CN111944775A - Modified 7 beta-hydroxysteroid dehydrogenase and application thereof - Google Patents

Modified 7 beta-hydroxysteroid dehydrogenase and application thereof Download PDF

Info

Publication number
CN111944775A
CN111944775A CN202010849118.7A CN202010849118A CN111944775A CN 111944775 A CN111944775 A CN 111944775A CN 202010849118 A CN202010849118 A CN 202010849118A CN 111944775 A CN111944775 A CN 111944775A
Authority
CN
China
Prior art keywords
glu
lys
gly
ala
ile
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202010849118.7A
Other languages
Chinese (zh)
Other versions
CN111944775B (en
Inventor
谢新开
杜好勉
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Suzhou Lead Biotechnology Co ltd
Original Assignee
Suzhou Lead Biotechnology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Suzhou Lead Biotechnology Co ltd filed Critical Suzhou Lead Biotechnology Co ltd
Priority to CN202010849118.7A priority Critical patent/CN111944775B/en
Publication of CN111944775A publication Critical patent/CN111944775A/en
Application granted granted Critical
Publication of CN111944775B publication Critical patent/CN111944775B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P33/00—Preparation of steroids
    • 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
    • C12P33/00—Preparation of steroids
    • C12P33/06—Hydroxylating
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01201—7-Beta-hydroxysteroid dehydrogenase (NADP+) (1.1.1.201)

Landscapes

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

Abstract

The present invention relates to a modified 7 beta-hydroxysteroid dehydrogenase (7 beta-HSDH). In particular, the modified 7 β -HSDH according to the invention has an improved activity in catalysing the reduction of 7-keto-lithocholic acid or derivatives thereof to ursodeoxycholic acid or derivatives thereof, compared to its starting 7 β -HSDH. The invention also relates to polynucleotides encoding the modified 7 β -HSDH of the invention, vectors and host cells expressing the modified 7 β -HSDH of the invention and methods of producing ursodeoxycholic acid and its derivatives using the modified 7 β -HSDH of the invention and host cells.

Description

Modified 7 beta-hydroxysteroid dehydrogenase and application thereof
Technical Field
The present invention relates to the field of enzyme engineering. In particular, the invention relates to a modified 7 beta-hydroxysteroid dehydrogenase (7 beta-HSDH) and its use in the production of ursodeoxycholic acid or its derivatives.
Background
Ursodeoxycholic acid (UDCA) is the main effective component contained in the rare Chinese medicine bear bile, is a steroid compound with the chemical name of 3 alpha, 7 beta-dihydroxy-5 beta-cholestane-24-acid, is a cholic acid analogue and is beneficial to the action of gallbladder. Can be used for treating cholelithiasis, cholestatic liver disease, fatty liver, various hepatitis, toxic liver disorder, cholecystitis, cholangitis, bile dyspepsia, bile reflux gastritis, eye diseases, etc.
UDCA is available from the gall bladder of artificially cultivated bears, but has low yields, limited sources, and animal protection problems. Therefore, the artificial synthesis of UDCA is of great significance. The synthesis method of UDCA mainly includes total chemical synthesis and chemo-enzymatic synthesis, and starting material is animal-derived Cholic Acid (CA) or deoxycholic acid (such as CDCA).
The classical chemical synthesis of UDCA involves the steps shown in formula I.
Figure BDA0002644136040000011
Among them, the reduction of 7-keto-lithocholic acid (7-keto-LCA) requires the use of metallic sodium or Pd/C catalytic hydrogenation, the selectivity is low, and the industrial scale-up production is not easy to control and is unsafe. Research shows that the reaction of the formula I can be catalyzed by 7 beta-hydroxy steroid dehydrogenase (7 beta-HSDH), and the reaction selectivity is high without forming byproducts.
However, there remains a need in the art to provide 7 β -HSDH having an increased enzymatic activity catalyzing the reduction of 7-keto-lithocholic acid or a derivative thereof to UDCA or a derivative thereof.
Disclosure of Invention
In a first aspect, the present invention provides a modified 7 β -hydroxysteroid dehydrogenase (7 β -HSDH) comprising an amino acid substitution at one or more positions compared to its starting 7 β -HSDH and having improved activity in catalyzing the reduction of 7-keto-lithocholic acid or a derivative thereof to ursodeoxycholic acid or a derivative thereof.
In some embodiments, the modified 7 β -HSDH comprises an amino acid substitution at position 203, preferably F, Y or L, and is numbered with reference to SEQ ID No. 1. In some embodiments, the modified 7 β -HSDH further comprises a substitution at one or more positions selected from positions 4, 18, 44, 55, 64, 102, 128, 196, and 238. Preferably, position 4 is substituted with S. Preferably, the substitution in position 18 is D. Preferably, the substitution at position 44 is R. Preferably, the substitution in position 55 is T. Preferably, the substitution in position 64 is R. Preferably, the substitution at position 102 is E. Preferably, the substitution at position 128 is H. Preferably, position 196 is substituted with N. Preferably, position 238 is substituted with R.
In some embodiments, the modified 7 β -HSDH comprises amino acid substitutions at positions 55 and 203, wherein the substitution at position 55 is T and the substitution at position 203 is F or Y. In some embodiments, the modified 7 β -HSDH comprises amino acid substitutions at positions 64 and 203, wherein the substitution at position 64 is R and the substitution at position 203 is F or Y. In some embodiments, the modified 7 β -HSDH comprises amino acid substitutions at positions 55, 64 and 203, wherein the substitution at position 55 is T, the substitution at position 64 is R, and the substitution at position 203 is F or Y.
In some embodiments, the starting 7 β -HSDH comprises the amino acid sequence of SEQ ID NO 1.
In some embodiments, the modified 7 β -HSDH comprises the amino acid sequence of one of SEQ ID NOs 2-9, 11, 12, 14, 15 and 17-29.
In a second aspect, the invention provides polynucleotides encoding the modified 7 β -HSDH of the invention, and vectors comprising the polynucleotides of the invention.
In a third aspect, the invention provides a host cell comprising a modified 7 β -HSDH of the invention, a polynucleotide encoding it, or a vector comprising said polynucleotide.
In a fourth aspect, the invention also provides a method of producing ursodeoxycholic acid or a derivative thereof, comprising contacting the modified 7 β -HSDH of the invention or the host cell of the invention with 7-keto-lithocholic acid or a derivative thereof.
Detailed Description
Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying structural and reaction formulae. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications and equivalents, which may be included within the scope of the invention as defined by the appended claims. One skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The present invention is not limited to the methods and materials described. If one or more of the cited documents, patents, and similar materials are different from or contradictory to the present application, including but not limited to defined terms, usage of terms, described techniques, etc., the present application controls. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although suitable methods and materials are described below, methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Unless otherwise indicated, the nomenclature used in this application is based on the IUPAC systematic nomenclature.
Modified 7 beta-hydroxysteroid dehydrogenase
As used herein, "7 β -hydroxysteroid dehydrogenase" and "7 β -HSDH" have the activity of catalyzing the reduction of 7-keto-lithocholic acid or a derivative thereof to ursodeoxycholic acid or a derivative thereof. The present invention provides modified 7 β -HSDH polypeptides having improved activity in catalyzing the reduction of 7-keto-lithocholic acid or a derivative thereof to ursodeoxycholic acid or a derivative thereof, compared to its starting 7 β -HSDH polypeptide.
As used herein, the term "peptide" means a chain of at least two amino acids linked by peptide bonds. The term "polypeptide" is used interchangeably herein with the term "protein" and refers to a chain containing ten or more amino acid residues. All peptide and polypeptide chemical formulas or sequences herein are written from left to right, representing the direction from the amino terminus to the carboxy terminus.
The term "amino acid" includes naturally occurring amino acids and unnatural amino acids in proteins. The one-letter and three-letter designations of the naturally occurring amino acids in proteins are used by conventional names in the art and can be found in Sambrook, et al (Molecular Cloning: A Laboratory Manual,2nd, ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
Figure BDA0002644136040000031
As used herein, the term "modification" refers to any modification to a polypeptide, such as a substitution, deletion, insertion, and/or addition of an amino acid.
In some embodiments, the modified 7 β -HSDH of the invention comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, or more amino acid substitutions compared to its starting 7 β -HSDH, wherein the modified 7 β -HSDH of the invention has improved activity in catalyzing the reduction of 7-keto-lithocholic acid or a derivative thereof to ursodeoxycholic acid or a derivative thereof compared to its starting 7 β -HSDH.
In some embodiments, the modified 7 β -HSDH comprises 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acid substitutions as compared to its starting 7 β -HSDH, wherein the modified 7 β -HSDH of the invention has improved activity in catalyzing the reduction of 7-keto-lithocholic acid or a derivative thereof to ursodeoxycholic acid or a derivative thereof as compared to its starting 7 β -HSDH.
In some embodiments, the modified 7 β -HSDH according to the invention comprises an amino acid substitution at position 203, as compared to its starting 7 β -HSDH, said position being numbered with reference to SEQ ID No. 1, preferably the substitution F, Y or L.
In some embodiments, the modified 7 β -HSDH of the invention further comprises an amino acid substitution at one or more positions selected from positions 4, 18, 44, 55, 64, 102, 128, 196 and 238. Preferably, position 4 is substituted with S. Preferably, the substitution in position 18 is D. Preferably, the substitution at position 44 is R. Preferably, the substitution in position 55 is T. Preferably, the substitution in position 64 is R. Preferably, the substitution at position 102 is E. Preferably, the substitution at position 128 is H. Preferably, position 196 is substituted with N. Preferably, position 238 is substituted with R.
In a preferred embodiment, the modified 7 β -HSDH according to the invention comprises amino acid substitutions at positions 55 and 203, wherein the substitution at position 55 is T and the substitution at position 203 is F or Y. In a preferred embodiment, the modified 7 β -HSDH according to the invention comprises amino acid substitutions at positions 64 and 203, wherein the substitution at position 64 is R and the substitution at position 203 is F or Y. More preferably, the modified 7 β -HSDH according to the invention comprises amino acid substitutions at positions 55, 64 and 203, wherein the substitution at position 55 is T, the substitution at position 64 is R, and the substitution at position 203 is F or Y. In some embodiments, the modified 7 β -HSDH further comprises an amino acid substitution at one or more positions selected from the group consisting of positions 4, 18, 44, 102, 128, 196, and 238, wherein the position 4 substitution is S, the position 18 substitution is D, the position 44 substitution is R, the position 102 substitution is E, the position 128 substitution is H, the position 196 substitution is N, and the position 238 substitution is R.
In some embodiments, the modified 7 β -HSDH of the invention has an amino acid substitution or combination of amino acid substitutions (positions numbered with reference to SEQ ID NO:1) compared to its starting 7 β -HSDH selected from:
55T；
64R；
102E；
128H；
203F；
203Y；
203L；
55T-203F；
55T-203L；
55T-203Y；
64R-203F；
64R-203L；
64R-203Y；
55T-64R-203F；
55T-64R-203L；
55T-64R-203Y；
55T-64R-203F-238R；
4S-55T-64R-203F；
18D-55T-64R-203F；
44R-55T-64R-203F；
55T-64R-196N-203F；
4S-55T-64R-203F-238R；
18D-55T-64R-203F-238R；
44R-55T-64R-203F-238R；
55T-64R-196N-203F-238R；
4S-18D-55T-64R-203F-238R；
4S-44R-55T-64R-203F-238R; and
4S-18D-55T-64R-196N-203F-238R。
herein, the 7 β -HSDH polypeptide on which the amino acid modification is based is referred to as the starting 7 β -HSDH. The starting 7 β -HSDH may be wild-type 7 β -HSDH or a variant of wild-type 7 β -HSDH. For example, if the modification is made starting from the polypeptide of SEQ ID NO. 1, the polypeptide of SEQ ID NO. 1 is "starting 7 β -HSDH" relative to the modified 7 β -HSDH; whereas if the modification is made starting from a variant polypeptide of SEQ ID NO:1 (e.g., SEQ ID NO:2-29), said variant polypeptide is a "starting 7 β -HSDH" relative to the modified 7 β -HSDH.
As used herein, the term "wild-type 7 β -HSDH" refers to the naturally occurring 7 β -HSDH. In some embodiments, the wild-type 7 β -HSDH is 7 β -HSDH from clostridium. In some embodiments, the wild-type 7 β -HSDH is set forth in SEQ ID NO:1, which is the amino acid sequence of 7 β -HSDH from Clostridium sp.
For purposes of the present invention, to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity is the number of identical positions/total number of positions (i.e., overlapping positions) × 100). Preferably, the two sequences are of the same length.
One skilled in the art will appreciate that different computer programs can be used to determine identity between two sequences.
"percent amino acid identity" or "percent amino acid sequence identity" refers to the comparison of amino acids of two polypeptides that, when optimally aligned, have approximately the specified percentage of amino acids that are identical. For example, "95% amino acid identity" refers to comparing the amino acids of two polypeptides, which are 95% identical when optimally aligned.
In some embodiments, the modified 7 β -HSDH polypeptide of the invention has at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, 90% or 95%, particularly preferably at least 96%, 97%, 98% or 99% sequence identity with SEQ ID No. 1.
In some embodiments, a modified 7 β -HSDH of the invention comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, or more amino acid substitutions as compared to its starting 7 β -HSDH (e.g., SEQ ID NO:1), wherein the modified 7 β -HSDH of the invention has improved activity in catalyzing the reduction of 7-keto-lithocholic acid or a derivative thereof to ursodeoxycholic acid or a derivative thereof as compared to its starting 7 β -HSDH. In some embodiments, the modified 7 β -HSDH comprises 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acid substitutions as compared to its starting 7 β -HSDH (e.g., SEQ ID NO: 1). In some embodiments, the modified 7 β -HSDH according to the invention comprises an amino acid substitution at position 203, as compared to its starting 7 β -HSDH, said position being numbered with reference to SEQ ID No. 1, preferably the substitution F, Y or L. In some embodiments, the modified 7 β -HSDH has a sequence identity of at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, 90% or 95%, particularly preferably at least 96%, 97%, 98% or 99% compared to its starting 7 β -HSDH.
In some embodiments, the modified 7 β -HSDH of the invention further comprises an amino acid substitution at one or more positions selected from positions 4, 18, 44, 55, 64, 102, 128, 196 and 238. Preferably, position 4 is substituted with S. Preferably, the substitution in position 18 is D. Preferably, the substitution at position 44 is R. Preferably, the substitution in position 55 is T. Preferably, the substitution in position 64 is R. Preferably, the substitution at position 102 is E. Preferably, the substitution at position 128 is H. Preferably, position 196 is substituted with N. Preferably, position 238 is substituted with R. In some embodiments, the modified 7 β -HSDH has a sequence identity of at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, 90% or 95%, particularly preferably at least 96%, 97%, 98% or 99% compared to its starting 7 β -HSDH.
In a preferred embodiment, the modified 7 β -HSDH according to the invention comprises amino acid substitutions at positions 55 and 203, wherein the substitution at position 55 is T and the substitution at position 203 is F or Y, said positions being numbered with reference to SEQ ID No. 1. In a preferred embodiment, the modified 7 β -HSDH according to the invention comprises amino acid substitutions at positions 64 and 203, wherein the substitution at position 64 is R and the substitution at position 203 is F or Y. More preferably, the modified 7 β -HSDH according to the invention comprises amino acid substitutions at positions 55, 64 and 203, wherein the substitution at position 55 is T, the substitution at position 64 is R, and the substitution at position 203 is F or Y. In some embodiments, the modified 7 β -HSDH further comprises an amino acid substitution at one or more positions selected from the group consisting of positions 4, 18, 44, 102, 128, 196, and 238, wherein the position 4 substitution is S, the position 18 substitution is D, the position 44 substitution is R, the position 102 substitution is E, the position 128 substitution is H, the position 196 substitution is N, and the position 238 substitution is R. Preferably, said modified 7 β -HSDH has a sequence identity of at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, 90% or 95%, especially preferably at least 96%, 97%, 98% or 99% compared to its starting 7 β -HSDH.
In some embodiments, the modified 7 β -HSDH of the invention comprises an amino acid substitution at position 203 compared to its starting 7 β -HSDH, said positions being numbered with reference to SEQ ID NO:1, preferably the substitution is F, Y or L, wherein the modified 7 β -HSDH of the invention has an improved activity in catalyzing the reduction of 7-keto-lithocholic acid or a derivative thereof to ursodeoxycholic acid or a derivative thereof compared to its starting 7 β -HSDH. In some embodiments, the starting 7 β -HSDH has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID No. 1. In some embodiments, the modified 7 β -HSDH of the invention further comprises an amino acid substitution at one or more positions selected from positions 4, 18, 44, 55, 64, 102, 128, 196 and 238. Preferably, position 4 is substituted with S. Preferably, the substitution in position 18 is D. Preferably, the substitution at position 44 is R. Preferably, the substitution in position 55 is T. Preferably, the substitution in position 64 is R. Preferably, the substitution at position 102 is E. Preferably, the substitution at position 128 is H. Preferably, position 196 is substituted with N. Preferably, position 238 is substituted with R. In a preferred embodiment, the modified 7 β -HSDH according to the invention comprises amino acid substitutions at positions 55 and 203, wherein the substitution at position 55 is T and the substitution at position 203 is F or Y, said positions being numbered with reference to SEQ ID No. 1. In a preferred embodiment, the modified 7 β -HSDH according to the invention comprises amino acid substitutions at positions 64 and 203, wherein the substitution at position 64 is R and the substitution at position 203 is F or Y. More preferably, the modified 7 β -HSDH according to the invention comprises amino acid substitutions at positions 55, 64 and 203, wherein the substitution at position 55 is T, the substitution at position 64 is R, and the substitution at position 203 is F or Y.
The term "conservative substitution", also referred to as substitution by a "homologous" amino acid residue, refers to a substitution in which the amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., an amino acid having a basic side chain (e.g., lysine, arginine, and histidine), an amino acid having an acidic side chain (e.g., aspartic acid, glutamic acid), an uncharged polar side chain amino acid (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), an nonpolar side chain amino acid (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), a β -branched side chain amino acid (e.g., threonine, valine, isoleucine), and an aromatic side chain amino acid (e.g., tyrosine, phenylalanine, tryptophan, histidine).
Conservative amino acid substitutions generally have minimal effect on the activity of the resulting protein. Such substitutions are described below. Conservative substitutions are those that replace an amino acid with an amino acid that is similar in size, hydrophobicity, charge, polarity, steric characteristics, aromaticity, and the like. Such substitutions are generally conservative when fine-tuning of the properties of the protein is desired.
As used herein, "homologous" amino acid residues refer to amino acid residues having similar chemical properties relating to hydrophobicity, charge, polarity, steric characteristics, aromaticity characteristics, and the like. Examples of amino acids that are homologous to each other include positively charged lysine, arginine, histidine, negatively charged glutamic acid, aspartic acid, hydrophobic glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, polar serine, threonine, cysteine, methionine, tryptophan, tyrosine, asparagine, glutamine, aromatic phenylalanine, tyrosine, tryptophan, serine and threonine of chemically similar side chain groups, or glutamine and asparagine, or leucine and isoleucine.
Examples of conservative amino acid substitutions in proteins include: ser for Ala, Lys for Arg, Gln or His for Asn, Glu for Asp, Ser for Cys, Asn for Gln, Asp for Glu, Pro for Gly, Asn or Gln for His, Leu or Val for Ile, Ile or Val for Leu, Arg or Gln for Lys, Leu or Ile for Met, Leu or Tyr for Phe, Thr for Ser, Ser for Thr, Tyr for Trp, Trp or Phe for Tyr, and Ile or Leu for Val.
In some embodiments, the modified 7 β -HSDH comprises the amino acid sequence of one of SEQ ID NOs 2-9, 11, 12, 14, 15 and 17-29; or said modified 7 β -HSDH comprises 1 to 10 amino acid substitutions, preferably conservative substitutions, in positions other than positions 4, 18, 44, 55, 64, 102, 128, 196, 203 and 238, compared to the amino acid sequence of one of positions 2 to 9, 11, 12, 14, 15 and 17 to 29, wherein the modified 7 β -HSDH of the invention has improved activity of reducing 7-keto-lithocholic acid or a derivative thereof to ursodeoxycholic acid or a derivative thereof, compared to its starting 7 β -HSDH. In some embodiments, the modified 7 β -HSDH comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, preferably conservative substitutions, at positions other than positions 4, 18, 44, 55, 64, 102, 128, 196, 203 and 238, compared to the amino acid sequence of one of SEQ ID NOs 13-65 and 69-72.
As used herein, enzymatic activity refers to the decrease in substrate or increase in product per unit time in a unit mass of an enzymatically catalyzed chemical reaction under certain conditions. For example, the activity of the modified 7 β -HSDH of the invention is expressed in terms of the amount of ursodeoxycholic acid or its derivatives produced per unit time under certain conditions and catalysis by the modified 7 β -HSDH per unit mass.
In this context, the activity of an enzyme may also refer to the relative activity of the enzyme, expressed as the ratio of the activity of the enzyme of interest to the activity of a given enzyme catalyzing the same reaction, e.g., percent relative activity.
In some embodiments, the activity of the modified 7 β -HSDH of the invention is expressed as a ratio to the activity of the 7 β -HSDH of SEQ ID NO: 1. In some embodiments, the modified 7 β -HSDH catalyzes the reduction of 7-keto-lithocholic acid or a derivative thereof to ursodeoxycholic acid or a derivative thereof with at least 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2-fold or more activity than the 7 β -HSDH of SEQ ID No. 1.
II, a polynucleotide encoding the modified 7 beta-HSDH.
As used herein, the term "polynucleotide" or "nucleic acid molecule" includes DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule may be single-stranded or double-stranded, preferably double-stranded DNA. The synthesis of the nucleic acid may use nucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such nucleotides can be used, for example, to prepare nucleic acids having altered base-pairing abilities or increased nuclease resistance.
The invention also provides polynucleotides encoding the modified 7 β -HSDH of the invention. Thus, in the present invention, the term modification also includes the genetic manipulation of a polynucleotide encoding a 7 β -HSDH polypeptide of the invention. The modification may be a substitution, deletion, insertion and/or addition of a nucleotide.
As used herein, the term "encoding" refers to a polynucleotide that directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or another start codon, such as GTG and TTG, and ends with a stop codon, such as TAA, TAG and TGA. The coding sequence may be a DNA, cDNA or recombinant nucleotide sequence.
In addition, nucleic acid molecules encompassing all or part of a nucleic acid sequence of the invention can be isolated by Polymerase Chain Reaction (PCR) using synthetic oligonucleotide primers designed based on sequence information contained in the sequence.
The polynucleotides of the invention may be amplified using cDNA, mRNA or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid thus amplified can be cloned into a suitable vector and characterized by DNA sequence analysis.
Polynucleotides of the invention can be prepared by standard synthetic techniques, for example using an automated DNA synthesizer.
The invention also relates to the complementary strand of the nucleic acid molecules described herein. A nucleic acid molecule that is complementary to another nucleotide sequence is a molecule that is sufficiently complementary to the nucleotide sequence that it can hybridize to the other nucleotide sequence, thereby forming a stable duplex.
As used herein, the term "hybridize" is a process in which nucleotide sequences that are at least about 90%, preferably at least about 95%, more preferably at least about 96%, and more preferably at least 98% homologous to each other typically remain hybridized to each other under the conditions of a given stringent hybridization and wash.
Those skilled in the art are aware of various conditions for hybridization, such as stringent hybridization conditions and highly stringent hybridization conditions. See, e.g., Sambrook et al, 1989, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Press, n.y.; and Ausubel et al, (eds.),1995, Current Protocols in Molecular Biology, John Wiley & Sons, N.Y..
Of course, polynucleotides of the invention do not include polynucleotides that hybridize only to poly A sequences (e.g., the 3' end of mRNA poly (A)) or to a complementary stretch of poly T (or U) residues.
Expression and production of modified 7 beta-HSDH
To express the modified 7 β -HSDH of the invention, nucleic acid constructs and vectors, such as expression vectors, comprising the polynucleotides of the invention are also provided.
As used herein, the term "expression" includes any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
The term "nucleic acid construct" refers to a nucleic acid molecule, either single-or double-stranded, that is isolated from a naturally occurring gene or modified to contain a nucleic acid segment that does not occur in nature. The term nucleic acid construct is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the invention.
The term "expression vector" refers herein to a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide of the present invention operably linked to additional nucleotides, e.g., control sequences, provided for expression of the polynucleotide. The expression vector includes a viral vector or a plasmid vector.
The term "control sequences" is meant herein to include all elements required or advantageous for expression of a polynucleotide encoding a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide, or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter and transcriptional and translational stop signals.
For example, the control sequence may be an appropriate promoter sequence, a nucleotide sequence which is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter sequence contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any nucleotide sequence which shows transcriptional activity in the host cell of choice, for example, the E.coli (Escherichia coli) lac operon. The promoters also include mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
The term "operably linked" refers herein to a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide sequence, whereby the control sequence directs the expression of the coding sequence of a polypeptide.
The polynucleotide encoding the polypeptide of the present invention may be subjected to various manipulations to allow the polypeptide to be expressed. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods are well known in the art.
For the identification and selection of host cells comprising the expression vectors of the invention, the vectors of the invention preferably contain one or more selectable markers that allow for easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides biocide or viral resistance, heavy metal resistance, supplements auxotrophy, and the like. For example, a bacterial selectable marker is the dal gene from Bacillus subtilis or Bacillus licheniformis, or a marker that confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance.
The vectors of the invention may be integrated into the host cell genome or may replicate autonomously in the cell, independent of the genome. Elements required for integration into the host cell genome or for autonomous replication are known in the art (see, e.g., Sambrook et al, 1989, supra).
Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" refer to the introduction of foreign nucleic acid (e.g., DNA) into a host cell by a variety of art-recognized techniques, as can be found, for example, in Sambrook et al, 1989; davis et al, Basic Methods in Molecular Biology (1986) and other laboratory manuals.
The present invention also relates to recombinant host cells comprising a polynucleotide of the present invention, which polynucleotide is advantageously used for the recombinant production of 7 β -HSDH polypeptide. A vector comprising a polynucleotide of the invention is introduced into a host cell, whereby the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector. The skilled worker is aware of conventional vectors and host cells for expressing proteins.
In some embodiments, the host cell of the invention is an E.coli cell, such as E.coli BL21(DE 3). In some embodiments, the expression vector is pET-30a (+).
Production of ursodeoxycholic acid and its derivatives
Furthermore, the present invention provides a method for producing ursodeoxycholic acid or a derivative thereof, comprising contacting the modified 7 β -HSDH or the host cell of the invention with 7-keto-lithocholic acid or a derivative thereof.
In some embodiments, the method of producing ursodeoxycholic acid or derivatives thereof of the present invention comprises the steps of:
(a) providing the activity of the modified 7 β -HSDH according to the invention and 7-keto-lithocholic acid or a derivative thereof to a reaction medium;
(b) incubating the reaction medium to reduce 7-keto-lithocholic acid or a derivative thereof to produce ursodeoxycholic acid or a derivative thereof;
(c) optionally, recovering ursodeoxycholic acid or derivatives thereof.
In some embodiments, glucose dehydrogenase, oxidized Nicotinamide Adenine Dinucleotide Phosphate (NADP) is added to the reaction medium. In some embodiments, the glucose dehydrogenase is derived from Bacillus subtilis and has the amino acid sequence shown in SEQ ID NO: 30.
In some embodiments, the reaction medium comprises tert-butanol and Triethanolamine (TEA) buffer. In one embodiment, the reaction medium comprises 14% t-butanol and 0.1M TEA buffer, ph 7.5.
In some embodiments, the incubation is performed at 25-40 ℃, preferably 28-35 ℃, e.g. 30 ℃ for 15 minutes to 1 hour, e.g. 0.5 hour.
Examples
The present invention will be more clearly understood by those skilled in the art from the following examples. It is to be understood that the examples are for illustration only and do not limit the scope of the invention.
Example 1: materials and methods
Unless otherwise specified, the experimental procedures used in the present invention are conventional, and the procedures for gene cloning are specifically described in Sambrook et al, 1989.
i) Reagents and instrumentation:
DNA Polymerase (PrimeSTAR Max DNA Polymerase) and DpnI endonuclease were purchased from TaKaRa;
the plasmid extraction kit is purchased from Axygen corporation;
7-ketolithocholic acid purchased from Mecanne under the product number A832268 with the purity of 98%;
oxidized Nicotinamide Adenine Dinucleotide Phosphate (NADP) was purchased from Aladdin, cat # N113163, 97% pure.
ii) vectors and strains: the expression vector used was pET-30a (+), the plasmid was purchased from Novagen, and the host cell used was E.coli BL21(DE3) purchased from Tiangen Biochemical technology, Inc. (Beijing).
iii) sequencing, primer synthesis and Gene Synthesis were performed by Suzhou Hongxin Biotechnology Ltd. Wherein the gene is constructed into a vector pET-30a after being synthesized.
iv) site-directed mutagenesis:
specific primer pairs are designed to introduce the desired substitution at the corresponding base at the amino acid position of the desired mutation. Mutants were prepared using the extracted pre-mutation plasmid (containing the wild-type 7. beta. -HSDH coding sequence, pET-30a (+) backbone) as a template, using the Methods described by Packer and Liu (Methods for the directed evolution of proteins. Nat. Rev Genet,2015,16(7): 379-394).
v) protein expression and preparation of enzyme solution:
escherichia coli cells transformed with a plasmid containing the gene of interest were inoculated into LB liquid medium (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, pH7.0) containing 50mg/L kanamycin, and incubated overnight with shaking at 37 ℃. The culture was transferred to TB liquid medium (peptone 12g/L, yeast extract 24g/L, glycerol 4mL/L, potassium dihydrogenphosphate 2.31g/L, dipotassium hydrogenphosphate 12.54g/L), incubated at 37 ℃ with shaking until OD600 reached 0.6-0.8, and incubated overnight at 30 ℃ with the addition of IPTG (final concentration of 0.4mM) to induce protein expression.
After incubation, the culture was centrifuged at 4,000g at 4 ℃ for 10min, and the supernatant was discarded to collect E.coli cells. The collected E.coli cells were resuspended in pre-cooled 20mL Phosphate Buffered Saline (PBS) pH7.0 and the E.coli cells were sonicated at 4 ℃. The cell disruption solution was centrifuged at 6,000g at 4 ℃ for 15min to remove the precipitate, and the resulting supernatant was used as an enzyme solution containing the recombinant enzyme for catalytic reaction. Or freeze drying the enzyme solution to obtain enzyme powder, and storing at 4 deg.C.
vi) enzyme Activity assay
The following system was prepared for determining the enzyme activity (1ml) of the 7 β -HSDH mutant:
7-keto-LCA20g/L, glucose 20g/L, oxidized Nicotinamide Adenine Dinucleotide Phosphate (NADP)0.04g/L, glucose dehydrogenase 0.4g/L and 7 beta-HSDH 0.4g/L, 14% tert-butanol and triethanolamine buffer (TEA)0.1M, pH 7.5. After a reaction at 30 ℃ for 0.5 hour, the produced ursodeoxycholic acid was measured by HPLC.
vii) HPLC analysis
The HPLC parameters were as follows:
Figure BDA0002644136040000121
example 2 preparation and testing of mutants of 7 β -HSDH of Clostridium sp
Mutants were prepared according to the method of example 1 using as template the nucleic acid encoding 7 β -HSDH from Clostridium sp. The resulting mutants are shown in table 1. The activities of SEQ ID NO. 1 and its mutants were determined according to the method in item vi) of example 1, and the results are shown in Table 1, where relative activities refer to activity of mutant/activity of SEQ ID NO. 1.
TABLE 1
Figure BDA0002644136040000122
Figure BDA0002644136040000131
Sequence listing
<110> Suzhou pilotage Biotechnology Ltd
<120> modified 7 beta-hydroxysteroid dehydrogenase and application thereof
<130> I2020TC4652CB
<160> 30
<170> PatentIn version 3.5
<210> 1
<211> 263
<212> PRT
<213> Clostridium sp. Marseille
<400> 1
Met Lys Leu Lys Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Lys Tyr Gly Val Lys His Leu Val Ile Lys
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Ala Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Lys Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 2
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 2
Met Lys Leu Lys Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Thr Tyr Gly Val Lys His Leu Val Ile Lys
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Ala Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Lys Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 3
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 3
Met Lys Leu Lys Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Lys Tyr Gly Val Lys His Leu Val Ile Arg
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Ala Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Lys Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 4
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 4
Met Lys Leu Lys Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Lys Tyr Gly Val Lys His Leu Val Ile Lys
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Glu Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Ala Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Lys Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 5
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 5
Met Lys Leu Lys Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Lys Tyr Gly Val Lys His Leu Val Ile Lys
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met His
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Ala Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Lys Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 6
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 6
Met Lys Leu Lys Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Lys Tyr Gly Val Lys His Leu Val Ile Lys
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Phe Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Lys Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 7
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 7
Met Lys Leu Lys Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Lys Tyr Gly Val Lys His Leu Val Ile Lys
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Tyr Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Lys Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 8
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 8
Met Lys Leu Lys Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Lys Tyr Gly Val Lys His Leu Val Ile Lys
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Leu Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Lys Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 9
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 9
Met Lys Leu Lys Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Thr Tyr Gly Val Lys His Leu Val Ile Lys
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Phe Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Lys Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 10
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 10
Met Lys Leu Lys Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Thr Tyr Gly Val Lys His Leu Val Ile Lys
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Leu Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Lys Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 11
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 11
Met Lys Leu Lys Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Thr Tyr Gly Val Lys His Leu Val Ile Lys
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Tyr Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Lys Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 12
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 12
Met Lys Leu Lys Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Lys Tyr Gly Val Lys His Leu Val Ile Arg
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Phe Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Lys Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 13
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 13
Met Lys Leu Lys Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Lys Tyr Gly Val Lys His Leu Val Ile Arg
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Leu Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Lys Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 14
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 14
Met Lys Leu Lys Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Lys Tyr Gly Val Lys His Leu Val Ile Arg
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Tyr Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Lys Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 15
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 15
Met Lys Leu Lys Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Thr Tyr Gly Val Lys His Leu Val Ile Arg
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Phe Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Lys Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 16
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 16
Met Lys Leu Lys Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Thr Tyr Gly Val Lys His Leu Val Ile Arg
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Leu Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Lys Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 17
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 17
Met Lys Leu Lys Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Thr Tyr Gly Val Lys His Leu Val Ile Arg
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Tyr Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Lys Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 18
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 18
Met Lys Leu Lys Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Thr Tyr Gly Val Lys His Leu Val Ile Arg
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Phe Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Arg Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 19
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 19
Met Lys Leu Ser Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Thr Tyr Gly Val Lys His Leu Val Ile Arg
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Phe Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Lys Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 20
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 20
Met Lys Leu Lys Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Asp Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Thr Tyr Gly Val Lys His Leu Val Ile Arg
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Phe Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Lys Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 21
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 21
Met Lys Leu Lys Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Arg Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Thr Tyr Gly Val Lys His Leu Val Ile Arg
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Phe Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Lys Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 22
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 22
Met Lys Leu Lys Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Thr Tyr Gly Val Lys His Leu Val Ile Arg
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Asn Asn Leu Pro Gly Gly Pro Phe Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Lys Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 23
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 23
Met Lys Leu Ser Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Thr Tyr Gly Val Lys His Leu Val Ile Arg
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Phe Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Arg Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 24
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 24
Met Lys Leu Lys Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Asp Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Thr Tyr Gly Val Lys His Leu Val Ile Arg
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Phe Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Arg Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 25
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 25
Met Lys Leu Lys Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Arg Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Thr Tyr Gly Val Lys His Leu Val Ile Arg
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Phe Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Arg Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 26
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 26
Met Lys Leu Lys Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Thr Tyr Gly Val Lys His Leu Val Ile Arg
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Asn Asn Leu Pro Gly Gly Pro Phe Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Arg Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 27
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 27
Met Lys Leu Ser Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Asp Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Thr Tyr Gly Val Lys His Leu Val Ile Arg
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Phe Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Arg Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 28
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 28
Met Lys Leu Ser Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Arg Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Thr Tyr Gly Val Lys His Leu Val Ile Arg
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Lys Asn Leu Pro Gly Gly Pro Phe Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Arg Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 29
<211> 263
<212> PRT
<213> Artificial Sequence
<220>
<223> mutant
<400> 29
Met Lys Leu Ser Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Asp Gly Val Gly Lys Ala Phe Cys Glu Lys Ile Ala Ser Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Met Leu Lys Ala Leu
35 40 45
Gly Glu Asp Ile Ser Ser Thr Tyr Gly Val Lys His Leu Val Ile Arg
50 55 60
Ala Asp Phe Ser Asp Pro Asn Ser Thr Asp Glu Ile Phe Glu Lys Thr
65 70 75 80
Lys Asp Leu Asp Met Gly Phe Met Ser Tyr Val Ala Cys Phe His Thr
85 90 95
Phe Gly Lys Leu Gln Asp Thr Pro Trp Glu Lys His Glu Gln Met Leu
100 105 110
Asn Val Asn Val Ile Thr Phe Leu Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ser Lys Gln Asp Arg Gly Ala Ile Ile Asn Val Ser Ser Leu
130 135 140
Thr Gly Ile Ser Ala Ser Pro Tyr Asn Ala Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ser Tyr Ile Leu Lys Leu Thr Glu Ala Val Ala Tyr Glu Ala Ser Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Ile Thr Pro
180 185 190
Ser Leu Leu Asn Asn Leu Pro Gly Gly Pro Phe Gly Glu Ala Val Met
195 200 205
Lys Ala Ala Leu Thr Pro Glu Ala Cys Val Glu Glu Ala Phe Glu Asn
210 215 220
Leu Gly Lys Lys Phe Ser Ile Ile Ala Gly Glu His Asn Arg Ala Ser
225 230 235 240
Ile His Asp Trp Lys Ala Asn His Thr Glu Asp Glu Phe Ile Ser Tyr
245 250 255
Met Gly Ser Phe Tyr Glu Arg
260
<210> 30
<211> 261
<212> PRT
<213> Bacillus subtilis
<400> 30
Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala
1 5 10 15
Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala
20 25 30
Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val
35 40 45
Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly
50 55 60
Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile
65 70 75 80
Lys Glu Phe Gly Thr Leu Asp Val Met Ile Asn Asn Ala Gly Leu Glu
85 90 95
Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val
100 105 110
Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile
115 120 125
Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser
130 135 140
Ser Val His Glu Val Ile Pro Trp Pro Leu Phe Val His Tyr Ala Ala
145 150 155 160
Ser Lys Gly Gly Ile Lys Leu Met Thr Glu Thr Leu Ala Leu Glu Tyr
165 170 175
Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn
180 185 190
Thr Pro Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp
195 200 205
Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile
210 215 220
Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr
225 230 235 240
Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Gln Tyr Pro Ser Phe
245 250 255
Gln Ala Gly Arg Gly
260

Claims (11)

1. A modified 7 beta-hydroxysteroid dehydrogenase (7 beta-HSDH),
comprising an amino acid substitution at position 203 compared to its starting 7 β -HSDH, said amino acid substitution at position 203 being F, Y or L, and optionally said modified 7 β -HSDH further comprises a substitution at one or more positions selected from the group consisting of positions 4, 18, 44, 55, 64, 102, 128, 196 and 238, wherein the amino acid positions are numbered with reference to SEQ ID NO:1,
wherein the modified 7 β -HSDH has improved activity in catalysing the reduction of 7-keto-lithocholic acid or a derivative thereof to ursodeoxycholic acid or a derivative thereof, compared to its starting 7 β -HSDH.
2. The modified 7 β -HSDH of claim 1, comprising one or more amino acid substitutions selected from: substitution at position 4 for S, substitution at position 18 for D, substitution at position 44 for R, substitution at position 55 for T, substitution at position 64 for R, substitution at position 102 for E, substitution at position 128 for H, substitution at position 196 for N and substitution at position 238 for R.
3. The modified 7 β -HSDH of claim 1 or 2, wherein said modified 7 β -HSDH comprises amino acid substitutions at positions 55 and 203, wherein the substitution at position 55 is T and the substitution at position 203 is F or Y.
4. The modified 7 β -HSDH of claim 1 or 2, wherein said modified 7 β -HSDH comprises amino acid substitutions at positions 64 and 203, wherein the substitution at position 64 is R and the substitution at position 203 is F or Y.
5. The modified 7 β -HSDH according to any one of claims 1 to 4, wherein said modified 7 β -HSDH comprises amino acid substitutions at positions 55, 64 and 203, wherein the substitution at position 55 is T, the substitution at position 64 is R, and the substitution at position 203 is F or Y.
6. The modified 7 β -HSDH according to any one of claims 1 to 5, wherein said starting 7 β -HSDH comprises the amino acid sequence of SEQ ID NO 1.
7. A modified 7 β -HSDH comprising the amino acid sequence of one of SEQ ID NOs 2-9, 11, 12, 14, 15 and 17-29.
8. A polynucleotide encoding the modified 7 β -HSDH according to any one of claims 1-7.
9. An expression vector comprising the polynucleotide of claim 8.
10. A host cell comprising the modified 7 β -HSDH according to any one of claims 1-7, the polynucleotide according to claim 8 or the vector according to claim 9.
11. A method of producing ursodeoxycholic acid or a derivative thereof, comprising contacting the modified 7 β -HSDH according to any one of claims 1-7 or the host cell of claim 10 with 7-keto-lithocholic acid or a derivative thereof.
CN202010849118.7A 2020-08-21 2020-08-21 Modified 7 beta-hydroxysteroid dehydrogenase and application thereof Active CN111944775B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010849118.7A CN111944775B (en) 2020-08-21 2020-08-21 Modified 7 beta-hydroxysteroid dehydrogenase and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010849118.7A CN111944775B (en) 2020-08-21 2020-08-21 Modified 7 beta-hydroxysteroid dehydrogenase and application thereof

Publications (2)

Publication Number Publication Date
CN111944775A true CN111944775A (en) 2020-11-17
CN111944775B CN111944775B (en) 2023-09-05

Family

ID=73359453

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010849118.7A Active CN111944775B (en) 2020-08-21 2020-08-21 Modified 7 beta-hydroxysteroid dehydrogenase and application thereof

Country Status (1)

Country Link
CN (1) CN111944775B (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114540338A (en) * 2020-11-25 2022-05-27 湖南引航生物科技有限公司 Immobilized modified 7 beta-hydroxysteroid dehydrogenase and application thereof
CN114657167A (en) * 2020-12-23 2022-06-24 苏州引航生物科技有限公司 Preparation method of decarboxylase and 5-hydroxytryptamine
WO2023075295A1 (en) * 2021-10-25 2023-05-04 에이스바이오팜 주식회사 Variant of ruminococcus gnavus strain-derived 7-beta-hsdh, improving conversion rate from 7-keto-lca to ursodeoxycholic acid, and method for producing ursodeoxycholic acid by using same

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107058250A (en) * 2017-01-05 2017-08-18 重庆大学 7 new beta hydroxysteroid dehydrogenase gene Y1 b 1
CN107287272A (en) * 2016-03-30 2017-10-24 上海中医药大学 A kind of preparation method of Tauro ursodesoxy cholic acid
CN109182284A (en) * 2018-09-28 2019-01-11 湖南福来格生物技术有限公司 A kind of 7beta-Hydroxysteroid dehydrogenase mutant, coded sequence, recombinant expression carrier, genetic engineering bacterium and application
CN110776572A (en) * 2019-11-14 2020-02-11 无锡佰翱得生物科学有限公司 Fusion protein of 7 β -HSDH enzyme and DPS

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107287272A (en) * 2016-03-30 2017-10-24 上海中医药大学 A kind of preparation method of Tauro ursodesoxy cholic acid
CN107058250A (en) * 2017-01-05 2017-08-18 重庆大学 7 new beta hydroxysteroid dehydrogenase gene Y1 b 1
CN109182284A (en) * 2018-09-28 2019-01-11 湖南福来格生物技术有限公司 A kind of 7beta-Hydroxysteroid dehydrogenase mutant, coded sequence, recombinant expression carrier, genetic engineering bacterium and application
CN110776572A (en) * 2019-11-14 2020-02-11 无锡佰翱得生物科学有限公司 Fusion protein of 7 β -HSDH enzyme and DPS

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114540338A (en) * 2020-11-25 2022-05-27 湖南引航生物科技有限公司 Immobilized modified 7 beta-hydroxysteroid dehydrogenase and application thereof
CN114540338B (en) * 2020-11-25 2023-09-05 湖南引航生物科技有限公司 Immobilized modified 7 beta-hydroxysteroid dehydrogenase and application thereof
CN114657167A (en) * 2020-12-23 2022-06-24 苏州引航生物科技有限公司 Preparation method of decarboxylase and 5-hydroxytryptamine
CN114657167B (en) * 2020-12-23 2023-09-05 苏州引航生物科技有限公司 Decarboxylase and preparation method of 5-hydroxytryptamine
WO2023075295A1 (en) * 2021-10-25 2023-05-04 에이스바이오팜 주식회사 Variant of ruminococcus gnavus strain-derived 7-beta-hsdh, improving conversion rate from 7-keto-lca to ursodeoxycholic acid, and method for producing ursodeoxycholic acid by using same

Also Published As

Publication number Publication date
CN111944775B (en) 2023-09-05

Similar Documents

Publication Publication Date Title
CN111944775B (en) Modified 7 beta-hydroxysteroid dehydrogenase and application thereof
Ferrandi et al. In search of sustainable chemical processes: cloning, recombinant expression, and functional characterization of the 7α-and 7β-hydroxysteroid dehydrogenases from Clostridium absonum
JP2007502114A (en) Improved glucose dehydrogenase polypeptides and related polynucleotides
DK2689015T3 (en) A process for the production of secreted polypeptides
CN110551701B (en) Carbonyl reductase mutant and application thereof in reduction of cyclopentadione compounds
CN111254126A (en) 7 α -hydroxysteroid dehydrogenase (St-2-2) mutant
US11667896B2 (en) Modified DAAO enzyme and application thereof
CN111500600B (en) 3-sterone-1, 2-dehydrogenase and gene sequence and application thereof
CN110791492B (en) Heat-resistant carbonic anhydrase mutants and compositions for capturing carbon dioxide containing the same
WO2021219124A1 (en) Modified threonine transaldolase and application thereof
WO2013052717A2 (en) Microorganisms and methods for producing acrylate and other products from homoserine
JP2011103792A (en) Glucose dehydrogenase, and method for producing the same
CN114752572B (en) Formate dehydrogenase mutant and application thereof
JP7492817B2 (en) Monoacylglycerol lipase variant and method for producing same
WO2024075674A1 (en) Microbial strain, protein, and method for producing gallic acid using microbial strain or protein
WO2023125769A1 (en) Modified glutamate dehydrogenase and application thereof
WO2022210228A1 (en) MODIFIED α-ISOPROPYLMALATE SYNTHASE
US20230332113A1 (en) Modified glutamate dehydrogenase and the use thereof
WO2006043555A1 (en) Reductase mutant for forming biopolymer
WO2023226978A1 (en) Microbial-derived sucrose synthase and use thereof
JP2024510328A (en) A variant of 7β-HSDH derived from a Ruminococcus gnabas strain that improves the conversion rate of 7-Keto-LCA to ursodeoxycholic acid, and a method for producing ursodeoxycholic acid using the same
JP2021052725A (en) Monoacylglycerol lipase variants and methods of making

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant