JP2006136314A - New modified phytase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To perform the functional change in a yeast phytase for creating a modified phytase improved in its stability in a stomach. <P>SOLUTION: A modified phytase gene is created by utilizing the function-changing technology of a protein. By using the modified phytase gene, a modified phytase-producing strain is constituted. By culturing the producing strain, the modified phytase is obtained. By evaluating the modified phytase, the modified phytase expected to show the improvement of stability in a porcine stomach is created. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a modified phytase in which the optimum pH of yeast phytase is shifted to the acid side and a gene encoding the modified phytase. Furthermore, the present invention relates to a modified phytase with improved pepsin resistance and a gene encoding the modified phytase. The present invention relates to a modified phytase that can be expected to improve stability in the stomach of pigs and a gene encoding the modified phytase. The present invention also relates to a method for creating a modified phytase. The animal feed composition containing the modified phytase is expected to improve performance as an animal feed.

  Most of the phosphorus in grains and seeds used for livestock feed such as pigs and chickens exists as phytic acid (inositol hexaphosphate, phytic phosphorus). Since these livestock cannot use phytic phosphorus, the phosphorus content in the excreta increases and contributes to pollution in rivers and lakes.

  Phytase is a phosphatase that hydrolyzes phytic acid and is present in plants, animals and microorganisms.

  From these points, phytase that hydrolyzes phytin phosphorus into phosphorus (effective phosphorus) that can be used by domestic animals such as pigs attracts attention, and Aspergillus fungus-derived phytase (hereinafter referred to as mold phytase) is produced as a feed additive.・ It is sold.

However, a relatively expensive point as a feed additive is an obstacle to the spread (Non-Patent Document 1). Further, mold phytase already on the market is also known to have the following problems (1) and (2).
(1) Phytase mainly acts in the stomach (Non-patent document 2), and the pH of porcine stomach contents is around 3.0 to 4.5 (Non-patent documents 3, 4, 5, 6). The optimum pH of phytase is 5.5, and its activity is significantly reduced at pH 3.5 (Non-patent Document 7).
(2) Feed additives containing phytase are usually used in pelletized form. In the pellet formation process, high-temperature treatment at 60 to 80 ° C. is performed, but most of the activity of mold phytase is lost at the temperature during the pellet formation (Non-patent Document 7).

  In order to compensate for the above drawbacks, it is necessary to increase the amount of mold phytase added to the feed, which leads to an increase in the cost of the feed. From this point of view, mold phytase that is already on the market is also undergoing functional modification aimed at reducing the dosage and resulting cost reduction associated with higher functionality. Attempts to improve activity (Non-Patent Document 8) and attempts to increase heat resistance (Non-Patent Document 9) have been reported. However, there is no report that it has been put to practical use.

  In addition to mold phytase, phytases derived from various organisms are known, and E. coli-derived phytase (hereinafter referred to as E. coli phytase) has been reported to try to improve the optimum pH and heat resistance as well as mold phytase. (Patent Document 1).

  On the other hand, Schwanniomyces yeast-derived phytase (hereinafter referred to as yeast phytase) is excellent in heat resistance unlike mold phytase and Escherichia coli phytase. Furthermore, the optimum pH of yeast phytase is in the vicinity of the stomach pH range of pigs, and it has been expected that yeast phytase has a potentially higher effect on pigs than mold phytase. Thus, in fact, yeast phytase or fungal phytase was added to pig feed, and the effect on phosphorus absorption from the feed was examined. As a result, although yeast phytase promoted phosphorus absorption, it has been found that its action is weaker than that of mold phytase (Non-patent Document 10). As a cause of this, it has been clarified that yeast phytase is inferior in gastric stability (Non-patent Document 11).

As described above, the present situation is that there is a demand for the development of next-generation high-performance phytase that is excellent in effect and economical efficiency for fungus phytase, Escherichia coli phytase, and yeast phytase.
Special table 2003-531574 gazette Japanese Patent Laid-Open No. 11-206368 Noboru Takizawa, BIO INDUSTRY, 16 (1), 72-77 (1999) AW Jongbloed et.al., J. Anim.Sci., 70, 1159-1168 (1992) FR Dintzis et.al., J. Anim.Sci., 73, 1138-1146 (1995) Z Yi et. Al., Animal. Feed. Sci. Technol., 61, 361-368 (1996) DC Regina et.al., J. Anim. Sci., 77, 2721-2729 (1999) FO Omogbenigun et.al., J. Anim. Sci., 81, 1806-1813 (2003) XG Lei et.al., Biotechnology Letters, 25, 1787-1794 (2003) EJ Mullaney et.al., Biochemical and Biophysical Research Communication., 297, 1016-1020 (2002) M Lehmann et.al., Protein Engineering, 13 (1), 49-57 (2000) A Tamura et.al., Animal Science Journal, 70 (6), 479-483 (1999) T Matsui et.al., J. Anim. Sci., 78, 94-99 (2000) B Honig et. Al., J. Phys. Chem., 97, 1101-1109 (1993) Y. Sakai et.al., J. Bacteriology, 173 (23), 7458-7463, (1991)

  Mold phytase, Escherichia coli phytase, and yeast phytase are known. However, mold phytase already on the market is insufficient in its effect, and a highly functional phytase excellent in effect and economy is demanded.

  It is an object of the present invention to create a modified phytase having improved properties as a feed additive enzyme by utilizing a protein functional modification technique.

  In order to solve the above problems, the present inventors have intensively studied and found that a known effect can be obtained by modifying the amino acid sequence of a conventionally known phytase according to several indices. Completed the invention.

That is, the present invention is as follows.
[1] A modified phytase obtained by modifying a Schwaniomyces genus yeast phytase, which improves the stability in the stomach.
[2] The modified phytase according to [1], wherein the optimum pH is shifted to the acid side by modification.
[3] The modified phytase according to [1], wherein pepsin resistance is improved by modification.
[4] The modified phytase according to any one of [1] to [3], wherein the amino acid sequence of Schwaniomyces yeast phytase is the amino acid sequence shown in SEQ ID NO: 2.
[5] The modified phytase according to any one of [1] to [4], which is created by substituting at least one of the acidic amino acids constituting the Schwaniomyces yeast phytase with another amino acid. .
[6] The modified phytase according to [5], wherein the acidic amino acid is aspartic acid at the 429th residue of the amino acid sequence shown in SEQ ID NO: 2.
[7] The modified phytase according to [6], wherein the other amino acid is asparagine.
[8] The modified phytase according to any one of [1] to [4], which is created by substituting at least one of the amino acids constituting the Schwaniomyces yeast phytase with a basic amino acid.
[9] The modified phytase according to [8], wherein the basic amino acid is lysine or arginine. [10] The modified phytase according to any one of [1] to [4], which is created by substituting at least one of the amino acids constituting Schwaniomyces yeast phytase with histidine.
[11] The modified phytase according to any one of [8] to [10], wherein the amino acid to be substituted is serine at the 292nd residue of the amino acid sequence shown in SEQ ID NO: 2.
[12] The positive charge in the electrostatic potential around pH 3.5 is weakened by substituting at least one of the amino acids constituting Schwaniomyces yeast phytase with another amino acid, [1] The modified phytase according to any one of to [4].
[13] The modified phytase according to [12], wherein the amino acid to be substituted is serine at the 82nd residue of the amino acid sequence shown in SEQ ID NO: 2.
[14] The modified phytase according to [13], wherein the other amino acid introduced by substitution at the 82nd residue is glutamic acid or aspartic acid.
[15] The modified phytase according to [12], wherein the amino acid to be substituted is serine at the 85th residue described in SEQ ID NO: 2.
[16] The modified phytase according to [15], wherein the other amino acid introduced by substitution at the 85th residue is glutamic acid or aspartic acid.
[17] The modified phytase according to [12], wherein the amino acid to be substituted is the 301st residue histidine described in SEQ ID NO: 2.
[18] The modified phytase according to [17], wherein the other amino acid introduced by substitution at the 301st residue is tylosin.
[19] The modified phytase according to [12], wherein the amino acid to be substituted is histidine at the 342nd residue described in SEQ ID NO: 2.
[20] The modified phytase according to [19], wherein the other amino acid introduced by substitution at the 342nd residue is glutamic acid or aspartic acid.
[21] The 82nd residue, 85th residue, 292nd residue, 301st residue, 342nd residue, and 429th residue of the amino acid sequence of the phytase described in SEQ ID NO: 2 The modified phytase according to any one of [1] to [4], which is created by substituting at least two amino acids selected from the above with other amino acids.
[22] As for the amino acid residue introduced by substitution, the 82nd residue of the amino acid sequence of the phytase set forth in SEQ ID NO: 2 is glutamic acid or aspartic acid, the 85th is glutamic acid or aspartic acid, and the 292 residue The modification is described in [21], wherein the group is lysine, arginine or histidine, the 301st residue is tylosin, the 342nd residue is glutamic acid or aspartic acid, and the 429th residue is asparagine. Phytase.
[23] The modification according to [21], wherein the amino acid residue introduced by substitution is glutamic acid at the 342nd residue and asparagine at the 429th residue of the amino acid sequence of the phytase described in SEQ ID NO: 2. Phytase.
[24] Regarding the amino acid residue introduced by substitution, the 292nd residue of the phytase amino acid sequence shown in SEQ ID NO: 2 is lysine, the 342nd residue is glutamic acid, and the 429th residue is The modified phytase according to [21], which is asparagine.
[25] Modification according to [21], wherein the amino acid residue introduced by substitution is lysine at residue 292 and tylosin at residue 301 of the amino acid sequence of phytase described in SEQ ID NO: 2. Phytase.
[26] Regarding the amino acid residue introduced by substitution, the 292nd residue of the phytase amino acid sequence shown in SEQ ID NO: 2 is lysine, the 301st residue is tylosin, and the 342nd residue is The modified phytase according to [21], which is glutamic acid.
[27] Regarding the amino acid residue introduced by substitution, the 292th residue of the phytase amino acid sequence shown in SEQ ID NO: 2 is lysine, the 301st residue is tylosin, and the 429th residue is The modified phytase according to [21], which is asparagine.
[28] A gene encoding the modified phytase according to any one of [1] to [27].
[29] A method of performing a functional modification of the phytase having the amino acid sequence of SEQ ID NO: 2 by site-specific modification, and as a result, creating a modified phytase with improved stability at gastric pH.
[30] A method for performing a functional modification of the phytase having the amino acid sequence of SEQ ID NO: 2 by site-specific modification, and as a result, creating a modified phytase having an optimum pH shifted to the acid side.
[31] A method for performing a functional modification of a phytase having the amino acid sequence of SEQ ID NO: 2 by site-specific modification and, as a result, creating a modified phytase with improved pepsin resistance.
[32] The method according to any one of [29] to [31], wherein acidic amino acids in the phytase having the amino acid sequence of SEQ ID NO: 2 are reduced.
[33] The method according to any one of [29] to [31], wherein the basic amino acid in the phytase having the amino acid sequence of SEQ ID NO: 2 is increased.
[34] The modified phytase according to any one of [29] to [33], wherein a modified phytase is created by weakening a positive charge at an electrostatic potential around pH 3.5 in the phytase having the amino acid sequence of SEQ ID NO: 2. Method.

  What was created in the present invention is a modified phytase gene and a modified phytase in which the optimum pH of yeast phytase is shifted to an acidic side from pH 4.5. What was created by the present invention is a modified phytase gene and a modified phytase with improved pepsin resistance. What was created by the present invention is a modified phytase gene and a modified phytase that can be expected to improve the stability in the stomach of pigs. By using the modified phytase of the present invention, it is possible to achieve a reduction in the amount of application due to higher functionality, and it is possible to reduce the cost of phytase occupying the feed.

  The method of the present invention can be widely applied to the enhancement of functions of other feed additive enzymes that act in the stomach, including phytase.

  Using the modified phytase gene of the present invention, a modified phytase-producing strain can be constructed by using a technique well known in the art.

  By culturing the constructed modified phytase-producing strain, the modified phytase can be secreted and produced in the culture solution.

  Recovery of the modified phytase in the culture solution may be performed according to a commonly used protein recovery method after removing the cells from the obtained culture solution by centrifugation or the like. Further, considering that the modified phytase of the present invention is effective as a feed additive enzyme for livestock, the crude purified phytase recovered from the culture solution can be used as a final product, and further, for the growth of livestock. The phytase containing a medium component can be used as a final product as long as there is no problem.

  First, a yeast phytase having an enzymatic characteristic different from that of mold phytase was selected as a target phytase for functional modification. Yeast phytase has an optimum pH in the pH range of the stomach of pigs, and is superior in heat resistance to mold phytase. However, the disadvantage of poor gastric stability is also known.

  In addition, the DNA sequence which codes a yeast phytase gene was shown to sequence number 1 of the sequence table. The amino acid sequence of yeast phytase is shown in SEQ ID NO: 2 in the sequence listing. In addition, an amino acid sequence having 70%, preferably 90% homology with the amino acid sequence of phytase described in SEQ ID NO: 2 in the sequence listing is also included in yeast phytase as long as it has phytase activity.

  This patent relates to improving the intragastric stability of yeast phytase.

First, we tried to analyze various factors affecting the stability of yeast phytase in the stomach.
The place where phytase acts is the stomach. Generally, the state in the stomach after ingesting phytase-added feed is a state in which feed, pepsin (digestive enzyme), and phytase are mixed. The gastric pH is 3.0 to 4.5.

  When examining factors affecting the stability of phytase in the stomach, it is common to cause pepsin, a digestive enzyme, to act in the stomach pH region in the presence of feed. It is known that soybean, corn, and the like, which are main ingredients of feed, have phytate as a substrate for phytase, and stabilize the phytase structure by forming a complex with phytase.

Therefore, in order to investigate the basic properties of phytase, we dared to evaluate the stability of phytase in an evaluation system without adding phytic acid as a substrate. As a result, surprisingly, even in the absence of pepsin, the phytase lysate was removed from the gastric pH range of pH 3.0,
It was found that the phytase activity was significantly reduced instantaneously only by adjusting the pH to 3.5 (Example 1).

  Next, the resistance of phytase to pepsin was examined. As described above, it is known that the pH region in the stomach of pigs is pH 3.0 to pH 4.5, but at pH 3.0 and pH 3.5, phytase is instantly inactivated. Therefore, the resistance to pepsin at pH 4.0 where phytase is stably present was examined. At pH 4.0, no significant decrease in yeast phytase activity was observed even after sufficient pepsin treatment (Example 1).

  Therefore, if the optimum pH of yeast phytase can be shifted to the acidic side, it is possible to improve the resistance to pepsin, resulting in improved gastric stability, which is considered a disadvantage of yeast phytase. It was considered possible to create a modified phytase.

  The functional modification of yeast phytase was performed as follows using a site-directed mutagenesis method (Example 2).

  In general, positive charges and negative charges on the surface of a protein are distributed in a well-balanced manner near neutrality, and a compact molecular shape is maintained stably due to their electrostatic attraction. At extreme pH, for example, pH 3.5 or less, acidic amino acids are protonated and negative charges are reduced, so that stabilization due to electrostatic interaction is lost and repulsion between positive charges becomes dominant and molecular It is assumed that the structure is loose and denatured.

  Therefore, as a first measure, in order to remove instability at pH 3.0 and pH 3.5, at least one of the acidic amino acids constituting yeast phytase is replaced with another amino acid, thereby making it acidic. We decided to reduce the number of amino acids.

  As a second policy, an attempt was made to reduce the influence of acidic amino acids present in yeast phytase by substituting at least one of the amino acids constituting yeast phytase with a basic amino acid.

  Next, the predicted electrostatic potential at pH 3.0 and pH 3.5 was actually calculated. As a result, it was found that the periphery of the phytase reaction site / substrate recognition site was extremely biased to a positive charge (FIG. 1). Therefore, as a third measure, in order to relieve the positive charge in this region, it was decided to substitute an acidic amino acid present in this region in a negative state at pH 3.0 and pH 3.5.

  The modification that reduces acidic amino acids in the first strategy can be achieved by substituting aspartic acid and glutamic acid, which are acidic amino acids constituting yeast phytase, with other amino acids.

  The modification that increases basic amino acids in the second policy can be achieved by substituting amino acids constituting yeast phytase that are not basic amino acids with lysine or arginine, which are basic amino acids.

  In the predicted electrostatic potential at pH 3.5 in the third measure, the modification that weakens the positive charge is an amino acid that constitutes yeast phytase and that exists in a neutral state at pH 3.5. This can be accomplished by substituting the amino acid present in the minus state in 5. Alternatively, it can be achieved by substituting amino acids constituting yeast phytase that are present in a positive state at pH 3.5 with amino acids that are neutral or present in a negative state at pH 3.5. The predicted electrostatic potential around pH 3.5 is calculated by calculating the predicted electrostatic potential around pH 3.5 when the Poisson-Boltzmann equation is numerically solved by software of Delphi et al. (Non-patent Document 12). Asked.

The modified phytase gene can be constructed by site-specific modification using, for example, QuickChange ^R Site-Directed Mutagenesis Kit (manufactured by STRATAGENE). That is, a set of complementary synthetic primers having mutations at the same location was prepared, and after annealing these primers with the template DNA, a PCR reaction was performed. As the template DNA, plasmid pPHY36 having a yeast phytase gene can be used (Patent Document 2).

By using the constructed modified phytase gene, a modified phytase expression plasmid can be constructed by using a technique well known in the art. As a preferred example of the method for constructing the expression plasmid, the following method may be mentioned. That is, a modified phytase expression plasmid can be constructed by binding the modified phytase gene downstream of the promoter of the alcohol oxidase gene (AOD1) of Candida boidinii using the modified phytase gene. Furthermore, in order to improve the expression efficiency, it is desirable to add a terminator downstream of the phytase gene. Examples of such terminator include the terminator of the AOD1 gene of Candida voidiny (Example 2).

  A modified phytase-producing strain can be constructed by transforming Candida voidinii with this plasmid using a known transformation method (Example 3). In order to discriminate the transformed production strain, it is desirable to link an appropriate selection marker in the recombinant plasmid. Examples of selection markers include genes related to biosynthesis of nucleic acids, genes related to amino acid synthesis, genes related to growth, genes related to drug resistance, and the like. In addition, since the recombinant plasmid of the present invention is retained in a transformed host cell, it contains a sequence that enables autonomous replication in the cell, or is integrated into the chromosome by homologous recombination. It is desirable to include a sequence having high homology with the sequence.

  The constructed modified phytase-producing strain can be cultured, and the modified phytase can be secreted and produced outside the cells of the recombinant Candoidoidinii. A variant was obtained from the culture supernatant and evaluated for various properties (Example 4).

  First, the activity of each variant at pH 3.5 and pH 4.5 was measured. Next, the activity value at pH 3.5 with respect to pH 4.5 (activity value at pH 3.5 / activity value at pH 4.5) was calculated, and this value was defined as the “acid shift value”. Since the optimum pH of yeast phytase is 4.5, it is considered that the optimum pH is shifted to the acidic side as the value is larger, and the intragastric pH region of pig (pH 3.0 to pH 4.5). Improvement in activity can be expected.

  In the first strategy, 55 acidic amino acids are present among 461 amino acids constituting yeast phytase. As a result of intensive studies among such many acidic amino acids, it was possible to create a variant 429 having an increased “acid shift value”, particularly by replacing the aspartic acid at the 429th residue. (Table 5). The amino acid to be substituted may be anything other than an acidic amino acid, and preferably includes asparagine.

  In the second strategy, as a result of intensive studies from among the 461 amino acid residues constituting yeast phytase, the “acid shift value” was increased by replacing serine at the 292th residue. A body 292 could be created (Table 5). The amino acid to be substituted is preferably a basic amino acid, and particularly preferred amino acids include lysine, asparagine, and glutamine.

  In the third policy, that is, in the policy of substituting with an acidic amino acid that exists in a negative state at pH 3.0 and pH 3.5, as a result of intensive studies, serine at the 82nd residue, lysine at the 85th residue, 301 It is possible to create a variant 82, a variant 85, a variant 301, and a variant 342 in which the “acid shift value” is increased by substituting histidine at the residue or histidine at the 342 residue. (Table 5). When the amino acid to be substituted is a basic amino acid that is positively charged at pH 3.0 and pH 3.5, the amino acid to be substituted may be anything other than the basic amino acid, aspartic acid, glutamic acid, glycine, tylosin, or Examples include cysteine. When the amino acid to be substituted is a neutral amino acid that is in a neutral state at pH 3.0 and pH 3.5, the amino acid to be substituted is preferably an acidic amino acid, and examples include aspartic acid and glutamic acid.

  The above-mentioned modified forms (modified body 82, modified body 85, modified body 292, modified body 301, modified body 342, modified body 429) have an optimum pH shifted from pH 4.5 to more acidic side than yeast phytase. It was thought that the activity in the stomach of pigs could be improved.

  Since yeast phytase has been found to be inferior in gastric stability, each modified product (modified product 82, modified product 85, modified product 292, modified product) in an environment assuming the stomach, that is, in the presence of feed and pepsin. Body 301, modified body 342, and modified body 429) were evaluated for pepsin resistance.

All of these modified phytases were confirmed to have improved pepsin resistance compared to yeast phytase (Table 7), and the modified phytase created in the present invention was found to be superior to yeast phytase. .

  Next, in order to examine the effect of the combination of substitution sites in the variants found in the present invention (variant 82, variant 85, variant 292, variant 301, variant 342, variant 429), Several variants containing at least two sites were constructed, and the acid shift value and pepsin resistance described above were evaluated. As an example, variant 36G09, variant 43C03, variant 135E05, variant 136D06, and variant 146H06 were constructed (Example 5). The variant 36G09 is a combination of the variant 342 and the variant 429, the variant 43C03 is a combination of the variant 292, the variant 342, and the variant 429, and the variant 135E05 is a combination of the variant 292, the variant 301, and It is a combination of variant 429. The variant 136D06 is a combination of the variant 292, the variant 301, and the variant 342, and 146H06 is a combination of the variant 292 and the variant 301.

  As a result, the modified body (modified body 36G09, modified body 43C03, modified body 135E05, modified body 136D06, modified body 146H06) including at least two newly constructed substitution sites is a modified body consisting of a single substitution site (modified) As well as yeast 82 phytase, the optimum pH is considered to be shifted from pH 4.5 to the acidic side, as in the case of the body 82, the modified body 85, the modified body 292, the modified body 301, the modified body 342, and the modified body 429). As a result, it was possible to expect an improvement in activity in the stomach of pigs (Table 6), and it was found that combinations of substitution sites in the variants found in the present invention are possible.

  In addition, all of the modified bodies (modified body 36G09, modified body 43C03, modified body 135E05, modified body 136D06, modified body 146H06) including at least two newly constructed substitution sites are all modified bodies (modified) As well as yeast 82 phytase (Table 8), as in the case of the body 82, the modified body 85, the modified body 292, the modified body 301, the modified body 342, and the modified body 429). It has been found that combinations of substitution sites constituting the variants found in the invention are possible. It should be noted that the modified body 135E05 and the modified bodies 136D06 and 146H06 had a large pepsin resistance value compared to the yeast phytase (Table 8), and it was particularly expected to improve the stability in the pig stomach.

  The combinations of substitution sites in the variants found in the present invention (variant 82, variant 85, variant 292, variant 301, variant 342, variant 429) were constructed in Example 5 as an example. As combinations other than the modified body (modified body 36G09, modified body 43C03, modified body 146H06, modified body 135E05, modified body 136D06), the following combinations can be given. That is, residues 82 and 85, residues 82 and 292, residues 82 and 301, residues 82 and 342, Residues 82 and 429, Residues 85 and 292, Residues 85 and 301, Residues 85 and 342, Residue 85 Residues and 429th Residues, 292th and 429th Residues, 301st and 342st Residues, 301st and 429th Residues, 342th Residue And residues 429, residues 82 and 85, residues 292, residues 82 and 85, residues 301, and 82 Residues 85 and 342, 82, 85 and 429, 82, 292 and 301, 82 Residues 292 and 342 Residues: 82nd residue, 292nd residue, 429th residue, 82nd residue, 301st residue, 342nd residue, 82nd residue, 301st residue And residues 429, 82, 342 and 429, 85, 292 and 301, 85 and 85 Residues 292 and 342, residues 85 and 292, 429, residues 85, 301 and 342, residue 85 Residues, 301st and 429th residues, 85th, 342nd and 429th residues, 301st, 342nd and 429th residues 82nd residue, 85th residue, 292nd residue and 301st residue, 82nd residue, 85th residue, 292nd residue and 342nd residue, Residues 82 and 85 Residues 92 and 429, residues 82 and 85, 301 and 342, residues 82 and 85 and 301 Residues at 429th residue, 82nd residue, 85th residue, 342nd residue, 429th residue, 82nd residue, 292nd residue, and 301st residue Residues 342, 82, 292, 301, 429, 82, 301, 342, and 342. Residues 429, 85, 292, 301 and 342, 85, 292, 301 and 429 Residue, 85th residue, 301st residue, 342nd residue and 429th residue, 292nd residue, 301st residue, 342nd residue and 429th residue , Residue 82 and residue 85 Residue, Residues 292, Residues 301, 342, Residues 82, 85, 292, 301, 429 82nd residue, 85th residue, 292nd residue, 301st residue, 342nd residue, 429th residue, 82nd residue, 292nd residue, and 301st residue, 342nd residue, 429th residue, 85th residue, 292nd residue, 301st residue, 342nd residue, 429th residue, 82nd residue The base, the 85th residue, the 292nd residue, the 301st residue, the 342nd residue, the 429th residue, etc. can be mentioned.

  Production of the modified phytase is carried out by culturing the constructed modified phytase-producing strain in a liquid medium containing phosphate or a phosphate compound while adding methanol, as shown in a known method (Patent Document 2), The modified phytase can be secreted into the medium.

  The modified phytase may be recovered according to a commonly used protein recovery method after removing the cells from the obtained culture solution by centrifugation or the like. The recovered modified phytase may be further purified. Moreover, considering that the modified phytase produced by the present invention is used as a livestock feed, the crude modified phytase recovered from the culture solution can be used as a final product, and further, there is a problem with the growth of livestock. In the absence of phytase, a phytase containing medium components can also be used as the final product.

  Hereinafter, analysis of factors affecting the gastric stability of yeast phytase, construction of a modified phytase gene, construction of a modified phytase production strain, and evaluation of the modified phytase will be described by way of specific examples. There are no restrictions.

(Analysis of factors affecting the intragastric stability of yeast phytase)
Yeast phytase has been reported to be less stable in the stomach in monogastric animals such as pigs. Therefore, we tried to analyze various factors affecting the stability in the stomach.
The state in the pig stomach after feeding the phytase-added enzyme is a state in which feed as a substrate, digestive enzyme (pepsin) and added phytase are mixed. There is a report that the pH range in the stomach of pigs is 3.0 to 4.5.

  Therefore, first, the stability of phytase in the intragastric pH region was evaluated.

  Using the culture supernatant of a transformant that secreted and produced yeast phytase, pH treatment was carried out by the following method, and the activity at each pH was measured.

The yeast culture supernatant was diluted 2 times with water, and then the solution was diluted 5 times with each 0.4 M pH solution (pH 3.0, pH 3.5, pH 4.0). Each pH solution was adjusted to each pH by adding acetate to 0.4M CH ₃ COONa. Divide the diluted solution of the culture supernatant into 5 Eppendorf tubes, 50 μl each, and store for 0, 10, 30, 60, 120 minutes at 37 ° C., respectively, and add 9 times the amount of water (450 μl) at the end of the reaction. The reaction was stopped by finally making it 10 times the amount. Further, this solution was diluted 10 times to obtain an enzyme solution.

  Using this solution, phytase activity was measured by the following method.

After 10 μl of 10 mM sodium phytate / 0.4 M CH ₃ COONa-acetete buffer (pH 4.5), 85 μl of H ₂ O and 5 μl of the enzyme solution were mixed, the mixture was reacted at 37 ° C. for 15 minutes. ₄ μl of this reaction solution was added to 200 μl of 5 mM H ₂ SO ₄ to stop the reaction, 40 μl of coloring solution was added to 160 μl of this solution, and the absorbance at 655 nm was measured. Activity was calculated from comparison with a standard curve. The coloring solution used was a mixture of solution A, solution B and solution C at a ratio of 1000: 250: 20 immediately before use.

In addition, Malachite Green 0.147 g / 20% (V / V) H ₂ SO ₄ 100 ml) was used as the A liquid, 7.5% sodium molybdate was used as the B liquid, and 11% Tween 20 was used as the C liquid.

The time course of phytase activity at each pH is shown in FIG. Yeast phytase was instantly inactivated when treated at pH 3.0. At pH 3.5, it was completely inactivated by treatment for 10 minutes. It was stable at pH 4.0. The activity decreased at each pH was not recovered even when the reaction solution was returned to pH 4.5, which is the optimum pH. In addition, as a result of examining the molecular size of phytase after each pH treatment by SDS-PAGE, there was no change in the molecular size, so deactivation at each pH is not due to decomposition but is a structural change. Was inferred.

Next, stability against pepsin was evaluated. As described above, phytase was very unstable at pH 3.0 and 3.5 in the stomach pH range of pigs, so pepsin treatment was performed at pH 4.0. Using the same culture supernatant as described above, pepsin treatment was performed by the following method, and phytase activity was measured. That is, yeast phytase was diluted 5 times with 4 times the amount of 0.4 M CH ₃ COONa-acetete buffer (pH 4.0) with respect to enzyme solution 1 to adjust the pH. 100 μl of this pH-adjusting enzyme solution was dispensed into five Eppendorf tubes. Next, 100 μl of pepsin solution was added and incubation started at 37 ° C. After 0, 10, 30, 60, and 120 minutes, 50 μl of the reaction solution was dispensed from each tube into 450 μl of water prepared separately to stop the reaction. In addition, the pepsin solution used what melt | dissolved 2660 mg of 3660 units / mg (made by SIGMA) in 1 ml of 10 mM HCl solution. As a control, a 10 mM HCl solution was used instead of the pepsin solution. The solution which stopped the reaction was further diluted 10 times with water, and the activity was measured. For the activity measurement, the method shown in Example 1 was used.

  As shown in FIG. 4, yeast phytase was not affected by pepsin treatment at pH 4.0.

  As described above, the effects of pH stability and pepsin as factors determining gastric stability were examined. As a result, it was confirmed that the activity of yeast phytase was significantly reduced only by adjusting the pH of the lysate to 3.0 or 3.5.

(Construction of modified phytase gene and creation of modified phytase expression plasmid)
The yeast phytase gene was modified using QuickChange ^R Site-Directed Mutagenesis Kit (manufactured by STRATAGENE). That is, a set of complementary synthetic primers having mutations at the same location was prepared, and after annealing these primers with the template DNA, a PCR reaction was performed. As template DNA, plasmid pPHY36 (patent document 3) having a yeast phytase gene was used. structure pPHY36 is, pBlueScript SK consists 2895 bp ^- in which (STRATAGENE Corp.) EcoRI of includes a DNA fragment of about 1800bp encoding the yeast phytase gene into the XhoI site.

  Complementary synthetic primers containing mutations are PM54 (SEQ ID NO: 3) and PM54R (SEQ ID NO: 4), PM34 (SEQ ID NO: 5) and PM34R (SEQ ID NO: 6), PM76 (SEQ ID NO: 7) and PM76R (sequence) No. 8), PM134 (SEQ ID NO: 9) and PM134R (SEQ ID NO: 10), PM63 (SEQ ID NO: 11) and PM63R (SEQ ID NO: 12), and PM22 (SEQ ID NO: 13) and PM22R (SEQ ID NO: 14) were used. Table 1 shows the relationship between the synthetic primer, primer sequence, and modification site.

PCR reaction conditions and the like were in accordance with the instruction manual. That is, 5 μl of x10 Reaction Buffer,
1 μl of 10 ng / μl pPHY36DNA, 12.5 μl each of 10 ng / μl synthetic primer,
dNTP mix 1μl, DW 17μl, PfuTurburbo DNA polymerase (2.5U / μl)
1 μl was added to prepare a reaction solution. The reaction is T-GRADIENT Thermocycler
1 cycle (30 seconds), [95 ° C (30 seconds), 55 ° C (1 minute) using 95 ° C (by BIOMETRA)
, And 68 ° C. (4.6 minutes)] were performed for 16 cycles.

  Competent high DH5 (manufactured by TOYOBO) was transformed with 5 μl of the reaction solution after PCR. A plasmid was prepared from the obtained transformant and used as a modified phytase plasmid. The base sequence of the modified phytase gene contained in the plasmid was determined using ABI PRISM 310 Genetic Analyzer.

  Of the phytase gene contained in each modified phytase plasmid, the base sequence of the portion different from the base sequence of yeast phytase is shown in italics in Table 2, and the plasmid (p82-1, p82-2, p82-3, p85-1, p292-1, p301-1, p342-1, and p429-1) are designated as variant 82, variant 85, variant 292, variant 301, variant 342, and variant 429, respectively, and yeast phytase Table 3 shows the amino acid sequences of parts different from those shown in italics.

  Next, the construction of the modified phytase expression plasmid was performed as follows.

  Modified phytase plasmids (p82-1, p85-1, p342-1, p292-1, p301-1, p429-1) and a phytase expression plasmid pNNPH60I were used. PNNPH60I was ligated using a linker having an XhoI site at one of two NotI cleavage sites before and after the phytase gene of the yeast phytase expression plasmid pNNPH60 (Patent Document 2) to create an XhoI site. It is.

  The yeast phytase expression plasmid p82-1EX was constructed as shown in FIG.

  First, the modified phytase plasmid p82-1 was cleaved with the restriction enzymes NotI and XhoI, and the DNA containing the modified phytase was recovered. The recovered DNA fragment was combined with the remaining vector portion excised from the yeast phytase gene contained between NotI and XhoI of the yeast phytase expression plasmid pNNPH60I using the T4 ligation kit to construct a modified phytase expression plasmid. Similarly, p85-1EX, p292-1EX, p342-1EX, and p429-1EX were constructed from the modified phytase plasmids p85-1, p292-1, p342-1, and p429-1, respectively.

  Table 4 shows the relationship between the modified phytase plasmid and the modified phytase expression plasmid.

(Creation and selection of modified phytase producing strain)
The uracil-requiring strain TK62 was obtained from Candida Boydinii S2 AOU-1. The Candida Boydini S2 AOU-1 strain was named MCI-040820 and was assigned to the National Institute of Advanced Industrial Science and Technology as the Patent Biological Depositary Center under the accession number FERM P-20199 on September 2, 2004. It has been deposited. Transformation was performed using this uracil-requiring strain. It has been clarified by Sakai et al. That a method for obtaining a uracil-requiring strain and that this uracil-requiring strain is a mutant strain of the URA3 gene (Non-patent Document 13). Transformation was performed as follows. The TK62 strain was cultured with shaking for about 24 hours to prepare a preculture solution. The obtained preculture solution (100 μl) was used and cultured in 100 ml of YPD medium for about 12 hours until OD ₆₁₀ was around 1.0. The culture solution was collected and suspended in 25 ml of lithium solution (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 3 mM lithium acetate).

  The cells were collected by centrifugation, suspended again in 5 ml of lithium solution, and then incubated at 30 ° C. for 1 hour with shaking to obtain competent cells. 1. Add 10 μl Calf thymus DNA heat-denatured in a 5 ml tube, 10 μl Calf thymus DNA, 20 μg / μl modified with EcoRV, 5 μl modified phytase expression vector, and 100 μl competent cells, mix gently, and 30 ° C., 30 Let stand for a minute. After standing, 700 μl of 45% PEG solution (45% (w / v) polyethylene glycol # 4000 (PEG), 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 3 mM lithium acetate) was added and mixed gently. Then, it was left still at 30 ° C. for 1 hour. Thereafter, a heat shock was applied at 42 ° C. for 5 minutes, and the mixture was allowed to stand at 30 ° C. for 5 minutes. After collecting cells by centrifugation, suspend in 1 ml of sterile water and apply 200 μl each to a YNB plate (0.67% Yeast Nitrogen base w / o Amino acid (Difco), 2% glucose, 2% agar). The cells were cultured at 0 ° C. for 3 days. Only bacterial cells transformed by this operation formed colonies. Transformants obtained by transforming the TK62 strain with modified phytase expression plasmids (p82-1EX, p85-1EX, p292-1EX, p301-1EX, p342-1EX, p429-1EX, respectively) 82, transformant 85, transformant 292, transformant 301, transformant 342, and transformant 429.

(Evaluation of various modifications)
The transformant constructed in Example 3 (transformant 82, transformant 85, transformant 292, transformant 301, transformant 342, transformant 429) was added to 20 ml of YPGM medium (1% yeast extract). , 2% polypeptone, 3% glycerol, 1.5% methanol) at 28 ° C. for 5 days.

  Next, an enzyme solution containing a modified phytase (modified body 82, modified body 85, modified body 292, transformed strain 301, modified body 342, modified body 429) is prepared from the same culture supernatant as described above. The phytase activity at pH 4.5 was measured using the method shown in Example 1.

  The activity value at pH 3.5 with respect to pH 4.5 (activity value at pH 3.5 / activity value at pH 4.5) was calculated. This value was defined as “acid shift value”. Since the optimum pH of yeast phytase is 4.5, it is considered that the optimum pH shifts to the acidic side as the value increases. The acid shift value of yeast phytase was 60.3. On the other hand, the acid shift values of the respective variants were 88.3, 73.9, 79.4, 61.5, 68.2, and 66.3, respectively. Table 5 shows the relative value of each variant when the acid shift value of yeast phytase is 100.

(Effects of combinations of various variants)
Next, the combination effect of the substitution sites of the variants (modified 82, modified 85, modified 292, modified 301, modified 342, modified 429) with high acid shift value found in the present invention was examined. In order to achieve this, several types of variants containing at least two or more substitution sites were constructed (variant 36G09, variant 43C03, variant 135E05, variant 136D06, variant 146H06). Note that variant 36G09 is a combination of variant 342 and variant 429, variant 43C03 is a combination of variant 292, variant 342 and variant 429, and variant 135E05 is a variant 292, variant 301 and A combination of the modification 429, the modification 136D06 is a combination of the modification 292, the modification 301, and the modification 342, and 146H06 is a combination of the modification 292 and the modification 301.

  Transformants that produce these variants (variant 36G09, variant 43C03, variant 135E05, variant 136D06, variant 146H06) (transformant 36G09, transformant 43C03, transformant 135E05, transformant) 136D06 and transformed strain 146H06) were obtained as follows.

  First, the transformed strain 36G09 was prepared using the modified phytase plasmid (p36G09) using the modified phytase plasmid (p342-1) DNA as a template and PM22 and PM22R as synthetic primers using the same method as shown in Example 2. -1) was constructed. Next, a modified phytase expression plasmid (p36G09-1EX) was constructed using the modified phytase plasmid (p36G09-1) and the phytase expression plasmid pNNPH60I. Using the obtained modified phytase expression plasmid (p36G09-1EX), the TK62 strain was transformed using the same method shown in Example 3 to obtain a transformed strain 36G09.

  The transformed strain 43C03 was also constructed in the same manner as described above using the modified phytase plasmid (p36G09-1) DNA as a template, PM76 and PM76R as synthetic primers, and a modified phytase plasmid (p43C03-1). Next, a modified phytase expression plasmid (p43C03-1EX) was constructed using the modified phytase plasmid (p43C03-1) and the phytase expression plasmid pNNPH60I. Using the obtained modified phytase expression plasmid (p43C03-1EX), the TK62 strain was transformed using the same method shown in Example 3 to obtain a transformed strain 43C03.

  The transformed strain 146H06 was also constructed in the same manner as described above using the modified phytase plasmid (p292-1) DNA as a template and PM134 and PM134R as synthetic primers, and a modified phytase plasmid (p146H06-1). Next, a modified phytase expression plasmid (p146H06-1EX) was constructed using the modified phytase plasmid (p146H06-1) and the phytase expression plasmid pNNPH60I. Using the obtained modified phytase expression plasmid (p146H06-1EX), a transformant 146H06 was obtained by transforming the TK62 strain using the same method as described in Example 3.

  For the transformed strain 135E05, a modified phytase plasmid (p135E05-1) was constructed using the modified phytase plasmid (p146H06-1) DNA as a template and PM22 and PM22R as synthetic primers using the same method as described above. Next, a modified phytase expression plasmid (p135E05-1EX) was constructed using the modified phytase plasmid (p135E05-1) and the phytase expression plasmid pNNPH60I. Using the obtained modified phytase expression plasmid (p135E05-1EX), the TK62 strain was transformed using the same method shown in Example 3 to obtain a transformed strain 135E05.

Similarly, the transformed strain 136D06 was prepared using the modified phytase plasmid (p136D06) using the modified phytase plasmid (p146H06-1) DNA as a template and PM63 and PM63R as synthetic primers using the same method as shown in Example 2. -1) was constructed. Next, a modified phytase expression plasmid (p136D06-1EX) was constructed using the modified phytase plasmid (p136D06-1) and the phytase expression plasmid pNNPH60I. Using the obtained modified phytase expression plasmid (p136D06-1EX), a transformant 136D06 was obtained by transforming the TK62 strain using the same method as described in Example 3.

  The obtained transformants (transformant 36G09, transformant 43C03, transformant 146H06, transformant 135E05, transformant 136D06) were cultured in the same manner as shown in Example 4, and the culture supernatant was used. Prepare an enzyme solution containing the modified phytase (modified 36G09, modified 43C03, modified 146H06, modified 135E05, modified 136D06) and measure the phytase activity at pH 3.5 and pH 4.5 using the method described above. The acid shift value was calculated. Table 6 shows the relative value of each variant when the acid shift value of yeast phytase is 100.

  As a result, a variant containing at least two or more newly constructed substitution sites (variant 36G09, variant 43C03, variant 146H06, variant 135E05, variant 136D06) is also a variant (modified) comprising a single modified site. As is the case with yeast phytase, it is considered that the optimum pH is shifted from pH 4.5 to the acidic side, as is the case with body 82, modified body 85, modified body 292, modified body 301, modified body 342, modified body 429). As a result, it can be expected that the activity in the stomach of pigs can be improved, and it has been found that the combination of the variants found in the present invention is possible.

[Test Example 1]
Next, since yeast phytase has been found to be inferior in gastric stability, each modified product (modified product 82, modified product) in an environment that assumes the stomach, that is, in the presence of feed and digestive enzyme (pepsin). The body 85, the modified body 292, the modified body 301, the modified body 342, and the modified body 429) were evaluated for pepsin resistance.

First, a feed preparation simulating pig food was prepared. The mixing ratio of the ingredients of the feed preparation is corn
75%, soybean meal 24% and rice bran 1% were used. The preparation of the feed preparation was performed as follows. Corn was crushed finely, mixed with soybean meal made fine in a mortar, rice bran was added and mixed well. 1 g of the feed preparation thus prepared was suspended in water to prepare a 5 ml solution. This solution was stored at 4 ° C. for 1-2 days, then centrifuged at 5000 rpm for 1 minute, and the collected supernatant was used as a food suspension.

The pepsin resistance test was performed by the following method. Yeast phytase enzyme solution in a solution containing 10 μl of the above-prepared food suspension, 0.4 μM CH ₃ COONa-acetate buffer pH 3.0 10 μl, pepsin solution (7320 U / ml 10 mM HCl) 0, 10, or 20 μl, 50 μl water 10 μl was finally added to make a 100 μl reaction solution. As a control, 20 μl of 10 mM HCl solution was added instead of pepsin solution. The reaction was carried out at 37 ° C., and after 0, 30, 60, 90, and 120 minutes, 4 μl each of the reaction solution was added to 200 μl of 5 mM H ₂ SO ₄ solution to stop the reaction. 40 μl of the coloring solution was added to 160 μl of this solution, and the absorbance at 655 nm was measured. The analysis was performed by the following method. The reaction time was plotted on the horizontal axis, and the activity (absorbance) was plotted on the vertical axis. The slope of the increase in absorbance in each reaction solution and the ratio of these to the control were calculated, and the value of the ratio was defined as the pepsin resistance value. The higher the value, the higher the resistance.

  The results for 10 μl and 20 μl of pepsin are shown in Table 7 as relative values when the value of yeast phytase is 100.

  From these results, it was confirmed that any modified phytase had improved pepsin resistance compared to yeast phytase.

[Test Example 2]
Next, in order to examine the combined effect of the above-mentioned modified forms, the substitution site contained in the modified form found in the present invention in an environment assuming the stomach, that is, in the presence of feed and digestive enzyme (pepsin) The pepsin resistance of the variants (36G09, 43C03, variants 146H06, 135E05, 136D06) containing at least two or more sites was evaluated. The results are shown in Table 8.

  As a result, all the variants (36G09, 43C03, 135E05, 136D06, 146H06) that contain at least two newly constructed substitution sites all consist of a single substitution site (variant 82, variant 85, modification). It can be confirmed that pepsin resistance is improved as compared to yeast phytase (Table 8), as in the case of the body 292, the modified body 301, the modified body 342, and the modified body 429). It has been found that combinations of substitution sites are possible. In particular, the modified body 135E05, the modified body 136D06, and the modified body 146H06 had higher pepsin resistance values than the yeast phytase (Table 8), and were expected to improve the stability in the stomach of pigs.

  By using the modified phytase of the present invention in a livestock feed, it is possible to reduce the application amount accompanying the increase in functionality, and as a result, the cost of phytase occupying the feed can be reduced. That is, this provides a highly functional feed additive enzyme excellent in effect and economy.

  The present invention further provides a method for creating a modified phytase that can be expected to improve the stability in the stomach of pigs, and is a high function of other feed additive enzymes that act in the stomach including phytase. The present invention can be widely applied to the production of feed, and provides a basic method for enhancing the function of a feed additive enzyme.

Predicted electrostatic potential of yeast phytase around pH 3.5. A three-dimensional model of yeast phytase showing the predicted electrostatic potential around pH 3.5. The phytase atom is shown in black and the positively charged region is shown in gray. Construction method of modified phytase expression plasmid (p82-1EX). Details of the construction method are described in Example 2. pPHY36 is a plasmid having a yeast phytase gene, pNNPH60I is a yeast phytase gene expression plasmid, p82-1 is a plasmid having a modified phytase gene, p82-1EX is a modified phytase gene expression plasmid, AOD P is a promoter of an alcohol oxidase gene, and AOD T is Each terminator of the alcohol oxidase gene is shown. It is a graph which shows the pH stability of yeast phytase. In the figure, the horizontal axis represents pH treatment time, and the vertical axis represents phytase activity. White circles indicate changes in activity over time when pH 3.0, rice marks at pH 3.5, and black triangles indicate pH 4.0. The activity was measured using the method shown in Example 1. It is a graph which shows the pepsin resistance of yeast phytase. In the figure, the horizontal axis represents pepsin treatment time, and the vertical axis represents the relative value of phytase activity. The white triangle mark shows the time course of activity when pepsin is applied at pH 4.0, and the black triangle mark shows the time course of phytase activity when it is acted at pH 4.0 without pepsin.

Claims (34)

  A modified phytase obtained by modifying Schwaniomyces genus yeast phytase, which improves the stability in the stomach.   The modified phytase according to claim 1, wherein the optimum pH is shifted to the acid side by modification.   The modified phytase according to claim 1, wherein pepsin resistance is improved by modification.   The modified phytase according to any one of claims 1 to 3, wherein the amino acid sequence of Schwaniomyces yeast phytase is the amino acid sequence shown in SEQ ID NO: 2.   The modified phytase according to any one of claims 1 to 4, which is created by substituting at least one of the acidic amino acids constituting Schwaniomyces yeast phytase with another amino acid.   6. The modified phytase according to claim 5, wherein the acidic amino acid is aspartic acid at the 429th residue of the amino acid sequence shown in SEQ ID NO: 2.   The modified phytase according to claim 6, wherein the other amino acid is asparagine.   The modified phytase according to any one of claims 1 to 4, which is created by substituting at least one of the amino acids constituting Schwaniomyces yeast phytase with a basic amino acid.   The modified phytase according to claim 8, wherein the basic amino acid is lysine or arginine.   The modified phytase according to any one of claims 1 to 4, which is created by substituting at least one of the amino acids constituting the Schwaniomyces yeast phytase with histidine.   The modified phytase according to any one of claims 8 to 10, wherein the amino acid to be substituted is serine at the 292nd residue of the amino acid sequence shown in SEQ ID NO: 2.   The positive charge in the electrostatic potential around pH 3.5 is weakened by substituting at least one amino acid constituting Schwaniomyces yeast phytase with another amino acid. The modified phytase according to any one of the above.   The modified phytase according to claim 12, wherein the amino acid to be substituted is serine at the 82nd residue of the amino acid sequence shown in SEQ ID NO: 2.   The modified phytase according to claim 13, wherein the other amino acid introduced by substitution at the 82nd residue is glutamic acid or aspartic acid.   The modified phytase according to claim 12, wherein the amino acid to be substituted is serine at the 85th residue as shown in SEQ ID NO: 2. The modified phytase according to claim 15, wherein the other amino acid introduced by substitution at the 85th residue is glutamic acid or aspartic acid.     The modified phytase according to claim 12, wherein the amino acid to be substituted is histidine at the 301st residue in the amino acid sequence shown in SEQ ID NO: 2.   The modified phytase according to claim 17, wherein the other amino acid introduced by substitution at the 301st residue is tylosin.   The modified phytase according to claim 12, wherein the amino acid to be substituted is histidine at the 342nd residue in SEQ ID NO: 2.   The modified phytase according to claim 19, wherein the other amino acid introduced by substitution at the 342nd residue is glutamic acid or aspartic acid.   Selected from the 82nd, 85th, 292th, 301th, 342th, and 429th residues of the amino acid sequence of the phytase set forth in SEQ ID NO: 2 The modified phytase according to any one of claims 1 to 4, which is created by substituting at least two or more amino acids with other amino acids.   The amino acid residue introduced by substitution is glutamic acid or aspartic acid at the 82nd residue in the amino acid sequence of phytase shown in SEQ ID NO: 2, 85th is glutamic acid or aspartic acid, and the 292nd residue is The modified phytase according to claim 21, which is lysine, arginine or histidine, the 301st residue is tylosin, the 342nd residue is glutamic acid or aspartic acid, and the 429th residue is asparagine.   The modified phytase according to claim 21, wherein the amino acid residue introduced by substitution is glutamic acid at the 342nd residue and asparagine at the 429th residue of the phytase shown in SEQ ID NO: 2.   The amino acid residue introduced by substitution is lysine at 292th residue of phytase shown in SEQ ID NO: 2, glutamic acid at 342th residue, and asparagine at 429th residue. Modified phytase.   The modified phytase according to claim 21, wherein the amino acid residue introduced by substitution is lysine at the 292th residue of the amino acid sequence of the phytase shown in SEQ ID NO: 2 and tylosin at the 301st residue.   The amino acid residue introduced by substitution is lysine in residue 292 of phytase described in SEQ ID NO: 2, tylosin in residue 301, and glutamic acid in residue 342. Modified phytase.   The amino acid residue introduced by substitution is lysine at residue 292 of phytase described in SEQ ID NO: 2, tylosin at residue 301, and asparagine at residue 429. Modified phytase.   A gene encoding the modified phytase according to any one of claims 1 to 27.   A method for performing a functional modification of a phytase having the amino acid sequence shown in SEQ ID NO: 2 by site-specific modification, thereby creating a modified phytase with improved stability at gastric pH.   A method for performing a functional modification of a phytase having the amino acid sequence of SEQ ID NO: 2 by site-specific modification, and as a result, creating a modified phytase having an optimum pH shifted to the acid side.   A method of performing functional modification of a phytase having the amino acid sequence of SEQ ID NO: 2 by site-specific modification, and as a result, creating a modified phytase with improved pepsin resistance.   32. The method according to any one of claims 29 to 31, wherein acidic amino acids in the phytase having the amino acid sequence set forth in SEQ ID NO: 2 are reduced.   32. The method according to any one of claims 29 to 31, wherein the basic amino acid in the phytase having the amino acid sequence of SEQ ID NO: 2 is increased.   34. The method according to any one of claims 29 to 33, wherein the modified phytase is created by attenuating a positive charge at an electrostatic potential around pH 3.5 in the phytase having the amino acid sequence of SEQ ID NO: 2.

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