Phe Gly Ala Glu Ala Val Ser Pro Asp
210 215 220
Lys Ile Tyr Asp Val Asp Cys Asp Ile Phe Ser Pro Cys Ala Leu Gly
225 230 235 240
Ala Ile Ile Asn Asp Glu Thr Ile Glu Arg Leu Thr Cys Lys Val Val
245 250 255
Ala Gly Ala Ala Leu Asn Gln Leu Lys Glu Glu Lys His Gly Glu Met
260 265 270
Leu Glu Gln Lys Gly Ile Ile Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Arg Asp
290 295 300
Arg Ala Met Lys Arg Val Glu Thr Ile Tyr Asp Asn Met Leu Lys Val
305 310 315 320
Phe Glu Ile Ala Lys Arg Asp Gly Ile Pro Ser Tyr Lys Ala Ala Asp
325 330 335
Arg Met Ala Glu Glu Arg Ile Ala Ala Met Arg Lys Thr Arg Ser Thr
340 345 350
Phe Leu Val Asn Gly Gln Ser Ile Leu Ser His Arg Leu Glu
355 360 365
<210> 9
<211> 380
<212> PRT
<213> Artificial sequence
<400> 9
Met Ser Leu Val Glu Lys Thr Ser Ile Ile Lys Asp Phe Thr Leu Phe
1 5 10 15
Glu Lys Met Ser Glu His Glu Gln Val Val Phe Cys Asn Asp Pro Ala
20 25 30
Thr Gly Leu Arg Ala Ile Ile Ala Ile His Asp Thr Thr Leu Gly Pro
35 40 45
Ala Leu Gly Gly Cys Arg Met Gln Pro Tyr Asn Ser Val Glu Glu Ala
50 55 60
Leu Glu Asp Ala Leu Arg Leu Ser Lys Gly Met Thr Tyr Ser Cys Ala
65 70 75 80
Ala Ser Asp Val Asp Phe Gly Gly Gly Lys Ala Val Ile Ile Gly Asp
85 90 95
Pro Gln Lys Asp Lys Ser Pro Glu Leu Phe Arg Ala Phe Gly Gln Phe
100 105 110
Val Asp Ser Leu Gly Gly Arg Phe Tyr Thr Gly Thr Asp Met Gly Thr
115 120 125
Asn Met Glu Asp Phe Ile His Ala Met Lys Glu Thr Asn Cys Ile Val
130 135 140
Gly Val Pro Glu Ala Tyr Gly Gly Gly Gly Asp Ser Ser Ile Pro Thr
145 150 155 160
Ala Met Gly Val Leu Tyr Gly Ile Lys Ala Thr Asn Lys Met Leu Phe
165 170 175
Gly Lys Asp Asp Leu Gly Gly Val Thr Tyr Ala Ile Gln Gly Leu Gly
180 185 190
Lys Val Gly Tyr Lys Val Ala Glu Gly Leu Leu Glu Glu Gly Ala His
195 200 205
Leu Phe Val Thr Asp Ile Asn Glu Gln Thr Leu Glu Ala Ile Gln Glu
210 215 220
Lys Ala Lys Thr Thr Ser Gly Ser Val Thr Val Val Ala Ser Asp Glu
225 230 235 240
Ile Tyr Ser Gln Glu Ala Asp Val Phe Val Pro Cys Ala Phe Gly Gly
245 250 255
Val Val Asn Asp Glu Thr Met Lys Gln Phe Lys Val Lys Ala Ile Ala
260 265 270
Gly Ser Ala Leu Asn Gln Leu Leu Thr Glu Asp His Gly Arg His Leu
275 280 285
Ala Asp Lys Gly Ile Leu Tyr Ala Pro Asp Tyr Ile Val Asn Ser Gly
290 295 300
Gly Leu Ile Gln Val Ala Asp Glu Leu Tyr Glu Val Asn Lys Glu Arg
305 310 315 320
Val Leu Ala Lys Thr Lys His Ile Tyr Asp Ala Ile Leu Glu Val Tyr
325 330 335
Gln Gln Ala Glu Leu Asp Gln Ile Thr Thr Met Glu Ala Ala Asn Arg
340 345 350
Met Cys Glu Gln Arg Met Ala Ala Arg Gly Arg Arg Asn Ser Phe Phe
355 360 365
Thr Ser Ser Val Lys Pro Lys Trp Asp Ile Arg Asn
370 375 380
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