WO2019107288A1 - 改変型トランスグルタミナーゼ - Google Patents
改変型トランスグルタミナーゼ Download PDFInfo
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- WO2019107288A1 WO2019107288A1 PCT/JP2018/043292 JP2018043292W WO2019107288A1 WO 2019107288 A1 WO2019107288 A1 WO 2019107288A1 JP 2018043292 W JP2018043292 W JP 2018043292W WO 2019107288 A1 WO2019107288 A1 WO 2019107288A1
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- amino acid
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Images
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
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/104—Aminoacyltransferases (2.3.2)
- C12N9/1044—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/02—Aminoacyltransferases (2.3.2)
- C12Y203/02013—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
Definitions
- the present invention relates to transglutaminase. Specifically, the present invention relates to a modified transglutaminase whose property has been changed or improved, its use, and the like.
- This application claims priority based on Japanese Patent Application No. 2017-231192 filed on Nov. 30, 2017, the entire contents of which are incorporated by reference.
- Transglutaminase is an enzyme that catalyzes an acyl transfer reaction of the ⁇ -carboxamide group of glutamine residue in a peptide chain, and when the ⁇ -amino group of lysine residue in protein acts as an acyl acceptor, a protein molecule Form an ⁇ - ( ⁇ -Gln) -Lys crosslink within or between molecules of Therefore, since the protein or peptide can be modified by using the action of transglutaminase, transglutaminase derived from Streptomyces (see, for example, Patent Document 1) binds meat, sausage, tofu, bread, It is used in the manufacture of noodles.
- transglutaminase in the fiber field, the medical field, the cosmetic field and the like as well as in the food field is being considered.
- attempts have been made to improve the characteristics (heat resistance, specific activity, substrate specificity, stability, etc.) of transglutaminase (eg, Patent Literatures 2, 3 and Non Patent Literatures 1 to 4). See).
- An object of the present invention is to find new mutations effective for the improvement of transglutaminase, and to provide highly useful modified transglutaminase (mutant) and its use.
- the mutation point is V6, the amino acid after substitution is Q, I, M, S, C, K, L, H, F, G, N, P, R, W or Y, the characteristic change due to amino acid substitution is temperature Reduced stability;
- the mutation point is R26, the amino acid after substitution is K, Q, M, H, Y, D, G, N, P or S, and the property change due to the amino acid substitution is temperature stability lowered;
- the mutation point is E28, the amino acid after substitution is V, Q, W, R, K, M, N, F, G, L,
- a recombinant DNA comprising the gene of [6] or [7].
- An enzyme preparation comprising the modified transglutaminase according to any one of [1] to [5].
- a process for preparing a modified transglutaminase which comprises the following steps (I) to (III): (I) preparing a nucleic acid encoding the amino acid sequence of the modified transglutaminase according to any one of [1] to [5]; (II) expressing the nucleic acid, and (III) recovering the expression product.
- Effective mutation point for temperature stability reduction Each mutation point was substituted by various amino acids, and the effective residual rate of the enzyme (mutant) after substitution was compared with a wild-type enzyme to identify an effective amino acid substitution. From the activity measurement value, TG (transglutaminase) activity per mL of culture solution of the mutant strain was calculated and used for evaluation.
- TG transglutaminase activity per mL of culture solution of the mutant strain was calculated and used for evaluation.
- the continuation of FIG. The continuation of FIG. The continuation of FIG. The continuation of FIG. The continuation of FIG. The continuation of FIG.
- Effective mutation point to improve heat resistance Each mutation point was substituted by various amino acids, and the effective residual rate of the enzyme (mutant) after substitution was compared with a wild-type enzyme to identify an effective amino acid substitution.
- TG transglutaminase activity per mL of culture solution of the mutant strain was calculated and used for evaluation.
- FIG. A mutation point effective for the improvement of antioxidant properties Each mutation point was substituted by various amino acids, and the effective residual rate of the enzyme (mutant) after substitution was compared with a wild-type enzyme to identify an effective amino acid substitution. From the activity measurement value, TG (transglutaminase) activity per mL of culture solution of the mutant strain was calculated and used for evaluation.
- the continuation of FIG. The continuation of FIG.
- Effective mutation point for improving reactivity Each mutation point was substituted by various amino acids, and the effective amino acid substitution was specified by comparing the activity of the enzyme (mutant) after substitution with a wild-type enzyme.
- TG transglutaminase activity per mL of culture solution of the mutant strain was calculated and used for evaluation.
- V6E / Y75F and D3P / T77Q are double mutants in which two amino acid substitutions were made. Effective mutation points for changes in two or more characteristics. Mutations (amino acid substitution) effective for complex improvement are shown at every mutation point. The continuation of FIG. The continuation of FIG. Effective mutation point for deamidation enzyme.
- Each mutation point is substituted with various amino acids, and after deamidation activity and transglutaminase activity of the enzyme (mutant) after substitution are calculated as relative values to wild type enzyme, deamidation activity (ratio to wild type) The value of / transglutaminase activity (ratio to wild type) was determined and used for evaluation.
- modified transglutaminase is an enzyme obtained by modifying or mutating reference transglutaminase (hereinafter referred to as “reference transglutaminase”).
- reference transglutaminase is a transglutaminase derived from Streptomyces mobaraensis, typically having the amino acid sequence of SEQ ID NO: 1.
- transglutaminase from Streptomyces mobaraensis refers to transglutaminase whose source is Streptomyces mobaraensis, and to the transglutaminase produced by Streptomyces mobaraensis or the genetic information of said transglutaminase It contains transglutaminase etc. which are expressed by other microorganisms etc. and used.
- modified transglutaminase is also referred to as modified enzyme or variant.
- each amino acid is indicated by one letter as follows.
- the position of the mutation point is specified by the number obtained by assigning the amino acid residue at the N-terminus of mature transglutaminase as the first position from the N-terminus toward the C-terminus.
- an amino acid residue to which an amino acid substitution is to be performed that is, a "mutation point” is expressed as a combination of a letter representing the type of amino acid and a number representing the position of the amino acid.
- mutations due to amino acid substitution are represented by the addition of one letter representing the type of amino acid after substitution to the right of the indication of mutation point. Therefore, for example, if valine at position 6 is a mutation point, it is expressed as "V6", and if it is a mutation in which valine at position 6 is substituted with glutamine, it is expressed as "V6Q".
- modified transglutaminase The first aspect of the present invention relates to modified transglutaminase (hereinafter referred to as "modified enzyme").
- modified enzyme of the present invention typically has an amino acid sequence comprising one or more specific amino acid substitutions (mutations) in the amino acid sequence of SEQ ID NO: 1. Due to this feature, the temperature stability reduction, the heat resistance improvement, the antioxidative property improvement, the reactivity improvement, the ratio of the deamidation activity to the transamidinase activity (transglutaminase activity relative to transglutaminase consisting of the amino acid sequence of SEQ ID NO: ) Or two or more of these changes in characteristics.
- the modified enzyme with reduced temperature stability has a practical advantage of preventing the food from being denatured in the heat inactivation step of the enzyme, and has high utility value in applications such as the production of yogurt and cheese.
- modified enzymes with improved heat resistance exhibit high activity even at high temperatures, and are particularly suitable for use in applications such as the synthesis of peptide compounds.
- a modified enzyme with improved antioxidative properties has advantages such as high storage stability, and can be expected to be effective in preventing inactivation during production, storage, and use of the enzyme.
- the modified enzyme having improved reactivity has advantages such as high reaction efficiency and reduced use of the enzyme, and the utility value is high regardless of the use.
- Deamidation enzyme modifies the substrate specificity, and in particular, leads to expansion of use (for example, application to solubilization of protein, emulsification, improvement of foamability, etc.) or creation.
- Transglutaminase is an enzyme that catalyzes an acyl transfer reaction between the ⁇ -carboxamide group of a glutamine residue in the peptide chain and a primary amine, but in the absence of a primary amine, water acts as an acyl acceptor Will catalyze a deamidation reaction that converts glutamine residues to glutamic acid residues.
- the deamidated enzyme-modified enzyme exhibits high deamidation catalytic ability and is more preferable as an enzyme used for deamidation.
- the amino acid sequence of SEQ ID NO: 1 is a sequence of transglutaminase derived from Streptomyces mobaraensis.
- containing an amino acid substitution means that a mutation point (ie, the position of an amino acid residue at which a specific amino acid substitution occurs) is an amino acid after substitution. Therefore, when the amino acid sequence containing the amino acid substitution (mutated amino acid sequence) is compared with the amino acid sequence of SEQ ID NO: 1 containing no amino acid substitution (reference amino acid sequence), differences in amino acid residues are found at the position of the amino acid substitution. It will be.
- Temperature stability can be evaluated based on, for example, residual activity when treated at 50 ° C. for 30 minutes.
- the modified enzyme with reduced temperature stability has a lower residual rate of activity than the reference transglutaminase.
- the activity residual rate of the modified enzyme is, for example, 90% or less, preferably 30% or less, more preferably 10% or less, of the activity residual rate of the reference transglutaminase.
- the heat resistance can be evaluated based on the residual activity when treated at, for example, 50 ° C. for 30 minutes, as with the temperature stability.
- the modified enzyme with improved heat resistance has a higher residual activity rate than the reference transglutaminase.
- the activity residual rate of the modified enzyme is, for example, 110% or more, preferably 120% or more, and more preferably 130% or more of the activity residual rate of the reference transglutaminase.
- the modified enzyme with improved antioxidative activity has a higher residual rate of activity than the reference transglutaminase.
- the activity residual rate of the modified enzyme is, for example, 110% or more, preferably 120% or more, and more preferably 130% or more of the activity residual rate of the reference transglutaminase.
- the modified enzyme with the above-mentioned properties is more useful than the standard transglutaminase in terms of the changed properties, but it is possible to reduce the amount used (amount of enzyme) Therefore, its reactivity should be high.
- the reactivity of the modified enzyme is preferably 50% or more of that of the reference transglutaminase.
- the reactivity can be evaluated, for example, based on the activity calculated by the measurement method described in the below-mentioned Examples. Modified enzymes with improved reactivity show higher activity than standard transglutaminase).
- the reactivity (activity) of the modified enzyme is 110% or more, preferably 120% or more, more preferably 130% or more of the reactivity (activity) of the reference transglutaminase.
- Deamidation can be assessed based on the ratio of deamidation activity (ratio to reference transglutaminase) to transglutaminase activity (ratio to reference transglutaminase) (deamidation activity / transglutaminase activity). Detailed evaluation method will be described later.
- the value of deamidation activity (relative to standard transglutaminase ratio) / transglutaminase activity (relative to standard transglutaminase ratio) exceeds 1, preferably 1.2 or more, more preferably 2 or more. More preferably, it will be 10 or more.
- amino acid substitutions (mutant points and amino acids after substitution) that bring about each characteristic change described above are listed.
- Amino Acid Substitution Effective to Decrease Temperature Stability> (1) The mutation point is V6, the amino acid after substitution is Q, I, M, S, C, K, L, H, F, G, N, P, R, W or Y (2) The mutation point is R26, and the amino acid after substitution is K, Q, M, H, Y, D, G, N, P or S (3) The mutation point is E28, and the amino acid after substitution is V, Q, W, R, K, M, N, F, G, L, P or Y (4) The mutation point is V30, and the amino acid after substitution is P, C, A, E, F, G, H, K, N, Q, R, W, Y or L (5) The mutation point is Y34, and the amino acid after substitution is A (6) The mutation point is Y42 and the amino acid after substitution is F, A, C, D, E, G, I,
- the mutation point is L60, and the amino acid after substitution is I, M, V, A, C, E, F, Q, S, T, W or Y. (10) The mutation point is Y62, and the amino acid after substitution is C, R, G, K or S (11) The mutation point is V65, and the amino acid after substitution is N, L, M, F, W or Y (12) The mutation point is V67, and the amino acid after substitution is L, N, A, C, M, Q or S (13) The mutation point is T68, and the amino acid after substitution is C, L, A, S, M, F, N, Q or Y.
- the mutation point is W69, and the amino acid after substitution is H, M, I, C, E, F, G, K, L, N, Q, R, S, T or V (15)
- the mutation point is Q74, and the amino acid after substitution is W, D, G or K (16)
- the mutation point is Y75, and the amino acid after substitution is R, Q, T or G (17)
- the mutation point is T77, and the amino acid after substitution is M, H, E, C or G (18)
- the mutation point is F85, and the amino acid after substitution is M (19)
- the mutation point is F90, and the amino acid after substitution is C, M, H, L or V (20)
- the mutation point is F108, and the amino acid after substitution is R, L, T, A, I, K, N or V (21)
- the mutation point is F117, and the amino acid after substitution is M or L (22)
- the mutation point is S199, and the amino acid after substitution is G, M, N, K or V (23)
- amino acid substitutions have a large degree of temperature stability decrease, and are more preferable amino acid substitutions.
- the following amino acid substitution is a highly preferable amino acid substitution, in which the degree of temperature stability reduction is particularly large and the reactivity is also high.
- the mutation point is D3 and the amino acid after substitution is Q, P, E, S or Y (35)
- the mutation point is R26, and the amino acid after substitution is V (36)
- the mutation point is Y34, and the amino acid after substitution is W (37)
- the mutation point is V67, and the amino acid after substitution is H (38)
- the mutation point is T68, and the amino acid after substitution is V or I (39)
- the mutation point is Q74, and the amino acid after substitution is F.
- the mutation point is T77, and the amino acid after substitution is Q.
- the mutation point is S199, and the amino acid after substitution is C or Q (42)
- the mutation point is T273, and the amino acid after substitution is Q.
- the mutation point is S284, and the amino acid after substitution is L, H, K, P or R (44)
- the mutation point is S299, and the amino acid after substitution is N.
- the mutation point is S303, and the amino acid after substitution is K
- amino acid substitutions have a large degree of improvement in heat resistance and are more preferable amino acid substitutions.
- the following amino acid substitution is a particularly preferred amino acid substitution with a particularly high degree of improvement in heat resistance.
- the following amino acid substitution is a highly preferable amino acid substitution, in which the degree of heat resistance improvement is particularly large and the reactivity is also high.
- the mutation point is D3, and the amino acid after substitution is E, Q, S or Y (47)
- the mutation point is V6, and the amino acid after substitution is P (48)
- the mutation point is R26, and the amino acid after substitution is M, K, W, C, Q, G, Y, E, T, N, D, I, S, P or V (49)
- Mutation point is E28, amino acid after substitution is L, M, K, C, V, R, W, G, N, F, Y or H (50)
- the mutation point is Y42, and the amino acid after substitution is L, N or F (51)
- the mutation point is E58, and the amino acid after substitution is Q, A, I, V, L, T, M, K, Y, W, F or R (52)
- the mutation point is V67, and the amino acid after substitution is T, S or A (53)
- the mutation point is T68, and the amino acid after substitution is L, I, V or M (54)
- the mutation point is Q74,
- the mutation point is S199, and the amino acid after substitution is C (57) The mutation point is T273, and the amino acid after substitution is E. (58) The mutation point is S284, and the amino acid after substitution is R or H (59) The mutation point is Y291, and the amino acid after substitution is I, S, F, L, C, N, V or M
- amino acid substitution is a particularly preferred amino acid substitution with a particularly high degree of antioxidative property improvement.
- V6P R26C, R26Q, R26G, R26Y, R26E, R26T, R26N, R26D, R26I, R26S, R26P, R26V E28V, E28R, E28W, E28G, E28N, E28F, E28Y, E28H Y42F E58I, E58V, E58L, E58T, E58M, E58K, E58Y, E58W, E58F, E58R T68I, T68V, T68M S284H
- the following amino acid substitution is a highly preferable amino acid substitution, in which the degree of improvement in antioxidant activity is particularly large and the reactivity is also high.
- R26C, R26Q, R26Y, R26E, R26T, R26N, R26I, R26S, R26V E28V, E28R, E28W, E28G, E28N, E28F, E28Y Y42F E58I, E58V, E58L, E58T, E58M, E58K, E58Y, E58W, E58F, E58R S284H
- the mutation point is S284, and the amino acid after substitution is M, K or V (61)
- the mutation point is F251, and the amino acid after substitution is Y.
- the mutation point is V6 and Y75, the amino acid after substitution at mutation point V6 is E, and the amino acid after substitution at mutation point Y75 is F
- the mutation point is D3 and T77, the amino acid after substitution at mutation point D3 is P, and the amino acid after substitution at mutation point T77 is Q
- amino acid substitutions have a large degree of improvement in reactivity, and are more preferable amino acid substitutions.
- the following amino acid substitution is a particularly preferred amino acid substitution with a particularly high degree of improvement in reactivity.
- amino acid substitutions resulted in more than one property change. That is, amino acid substitutions effective for complex improvement were identified.
- amino acid substitutions (mutation points and amino acids after substitution) successfully identified are listed together with the corresponding property changes.
- mutation point is D3, the amino acid after substitution is Q, S or Y, the property change due to the amino acid substitution is heat resistance improvement and the antioxidant improvement (65) the mutation point is D3, the amino acid after substitution is P, amino acid substitution Heat resistance improvement and reactivity improvement (66) mutation point is D3, amino acid after substitution is E, property change due to amino acid substitution is heat resistance improvement, antioxidant property improvement and reactivity improvement (67) mutation point V6, Amino acid after substitution is P, Characteristic change due to amino acid substitution is temperature stability reduction and antioxidant improvement (68) Mutation point is R26, Amino acid after substitution is K, Q, M, Y, D, G, N , P or S, property change due to amino acid substitution decreases temperature stability and antioxidant property improvement (69) The mutation point is R26, amino acid after substitution is V, property change due to amino acid substitution improves heat resistance property and antioxidant property ( 70) The mutation point is R26, the amino acid after substitution is H, the property change due to the amino acid substitution is temperature stability reduction, Oxidation improvement and re
- the mutation point is R5, and the amino acid after substitution is Q, P, C, M, I, V, Y, F, S, N, T or G (93)
- the mutation point is Y34, and the amino acid after substitution is H or M (94)
- the mutation point is W59, and the amino acid after substitution is K or E (95)
- the mutation point is S61, the amino acid after substitution is T, V, Y, D, I, L, N, E, M, F, W, Q, P, H or K (96)
- the mutation point is Y62, and the amino acid after substitution is D or P (97)
- the mutation point is G63, and the amino acid after substitution is D, Q, Y, R, K, H, P, M, N, C, F, L, W, T, V or S (98)
- the mutation point is V65, and the amino acid after substitution is C, T or K (99)
- the mutation point is G66, and the amino acid after substitution is W or K
- the mutation point is F108, and the amino acid after substitution is Q, C, E, S or N (105)
- the mutation point is F117, and the amino acid after substitution is C, I, W, V, A, G, T or N (106)
- the mutation point is Y198, and the amino acid after substitution is F, H, L, S, M or I (107)
- the mutation point is S199, and the amino acid after substitution is Q or P (108)
- the mutation point is K200, and the amino acid after substitution is Y, M, I, R, L or V (109)
- the mutation point is H201, and the amino acid after substitution is K, R, M, L or Q (110)
- the mutation point is F202, and the amino acid after substitution is Y, M, H, V or C (111)
- the mutation point is W203 and the amino acid after substitution is Y (112)
- the mutation point is F223, and the amino acid after substitution is Y, M, L, W, V, H, G or A (113)
- the mutation point is W258, and the amino acid after substitution is Y, F or N.
- the mutation point is T273, and the amino acid after substitution is N.
- the mutation point is G275, the amino acid after substitution is A, C, S, V, K, I, F, H, R, L, P, Q, Y, N, M or T (122)
- the mutation point is N276, and the amino acid after substitution is G, M, Q or H (123)
- the mutation point is H277, and the amino acid after substitution is N, S, Q, C, E, K, D, I, R, M, P, L, W, V, Y, G, F, or T (124)
- the mutation point is Y278, and the amino acid after substitution is P or D (125)
- the mutation point is H279, and the amino acid after substitution is N (126)
- the mutation point is S284, and the amino acid after substitution is C.
- the mutation point is M288, and the amino acid after substitution is V, Q or T (128)
- the mutation point is V290, and the amino acid after substitution is T, C, L, M, A, S or N (129)
- the mutation point is Y291, and the amino acid after substitution is H, T, E or G (130)
- the mutation point is W298, and the amino acid after substitution is F, Y, I, L or M (131)
- the mutation point is S299, and the amino acid after substitution is N or P (132)
- the mutation point is Y302, and the amino acid after substitution is L, H, M, V, C, T, P, S or W (133)
- the mutation point is S303, and the amino acid after substitution is I, Q, D, H or E (134)
- the mutation point is F305, and the amino acid after substitution is W, H or Y
- amino acid substitutions are more preferable amino acid substitutions because the degree of deamidation is large.
- amino acid substitutions are particularly favored amino acid substitutions with a particularly high degree of deamidation.
- transglutaminase consisting of an amino acid sequence of any of SEQ ID NOs: 2 to 10 (in order, R26N mutant, R26Y mutant, R26S mutant, E28N mutant, E28G mutant, E28F And E28Y mutant, E58R mutant and E58L mutant correspond to each other. As shown in Examples described later, it has been confirmed that these mutants have reduced temperature stability and improved antioxidative properties.
- the mutated protein when a part of the amino acid sequence of a certain protein is mutated, the mutated protein may have the same function as the protein before mutation. That is, the mutation of the amino acid sequence does not substantially affect the function of the protein, and the function of the protein may be maintained before and after the mutation.
- the identity of the amino acid sequences of the two proteins is high, the probability that the two exhibit the same properties is high.
- the amino acid sequence of the modified enzyme ie, the “amino acid sequence including the amino acid substitution of any of the above (1) to (134) in the amino acid sequence of SEQ ID NO: 1 (the amino acid sequence Of the amino acid sequences of SEQ ID NOs: 2 to 10) are not completely identical (ie, 100% identity), but have an amino acid sequence showing high identity, and are those that exhibit the desired property change.
- it can be regarded as an enzyme substantially identical to the above-mentioned modified enzyme (substantially identical transglutaminase).
- the identity is 70% or more, 80% or more, 82% or more, 85% or more, 90% or more, 93% or more, 95% or more, 98% or 99% or more. The higher the identity, the better. Therefore, in the most preferred embodiment, the identity is 99% or more.
- identity standard is the amino acid sequence of SEQ ID NO: 2 at a position other than position N
- identity standard is the amino acid sequence of SEQ ID NO 3 at a position other than position Y Y
- identity standard Is a position other than position S when the amino acid sequence of SEQ ID NO: 4
- a position other than position N when the reference of identity is the amino acid sequence of SEQ ID NO: 5
- the reference of identity is the amino acid sequence of SEQ ID NO: 6
- the same identity as the amino acid sequence of SEQ ID NO: 2 (70% or more, 80% or more, 82% or more, 85% or more, 90% or more, 93% or more, 95% or more, 98% or 99%
- the 26th amino acid is N.
- the amino acid at position 26 is Y in the amino acid sequence showing the above identity with the amino acid sequence of SEQ ID NO: 3 and the amino acid at position 26 is S in the amino acid sequence showing the above identity with the amino acid sequence of SEQ ID NO.
- the 28th amino acid is N in the amino acid sequence showing the above identity with the amino acid sequence of SEQ ID NO: 6
- Amino acid at position 28 is F in the amino acid sequence showing the above identity with the amino acid sequence of No. 7, and Y at position 28 in the amino acid sequence showing the above identity with the amino acid sequence of SEQ ID NO. 8
- the amino acid at position 58 is R in the amino acid sequence showing the above identity with the amino acid sequence of SEQ ID NO: 4
- the amino acid at position 58 is L in the amino acid sequence showing the above identity with the amino acid sequence of SEQ ID NO: 10.
- the "slight difference in amino acid sequence” herein is caused by amino acid deletion, substitution, addition, insertion, or a combination thereof. Typically, deletion, substitution, or one to a few (upper limit is, for example, three) of one to several (upper limit is, for example, three, five, seven, ten) amino acids constituting the amino acid sequence. It means that a mutation (change) occurs in the amino acid sequence by the addition, insertion or combination of 5, 7 or 10 amino acids. "Slight differences in amino acid sequence” are preferably caused by conservative amino acid substitutions. The term “conservative amino acid substitution” as used herein refers to substitution of an amino acid residue for an amino acid residue having a side chain with similar properties.
- the amino acid residue is a basic side chain (eg, lysine, arginine, histidine), an acidic side chain (eg, aspartic acid, glutamic acid), an uncharged polar side chain (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine) depending on its side chain.
- a basic side chain eg, lysine, arginine, histidine
- an acidic side chain eg, aspartic acid, glutamic acid
- an uncharged polar side chain eg, glycine, asparagine, glutamine, serine, threonine, tyrosine
- Cysteine nonpolar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), ⁇ branched side chains (eg, threonine, valine, isoleucine), aromatic side chains (eg, tyrosine, phenylalanine, As tryptophan, histidine), it is divided into several families. Conservative amino acid substitutions are preferably substitutions between amino acid residues within the same family.
- SEQ ID NO: 1 the active residue of transglutaminase (SEQ ID NO: 1) derived from Streptomyces mobaraensis is known to be a cysteine at position 64, there is no effect on the acid residue upon mutation. You should
- the identity (%) of two amino acid sequences or two base sequences can be determined, for example, by the following procedure.
- the two sequences are aligned (eg, gaps may be introduced into the first sequence to optimize the alignment with the second sequence) for optimal comparison.
- the molecule at a particular position of the first sequence amino acid residue or nucleotide
- the molecule at that position is said to be identical.
- Gapped BLAST as described in Altschul et al. (1997) Amino Acids Research 25 (17): 3389-3402 is available to obtain gap alignments for comparison.
- default parameters of corresponding programs eg, XBLAST and NBLAST can be used. Please refer to http://www.ncbi.nlm.nih.gov for details.
- gap weighting 12, 10, 8, 6, or 4
- a transglutaminase consisting of the amino acid sequence of SEQ ID NO: 1, ie, a transglutaminase derived from Streptomyces mobaraensis, is mutated (by substitution of any amino acid in any of the above (1) to (134)). It becomes a modified enzyme of the invention.
- the above substantially identical transglutaminase may be further mutated by adding a mutation to the transglutaminase consisting of the amino acid sequence of SEQ ID NO: 1 (amino acid substitution in any of the above (1) to (134));
- a mutation to the transglutaminase consisting of the amino acid sequence of SEQ ID NO: 1 amino acid substitution in any of the above (1) to (134)
- transglutaminase consisting of an amino acid sequence having high identity with the amino acid sequence of SEQ ID NO: 1 such as transglutaminase derived from the same genera with Streptomyces mobaraensis strain producing transglutaminase consisting of amino acid sequences
- it can be obtained by adding a mutation to what is obtained by the mutation.
- the mutation point in the present invention in the amino acid sequence having high identity to the amino acid sequence of SEQ ID NO: 1, the mutation point in the present invention (mutation point in any amino acid substitution of (1) to (134) above).
- the amino acid residue corresponding to the amino acid residue is to be substituted.
- transglutaminase consisting of an amino acid sequence having high identity with the amino acid sequence of SEQ ID NO: 1
- Streptomyces mobaraensis (formerly Streptoverticillium ladakanum) was classified.
- amino acid residues means that the proteins (enzymes) being compared have an equal contribution to the exertion of its function.
- amino acid sequence to be compared with the amino acid sequence of the reference transglutaminase (amino acid sequence of SEQ ID NO: 1) can be optimally compared while considering partial homology of the primary structure (amino acid sequence). Identify the amino acid at the position corresponding to the specific amino acid in the reference amino acid sequence as the “corresponding amino acid” when aligning (this may introduce gaps and optimize alignment as necessary) be able to.
- amino acids can be identified by comparison of three-dimensional structures (three-dimensional structures).
- the three-dimensional structural information of the mutation target enzyme can be obtained, for example, from Protein Data Bank (http://www.pdbj.org/index_j.html).
- Crystallize the protein is indispensable for determining the three-dimensional structure, but in addition to this, it has industrial utility as a method for purifying proteins with high purity and a method for high density and stable storage. In this case, it is preferable to crystallize a protein bound to a substrate or its analog compound as a ligand.
- the produced crystal is irradiated with X-rays to collect diffraction data. Protein crystals are often damaged by X-ray irradiation and the diffraction ability is degraded in many cases.
- phase information is required in addition to diffraction data.
- the crystal structure of the related protein is unknown, it is impossible to determine the structure by molecular replacement, and the phase problem must be solved by heavy atom isomorphism.
- the heavy atom isomorphous substitution method is a method of introducing a metal atom having a large atomic number such as mercury or platinum into a crystal, and obtaining phase information using the contribution of the metal atom to the X-ray diffraction data of the large X-ray scattering ability. .
- the determined phase can be improved by smoothing the electron density of the solvent region in the crystal. As water molecules in the solvent region have large fluctuations, the electron density is hardly observed, so by approximating the electron density in this region to 0, it is possible to approach the true electron density and thus the phase is improved. .
- the phase can be further improved significantly by averaging the electron density of these molecules.
- the model of the protein is fitted to the electron density map calculated using the phase thus improved.
- This process is performed on computer graphics using a program such as QUANTA of MSI (USA).
- structure refinement is performed using a program such as MSI's X-PLOR, and structural analysis is completed.
- crystal structure of a related protein is known for a target protein, it can be determined by the molecular replacement method using atomic coordinates of the known protein.
- Molecular replacement and structure refinement can be performed using the program CNS_SOLVE ver.
- the second aspect of the present invention provides a nucleic acid related to the modified enzyme of the present invention. That is, use as a primer for amplifying or mutating a nucleic acid encoding a modified enzyme, a nucleic acid that can be used as a probe for identifying a nucleic acid encoding a modified enzyme, or a nucleic acid encoding a modified enzyme A nucleic acid capable of
- the gene encoding the modified enzyme is typically utilized for the preparation of the modified enzyme. According to a genetic engineering preparation method using a gene encoding a modified enzyme, it is possible to obtain a more homogeneous modified enzyme. The method is also suitable for preparing a large amount of modified enzymes.
- the use of the gene encoding the modified enzyme is not limited to the preparation of the modified enzyme.
- the nucleic acid can also be used as a tool for experiments aimed at elucidating the mechanism of action of a modified enzyme, or as a tool for designing or producing further variants of the enzyme.
- the term "gene encoding a modified enzyme” refers to a nucleic acid from which the modified enzyme is obtained when it is expressed, and has a base sequence corresponding to the amino acid sequence of the modified enzyme.
- a nucleic acid includes, of course, a nucleic acid obtained by adding a sequence not encoding an amino acid sequence to such a nucleic acid. Also, degeneracy of codons is considered.
- SEQ ID NOs: 18 to 26 Examples of sequences (base sequences) of genes encoding modified enzymes are shown in SEQ ID NOs: 18 to 26. These sequences encode the variants shown in the examples below, as described below.
- the gene of the present invention When the gene of the present invention is expressed in a host, usually, 5 'of the above sequence (for example, any one of SEQ ID NOs: 18 to 26) for structural stabilization of the modified enzyme which is the expression product, etc.
- a gene construct in which a sequence encoding a propeptide (prosequence) is added to the terminal side will be introduced into the host.
- a gene contract is prepared in which a sequence encoding a presequence (signal sequence) is added to the 5 'end of the sequence encoding the prosequence.
- the gene construct When the gene construct is used, it is expressed as a prepro-type transglutaminase in which a pre-sequence and a pro-sequence are linked, and then cleavage of the pre-sequence (conversion to pro-type transglutaminase) and cleavage of the pro-sequence lead to mature transglutaminase Is obtained.
- the sequence encoding the presequence and the sequence encoding the prosequence the original sequence, that is, the sequence of the reference transglutaminase (transglutaminase before modification) may be used.
- a specific example of the presequence is SEQ ID NO: 27 (a presequence of transglutaminase from Streptomyces mobaraensis), a specific example of a pro sequence is SEQ ID NO: 28 (a transglutaminase prosequence from Streptomyces mobaraensis), a presequence
- a specific example of the sequence encoding SEQ ID NO: 29 (a sequence encoding a pre-sequence of transglutaminase from Streptomyces mobaraensis)
- a specific example of a sequence encoding the pro sequence SEQ ID NO: 30 (derived from Streptomyces mobaraensis)
- the nucleic acid of the present invention refers to the sequence information disclosed in the specification or the attached sequence listing, and uses standard genetic engineering techniques, molecular biological techniques, biochemical techniques, chemical synthesis, etc. It can be prepared in an isolated state.
- nucleic acid which differs in the base sequence in part of the equivalent protein when compared with the base sequence of the gene encoding the modified enzyme of the present invention (hereinafter referred to as It is also referred to as “homologous nucleic acid”, and a nucleotide sequence that defines homologous nucleic acid is also referred to as “homologous nucleotide sequence”.
- a homologous nucleic acid is a modified enzyme consisting of a nucleotide sequence including substitution, deletion, insertion, addition, or inversion of one or more bases based on the nucleotide sequence of the nucleic acid encoding the modified enzyme of the present invention And DNA encoding a protein having a characteristic enzyme activity (ie, transglutaminase activity). Substitutions or deletions of bases may occur at a plurality of sites.
- “plural” also varies depending on the position and type of amino acid residue in the three-dimensional structure of the protein encoded by the nucleic acid, but is, for example, 2 to 40 bases, preferably 2 to 20 bases, more preferably 2 to 10 bases It is.
- the homologous nucleic acid is, for example, 60% or more, preferably 70% or more, more preferably 80% or more, still more preferably 85% or more, still more preferably about 90% or more, still more preferably, relative to the reference base sequence. Have an identity of at least 95%, most preferably at least 99%.
- the homologous nucleic acid as described above can be treated, for example, by restriction enzyme treatment, treatment with exonuclease or DNA ligase, site-directed mutagenesis (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York), or random mutations. It is obtained by introduction of a mutation by mutagenesis (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York). Also, the homologous nucleic acid can be obtained by other methods such as ultraviolet irradiation.
- nucleic acid having a base sequence complementary to the base sequence of a gene encoding the modified enzyme of the present invention relates to a nucleic acid having a base sequence complementary to the base sequence of a gene encoding the modified enzyme of the present invention.
- the base sequence of the gene encoding the modified enzyme of the present invention or at least about 60%, 70%, 80%, 90%, 95%, relative to the base sequence complementary thereto.
- a nucleic acid having a 99%, 99.9% identical base sequence is provided.
- Yet another aspect of the present invention is a nucleic acid having a base sequence that hybridizes under stringent conditions to a base sequence of a gene encoding the modified enzyme of the present invention or a base sequence complementary to the homologous base sequence.
- stringent conditions refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed.
- Such stringent conditions are known to those skilled in the art and include, for example, Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York) and Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987). It can be set by referring to.
- hybridization solution 50% formamide, 10 ⁇ SSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0), 5 ⁇ Denhardt solution, 1% SDS, 10% dextran sulfate, 10 ⁇ g / ml denatured Incubation with salmon sperm DNA, 50 mM phosphate buffer (pH 7.5) at about 42 ° C. to about 50 ° C., followed by washing with 0.1 ⁇ SSC, 0.1% SDS at about 65 ° C. to about 70 ° C. Can be mentioned.
- More preferable stringent conditions are, for example, 50% formamide, 5 ⁇ SSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0) as a hybridization solution, 1 ⁇ Denhardt's solution, 1% SDS, 10% dextran sulfate, 10 ⁇ g / ml Conditions using denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5)) can be mentioned.
- nucleic acid having a base sequence of a gene encoding the modified enzyme of the present invention, or a part of the base sequence complementary thereto.
- a nucleic acid fragment can be used for detecting, identifying, and / or amplifying a nucleic acid having a base sequence of a gene encoding the modified enzyme of the present invention.
- the nucleic acid fragment is, for example, a contiguous nucleotide portion (eg, about 10 to about 100 bases in length, preferably about 20 to about 100 bases in length, more preferably about 30 to about 100 bases in length) in the base sequence of the gene encoding the modified enzyme of the present invention Are designed to include at least a portion that hybridizes to 100 bases).
- nucleic acid fragments can be labeled.
- labeling for example, fluorescent substances, enzymes, radioactive isotopes can be used.
- Yet another aspect of the present invention relates to a recombinant DNA comprising a gene of the present invention (a gene encoding a modified enzyme).
- the recombinant DNA of the present invention is provided, for example, in the form of a vector.
- vector refers to a nucleic acid molecule capable of transporting a nucleic acid inserted therein into a target such as a cell.
- an appropriate vector is selected.
- E. coli As a vector having E. coli as a host, M13 phage or its variant, ⁇ phage or its variant, pBR322 or its variant (pB325, pAT153, pUC8 etc.), pET21 etc., as a vector having yeast as host pYepSec1, pMFa
- yeast As a vector having yeast as host pYepSec1, pMFa
- yeast pYepSec1
- yeast Examples of vectors that can use insect cells as a host include pAc and pVL, and examples of vectors that use mammalian cells as a host include pCDM8 and pMT2PC.
- the vector of the present invention is preferably an expression vector.
- An "expression vector” refers to a vector capable of introducing a nucleic acid inserted therein into a target cell (host cell) and capable of being expressed in the cell.
- An expression vector usually contains a promoter sequence necessary for expression of the inserted nucleic acid, an enhancer sequence for promoting expression, and the like.
- Expression vectors containing a selectable marker can also be used. When such an expression vector is used, the presence or absence (and its extent) of introduction of the expression vector can be confirmed by using a selection marker.
- Insertion of the nucleic acid of the present invention into a vector, insertion of a selectable marker gene (if necessary), insertion of a promoter (if necessary), etc. can be performed using standard recombinant DNA techniques (eg, Molecular Cloning, Third Edition, 1.84, Cold This method can be performed using known methods using restriction enzymes and DNA ligase, which can be referred to Spring Harbor Laboratory Press, New York.
- Host cells include, for ease of handling, Aspergillus oryzae (eg, Aspergillus oryzae), Bacillus bacteria (eg, Bacillus subtilis, Bacillus licheniformis, Bacillus aminolikfaciens), Brevibacillus bacteria (eg, Brevibacillus) -A microorganism such as C. sinensis, E. coli (Escherichia coli), or budding yeast (Saccharomyces cerevisiae) can be used, but it may be a host cell in which the recombinant DNA can be replicated and the modified enzyme gene can be expressed. Available.
- E. oryzae eg, Aspergillus oryzae
- Bacillus bacteria eg, Bacillus subtilis, Bacillus licheniformis, Bacillus aminolikfaciens
- Brevibacillus bacteria eg, Brevibacillus
- -A microorganism such as C. sinensis
- Escherichia coli Esichia coli
- budding yeast Sacharomyces cerevisiae
- Streptomyces genus microorganisms for example, Streptomyces morabaensis
- E. coli E. coli BL21 (DE3) can be mentioned when using a T7 promoter
- E. coli JM109 can be mentioned otherwise.
- examples of budding yeast include budding yeast SHY2, budding yeast AH22 or budding yeast INVSc1 (Invitrogen).
- microorganism that is, a transformant
- the microorganism of the present invention can be obtained by transfection or transformation using the vector of the present invention.
- calcium chloride method Frnal of Molecular Biology (J. Mol. Biol.), Vol. 53, p. 159 (1970)
- Hanahan method Journal of Molecular Biology, vol. 166, p. 557. (1983), SEM (Gene, 96, 23 (1990)), Chung et al.
- microorganism of the present invention can be used to produce the modified enzyme of the present invention.
- the modified enzyme of the present invention is provided, for example, in the form of an enzyme agent.
- the enzyme agent is an active ingredient (the modified enzyme of the present invention), an excipient, a buffer, a suspension, a stabilizer, a preservative, a preservative, a preservative, a saline solution, various proteins, various protein degradation products, Various extracts, various salts, various antioxidants, cysteine, glutathione, sodium glutamate, sodium inosinate, sodium guanylate, calcium carbonate shell, silicon dioxide and the like may be contained.
- starch dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, glycerol and the like can be used.
- phosphate, citrate, acetate and the like can be used.
- propylene glycol, ascorbic acid and the like can be used.
- preservatives phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methyl paraben and the like can be used.
- proteins are soy protein, wheat protein, corn protein, milk protein, animal-derived protein and the like.
- extracts are meat extract, plant extract, yeast extract and the like.
- salts are chloride, phosphate, polyphosphate, pyrophosphate, citrate, lactate, carbonate and the like.
- antioxidants are L-ascorbate, sodium bisulfite and the like.
- the form of the enzyme preparation of the present invention is not particularly limited.
- a further aspect of the present invention relates to methods of preparing modified enzymes.
- the modified enzyme of the present invention is prepared by genetic engineering techniques.
- a nucleic acid encoding the amino acid sequence of the modified enzyme of the present invention (for example, any one of SEQ ID NOs: 2 to 10) is prepared (step (I)).
- nucleic acid encoding a specific amino acid sequence is a nucleic acid from which a polypeptide having the amino acid sequence is obtained when it is expressed, and it goes without saying that a nucleic acid consisting of a nucleotide sequence corresponding to the amino acid sequence
- an extra sequence a sequence encoding an amino acid sequence or a sequence not encoding an amino acid sequence
- degeneracy of codons is considered.
- nucleic acid encoding the amino acid sequence of the modified enzyme of the present invention refers to the sequence information disclosed in the specification or the attached sequence listing, and uses standard genetic engineering techniques, molecular biological techniques, biochemistry It can be prepared in an isolated state by using a chemical method or the like.
- the amino acid sequence of the modified enzyme of the present invention is obtained by mutating the amino acid sequence of the reference transglutaminase. Therefore, "a nucleic acid encoding the amino acid sequence of the modified enzyme of the present invention” can also be obtained by adding the necessary mutation to the gene encoding the reference transglutaminase.
- regiospecific sequence substitution Many methods for regiospecific sequence substitution are known in the art (see, eg, Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, New York), from which a suitable method is selected. Can be used.
- a site-directed mutagenesis method a site-directed amino acid saturation mutation can be employed.
- the regiospecific amino acid saturation mutation method is a "Semi-rational, semi-random" method which estimates the position to which the function to be sought relates based on the three-dimensional structure of a protein, and introduces an amino acid saturation mutation (J. Mol. Biol. 331, 585-592 (2003)).
- a regiospecific amino acid saturation mutation using a kit such as Quick change (Stratagene), Overlap extension PCR (Nucleic Acid Res. 16, 7351-7367 (1988)).
- Taq polymerase etc. can be used for the DNA polymerase used for PCR.
- a highly accurate DNA polymerase such as KOD-PLUS- (Toyobo Co., Ltd.) or Pfu turbo (Stratagene).
- step (II) the prepared nucleic acid is expressed (step (II)).
- an expression vector into which the above-mentioned nucleic acid is inserted is prepared, and this is used to transform a host cell.
- the transformant is cultured under conditions in which the modified enzyme that is the expression product is produced. Cultivation of transformants may be performed according to a conventional method.
- the carbon source used in the medium may be any assimilable carbon compound such as glucose, sucrose, lactose, maltose, molasses, pyruvic acid and the like.
- the nitrogen source may be any available nitrogen compound, such as peptone, meat extract, yeast extract, casein hydrolyzate, soybean meal alkaline extract and the like.
- salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, zinc, specific amino acids, specific vitamins and the like are used as needed.
- the culture temperature can be set in the range of 30 ° C. to 40 ° C. (preferably around 33 to 37 ° C.).
- the culture time can be set in consideration of the growth characteristics of the transformant to be cultured, the production characteristics of the modified enzyme, and the like.
- the pH of the medium is adjusted within the range in which the transformant grows and the enzyme is produced.
- the pH of the culture medium is about 6.0 to 9.0 (preferably, around pH 7.0).
- the expression product (modified enzyme) is recovered (step (III)).
- the culture solution containing the cultured cells can be used as an enzyme solution as it is or after concentration, removal of impurities, etc.
- the expression product is once recovered from the culture solution or cells. If the expression product is a secretory protein, it can be recovered from the culture solution, and from the cell body otherwise.
- the culture supernatant is filtered and centrifuged to remove insolubles, and then concentrated under reduced pressure, membrane concentration, salting out using ammonium sulfate or sodium sulfate, methanol, ethanol, acetone or the like Fractional precipitation method, dialysis, heat treatment, isoelectric point treatment, gel filtration or adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.
- the cells are collected by filtering, centrifuging or the like the culture solution, and then the cells are subjected to pressure treatment, ultrasonic treatment, physical disruption treatment or other mechanical method or lysozyme After disruption by an enzymatic method such as by the like, separation and purification are performed as described above to obtain a purified product of the modified enzyme.
- the purified enzyme obtained as described above may be dissolved in advance in phosphate buffer, triethanolamine buffer, Tris-HCl buffer or GOOD buffer.
- phosphate buffer, triethanolamine buffer can be used.
- examples of the GOOD buffer include PIPES, MES and MOPS.
- gene expression to expression product is recovered using a suitable host-vector system, but a cell-free synthesis system may be used.
- “cell-free synthesis system (cell-free transcription system, cell-free transcription / translation system)” does not use living cells, but includes ribosomes derived from living cells (or obtained by genetic engineering techniques)
- a cell extract obtained by purifying the cell lysate as needed is used.
- Cell extracts generally contain ribosomes necessary for protein synthesis, various factors such as initiation factors, and various enzymes such as tRNA. At the time of protein synthesis, various amino acids, energy sources such as ATP and GTP, creatine phosphate and other substances necessary for protein synthesis are added to the cell extract. Of course, at the time of protein synthesis, separately prepared ribosomes, various factors, and / or various enzymes may be supplemented as necessary.
- cell-free transcription / translation system is used interchangeably with cell-free protein synthesis system, in vitro translation system or in vitro transcription / translation system.
- RNA is used as a template to synthesize proteins.
- total RNA, mRNA, in vitro transcript and the like are used.
- DNA is used as a template.
- the template DNA should contain a ribosome binding region and preferably also contain a suitable terminator sequence.
- in vitro transcription / translation system conditions are set in which factors necessary for each reaction are added so that the transcription reaction and the translation reaction proceed continuously.
- E. coli BL21 (DE3) is transformed with the ligation reaction solution (11 ⁇ L / tube), and cultured in LB medium containing ampicillin (37 ° C., overnight).
- ⁇ Activity measurement method> Dilute the maturation enzyme with 200 mM Tris-HCl pH 6.0 to the appropriate concentration (sample solution). 100 ⁇ L of the substrate solution (R-1) is added to 10 ⁇ L of the sample solution, mixed, and reacted at 37 ° C. for 10 minutes. The reaction is stopped by adding 100 ⁇ L of a coloring solution (R-2) to form an Fe complex, and then the absorbance at 525 nm is measured. As a control, the absorbance of the reaction solution is similarly measured using an enzyme solution which has been heat-inactivated in advance, and the difference in absorbance from the sample solution is determined.
- a calibration curve is prepared using L-glutamic acid- ⁇ -monohydroxamic acid instead of the enzyme solution, and the amount of hydroxamic acid generated is determined from the difference in absorbance.
- the enzyme activity that produces 1 ⁇ mol of hydroxamic acid per minute is defined as 1 unit (1 U).
- Substrate solution (R-2)) Mix 30 mL of 3 M hydrochloric acid solution, 30 mL of 12% trichloroacetic acid solution, and 30 mL of 5% iron (III) chloride solution.
- the matured enzyme is diluted 2-fold with 200 mM Tris-HCl pH 6.0 and treated for 30 minutes at a predetermined temperature (20 ° C., 30 ° C., 40 ° C., 50 ° C., 60 ° C.) Do. After treatment, the activity is measured, and the residual rate of activity is calculated by comparing with the activity before treatment.
- Residual activity rate (%) (enzyme activity after heat treatment) / (enzyme activity before heat treatment) ⁇ 100
- Relative to wild type (%) (residual activity of mutant enzyme) / (residual activity of wild type enzyme) ⁇ 100
- the evaluation result of temperature stability is shown in FIG.
- the following (1) to (33) having a wild type (%) to 90% or less were judged as effective amino acid substitutions.
- the mutation point is V6, the amino acid after substitution is Q, I, M, S, C, K, L, H, F, G, N, P, R, W or Y (2)
- the mutation point is R26, and the amino acid after substitution is K, Q, M, H, Y, D, G, N, P or S (3)
- the mutation point is E28, and the amino acid after substitution is V, Q, W, R, K, M, N, F, G, L, P or Y (4)
- the mutation point is V30, and the amino acid after substitution is P, C, A, E, F, G, H, K, N, Q, R, W, Y or L (5)
- the mutation point is Y34, and the amino acid after substitution is A (6)
- the mutation point is Y42 and the amino acid after substitution is F, A, C, D,
- the mutation point is L60, and the amino acid after substitution is I, M, V, A, C, E, F, Q, S, T, W or Y. (10) The mutation point is Y62, and the amino acid after substitution is C, R, G, K or S (11) The mutation point is V65, and the amino acid after substitution is N, L, M, F, W or Y (12) The mutation point is V67, and the amino acid after substitution is L, N, A, C, M, Q or S (13) The mutation point is T68, and the amino acid after substitution is C, L, A, S, M, F, N, Q or Y.
- the mutation point is W69, and the amino acid after substitution is H, M, I, C, E, F, G, K, L, N, Q, R, S, T or V (15)
- the mutation point is Q74, and the amino acid after substitution is W, D, G or K (16)
- the mutation point is Y75, and the amino acid after substitution is R, Q, T or G (17)
- the mutation point is T77, and the amino acid after substitution is M, H, E, C or G (18)
- the mutation point is F85, and the amino acid after substitution is M (19)
- the mutation point is F90, and the amino acid after substitution is C, M, H, L or V (20)
- the mutation point is F108, and the amino acid after substitution is R, L, T, A, I, K, N or V (21)
- the mutation point is F117, and the amino acid after substitution is M or L (22)
- the mutation point is S199, and the amino acid after substitution is G, M, N, K or V (23)
- amino acid substitutions having a wild type (%) of 30% or less were identified.
- amino acid substitutions having a wild type (%) of 10% or less were identified.
- the evaluation result of heat resistance is shown in FIG. The following (34) to (45) whose wild type (%) was 110% or more were judged as effective amino acid substitutions.
- (34) The mutation point is D3 and the amino acid after substitution is Q, P, E, S or Y (35) The mutation point is R26, and the amino acid after substitution is V (36) The mutation point is Y34, and the amino acid after substitution is W (37) The mutation point is V67, and the amino acid after substitution is H (38) The mutation point is T68, and the amino acid after substitution is V or I (39) The mutation point is Q74, and the amino acid after substitution is F. (40) The mutation point is T77, and the amino acid after substitution is Q.
- the mutation point is S199, and the amino acid after substitution is C or Q (42)
- the mutation point is T273, and the amino acid after substitution is Q.
- the mutation point is S284, and the amino acid after substitution is L, H, K, P or R (44)
- the mutation point is S299, and the amino acid after substitution is N.
- the mutation point is S303, and the amino acid after substitution is K
- amino acid substitutions having a wild type (%) of 120% or more were identified.
- amino acid substitutions having a wild type (%) of 130% or more were identified.
- Hydrogen peroxide (30-35%) (Wako, reagent grade) is diluted with 200 mM Tris-HCl pH 6.0 to prepare hydrogen peroxide solution (0.03%) as an oxidizing agent.
- the mature enzyme is diluted 2-fold with hydrogen peroxide solution and then reacted at 30 ° C. for 1 hour. Activity is measured at the degree of reaction, and the residual rate of activity is calculated by comparing with the activity before treatment.
- Residual activity rate (%) (enzyme activity after hydrogen peroxide treatment) / (enzyme activity before treatment) ⁇ 100
- Relative to wild type (%) (residual activity of mutant enzyme) / (residual activity of wild type enzyme) ⁇ 100
- the evaluation results of the antioxidant properties are shown in FIG. The following (46) to (59) whose wild type (%) was 110% or more were judged as effective amino acid substitutions.
- (46) The mutation point is D3, and the amino acid after substitution is E, Q, S or Y (47)
- the mutation point is V6, and the amino acid after substitution is P (48)
- the mutation point is R26, and the amino acid after substitution is M, K, W, C, Q, G, Y, E, T, N, D, I, S, P or V (49)
- Mutation point is E28, amino acid after substitution is L, M, K, C, V, R, W, G, N, F, Y or H (50)
- the mutation point is Y42, and the amino acid after substitution is L, N or F (51)
- the mutation point is E58, and the amino acid after substitution is Q, A, I, V, L, T, M, K, Y, W, F or R (52)
- the mutation point is V67, and the amino
- the mutation point is S199, and the amino acid after substitution is C (57) The mutation point is T273, and the amino acid after substitution is E. (58) The mutation point is S284, and the amino acid after substitution is R or H (59) The mutation point is Y291, and the amino acid after substitution is I, S, F, L, C, N, V or M
- amino acid substitutions having a wild type (%) of 130% or more were identified.
- the protein concentration is calculated by measuring the absorbance at 280 nm of the sample solution, and the specific activity is calculated from the measured value of the activity and the protein concentration.
- the evaluation results of the reactivity are shown in FIG. The following (60) to (63) whose wild type (%) was 110% or more were judged as effective amino acid substitutions.
- the mutation point is S284, and the amino acid after substitution is M, K or V (61)
- the mutation point is F251, and the amino acid after substitution is Y.
- the mutation point is V6 and Y75, the amino acid after substitution at mutation point V6 is E, and the amino acid after substitution at mutation point Y75 is F
- the mutation point is D3 and T77, the amino acid after substitution at mutation point D3 is P, and the amino acid after substitution at mutation point T77 is Q
- amino acid substitutions having a wild type (%) of 120% or more were identified.
- amino acid substitutions having a wild type (%) of 130% or more were identified. Double mutation of V6E and Y75F Double mutation of D3P and T77Q
- the mutation point is D3, the amino acid after substitution is Q, S or Y, the property change due to the amino acid substitution is heat resistance improvement and the antioxidant improvement (65) the mutation point is D3, the amino acid after substitution is P, amino acid substitution Heat resistance improvement and reactivity improvement (66) mutation point is D3, amino acid after substitution is E, property change due to amino acid substitution is heat resistance improvement, antioxidant property improvement and reactivity improvement (67) mutation point V6, Amino acid after substitution is P, Characteristic change due to amino acid substitution is temperature stability reduction and antioxidant improvement (68) Mutation point is R26, Amino acid after substitution is K, Q, M, Y, D, G, N , P or S, property change due to amino acid substitution decreases temperature stability and antioxidant property improvement (69) The mutation point is R26, amino acid after substitution is V, property change due to amino acid substitution improves heat resistance property and antioxidant property ( 70) The mutation point is D3, the amino acid after substitution is Q, S or Y, the property change due to the amino acid substitution is heat resistance improvement and antioxidant improvement (65) the
- amino acid substitutions involve two characteristic changes that can be said to be contradictory: a decrease in temperature stability and an improvement in antioxidant properties.
- the identification of such amino acid substitutions is an unexpected outcome. Modified enzymes with such characteristic amino acid substitutions are expected to be used in food applications where the enzymes need to be inactivated at low temperatures.
- the maturation enzyme is diluted with 200 mM potassium phosphate buffer (pH 7.0) to an appropriate concentration (sample solution).
- the substrate solution 1% Z-Gln-Gly, 200 mM potassium phosphate buffer, pH 7.0
- the substrate solution 1% Z-Gln-Gly, 200 mM potassium phosphate buffer, pH 7.0
- the substrate solution 1% Z-Gln-Gly, 200 mM potassium phosphate buffer, pH 7.0
- 40 ⁇ L of color-forming reagent A, 20 ⁇ L of color-forming reagent B, and 40 ⁇ L of color-forming reagent C are added to 20 ⁇ L of the reaction sample, and reacted at 37 ° C. for 10 minutes.
- a supernatant liquid is collect
- deamidation enzyme The evaluation results of deamidation enzyme are shown in FIG. If the value of deamidation activity / transglutaminase activity exceeds 1, it means that deamidation has occurred.
- the following (92) to (134) were judged to be effective amino acid substitutions.
- the mutation point is R5, and the amino acid after substitution is Q, P, C, M, I, V, Y, F, S, N, T or G (93)
- the mutation point is Y34, and the amino acid after substitution is H or M (94)
- the mutation point is W59, and the amino acid after substitution is K or E (95)
- the mutation point is S61, the amino acid after substitution is T, V, Y, D, I, L, N, E, M, F, W, Q, P, H or K (96)
- the mutation point is Y62, and the amino acid after substitution is D or P (97)
- the mutation point is G63, and the amino acid after substitution is D, Q, Y, R, K, H, P, M, N, C, F, L, W, T, V or S (98)
- the mutation point is V65, and the amino acid after substitution is C, T or K (99)
- the mutation point is G66, and the amino acid after substitution is W or K (100)
- the mutation point is F108, and the amino acid after substitution is Q, C, E, S or N (105)
- the mutation point is F117, and the amino acid after substitution is C, I, W, V, A, G, T or N (106)
- the mutation point is Y198, and the amino acid after substitution is F, H, L, S, M or I (107)
- the mutation point is S199, and the amino acid after substitution is Q or P (108)
- the mutation point is K200, and the amino acid after substitution is Y, M, I, R, L or V (109)
- the mutation point is H201, and the amino acid after substitution is K, R, M, L or Q (110)
- the mutation point is F202, and the amino acid after substitution is Y, M, H, V or C (111)
- the mutation point is W203 and the amino acid after substitution is Y (112)
- the mutation point is F223, and the amino acid after substitution is Y, M, L, W, V, H, G or A (113)
- the mutation point is W258, and the amino acid after substitution is Y, F or N.
- the mutation point is T273, and the amino acid after substitution is N.
- the mutation point is G275, the amino acid after substitution is A, C, S, V, K, I, F, H, R, L, P, Q, Y, N, M or T (122)
- the mutation point is N276, and the amino acid after substitution is G, M, Q or H (123)
- the mutation point is H277, and the amino acid after substitution is N, S, Q, C, E, K, D, I, R, M, P, L, W, V, Y, G, F, or T (124)
- the mutation point is Y278, and the amino acid after substitution is P or D (125)
- the mutation point is H279, and the amino acid after substitution is N (126)
- the mutation point is S284, and the amino acid after substitution is C.
- the mutation point is M288, and the amino acid after substitution is V, Q or T (128)
- the mutation point is V290, and the amino acid after substitution is T, C, L, M, A, S or N (129)
- the mutation point is Y291, and the amino acid after substitution is H, T, E or G (130)
- the mutation point is W298, and the amino acid after substitution is F, Y, I, L or M (131)
- the mutation point is S299, and the amino acid after substitution is N or P (132)
- the mutation point is Y302, and the amino acid after substitution is L, H, M, V, C, T, P, S or W (133)
- the mutation point is S303, and the amino acid after substitution is I, Q, D, H or E (134)
- the mutation point is F305, and the amino acid after substitution is W, H or Y
- amino acid substitutions having a deamidation activity / transglutaminase activity value of 1.2 or more were identified.
- amino acid substitutions having a deamidation activity / transglutaminase activity value of 2 or more were identified.
- amino acid substitutions having a deamidation activity / transglutaminase activity value of 10 or more were identified.
- the modified transglutaminase of the present invention has improved properties that are practically important, and has high industrial value. Therefore, it is expected to be applied to new applications as well as to existing applications.
- SEQ ID NO: 2 Description of artificial sequence: R26N mutant SEQ ID NO: 3: Description of artificial sequence: R26Y mutant SEQ ID NO: 4: Description of artificial sequence: R26S mutant SEQ ID NO: 5: Description of artificial sequence: E28N mutant SEQ ID NO: 6: Description of artificial sequence: E28G mutant SEQ ID NO: 7: Description of artificial sequence: E28 F mutant SEQ ID NO: 8: Description of artificial sequence: E28 Y mutant SEQ ID NO: 9: Description of artificial sequence: E58R mutant SEQ ID NO: 10 Description of artificial sequences: E58L mutant SEQ ID NO: 18: Description of artificial sequences: R26N mutant SEQ ID NO: 19: Description of artificial sequences: R26Y mutant SEQ ID NO: 20: Description of artificial sequences: R26 S mutant SEQ ID NO: 21: artificial sequences Description of: E28N mutant SEQ ID NO: 22: Description of artificial sequence: E28G mutant SEQ ID NO: 23: Description of artificial sequence: E28F mutant SEQ ID NO: 24: Description of artificial
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Abstract
Description
[1]配列番号1のアミノ酸配列において、以下の(1)~(134)の中のいずれか一つのアミノ酸置換を含むアミノ酸配列、又は該アミノ酸配列と80%以上の同一性を示すアミノ酸配列(但し、前記アミノ酸置換の位置以外の部分でアミノ酸配列の相違が生じている)を有し、前記アミノ酸置換に対応した特性変化が認められる改変型トランスグルタミナーゼ:
(1)変異点がV6、置換後のアミノ酸がQ、I、M、S、C、K、L、H、F、G、N、P、R、W又はY、アミノ酸置換による特性変化が温度安定性低下;
(2)変異点がR26、置換後のアミノ酸がK、Q、M、H、Y、D、G、N、P又はS、アミノ酸置換による特性変化が温度安定性低下;
(3)変異点がE28、置換後のアミノ酸がV、Q、W、R、K、M、N、F、G、L、P又はY、アミノ酸置換による特性変化が温度安定性低下;
(4)変異点がV30、置換後のアミノ酸がP、C、A、E、F、G、H、K、N、Q、R、W、Y又はL、アミノ酸置換による特性変化が温度安定性低下;
(5)変異点がY34、置換後のアミノ酸がA、アミノ酸置換による特性変化が温度安定性低下;
(6)変異点がY42、置換後のアミノ酸がF、A、C、D、E、G、I、L、M、Q、S、T、V又はW、アミノ酸置換による特性変化が温度安定性低下;
(7)変異点がE58、置換後のアミノ酸がY、M、A、F、I、V、R、K、N、L、S、Q、G又はH、アミノ酸置換による特性変化が温度安定性低下;
(8)変異点がW59、置換後のアミノ酸がR、N、Y、A、S、I、V、D、G又はP、アミノ酸置換による特性変化が温度安定性低下;
(9)変異点がL60、置換後のアミノ酸がI、M、V、A、C、E、F、Q、S、T、W又はY、アミノ酸置換による特性変化が温度安定性低下;
(10)変異点がY62、置換後のアミノ酸がC、R、G、K又はS、アミノ酸置換による特性変化が温度安定性低下;
(11)変異点がV65、置換後のアミノ酸がN、L、M、F、W又はY、アミノ酸置換による特性変化が温度安定性低下;
(12)変異点がV67、置換後のアミノ酸がL、N、A、C、M、Q又はS、アミノ酸置換による特性変化が温度安定性低下;
(13)変異点がT68、置換後のアミノ酸がC、L、A、S、M、F、N、Q又はY、アミノ酸置換による特性変化が温度安定性低下;
(14)変異点がW69、置換後のアミノ酸がH、M、I、C、E、F、G、K、L、N、Q、R、S、T又はV、アミノ酸置換による特性変化が温度安定性低下;
(15)変異点がQ74、置換後のアミノ酸がW、D、G又はK、アミノ酸置換による特性変化が温度安定性低下;
(16)変異点がY75、置換後のアミノ酸がR、Q、T又はG、アミノ酸置換による特性変化が温度安定性低下;
(17)変異点がT77、置換後のアミノ酸がM、H、E、C又はG、アミノ酸置換による特性変化が温度安定性低下;
(18)変異点がF85、置換後のアミノ酸がM、アミノ酸置換による特性変化が温度安定性低下;
(19)変異点がF90、置換後のアミノ酸がC、M、H、L又はV、アミノ酸置換による特性変化が温度安定性低下;
(20)変異点がF108、置換後のアミノ酸がR、L、T、A、I、K、N又はV、アミノ酸置換による特性変化が温度安定性低下;
(21)変異点がF117、置換後のアミノ酸がM又はL、アミノ酸置換による特性変化が温度安定性低下;
(22)変異点がS199、置換後のアミノ酸がG、M、N、K又はV、アミノ酸置換による特性変化が温度安定性低下;
(23)変異点がF202、置換後のアミノ酸がL又はW、アミノ酸置換による特性変化が温度安定性低下;
(24)変異点がW203、置換後のアミノ酸がF、アミノ酸置換による特性変化が温度安定性低下;
(25)変異点がF254、置換後のアミノ酸がY又はM、アミノ酸置換による特性変化が温度安定性低下;
(26)変異点がT273、置換後のアミノ酸がI、V、M、C、S、L、R、G、A、E、F、Y、D、K、W又はH、アミノ酸置換による特性変化が温度安定性低下;
(27)変異点がN276、置換後のアミノ酸がC、E、K、L、S、T又はV、アミノ酸置換による特性変化が温度安定性低下;
(28)変異点がY278、置換後のアミノ酸がM、L、H、I、K、R、W、C、G、N、Q、S、T又はV、アミノ酸置換による特性変化が温度安定性低下;
(29)変異点がS284、置換後のアミノ酸がW、Y、M、F又はNアミノ酸置換による特性変化が温度安定性低下;
(30)変異点がY291、置換後のアミノ酸がI、W、L、A、C、K、N、Q、R、S又はV、アミノ酸置換による特性変化が温度安定性低下;
(31)変異点がS299、置換後のアミノ酸がI、Y、V、K、M、Q、A、F、G又はE、アミノ酸置換による特性変化が温度安定性低下;
(32)変異点がS303、置換後のアミノ酸がN、G、C、V、P又はY、アミノ酸置換による特性変化が温度安定性低下;
(33)変異点がY310、置換後のアミノ酸がC、M又はI、アミノ酸置換による特性変化が温度安定性低下;
(34)変異点がD3、置換後のアミノ酸がQ、P、E、S又はY、アミノ酸置換による特性変化が耐熱性向上;
(35)変異点がR26、置換後のアミノ酸がV、アミノ酸置換による特性変化が耐熱性向上;
(36)変異点がY34、置換後のアミノ酸がW、アミノ酸置換による特性変化が耐熱性向上;
(37)変異点がV67、置換後のアミノ酸がH、アミノ酸置換による特性変化が耐熱性向上;
(38)変異点がT68、置換後のアミノ酸がV又はI、アミノ酸置換による特性変化が耐熱性向上;
(39)変異点がQ74、置換後のアミノ酸がF、アミノ酸置換による特性変化が耐熱性向上;
(40)変異点がT77、置換後のアミノ酸がQ、アミノ酸置換による特性変化が耐熱性向上;
(41)変異点がS199、置換後のアミノ酸がC又はQ、アミノ酸置換による特性変化が耐熱性向上;
(42)変異点がT273、置換後のアミノ酸がQ、アミノ酸置換による特性変化が耐熱性向上;
(43)変異点がS284、置換後のアミノ酸がL、H、K、P又はR、アミノ酸置換による特性変化が耐熱性向上;
(44)変異点がS299、置換後のアミノ酸がN、アミノ酸置換による特性変化が耐熱性向上;
(45)変異点がS303、置換後のアミノ酸がK、アミノ酸置換による特性変化が耐熱性向上;
(46)変異点がD3、置換後のアミノ酸がE、Q、S又はY、アミノ酸置換による特性変化が抗酸化性向上;
(47)変異点がV6、置換後のアミノ酸がP、アミノ酸置換による特性変化が抗酸化性向上;
(48)変異点がR26、置換後のアミノ酸がM、K、W、C、Q、G、Y、E、T、N、D、I、S、P又はV、アミノ酸置換による特性変化が抗酸化性向上;
(49)変異点がE28、置換後のアミノ酸がL、M、K、C、V、R、W、G、N、F、Y又はH、アミノ酸置換による特性変化が抗酸化性向上;
(50)変異点がY42、置換後のアミノ酸がL、N又はF、アミノ酸置換による特性変化が抗酸化性向上;
(51)変異点がE58、置換後のアミノ酸がQ、A、I、V、L、T、M、K、Y、W、F又はR、アミノ酸置換による特性変化が抗酸化性向上;
(52)変異点がV67、置換後のアミノ酸がT、S又はA、アミノ酸置換による特性変化が抗酸化性向上;
(53)変異点がT68、置換後のアミノ酸がL、I、V又はM、アミノ酸置換による特性変化が抗酸化性向上;
(54)変異点がQ74、置換後のアミノ酸がV、アミノ酸置換による特性変化が抗酸化性向上;
(55)変異点がT77、置換後のアミノ酸がD、アミノ酸置換による特性変化が抗酸化性向上;
(56)変異点がS199、置換後のアミノ酸がC、アミノ酸置換による特性変化が抗酸化性向上;
(57)変異点がT273、置換後のアミノ酸がE、アミノ酸置換による特性変化が抗酸化性向上;
(58)変異点がS284、置換後のアミノ酸がR又はH、アミノ酸置換による特性変化が抗酸化性向上;
(59)変異点がY291、置換後のアミノ酸がI、S、F、L、C、N、V又はM、アミノ酸置換による特性変化が抗酸化性向上;
(60)変異点がS284、置換後のアミノ酸がM、K又はV、アミノ酸置換による特性変化が反応性向上;
(61)変異点がF251、置換後のアミノ酸がY、アミノ酸置換による特性変化が反応性向上;
(62)変異点がV6とY75、変異点V6の置換後のアミノ酸がE、変異点Y75の置換後のアミノ酸がF、アミノ酸置換による特性変化が反応性向上;
(63)変異点がD3とT77、変異点D3の置換後のアミノ酸がP、変異点T77の置換後のアミノ酸がQ、アミノ酸置換による特性変化が反応性向上;
(64)変異点がD3、置換後のアミノ酸がQ、S又はY、アミノ酸置換による特性変化が耐熱性向上及び抗酸化性向上;
(65)変異点がD3、置換後のアミノ酸がP、アミノ酸置換による特性変化が耐熱性向上及び反応性向上;
(66)変異点がD3、置換後のアミノ酸がE、アミノ酸置換による特性変化が耐熱性向上、抗酸化性向上及び反応性向上;
(67)変異点がV6、置換後のアミノ酸がP、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上;
(68)変異点がR26、置換後のアミノ酸がK、Q、M、Y、D、G、N、P又はS、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上;
(69)変異点がR26、置換後のアミノ酸がV、アミノ酸置換による特性変化が耐熱性向上及び抗酸化性向上;
(70)変異点がR26、置換後のアミノ酸がH、アミノ酸置換による特性変化が温度安定性低下、抗酸化性向上及び反応性向上;
(71)変異点がE28、置換後のアミノ酸がV、R、K、M、N、F、G、L又はY、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上;
(72)変異点がV30、置換後のアミノ酸がP、G、N、R又はY、アミノ酸置換による特性変化が温度安定性低下及び反応性向上;
(73)変異点がY42、置換後のアミノ酸がF、L又はM、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上;
(74)変異点がE58、置換後のアミノ酸がY、M、A、F、I、V、R、K、L又はQ、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上;
(75)変異点がL60、置換後のアミノ酸がI、アミノ酸置換による特性変化が温度安定性低下及び反応性向上;
(76)変異点がV67、置換後のアミノ酸がA又はS、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上;
(77)変異点がT68、置換後のアミノ酸がC、L又はM、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上;
(78)変異点がT68、置換後のアミノ酸がV又はI、アミノ酸置換による特性変化が耐熱性向上及び抗酸化性向上;
(79)変異点がQ74、置換後のアミノ酸がV、アミノ酸置換による特性変化が抗酸化性向上及び反応性向上;
(80)変異点がT77、置換後のアミノ酸がC、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上;
(81)変異点がT77、置換後のアミノ酸がE、アミノ酸置換による特性変化が温度安定性低下及び反応性向上;
(82)変異点がT77、置換後のアミノ酸がD、アミノ酸置換による特性変化が抗酸化性向上及び反応性向上;
(83)変異点がS199、置換後のアミノ酸がG、アミノ酸置換による特性変化が温度安定性低下及び反応性向上;
(84)変異点がS199、置換後のアミノ酸がC、アミノ酸置換による特性変化が耐熱性向上、抗酸化性向上及び反応性向上;
(85)変異点がT273、置換後のアミノ酸がE、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上;
(86)変異点がS284、置換後のアミノ酸がM又はF、アミノ酸置換による特性変化が温度安定性低下及び反応性向上;
(87)変異点がS284、置換後のアミノ酸がL又はK、アミノ酸置換による特性変化が耐熱性向上及び反応性向上;
(88)変異点がS284、置換後のアミノ酸がH又はR、アミノ酸置換による特性変化が耐熱性向上及び抗酸化性向上;
(89)変異点がY291、置換後のアミノ酸がI、L、C、N、S又はV、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上;
(90)変異点がS299、置換後のアミノ酸がV、K、M又はA、アミノ酸置換による特性変化が温度安定性低下及び反応性向上;
(91)変異点がS303、置換後のアミノ酸がK、アミノ酸置換による特性変化が耐熱性向上及び反応性向上;
(92)変異点がR5、置換後のアミノ酸がQ、P、C、M、I、V、Y、F、S、N、T又はG、アミノ酸置換による特性変化が脱アミド酵素化;
(93)変異点がY34、置換後のアミノ酸がH又はM、アミノ酸置換による特性変化が脱アミド酵素化;
(94)変異点がW59、置換後のアミノ酸がK又はE、アミノ酸置換による特性変化が脱アミド酵素化;
(95)変異点がS61、置換後のアミノ酸がT、V、Y、D、I、L、N、E、M、F、W、Q、P、H又はK、アミノ酸置換による特性変化が脱アミド酵素化;
(96)変異点がY62、置換後のアミノ酸がD又はP、アミノ酸置換による特性変化が脱アミド酵素化;
(97)変異点がG63、置換後のアミノ酸がD、Q、Y、R、K、H、P、M、N、C、F、L、W、T、V又はS、アミノ酸置換による特性変化が脱アミド酵素化;
(98)変異点がV65、置換後のアミノ酸がC、T又はK、アミノ酸置換による特性変化が脱アミド酵素化;
(99)変異点がG66、置換後のアミノ酸がW又はK、アミノ酸置換による特性変化が脱アミド酵素化;
(100)変異点がT68、置換後のアミノ酸がP又はH、アミノ酸置換による特性変化が脱アミド酵素化;
(101)変異点がW69、置換後のアミノ酸がY、D又はP、アミノ酸置換による特性変化が脱アミド酵素化;
(102)変異点がQ74、置換後のアミノ酸がR又はP、アミノ酸置換による特性変化が脱アミド酵素化;
(103)変異点がF85、置換後のアミノ酸がY、W、L、I、Q、C、T、A、V、S、R、H又はN、アミノ酸置換による特性変化が脱アミド酵素化;
(104)変異点がF108、置換後のアミノ酸がQ、C、E、S又はN、アミノ酸置換による特性変化が脱アミド酵素化;
(105)変異点がF117、置換後のアミノ酸がC、I、W、V、A、G、T又はN、アミノ酸置換による特性変化が脱アミド酵素化;
(106)変異点がY198、置換後のアミノ酸がF、H、L、S、M又はI、アミノ酸置換による特性変化が脱アミド酵素化;
(107)変異点がS199、置換後のアミノ酸がQ又はP、アミノ酸置換による特性変化が脱アミド酵素化;
(108)変異点がK200、置換後のアミノ酸がY、M、I、R、L又はV、アミノ酸置換による特性変化が脱アミド酵素化;
(109)変異点がH201、置換後のアミノ酸がK、R、M、L又はQ、アミノ酸置換による特性変化が脱アミド酵素化;
(110)変異点がF202、置換後のアミノ酸がY、M、H、V又はC、アミノ酸置換による特性変化が脱アミド酵素化;
(111)変異点がW203、置換後のアミノ酸がY、アミノ酸置換による特性変化が脱アミド酵素化;
(112)変異点がF223、置換後のアミノ酸がY、M、L、W、V、H、G又はA、アミノ酸置換による特性変化が脱アミド酵素化;
(113)変異点がR238、置換後のアミノ酸がA、D、V、G、H又はS、アミノ酸置換による特性変化が脱アミド酵素化;
(114)変異点がF251、置換後のアミノ酸がH、N、C、M、R、S、A、T、L、P、G、V又はQ、アミノ酸置換による特性変化が脱アミド酵素化;
(115)変異点がV252、置換後のアミノ酸がM、L、T又はC、アミノ酸置換による特性変化が脱アミド酵素化;
(116)変異点がN253、置換後のアミノ酸がT、V、K、C又はR、アミノ酸置換による特性変化が脱アミド酵素化;
(117)変異点がY256、置換後のアミノ酸がL、V又はI、アミノ酸置換による特性変化が脱アミド酵素化;
(118)変異点がG257、置換後のアミノ酸がA又はC、アミノ酸置換による特性変化が脱アミド酵素化;
(119)変異点がW258、置換後のアミノ酸がY、F又はN、アミノ酸置換による特性変化が脱アミド酵素化;
(120)変異点がT273、置換後のアミノ酸がN、アミノ酸置換による特性変化が脱アミド酵素化;
(121)変異点がG275、置換後のアミノ酸がA、C、S、V、K、I、F、H、R、L、P、Q、Y、N、M又はT、アミノ酸置換による特性変化が脱アミド酵素化;
(122)変異点がN276、置換後のアミノ酸がG、M、Q又はH、アミノ酸置換による特性変化が脱アミド酵素化;
(123)変異点がH277、置換後のアミノ酸がN、S、Q、C、E、K、D、I、R、M、P、L、W、V、Y、G、F又はT、アミノ酸置換による特性変化が脱アミド酵素化;
(124)変異点がY278、置換後のアミノ酸がP又はD、アミノ酸置換による特性変化が脱アミド酵素化;
(125)変異点がH279、置換後のアミノ酸がN、アミノ酸置換による特性変化が脱アミド酵素化;
(126)変異点がS284、置換後のアミノ酸がC、アミノ酸置換による特性変化が脱アミド酵素化;
(127)変異点がM288、置換後のアミノ酸がV、Q又はT、アミノ酸置換による特性変化が脱アミド酵素化;
(128)変異点がV290、置換後のアミノ酸がT、C、L、M、A、S又はN、アミノ酸置換による特性変化が脱アミド酵素化;
(129)変異点がY291、置換後のアミノ酸がH、T、E又はG、アミノ酸置換による特性変化が脱アミド酵素化;
(130)変異点がW298、置換後のアミノ酸がF、Y、I、L又はM、アミノ酸置換による特性変化が脱アミド酵素化;
(131)変異点がS299、置換後のアミノ酸がN又はP、アミノ酸置換による特性変化が脱アミド酵素化;
(132)変異点がY302、置換後のアミノ酸がL、H、M、V、C、T、P、S又はW、アミノ酸置換による特性変化が脱アミド酵素化;
(133)変異点がS303、置換後のアミノ酸がI、Q、D、H又はE、アミノ酸置換による特性変化が脱アミド酵素化;
(134)変異点がF305、置換後のアミノ酸がW、H又はY、アミノ酸置換による特性変化が脱アミド酵素化。
[2]前記同一性が82%以上である、[1]に記載の改変型トランスグルタミナーゼ。
[3]前記同一性が85%以上である、[1]に記載の改変型トランスグルタミナーゼ。
[4]前記同一性が90%以上である、[1]に記載の改変型トランスグルタミナーゼ。
[5]配列番号2~10のいずれかのアミノ酸配列からなる、[1]に記載の改変型トランスグルタミナーゼ。
[6][1]~[5]のいずれか一項に記載の改変型トランスグルタミナーゼをコードする遺伝子。
[7]配列番号18~26のいずれかの塩基配列を含む、[6]に記載の遺伝子。
[8][6]又は[7]に記載の遺伝子を含む組換えDNA。
[9][8]に記載の組換えDNAを保有する微生物。
[10][1]~[5]のいずれか一項に記載の改変型トランスグルタミナーゼを含む酵素剤。
[11]以下のステップ(I)~(III)を含む、改変型トランスグルタミナーゼの調製法:
(I)[1]~[5]のいずれか一項の改変型トランスグルタミナーゼのアミノ酸配列をコードする核酸を用意するステップ;
(II)前記核酸を発現させるステップ、及び
(III)発現産物を回収するステップ。
[12]前記アミノ酸配列が配列番号2~10のいずれかのアミノ酸配列である、[11]に記載の調製法。
[13]前記核酸が、配列番号18~26のいずれかの塩基配列を含む、[12]に記載の調製法。
(用語)
用語「改変型トランスグルタミナーゼ」とは、基準となるトランスグルタミナーゼ(以下、「基準トランスグルタミナーゼ」と呼ぶ)を改変ないし変異して得られる酵素である。基準トランスグルタミナーゼは、典型的には、配列番号1のアミノ酸配列を有する、ストレプトマイセス・モバラエンシス(Streptomyces mobaraensis)由来のトランスグルタミナーゼである。
メチオニン:M、セリン:S、アラニン:A、トレオニン:T、バリン:V、チロシン:Y、ロイシン:L、アスパラギン:N、イソロイシン:I、グルタミン:Q、プロリン:P、アスパラギン酸:D、フェニルアラニン:F、グルタミン酸:E、トリプトファン:W、リジン:K、システイン:C、アルギニン:R、グリシン:G、ヒスチジン:H
本発明の第1の局面は改変型トランスグルタミナーゼ(以下、「改変型酵素」と呼ぶ)に関する。本発明の改変型酵素は、典型的には、配列番号1のアミノ酸配列において、一又は二以上の特定のアミノ酸置換(変異)を含むアミノ酸配列を有する。当該特徴により、配列番号1のアミノ酸配列からなるトランスグルタミナーゼに対して、温度安定性低下、耐熱性向上、抗酸化性向上、反応性向上、脱アミド酵素化(トランスグルタミナーゼ活性に対する脱アミド活性の比率の向上)或いはこれらの中の二つ以上の特性変化が認められる。温度安定性が低下した改変型酵素は酵素の熱失活工程における食品の変性防止という実用上の利点があり、例えばヨーグルトやチーズの製造等の用途での利用価値が高い。一方、耐熱性が向上した改変型酵素は、高温下においても高い活性を発揮し、例えばペプチド化合物の合成等の用途での使用に特に適する。また、抗酸化性が向上した改変型酵素は、高い保存安定性を有する等の利点があり、酵素の製造、保存、使用の際の失活を防ぐ効果が期待できる。反応性が向上した改変型酵素については、高い反応効率を望める、酵素使用量を低減できる等の利点があり、用途を問わずその利用価値が高い。脱アミド酵素化は基質特異性を改変するものであり、特に、用途の拡大(例えばタンパク質の可溶化、乳化、起泡性の向上等の用途への利用)ないし創出に繋がる。トランスグルタミナーゼはペプチド鎖内にあるグルタミン残基のγ-カルボキシルアミド基と1級アミンとの間のアシル転移反応を触媒する酵素であるが、1級アミンが存在しないと水がアシル受容体として作用し、グルタミン残基をグルタミン酸残基に変換する脱アミド反応を触媒することになる。脱アミド酵素化された改変型酵素は高い脱アミド化触媒能を示し、脱アミド化に利用される酵素としてより好ましいものとなる。尚、配列番号1のアミノ酸配列は、ストレプトマイセス・モバラエンシス由来のトランスグルタミナーゼの配列である。
活性残存率(%)=(熱処理後の酵素活性)/(熱処理前の酵素活性) ×100
活性残存率(%)=(過酸化水素処理後の酵素活性)/(処理前の酵素活性) ×100
<温度安定性低下に有効なアミノ酸置換>
(1)変異点がV6、置換後のアミノ酸がQ、I、M、S、C、K、L、H、F、G、N、P、R、W又はY
(2)変異点がR26、置換後のアミノ酸がK、Q、M、H、Y、D、G、N、P又はS
(3)変異点がE28、置換後のアミノ酸がV、Q、W、R、K、M、N、F、G、L、P又はY
(4)変異点がV30、置換後のアミノ酸がP、C、A、E、F、G、H、K、N、Q、R、W、Y又はL
(5)変異点がY34、置換後のアミノ酸がA
(6)変異点がY42、置換後のアミノ酸がF、A、C、D、E、G、I、L、M、Q、S、T、V又はW
(7)変異点がE58、置換後のアミノ酸がY、M、A、F、I、V、R、K、N、L、S、Q、G又はH
(8)変異点がW59、置換後のアミノ酸がR、N、Y、A、S、I、V、D、G又はP
(9)変異点がL60、置換後のアミノ酸がI、M、V、A、C、E、F、Q、S、T、W又はY
(10)変異点がY62、置換後のアミノ酸がC、R、G、K又はS
(11)変異点がV65、置換後のアミノ酸がN、L、M、F、W又はY
(12)変異点がV67、置換後のアミノ酸がL、N、A、C、M、Q又はS
(13)変異点がT68、置換後のアミノ酸がC、L、A、S、M、F、N、Q又はY
(14)変異点がW69、置換後のアミノ酸がH、M、I、C、E、F、G、K、L、N、Q、R、S、T又はV
(15)変異点がQ74、置換後のアミノ酸がW、D、G又はK
(16)変異点がY75、置換後のアミノ酸がR、Q、T又はG
(17)変異点がT77、置換後のアミノ酸がM、H、E、C又はG
(18)変異点がF85、置換後のアミノ酸がM
(19)変異点がF90、置換後のアミノ酸がC、M、H、L又はV
(20)変異点がF108、置換後のアミノ酸がR、L、T、A、I、K、N又はV
(21)変異点がF117、置換後のアミノ酸がM又はL
(22)変異点がS199、置換後のアミノ酸がG、M、N、K又はV
(23)変異点がF202、置換後のアミノ酸がL又はW
(24)変異点がW203、置換後のアミノ酸がF
(25)変異点がF254、置換後のアミノ酸がY又はM
(26)変異点がT273、置換後のアミノ酸がI、V、M、C、S、L、R、G、A、E、F、Y、D、K、W又はH
(27)変異点がN276、置換後のアミノ酸がC、E、K、L、S、T又はV
(28)変異点がY278、置換後のアミノ酸がM、L、H、I、K、R、W、C、G、N、Q、S、T又はV
(29)変異点がS284、置換後のアミノ酸がW、Y、M、F又はN
(30)変異点がY291、置換後のアミノ酸がI、W、L、A、C、K、N、Q、R、S又はV
(31)変異点がS299、置換後のアミノ酸がI、Y、V、K、M、Q、A、F、G又はE
(32)変異点がS303、置換後のアミノ酸がN、G、C、V、P又はY
(33)変異点がY310、置換後のアミノ酸がC、M又はI
V6S、V6C、V6K、V6L、V6H、V6F、V6G、V6N、V6P、V6R、V6W、V6Y
R26M、R26H、R26Y、R26D、R26G、R26N、R26P、R26S
E28N、E28F、E28G、E28L、E28P、E28Y
V30P、V30C、V30A、V30E、V30F、V30G、V30H、V30K、V30N、V30Q、V30R、V30W、V30Y、V30L
Y34A
Y42A、Y42C、Y42D、Y42E、Y42G、Y42I、Y42L、Y42M、Y42Q、Y42S、Y42T、Y42V、Y42W
E58G、E58H
W59S、W59I、W59V、W59D、W59G、W59P
L60M、L60V、L60A、L60C、L60E、L60F、L60Q、L60S、L60T、L60W、L60Y
Y62C、Y62R、Y62G、Y62K、Y62S
V65N、V65L、V65M、V65F、V65W、V65Y
V67L、V67N、V67A、V67C、V67M、V67Q、V67S
T68M、T68F、T68N、T68Q、T68Y
W69M、W69I、W69C、W69E、W69F、W69G、W69K、W69L、W69N、W69Q、W69R、W69S、W69T、W69V
Y75T、Y75G
F85M
F90V
F108T、F108A、F108I、F108K、F108N、F108V
F117L
S199K、S199V
F202W
T273L、T273R、T273G、T273A、T273E、T273F、T273Y、T273D、T273K、T273W、T273H
N276C、N276E、N276K、N276L、N276T、N276V、N276S
Y278L、Y278H、Y278I、Y278K、Y278R、Y278W、Y278C、Y278G、Y278N、Y278Q、Y278S、Y278T、Y278V
Y291W、Y291L、Y291A、Y291C、Y291K、Y291N、Y291Q、Y291R、Y291S、Y291V
S299E
Y310I
V6H、V6F、V6G、V6N、V6P、V6R、V6W、V6Y
R26H、R26Y、R26D、R26G、R26N、R26P、R26S
E28F、E28G、E28L、E28P、E28Y
V30C、V30A、V30E、V30F、V30G、V30H、V30K、V30N、V30Q、V30R、V30W、V30Y、V30L
Y34A
Y42A、Y42C、Y42D、Y42E、Y42G、Y42I、Y42L、Y42M、Y42Q、Y42S、Y42T、Y42V、Y42W
W59I、W59V、W59D、W59G、W59P
L60V、L60A、L60C、L60E、L60F、L60Q、L60S、L60T、L60W、L60Y
Y62C、Y62R、Y62G、Y62K、Y62S
V65M、V65F、V65W、V65Y
V67A、V67C、V67M、V67Q、V67S
T68F、T68N、T68Q、T68Y
W69M、W69I、W69C、W69E、W69F、W69G、W69K、W69L、W69N、W69Q、W69R、W69S、W69T、W69V
Y75T、Y75G
F108A、F108I、F108K、F108N、F108V
F117L
F202W
T273F、T273Y、T273D、T273K、T273W、T273H
N276C、N276E、N276K、N276L、N276S、N276T、N276V
Y278K、Y278R、Y278W、Y278C、Y278G、Y278N、Y278Q、Y278S、Y278T、Y278V
Y291L、Y291A、Y291C、Y291K、Y291N、Y291Q、Y291R、Y291S、Y291V
S299E
V6H、V6G、V6N、V6W
R26H、R26Y、R26N、R26S
E28F、E28G、E28L、E28Y
V30C、V30A、V30E、V30F、V30G、V30H、V30K、V30N、V30Q、V30R、V30W、V30Y、V30L
Y42C、Y42L、Y42M、Y42Q、Y42S、Y42W
W59I、W59V、W59G
L60V、L60C、L60F
Y62C、Y62R
V67C、V67M
W69M、W69I、W69C、W69F、W69L、W69T、W69V
Y75T
T273F、T273Y、T273H
Y278W
Y291L、Y291K、Y291R
(34)変異点がD3、置換後のアミノ酸がQ、P、E、S又はY
(35)変異点がR26、置換後のアミノ酸がV
(36)変異点がY34、置換後のアミノ酸がW
(37)変異点がV67、置換後のアミノ酸がH
(38)変異点がT68、置換後のアミノ酸がV又はI
(39)変異点がQ74、置換後のアミノ酸がF
(40)変異点がT77、置換後のアミノ酸がQ
(41)変異点がS199、置換後のアミノ酸がC又はQ
(42)変異点がT273、置換後のアミノ酸がQ
(43)変異点がS284、置換後のアミノ酸がL、H、K、P又はR
(44)変異点がS299、置換後のアミノ酸がN
(45)変異点がS303、置換後のアミノ酸がK
D3P、D3E、D3S、D3Y
R26V
Y34W
V67H
T77Q
S199C、S199Q
T273Q
S284H、S284K、S284P、S284R
S303K
D3E、D3S、D3Y
R26V
Y34W
V67H
S199Q
T273Q
S284H、S284K、S284P、S284R
D3E、D3S、D3Y
R26V
Y34W
T273Q
S284H、S284K、S284P、S284R
(46)変異点がD3、置換後のアミノ酸がE、Q、S又はY
(47)変異点がV6、置換後のアミノ酸がP
(48)変異点がR26、置換後のアミノ酸がM、K、W、C、Q、G、Y、E、T、N、D、I、S、P又はV
(49)変異点がE28、置換後のアミノ酸がL、M、K、C、V、R、W、G、N、F、Y又はH
(50)変異点がY42、置換後のアミノ酸がL、N又はF
(51)変異点がE58、置換後のアミノ酸がQ、A、I、V、L、T、M、K、Y、W、F又はR
(52)変異点がV67、置換後のアミノ酸がT、S又はA
(53)変異点がT68、置換後のアミノ酸がL、I、V又はM
(54)変異点がQ74、置換後のアミノ酸がV
(55)変異点がT77、置換後のアミノ酸がD
(56)変異点がS199、置換後のアミノ酸がC
(57)変異点がT273、置換後のアミノ酸がE
(58)変異点がS284、置換後のアミノ酸がR又はH
(59)変異点がY291、置換後のアミノ酸がI、S、F、L、C、N、V又はM
D3S、D3Y
V6P
R26W、R26C、R26Q、R26G、R26Y、R26E、R26T、R26N、R26D、R26I、R26S、R26P、R26V
E28L、E28M、E28K、E28C、E28V、E28R、E28W、E28G、E28N、E28F、E28Y、E28H
E58A、E58I、E58V、E58L、E58T、E58M、E58K、E58Y、E58W、E58F、E58R
V67S、V67A
T68I、T68V、T68M
T273E
S284R、S284H
Y291C、Y291N、Y291V、Y291M
V6P
R26C、R26Q、R26G、R26Y、R26E、R26T、R26N、R26D、R26I、R26S、R26P、R26V
E28V、E28R、E28W、E28G、E28N、E28F、E28Y、E28H
Y42F
E58I、E58V、E58L、E58T、E58M、E58K、E58Y、E58W、E58F、E58R
T68I、T68V、T68M
S284H
R26C、R26Q、R26Y、R26E、R26T、R26N、R26I、R26S、R26V
E28V、E28R、E28W、E28G、E28N、E28F、E28Y
Y42F
E58I、E58V、E58L、E58T、E58M、E58K、E58Y、E58W、E58F、E58R
S284H
(60)変異点がS284、置換後のアミノ酸がM、K又はV
(61)変異点がF251、置換後のアミノ酸がY
(62)変異点がV6とY75、変異点V6の置換後のアミノ酸がE、変異点Y75の置換後のアミノ酸がF
(63)変異点がD3とT77、変異点D3の置換後のアミノ酸がP、変異点T77の置換後のアミノ酸がQ
S284V
F251Y
V6EとY75Fの二重変異
D3PとT77Qの二重変異
V6EとY75Fの二重変異
D3PとT77Qの二重変異
(64)変異点がD3、置換後のアミノ酸がQ、S又はY、アミノ酸置換による特性変化が耐熱性向上及び抗酸化性向上
(65)変異点がD3、置換後のアミノ酸がP、アミノ酸置換による特性変化が耐熱性向上及び反応性向上
(66)変異点がD3、置換後のアミノ酸がE、アミノ酸置換による特性変化が耐熱性向上、抗酸化性向上及び反応性向上
(67)変異点がV6、置換後のアミノ酸がP、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上
(68)変異点がR26、置換後のアミノ酸がK、Q、M、Y、D、G、N、P又はS、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上
(69)変異点がR26、置換後のアミノ酸がV、アミノ酸置換による特性変化が耐熱性向上及び抗酸化性向上
(70)変異点がR26、置換後のアミノ酸がH、アミノ酸置換による特性変化が温度安定性低下、抗酸化性向上及び反応性向上
(71)変異点がE28、置換後のアミノ酸がV、R、K、M、N、F、G、L又はY、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上
(72)変異点がV30、置換後のアミノ酸がP、G、N、R又はY、アミノ酸置換による特性変化が温度安定性低下及び反応性向上
(73)変異点がY42、置換後のアミノ酸がF、L又はM、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上
(74)変異点がE58、置換後のアミノ酸がY、M、A、F、I、V、R、K、L又はQ、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上
(75)変異点がL60、置換後のアミノ酸がI、アミノ酸置換による特性変化が温度安定性低下及び反応性向上
(76)変異点がV67、置換後のアミノ酸がA又はS、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上
(77)変異点がT68、置換後のアミノ酸がC、L又はM、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上
(78)変異点がT68、置換後のアミノ酸がV又はI、アミノ酸置換による特性変化が耐熱性向上及び抗酸化性向上
(79)変異点がQ74、置換後のアミノ酸がV、アミノ酸置換による特性変化が抗酸化性向上及び反応性向上
(80)変異点がT77、置換後のアミノ酸がC、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上
(81)変異点がT77、置換後のアミノ酸がE、アミノ酸置換による特性変化が温度安定性低下及び反応性向上
(82)変異点がT77、置換後のアミノ酸がD、アミノ酸置換による特性変化が抗酸化性向上及び反応性向上
(83)変異点がS199、置換後のアミノ酸がG、アミノ酸置換による特性変化が温度安定性低下及び反応性向上
(84)変異点がS199、置換後のアミノ酸がC、アミノ酸置換による特性変化が耐熱性向上、抗酸化性向上及び反応性向上
(85)変異点がT273、置換後のアミノ酸がE、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上
(86)変異点がS284、置換後のアミノ酸がM又はF、アミノ酸置換による特性変化が温度安定性低下及び反応性向上
(87)変異点がS284、置換後のアミノ酸がL又はK、アミノ酸置換による特性変化が耐熱性向上及び反応性向上
(88)変異点がS284、置換後のアミノ酸がH又はR、アミノ酸置換による特性変化が耐熱性向上及び抗酸化性向上
(89)変異点がY291、置換後のアミノ酸がI、L、C、N、S又はV、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上
(90)変異点がS299、置換後のアミノ酸がV、K、M又はA、アミノ酸置換による特性変化が温度安定性低下及び反応性向上
(91)変異点がS303、置換後のアミノ酸がK、アミノ酸置換による特性変化が耐熱性向上及び反応性向上
V6P
R26K、R26Q、R26M、R26Y、R26D、R26G、R26N、R26P、R26S
E28V、E28R、E28K、E28M、E28N、E28F、E28G、E28L、E28Y
Y42F、Y42L、Y42M
E58Y、E58M、E58A、E58F、E58I、E58V、E58R、E58K、E58L、E58Q
V67A、V67S
T68C、T68L、T68M
T77C
T273E
Y291I、Y291L、Y291C、Y291N、Y291S、Y291V
(92)変異点がR5、置換後のアミノ酸がQ、P、C、M、I、V、Y、F、S、N、T又はG
(93)変異点がY34、置換後のアミノ酸がH又はM
(94)変異点がW59、置換後のアミノ酸がK又はE
(95)変異点がS61、置換後のアミノ酸がT、V、Y、D、I、L、N、E、M、F、W、Q、P、H又はK
(96)変異点がY62、置換後のアミノ酸がD又はP
(97)変異点がG63、置換後のアミノ酸がD、Q、Y、R、K、H、P、M、N、C、F、L、W、T、V又はS
(98)変異点がV65、置換後のアミノ酸がC、T又はK
(99)変異点がG66、置換後のアミノ酸がW又はK
(100)変異点がT68、置換後のアミノ酸がP又はH
(101)変異点がW69、置換後のアミノ酸がY、D又はP
(102)変異点がQ74、置換後のアミノ酸がR又はP
(103)変異点がF85、置換後のアミノ酸がY、W、L、I、Q、C、T、A、V、S、R、H又はN
(104)変異点がF108、置換後のアミノ酸がQ、C、E、S又はN
(105)変異点がF117、置換後のアミノ酸がC、I、W、V、A、G、T又はN
(106)変異点がY198、置換後のアミノ酸がF、H、L、S、M又はI
(107)変異点がS199、置換後のアミノ酸がQ又はP
(108)変異点がK200、置換後のアミノ酸がY、M、I、R、L又はV
(109)変異点がH201、置換後のアミノ酸がK、R、M、L又はQ
(110)変異点がF202、置換後のアミノ酸がY、M、H、V又はC
(111)変異点がW203、置換後のアミノ酸がY
(112)変異点がF223、置換後のアミノ酸がY、M、L、W、V、H、G又はA
(113)変異点がR238、置換後のアミノ酸がA、D、V、G、H又はS
(114)変異点がF251、置換後のアミノ酸がH、N、C、M、R、S、A、T、L、P、G、V又はQ
(115)変異点がV252、置換後のアミノ酸がM、L、T又はC
(116)変異点がN253、置換後のアミノ酸がT、V、K、C又はR
(117)変異点がY256、置換後のアミノ酸がL、V又はI
(118)変異点がG257、置換後のアミノ酸がA又はC
(119)変異点がW258、置換後のアミノ酸がY、F又はN
(120)変異点がT273、置換後のアミノ酸がN
(121)変異点がG275、置換後のアミノ酸がA、C、S、V、K、I、F、H、R、L、P、Q、Y、N、M又はT
(122)変異点がN276、置換後のアミノ酸がG、M、Q又はH
(123)変異点がH277、置換後のアミノ酸がN、S、Q、C、E、K、D、I、R、M、P、L、W、V、Y、G、F又はT
(124)変異点がY278、置換後のアミノ酸がP又はD
(125)変異点がH279、置換後のアミノ酸がN
(126)変異点がS284、置換後のアミノ酸がC
(127)変異点がM288、置換後のアミノ酸がV、Q又はT
(128)変異点がV290、置換後のアミノ酸がT、C、L、M、A、S又はN
(129)変異点がY291、置換後のアミノ酸がH、T、E又はG
(130)変異点がW298、置換後のアミノ酸がF、Y、I、L又はM
(131)変異点がS299、置換後のアミノ酸がN又はP
(132)変異点がY302、置換後のアミノ酸がL、H、M、V、C、T、P、S又はW
(133)変異点がS303、置換後のアミノ酸がI、Q、D、H又はE
(134)変異点がF305、置換後のアミノ酸がW、H又はY
R5Q、R5P、R5C、R5M、R5I、R5V、R5Y、R5F、R5S、R5N、R5T、R5G
Y34H、Y34M
W59K、W59E
S61T、S61V、S61Y、S61D、S61I、S61L、S61N、S61E、S61M、S61F、S61W、S61Q、S61P、S61H、S61K
Y62D、Y62P
G63D、G63Q、G63Y、G63R、G63K、G63H、G63P、G63M、G63N、G63C、G63F、G63L、G63W、G63T、G63V、G63S
V65C、V65T、V65K
G66W、G66K
T68P、T68H
W69Y、W69D、W69P
Q74R、Q74P
F85Y、F85W、F85L、F85I、F85Q、F85C、F85T、F85A、F85V、F85S、F85R、F85H、F85N
F108Q、F108C、F108E、F108S、F108N
F117C、F117I、F117W、F117V、F117A、F117G、F117T、F117N
Y198F、Y198H、Y198L、Y198S、Y198M、Y198I
S199Q、S199P
K200Y、K200M、K200I、K200R、K200L、K200V
H201K、H201R、H201M、H201L、H201Q
F202Y、F202M、F202H、F202V、F202C
W203Y
F223Y、F223M、F223L、F223W、F223V、F223H、F223G、F223A
R238A、R238D、R238V、R238G、R238H、R238S
F251H、F251N、F251C、F251M、F251R、F251S、F251A、F251T、F251L、F251P、F251G、F251V、F251Q
V252M、V252L、V252T、V252C
N253T、N253V、N253K、N253C、N253R
Y256L、Y256V、Y256I
G257A、G257C
W258Y、W258F、W258N
T273N
G275A、G275C、G275S、G275V、G275K、G275I、G275F、G275H、G275R、G275L、G275P、G275Q、G275Y、G275N、G275M、G275T
N276G、N276M、N276Q、N276H
H277N、H277S、H277Q、H277C、H277E、H277K、H277D、H277I、H277R、H277M、H277P、H277L、H277W、H277V、H277Y、H277G、H277F、H277T
Y278P、Y278D
H279N
S284C
M288V、M288Q、M288T
V290T、V290C、V290L、V290M、V290A、V290S、V290N
Y291H、Y291T、Y291E、Y291G
W298F、W298Y、W298I、W298L、W298M
S299N、S299P
Y302L、Y302H、Y302M、Y302V、Y302C、Y302T、Y302P、Y302S、Y302W
S303I、S303Q、S303D、S303H、S303E
F305W、F305H、F305Y
R5Y、R5F、R5S、R5N、R5T、R5G
Y34M
S61T、S61V、S61Y、S61D、S61I、S61L、S61N、S61E、S61M、S61F、S61W、S61Q、S61P、S61H、S61K
Y62P
G63D、G63Q、G63Y、G63R、G63K、G63H、G63P、G63M、G63N、G63C、G63F、G63L、G63W、G63T、G63V、G63S
V65C、V65T、V65K
G66K
T68P、T68H
W69D、W69P
F85L、F85I、F85Q、F85C、F85T、F85A、F85V、F85S、F85R、F85H、F85N
F108C、F108E、F108S、F108N
F117C、F117I、F117W、F117V、F117A、F117G、F117T、F117N
Y198L、Y198S、Y198M、Y198I
S199Q、S199P
K200Y、K200M、K200I、K200R、K200L、K200V
H201K、H201R、H201M、H201L、H201Q
F202M、F202H、F202V、F202C
W203Y
F223M、F223L、F223W、F223V、F223H、F223G、F223A
R238H、R238S
F251N、F251C、F251M、F251R、F251S、F251A、F251T、F251L、F251P、F251G、F251V、F251Q
V252L、V252T、V252C
N253T、N253V、N253K、N253C、N253R
Y256L、Y256V、Y256I
G257A、G257C
W258Y、W258F、W258N
T273N
G275C、G275S、G275V、G275K、G275I、G275F、G275H、G275R、G275L、G275P、G275Q、G275Y、G275N、G275M、G275T
N276G、N276M、N276Q、N276H
H277S、H277Q、H277C、H277E、H277K、H277D、H277I、H277R、H277M、H277P、H277L、H277W、H277V、H277Y、H277G、H277F、H277T
Y278P、Y278D
M288Q、M288T
V290M、V290A、V290S、V290N
Y291T、Y291E、Y291G
W298F、W298Y、W298I、W298L、W298M
S299P
Y302L、Y302H、Y302M、Y302V、Y302C、Y302T、Y302P、Y302S、Y302W
F305H、F305Y
S61Y、S61D、S61I、S61L、S61N、S61E、S61M、S61F、S61W、S61Q、S61P、S61H、S61K
Y62P
G63D、G63Q、G63Y、G63R、G63K、G63H、G63P、G63M、G63N、G63C、G63F、G63L、G63W、G63T、G63V、G63S
V65T、V65K
G66K
T68P、T68H
W69D、W69P
F85H、F85N
F117G、F117T、F117N
Y198S、Y198M、Y198I
S199P
K200M、K200I、K200R、K200L、K200V
H201M、H201L、H201Q
F202V、F202C
W203Y
F223W、F223V、F223H、F223G、F223A
F251L、F251P、F251G、F251V、F251Q
N253T、N253V、N253K、N253C、N253R
Y256L、Y256V、Y256I
G257C
W258N
G275V、G275K、G275I、G275F、G275H、G275R、G275L、G275P、G275Q、G275Y、G275N、G275M、G275T
N276G、N276M、N276Q、N276H
H277S、H277Q、H277C、H277E、H277K、H277D、H277I、H277R、H277M、H277P、H277L、H277W、H277V、H277Y、H277G、H277F、H277T
Y278P、Y278D
M288T
V290S、V290N
Y291G
W298Y、W298I、W298L、W298M
F305Y
(1)タンパク質を結晶化する。結晶化は、立体構造決定のためには欠かせないが、それ以外にも、タンパク質の高純度の精製法、高密度で安定な保存法として産業上の有用性もある。この場合、リガンドとして基質もしくはそのアナログ化合物を結合したタンパク質を結晶化すると良い。
(2)作製した結晶にX線を照射して回折データを収集する。なお、タンパク質結晶はX線照射によりダメージを受け回折能が劣化するケースが多々ある。その場合、結晶を急激に-173℃程度に冷却し、その状態で回折データを収集する低温測定技術が最近普及してきた。なお、最終的に、構造決定に利用する高分解能データを収集するために、輝度の高いシンクロトロン放射光が利用される。
(3)結晶構造解析を行うには、回折データに加えて、位相情報が必要になる。目的のタンパク質に対して、類縁のタンパク質の結晶構造が未知の場合、分子置換法で構造決定することは不可能であり、重原子同型置換法により位相問題が解決されなくてはならない。重原子同型置換法は、水銀や白金等原子番号が大きな金属原子を結晶に導入し、金属原子の大きなX線散乱能のX線回折データへの寄与を利用して位相情報を得る方法である。決定された位相は、結晶中の溶媒領域の電子密度を平滑化することにより改善することが可能である。溶媒領域の水分子は揺らぎが大きいために電子密度がほとんど観測されないので、この領域の電子密度を0に近似することにより、真の電子密度に近づくことができ、ひいては位相が改善されるのである。また、非対称単位に複数の分子が含まれている場合、これらの分子の電子密度を平均化することにより位相が更に大幅に改善される。このようにして改善された位相を用いて計算した電子密度図にタンパク質のモデルをフィットさせる。このプロセスは、コンピューターグラフィックス上で、MSI社(アメリカ)のQUANTA等のプログラムを用いて行われる。この後、MSI社のX-PLOR等のプログラムを用いて、構造精密化を行い、構造解析は完了する。目的のタンパク質に対して、類縁のタンパク質の結晶構造が既知の場合は、既知タンパク質の原子座標を用いて分子置換法により決定できる。分子置換と構造精密化はプログラム CNS_SOLVE ver.11などを用いて行うことができる。
本発明の第2の局面は本発明の改変型酵素に関連する核酸を提供する。即ち、改変型酵素をコードする遺伝子、改変型酵素をコードする核酸を同定するためのプローブとして用いることができる核酸、改変型酵素をコードする核酸を増幅又は突然変異等させるためのプライマーとして用いることができる核酸が提供される。
配列番号18:R26N変異体
配列番号19:R26Y変異体
配列番号20:R26S変異体
配列番号21:E28N変異体
配列番号22:E28G変異体
配列番号23:E28F変異体
配列番号24:E28Y変異体
配列番号25:E58R変異体
配列番号26:E58L変異体
本発明の改変型酵素は例えば酵素剤の形態で提供される。酵素剤は、有効成分(本発明の改変型酵素)の他、賦形剤、緩衝剤、懸濁剤、安定剤、保存剤、防腐剤、生理食塩水、各種タンパク質、各種タンパク質の分解物、各種エキス類、各種塩類、各種酸化防止剤、システイン、グルタチオン、グルタミン酸ナトリウム、イノシン酸ナトリウム、グアニル酸ナトリウム、貝殻焼成カルシウム、二酸化ケイ素などを含有していてもよい。賦形剤としてはデンプン、デキストリン、マルトース、トレハロース、乳糖、D-グルコース、ソルビトール、D-マンニトール、白糖、グリセロール等を用いることができる。緩衝剤としてはリン酸塩、クエン酸塩、酢酸塩等を用いることができる。安定剤としてはプロピレングリコール、アスコルビン酸等を用いることができる。保存剤としてはフェノール、塩化ベンザルコニウム、ベンジルアルコール、クロロブタノール、メチルパラベン等を用いることができる。防腐剤としてはエタノール、塩化ベンザルコニウム、パラオキシ安息香酸、クロロブタノール等を用いることができる。タンパク質の例は、大豆タンパク質、小麦タンパク質、トウモロコシタンパク質、乳タンパク質、動物由来タンパク質などである。エキス類の例は肉エキス、植物エキス、酵母エキスなどである。塩類の例は、塩化塩、リン酸塩、ポリリン酸塩、ピロリン酸塩、クエン酸塩、乳酸塩、炭酸塩などである。酸化防止剤の例は、L-アスコルビン酸塩、亜硫酸水素ナトリウムなどである。本発明の酵素製剤の形状は特に限定されず、例えば粉末状、顆粒状、液体状、カプセル状等である。
本発明の更なる局面は改変型酵素の調製法に関する。本発明の調製法の一態様では、本発明の改変型酵素を遺伝子工学的手法で調製する。この態様の場合、本発明の改変型酵素のアミノ酸配列(例えば配列番号2~10のいずれか)をコードする核酸を用意する(ステップ(I))。ここで、「特定のアミノ酸配列をコードする核酸」は、それを発現させた場合に当該アミノ酸配列を有するポリペプチドが得られる核酸であり、当該アミノ酸配列に対応する塩基配列からなる核酸は勿論のこと、そのような核酸に余分な配列(アミノ酸配列をコードする配列であっても、アミノ酸配列をコードしない配列であってもよい)が付加されていてもよい。また、コドンの縮重も考慮される。「本発明の改変型酵素のアミノ酸配列をコードする核酸」は、本明細書又は添付の配列表が開示する配列情報を参考にし、標準的な遺伝子工学的手法、分子生物学的手法、生化学的手法などを用いることによって、単離された状態に調製することができる。ここで、本発明の改変型酵素のアミノ酸配列は基準トランスグルタミナーゼのアミノ酸配列に変異を施したものである。従って、基準トランスグルタミナーゼをコードする遺伝子に対して必要な変異を加えることによっても、「本発明の改変型酵素のアミノ酸配列をコードする核酸」を得ることができる。位置特異的塩基配列置換のための方法は当該技術分野において数多く知られており(例えば、Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, New Yorkを参照)、その中から適切な方法を選択して用いることができる。位置特異的変異導入法として、位置特異的アミノ酸飽和変異法を採用することができる。位置特異的アミノ酸飽和変異法は、タンパクの立体構造を基に、求める機能の関与する位置を推定し、アミノ酸飽和変異を導入する「Semi-rational,semi-random」手法である(J.Mol.Biol.331,585-592(2003))。例えば、Quick change(ストラタジーン社)等のキット、Overlap extention PCR(Nucleic Acid Res. 16,7351-7367(1988))を用いて位置特異的アミノ酸飽和変異を導入することが可能である。PCRに用いるDNAポリメラーゼはTaqポリメラーゼ等を用いることができる。但し、KOD-PLUS-(東洋紡社)、Pfu turbo(ストラタジーン社)などの精度の高いDNAポリメラーゼを用いることが好ましい。
有用性の高い改変型のトランスグルタミナーゼの取得を目指し、蛋白質工学を用い、ストレプトマイセス・モバラエンシス由来のトランスグルタミナーゼ(野生型酵素。配列番号1)の酵素機能の改良(温度安定性の低減、耐熱性の向上、抗酸化性の向上、反応性の向上、脱アミド酵素化)を試みた。まず、アミノ酸置換による変異を導入することにし、CASTing library(例えばAngew Chem Int Ed Engl. 2006 Feb 13;45(8):1236-41.を参照)及びアラニンスキャニング(Ala scanning)(例えばJ Mol Biol. 1995 Feb 17;246(2):317-30.を参照)を利用して変異点の候補を選出した。次に、以下の方法で各変異点に変異を導入し、変異酵素を調製した。
1-1.変異の導入
(1)変異導入用のPCRプライマーを設計する。
(2)各変異点用のプライマーセットを用い、トランスグルタミナーゼ遺伝子(配列番号17)が組み込まれたプラスミドpET20bを鋳型としてPCR(98℃で1分の反応の後、98℃で10秒、60℃で15秒、68℃で2分の反応を15サイクル行い、68℃で5分反応させた後、4℃で放置)。
(3)PCR反応液(25μL/チューブ)にDpnI(1.5μL/チューブ)を添加して処理(37℃、3時間以上)する。
(4)T4キナーゼを用い、ライゲーション処理(16℃、一晩)する。
(5)ライゲーション反応液(11μL/チューブ)でE.coli BL21(DE3)を形質転換し、アンピシリン含有LB培地で培養(37℃、一晩)する。
(1)変異が導入された株(変異株)をアンピシリン含有TB培地に植菌し、33℃、48時間培養する。培養開始から24時間の時点でIPTG(最終濃度0.1mM)を添加する。
(2)培養液を遠心分離(3,000 g × 10分)した後、上澄みを除去して菌体を回収する。
(3)溶菌剤を添加して菌体を溶菌させる。
(4)溶菌液を遠心分離(3,000 g × 10分)した後、上澄みを回収して酵素抽出液とする。
(1)酵素抽出液と2mg/mLプロテアーゼ(ディスパーゼ)溶液を等量混合し、反応(30℃、2時間以上)させる。
(2)混合液を遠心分離(3,000 g × 10分)した後、上澄みを回収して成熟体化酵素とする。
調製した各変異酵素の特性を評価する際には以下の活性測定法を用いた。
<活性測定法>
成熟体化酵素を200mM Tris-HCl pH6.0で適当な濃度に希釈する(サンプル溶液)。サンプル溶液10μLに基質溶液(R-1) 100μLを添加し混合した後、37℃で10分間反応させる。発色溶液(R-2) 100μLを加えて反応を停止させるともにFe錯体を形成させた後、525nmの吸光度を測定する。対照として、予め熱失活させた酵素液を用いて同様に反応させたものの吸光度を測定し、サンプル溶液との吸光度差を求める。別途、酵素液の代わりにL-グルタミン酸-γ-モノヒドロキサム酸を用いて検量線を作成し、前記吸光度差より生成されたヒドロキサム酸の量を求める。1分間に1μモルのヒドロキサム酸を生成する酵素活性を1単位(1U)とする。
(基質溶液(R-1))
2-アミノ-2-ヒドロキシメチル-1.3-プロパンジオール 2.42g、塩酸ヒドロキシアンモニウム 0.70g、還元型グルタチオン 0.31g、Z-Gln-Gly (ベンジルオキシカルボニル-L-グルタミニルグリシン) 1.01gを蒸留水に溶解し総量100 mLとする(pH 6.0)。
(基質溶液(R-2))
3M 塩酸溶液 30 mL、12% トリクロロ酢酸溶液 30 mL、5% 塩化鉄(III)溶液 30 mLを混合する。
成熟体化酵素を200mM Tris-HCl pH6.0で2倍希釈し、所定の温度(20℃、30℃、40℃、50℃、60℃)にて30分間処理する。処理後に活性測定し、処理前の活性と比較することで、活性の残存率を算出する。
活性残存率(%)=(熱処理後の酵素活性)/(熱処理前の酵素活性)×100
対野生型(%)=(変異酵素の活性残存率)/(野生型酵素の活性残存率)×100
(1)変異点がV6、置換後のアミノ酸がQ、I、M、S、C、K、L、H、F、G、N、P、R、W又はY
(2)変異点がR26、置換後のアミノ酸がK、Q、M、H、Y、D、G、N、P又はS
(3)変異点がE28、置換後のアミノ酸がV、Q、W、R、K、M、N、F、G、L、P又はY
(4)変異点がV30、置換後のアミノ酸がP、C、A、E、F、G、H、K、N、Q、R、W、Y又はL
(5)変異点がY34、置換後のアミノ酸がA
(6)変異点がY42、置換後のアミノ酸がF、A、C、D、E、G、I、L、M、Q、S、T、V又はW
(7)変異点がE58、置換後のアミノ酸がY、M、A、F、I、V、R、K、N、L、S、Q、G又はH
(8)変異点がW59、置換後のアミノ酸がR、N、Y、A、S、I、V、D、G又はP
(9)変異点がL60、置換後のアミノ酸がI、M、V、A、C、E、F、Q、S、T、W又はY
(10)変異点がY62、置換後のアミノ酸がC、R、G、K又はS
(11)変異点がV65、置換後のアミノ酸がN、L、M、F、W又はY
(12)変異点がV67、置換後のアミノ酸がL、N、A、C、M、Q又はS
(13)変異点がT68、置換後のアミノ酸がC、L、A、S、M、F、N、Q又はY
(14)変異点がW69、置換後のアミノ酸がH、M、I、C、E、F、G、K、L、N、Q、R、S、T又はV
(15)変異点がQ74、置換後のアミノ酸がW、D、G又はK
(16)変異点がY75、置換後のアミノ酸がR、Q、T又はG
(17)変異点がT77、置換後のアミノ酸がM、H、E、C又はG
(18)変異点がF85、置換後のアミノ酸がM
(19)変異点がF90、置換後のアミノ酸がC、M、H、L又はV
(20)変異点がF108、置換後のアミノ酸がR、L、T、A、I、K、N又はV
(21)変異点がF117、置換後のアミノ酸がM又はL
(22)変異点がS199、置換後のアミノ酸がG、M、N、K又はV
(23)変異点がF202、置換後のアミノ酸がL又はW
(24)変異点がW203、置換後のアミノ酸がF
(25)変異点がF254、置換後のアミノ酸がY又はM
(26)変異点がT273、置換後のアミノ酸がI、V、M、C、S、L、R、G、A、E、F、Y、D、K、W又はH
(27)変異点がN276、置換後のアミノ酸がC、E、K、L、S、T又はV
(28)変異点がY278、置換後のアミノ酸がM、L、H、I、K、R、W、C、G、N、Q、S、T又はV
(29)変異点がS284、置換後のアミノ酸がW、Y、M、F又はN
(30)変異点がY291、置換後のアミノ酸がI、W、L、A、C、K、N、Q、R、S又はV
(31)変異点がS299、置換後のアミノ酸がI、Y、V、K、M、Q、A、F、G又はE
(32)変異点がS303、置換後のアミノ酸がN、G、C、V、P又はY
(33)変異点がY310、置換後のアミノ酸がC、M又はI
V6S、V6C、V6K、V6L、V6H、V6F、V6G、V6N、V6P、V6R、V6W、V6Y
R26M、R26H、R26Y、R26D、R26G、R26N、R26P、R26S
E28N、E28F、E28G、E28L、E28P、E28Y
V30P、V30C、V30A、V30E、V30F、V30G、V30H、V30K、V30N、V30Q、V30R、V30W、V30Y、V30L
Y34A
Y42A、Y42C、Y42D、Y42E、Y42G、Y42I、Y42L、Y42M、Y42Q、Y42S、Y42T、Y42V、Y42W
E58G、E58H
W59S、W59I、W59V、W59D、W59G、W59P
L60M、L60V、L60A、L60C、L60E、L60F、L60Q、L60S、L60T、L60W、L60Y
Y62C、Y62R、Y62G、Y62K、Y62S
V65N、V65L、V65M、V65F、V65W、V65Y
V67L、V67N、V67A、V67C、V67M、V67Q、V67S
T68M、T68F、T68N、T68Q、T68Y
W69M、W69I、W69C、W69E、W69F、W69G、W69K、W69L、W69N、W69Q、W69R、W69S、W69T、W69V
Y75T、Y75G
F85M
F90V
F108T、F108A、F108I、F108K、F108N、F108V
F117L
S199K、S199V
F202W
T273L、T273R、T273G、T273A、T273E、T273F、T273Y、T273D、T273K、T273W、T273H
N276C、N276E、N276K、N276L、N276T、N276V、N276S
Y278L、Y278H、Y278I、Y278K、Y278R、Y278W、Y278C、Y278G、Y278N、Y278Q、Y278S、Y278T、Y278V
Y291W、Y291L、Y291A、Y291C、Y291K、Y291N、Y291Q、Y291R、Y291S、Y291V
S299E
Y310I
V6H、V6F、V6G、V6N、V6P、V6R、V6W、V6Y
R26H、R26Y、R26D、R26G、R26N、R26P、R26S
E28F、E28G、E28L、E28P、E28Y
V30C、V30A、V30E、V30F、V30G、V30H、V30K、V30N、V30Q、V30R、V30W、V30Y、V30L
Y34A
Y42A、Y42C、Y42D、Y42E、Y42G、Y42I、Y42L、Y42M、Y42Q、Y42S、Y42T、Y42V、Y42W
W59I、W59V、W59D、W59G、W59P
L60V、L60A、L60C、L60E、L60F、L60Q、L60S、L60T、L60W、L60Y
Y62C、Y62R、Y62G、Y62K、Y62S
V65M、V65F、V65W、V65Y
V67A、V67C、V67M、V67Q、V67S
T68F、T68N、T68Q、T68Y
W69M、W69I、W69C、W69E、W69F、W69G、W69K、W69L、W69N、W69Q、W69R、W69S、W69T、W69V
Y75T、Y75G
F108A、F108I、F108K、F108N、F108V
F117L
F202W
T273F、T273Y、T273D、T273K、T273W、T273H
N276C、N276E、N276K、N276L、N276S、N276T、N276V
Y278K、Y278R、Y278W、Y278C、Y278G、Y278N、Y278Q、Y278S、Y278T、Y278V
Y291L、Y291A、Y291C、Y291K、Y291N、Y291Q、Y291R、Y291S、Y291V
S299E
(34)変異点がD3、置換後のアミノ酸がQ、P、E、S又はY
(35)変異点がR26、置換後のアミノ酸がV
(36)変異点がY34、置換後のアミノ酸がW
(37)変異点がV67、置換後のアミノ酸がH
(38)変異点がT68、置換後のアミノ酸がV又はI
(39)変異点がQ74、置換後のアミノ酸がF
(40)変異点がT77、置換後のアミノ酸がQ
(41)変異点がS199、置換後のアミノ酸がC又はQ
(42)変異点がT273、置換後のアミノ酸がQ
(43)変異点がS284、置換後のアミノ酸がL、H、K、P又はR
(44)変異点がS299、置換後のアミノ酸がN
(45)変異点がS303、置換後のアミノ酸がK
D3P、D3E、D3S、D3Y
R26V
Y34W
V67H
T77Q
S199C、S199Q
T273Q
S284H、S284K、S284P、S284R
S303K
D3E、D3S、D3Y
R26V
Y34W
V67H
S199Q
T273Q
S284H、S284K、S284P、S284R
過酸化水素(30~35%)(Wako, 試薬特級)を200mM Tris-HCl pH6.0で希釈し、酸化剤としての過酸化水素溶液(0.03%)を調製する。成熟体酵素を過酸化水素溶液で2倍希釈した後、30℃で1時間反応させる。反応度に活性測定し、処理前の活性と比較することで、活性の残存率を算出する。
活性残存率(%)=(過酸化水素処理後の酵素活性)/(処理前の酵素活性) ×100
対野生型(%)=(変異酵素の活性残存率)/(野生型酵素の活性残存率) ×100
(46)変異点がD3、置換後のアミノ酸がE、Q、S又はY
(47)変異点がV6、置換後のアミノ酸がP
(48)変異点がR26、置換後のアミノ酸がM、K、W、C、Q、G、Y、E、T、N、D、I、S、P又はV
(49)変異点がE28、置換後のアミノ酸がL、M、K、C、V、R、W、G、N、F、Y又はH
(50)変異点がY42、置換後のアミノ酸がL、N又はF
(51)変異点がE58、置換後のアミノ酸がQ、A、I、V、L、T、M、K、Y、W、F又はR
(52)変異点がV67、置換後のアミノ酸がT、S又はA
(53)変異点がT68、置換後のアミノ酸がL、I、V又はM
(54)変異点がQ74、置換後のアミノ酸がV
(55)変異点がT77、置換後のアミノ酸がD
(56)変異点がS199、置換後のアミノ酸がC
(57)変異点がT273、置換後のアミノ酸がE
(58)変異点がS284、置換後のアミノ酸がR又はH
(59)変異点がY291、置換後のアミノ酸がI、S、F、L、C、N、V又はM
D3S、D3Y
V6P
R26W、R26C、R26Q、R26G、R26Y、R26E、R26T、R26N、R26D、R26I、R26S、R26P、R26V
E28L、E28M、E28K、E28C、E28V、E28R、E28W、E28G、E28N、E28F、E28Y、E28H
E58A、E58I、E58V、E58L、E58T、E58M、E58K、E58Y、E58W、E58F、E58R
V67S、V67A
T68I、T68V、T68M
T273E
S284R、S284H
Y291C、Y291N、Y291V、Y291M
V6P
R26C、R26Q、R26G、R26Y、R26E、R26T、R26N、R26D、R26I、R26S、R26P、R26V
E28V、E28R、E28W、E28G、E28N、E28F、E28Y、E28H
Y42F
E58I、E58V、E58L、E58T、E58M、E58K、E58Y、E58W、E58F、E58R
T68I、T68V、T68M
S284H
成熟体化酵素を200mM Tris-HCl pH6.0で適当な濃度に希釈し(サンプル溶液)、活性測定する。野生型に対する相対値を以下の計算式で算出し、反応性の向上に有効なアミノ酸置換を特定した。
対野生型(%)=(変異酵素の活性)/(野生型酵素の活性) ×100
(60)変異点がS284、置換後のアミノ酸がM、K又はV
(61)変異点がF251、置換後のアミノ酸がY
(62)変異点がV6とY75、変異点V6の置換後のアミノ酸がE、変異点Y75の置換後のアミノ酸がF
(63)変異点がD3とT77、変異点D3の置換後のアミノ酸がP、変異点T77の置換後のアミノ酸がQ
S284V
F251Y
V6EとY75Fの二重変異
D3PとT77Qの二重変異
V6EとY75Fの二重変異
D3PとT77Qの二重変異
(64)変異点がD3、置換後のアミノ酸がQ、S又はY、アミノ酸置換による特性変化が耐熱性向上及び抗酸化性向上
(65)変異点がD3、置換後のアミノ酸がP、アミノ酸置換による特性変化が耐熱性向上及び反応性向上
(66)変異点がD3、置換後のアミノ酸がE、アミノ酸置換による特性変化が耐熱性向上、抗酸化性向上及び反応性向上
(67)変異点がV6、置換後のアミノ酸がP、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上
(68)変異点がR26、置換後のアミノ酸がK、Q、M、Y、D、G、N、P又はS、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上
(69)変異点がR26、置換後のアミノ酸がV、アミノ酸置換による特性変化が耐熱性向上及び抗酸化性向上
(70)変異点がR26、置換後のアミノ酸がH、アミノ酸置換による特性変化が温度安定性低下、抗酸化性向上及び反応性向上
(71)変異点がE28、置換後のアミノ酸がV、S、R、K、M、N、F、G、L又はY、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上
(72)変異点がV30、置換後のアミノ酸がP、G、N、R又はY、アミノ酸置換による特性変化が温度安定性低下及び反応性向上
(73)変異点がY42、置換後のアミノ酸がF、L又はM、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上
(74)変異点がE58、置換後のアミノ酸がY、M、A、F、I、V、R、K、L又はQ、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上
(75)変異点がL60、置換後のアミノ酸がI、アミノ酸置換による特性変化が温度安定性低下及び反応性向上
(76)変異点がV67、置換後のアミノ酸がA又はS、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上
(77)変異点がT68、置換後のアミノ酸がC、L又はM、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上
(78)変異点がT68、置換後のアミノ酸がV又はI、アミノ酸置換による特性変化が耐熱性向上及び抗酸化性向上
(79)変異点がQ74、置換後のアミノ酸がV、アミノ酸置換による特性変化が抗酸化性向上及び反応性向上
(80)変異点がT77、置換後のアミノ酸がC、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上
(81)変異点がT77、置換後のアミノ酸がE、アミノ酸置換による特性変化が温度安定性低下及び反応性向上
(82)変異点がT77、置換後のアミノ酸がD、アミノ酸置換による特性変化が抗酸化性向上及び反応性向上
(83)変異点がS199、置換後のアミノ酸がG、アミノ酸置換による特性変化が温度安定性低下及び反応性向上
(84)変異点がS199、置換後のアミノ酸がC、アミノ酸置換による特性変化が耐熱性向上、抗酸化性向上及び反応性向上
(85)変異点がT273、置換後のアミノ酸がE、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上
(86)変異点がS284、置換後のアミノ酸がM又はF、アミノ酸置換による特性変化が温度安定性低下及び反応性向上
(87)変異点がS284、置換後のアミノ酸がL又はK、アミノ酸置換による特性変化が耐熱性向上及び反応性向上
(88)変異点がS284、置換後のアミノ酸がH又はR、アミノ酸置換による特性変化が耐熱性向上及び抗酸化性向上
(89)変異点がY291、置換後のアミノ酸がI、L、C、N、S又はV、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上
(90)変異点がS299、置換後のアミノ酸がV、K、M又はA、アミノ酸置換による特性変化が温度安定性低下及び反応性向上
(91)変異点がS303、置換後のアミノ酸がK、アミノ酸置換による特性変化が耐熱性向上及び反応性向上
R26K、R26Q、R26M、R26Y、R26D、R26G、R26N、R26P、R26S
E28V、E28R、E28K、E28M、E28N、E28F、E28G、E28L、E28Y
Y42F、Y42L、Y42M
E58Y、E58M、E58A、E58F、E58I、E58V、E58R、E58K、E58L、E58Q
V67A、V67S
T68C、T68L、T68M
T77C
T273E
Y291I、Y291L、Y291C、Y291N、Y291S、Y291V
成熟体酵素の脱アミド活性とトランスグルタミナーゼ活性の各々を、野生型酵素に対する相対値として以下の計算式で算出した後、脱アミド活性(対野生型比率)/トランスグルタミナーゼ活性(対野生型比率)の値によって、基質特異性の変化(即ち、脱アミド酵素化)の程度を評価し、脱アミド酵素化に有効なアミノ酸置換を特定した。尚、脱アミド活性は以下の方法で測定し、トランスグルタミナーゼ活性については、基質溶液(R-1)を用いた上記の活性測定法で測定した。
脱アミド活性(対野生型比率)(%)=(変異酵素の脱アミド活性)/(野生型酵素の脱アミド活性)×100
トランスグルタミナーゼ活性(対野生型比率)(%)=(変異酵素のトランスグルタミナーゼ活性)/(野生型酵素のトランスグルタミナーゼ活性)×100
成熟体化酵素を200mMリン酸カリウム緩衝液(pH7.0)で適当な濃度に希釈する(サンプル溶液)。サンプル溶液20μLに基質溶液(1% Z-Gln-Gly、200mMリン酸カリウム緩衝液、pH7.0)を80μL添加し混合した後、37℃で24時間反応させる。アンモニアテストワコー(WAKO)を用いて、反応サンプル20μLに発色試薬A 40μL、発色試薬B 20μL、発色試薬C 40μLを添加して、37℃で10分反応させる。発色サンプル50μLと除蛋白液50μLを混合し、遠心分離した後、上澄みを回収して、630nmの吸光度を測定する。
(92)変異点がR5、置換後のアミノ酸がQ、P、C、M、I、V、Y、F、S、N、T又はG
(93)変異点がY34、置換後のアミノ酸がH又はM
(94)変異点がW59、置換後のアミノ酸がK又はE
(95)変異点がS61、置換後のアミノ酸がT、V、Y、D、I、L、N、E、M、F、W、Q、P、H又はK
(96)変異点がY62、置換後のアミノ酸がD又はP
(97)変異点がG63、置換後のアミノ酸がD、Q、Y、R、K、H、P、M、N、C、F、L、W、T、V又はS
(98)変異点がV65、置換後のアミノ酸がC、T又はK
(99)変異点がG66、置換後のアミノ酸がW又はK
(100)変異点がT68、置換後のアミノ酸がP又はH
(101)変異点がW69、置換後のアミノ酸がY、D又はP
(102)変異点がQ74、置換後のアミノ酸がR又はP
(103)変異点がF85、置換後のアミノ酸がY、W、L、I、Q、C、T、A、V、S、R、H又はN
(104)変異点がF108、置換後のアミノ酸がQ、C、E、S又はN
(105)変異点がF117、置換後のアミノ酸がC、I、W、V、A、G、T又はN
(106)変異点がY198、置換後のアミノ酸がF、H、L、S、M又はI
(107)変異点がS199、置換後のアミノ酸がQ又はP
(108)変異点がK200、置換後のアミノ酸がY、M、I、R、L又はV
(109)変異点がH201、置換後のアミノ酸がK、R、M、L又はQ
(110)変異点がF202、置換後のアミノ酸がY、M、H、V又はC
(111)変異点がW203、置換後のアミノ酸がY
(112)変異点がF223、置換後のアミノ酸がY、M、L、W、V、H、G又はA
(113)変異点がR238、置換後のアミノ酸がA、D、V、G、H又はS
(114)変異点がF251、置換後のアミノ酸がH、N、C、M、R、S、A、T、L、P、G、V又はQ
(115)変異点がV252、置換後のアミノ酸がM、L、T又はC
(116)変異点がN253、置換後のアミノ酸がT、V、K、C又はR
(117)変異点がY256、置換後のアミノ酸がL、V又はI
(118)変異点がG257、置換後のアミノ酸がA又はC
(119)変異点がW258、置換後のアミノ酸がY、F又はN
(120)変異点がT273、置換後のアミノ酸がN
(121)変異点がG275、置換後のアミノ酸がA、C、S、V、K、I、F、H、R、L、P、Q、Y、N、M又はT
(122)変異点がN276、置換後のアミノ酸がG、M、Q又はH
(123)変異点がH277、置換後のアミノ酸がN、S、Q、C、E、K、D、I、R、M、P、L、W、V、Y、G、F又はT
(124)変異点がY278、置換後のアミノ酸がP又はD
(125)変異点がH279、置換後のアミノ酸がN
(126)変異点がS284、置換後のアミノ酸がC
(127)変異点がM288、置換後のアミノ酸がV、Q又はT
(128)変異点がV290、置換後のアミノ酸がT、C、L、M、A、S又はN
(129)変異点がY291、置換後のアミノ酸がH、T、E又はG
(130)変異点がW298、置換後のアミノ酸がF、Y、I、L又はM
(131)変異点がS299、置換後のアミノ酸がN又はP
(132)変異点がY302、置換後のアミノ酸がL、H、M、V、C、T、P、S又はW
(133)変異点がS303、置換後のアミノ酸がI、Q、D、H又はE
(134)変異点がF305、置換後のアミノ酸がW、H又はY
R5Q、R5P、R5C、R5M、R5I、R5V、R5Y、R5F、R5S、R5N、R5T、R5G
Y34H、Y34M
W59K、W59E
S61T、S61V、S61Y、S61D、S61I、S61L、S61N、S61E、S61M、S61F、S61W、S61Q、S61P、S61H、S61K
Y62D、Y62P
G63D、G63Q、G63Y、G63R、G63K、G63H、G63P、G63M、G63N、G63C、G63F、G63L、G63W、G63T、G63V、G63S
V65C、V65T、V65K
G66W、G66K
T68P、T68H
W69Y、W69D、W69P
Q74R、Q74P
F85Y、F85W、F85L、F85I、F85Q、F85C、F85T、F85A、F85V、F85S、F85R、F85H、F85N
F108Q、F108C、F108E、F108S、F108N
F117C、F117I、F117W、F117V、F117A、F117G、F117T、F117N
Y198F、Y198H、Y198L、Y198S、Y198M、Y198I
S199Q、S199P
K200Y、K200M、K200I、K200R、K200L、K200V
H201K、H201R、H201M、H201L、H201Q
F202Y、F202M、F202H、F202V、F202C
W203Y
F223Y、F223M、F223L、F223W、F223V、F223H、F223G、F223A
R238A、R238D、R238V、R238G、R238H、R238S
F251H、F251N、F251C、F251M、F251R、F251S、F251A、F251T、F251L、F251P、F251G、F251V、F251Q
V252M、V252L、V252T、V252C
N253T、N253V、N253K、N253C、N253R
Y256L、Y256V、Y256I
G257A、G257C
W258Y、W258F、W258N
T273N
G275A、G275C、G275S、G275V、G275K、G275I、G275F、G275H、G275R、G275L、G275P、G275Q、G275Y、G275N、G275M、G275T
N276G、N276M、N276Q、N276H
H277N、H277S、H277Q、H277C、H277E、H277K、H277D、H277I、H277R、H277M、H277P、H277L、H277W、H277V、H277Y、H277G、H277F、H277T
Y278P、Y278D
H279N
S284C
M288V、M288Q、M288T
V290T、V290C、V290L、V290M、V290A、V290S、V290N
Y291H、Y291T、Y291E、Y291G
W298F、W298Y、W298I、W298L、W298M
S299N、S299P
Y302L、Y302H、Y302M、Y302V、Y302C、Y302T、Y302P、Y302S、Y302W
S303I、S303Q、S303D、S303H、S303E
F305W、F305H、F305Y
R5Y、R5F、R5S、R5N、R5T、R5G
Y34M
S61T、S61V、S61Y、S61D、S61I、S61L、S61N、S61E、S61M、S61F、S61W、S61Q、S61P、S61H、S61K
Y62P
G63D、G63Q、G63Y、G63R、G63K、G63H、G63P、G63M、G63N、G63C、G63F、G63L、G63W、G63T、G63V、G63S
V65C、V65T、V65K
G66K
T68P、T68H
W69D、W69P
F85L、F85I、F85Q、F85C、F85T、F85A、F85V、F85S、F85R、F85H、F85N
F108C、F108E、F108S、F108N
F117C、F117I、F117W、F117V、F117A、F117G、F117T、F117N
Y198L、Y198S、Y198M、Y198I
S199Q、S199P
K200Y、K200M、K200I、K200R、K200L、K200V
H201K、H201R、H201M、H201L、H201Q
F202M、F202H、F202V、F202C
W203Y
F223M、F223L、F223W、F223V、F223H、F223G、F223A
R238H、R238S
F251N、F251C、F251M、F251R、F251S、F251A、F251T、F251L、F251P、F251G、F251V、F251Q
V252L、V252T、V252C
N253T、N253V、N253K、N253C、N253R
Y256L、Y256V、Y256I
G257A、G257C
W258Y、W258F、W258N
T273N
G275C、G275S、G275V、G275K、G275I、G275F、G275H、G275R、G275L、G275P、G275Q、G275Y、G275N、G275M、G275T
N276G、N276M、N276Q、N276H
H277S、H277Q、H277C、H277E、H277K、H277D、H277I、H277R、H277M、H277P、H277L、H277W、H277V、H277Y、H277G、H277F、H277T
Y278P、Y278D
M288Q、M288T
V290M、V290A、V290S、V290N
Y291T、Y291E、Y291G
W298F、W298Y、W298I、W298L、W298M
S299P
Y302L、Y302H、Y302M、Y302V、Y302C、Y302T、Y302P、Y302S、Y302W
F305H、F305Y
S61Y、S61D、S61I、S61L、S61N、S61E、S61M、S61F、S61W、S61Q、S61P、S61H、S61K
Y62P
G63D、G63Q、G63Y、G63R、G63K、G63H、G63P、G63M、G63N、G63C、G63F、G63L、G63W、G63T、G63V、G63S
V65T、V65K
G66K
T68P、T68H
W69D、W69P
F85H、F85N
F117G、F117T、F117N
Y198S、Y198M、Y198I
S199P
K200M、K200I、K200R、K200L、K200V
H201M、H201L、H201Q
F202V、F202C
W203Y
F223W、F223V、F223H、F223G、F223A
F251L、F251P、F251G、F251V、F251Q
N253T、N253V、N253K、N253C、N253R
Y256L、Y256V、Y256I
G257C
W258N
G275V、G275K、G275I、G275F、G275H、G275R、G275L、G275P、G275Q、G275Y、G275N、G275M、G275T
N276G、N276M、N276Q、N276H
H277S、H277Q、H277C、H277E、H277K、H277D、H277I、H277R、H277M、H277P、H277L、H277W、H277V、H277Y、H277G、H277F、H277T
Y278P、Y278D
M288T
V290S、V290N
Y291G
W298Y、W298I、W298L、W298M
F305Y
配列番号3:人工配列の説明:R26Y変異体
配列番号4:人工配列の説明:R26S変異体
配列番号5:人工配列の説明:E28N変異体
配列番号6:人工配列の説明:E28G変異体
配列番号7:人工配列の説明:E28F変異体
配列番号8:人工配列の説明:E28Y変異体
配列番号9:人工配列の説明:E58R変異体
配列番号10:人工配列の説明:E58L変異体
配列番号18:人工配列の説明:R26N変異体
配列番号19:人工配列の説明:R26Y変異体
配列番号20:人工配列の説明:R26S変異体
配列番号21:人工配列の説明:E28N変異体
配列番号22:人工配列の説明:E28G変異体
配列番号23:人工配列の説明:E28F変異体
配列番号24:人工配列の説明:E28Y変異体
配列番号25:人工配列の説明:E58R変異体
配列番号26:人工配列の説明:E58L変異体
Claims (13)
- 配列番号1のアミノ酸配列において、以下の(1)~(134)の中のいずれか一つのアミノ酸置換を含むアミノ酸配列、又は該アミノ酸配列と80%以上の同一性を示すアミノ酸配列(但し、前記アミノ酸置換の位置以外の部分でアミノ酸配列の相違が生じている)を有し、前記アミノ酸置換に対応した特性変化が認められる改変型トランスグルタミナーゼ:
(1)変異点がV6、置換後のアミノ酸がQ、I、M、S、C、K、L、H、F、G、N、P、R、W又はY、アミノ酸置換による特性変化が温度安定性低下;
(2)変異点がR26、置換後のアミノ酸がK、Q、M、H、Y、D、G、N、P又はS、アミノ酸置換による特性変化が温度安定性低下;
(3)変異点がE28、置換後のアミノ酸がV、Q、W、R、K、M、N、F、G、L、P又はY、アミノ酸置換による特性変化が温度安定性低下;
(4)変異点がV30、置換後のアミノ酸がP、C、A、E、F、G、H、K、N、Q、R、W、Y又はL、アミノ酸置換による特性変化が温度安定性低下;
(5)変異点がY34、置換後のアミノ酸がA、アミノ酸置換による特性変化が温度安定性低下;
(6)変異点がY42、置換後のアミノ酸がF、A、C、D、E、G、I、L、M、Q、S、T、V又はW、アミノ酸置換による特性変化が温度安定性低下;
(7)変異点がE58、置換後のアミノ酸がY、M、A、F、I、V、R、K、N、L、S、Q、G又はH、アミノ酸置換による特性変化が温度安定性低下;
(8)変異点がW59、置換後のアミノ酸がR、N、Y、A、S、I、V、D、G又はP、アミノ酸置換による特性変化が温度安定性低下;
(9)変異点がL60、置換後のアミノ酸がI、M、V、A、C、E、F、Q、S、T、W又はY、アミノ酸置換による特性変化が温度安定性低下;
(10)変異点がY62、置換後のアミノ酸がC、R、G、K又はS、アミノ酸置換による特性変化が温度安定性低下;
(11)変異点がV65、置換後のアミノ酸がN、L、M、F、W又はY、アミノ酸置換による特性変化が温度安定性低下;
(12)変異点がV67、置換後のアミノ酸がL、N、A、C、M、Q又はS、アミノ酸置換による特性変化が温度安定性低下;
(13)変異点がT68、置換後のアミノ酸がC、L、A、S、M、F、N、Q又はY、アミノ酸置換による特性変化が温度安定性低下;
(14)変異点がW69、置換後のアミノ酸がH、M、I、C、E、F、G、K、L、N、Q、R、S、T又はV、アミノ酸置換による特性変化が温度安定性低下;
(15)変異点がQ74、置換後のアミノ酸がW、D、G又はK、アミノ酸置換による特性変化が温度安定性低下;
(16)変異点がY75、置換後のアミノ酸がR、Q、T又はG、アミノ酸置換による特性変化が温度安定性低下;
(17)変異点がT77、置換後のアミノ酸がM、H、E、C又はG、アミノ酸置換による特性変化が温度安定性低下;
(18)変異点がF85、置換後のアミノ酸がM、アミノ酸置換による特性変化が温度安定性低下;
(19)変異点がF90、置換後のアミノ酸がC、M、H、L又はV、アミノ酸置換による特性変化が温度安定性低下;
(20)変異点がF108、置換後のアミノ酸がR、L、T、A、I、K、N又はV、アミノ酸置換による特性変化が温度安定性低下;
(21)変異点がF117、置換後のアミノ酸がM又はL、アミノ酸置換による特性変化が温度安定性低下;
(22)変異点がS199、置換後のアミノ酸がG、M、N、K又はV、アミノ酸置換による特性変化が温度安定性低下;
(23)変異点がF202、置換後のアミノ酸がL又はW、アミノ酸置換による特性変化が温度安定性低下;
(24)変異点がW203、置換後のアミノ酸がF、アミノ酸置換による特性変化が温度安定性低下;
(25)変異点がF254、置換後のアミノ酸がY又はM、アミノ酸置換による特性変化が温度安定性低下;
(26)変異点がT273、置換後のアミノ酸がI、V、M、C、S、L、R、G、A、E、F、Y、D、K、W又はH、アミノ酸置換による特性変化が温度安定性低下;
(27)変異点がN276、置換後のアミノ酸がC、E、K、L、S、T又はV、アミノ酸置換による特性変化が温度安定性低下;
(28)変異点がY278、置換後のアミノ酸がM、L、H、I、K、R、W、C、G、N、Q、S、T又はV、アミノ酸置換による特性変化が温度安定性低下;
(29)変異点がS284、置換後のアミノ酸がW、Y、M、F又はNアミノ酸置換による特性変化が温度安定性低下;
(30)変異点がY291、置換後のアミノ酸がI、W、L、A、C、K、N、Q、R、S又はV、アミノ酸置換による特性変化が温度安定性低下;
(31)変異点がS299、置換後のアミノ酸がI、Y、V、K、M、Q、A、F、G又はE、アミノ酸置換による特性変化が温度安定性低下;
(32)変異点がS303、置換後のアミノ酸がN、G、C、V、P又はY、アミノ酸置換による特性変化が温度安定性低下;
(33)変異点がY310、置換後のアミノ酸がC、M又はI、アミノ酸置換による特性変化が温度安定性低下;
(34)変異点がD3、置換後のアミノ酸がQ、P、E、S又はY、アミノ酸置換による特性変化が耐熱性向上;
(35)変異点がR26、置換後のアミノ酸がV、アミノ酸置換による特性変化が耐熱性向上;
(36)変異点がY34、置換後のアミノ酸がW、アミノ酸置換による特性変化が耐熱性向上;
(37)変異点がV67、置換後のアミノ酸がH、アミノ酸置換による特性変化が耐熱性向上;
(38)変異点がT68、置換後のアミノ酸がV又はI、アミノ酸置換による特性変化が耐熱性向上;
(39)変異点がQ74、置換後のアミノ酸がF、アミノ酸置換による特性変化が耐熱性向上;
(40)変異点がT77、置換後のアミノ酸がQ、アミノ酸置換による特性変化が耐熱性向上;
(41)変異点がS199、置換後のアミノ酸がC又はQ、アミノ酸置換による特性変化が耐熱性向上;
(42)変異点がT273、置換後のアミノ酸がQ、アミノ酸置換による特性変化が耐熱性向上;
(43)変異点がS284、置換後のアミノ酸がL、H、K、P又はR、アミノ酸置換による特性変化が耐熱性向上;
(44)変異点がS299、置換後のアミノ酸がN、アミノ酸置換による特性変化が耐熱性向上;
(45)変異点がS303、置換後のアミノ酸がK、アミノ酸置換による特性変化が耐熱性向上;
(46)変異点がD3、置換後のアミノ酸がE、Q、S又はY、アミノ酸置換による特性変化が抗酸化性向上;
(47)変異点がV6、置換後のアミノ酸がP、アミノ酸置換による特性変化が抗酸化性向上;
(48)変異点がR26、置換後のアミノ酸がM、K、W、C、Q、G、Y、E、T、N、D、I、S、P又はV、アミノ酸置換による特性変化が抗酸化性向上;
(49)変異点がE28、置換後のアミノ酸がL、M、K、C、V、R、W、G、N、F、Y又はH、アミノ酸置換による特性変化が抗酸化性向上;
(50)変異点がY42、置換後のアミノ酸がL、N又はF、アミノ酸置換による特性変化が抗酸化性向上;
(51)変異点がE58、置換後のアミノ酸がQ、A、I、V、L、T、M、K、Y、W、F又はR、アミノ酸置換による特性変化が抗酸化性向上;
(52)変異点がV67、置換後のアミノ酸がT、S又はA、アミノ酸置換による特性変化が抗酸化性向上;
(53)変異点がT68、置換後のアミノ酸がL、I、V又はM、アミノ酸置換による特性変化が抗酸化性向上;
(54)変異点がQ74、置換後のアミノ酸がV、アミノ酸置換による特性変化が抗酸化性向上;
(55)変異点がT77、置換後のアミノ酸がD、アミノ酸置換による特性変化が抗酸化性向上;
(56)変異点がS199、置換後のアミノ酸がC、アミノ酸置換による特性変化が抗酸化性向上;
(57)変異点がT273、置換後のアミノ酸がE、アミノ酸置換による特性変化が抗酸化性向上;
(58)変異点がS284、置換後のアミノ酸がR又はH、アミノ酸置換による特性変化が抗酸化性向上;
(59)変異点がY291、置換後のアミノ酸がI、S、F、L、C、N、V又はM、アミノ酸置換による特性変化が抗酸化性向上;
(60)変異点がS284、置換後のアミノ酸がM、K又はV、アミノ酸置換による特性変化が反応性向上;
(61)変異点がF251、置換後のアミノ酸がY、アミノ酸置換による特性変化が反応性向上;
(62)変異点がV6とY75、変異点V6の置換後のアミノ酸がE、変異点Y75の置換後のアミノ酸がF、アミノ酸置換による特性変化が反応性向上;
(63)変異点がD3とT77、変異点D3の置換後のアミノ酸がP、変異点T77の置換後のアミノ酸がQ、アミノ酸置換による特性変化が反応性向上;
(64)変異点がD3、置換後のアミノ酸がQ、S又はY、アミノ酸置換による特性変化が耐熱性向上及び抗酸化性向上;
(65)変異点がD3、置換後のアミノ酸がP、アミノ酸置換による特性変化が耐熱性向上及び反応性向上;
(66)変異点がD3、置換後のアミノ酸がE、アミノ酸置換による特性変化が耐熱性向上、抗酸化性向上及び反応性向上;
(67)変異点がV6、置換後のアミノ酸がP、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上;
(68)変異点がR26、置換後のアミノ酸がK、Q、M、Y、D、G、N、P又はS、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上;
(69)変異点がR26、置換後のアミノ酸がV、アミノ酸置換による特性変化が耐熱性向上及び抗酸化性向上;
(70)変異点がR26、置換後のアミノ酸がH、アミノ酸置換による特性変化が温度安定性低下、抗酸化性向上及び反応性向上;
(71)変異点がE28、置換後のアミノ酸がV、R、K、M、N、F、G、L又はY、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上;
(72)変異点がV30、置換後のアミノ酸がP、G、N、R又はY、アミノ酸置換による特性変化が温度安定性低下及び反応性向上;
(73)変異点がY42、置換後のアミノ酸がF、L又はM、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上;
(74)変異点がE58、置換後のアミノ酸がY、M、A、F、I、V、R、K、L又はQ、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上;
(75)変異点がL60、置換後のアミノ酸がI、アミノ酸置換による特性変化が温度安定性低下及び反応性向上;
(76)変異点がV67、置換後のアミノ酸がA又はS、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上;
(77)変異点がT68、置換後のアミノ酸がC、L又はM、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上;
(78)変異点がT68、置換後のアミノ酸がV又はI、アミノ酸置換による特性変化が耐熱性向上及び抗酸化性向上;
(79)変異点がQ74、置換後のアミノ酸がV、アミノ酸置換による特性変化が抗酸化性向上及び反応性向上;
(80)変異点がT77、置換後のアミノ酸がC、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上;
(81)変異点がT77、置換後のアミノ酸がE、アミノ酸置換による特性変化が温度安定性低下及び反応性向上;
(82)変異点がT77、置換後のアミノ酸がD、アミノ酸置換による特性変化が抗酸化性向上及び反応性向上;
(83)変異点がS199、置換後のアミノ酸がG、アミノ酸置換による特性変化が温度安定性低下及び反応性向上;
(84)変異点がS199、置換後のアミノ酸がC、アミノ酸置換による特性変化が耐熱性向上、抗酸化性向上及び反応性向上;
(85)変異点がT273、置換後のアミノ酸がE、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上;
(86)変異点がS284、置換後のアミノ酸がM又はF、アミノ酸置換による特性変化が温度安定性低下及び反応性向上;
(87)変異点がS284、置換後のアミノ酸がL又はK、アミノ酸置換による特性変化が耐熱性向上及び反応性向上;
(88)変異点がS284、置換後のアミノ酸がH又はR、アミノ酸置換による特性変化が耐熱性向上及び抗酸化性向上;
(89)変異点がY291、置換後のアミノ酸がI、L、C、N、S又はV、アミノ酸置換による特性変化が温度安定性低下及び抗酸化性向上;
(90)変異点がS299、置換後のアミノ酸がV、K、M又はA、アミノ酸置換による特性変化が温度安定性低下及び反応性向上;
(91)変異点がS303、置換後のアミノ酸がK、アミノ酸置換による特性変化が耐熱性向上及び反応性向上;
(92)変異点がR5、置換後のアミノ酸がQ、P、C、M、I、V、Y、F、S、N、T又はG、アミノ酸置換による特性変化が脱アミド酵素化;
(93)変異点がY34、置換後のアミノ酸がH又はM、アミノ酸置換による特性変化が脱アミド酵素化;
(94)変異点がW59、置換後のアミノ酸がK又はE、アミノ酸置換による特性変化が脱アミド酵素化;
(95)変異点がS61、置換後のアミノ酸がT、V、Y、D、I、L、N、E、M、F、W、Q、P、H又はK、アミノ酸置換による特性変化が脱アミド酵素化;
(96)変異点がY62、置換後のアミノ酸がD又はP、アミノ酸置換による特性変化が脱アミド酵素化;
(97)変異点がG63、置換後のアミノ酸がD、Q、Y、R、K、H、P、M、N、C、F、L、W、T、V又はS、アミノ酸置換による特性変化が脱アミド酵素化;
(98)変異点がV65、置換後のアミノ酸がC、T又はK、アミノ酸置換による特性変化が脱アミド酵素化;
(99)変異点がG66、置換後のアミノ酸がW又はK、アミノ酸置換による特性変化が脱アミド酵素化;
(100)変異点がT68、置換後のアミノ酸がP又はH、アミノ酸置換による特性変化が脱アミド酵素化;
(101)変異点がW69、置換後のアミノ酸がY、D又はP、アミノ酸置換による特性変化が脱アミド酵素化;
(102)変異点がQ74、置換後のアミノ酸がR又はP、アミノ酸置換による特性変化が脱アミド酵素化;
(103)変異点がF85、置換後のアミノ酸がY、W、L、I、Q、C、T、A、V、S、R、H又はN、アミノ酸置換による特性変化が脱アミド酵素化;
(104)変異点がF108、置換後のアミノ酸がQ、C、E、S又はN、アミノ酸置換による特性変化が脱アミド酵素化;
(105)変異点がF117、置換後のアミノ酸がC、I、W、V、A、G、T又はN、アミノ酸置換による特性変化が脱アミド酵素化;
(106)変異点がY198、置換後のアミノ酸がF、H、L、S、M又はI、アミノ酸置換による特性変化が脱アミド酵素化;
(107)変異点がS199、置換後のアミノ酸がQ又はP、アミノ酸置換による特性変化が脱アミド酵素化;
(108)変異点がK200、置換後のアミノ酸がY、M、I、R、L又はV、アミノ酸置換による特性変化が脱アミド酵素化;
(109)変異点がH201、置換後のアミノ酸がK、R、M、L又はQ、アミノ酸置換による特性変化が脱アミド酵素化;
(110)変異点がF202、置換後のアミノ酸がY、M、H、V又はC、アミノ酸置換による特性変化が脱アミド酵素化;
(111)変異点がW203、置換後のアミノ酸がY、アミノ酸置換による特性変化が脱アミド酵素化;
(112)変異点がF223、置換後のアミノ酸がY、M、L、W、V、H、G又はA、アミノ酸置換による特性変化が脱アミド酵素化;
(113)変異点がR238、置換後のアミノ酸がA、D、V、G、H又はS、アミノ酸置換による特性変化が脱アミド酵素化;
(114)変異点がF251、置換後のアミノ酸がH、N、C、M、R、S、A、T、L、P、G、V又はQ、アミノ酸置換による特性変化が脱アミド酵素化;
(115)変異点がV252、置換後のアミノ酸がM、L、T又はC、アミノ酸置換による特性変化が脱アミド酵素化;
(116)変異点がN253、置換後のアミノ酸がT、V、K、C又はR、アミノ酸置換による特性変化が脱アミド酵素化;
(117)変異点がY256、置換後のアミノ酸がL、V又はI、アミノ酸置換による特性変化が脱アミド酵素化;
(118)変異点がG257、置換後のアミノ酸がA又はC、アミノ酸置換による特性変化が脱アミド酵素化;
(119)変異点がW258、置換後のアミノ酸がY、F又はN、アミノ酸置換による特性変化が脱アミド酵素化;
(120)変異点がT273、置換後のアミノ酸がN、アミノ酸置換による特性変化が脱アミド酵素化;
(121)変異点がG275、置換後のアミノ酸がA、C、S、V、K、I、F、H、R、L、P、Q、Y、N、M又はT、アミノ酸置換による特性変化が脱アミド酵素化;
(122)変異点がN276、置換後のアミノ酸がG、M、Q又はH、アミノ酸置換による特性変化が脱アミド酵素化;
(123)変異点がH277、置換後のアミノ酸がN、S、Q、C、E、K、D、I、R、M、P、L、W、V、Y、G、F又はT、アミノ酸置換による特性変化が脱アミド酵素化;
(124)変異点がY278、置換後のアミノ酸がP又はD、アミノ酸置換による特性変化が脱アミド酵素化;
(125)変異点がH279、置換後のアミノ酸がN、アミノ酸置換による特性変化が脱アミド酵素化;
(126)変異点がS284、置換後のアミノ酸がC、アミノ酸置換による特性変化が脱アミド酵素化;
(127)変異点がM288、置換後のアミノ酸がV、Q又はT、アミノ酸置換による特性変化が脱アミド酵素化;
(128)変異点がV290、置換後のアミノ酸がT、C、L、M、A、S又はN、アミノ酸置換による特性変化が脱アミド酵素化;
(129)変異点がY291、置換後のアミノ酸がH、T、E又はG、アミノ酸置換による特性変化が脱アミド酵素化;
(130)変異点がW298、置換後のアミノ酸がF、Y、I、L又はM、アミノ酸置換による特性変化が脱アミド酵素化;
(131)変異点がS299、置換後のアミノ酸がN又はP、アミノ酸置換による特性変化が脱アミド酵素化;
(132)変異点がY302、置換後のアミノ酸がL、H、M、V、C、T、P、S又はW、アミノ酸置換による特性変化が脱アミド酵素化;
(133)変異点がS303、置換後のアミノ酸がI、Q、D、H又はE、アミノ酸置換による特性変化が脱アミド酵素化;
(134)変異点がF305、置換後のアミノ酸がW、H又はY、アミノ酸置換による特性変化が脱アミド酵素化。 - 前記同一性が82%以上である、請求項1に記載の改変型トランスグルタミナーゼ。
- 前記同一性が85%以上である、請求項1に記載の改変型トランスグルタミナーゼ。
- 前記同一性が90%以上である、請求項1に記載の改変型トランスグルタミナーゼ。
- 配列番号2~10のいずれかのアミノ酸配列からなる、請求項1に記載の改変型トランスグルタミナーゼ。
- 請求項1~5のいずれか一項に記載の改変型トランスグルタミナーゼをコードする遺伝子。
- 配列番号18~26のいずれかの塩基配列を含む、請求項6に記載の遺伝子。
- 請求項6又は7に記載の遺伝子を含む組換えDNA。
- 請求項8に記載の組換えDNAを保有する微生物。
- 請求項1~5のいずれか一項に記載の改変型トランスグルタミナーゼを含む酵素剤。
- 以下のステップ(I)~(III)を含む、改変型トランスグルタミナーゼの調製法:
(I)請求項1~5のいずれか一項の改変型トランスグルタミナーゼのアミノ酸配列をコードする核酸を用意するステップ;
(II)前記核酸を発現させるステップ、及び
(III)発現産物を回収するステップ。 - 前記アミノ酸配列が配列番号2~10のいずれかのアミノ酸配列である、請求項11に記載の調製法。
- 前記核酸が、配列番号18~26のいずれかの塩基配列を含む、請求項12に記載の調製法。
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EP18883947.6A EP3719131A4 (en) | 2017-11-30 | 2018-11-22 | MODIFIED TRANSGLUTAMINASE |
CN201880076952.8A CN111386346A (zh) | 2017-11-30 | 2018-11-22 | 修饰型转谷氨酰胺酶 |
US16/763,779 US20200370026A1 (en) | 2017-11-30 | 2018-11-22 | Modified transglutaminase |
BR112020009793-4A BR112020009793A2 (pt) | 2017-11-30 | 2018-11-22 | transglutaminase modificada |
JP2019557205A JPWO2019107288A1 (ja) | 2017-11-30 | 2018-11-22 | 改変型トランスグルタミナーゼ |
US18/212,427 US20240052323A1 (en) | 2017-11-30 | 2023-06-21 | Modified transglutaminase |
JP2023120137A JP2023129554A (ja) | 2017-11-30 | 2023-07-24 | 改変型トランスグルタミナーゼ |
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WO2021177392A1 (ja) | 2020-03-04 | 2021-09-10 | 味の素株式会社 | 変異型トランスグルタミナーゼ |
WO2021177181A1 (ja) | 2020-03-03 | 2021-09-10 | 天野エンザイム株式会社 | 改変型トランスグルタミナーゼ |
WO2021231705A1 (en) * | 2020-05-13 | 2021-11-18 | Curie Co. Inc. | Transglutaminase variants and applications of use thereof |
WO2021256518A1 (ja) | 2020-06-18 | 2021-12-23 | 天野エンザイム株式会社 | 新規トランスグルタミナーゼ |
WO2022071061A1 (ja) | 2020-09-29 | 2022-04-07 | 味の素株式会社 | 変異型トランスグルタミナーゼ |
WO2023058765A1 (ja) | 2021-10-07 | 2023-04-13 | 天野エンザイム株式会社 | 改変型トランスグルタミナーゼ |
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CN111593038B (zh) * | 2020-06-23 | 2022-02-15 | 江南大学 | 一种稳定性提高的谷氨酰胺酶突变体 |
CN113999862A (zh) * | 2020-07-28 | 2022-02-01 | 四川大学 | 一种谷氨酰胺转氨酶的异源表达及其应用 |
CN112553177B (zh) * | 2020-12-29 | 2022-04-01 | 江南大学 | 一种热稳定性提高的谷氨酰胺转氨酶变体 |
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WO2021231705A1 (en) * | 2020-05-13 | 2021-11-18 | Curie Co. Inc. | Transglutaminase variants and applications of use thereof |
WO2021256518A1 (ja) | 2020-06-18 | 2021-12-23 | 天野エンザイム株式会社 | 新規トランスグルタミナーゼ |
WO2022071061A1 (ja) | 2020-09-29 | 2022-04-07 | 味の素株式会社 | 変異型トランスグルタミナーゼ |
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BR112020009793A2 (pt) | 2020-11-03 |
US20240052323A1 (en) | 2024-02-15 |
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