Heat-resistant antifungal protein, drug containing the same as an active ingredient, and use thereof

Abstract

To provide antifungal proteins prepared by imparting heat tolerance to a lead compound Gazyumaru (Ficus microcarpa) latex-derived chitinase B (GlxChiB), with expectation for formulation.SOLUTION: Disclosed is a protein represented by a formula b-ln-a where b represents a chitin binding domain of 35-45 amino acids, ln represents a linker moiety of 4-20 amino acids, a represents a catalytic domain of a specific amino sequence comprising 221-269 amino acids, where the protein is characterized by an amino acid sequence in which at least a proline is substituted or inserted in the loop region in the tertiary structure.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to a heat-resistant antifungal protein and a drug containing the same as an active ingredient. More specifically, the present invention relates to a modified enzyme of Gazyumaru latex chitinase-B (GlxChiB) and a drug containing the same as an active ingredient.

Background Art

[0002] Chitinase (EC 3.2.1.14), which hydrolyzes chitin contained in crustaceans, insects, fungi, etc., is widely distributed in the biological world. Among these, chitinases derived from plants have the property of decomposing the cell wall chitin of fungi, which account for more than 80% of plant pathogenic microorganisms. As a result, some plant-derived chitinases function as biodefense proteins to prevent the invasion and growth of fungi, etc., and actually exhibit antifungal activity. On the other hand, there are few reports of chitinases derived from microorganisms that exhibit antifungal activity. In addition, it has been confirmed that the few chitinases (only from microorganisms) commercially available as enzyme preparations have no antifungal activity.

[0003] The inventors have focused on plant-derived chitinases and reported the discovery of useful chitinases (Non-Patent Document 1). That is, the inventors screened chitinases from various plants and found a chitinase (Gazyumaru latex chitinase-B: GlxChiB) that exhibits extremely strong antifungal activity from the latex of Gazyumaru (scientific name: Ficus microcarpa), an evergreen tall tree of the Moraceae family distributed in subtropical and tropical regions. In addition, the inventors have succeeded in purifying such GlxChiB, cDNA cloning, and producing a recombinant using Escherichia coli.

Prior Art Documents

Non-Patent Documents

[0004] [Non-Patent Document 1] Toki Taira et al., Biosci Biotechnol Biochem. 2005 ; 69 (4) :811-818 [Disclosure of the Invention] [Problems to be Solved by the Invention]

[0005] However, while GlxChiB has strong antifungal activity, it has problems. That is, GlxChiB is unstable to heat, and when formulating GlxChiB as an active ingredient, there was a concern that GlxChiB would be denatured during the formulation process, resulting in a decrease in antifungal activity. From this, the inventors considered that when GlxChiB is used as an active ingredient of an antifungal agent, it is necessary to improve the thermal stability of GlxChiB and maintain its antifungal activity.

[0006] Against this background, in the present invention, using GlxChiB as a lead compound, the object is to develop an antifungal protein that imparts heat resistance to it and can be expected to be formulated. [Means for Solving the Problems]

[0007] As a result of intensive research, the inventors performed three-dimensional structure analysis of GlxChiB and succeeded in improving its stability to heat by introducing proline into the loop region in the catalytic active domain of such a protein, thereby completing the invention. Furthermore, the inventors succeeded in further improving the thermal stability by introducing a disulfide bond or arranging an ionic bond at a position away from the active center of the catalytic active domain, thereby completing the invention.

[0008] The present invention comprises the following constitution. The first constitution of the present invention is a protein represented by the following formula 1, b-ln-a…Formula 1 (In the formula, b is a chitin-binding domain, ln is a linker portion, and a is a catalytic domain) In the formula, b is represented by an amino acid sequence of 35 to 45, ln is represented by an amino acid sequence of 4 to 20, a is the amino acid sequence of 221 to 269 having 90% or more identity with SEQ ID NO: 3 or SEQ ID NO: 3, and is a protein characterized by being represented by an amino acid sequence in which at least proline is substituted or inserted in the loop region of the tertiary structure in any of these sequences.

[0009] The second configuration of the present invention is the protein according to the first configuration, wherein the loop region in a is the 15th to 30th, 41st to 49th, 69th to 121st, 122nd to 132nd, 133rd to 139th, 160th to 167th, 174th to 179th, 186th to 190th, 204th to 210th, 226th to 243rd positions in SEQ ID NO: 3. The third configuration of the present invention is the protein according to the first configuration, wherein the loop region in a is the 69th to 121st, 122nd to 132nd, 133rd to 139th, 160th to 167th, 174th to 179th, 186th to 190th, 204th to 210th, 226th to 243rd positions in SEQ ID NO: 3. The fourth configuration of the present invention is the protein according to the first or second configuration, wherein the proline substitution in a is a substitution of any one or more of the amino acids indicated by the 20th, 44th, 73rd, 107th, 123rd, 127th, 134th, 137th, 189th, 210th, 228th positions in SEQ ID NO: 3. The fifth configuration of the present invention is the protein according to any one of the first to third configurations, wherein the proline substitution in a is a substitution of any one or more of the amino acids indicated by the 73rd, 107th, 127th, 189th, 210th positions in SEQ ID NO: 3. The sixth configuration of the present invention is the protein according to any one of the first to fourth, wherein the proline substitution at a is a substitution of all of the amino acids represented by the 73rd, 107th, 127th, 189th, and 210th amino acids in SEQ ID NO: 3.

[0010] The seventh configuration of the present invention is the protein according to any one of the first to sixth configurations, wherein in a, the 39th amino acid in SEQ ID NO: 3 is substituted with cysteine. The eighth configuration of the present invention is the protein according to any one of the first to seventh configurations, wherein in a, the 206th amino acid in SEQ ID NO: 3 is substituted with any one of lysine, arginine, and histidine, the 209th amino acid is substituted with aspartic acid, and the 211th amino acid is substituted with arginine.

[0011] The ninth configuration of the present invention is the protein according to any one of the first to eighth configurations, wherein b includes any one of SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21. The tenth configuration of the present invention is the protein according to the ninth configuration, wherein b is represented by the amino acid sequence of SEQ ID NO: 2. The eleventh configuration of the present invention is the protein according to any one of the first to tenth configurations, wherein ln is represented by the amino acid sequence of SEQ ID NO: 4.

[0012] The twelfth configuration of the present invention is the protein according to the first configuration, which is represented by any one of the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10. The thirteenth configuration of the present invention is the protein according to the first configuration, which is represented by any one of the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14.

[0013] The fourteenth configuration of the present invention is a cell line that produces the protein according to any one of the first to thirteenth configurations. The fifteenth configuration of the present invention is the cell line described in the thirteenth configuration, which is Escherichia coli transformed with a recombinant vector containing DNA encoding any of the proteins of the first to thirteenth configurations. The sixteenth configuration of the present invention is an antifungal agent containing the protein represented by the first to thirteenth configurations as an active ingredient.

Effects of the Invention

[0014] According to the present invention, it has become possible to provide an antifungal protein that uses GlxChiB as a lead compound, imparts heat resistance to it, and can be expected to be formulated.

Brief Description of the Drawings

[0015]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Modes for Carrying Out the Invention

[0016] The present invention will be described in detail.

[0017] The protein of the present invention is a protein represented by the following formula 1, b-ln-a... Formula 1 (wherein, b is a chitin-binding domain, ln is a linker portion, and a is a catalytic domain) In the formula, b is represented by an amino acid sequence of 35 to 45, ln is represented by an amino acid sequence of 4 to 20, a is the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence of 221 to 269 having 90% or more identity with SEQ ID NO: 3, characterized in that it is represented by an amino acid sequence in which at least proline is substituted or inserted in the loop region of the tertiary structure in any of these sequences. That is, such a protein is a derivative having the basic structure of gazyumaru latex chitinase (Gazyumaru latex chitinase-B: GlxChiB, SEQ ID NO: 1), and is a protein with improved heat resistance while having properties such as antifungal activity possessed by GlxChiB. Such a protein can be expected to be used as an antifungal agent such as an antifungal agent having this as an active ingredient. In addition, for the protein represented by the above formula or the amino acid sequence subordinate thereto in the present invention, a protein that exhibits the same antifungal effect by substituting, deleting, or inserting a part of the amino acid sequence thereof without objectively recognizing a particularly excellent effect can be evaluated as an equivalent compound.

[0018] b is an amino acid sequence that functions as a chitin-binding domain. As such a chitin-binding domain, an amino acid sequence called class I chitinase or class IV chitinase is known, and these are known to have an amino acid sequence of 35 to 42. From these, b does not particularly need to be limited as long as it functions as a chitin-binding domain and has an amino acid sequence length that does not inhibit this function, and can have various sequences. As such a chitin-binding domain, for example, a sequence containing an amino acid sequence called class I chitinase or class IV chitinase may be used. Examples of class I chitinase include amino acid sequences such as SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, etc., and examples of class IV chitinase include amino acid sequences such as SEQ ID NO: 21, etc.

[0019] As b, it is preferably a sequence containing SEQ ID NO: 2, which is the chitin-binding domain in GlxChiB. Thereby, the protein according to the present invention can have an amino acid sequence similar to GlxChiB, and stable exhibition of the antifungal or antibacterial action of the protein of the present invention can be expected. In such a case, using SEQ ID NO: 2 as the basic sequence, any sequence that does not inhibit this function can be added. Such a sequence may be 45 amino acid residues, which is a length added by about 13% from 40 amino acid residues of SEQ ID NO: 2, preferably 44 amino acid residues, which is a length added by about 10%, more preferably 43 amino acid residues, which is a length added by about 8%, and most preferably 42 amino acid residues, which is a length added by about 5%. Further, such added amino acids may be added to either the C-terminus or the N-terminus of SEQ ID NO: 2, or may be added to both termini.

[0020] Ln is an amino acid sequence that functions as a linker portion, and functions as an amino acid sequence that connects b, which is a chitin-binding domain, and a, which is a catalytic domain. Ln does not particularly need to be limited as long as it serves as such a linker and does not impair the functions of a and b respectively, and can be various amino acid sequences. As such a linker portion, Ln may typically be composed of an amino acid sequence of any length from 4 to 20, preferably from 4 to 18, more preferably from 4 to 15, and most preferably from 4 to 12. In addition, ln is preferably composed of neutral amino acids so as not to impair the functionality of a and b, and may also contain one or more cysteines to improve heat resistance. In such a case, the same number of cysteines corresponding to the cysteines substitutionally introduced into the catalytic domain or the chitin-binding domain can be substitutionally introduced, etc. Ln can typically be the sequence represented by SEQ ID NO: 4, or a sequence with any amino acid added thereto.

[0021] A is an amino acid sequence that functions as a catalytic domain. In the present invention, the catalytic domain is defined as an amino acid sequence having at least a function of decomposing chitin. That is, the catalytic domain can exhibit antibacterial activity or antifungal activity by a chitin-decomposing function similar to GlxChiB, and is defined as an amino acid sequence having these functions or active sites. Note that the catalytic domain has such a chitin-decomposing function, but it is not necessary to be limited thereto, and it may have other functions.

[0022] As the basic structure of a, it has the amino acid sequence of SEQ ID NO: 3, which is the catalytic domain in GlxChiB, or an amino acid sequence having identity therewith, and proline is introduced into the loop region of such a tertiary structure. In the present invention, the "amino acid sequence having identity with SEQ ID NO: 3" means that, compared with SEQ ID NO: 3, 90% or more of the amino acid sequences match, and the function as the catalytic domain of SEQ ID NO: 3 is provided to an equivalent or higher level, or to a degree that does not impair it. The function of the catalytic domain is intended to have the chitin-decomposing activity of GlxChiB of SEQ ID NO: 3, and as a criterion for an equivalent or higher level of the same function, it is to have 90% or more of the chitin-decomposing activity, and as a criterion for a degree that does not impair the same function, it is intended to be from 80% to less than 90%. As such an amino acid sequence, typically, it can be an amino acid sequence of positions 221 to 269 having 90% or more identity with SEQ ID NO: 3, more preferably an amino acid sequence of positions 233 to 257 having 95% or more identity with SEQ ID NO: 3, still more preferably an amino acid sequence of positions 241 to 249 having 98% or more identity with SEQ ID NO: 3, and most preferably an amino acid sequence of positions 243 to 247 having 99% or more identity with SEQ ID NO: 3.

[0023] In the present invention, the loop region refers to a movable region that does not have a helix structure or a sheet structure in the three-dimensional structure of a protein and connects these (hereinafter, simply abbreviated as "pure loop region"), or a terminal helix region adjacent to a loop region that can be treated equivalently to the loop region (hereinafter, simply abbreviated as "loop-equivalent region"). These pure loop regions or loop-equivalent regions can be determined by the common general knowledge of those skilled in the art. That is, if the amino acid sequence of a before proline introduction (or the entire amino acid sequence as a protein) is determined, based on various conditions such as temperature, pH, and composition assuming the intended use, by performing a three-dimensional structure prediction of the protein using a computer, the pure loop region and the helix region (loop-equivalent region) adjacent thereto can be determined. The sites for introducing proline can be appropriately determined from these pure loop regions and loop-equivalent regions. Finally, the protein with proline introduced can be produced and confirmed by actual measurement using X-ray crystal structure analysis or the like.

[0024] In the present invention, examples of the pure loop region include the sequence regions of positions 15 to 30, 41 to 49, 69 to 121, 133 to 139, 160 to 167, 174 to 179, 186 to 190, 204 to 210, and 226 to 243 in SEQ ID NO: 3. Examples of the loop-equivalent region include the sequence region of positions 122 to 132 in SEQ ID NO: 3. In a, it is preferable that the loop regions be the 69th to 121st, 122nd to 132nd, 133rd to 139th, 160th to 167th, 174th to 179th, 186th to 190th, 204th to 210th, and 226th to 243rd positions in SEQ ID NO: 3. This has the effect of making it easier to produce a protein that improves thermal stability without impairing activity.

[0025] Regarding the substitution and introduction of proline in a, typically, substitution and introduction may be performed for any one or more of the amino acids represented by the 20th, 44th, 73rd, 107th, 123rd, 127th, 134th, 137th, 189th, 210th, and 228th positions in SEQ ID NO: 3. These amino acids are in the loop region in a (or the helix region adjacent thereto; see Figure 2), and have the effect of being able to improve heat resistance by substituting and introducing proline while maintaining catalytic activity. Regarding the substitution and introduction of proline, more preferably, any one or more of the amino acids represented by the 73rd, 107th, 127th, 189th, and 210th positions in SEQ ID NO: 3 may be substituted and introduced with proline (except when all amino acid Xs are substituted or unsubstituted in SEQ ID NO: 5). Most preferably, all of these amino acids may be substituted and introduced with proline (corresponding to SEQ ID NO: 5 when all amino acid Xs are substituted with P).

[0026] In a, it is preferable that the 39th amino acid in SEQ ID NO: 3 be substituted with cysteine (see Figure 3). This enables substitution and introduction of cysteine corresponding to other sequence portions (for example, ln) to form a disulfide bond in the protein, and has the effect of improving the thermal stability of the protein. In such a case, typically, cysteine may be substituted and introduced into ln, and for example, the 4th glycine in SEQ ID NO: 4 may be substituted with cysteine as such a sequence.

[0027] In a, it is preferable that the 206th amino acid at SEQ ID NO: 3 is a basic amino acid (lysine, arginine) or histidine, the 209th amino acid is aspartic acid, and the 211th amino acid is arginine (see Figure 4). Thereby, it becomes possible to form a good salt bond with these three amino acids, and such salt bond formation has the effect of further improving the thermal stability of the protein. As described above, in a, the thermal stability can be improved by substitution with proline or cysteine, or introduction of a salt bond site. Such an a sequence can be represented as SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. SEQ ID NO: 5... Proline substitution-introduced active site (at least one of the 73rd, 107th, 127th, 189th, and 210th positions in SEQ ID NO: 3, which is the active site of GlxChiB, is substituted with proline) SEQ ID NO: 7... Proline and cysteine substitution-introduced active site (at least one of the 73rd, 107th, 127th, 189th, and 210th positions in SEQ ID NO: 3, which is the active site of GlxChiB, is substituted with proline. In addition, the 39th amino acid is substituted with cysteine) SEQ ID NO: 9... Proline and salt bond site-introduced active site (at least one of the 73rd, 107th, 127th, 189th, and 210th positions in SEQ ID NO: 3, which is the active site of GlxChiB, is substituted with proline. In addition, the 209th position is substituted with lysine or histidine, and the 211th position is substituted with arginine)

[0028] Next, the following amino acid sequences are exemplified as proteins having a specific sequence with GlxChiB as the basic structure in the present invention. SEQ ID NO: 6... Proline substitution-introduced derivative (an amino acid sequence in which any one or more of the 117th, 151st, 171st, 233rd, and 254th positions in the WT of GlxChiB are substituted with proline) SEQ ID NO: 8… Proline and cysteine substitution-introduced variant (in addition to the substitution-introduced variant of SEQ ID NO: 6, the amino acid sequences in which the 44th and 83rd amino acids are substituted and introduced with cysteine) SEQ ID NO: 10… Proline, cysteine, salt-bonding site substitution-introduced variant (in addition to the substitution-introduced variant of SEQ ID NO: 8, the amino acid sequence in which the 250th amino acid is lysine or histidine, the 253rd amino acid is aspartic acid, and the 255th amino acid is arginine) SEQ ID NO: 11… Proline penta-mutant (the amino acid sequence in which all of the 117th, 151st, 171st, 233rd, and 254th amino acids in the WT of GlxChiB are substituted and introduced with proline. Hereinafter, represented as "mt5") SEQ ID NO: 12… Proline and cysteine substitution-introduced variant (in addition to the substitution-introduced variant of SEQ ID NO: 11, the amino acid sequence in which the 44th and 83rd amino acids are substituted and introduced with cysteine. Hereinafter, represented as "mt5ss") SEQ ID NO: 13… Proline, cysteine, KDR substitution-introduced variant (in addition to the substitution-introduced variant of SEQ ID NO: 12, the amino acid sequence in which the 250th amino acid is lysine, the 253rd amino acid is aspartic acid, and the 255th amino acid is arginine. Hereinafter, represented as "mt5ss / KDR") SEQ ID NO: 14… Proline, cysteine, HDR substitution-introduced variant (in addition to the substitution-introduced variant of SEQ ID NO: 12, the amino acid sequence in which the 250th amino acid is histidine, the 253rd amino acid is aspartic acid, and the 255th amino acid is arginine. Hereinafter, represented as "mt5ss / HDR")

[0029] The protein of the present invention can be produced by any commonly used method. Preferably, as exemplified in FIG. 1, a culture method using a transformed cell line (E. coli) can be mentioned. That is, a recombinant vector containing DNA encoding the corresponding protein is introduced into E. coli for transformation, and after culturing and inducing expression, the protein component is purified to produce the protein of the present invention.

[0030] The protein of the present invention can be used as an antifungal agent with this as the active ingredient. In such a case, as long as the protein of the present invention acts as the active ingredient, there is no particular need for limitation, and it can be used in various forms such as spraying by liquid spraying and powder particles. Further, when the protein of the present invention is used as the active ingredient, it is not necessarily limited to the chemical form of the protein itself. That is, the protein used as an antifungal agent includes compounds that can be evaluated as equivalent to the protein of the present invention when functioning as an antifungal agent in view of the gist of the present invention. For example, with regard to chemical modifications without amino acid substitution such as stabilization for formulation and extension of the effective period, they can be evaluated as equivalent to the protein of the present invention.

Examples

[0031] <<Experimental Example 1>> <1. GlxChiB Mutant> Mutants of GlxChiB were prepared by the method shown in FIG. 1. The amino acid substitution sites, substituted amino acids, and functional regions of the main mutants are shown in Table 1. In Table 1, the wild type (SEQ ID NO: 1) of GlxChiB is used as the basic sequence, and the substitution sites and substituted amino acids in this sequence are shown for each mutant. In addition, the substitution sites in the catalytic domain (SEQ ID NO: 3) are also described together.

Table 1

[0032] Hereinafter, the amino acid substitution sites will be represented as “(one-letter notation of the amino acid before substitution)(number of the substitution site)(one-letter notation of the amino acid after substitution)” as necessary. For example, in WT, if it is “A117P”, it means that “alanine (A) at the 117th amino acid of WT (SEQ ID NO: 1) is substituted with proline (P)”. When there are multiple substitution sites, these are shown together with “ / ”.

[0033] <2. Experimental Method> [Measurement of Chitinase Activity] Using glycol chitin, the method of Imoto and Yagishita et al. (Imoto T., Yagishita K., Agr. Biol. Chem. 1971; 35: 1154-1156.) was followed. 10 μL of the enzyme sample solution was added to 250 μL of each 0.1 M buffer containing 0.2% glycol chitin and kept at 37 °C for 15 minutes. To this, 1 mL of a 0.05% potassium ferricyanide solution containing 0.5 M sodium carbonate was added and boiled for 15 minutes. After cooling in water, the absorbance at 420 nm was measured (absorbance A). Also, a reaction solution obtained by adding the same amount of distilled water as the sample solution to the reaction system was reacted in the same manner, and the absorbance at 420 nm was measured (absorbance B). The value obtained by subtracting A from B (at 420) was taken as the chitinase activity. 1 unit was defined as the amount that liberates 1 μmol of N-acetylglucosamine per minute at 37 °C.

[0034] [SDS-PAGE] SDS-PAGE was performed using a 15% polyacrylamide gel after boiling the sample for 3 minutes in the presence of 0.1% SDS and 5% β-mercaptoethanol. Coomassie brilliant blue R-250 (CBB) was used for staining the proteins after electrophoresis.

[0035] [Protein Quantification] Protein quantification was measured by the bicinchoninic acid method (BCA method). A calibration curve was prepared using bovine serum albumin, and it was performed using the BCA Protein Assay Reagent Kit from PIERCE.

[0036] [Thermostability Evaluation] (1) Measurement of the Tm value by differential scanning calorimetry (DSC) The mutant samples were all dialyzed against 10 mM sodium phosphate buffer (pH 7.0), prepared to a protein concentration of 0.5 mg / ml, and subjected to DSC measurement. For DSC measurement, Nano DSC (TA Instruments) was used, and the same buffer was used in the blank cell. The samples were heated at a heating rate of 1 °C / min (25 - 125 °C) to measure the specific heat associated with the denaturation of the enzyme protein. The temperature corresponding to the peak of the specific heat curve was defined as the denaturation temperature (Tm) of the enzyme protein. (2) Measurement of half-life at 60 °C or 65 °C After incubating the recombinant protein at 60 °C or 65 °C for 5 to 120 minutes, the residual activity was measured by the method of measuring chitinase activity, and the time (half-life) when the residual activity reached 50% was calculated.

[0037] [Measurement of antifungal activity] Potato Dextrose Agar (PDA) containing 1.5% agar and 1.5% glucose was autoclaved to prepare plates. Prior to this, the filamentous fungus T. viride was cultured on the PDA plates, and the site where the mycelium grew uniformly was extracted with a cork borer (diameter 4 mm). The extracted agar was placed at equal intervals on another PDA plate, and a certain amount of the sample was added onto the placed agar. After leaving it at room temperature for 12 hours, it was scanned using an image scanner and saved as a digital image. The area of the extended mycelium was counted as the number of pixels using image analysis software. Based on the area of the extended mycelium (A) in the sample-added group and the area of the extended mycelium (B) in the water-added group as a blank, the inhibition value was calculated using the following formula. (Formula) Inhibition rate (%) = (B - A) × 100 / B

[0038] <3. Experimental results> 1. In the loop region, the results of the heat resistance evaluation when one proline was introduced are shown in Table 2. Regarding the substitution sites in the table, they are shown based on the catalytic domain sequence. (1) In all mutants, the Tm increased compared to WT. (2) The half-life at 60 °C was extended for each variant, and the half-life was extended by about 1.4 to 2.5 times. (3) From these results, it was found that the introduction of proline improves the thermal stability.

[0039] 2. Furthermore, the results of the heat resistance evaluation of the variant with proline substituted and introduced at five positions (proline penta-variant, mt5) and the variant with cysteine substituted and introduced at two positions (ss) are shown in Table 2, Figures 5 and 6. The substitution positions in the table are shown based on the sequence of WT. (1) The Tm values of WT and G44C / K83C (ss) were the same, but in the half-life at 60 °C (Figure 5 and Table 2), G44C / K83C (ss) was 14.5 minutes compared to 6.9 minutes for WT. From this, it was found that the introduction of cysteine resulted in an extension of the half-life by about 2.1 times, indicating an improvement in thermal stability. (2) The Tm value increased for mt5 compared to WT. The half-life of mt5 was 47.4 minutes compared to 6.9 minutes for WT. From this, it was found that the introduction of proline at five positions resulted in an extension of the half-life by about 6.9 times, indicating a significant improvement in thermal stability. (3) Furthermore, in mt5ss with cysteine introduced into mt5, no increase in the Tm value was observed compared to mt5, but the half-life of mt5ss was 73.8 minutes, about 1.6 times that of mt5 and about 10.7 times that of WT. From these results, it was confirmed that the introduction of ss improved the thermal stability in mt5, and it was found that a dramatic improvement in thermal stability can be achieved by combining the introduction of proline and cysteine. (4) Furthermore, in two mutants (mt5ss / HDR and mt5ss / KDR) in which the 250th, 253rd, and 255th amino acids of WT were replaced for the purpose of introducing salt bridges into mt5ss, the Tm values of both mutants were higher than those of mt5 and mt5ss, with mt5ss / KDR being 69.1 °C and mt5ss / HDR being 71.1 °C, indicating that their thermal stability was improved compared to these. When compared with the Tm value of the wild type (64.2 °C), mt5ss / KDR and mt5ss / HDR showed significant increases of 4.9 °C and 6.9 °C, respectively. Also, the half-lives of mt5ss / KDR and mt5ss / HDR at 60 °C (Figure 5 and Table 2) were 104.7 minutes and 103.9 minutes, respectively, both being approximately 1.4 times that of mt5ss and approximately 15 times that of WT. Furthermore, the half-lives of mt5ss / KDR and mt5ss / HDR at 65 °C (Figure 6 and Table 2) were 33.2 minutes and 63.3 minutes, respectively, showing an increase of one order of magnitude compared to mt5ss (3.9 minutes) and nearly two orders of magnitude compared to WT (0.7 minutes). From these results, it was confirmed that the introduction of salt bridges improved the thermal stability of mt5ss, and it was found that a further dramatic improvement in thermal stability could be achieved by combining proline introduction, cysteine introduction, and salt bridge introduction.

[0040] Table 2 shows the results of examining the enzyme activity and antifungal activity. (1) In terms of enzyme activity, compared with 5.40×10 9 (U / mol) of WT, G44C / K83C(ss) was 6.06×10 9 (U / mol), mt5ss was 5.88×10 9 (U / mol), mt5ss / KDR was 4.31×10 9 (U / mol), and mt5ss / HDR was 5.13×10 9 (U / mol), all showing enzyme activities comparable to that of WT. (2) The IC 50It was found that, with respect to 1.56 μM of WT, G44C / K83C(ss) exhibited antifungal activity equivalent to 1.58 μM. On the other hand, mt5ss showed a value of 0.69 μM, which was lower than that of WT, indicating that it had better antifungal activity than WT. Also, the IC 50 values of mt5ss / KDR and mt5ss / HDR were 1.58 and 1.94, respectively, and it was found that they exhibited antifungal activity almost equivalent to that of WT. (3) The Tm values in Table 2 were measured using a differential scanning calorimeter (DSC) by the method described in the above [Heat resistance evaluation] (1) "Measurement of Tm value by differential scanning calorimeter (DSC)". Also, the specific activity of chitinase activity in Table 2 was measured by the method described in the above <2. Experimental method> [Chitinase activity measurement]. [Table 2]

Claims

1. A protein represented by the following formula (1): b-ln-a... Formula (1) (wherein, b is a chitin-binding domain derived from class I chitinase or class IV chitinase, ln is a linker portion, and a is a catalytic domain). In the formula, b is represented by an amino acid sequence of 35 to 45,[[]] ln is represented by an amino acid sequence of 4 to 20,[[]] a is an amino acid sequence of 221 to 269 having 90% or more identity with SEQ ID NO: 3 or SEQ ID NO: 3, In any of these sequences, an amino acid sequence in which the amino acid residue corresponding to any one of the 73rd, 107th, 127th, 189th, and 210th amino acids in the amino acid sequence of SEQ ID NO: 3 or a plurality of amino acid residues thereof are substituted with proline, represented by having a longer half-life at 60°C and having antifungal activity than chitinase B derived from gazumal emulsion consisting of the amino acid sequence of SEQ ID NO: 1 A protein characterized by the above.

2. The protein according to claim 1, wherein, in a, the amino acid residue corresponding to the 206th amino acid in the amino acid sequence of SEQ ID NO: 3 is any one of lysine, arginine, and histidine, the amino acid residue corresponding to the 209th amino acid is aspartic acid, and the amino acid residue corresponding to the 211th amino acid is arginine, and these are substituted.

3. The protein according to claim 1 or 2, wherein b contains any one of SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO:

21.

4. The protein according to any one of claims 1 to 3, wherein ln is represented by the amino acid sequence of SEQ ID NO:

4.

5. The protein according to claim 1, wherein a is represented by the amino acid sequence of SEQ ID NO:

5.

6. The protein according to claim 1, wherein a is represented by the amino acid sequence of SEQ ID NO:

9.

7. The protein according to claim 1, represented by any one of the amino acid sequences of SEQ ID NO: 8 and SEQ ID NO:

10.

8. The protein according to claim 1, represented by any one of the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO:

14.

9. A cell line producing the protein according to any one of claims 1 to 8.

10. The cell line according to claim 9, wherein a recombinant vector containing DNA encoding the protein according to any one of claims 1 to 8 is transformed into Escherichia coli.

11. ​ An antifungal agent comprising, as an active ingredient, any one of the proteins according to claims 1 to 8.

Images