CN115466756A - Transaminase, immobilized transaminase and application of transaminase to preparation of sitagliptin - Google Patents
Transaminase, immobilized transaminase and application of transaminase to preparation of sitagliptin Download PDFInfo
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- CN115466756A CN115466756A CN202110655349.9A CN202110655349A CN115466756A CN 115466756 A CN115466756 A CN 115466756A CN 202110655349 A CN202110655349 A CN 202110655349A CN 115466756 A CN115466756 A CN 115466756A
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- seq
- leu
- val
- gly
- ile
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- 108090000340 Transaminases Proteins 0.000 title claims abstract description 143
- 102000003929 Transaminases Human genes 0.000 title claims abstract description 139
- MFFMDFFZMYYVKS-SECBINFHSA-N sitagliptin Chemical compound C([C@H](CC(=O)N1CC=2N(C(=NN=2)C(F)(F)F)CC1)N)C1=CC(F)=C(F)C=C1F MFFMDFFZMYYVKS-SECBINFHSA-N 0.000 title claims abstract description 43
- 229960004034 sitagliptin Drugs 0.000 title claims abstract description 41
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 238000006243 chemical reaction Methods 0.000 claims abstract description 59
- 239000000758 substrate Substances 0.000 claims abstract description 36
- 229920005989 resin Polymers 0.000 claims abstract description 30
- 239000011347 resin Substances 0.000 claims abstract description 30
- FZRKAZHKEDOPNN-UHFFFAOYSA-N Nitric oxide anion Chemical compound O=[N-] FZRKAZHKEDOPNN-UHFFFAOYSA-N 0.000 claims abstract description 26
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 8
- 238000004519 manufacturing process Methods 0.000 claims abstract description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 11
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 45
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 claims description 38
- 239000002773 nucleotide Substances 0.000 claims description 32
- 125000003729 nucleotide group Chemical group 0.000 claims description 32
- 239000000243 solution Substances 0.000 claims description 22
- 235000007682 pyridoxal 5'-phosphate Nutrition 0.000 claims description 19
- 239000011589 pyridoxal 5'-phosphate Substances 0.000 claims description 19
- 229960001327 pyridoxal phosphate Drugs 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 102000004190 Enzymes Human genes 0.000 claims description 13
- 108090000790 Enzymes Proteins 0.000 claims description 13
- JJWLVOIRVHMVIS-UHFFFAOYSA-N isopropylamine Chemical group CC(C)N JJWLVOIRVHMVIS-UHFFFAOYSA-N 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 13
- 239000007810 chemical reaction solvent Substances 0.000 claims description 12
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 12
- 239000007864 aqueous solution Substances 0.000 claims description 10
- 229960004115 sitagliptin phosphate Drugs 0.000 claims description 10
- RTZRUVMEWWPNRR-UHFFFAOYSA-N tert-butyl n-(3-iodo-1h-pyrrolo[2,3-b]pyridin-5-yl)carbamate Chemical compound CC(C)(C)OC(=O)NC1=CN=C2NC=C(I)C2=C1 RTZRUVMEWWPNRR-UHFFFAOYSA-N 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 9
- QAEDTLFWHIEVPK-UHFFFAOYSA-N 1-[3-(trifluoromethyl)-6,8-dihydro-5h-[1,2,4]triazolo[4,3-a]pyrazin-7-yl]-4-(2,4,5-trifluorophenyl)butane-1,3-dione Chemical group C1=C(F)C(F)=CC(F)=C1CC(=O)CC(=O)N1CC2=NN=C(C(F)(F)F)N2CC1 QAEDTLFWHIEVPK-UHFFFAOYSA-N 0.000 claims description 8
- 230000001580 bacterial effect Effects 0.000 claims description 6
- 239000013604 expression vector Substances 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 108091033319 polynucleotide Proteins 0.000 claims description 6
- 239000002157 polynucleotide Substances 0.000 claims description 6
- 102000040430 polynucleotide Human genes 0.000 claims description 6
- 238000003259 recombinant expression Methods 0.000 claims description 6
- 239000003822 epoxy resin Substances 0.000 claims description 5
- 229920000647 polyepoxide Polymers 0.000 claims description 5
- 239000010802 sludge Substances 0.000 claims description 5
- 241000672609 Escherichia coli BL21 Species 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 241000588724 Escherichia coli Species 0.000 claims description 2
- 239000013612 plasmid Substances 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 230000035772 mutation Effects 0.000 abstract description 5
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- WJPYOCIWVYDFDT-UHFFFAOYSA-N ethyl 3-oxo-4-(2,4,5-trifluorophenyl)butanoate Chemical compound CCOC(=O)CC(=O)CC1=CC(F)=C(F)C=C1F WJPYOCIWVYDFDT-UHFFFAOYSA-N 0.000 description 1
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- 230000003914 insulin secretion Effects 0.000 description 1
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Classifications
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- 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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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- 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
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- 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
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
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- 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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/14—Nitrogen or oxygen as hetero atom and at least one other diverse hetero ring atom in the same ring
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
Abstract
The invention provides an application of immobilized transaminase in preparation of sitagliptin and/or (R) -3-amino-1-morpholine-4- (2,4,5-trifluorophenyl) -1-butanone; the immobilized transaminase comprises resin and transaminase, and the amino acid sequence of the transaminase is shown as SEQ ID NO 5, SEQ ID NO 45, SEQ ID NO 47, SEQ ID NO 49 or variants thereof; the variant is that the R mutation at the 241 th position of the amino acid sequence shown as SEQ ID NO. 5 is A, H, N, S, L, T, C or M. The invention also provides immobilized transaminase, a transaminase, and a preparation method and application thereof. After the transaminase is prepared into the immobilized transaminase, the immobilized transaminase is used for catalyzing a ketoamide substrate to produce sitagliptin or an intermediate thereof, and has the advantages of high conversion rate, good stereoselectivity, good stability, improvement of reusability and simpler operation, so that the production cost is reduced, and the immobilized transaminase is beneficial to industrial production.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to transaminase and immobilized transaminase, application of the immobilized transaminase in preparation of sitagliptin or an intermediate thereof, and a preparation method of the sitagliptin or the intermediate thereof.
Background
Diabetes is a metabolic disease characterized by hyperglycemia accompanied by metabolic disorders of proteins, sugars, and fats, which occur due to altered insulin secretion, resulting in insulin deficiency and impaired action, or decreased insulin activity, or both. The degree of harm of diabetes and its complications to human health is the third place after cardiovascular diseases and tumors, and becomes an important disease harming human health. Of the four types of diabetes, type II diabetes accounts for over 90%, most of which are seen in middle aged and elderly people over 30 years old, and the cause of the disease is mainly due to the insensitivity of the body to insulin.
Sitagliptin phosphate (Sitagliptin phosphate) is the first dipeptidyl enzyme-IV (DPP-4) inhibitor approved by FDA for marketing in 2006 for the treatment of type II diabetes. The composition has obvious blood sugar reducing effect when being used singly or used together with metformin and pioglitazone, and has the advantages of safe taking, good tolerance and few adverse reactions.
U.S. Pat. No. 3,3534,3534 discloses that the transaminase obtained by modifying the transaminase from Arthrobacter by Codexes catalyzes 4-oxo-4- [3- (trifluoromethyl) -5,6-dihydro- [1,2,4] triazolo [4,3-a ] pyrazine-7- (8H) -yl ] -1- (2,4,5-trifluorophenyl) butan-2-one to obtain sitagliptin ((2R) -4-oxo-4- [3- (trifluoromethyl) -5,6-dihydro- [1,2,4] triazolo [4,3-a ] pyrazine-7- (8H) -yl ] -1- (2,4,5-trifluorophenyl) butan-2-amine), and the sitagliptin is further phosphorylated to obtain sitagliptin phosphate. The engineered transaminase was capable of converting the substrate 4-oxo-4- [3- (trifluoromethyl) -5,6-dihydro- [1,2,4] triazolo [4,3-a ] pyrazin-7- (8H) -yl ] -1- (2,4,5-trifluorophenyl) butan-2-one (abbreviated as sitagliptin herein) to a detectable level at 210nm by HPLC-UV under conditions of 2g/L of ketoamide substrate, 0.5M isopropylamine, 22 ℃,5%DMSO, 100. Mu.M pyridoxal phosphate (PLP) and 20mg/mL of the transaminase polypeptide. Wherein the catalytic conversion rate of the mutant (SEQ ID NO: 110) with the best effect can reach 90-95%.
US9617573 continuing with the modification of SEQ ID NO:110 in US8293507, the resulting mutant was capable of converting 4-oxo-4- [3- (trifluoromethyl) -5,6-dihydro- [1,2,4] triazolo [4,3-a ] pyrazin-7- (8H) -yl ] -1- (2,4,5-trifluorophenyl) butan-2-one to sitagliptin at an activity at least 1.2 times that of SEQ ID NO:110 under conditions of 50g/L of ketoamide substrate, 1.5M isopropylamine (isopropylamine), 55 ℃,50 ℃ DMSO (v/v), 1mM pyridoxalphosphate. Wherein the catalytic conversion rate of the mutant (SEQ ID NO: 130) with the best effect can reach 90-95%.
However, these transaminase mutants are unstable in 100% organic solvents, and thus, the liquid enzyme is immobilized to obtain an immobilized enzyme, so as to improve the stability of the transaminase in organic solvents. As disclosed in US9587229 for immobilization of SEQ ID NO:110 in US8293507 on SEPABEADS EXE120 resin, the results show that the SEPABEADS EXE120 immobilized enzyme of SEQ ID NO:110 is capable of catalyzing 100g/L of a substrate of 4-oxo-4- [3- (trifluoromethyl) -5,6-dihydro- [1,2,4] triazolo [4,3-a ] pyrazin-7- (8H) -yl ] -1- (2,4,5-trifluorophenyl) butan-2-one to sitagliptin in a water saturated IPAc (isopropyl acetate) solvent with an ee value above 99.9%. However, US9587229 does not study the reusability (number of application batches) of the immobilized enzyme, and the reaction solvent is a water-saturated IPAc (isopropyl acetate) solvent, when the solvent is used for production, IPAc needs to be saturated with water in advance, so the operation is more complicated, and if the solvent is used for multiple batches of application reactions, the water content in the reaction system is not easy to control, the water activity of the immobilized enzyme is affected, and the immobilized enzyme is easy to inactivate and denature.
WO2019011236A1 reports that 1-morpholine-4- (2,4,5-trifluorophenyl) -1,3-butanedione (also referred to as morpholine dione in the invention) can be prepared into (R) -3-amino-1-morpholine-4- (2,4,5-trifluorophenyl) -1-butanone through transaminase catalysis, and then sitagliptin is prepared through multi-step reaction.
Disclosure of Invention
The invention aims to overcome the defects that in the prior art, when transaminase is prepared into immobilized enzyme, the stability is still poor when the transaminase is used in a reaction system, and further when the transaminase is used for catalyzing a ketoamide substrate to produce sitagliptin or an intermediate thereof, the conversion rate is not high, the transaminase cannot be recycled, and the like, and provides the transaminase, the immobilized transaminase and the application of the transaminase in preparing the sitagliptin or the intermediate thereof. After the transaminase is prepared into the immobilized transaminase, the immobilized transaminase is used for catalyzing a ketoamide substrate to produce sitagliptin or an intermediate thereof, and has the advantages of high conversion rate, good stereoselectivity, good stability, improved reusability and simpler operation, thereby reducing the production cost and being beneficial to industrial production.
The present inventors have conducted extensive studies on transaminases in the prior art, and found that when certain specific sites are mutated, the obtained transaminase is immobilized, and then used for catalyzing a ketoamide substrate, the transaminase has higher stability and higher conversion rate, and the transaminase is used for producing sitagliptin or an intermediate thereof at a lower cost.
In order to solve the technical problems, the invention provides a use of immobilized transaminase in the preparation of sitagliptin and/or (R) -3-amino-1-morpholine-4- (2,4,5-trifluorophenyl) -1-butanone;
wherein the immobilized transaminase comprises a resin and a transaminase, and the amino acid sequence of the transaminase is shown in SEQ ID NO 5, SEQ ID NO 45, SEQ ID NO 47, SEQ ID NO 49 or variants thereof; the variant is that the R mutation at the 241 th position of the amino acid sequence shown as SEQ ID NO. 5 is A, H, N, S, L, T, C or M.
Preferably, the amino acid sequence of the variant is shown as SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 21, SEQ ID NO. 25, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41 or SEQ ID NO. 43. More preferably, the nucleotide sequence of the transaminase is preferably as shown in SEQ ID NO 6, SEQ ID NO 12, SEQ ID NO 16, SEQ ID NO 22, SEQ ID NO 26, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48 or SEQ ID NO 50;
preferably, the transaminase is attached to the resin by covalent bonding.
Preferably, the resin is preferably an epoxy resin, preferably
And (4) HFA. In a preferred embodiment, the resin is purchased from Viton, suzhou, chromatography, separation and purification, inc
An HFA resin.
Preferably, the immobilized transaminase has a concentration of the resin-borne transaminase of 150 to 300mg/g (i.e., a mass of 150 to 300mg per gram of the resin-borne transaminase), for example 250mg/g.
Preferably, said use comprises the step of catalyzing a ketoamide substrate with said immobilized transaminase in a reaction solvent in the presence of an amino donor to obtain said sitagliptin and/or (R) -3-amino-1-morpholine-4- (2,4,5-trifluorophenyl) -1-butanone.
More preferably, the reaction solvent is an aqueous isopropanol solution, preferably, the volume content of water in the aqueous isopropanol solution (the volume ratio of water to the whole solution) is 2-20%, and more preferably, the prepared reaction system further comprises a co-factor of transaminase, such as pyridoxal phosphate, and the concentration of the co-factor in the whole reaction system is preferably 0.5-5 mg/mL.
More preferably, the ketoamide substrate is 4-oxo-4- [3- (trifluoromethyl) -5,6-dihydro- [1,2,4] triazolo [4,3-a ] pyrazin-7- (8H) -yl ] -1- (2,4,5-trifluorophenyl) butan-2-one and/or 1-morpholine-4- (2,4,5-trifluorophenyl) -1,3-butanedione.
In the invention, the 4-oxo-4- [3- (trifluoromethyl) -5,6-dihydro- [1,2,4] triazolo [4,3-a ] pyrazin-7- (8H) -yl ] -1- (2,4,5-trifluorophenyl) butan-2-one (also referred to as sitaxdione in the invention) has the following specific structural formula:
in the invention, the specific structural formula of the 1-morpholine-4- (2,4,5-trifluorophenyl) -1,3-butanedione (also referred to as morpholine dione in the invention) is as follows:
more preferably, the amino donor is isopropylamine.
More preferably, the molar ratio of the amino donor to the ketoamide substrate is 1:1-5:1.
More preferably, the concentration of the ketoamide substrate in the whole reaction system is 20-200 g/L.
More preferably, the mass ratio of the immobilized transaminase to the ketoamide substrate is 1:1 to 6:1, e.g., 3, 2.5.
More preferably, the temperature of the reaction is 30 to 60 ℃, preferably 45 ℃.
In order to solve the above technical problems, the second aspect of the present invention provides an immobilized transaminase comprising a resin and a transaminase, the transaminase having an amino acid sequence shown in SEQ ID NO 5, SEQ ID NO 45, SEQ ID NO 47, SEQ ID NO 49 or variants thereof; the variant is that the R mutation at position 241 of the amino acid sequence shown as SEQ ID NO. 5 is A, H, N, S, L, T, C or M.
Preferably, the amino acid sequence of the variant is shown as SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 21, SEQ ID NO. 25, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41 or SEQ ID NO. 43. More preferably, the nucleotide sequence of the transaminase is preferably as shown in SEQ ID NO 6, 12, 16, 22, 26, 38, 40, 42, 44, 46, 48 or 50.
Preferably, the transaminase is attached to the resin by covalent bonding.
Preferably, the resin is preferably an epoxy resin, preferably
HFA. In a preferred embodiment, the resin is purchased from Viton, suzhou, chromatography, separation and purification, inc
HFA resin.
Preferably, the immobilized transaminase has a concentration of the resin-borne transaminase of 150 to 300mg/g (i.e., a mass of 150 to 300mg per gram of the resin-borne transaminase), for example 250mg/g. In order to solve the above technical problems, the third aspect of the present invention provides a method for preparing an immobilized transaminase, which comprises:
1) Contacting the solution of transaminase enzyme with a resin to form an immobilized transaminase enzyme, the transaminase enzyme having an amino acid sequence set forth in SEQ ID NO 5, SEQ ID NO 45, SEQ ID NO 47, SEQ ID NO 49, or variants thereof; the variant is that the R at the 241 th position of the amino acid sequence shown as SEQ ID NO. 5 is mutated into A, H, N, S, L, T, C or M;
2) Filtering and washing the immobilized transaminase.
Preferably, the amino acid sequence of the variant is shown as SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 21, SEQ ID NO. 25, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41 or SEQ ID NO. 43; the preferred nucleotide sequence of the transaminase is shown in SEQ ID NO 6, SEQ ID NO 12, SEQ ID NO 16, SEQ ID NO 22, SEQ ID NO 26, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48 or SEQ ID NO 50.
Preferably, the transaminase is attached to the resin by covalent bonding.
Preferably, the resin is preferably an epoxy resin, preferably
HFA. In a preferred embodiment, the resin is purchased from Viton, suzhou, chromatography, separation and purification, inc
HFA resin.
Preferably, the transaminase is present in the form of transaminase bacterial sludge, and the mass ratio of the transaminase bacterial sludge to the resin is 3:1-1:2, such as 5:4.
Preferably, the temperature of the reaction is 10 to 40 ℃, preferably 20 to 25 ℃.
Preferably, the reaction time is 10 to 30 hours, preferably 20 to 25 hours.
Preferably, the reaction is carried out with stirring, preferably at a rate of 50 to 300rpm, for example 150 or 200rpm.
In a preferred embodiment, the immobilized transaminase is prepared by the following method: adding 5g transaminase (e.g. in the form of bacterial sludge) into dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (e.g. 50mL in volume, 100mM in concentration), homogenizing under high pressure, crushing cells, centrifuging, collecting supernatant enzyme solution, adding 10g K 2 HPO 4 ·3H 2 O、1g KH 2 PO 4 And 0.01g of PLP, dissolved with stirring, 4g of which are added
HFA resin (purchased from Suzhou Vietnam chromatography separation and purification Co., ltd.) is fixed, the temperature is 20-25 ℃, the shaking table is used for fixing for 20-25 h at 200rpm, and the immobilized enzyme is obtained through suction filtration, deionization washing and suction filtration.
In order to solve the above technical problems, the fourth aspect of the present invention provides a method for preparing sitagliptin and/or (R) -3-amino-1-morpholine-4- (2,4,5-trifluorophenyl) -1-butanone, comprising the step of catalyzing a ketoamide substrate with the immobilized transaminase according to the second aspect of the present invention in a reaction solvent in the presence of an amino donor to obtain sitagliptin and/or (R) -3-amino-1-morpholine-4- (2,4,5-trifluorophenyl) -1-butanone.
Preferably, the ketoamide substrate is 4-oxo-4- [3- (trifluoromethyl) -5,6-dihydro- [1,2,4] triazolo [4,3-a ] pyrazin-7- (8H) -yl ] -1- (2,4,5-trifluorophenyl) butan-2-one and/or 1-morpholine-4- (2,4,5-trifluorophenyl) -1,3-butanedione.
Preferably, the reaction solvent is an aqueous isopropanol solution.
Preferably, the amino donor is isopropylamine.
Preferably, the molar ratio of the amino donor to the ketoamide substrate is 1:1-5:1.
Preferably, when the reaction solvent is an aqueous isopropanol solution, the volume content of water (the volume ratio of water to the whole solution) is 2-20%.
Preferably, the concentration of the ketoamide substrate in the whole reaction system is 20 g/L-200 g/L.
Preferably, the mass ratio of immobilized transaminase to substrate is 1:1 to 6:1, e.g. 3.
Preferably, the prepared reaction system also comprises a co-factor of transaminase, such as pyridoxal phosphate, and the concentration of the co-factor in the whole reaction system is preferably 0.5-5 mg/mL.
Preferably, the temperature of the reaction is 30-60 ℃, preferably 45 ℃.
In order to solve the above technical problems, the fifth aspect of the present invention provides a transaminase whose amino acid sequence is represented by SEQ ID NO. 5, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49 or variants thereof; the variant is a mutation from R to A, H, N, S, L, T, C or M at position 241 of the amino acid sequence shown in SEQ ID NO: 5.
Preferably, the amino acid sequence of the variant is shown as SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 21, SEQ ID NO. 25, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41 or SEQ ID NO. 43;
more preferably, the nucleotide sequence of the transaminase is shown as SEQ ID NO 6, SEQ ID NO 12, SEQ ID NO 16, SEQ ID NO 22, SEQ ID NO 26, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48 or SEQ ID NO 50.
In order to solve the above technical problems, a sixth aspect of the present invention provides a polynucleotide encoding the transaminase according to the fifth aspect of the present invention.
In order to solve the above technical problems, the seventh aspect of the present invention provides a recombinant expression vector comprising the polynucleotide according to the sixth aspect of the present invention.
Preferably, the backbone of the recombinant expression vector is plasmid pET21a.
In order to solve the above technical problems, an eighth aspect of the present invention provides a transformant obtained by introducing the polynucleotide according to the sixth aspect of the present invention or the recombinant expression vector according to the seventh aspect of the present invention into a host.
Preferably, the host is escherichia coli; escherichia coli BL21 is preferred.
In order to solve the above technical problems, a ninth aspect of the present invention provides a use of the transaminase according to the fifth aspect of the present invention in preparing sitagliptin and/or (R) -3-amino-1-morpholine-4- (2,4,5-trifluorophenyl) -1-butanone.
Preferably, the use comprises the step of catalyzing a ketoamide substrate with a transaminase in a reaction solvent in the presence of an amino donor to obtain the sitagliptin and/or (R) -3-amino-1-morpholine-4- (2,4,5-trifluorophenyl) -1-butanone.
More preferably, the reaction solvent is an aqueous isopropanol solution, preferably, the volume content of water in the aqueous isopropanol solution (the volume ratio of water to the whole solution) is 2-20%, and more preferably, the prepared reaction system further comprises a co-factor of transaminase, such as pyridoxal phosphate, and the concentration of the co-factor in the whole reaction system is preferably 0.5-5 mg/mL.
More preferably, the ketoamide substrate is 4-oxo-4- [3- (trifluoromethyl) -5,6-dihydro- [1,2,4] triazolo [4,3-a ] pyrazin-7- (8H) -yl ] -1- (2,4,5-trifluorophenyl) butan-2-one and/or 1-morpholine-4- (2,4,5-trifluorophenyl) -1,3-butanedione.
More preferably, the amino donor is isopropylamine.
More preferably, the molar ratio of the amino donor to the ketoamide substrate is 1:1 to 5:1.
More preferably, the concentration of the ketoamide substrate in the whole reaction system is 20-200 g/L.
More preferably, the mass ratio of said transaminase to said ketoamide substrate is 1:1 to 6:1, e.g., 3.
More preferably, the transaminase is present as an immobilized transaminase.
More preferably, the prepared reaction system also comprises a co-factor of transaminase, such as pyridoxal phosphate, and the concentration of the co-factor in the whole reaction system is preferably 0.5-5 mg/mL.
More preferably, the temperature of the reaction is 30 to 60 ℃, preferably 45 ℃.
In the present invention, the amount of isopropanol in the aqueous isopropanol solution is sufficient to dissolve the substrate completely. The volume content of water in the isopropanol aqueous solution (the volume ratio of water to the whole solution) can be 2-20%, if the water is too little, the immobilized enzyme can be deactivated, and if the water is too much, the substrate can not be completely dissolved.
In one aspect, the invention further provides a preparation method of sitagliptin phosphate, which comprises the following steps:
(1) Preparing sitagliptin and/or (R) -3-amino-1-morpholine-4- (2,4,5-trifluorophenyl) -1-butanone according to the preparation method according to the fourth aspect of the invention;
(2) And (R) -3-amino-1-morpholine-4- (2,4,5-trifluorophenyl) -1-butanone prepared in the step (1) is reacted to obtain sitagliptin phosphate.
Preferably, the sitagliptin phosphate is sitagliptin phosphate monohydrate.
In a certain aspect, the invention also provides an enzyme preparation comprising an immobilized transaminase according to the second aspect of the invention or a transaminase according to the fifth aspect of the invention;
preferably, the enzyme preparation further comprises a co-factor for transaminase, such as pyridoxal phosphate.
In the present invention, the enzyme preparation may be generally an enzyme preparation obtained from a transformant host cell containing transaminase obtained from a culture or a culture solution thereof, or a product obtained by processing the same; wherein the product is an extract obtained from a transformant host cell, an isolated product obtained by isolating or purifying transaminase in the extract, or an immobilized product obtained by immobilizing a transformant cell and an extract thereof or an isolated product of the extract.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows: after the transaminase is prepared into the immobilized transaminase, the immobilized transaminase is used for catalyzing a ketoamide substrate to produce a sitagliptin intermediate, and the immobilized transaminase has the advantages of high conversion rate, good stereoselectivity, good stability, improved reusability and simpler operation, thereby reducing the production cost and being beneficial to industrial production.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
The experimental methods in the invention are conventional methods unless otherwise specified, and the gene cloning operation can be specifically referred to in molecular cloning experimental guidelines compiled by J. SammBruk et al.
The abbreviations of the amino acids in the present invention are those conventional in the art unless otherwise specified, and the amino acids corresponding to the specific abbreviations are shown in Table 1.
TABLE 1
The codons corresponding to the amino acids are also conventional in the art, and the corresponding relationship between specific amino acids and codons is shown in table 2.
TABLE 2
Pet21a was purchased from Novagen, inc.; ndeI enzyme, hindIII enzyme were purchased from Thermo Fisher, BL21 competent cells from Changshu Biotech, inc., beijing ancient cooking.
Conversion HPLC method: a chromatographic column: c18 column 4.6X 250mm,5 μm; a detector: UV268nm; column temperature: 40 ℃; flow rate: 0.8mL/min; sample introduction amount: 20 mu L of the solution; mobile phase A: water, acetonitrile, formic acid and ammonia water = 950: 50: 0.5, the pH value is 3.60-3.80, if the pH value is not reached, the pH value is adjusted to 3.70 by 10% ammonia water or 10% formic acid; ammonium formate can also be used to adjust the pH to 3.70; and (3) mobile phase B: water acetonitrile = 20: 80; gradient elution: 100% A (0.01 min), 60% A +40% B (20 min), 60% A +40% B (40 min), 100% A (50 min), 100% A (60 min).
Morpholine dione control retention time: 27.820min; (R) -3-amino-1-morpholine-4- (2,4,5-trifluorophenyl) -1-butanone control retention time: 14.856min;
sitagliptin control retention time: 34.715min;
sitagliptin control (purchased from beijing ying luck science ltd) retention time: 17.705min.
Wherein, the morpholine diketone substrate raw material and the reference product (R) -3-amino-1-morpholine-4- (2,4,5-trifluorophenyl) -1-butanone are synthesized by the company, and the synthesis method refers to WO2019011236A1; the 3-amino-1-morpholine-4- (2,4,5-trifluorophenyl) -1-butanone racemate is synthesized by a laboratory, and is prepared by amination and catalytic hydrogenation of morpholine diketone. Determination of configuration of control (R) -3-amino-1-morpholine-4- (2,4,5-trifluorophenyl) -1-butanone: (R) -3-amino-1-morpholine-4- (2,4,5-trifluorophenyl) -1-butanone may be further reacted to prepare (3R) -N-t-butoxycarbonyl-3-amino-4- (2,4,5-trifluorophenyl) -butyric acid (see WO2019011236A 1), which may be determined to be R configuration by reference to a standard of (3R) -N-t-butoxycarbonyl-3-amino-4- (2,4,5-trifluorophenyl) -butyric acid, available from Anhui Kangyao pharmaceutical Limited liability company.
HPLC method for configuration determination:
a chromatographic column: daicel Chiralpak AD-H (4.6 mm 250mm,5 μm); mobile phase: n-hexane: isopropanol = 90; detection wavelength: 210nm; flow rate: 1.0ml/min; sample introduction volume: 10 mul; column temperature: 25 ℃; operating time: and (4) 40min.
The sitagliptin substrate raw material is synthesized by the laboratory, the synthetic method refers to CN100430397C, the sitagliptin racemate is synthesized by the laboratory, and the sitagliptin substrate raw material is prepared by amination and catalytic hydrogenation of the sitagliptin.
The ee value of the product is detected by a chiral HPLC method as follows:
and (3) chromatographic column: a Daicel ChiralpakAD-H column of 4.6 mm. Times.250mm, 5 μm; mobile phase: n-hexane: isopropanol: diethylamine = 40; a detector: UV268nm; column temperature: 25 ℃; flow rate: 0.8mL/min; sample introduction amount: 10 μ L.
3-amino-1-morpholine-4- (2,4,5-trifluorophenyl) -1-butanone racemate control retention time: 10.290min and 28.087min;
(R) -3-amino-1-morpholine-4- (2,4,5-trifluorophenyl) -1-butanone control retention time: 28.093min.
Sitagliptin racemate control retention time: 14.172min and 17.702min;
sitagliptin control retention time: 17.665min.
EXAMPLE 1 preparation of transaminase mutant enzyme solution
According to the reported coding genes of Enz.1 (SEQ ID NO:1, namely SEQ ID NO:110 in US 8293507) and Enz.2 (SEQ ID NO:3, namely SEQ ID NO:130 in US 9617573), the whole gene is synthesized, the restriction sites NdeI and HindIII are used, and the vector pET21a is obtained. The gene synthesis company is Suzhou Jin Weizhi Biotechnology GmbH (Suzhou Industrial park Star lake street 218 Bionanometer technology park C3). The synthesized transaminase gene is transformed into host escherichia coli BL21 competent cells to obtain an engineering strain containing transaminase Enz.1 and Enz.2 genes.
Similarly, the genes of transaminases Enz.3 to Enz.25 engineered in Table 3, the cleavage sites NdeI and HindIII, and the vector pET21a were used. The synthesized transaminase gene is transformed into host escherichia coli BL21 competent cells to obtain engineering bacteria Enz.3-Enz.25 containing the transaminase gene in the table 3.
TABLE 3
After the engineering bacteria containing transaminase genes are activated by plating and streaking, single colony is selected and inoculated into 5ml LB liquid culture medium containing 50 mug/ml kanamycin, and shake culture is carried out for 12h at 37 ℃. Was inoculated into 150ml of fresh LB liquid medium containing 50. Mu.g/ml of kanamycin at an inoculum size of 2% (v/v), shaken at 37 ℃ until the OD600 reached about 0.8, IPTG was added to a final concentration of 0.5mM, and induced at 18 ℃ for 16 hours. After the culture is finished, the culture solution is centrifuged at 10,000rpm for 10min, the supernatant is discarded, and the thalli are collected and stored in an ultra-low temperature refrigerator at minus 80 ℃ for standby.
EXAMPLE 2 preparation of immobilized transaminase mutants
Mixing 5g transaminase mutant bacterial mud with 50mL 100mM dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, homogenizing under high pressure, breaking cells, centrifuging, collecting supernatant enzyme solution, adding 10g K 2 HPO 4 ·3H 2 O、1g KH 2 PO 4 And 0.01g of PLP, dissolved with stirring,adding 4g of
HFA resin (purchased from Suzhou Vietnam chromatography separation and purification Co., ltd.) is fixed, the temperature is 20-25 ℃, the shaking table is used for fixing for 20-25 h at 200rpm, and the immobilized enzyme is obtained through suction filtration, deionization washing and suction filtration.
Example 3 immobilized enzymes of respective transaminase mutants for catalyzing morpholino-diketones
In a triangular flask, 10mL of isopropanol, 0.5g of morpholinodione, 0.8mL of a PLP aqueous solution of 16mg/mL, 0.25mL of isopropylamine and 1g of the immobilized enzyme obtained in example 2 were charged, subjected to shake reaction at 45 ℃ and 200rpm, and sampled for 4 hours and 24 hours to examine the conversion. The results of the measurements are shown in Table 4 below.
Among them, the conversion rate was measured by HPLC as described above in the section of the HPLC method for conversion rate. The retention time of each substrate and product was found to be consistent with the respective control.
TABLE 4
The results show that the conversion rates of Enz.3, enz.6, enz.8, enz.11, enz.13, enz.19, enz.20, enz.21, enz.22, enz.23, enz.24 and Enz.25 are all superior to those of Enz.2 and Enz.1, wherein the conversion rates of Enz.6, enz.11 and Enz.24 in 24 hours can all reach more than 80%.
Example 4 immobilized enzyme Enz.11-
HFA multi-batch catalytic morpholine dione
To a flask, 25mL of isopropanol, 2.5g of morpholinodione, 2mL of a PLP aqueous solution of 16mg/mL, 1.24mL of isopropylamine and 7.5g of the immobilized enzyme prepared in example 2 were added, and the mixture was subjected to a shake reaction at 45 ℃ and 200rpm for 24 hours, followed by sampling and detecting the conversion rate. Filtering the reaction liquid to obtain immobilized enzyme, continuously adding 25mL of isopropanol, 2.5g of morpholine diketone, 2mL of 16mg/mL PLP aqueous solution and 1.24mL of isopropylamine, reacting at 45 ℃ with a shaking table at 200rpm, and sampling for 24 hours to detect the conversion rate. The immobilized enzyme is repeatedly applied according to the method, and the result shows that the immobilized enzyme is applied for 10 batches of reactions, the conversion rate is still kept above 90%, the ee value is more than 99.9%, and the immobilized enzyme is relatively stable. While the conversion rate of Enz.1 and Enz.2 which are applied for 3 times under the same condition is below 50 percent, so that the method is not suitable for catalyzing morpholine diketone. The specific application results are shown in Table 5 below.
TABLE 5
Example 5 immobilized enzymes of respective transaminase mutants for catalyzing sitobinone
In a triangular flask, 10mL of isopropyl alcohol, 0.5g of sitobianone, 0.8mL of 16mg/mL of an aqueous solution of PLP, 0.23mL of isopropylamine and 1g of the immobilized enzyme obtained in example 2 were charged, and subjected to a shaking reaction at 45 ℃ and 200rpm, and sampling was performed for 4 hours and 24 hours to determine the conversion by HPLC, and the results are shown in Table 6 below.
Among them, the conversion rate was measured by HPLC as described above in the section of the conversion rate HPLC method. The retention time of each substrate and product was determined to be consistent with the respective control.
TABLE 6
The results show that the conversion rates of Enz.3, enz.6, enz.11, enz.13, enz.19, enz.20, enz.21 and Enz.22 are all better than those of Enz.1 and Enz.2, wherein the effect of Enz.11 is optimal.
The protein concentration of the better immobilized enzymes is measured, and the detection method comprises the following steps: the change of the absorbance at 595nm of the enzyme solution before and after immobilization was measured by the Bradford method, and the protein concentration was calculated, and the measurement results are shown in Table 7 below:
TABLE 7
Example 6 immobilized enzyme Enz.11-
HFA multi-batch catalysis of sitobianone
To a flask, 25mL of isopropanol, 2.5g of sitaxadione, 2mL of 16mg/mL of an aqueous PLP solution, 1.135mL of isopropylamine and 6.25g of the immobilized enzyme prepared in example 2 were added, and the mixture was subjected to a shaking reaction at 45 ℃ and 200rpm for 24 hours, followed by sampling and measuring the conversion rate. Filtering the reaction liquid to obtain immobilized enzyme, continuously adding 25mL of isopropanol, 2.5g of sitodione, 2mL of 16mg/mL of PLP aqueous solution and 1.135mL of isopropylamine, reacting at 45 ℃ with a shaking table at 200rpm, and sampling for 24 hours to detect the conversion rate. The immobilized enzyme is repeatedly used according to the method, and the result shows that the immobilized enzyme Enz.11-
The HFA is mechanically applied for 10 batches of reactions, the conversion rate is still kept above 95 percent, and the ee value is>99.9 percent, and the immobilized enzyme is relatively stable. The specific application results are shown in Table 8 below.
TABLE 8
* : conversion compared to enz.1; #: conversion comparison with Enz.2
The above results show that the transaminase mutations of the present invention show superior effects on the conversion rate of the immobilized enzyme catalyzed sitagliptin than the conversion rates of the prior art Enz.1 and Enz.2 when used for immobilized enzyme catalysis, and the difference is statistically significant (p values are both less than 0.001, wherein the p value is calculated by T-Test two-tailed experiment).
Example 7 preparation of sitagliptin phosphate monohydrate
After the reaction of example 6 was completed, the immobilized enzyme and the filtrate were obtained by filtration, and the filtrate was concentrated and dried at 60 ℃ to obtain a concentrate. Dissolving in 100mL of dichloromethane, adding 100mL of purified water, stirring, adjusting pH to 2-3 with 30% concentrated hydrochloric acid, and standing for layering. Adding 100mL of dichloromethane into the water phase, stirring, adding 30% sodium hydroxide solution to adjust the pH value to 11, and standing for layering. The aqueous phase was added again with 100mL of methylene chloride, extracted with stirring, and allowed to stand for separation. The organic phases of the two alkali extractions are combined. Concentrating at 60 ℃, adding 120mL of isopropanol into the concentrate, stirring for dissolving, adding 10.6g of 85% phosphoric acid, heating to 75 ℃, stirring for dissolving, slowly cooling to separate out the sitagliptin phosphate crystals, keeping the temperature at 5 ℃ for 2h, filtering, and drying the filter cake at 60 ℃ to obtain the sitagliptin phosphate monohydrate.
SEQUENCE LISTING
<110> chess Ke Lai Biotechnology (Shanghai) GmbH
<120> transaminase, immobilized transaminase and use for the preparation of sitagliptin
<130> P20014045C
<160> 50
<170> PatentIn version 3.5
<210> 1
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.1 amino acids
<400> 1
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Ser Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Met Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Ser Ala Cys Pro Tyr Gln Trp Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Val Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Cys Asp Asn Leu Leu Ala Glu Gly Pro Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Ala Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 2
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.1 nucleotides
<400> 2
atggctttct ctgctgacac cccggaaatc gtttacaccc acgacaccgg tctggactac 60
atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120
atcgaaggtg ctttcgttcc gccgtctgaa gctcgtatct ctatcttcga ccagggtttc 180
tacacctctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240
ggtgaccaca tcgaacgtct gttctctaac gctgaatcta tccgtctgat cccgccgctg 300
acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360
gctatggtta ccgttaccat cacccgtggt tactcttcta ccccgttcga acgtgacatc 420
accaaacacc gtccgcaggt ttacatgtct gcttgcccgt accagtggat cgttccgttc 480
gaccgtatcc gtgacggtgt tcacctgatg gttgctcagt ctgttcgtcg taccccgcgt 540
tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctatccag 600
gaaacccacg accgtggttt cgaactgccg ctgctgctgg actgcgacaa cctgctggct 660
gaaggtccgg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720
gctgctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780
gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840
tgctctaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900
ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960
ccgtcttctc tgctgacccc ggttcagtac 990
<210> 3
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.2 amino acids
<400> 3
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Gly Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Pro Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Met Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Phe Ala Ser Pro Tyr Leu Gln Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Val Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Gly Asp Asn Leu Leu Ala Glu Gly Pro Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Ala Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 4
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.2 nucleotides
<400> 4
atggctttct ctgctgacac cccggaaatc gtttacaccc acgacaccgg tctggactac 60
atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120
atcggtggtg ctttcgttcc gccgtctgaa gctcgtatcc cgatcttcga ccagggtttc 180
tacacctctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240
ggtgaccaca tcgaacgtct gttctctaac gctgaatcta tccgtctgat cccgccgctg 300
acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360
gctatggtta ccgttaccat cacccgtggt tactcttcta ccccgttcga acgtgacatc 420
accaaacacc gtccgcaggt ttacatgttc gcttctccgt acctgcagat cgttccgttc 480
gaccgtatcc gtgacggtgt tcacctgatg gttgctcagt ctgttcgtcg taccccgcgt 540
tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctatccag 600
gaaacccacg accgtggttt cgaactgccg ctgctgctgg acggtgacaa cctgctggct 660
gaaggtccgg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720
gctgctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780
gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840
tgctctaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900
ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960
ccgtcttctc tgctgacccc ggttcagtac 990
<210> 5
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.3 amino acids
<400> 5
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Gly Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Pro Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Gln Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Phe Ala Ser Pro Tyr Leu Gln Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Ile Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Gly Asp Asn Leu Leu Ala Glu Gly Thr Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Arg Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 6
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.3 nucleotides
<400> 6
atggctttct ctgctgacac cccggaaatc gtttacaccc acgacaccgg tctggactac 60
atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120
atcggtggtg ctttcgttcc gccgtctgaa gctcgtatcc cgatcttcga ccagggtttc 180
tacacctctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240
ggtgaccaca tcgaacgtct gttctctaac gctgaatcta tccgtctgat cccgccgctg 300
acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360
gctcaggtta ccgttaccat cacccgtggt tactcttcta ccccgttcga acgtgacatc 420
accaaacacc gtccgcaggt ttacatgttc gcttctccgt acctgcagat cgttccgttc 480
gaccgtatcc gtgacggtgt tcacctgatg atcgctcagt ctgttcgtcg taccccgcgt 540
tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctatccag 600
gaaacccacg accgtggttt cgaactgccg ctgctgctgg acggtgacaa cctgctggct 660
gaaggtaccg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720
cgtgctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780
gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840
tgctctaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900
ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960
ccgtcttctc tgctgacccc ggttcagtac 990
<210> 7
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.4 amino acids
<400> 7
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Gly Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Pro Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Gln Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Phe Ala Ser Pro Tyr Leu Gln Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Ile Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Gly Asp Asn Leu Leu Ala Glu Gly Thr Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Glu Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 8
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.4 nucleotides
<400> 8
atggctttct ctgctgacac cccggaaatc gtttacaccc acgacaccgg tctggactac 60
atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120
atcggtggtg ctttcgttcc gccgtctgaa gctcgtatcc cgatcttcga ccagggtttc 180
tacacctctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240
ggtgaccaca tcgaacgtct gttctctaac gctgaatcta tccgtctgat cccgccgctg 300
acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360
gctcaggtta ccgttaccat cacccgtggt tactcttcta ccccgttcga acgtgacatc 420
accaaacacc gtccgcaggt ttacatgttc gcttctccgt acctgcagat cgttccgttc 480
gaccgtatcc gtgacggtgt tcacctgatg atcgctcagt ctgttcgtcg taccccgcgt 540
tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctatccag 600
gaaacccacg accgtggttt cgaactgccg ctgctgctgg acggtgacaa cctgctggct 660
gaaggtaccg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720
gaagctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780
gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840
tgctctaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900
ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960
ccgtcttctc tgctgacccc ggttcagtac 990
<210> 9
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.5 amino acids
<400> 9
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Gly Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Pro Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Gln Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Phe Ala Ser Pro Tyr Leu Gln Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Ile Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Gly Asp Asn Leu Leu Ala Glu Gly Thr Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Trp Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 10
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.5 nucleotides
<400> 10
atggctttct ctgctgacac cccggaaatc gtttacaccc acgacaccgg tctggactac 60
atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120
atcggtggtg ctttcgttcc gccgtctgaa gctcgtatcc cgatcttcga ccagggtttc 180
tacacctctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240
ggtgaccaca tcgaacgtct gttctctaac gctgaatcta tccgtctgat cccgccgctg 300
acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360
gctcaggtta ccgttaccat cacccgtggt tactcttcta ccccgttcga acgtgacatc 420
accaaacacc gtccgcaggt ttacatgttc gcttctccgt acctgcagat cgttccgttc 480
gaccgtatcc gtgacggtgt tcacctgatg atcgctcagt ctgttcgtcg taccccgcgt 540
tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctatccag 600
gaaacccacg accgtggttt cgaactgccg ctgctgctgg acggtgacaa cctgctggct 660
gaaggtaccg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720
tgggctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780
gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840
tgctctaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900
ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960
ccgtcttctc tgctgacccc ggttcagtac 990
<210> 11
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.6 amino acids
<400> 11
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Gly Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Pro Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Gln Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Phe Ala Ser Pro Tyr Leu Gln Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Ile Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Gly Asp Asn Leu Leu Ala Glu Gly Thr Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Ala Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 12
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.6 nucleotides
<400> 12
atggctttct ctgctgacac cccggaaatc gtttacaccc acgacaccgg tctggactac 60
atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120
atcggtggtg ctttcgttcc gccgtctgaa gctcgtatcc cgatcttcga ccagggtttc 180
tacacctctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240
ggtgaccaca tcgaacgtct gttctctaac gctgaatcta tccgtctgat cccgccgctg 300
acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360
gctcaggtta ccgttaccat cacccgtggt tactcttcta ccccgttcga acgtgacatc 420
accaaacacc gtccgcaggt ttacatgttc gcttctccgt acctgcagat cgttccgttc 480
gaccgtatcc gtgacggtgt tcacctgatg atcgctcagt ctgttcgtcg taccccgcgt 540
tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctatccag 600
gaaacccacg accgtggttt cgaactgccg ctgctgctgg acggtgacaa cctgctggct 660
gaaggtaccg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720
gctgctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780
gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840
tgctctaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900
ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960
ccgtcttctc tgctgacccc ggttcagtac 990
<210> 13
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.7 amino acids
<400> 13
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Gly Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Pro Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Gln Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Phe Ala Ser Pro Tyr Leu Gln Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Ile Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Gly Asp Asn Leu Leu Ala Glu Gly Thr Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Lys Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 14
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.7 nucleotides
<400> 14
atggctttct ctgctgacac cccggaaatc gtttacaccc acgacaccgg tctggactac 60
atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120
atcggtggtg ctttcgttcc gccgtctgaa gctcgtatcc cgatcttcga ccagggtttc 180
tacacctctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240
ggtgaccaca tcgaacgtct gttctctaac gctgaatcta tccgtctgat cccgccgctg 300
acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360
gctcaggtta ccgttaccat cacccgtggt tactcttcta ccccgttcga acgtgacatc 420
accaaacacc gtccgcaggt ttacatgttc gcttctccgt acctgcagat cgttccgttc 480
gaccgtatcc gtgacggtgt tcacctgatg atcgctcagt ctgttcgtcg taccccgcgt 540
tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctatccag 600
gaaacccacg accgtggttt cgaactgccg ctgctgctgg acggtgacaa cctgctggct 660
gaaggtaccg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720
aaagctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780
gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840
tgctctaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900
ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960
ccgtcttctc tgctgacccc ggttcagtac 990
<210> 15
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.8 amino acid
<400> 15
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Gly Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Pro Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Gln Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Phe Ala Ser Pro Tyr Leu Gln Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Ile Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Gly Asp Asn Leu Leu Ala Glu Gly Thr Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
His Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 16
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.8 nucleotides
<400> 16
atggctttct ctgctgacac cccggaaatc gtttacaccc acgacaccgg tctggactac 60
atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120
atcggtggtg ctttcgttcc gccgtctgaa gctcgtatcc cgatcttcga ccagggtttc 180
tacacctctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240
ggtgaccaca tcgaacgtct gttctctaac gctgaatcta tccgtctgat cccgccgctg 300
acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360
gctcaggtta ccgttaccat cacccgtggt tactcttcta ccccgttcga acgtgacatc 420
accaaacacc gtccgcaggt ttacatgttc gcttctccgt acctgcagat cgttccgttc 480
gaccgtatcc gtgacggtgt tcacctgatg atcgctcagt ctgttcgtcg taccccgcgt 540
tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctatccag 600
gaaacccacg accgtggttt cgaactgccg ctgctgctgg acggtgacaa cctgctggct 660
gaaggtaccg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720
cacgctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780
gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840
tgctctaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900
ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960
ccgtcttctc tgctgacccc ggttcagtac 990
<210> 17
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.9 amino acids
<400> 17
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Gly Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Pro Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Gln Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Phe Ala Ser Pro Tyr Leu Gln Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Ile Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Gly Asp Asn Leu Leu Ala Glu Gly Thr Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Tyr Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 18
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.9 nucleotides
<400> 18
atggctttct ctgctgacac cccggaaatc gtttacaccc acgacaccgg tctggactac 60
atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120
atcggtggtg ctttcgttcc gccgtctgaa gctcgtatcc cgatcttcga ccagggtttc 180
tacacctctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240
ggtgaccaca tcgaacgtct gttctctaac gctgaatcta tccgtctgat cccgccgctg 300
acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360
gctcaggtta ccgttaccat cacccgtggt tactcttcta ccccgttcga acgtgacatc 420
accaaacacc gtccgcaggt ttacatgttc gcttctccgt acctgcagat cgttccgttc 480
gaccgtatcc gtgacggtgt tcacctgatg atcgctcagt ctgttcgtcg taccccgcgt 540
tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctatccag 600
gaaacccacg accgtggttt cgaactgccg ctgctgctgg acggtgacaa cctgctggct 660
gaaggtaccg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720
tacgctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780
gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840
tgctctaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900
ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960
ccgtcttctc tgctgacccc ggttcagtac 990
<210> 19
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.10 amino acids
<400> 19
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Gly Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Pro Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Gln Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Phe Ala Ser Pro Tyr Leu Gln Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Ile Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Gly Asp Asn Leu Leu Ala Glu Gly Thr Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Gln Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 20
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.10 nucleotides
<400> 20
atggctttct ctgctgacac cccggaaatc gtttacaccc acgacaccgg tctggactac 60
atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120
atcggtggtg ctttcgttcc gccgtctgaa gctcgtatcc cgatcttcga ccagggtttc 180
tacacctctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240
ggtgaccaca tcgaacgtct gttctctaac gctgaatcta tccgtctgat cccgccgctg 300
acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360
gctcaggtta ccgttaccat cacccgtggt tactcttcta ccccgttcga acgtgacatc 420
accaaacacc gtccgcaggt ttacatgttc gcttctccgt acctgcagat cgttccgttc 480
gaccgtatcc gtgacggtgt tcacctgatg atcgctcagt ctgttcgtcg taccccgcgt 540
tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctatccag 600
gaaacccacg accgtggttt cgaactgccg ctgctgctgg acggtgacaa cctgctggct 660
gaaggtaccg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720
caggctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780
gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840
tgctctaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900
ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960
ccgtcttctc tgctgacccc ggttcagtac 990
<210> 21
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.11 amino acids
<400> 21
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Gly Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Pro Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Gln Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Phe Ala Ser Pro Tyr Leu Gln Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Ile Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Gly Asp Asn Leu Leu Ala Glu Gly Thr Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Asn Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 22
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.11 nucleotides
<400> 22
atggctttct ctgctgacac cccggaaatc gtttacaccc acgacaccgg tctggactac 60
atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120
atcggtggtg ctttcgttcc gccgtctgaa gctcgtatcc cgatcttcga ccagggtttc 180
tacacctctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240
ggtgaccaca tcgaacgtct gttctctaac gctgaatcta tccgtctgat cccgccgctg 300
acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360
gctcaggtta ccgttaccat cacccgtggt tactcttcta ccccgttcga acgtgacatc 420
accaaacacc gtccgcaggt ttacatgttc gcttctccgt acctgcagat cgttccgttc 480
gaccgtatcc gtgacggtgt tcacctgatg atcgctcagt ctgttcgtcg taccccgcgt 540
tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctatccag 600
gaaacccacg accgtggttt cgaactgccg ctgctgctgg acggtgacaa cctgctggct 660
gaaggtaccg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720
aacgctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780
gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840
tgctctaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900
ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960
ccgtcttctc tgctgacccc ggttcagtac 990
<210> 23
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.12 amino acids
<400> 23
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Gly Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Pro Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Gln Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Phe Ala Ser Pro Tyr Leu Gln Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Ile Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Gly Asp Asn Leu Leu Ala Glu Gly Thr Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Pro Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 24
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.12 nucleotides
<400> 24
atggctttct ctgctgacac cccggaaatc gtttacaccc acgacaccgg tctggactac 60
atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120
atcggtggtg ctttcgttcc gccgtctgaa gctcgtatcc cgatcttcga ccagggtttc 180
tacacctctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240
ggtgaccaca tcgaacgtct gttctctaac gctgaatcta tccgtctgat cccgccgctg 300
acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360
gctcaggtta ccgttaccat cacccgtggt tactcttcta ccccgttcga acgtgacatc 420
accaaacacc gtccgcaggt ttacatgttc gcttctccgt acctgcagat cgttccgttc 480
gaccgtatcc gtgacggtgt tcacctgatg atcgctcagt ctgttcgtcg taccccgcgt 540
tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctatccag 600
gaaacccacg accgtggttt cgaactgccg ctgctgctgg acggtgacaa cctgctggct 660
gaaggtaccg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720
ccggctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780
gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840
tgctctaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900
ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960
ccgtcttctc tgctgacccc ggttcagtac 990
<210> 25
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.13 amino acids
<400> 25
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Gly Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Pro Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Gln Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Phe Ala Ser Pro Tyr Leu Gln Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Ile Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Gly Asp Asn Leu Leu Ala Glu Gly Thr Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Ser Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 26
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.13 nucleotides
<400> 26
atggctttct ctgctgacac cccggaaatc gtttacaccc acgacaccgg tctggactac 60
atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120
atcggtggtg ctttcgttcc gccgtctgaa gctcgtatcc cgatcttcga ccagggtttc 180
tacacctctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240
ggtgaccaca tcgaacgtct gttctctaac gctgaatcta tccgtctgat cccgccgctg 300
acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360
gctcaggtta ccgttaccat cacccgtggt tactcttcta ccccgttcga acgtgacatc 420
accaaacacc gtccgcaggt ttacatgttc gcttctccgt acctgcagat cgttccgttc 480
gaccgtatcc gtgacggtgt tcacctgatg atcgctcagt ctgttcgtcg taccccgcgt 540
tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctatccag 600
gaaacccacg accgtggttt cgaactgccg ctgctgctgg acggtgacaa cctgctggct 660
gaaggtaccg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720
tctgctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780
gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840
tgctctaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900
ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960
ccgtcttctc tgctgacccc ggttcagtac 990
<210> 27
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.14 amino acids
<400> 27
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Gly Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Pro Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Gln Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Phe Ala Ser Pro Tyr Leu Gln Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Ile Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Gly Asp Asn Leu Leu Ala Glu Gly Thr Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Asp Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 28
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.14 nucleotides
<400> 28
atggctttct ctgctgacac cccggaaatc gtttacaccc acgacaccgg tctggactac 60
atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120
atcggtggtg ctttcgttcc gccgtctgaa gctcgtatcc cgatcttcga ccagggtttc 180
tacacctctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240
ggtgaccaca tcgaacgtct gttctctaac gctgaatcta tccgtctgat cccgccgctg 300
acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360
gctcaggtta ccgttaccat cacccgtggt tactcttcta ccccgttcga acgtgacatc 420
accaaacacc gtccgcaggt ttacatgttc gcttctccgt acctgcagat cgttccgttc 480
gaccgtatcc gtgacggtgt tcacctgatg atcgctcagt ctgttcgtcg taccccgcgt 540
tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctatccag 600
gaaacccacg accgtggttt cgaactgccg ctgctgctgg acggtgacaa cctgctggct 660
gaaggtaccg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720
gacgctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780
gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840
tgctctaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900
ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960
ccgtcttctc tgctgacccc ggttcagtac 990
<210> 29
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.15 amino acids
<400> 29
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Gly Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Pro Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Gln Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Phe Ala Ser Pro Tyr Leu Gln Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Ile Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Gly Asp Asn Leu Leu Ala Glu Gly Thr Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Gly Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 30
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.15 nucleotides
<400> 30
atggctttct ctgctgacac cccggaaatc gtttacaccc acgacaccgg tctggactac 60
atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120
atcggtggtg ctttcgttcc gccgtctgaa gctcgtatcc cgatcttcga ccagggtttc 180
tacacctctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240
ggtgaccaca tcgaacgtct gttctctaac gctgaatcta tccgtctgat cccgccgctg 300
acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360
gctcaggtta ccgttaccat cacccgtggt tactcttcta ccccgttcga acgtgacatc 420
accaaacacc gtccgcaggt ttacatgttc gcttctccgt acctgcagat cgttccgttc 480
gaccgtatcc gtgacggtgt tcacctgatg atcgctcagt ctgttcgtcg taccccgcgt 540
tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctatccag 600
gaaacccacg accgtggttt cgaactgccg ctgctgctgg acggtgacaa cctgctggct 660
gaaggtaccg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720
ggtgctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780
gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840
tgctctaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900
ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960
ccgtcttctc tgctgacccc ggttcagtac 990
<210> 31
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.16 amino acids
<400> 31
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Gly Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Pro Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Gln Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Phe Ala Ser Pro Tyr Leu Gln Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Ile Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Gly Asp Asn Leu Leu Ala Glu Gly Thr Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Ile Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 32
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.16 nucleotides
<400> 32
atggctttct ctgctgacac cccggaaatc gtttacaccc acgacaccgg tctggactac 60
atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120
atcggtggtg ctttcgttcc gccgtctgaa gctcgtatcc cgatcttcga ccagggtttc 180
tacacctctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240
ggtgaccaca tcgaacgtct gttctctaac gctgaatcta tccgtctgat cccgccgctg 300
acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360
gctcaggtta ccgttaccat cacccgtggt tactcttcta ccccgttcga acgtgacatc 420
accaaacacc gtccgcaggt ttacatgttc gcttctccgt acctgcagat cgttccgttc 480
gaccgtatcc gtgacggtgt tcacctgatg atcgctcagt ctgttcgtcg taccccgcgt 540
tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctatccag 600
gaaacccacg accgtggttt cgaactgccg ctgctgctgg acggtgacaa cctgctggct 660
gaaggtaccg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720
atcgctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780
gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840
tgctctaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900
ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960
ccgtcttctc tgctgacccc ggttcagtac 990
<210> 33
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.17 amino acids
<400> 33
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Gly Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Pro Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Gln Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Phe Ala Ser Pro Tyr Leu Gln Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Ile Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Gly Asp Asn Leu Leu Ala Glu Gly Thr Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Phe Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 34
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.17 nucleotides
<400> 34
atggctttct ctgctgacac cccggaaatc gtttacaccc acgacaccgg tctggactac 60
atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120
atcggtggtg ctttcgttcc gccgtctgaa gctcgtatcc cgatcttcga ccagggtttc 180
tacacctctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240
ggtgaccaca tcgaacgtct gttctctaac gctgaatcta tccgtctgat cccgccgctg 300
acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360
gctcaggtta ccgttaccat cacccgtggt tactcttcta ccccgttcga acgtgacatc 420
accaaacacc gtccgcaggt ttacatgttc gcttctccgt acctgcagat cgttccgttc 480
gaccgtatcc gtgacggtgt tcacctgatg atcgctcagt ctgttcgtcg taccccgcgt 540
tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctatccag 600
gaaacccacg accgtggttt cgaactgccg ctgctgctgg acggtgacaa cctgctggct 660
gaaggtaccg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720
ttcgctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780
gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840
tgctctaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900
ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960
ccgtcttctc tgctgacccc ggttcagtac 990
<210> 35
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.18 amino acids
<400> 35
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Gly Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Pro Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Gln Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Phe Ala Ser Pro Tyr Leu Gln Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Ile Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Gly Asp Asn Leu Leu Ala Glu Gly Thr Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Val Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 36
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.18 nucleotides
<400> 36
atggctttct ctgctgacac cccggaaatc gtttacaccc acgacaccgg tctggactac 60
atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120
atcggtggtg ctttcgttcc gccgtctgaa gctcgtatcc cgatcttcga ccagggtttc 180
tacacctctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240
ggtgaccaca tcgaacgtct gttctctaac gctgaatcta tccgtctgat cccgccgctg 300
acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360
gctcaggtta ccgttaccat cacccgtggt tactcttcta ccccgttcga acgtgacatc 420
accaaacacc gtccgcaggt ttacatgttc gcttctccgt acctgcagat cgttccgttc 480
gaccgtatcc gtgacggtgt tcacctgatg atcgctcagt ctgttcgtcg taccccgcgt 540
tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctatccag 600
gaaacccacg accgtggttt cgaactgccg ctgctgctgg acggtgacaa cctgctggct 660
gaaggtaccg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720
gttgctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780
gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840
tgctctaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900
ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960
ccgtcttctc tgctgacccc ggttcagtac 990
<210> 37
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.19 amino acids
<400> 37
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Gly Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Pro Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Gln Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Phe Ala Ser Pro Tyr Leu Gln Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Ile Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Gly Asp Asn Leu Leu Ala Glu Gly Thr Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Leu Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 38
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.19 nucleotides
<400> 38
atggctttct ctgctgacac cccggaaatc gtttacaccc acgacaccgg tctggactac 60
atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120
atcggtggtg ctttcgttcc gccgtctgaa gctcgtatcc cgatcttcga ccagggtttc 180
tacacctctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240
ggtgaccaca tcgaacgtct gttctctaac gctgaatcta tccgtctgat cccgccgctg 300
acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360
gctcaggtta ccgttaccat cacccgtggt tactcttcta ccccgttcga acgtgacatc 420
accaaacacc gtccgcaggt ttacatgttc gcttctccgt acctgcagat cgttccgttc 480
gaccgtatcc gtgacggtgt tcacctgatg atcgctcagt ctgttcgtcg taccccgcgt 540
tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctatccag 600
gaaacccacg accgtggttt cgaactgccg ctgctgctgg acggtgacaa cctgctggct 660
gaaggtaccg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720
ctggctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780
gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840
tgctctaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900
ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960
ccgtcttctc tgctgacccc ggttcagtac 990
<210> 39
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.20 amino acids
<400> 39
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Gly Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Pro Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Gln Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Phe Ala Ser Pro Tyr Leu Gln Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Ile Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Gly Asp Asn Leu Leu Ala Glu Gly Thr Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Thr Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 40
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.20 nucleotides
<400> 40
atggctttct ctgctgacac cccggaaatc gtttacaccc acgacaccgg tctggactac 60
atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120
atcggtggtg ctttcgttcc gccgtctgaa gctcgtatcc cgatcttcga ccagggtttc 180
tacacctctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240
ggtgaccaca tcgaacgtct gttctctaac gctgaatcta tccgtctgat cccgccgctg 300
acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360
gctcaggtta ccgttaccat cacccgtggt tactcttcta ccccgttcga acgtgacatc 420
accaaacacc gtccgcaggt ttacatgttc gcttctccgt acctgcagat cgttccgttc 480
gaccgtatcc gtgacggtgt tcacctgatg atcgctcagt ctgttcgtcg taccccgcgt 540
tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctatccag 600
gaaacccacg accgtggttt cgaactgccg ctgctgctgg acggtgacaa cctgctggct 660
gaaggtaccg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720
accgctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780
gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840
tgctctaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900
ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960
ccgtcttctc tgctgacccc ggttcagtac 990
<210> 41
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.21 amino acids
<400> 41
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Gly Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Pro Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Gln Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Phe Ala Ser Pro Tyr Leu Gln Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Ile Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Gly Asp Asn Leu Leu Ala Glu Gly Thr Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Cys Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 42
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.21 nucleotides
<400> 42
atggctttct ctgctgacac cccggaaatc gtttacaccc acgacaccgg tctggactac 60
atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120
atcggtggtg ctttcgttcc gccgtctgaa gctcgtatcc cgatcttcga ccagggtttc 180
tacacctctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240
ggtgaccaca tcgaacgtct gttctctaac gctgaatcta tccgtctgat cccgccgctg 300
acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360
gctcaggtta ccgttaccat cacccgtggt tactcttcta ccccgttcga acgtgacatc 420
accaaacacc gtccgcaggt ttacatgttc gcttctccgt acctgcagat cgttccgttc 480
gaccgtatcc gtgacggtgt tcacctgatg atcgctcagt ctgttcgtcg taccccgcgt 540
tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctatccag 600
gaaacccacg accgtggttt cgaactgccg ctgctgctgg acggtgacaa cctgctggct 660
gaaggtaccg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720
tgcgctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780
gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840
tgctctaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900
ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960
ccgtcttctc tgctgacccc ggttcagtac 990
<210> 43
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.22 amino acids
<400> 43
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Gly Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Pro Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Gln Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Phe Ala Ser Pro Tyr Leu Gln Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Ile Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Gly Asp Asn Leu Leu Ala Glu Gly Thr Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Met Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 44
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.22 nucleotides
<400> 44
atggctttct ctgctgacac cccggaaatc gtttacaccc acgacaccgg tctggactac 60
atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120
atcggtggtg ctttcgttcc gccgtctgaa gctcgtatcc cgatcttcga ccagggtttc 180
tacacctctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240
ggtgaccaca tcgaacgtct gttctctaac gctgaatcta tccgtctgat cccgccgctg 300
acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360
gctcaggtta ccgttaccat cacccgtggt tactcttcta ccccgttcga acgtgacatc 420
accaaacacc gtccgcaggt ttacatgttc gcttctccgt acctgcagat cgttccgttc 480
gaccgtatcc gtgacggtgt tcacctgatg atcgctcagt ctgttcgtcg taccccgcgt 540
tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctatccag 600
gaaacccacg accgtggttt cgaactgccg ctgctgctgg acggtgacaa cctgctggct 660
gaaggtaccg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720
atggctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780
gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840
tgctctaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900
ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960
ccgtcttctc tgctgacccc ggttcagtac 990
<210> 45
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.23 amino acids
<400> 45
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Ser Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Phe Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Ser Ala Cys Pro Tyr Gln Trp Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Val Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Cys Asp Asn Leu Leu Ala Glu Gly Cys Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Ala Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 46
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.23 nucleotides
<400> 46
atggctttct ctgctgacac cccggaaatc gtttacaccc acgacaccgg tctggactac 60
atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120
atcgaaggtg ctttcgttcc gccgtctgaa gctcgtatct ctatcttcga ccagggtttc 180
tacacctctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240
ggtgaccaca tcgaacgtct gttctctaac gctgaatcta tccgtctgat cccgccgctg 300
acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360
gctttcgtta ccgttaccat cacccgtggt tactcttcta ccccgttcga acgtgacatc 420
accaaacacc gtccgcaggt ttacatgtct gcttgcccgt accagtggat cgttccgttc 480
gaccgtatcc gtgacggtgt tcacctgatg gttgctcagt ctgttcgtcg taccccgcgt 540
tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctatccag 600
gaaacccacg accgtggttt cgaactgccg ctgctgctgg actgcgacaa cctgctggct 660
gaaggttgcg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720
gctgctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780
gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840
tgctctaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900
ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960
ccgtcttctc tgctgacccc ggttcagtac 990
<210> 47
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.24 amino acids
<400> 47
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Ser Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Gln Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Ser Ala Cys Pro Tyr Gln Trp Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Val Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Cys Asp Asn Leu Leu Ala Glu Gly Thr Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Asn Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 48
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.24 nucleotides
<400> 48
atggctttct ctgctgacac cccggaaatc gtttacaccc acgacaccgg tctggactac 60
atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120
atcgaaggtg ctttcgttcc gccgtctgaa gctcgtatct ctatcttcga ccagggtttc 180
tacacctctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240
ggtgaccaca tcgaacgtct gttctctaac gctgaatcta tccgtctgat cccgccgctg 300
acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360
gctcaggtta ccgttaccat cacccgtggt tactcttcta ccccgttcga acgtgacatc 420
accaaacacc gtccgcaggt ttacatgtct gcttgcccgt accagtggat cgttccgttc 480
gaccgtatcc gtgacggtgt tcacctgatg gttgctcagt ctgttcgtcg taccccgcgt 540
tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctatccag 600
gaaacccacg accgtggttt cgaactgccg ctgctgctgg actgcgacaa cctgctggct 660
gaaggtaccg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720
aacgctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780
gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840
tgctctaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900
ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960
ccgtcttctc tgctgacccc ggttcagtac 990
<210> 49
<211> 330
<212> PRT
<213> Artificial Sequence
<220>
<223> Enz.25 amino acids
<400> 49
Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Pro Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Gln Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Ser Ala Cys Pro Tyr Gln Trp Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Ile Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Cys Asp Asn Leu Leu Ala Glu Gly Thr Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Arg Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 50
<211> 990
<212> DNA
<213> Artificial Sequence
<220>
<223> Enz.25 nucleotides
<400> 50
atggctttct ctgctgacac cccggaaatc gtttacaccc acgacaccgg tctggactac 60
atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120
atcgaaggtg ctttcgttcc gccgtctgaa gctcgtatcc cgatcttcga ccagggtttc 180
tacacctctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240
ggtgaccaca tcgaacgtct gttctctaac gctgaatcta tccgtctgat cccgccgctg 300
acccaggacg aagttaaaga aatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360
gctcaggtta ccgttaccat cacccgtggt tactcttcta ccccgttcga acgtgacatc 420
accaaacacc gtccgcaggt ttacatgtct gcttgcccgt accagtggat cgttccgttc 480
gaccgtatcc gtgacggtgt tcacctgatg atcgctcagt ctgttcgtcg taccccgcgt 540
tcttctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgctatccag 600
gaaacccacg accgtggttt cgaactgccg ctgctgctgg actgcgacaa cctgctggct 660
gaaggtaccg gtttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720
cgtgctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780
gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840
tgctctaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900
ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960
ccgtcttctc tgctgacccc ggttcagtac 990
Claims (11)
1. Use of an immobilized transaminase in the preparation of sitagliptin and/or (R) -3-amino-1-morpholine-4- (2,4,5-trifluorophenyl) -1-butanone;
wherein the immobilized transaminase comprises a resin and a transaminase, and the amino acid sequence of the transaminase is shown in SEQ ID NO 5, SEQ ID NO 45, SEQ ID NO 47, SEQ ID NO 49 or variants thereof; the variant is that the R at the 241 th site of the amino acid sequence shown as SEQ ID NO. 5 is mutated into A, H, N, S, L, T, C or M;
preferably:
the amino acid sequence of the variant is shown as SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 21, SEQ ID NO. 25, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41 or SEQ ID NO. 43; the nucleotide sequence of the transaminase is preferably shown as SEQ ID NO 6, SEQ ID NO 12, SEQ ID NO 16, SEQ ID NO 22, SEQ ID NO 26, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48 or SEQ ID NO 50; and/or, said transaminase is attached to said resin by covalent bonding; and/or the resin is epoxy resin, preferably
And/or the reaction solvent used in the preparation is an isopropanol aqueous solution, and preferably the volume content of water in the isopropanol aqueous solution is 2-20%; and/or, the prepared reaction system also comprises a co-factor of transaminase, such as pyridoxal phosphate, the concentration of the co-factor is preferably 0.5-5 mg/mL; and/or the immobilized transaminase has a concentration of the transaminase immobilized on the resin of 150 to 300mg/g, e.g., 250mg/g.
2. An immobilized transaminase comprising a resin and a transaminase, the transaminase having an amino acid sequence set forth in SEQ ID NO 5, SEQ ID NO 45, SEQ ID NO 47, SEQ ID NO 49, or variants thereof; the variant is that the R at the 241 th site of the amino acid sequence shown as SEQ ID NO. 5 is mutated into A, H, N, S, L, T, C or M;
preferably:
the amino acid sequence of the variant is shown in SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 21, SEQ ID NO. 25, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41 or SEQ ID NO. 43; the nucleotide sequence of the transaminase is preferably shown as SEQ ID NO 6, SEQ ID NO 12, SEQ ID NO 16, SEQ ID NO 22, SEQ ID NO 26, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48 or SEQ ID NO 50; and/or, said transaminase is attached to said resin by covalent bonding; and/or the resin is epoxy resin, preferably
And/or the immobilized transaminase has a concentration of the transaminase immobilized on the resin of 150 to 300mg/g, e.g., 250mg/g.
3. A process for preparing an immobilized transaminase of claim 2, comprising:
1) Contacting a solution of the transaminase enzyme with the resin to react to form an immobilized transaminase enzyme;
2) Filtering and washing the immobilized transaminase;
preferably:
the transaminase exists in the form of transaminase bacterial sludge, and the mass ratio of the transaminase bacterial sludge to the resin is 3:1-1:2, such as 5:4; and/or the reaction temperature is 10-40 ℃, preferably 20-25 ℃; and/or the reaction time is 10-30 h, preferably 20-25 h; and/or the reaction is carried out under stirring, preferably at a rate of 50 to 300rpm, for example 150 or 200rpm.
4. A process for the preparation of sitagliptin and/or (R) -3-amino-1-morpholine-4- (2,4,5-trifluorophenyl) -1-butanone comprising the step of catalyzing a ketoamide substrate with an immobilized transaminase in a reaction solvent in the presence of an amino donor to yield sitagliptin and/or (R) -3-amino-1-morpholine-4- (2,4,5-trifluorophenyl) -1-butanone;
wherein the immobilized transaminase is as described in claim 2;
preferably, the ketoamide substrate is 4-oxo-4- [3- (trifluoromethyl) -5,6-dihydro- [1,2,4] triazolo [4,3-a ] pyrazin-7- (8H) -yl ] -1- (2,4,5-trifluorophenyl) butan-2-one and/or 1-morpholine-4- (2,4,5-trifluorophenyl) -1,3-butanedione;
and/or the reaction solvent is an isopropanol aqueous solution;
and/or the amino donor is isopropylamine;
and/or the molar ratio of the amino donor to the ketoamide substrate is 1:1-5:1;
and/or, when the reaction solvent is isopropanol aqueous solution, the volume content of water is 2-20%;
and/or the concentration of the ketoamide substrate is 20-200 g/L;
and/or the mass ratio of the immobilized transaminase to the ketoamide substrate is 1:1 to 6:1, e.g., 3;
and/or, the prepared reaction system also comprises a co-factor of transaminase, such as pyridoxal phosphate, the concentration of the co-factor is preferably 0.5-5 mg/mL;
and/or the temperature of the reaction is 30-60 ℃, preferably 45 ℃.
5. A transaminase, characterized in that the amino acid sequence of the transaminase is shown in SEQ ID No. 5, SEQ ID No. 45, SEQ ID No. 47, SEQ ID No. 49 or variants thereof; the variant is characterized in that the 241 th position of the amino acid sequence shown as SEQ ID NO. 5 is mutated from R to A, H, N, S, L, T, C or M;
preferably, the amino acid sequence of the variant is shown as SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 21, SEQ ID NO. 25, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41 or SEQ ID NO. 43;
more preferably, the nucleotide sequence of the transaminase is shown as SEQ ID NO 6, SEQ ID NO 12, SEQ ID NO 16, SEQ ID NO 22, SEQ ID NO 26, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48 or SEQ ID NO 50.
6. A polynucleotide encoding the transaminase enzyme of claim 5.
7. A recombinant expression vector comprising the polynucleotide of claim 6;
preferably, the backbone of the recombinant expression vector is plasmid pET21a.
8. A transformant obtained by introducing the polynucleotide of claim 6 or the recombinant expression vector of claim 7 into a host;
preferably, the host is escherichia coli; escherichia coli BL21 is preferred.
9. Use of a transaminase of claim 5 in the preparation of sitagliptin and/or (R) -3-amino-1-morpholine-4- (2,4,5-trifluorophenyl) -1-butanone.
10. A preparation method of sitagliptin phosphate, which is characterized by comprising the following steps:
(1) Preparing sitagliptin and/or (R) -3-amino-1-morpholine-4- (2,4,5-trifluorophenyl) -1-butanone according to the process of claim 4;
(2) Reacting sitagliptin and/or (R) -3-amino-1-morpholine-4- (2,4,5-trifluorophenyl) -1-butanone prepared in the step (1) to obtain sitagliptin phosphate;
preferably, the sitagliptin phosphate is sitagliptin phosphate monohydrate.
11. An enzyme preparation comprising an immobilized transaminase of claim 2 or a transaminase of claim 5;
preferably, the enzyme preparation further comprises a co-factor for transaminase, such as pyridoxal phosphate.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110655349.9A CN115466756A (en) | 2021-06-11 | 2021-06-11 | Transaminase, immobilized transaminase and application of transaminase to preparation of sitagliptin |
| PCT/CN2022/092053 WO2022257686A1 (en) | 2021-06-11 | 2022-05-10 | Transaminase, immobilized transaminase and use for preparing sitagliptin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110655349.9A CN115466756A (en) | 2021-06-11 | 2021-06-11 | Transaminase, immobilized transaminase and application of transaminase to preparation of sitagliptin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115466756A true CN115466756A (en) | 2022-12-13 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110655349.9A Pending CN115466756A (en) | 2021-06-11 | 2021-06-11 | Transaminase, immobilized transaminase and application of transaminase to preparation of sitagliptin |
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| CN (1) | CN115466756A (en) |
| WO (1) | WO2022257686A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117467733A (en) * | 2023-12-27 | 2024-01-30 | 北京元延医药科技股份有限公司 | High chiral purity sitagliptin and method for preparing same by using immobilized transaminase |
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| CN116024083B (en) * | 2023-02-22 | 2023-06-13 | 凯莱英生命科学技术(天津)有限公司 | Device and method for preparing chiral amine compound by continuous flow reaction |
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| CA2838744C (en) * | 2011-06-24 | 2020-10-13 | Merck Sharp & Dohme Corp. | Immobilized transaminases and process for making and using immobilized transaminase |
| DK2961844T3 (en) * | 2013-02-28 | 2019-01-02 | Codexis Inc | MODIFIED TRANSAMINASE POLYPEPTIDES FOR INDUSTRIAL BIOCATALYSIS |
| CN105219745B (en) * | 2014-06-12 | 2018-12-04 | 上海弈柯莱生物医药科技有限公司 | A kind of immobilization transaminase and its application in synthesis sitagliptin intermediate |
| ES2733477T3 (en) * | 2017-07-04 | 2019-11-29 | Fis Fabbrica Italiana Sintetici Spa | Effective procedure for the preparation of sitagliptin through a very effective preparation of the intermediate 2,4,5-trifluorophenylacetic acid |
| WO2019207443A1 (en) * | 2018-04-24 | 2019-10-31 | Unichem Laboratories Ltd | An enzymatic process for the preparation of (r)-sitagliptin |
| CN108586346B (en) * | 2018-05-10 | 2019-10-01 | 北京富盛嘉华医药科技有限公司 | A kind of method that biocatalysis synthesizes sitagliptin and its intermediate |
| EP3604507A1 (en) * | 2018-07-30 | 2020-02-05 | University College Cork-National University of Ireland, Cork | An omega-transaminase enzyme |
| US20220380817A1 (en) * | 2019-10-25 | 2022-12-01 | Asymchem Laboratories (Fuxin) Co., Ltd. | Transaminase mutant and use thereof |
| CN113061594B (en) * | 2019-12-31 | 2023-11-24 | 弈柯莱生物科技(集团)股份有限公司 | Transaminase mutants, immobilized transaminases and use for preparing sitagliptin |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117467733A (en) * | 2023-12-27 | 2024-01-30 | 北京元延医药科技股份有限公司 | High chiral purity sitagliptin and method for preparing same by using immobilized transaminase |
| CN117467733B (en) * | 2023-12-27 | 2024-03-12 | 北京元延医药科技股份有限公司 | High chiral purity sitagliptin and method for preparing same by using immobilized transaminase |
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