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    • 专利摘要:
    PURPOSE: A phytase gene isolated from Bacillus coagulans(KCTC 1823) is provided, thereby producing phytase having improved phytate-decomposability. Therefore, of phytate form phosphorous in foods can be available. CONSTITUTION: A phytase gene isolated from Bacillus coagulans(KCTC 1823) has the nucleotide sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2. An expression vector contains the phytase gene of SEQ ID NO: 1. A host cell is transformed with the expression vector containing the phytase gene of SEQ ID NO: 1. A method for producing phytase comprises culturing the transformed host cell under suitable conditions and recovering phytase from the host cell or the cultured medium.
    公开号:KR20030050524A
    申请号:KR1020010080964
    申请日:2001-12-18
    公开日:2003-06-25
    发明作者:신용철；전영중；정경화；주은진；박철
    申请人:아미코젠주식회사；
    IPC主号:
    专利说明:

    A novel phytase gene from Bacillus coagulans KCTC 1823}
    [11] The present invention relates to a novel phytase gene derived from Bacillus coagulans KCTC 1823 strain and a gene product thereof, and more specifically, to a phytase from Bacillus coagulans KCTC 1823 strain. The present invention relates to a method for producing a phytase by separating a novel gene encoding and cloning it into a foreign microorganism, and then using the prepared phytase in the preparation of processed foods containing animal feed additives and phytate.
    [12] Phytic acid (phytate, myo- inositol 1,2,3,4,5,6-hexakisphosphate, IP6) is present as a major storage form of phosphate in plant seeds such as soybeans and barley corn. Accounted for 2% ( Crit. Rev. Food Sci. Nutr ., 16 : 49-114 (1982)). The phytic acid is a compound in which six phosphoric acids are bound to myo-inositol, and is mainly present in the form of a salt in which minerals such as calcium, magnesium, iron, and zinc are bound, and phytase (EC 3.1.3.8). Is an enzyme that can liberate phosphorus by hydrolyzing phytic acid. However, most compounded feeds for pigs and poultry are mainly composed of vegetable grains or by-products and greens. About 70% of phosphorus in vegetable feed is present in the form of phytic acid, but monogastric such as chicken and pigs (monogastric) Since animals do not have phytase in their intestines, they release phytic acid as manure, so they need to supply sufficient amounts of phosphorus for animal growth from the outside. In addition, phytic acid, which is not digested by a unit animal and is excreted in manure, is enzymatically decomposed by microorganisms in soil or water, causing eutrophication due to phosphorus influx in aquatic environment. It is emerging as a pollution problem. In addition, phytic acid not only forms strong complexes with various metal ions (Ca 2+ , Zn 2+ , Mg 2+ and Fe 2+ ) that are micronutrients, but also binds to various proteins in grains to act as a protease. Because of its inhibition, phytic acid in grains acts as an anti-nutritional factor, causing inhibition of digestion and absorption of proteins and important minerals in the body of livestock and humans, including animals ( J. Food Sci ., 46 : 546 (1981)).
    [13] As a viable alternative to solve this problem, a method of using phytase, a phytic acid degrading enzyme, has been applied as a feed additive. When phytase is added to feed, it significantly reduces the amount of phosphate added, and increases the efficiency and protein digestibility of inorganic metal ions in livestock, thereby increasing productivity and reducing excretion of effective phosphorus by 50%. . With this in mind, the Gumi region, including the Netherlands, is inducing the addition of phytase to feed for the purpose of reducing phosphorus emissions from livestock waste, and the use of phytase in domestic feed is also increasing. Particularly in the 21st century, global environmental problems have become an important factor in all industries and sectors, and in many countries, there is a trend to regulate the phosphorus content of livestock wastewater. Therefore, the usefulness of phytase will be increased in the future, and thus the market for phytase enzyme is expected to expand rapidly. On the other hand, phytase is expected to be applied not only as a feed additive for livestock, but also to be used in the manufacture of processed foods containing phytic acid, and to be applied to infant foods for enhancing digestibility.
    [14] Currently, fungus-derived phytases are manufactured and sold by BASF and Roche as feed additives. Efforts are being made to continuously discover and develop phytase gene sources that are more suitable as feed additives for unit animals around the world.
    [15] Accordingly, the present inventors hundreds of species, such as fungi, actinomycetes, bacteria, etc. to obtain other phytase gene source of novel properties superior to the conventional phytase for use in the production of feed additives and phytic acid-containing grain processed food for unit animals, etc. Among these strains, new phytase-producing microorganisms were identified, and new phytase was produced from the Bacillus coagulans KCTC 1823 strain, a test bacterium. After separating the phytase gene from the strain by using the nucleotide sequence of the gene and the amino acid sequence of the protein translated therefrom was determined to be a novel gene. In addition, as a result of purifying the phytase produced by the strain and recombinant E. coli to examine the enzyme properties, it was confirmed that the reaction properties are different from the existing phytase, a new enzyme to complete the present invention.
    [16] Accordingly, an object of the present invention is to provide a nucleotide sequence of the novel phytase gene.
    [17] Another object of the present invention is to provide an amino acid sequence of a protein translated from the base sequence.
    [18] Still another object of the present invention is to provide a recombinant expression vector comprising the gene.
    [19] Still another object of the present invention is to provide a transformant transformed with the recombinant expression vector.
    [20] Still another object of the present invention is to provide a method for preparing phytase, which comprises culturing the transformant.
    [21] Still another object of the present invention is to provide a feed additive and a grain processed food comprising the prepared phytase.
    [22] Hereinafter, the configuration of the present invention will be described in detail. Other objects and advantages of the invention will be more clearly understood through the following examples.
    [1] 1 is a graph showing soy flour resolution using phytase produced by Bacillus coagulans of the present invention.
    [2] FIG. 2 shows Coomassie staining by electrophoresis of phytase purified from Bacillus coagulans culture. FIG.
    [3] Figure 3 is a graph showing the relative activity and stability of the purified phytase (a) and the thermal stability (b) with CaCl 2 addition with the change of reaction temperature.
    [4] 4 is a graph showing the relative activity and stability of purified phytase according to the change of reaction pH.
    [5] 5 is a TLC diagram showing a reaction product generated from phytic acid and inositol phosphates using the present phytase.
    [6] 6 is a diagram showing a Linewever-Burk plot of phytic acid of the present invention phytase.
    [7] Figure 7 shows the nucleotide sequence of the gene encoding Bacillus coagulans phytase.
    [8] Figure 8 shows the amino acid sequence of a protein translated from the nucleotide sequence of the Bacillus coagulans phytase gene.
    [9] Figure 9 shows the recombinant plasmids pBSK / phyJ and pJH27 / phyJ containing the Bacillus coagulans phytase gene.
    [10] 10 is a graph showing the phytase productivity pattern in LB medium of recombinant Bacillus subtilis strains.
    [23] The present invention relates to a DNA comprising a DNA sequence encoding a novel phytase derived from Bacillus coagulans KCTC 1823 strain which can be used for the production of livestock feed additives and phytic acid-containing grain processed foods; An amino acid sequence of the phytase gene encoding protein and a derivative sequence functionally equivalent thereto; A recombinant expression vector comprising the DNA sequence; A host cell transformed with the DNA sequence or vector; A recombinant expression vector is prepared by inserting the DNA sequence of the gene into a vector capable of expressing in a suitable host cell, and transforming the gene DNA sequence or the recombinant expression vector into a suitable host cell by a method known in the art. And culturing the transformed host cell under suitable growth conditions and isolating the produced phytase from the host cell or culture medium. A food or feed composition comprising the phytase and / or host cell culture produced thereby.
    [24] Separation and purification of phytase enzyme can be used by using the protein separation method by a variety of chromatographic methods using the properties of phytase previously known or modified slightly to suit the purpose of the experiment. The purity assay of the phytase of the present invention purely separated by the chromatographic method was demonstrated using electrophoresis. The phytase molecular weight was determined by moving with standard molecular weight material and comparing the travel distance. The inventors found that the sample of the active fraction that passed through the Heparin Sepharose column showed a single band at the position of about 44,000 daltons when subjected to electrophoresis, so that the phytase was purified purely, and its molecular weight was about 44,000 Daltons. . As a result of investigating changes in enzyme activity for the temperature, pH, etc., which determine the phytase enzyme activity of the present invention, the optimum reaction temperature is about 40 ° C., and the same as the activity of the enzyme solution without heat treatment even after heat treatment for 10 min. It was rapidly inactivated at the temperature above ℃, and when 5mM CaCl 2 was added to the enzyme reaction, it showed more than 50% of remaining activity even at 70 ℃, showed the optimum activity at pH 7.0, and showed more than 90% activity between pH 5.0 and 11.0. It was found to be relatively stable at pH in this range.
    [25] In order to investigate the substrate specificity of the phytase of the present invention, the enzyme activity was measured by using substrates having various phosphate ester bonds. As a result, the phytase of the present invention hydrolyzed phytic acid and ADP and ATP. The enzyme activity was about 12% and 3%, respectively, compared to that of phytic acid. Sodium pyrophosphate, α-glycero-phosphate, and β-glycerophosphate were shown. (β-glycerophosphate), α- naphthyl phosphate (α-naphthylphosphate), did not exhibit an enzyme activity or the like for p- nitrophenyl phosphate (p -nitrophenyl phosphate). In order to investigate the cleavage specificity of the present invention, the cleavage specificity of the phytase was examined using phytic acid and various inositol phosphate substrates. As a result, the phytase of the present invention cleaves three of the six phosphorus bound to the phytic acid. Could.
    [26] As a result of measuring enzyme activity using soybean meal as a substrate, the phytase of phytase of the present invention was higher than that of other Bacillus phytase. Found to be excellent. Therefore, the phytase of the present invention may be effectively used in feed industry or phytic acid-containing grain processed food industry.
    [27] The nucleotide sequence of the gene encoding the phytase derived from Bacillus coagulans KCTC 1823 strain was determined and shown in FIG. As shown in FIG. 7, the gene consists of 1,149 bases (SEQ ID NO: 1), and the gene encodes 382 amino acids as shown in FIG. 8 (SEQ ID NO: 2). On the other hand, the phytase of the present invention had a signal sequence consisting of 30 amino acids responsible for extracellular secretion in the amino acid sequence of the gene product.
    [28] The present invention relates to an isolated nucleic acid or functional derivative thereof encoding a phytase having one or more of the above characteristics. Preferably, the nucleic acid may comprise DNA according to SEQ ID NO: 1 or functional derivatives thereof, or may be hybridized with DNA or functional derivatives thereof according to SEQ ID NO: 1. The nucleic acid is preferably a DNA molecule. In the context of the present invention, "functional equivalent" thereof in reference to a nucleic acid or gene means a derivative of nucleic acid having the functional properties of the nucleic acid encoding the phytase. That is, a functional equivalent or derivative of a nucleic acid encoding a phytase according to the invention encodes a phytase having the characteristics according to the invention. Nucleic acid derivatives encoding phytase according to the present invention are described, for example, by site-specific mutagenesis (Botstein, D., Shortle, D., 1985, Science 229: 1193-1201, Myers, RM, Lerman, LS, Maniatis, T., 1985, Science 229: 242-247 et al., Error-prone PCR (e.g. Leung, DW, Chen, E., Goeddel, DV, 1989, Technique 1: 11-15; Eckert, K, A , Kunkel, TA, 1991, PCR Methods Applic. 1: 17-24; Cadwell, RC, Joyce, GF, 1992, PCR Methods Applic. 2: 28-33), and / or chemically derived in the art. By mutagenesis techniques (eg, Elander, RP 'Microbial screening, Selection and Strain Improvement' in Basic Biotechnology, J. Bu'lock and B. Kristiansen Eds., Academic Press, New York, 1987, 217). The resulting mutants are included.
    [29] The present invention also relates to a method for producing a nucleic acid according to the present invention, wherein the method recovers the nucleic acid by hybridizing a probe containing the nucleic acid to a sample suspected of containing the nucleic acid under standard conditions. Characterized in that. Standard techniques using probes for the hybridization include Southern blotting (e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nd. Edition, Cold Spring Harbor Laboratory Press, 1989) and PCR and RT-PCR (e.g. PCR Protocols: A Guide to Methods and Applications, Innis, MA, Gelfand, DH, Sninsky, JJ and White, TJ Eds., Academic Press New York, 1990).
    [30] In the present invention, the phytase gene having a phytic acid degrading activity may be separated from the Bacillus coagulans KCTC 1823 strain according to a conventional gene separation method. After synthesizing primer DNA using a DNA synthesizer, a vector capable of amplifying the phytase gene fragment and inserting the fragment by the polymerase chain reaction method using the synthetic primer. Using the gene library to prepare and measure the phytic acid degradation activity can be selected for clones with phytase gene. Methods for isolation and identification of genes of the invention are described in the Examples. Those skilled in the art will appreciate that the present invention is a phytase, as defined, for example, by Sambrook, et al. Molecular Cloning: A Laboratory Manual, 2ed.Vol. 1. pp. 101-104, Cold Spring Harbor Laboratory Press (1989). It will be apparent that gene sequences also include such DNA or RNA sequences that can be hybridized.
    [31] Accordingly, the nucleic acid molecules to be interpreted according to the present invention are prepared by the above-described method as well as nucleic acid molecules having sequences that hybridize to the nucleic acid sequences of the present invention and sequences due to codon degeneracy of the genetic code. It also includes molecules having sequences inductively inferred from phytase gene sequences or amino acid sequences.
    [32] In a preferred embodiment, the phytase according to the invention comprises an amino acid sequence of SEQ ID NO: 2 or a derivative functionally equivalent thereto. In the present specification, "functionally equivalent derivative" means a phytase derivative having the functional properties of the phytase according to the present invention. That is, functional derivatives of phytase have the general characteristics of phytase according to the present invention, and are natural, synthetic or recombinant peptides, peptide fragments, mutants or variants in which one or more amino acids are deleted, substituted or added. It can be said to encompass all of them. Thus, the present invention includes not only the phytase enzyme amino acid sequence (SEQ ID NO: 2), but also functionally equivalent sequences substituted with other amino acid residues in the sequence according to a latent change. For example, one or more amino acids in the sequence may be substituted for other amino acid (s) of similar polarity that function functionally equivalent (silent change). Amino acid substitutions in the sequence may be selected from other members of the class to which the amino acid belongs. For example, nonpolar, hydrophobic amino acid classes include alanine, valine, leucine, isoleucine, phenylalanine, valine, tryptophan, proline, and methionine. Polar, neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged, basic amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Also included in the scope of the present invention are fragments or derivatives thereof having a range of homology between the phytase enzyme protein and amino acid sequence of the present invention, such as the same or similar biological activity within the range of 90-100%. .
    [33] When comparing the nucleotide sequence homology of the present invention phytase gene with other known genes, it was confirmed that the gene of the present invention is a novel gene showing different homology with other known phytase genes. For example, the sequence of the phyC gene from Bacillus subtilis VTT E-68013 (GenBank AF029053) and the phy gene from Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) DS11 (GenBank U85968) were 72% homologous, respectively. In addition, there was little sequence homology with other phytase genes, including the fungal phytase gene. However, the phytase gene sequence of the present invention showed a very high homology of 98% with that of the yzxA gene derived from Bacillus subtilis 168 (GenBank AF015775), but the yzxA gene ( yodV derived from Bacillus subtilis 168 strain). The gene (also known as a gene) is a putative gene found through sequencing as the genome sequence of the Bacillus subtilis 168 strain is revealed, and the presence and function of the gene product are unknown. Furthermore, the Bacillus coagulans strain, the phytase gene donor of the present invention, is a gene bank (Korean Collection for Type Cultures (KCTC)) strain and differs from the Bacillus subtilis 168 strain and through the present invention Bacillus coagulans phytase. By discovering the function of the gene product, the present invention has been reached.
    [34] The present invention includes not only the nucleic acid base sequences described above, but also recombinant expression vectors into which DNA having such base sequences is inserted. Those skilled in the art can prepare expression vectors encoding phytase enzymes using appropriate transcriptional / translational regulatory sequences and phytase-encoding nucleic acid sequences according to any method known in the art for inserting DNA fragments into a vector. The recombinant expression vector may be any vector as long as it functions in a selected host. For example, plasmid, cosmid, and the like, which are conventional expression vectors known in the art, may be used. Methods for constructing expression vectors are known per se and are disclosed, for example, in Sambrook et al. (Molecular Cloning, Cold Spring Harbor Laboratory (1989)).
    [35] The recombinant expression vector thus prepared can transform host cells. Suitable expression cells of recombinant DNA include E. coli and Bacillus bacteria, actinomycetes, yeasts, fungi, animal cells, insect cells, or plant cells. Can be used. For example, a host cell transformed with the above-described DNA nucleic acid or expression vector is a probiotic lactic acid bacterium such as E. coli, Bacillus sp., Lactobacillus or Lactococcus. , Aspergillus sp., Saccharomyces sp., Humicola sp., Pichia sp., Trichoderma, and soybeans or corn. Can be.
    [36] The phytase may be prepared by culturing the host cell transformed with the recombinant expression vector into which the phytase enzyme coding gene of the present invention or a gene having a sequence having a function equivalent thereto is inserted in an appropriate medium and conditions. One skilled in the art can utilize the phytic acid activity as described above or prepare the desired product in the form of a culture or composition comprising the phytase prepared by the above method, or in a state in which the isolated enzyme is freed or immobilized.
    [37] The invention also relates to the use of the phytase according to the invention in food or animal feed. The food or animal feed includes phytase having an activity in the digestive tract as an additive, and the animal preferably comprises aquatic animals including birds, cattle, sheep and other ruminants, pigs, fish and shrimps. It is selected from the group, wherein the additive is preferably kept active during the food or feed processing process. The present invention also relates to a food or animal feed comprising Bacillus coagulans KCTC 1823 capable of expressing phytase according to the present invention, host cells or spores of host cells transformed with the gene of the present invention. In addition, the present invention relates to a method for producing a food or animal feed, wherein the method is characterized in that the phytase according to the present invention is mixed with the food or animal feed. The phytase may be added in the form of a dry matter before treatment or in liquid form. In addition, the method is characterized in that Bacillus coagulans KCTC 1823, a host cell or spores of the host cell, which can express the phytase according to the present invention, is added to the food or animal feed.
    [38] The invention also relates to the use of the piases according to the invention in the production of inositol and inorganic phosphates with or without the aid of a phosphatase. The present invention also relates to a method for reducing phosphorus levels in animal manure, which method is characterized by providing an animal feed according to the present invention in an amount capable of effectively converting the phytic acid contained in the animal feed. That is, the phytase of the present invention can be industrially applied to agriculture and food. For example, the phytase of the present invention is added to feeds for unit animals such as pigs and chickens to drastically reduce the amount of phosphate added to feeds, and to increase productivity by increasing the efficiency and protein digestibility of phosphorus and inorganic metal ions in livestock. The excretion of effective phosphorus can be reduced. In particular, as the trend of regulating the phosphorus content of livestock wastewater in the world including Korea, the problem of lowering the basic phosphorus content of pollutant sources becomes an important matter in economic and environmental aspects. The use of phytase is therefore expected to be essential. In addition, phytic acid in grains strongly forms complexes with various metal ions, which are micronutrients, and binds to various proteins in grains to inhibit the action of proteolytic enzymes. Since it causes the inhibition of digestion and absorption of important minerals, the present invention is applicable to the food industry, such as the development of drinks for the improvement of digestibility by applying the present invention phytase to grain processed foods.
    [39] The present invention comprises the steps of purifying phytase and characterizing purified enzymes in Bacillus coagulans KCTC 1823 strain; Isolation of the phytase gene from Bacillus coagulans KCTC 1823 strain; Sequencing and analyzing the cloned gene; Recombinant phytase production in Escherichia coli and Bacillus subtilis strains.
    [40] Hereinafter, the configuration and operation of the present invention will be described in more detail with reference to Examples. However, the following examples are only for the purpose of illustrating the present invention and the scope of the present invention is not limited to the following examples.
    [41] Example 1 Purification of Phytase Produced by Bacillus Coagulans Strain
    [42] In order to search for novel phytase-producing strains, the enzyme activity characteristics, heat resistance and acid resistance of phytase produced by strains isolated from soil samples in Gyeongnam and Bacillus strains distributed by KCTC were analyzed. By measuring, Bacillus coagulans KCTC 1823 strains were selected that produce a phytase that is relatively suitable for feed additive use. There is no report of phytase production in the Bacillus coagulans KCTC 1823 strain, which is a test bacterium, and the reaction properties of the phytase produced by the strain are different from those of the known phytase. It came to invention. In particular, as a result of investigating the ability to decompose phytic acid in real grains using soybean meal, as shown in FIG. Found to be excellent. Such a result means that the phytase of the present invention can be effectively used in livestock feed industry or phytic acid-containing grain processed food industry. Therefore, the phytase was purified from the culture supernatant of the strain in order to investigate the physical properties and enzyme reaction characteristics of the phytase produced by the Bacillus coagulans KCTC 1823 strain.
    [43] Bacillus coagulans KCTC 1823 strain was inoculated in PSM medium (10 g peptone, 5 g beef extract, 10 g D-glucose, 5 g calcium phytate, 1 g MgSO 4 · 7H 2 O, 1 g CaCl 2 ) After shaking for 2 days at the culture culture was centrifuged for 20 minutes at 8,000 rpm to take the culture supernatant. Then, the culture supernatant was concentrated by ultrafiltration and diafiltration was performed by adding 10 mM Tris-Cl (pH 7.0) buffer containing 1 mM CaCl 2 . Q-Sepharose Fast Flow column chromatography was performed with the crude enzyme solution prepared in this manner. After passing the coenzyme solution through the Q-Sepharose Fast Flow column, washing it with 10 mM Tris-Cl (pH 7.0) buffer containing 1 mM CaCl 2 to remove unadsorbed proteins and applying a 0 to 0.5 M NaCl gradient. A phytase active fraction was eluted at around 0.3 M NaCl concentration. The eluted enzyme solution was collected and dialyzed in 10 mM Tris-Cl (pH 7.0) buffer containing 1 mM CaCl 2 . Thereafter, the dialysate was passed through a Heparin Sepharose column, washed with 10 mM Tris-Cl (pH 7.0) buffer containing 1 mM CaCl 2 , and a 0-0.5 M NaCl gradient was applied to the vicinity of 0.15-0.2 M NaCl concentration. The phytase active fraction was eluted. The enzyme activity fractions were then collected and dialyzed in 10 mM Tris-Cl (pH 7.0) buffer containing 1 mM CaCl 2 to purify the phytase produced by Bacillus coagulans.
    [44] When Coomassie staining was carried out by electrophoresis of the active fraction obtained by the above process, as shown in Figure 2, the sample of the active fraction passed through the Heparin Sepharose column shows a single band at a position of about 44,000 Daltons, the phytase is pure It was purified and found to have a molecular weight of about 44,000 Daltons.
    [45] In addition, about 32% of the crude enzyme solution activity was recovered by the above purification process, and the degree of purification was increased by 47 times, and the specific activity was about 16 units per mg of purified protein. At this time, the phytase activity was measured by the method of Choi et al . ( J. Microbiol. Biotechnol , 9 : 223-226 (1999)) to measure the amount of inorganic phosphate liberated by the enzymatic reaction from the substrate. The phytase enzyme activity was measured in this method by mixing 900 μl of 2 mM sodium phytate dissolved in 100 mM Tris-Cl (pH 7.0) buffer solution with 100 μl of the appropriately diluted enzyme solution and reacting at 37 ° C. for 30 minutes. 750 μl of 5% trichloroacetic acid solution was added to stop the reaction. At this time, 100 μl of distilled water was added instead of the enzyme solution as a control. Thereafter, 1.5 ml of a coloring reagent (a 4: 1 mixture of 2.5% ammonium molybdate solution and 2.5% FeSO 4 solution dissolved in 5.5% sulfuric acid) was added to measure absorbance at 700 nm. One unit of enzyme was defined as the amount of enzyme that releases 1 μmol of inorganic phosphorus in one minute.
    [46] Example 2 Properties of Phytase Purified from Bacillus Coagulans Culture
    [47] Example 2-1. Optimum Reaction Temperature and Thermal Stability of Phytase
    [48] In order to investigate the optimum reaction temperature and thermal stability of the purified phytase, the enzyme activity was measured at various temperatures and the relative activity is shown in FIG. 3. As shown in FIG. 3 (a), the optimum reaction temperature of the purified enzyme was 40 ° C., and the residual activity was measured after 10 minutes at each temperature. It was rapidly inactivated at the above temperature. However, as shown in FIG. 3 (b), as the concentration of CaCl 2 was increased in the enzyme reaction solution, the thermal stability of the enzyme increased. When 5 mM CaCl 2 was added to the enzyme reaction solution, the residual activity was higher than 50% even at 70 ° C. .
    [49] Example 2-2. Optimum pH and pH Stability of Phytase
    [50] In order to determine the optimal pH of the purified phytase, enzyme activity was measured at various pH conditions (FIG. 4). In addition, in order to investigate the pH stability of the enzyme was mixed with the enzyme solution in 100 mM buffer prepared for each pH and left for 1 hour at 37 ℃ was measured residual activity (Fig. 4). As a result, the purified phytase showed optimum activity at pH 7.0, and showed at least 90% of activity between pH 5.0 and 11.0, indicating that it was relatively stable at this range of pH.
    [51] Example 2-3. Substrate Specificity of Phytase
    [52] In order to investigate the substrate specificity of purified phytase, enzyme activity was measured using substrates with various phosphate ester bonds. As a result, the phytase of the present invention hydrolyzed phytic acid best and showed about 12% and 3% enzymatic activity for ADP and ATP, respectively, compared to the activity for phytic acid. However, the phytase of the present invention did not show enzymatic activity against sodium pyrophosphate, α-glycerophosphate, β-glycerophosphate, α-naphthylphosphate, p- nitrophenyl phosphate, and the like.
    [53] Example 2-4. Investigation of cleavage specificity of phytase
    [54] The cleavage specificity of phytase was investigated using phytic acid and various inositol phosphates as substrates using thin-layer chromatography. As a result, as shown in FIG. 5 (a), as the reaction time elapses, the purified enzyme decomposes phosphate, which is bound to phytic acid, and the amount of free phosphate increases gradually, and as a final product, myo-Inositol. -tris-phosphate was produced. That is, three of the six phosphorus to which the phytase of the present invention is bound to phytic acid can be cleaved. Also, phosphorus in Ins (1) P and Ins (1,4) P2 could not be cleaved; one Ins (1,4,5) P3 and one Ins (3,4,5,6) P4 Phosphorus was cleaved (FIG. 5 (b)).
    [55] Example 2-5. Kinetic Parameters of Purified Phytase
    [56] The kinetic parameters of purified phytase for phytic acid were determined by Lineweaver-Burk plot method. As shown in FIG. 6, K m and V max were 0.77 mM and 0.244 mmole / min / mg, respectively.
    [57] Example 2-6. N-terminal amino acid sequence of purified phytase
    [58] Analysis of the N-terminal amino acid sequence of the purified phytase revealed glutamic acid-glutamic acid-histidine-histidine-phenylalanine-lysine-valine-threonine-alanine-histidine-threonine-glutamic acid-threonine-aspartic acid-proline-valine. When the N-terminal amino acid sequence was compared with the amino acid sequences of other known proteins, the N-terminal amino acid sequence of the phytase of the present invention was identical to that of the yzxA gene product of the Bacillus subtilis 168 strain. It did not show homology with the amino acid sequences of other known proteins, including fungi including Aspergillus, Bacillus, and phytase derived from bacteria, including E. coli. However, the yzxA gene derived from the Bacillus subtilis 168 strain (also called the yodV gene) is a putative gene whose function is not revealed through sequencing analysis as the genome sequence of the Bacillus subtilis 168 strain is revealed. (Nature, 390 : 249-256 (1997)).
    [59] Example 3 Isolation of Phytase Gene from Bacillus Coagulans KCTC 1823 Strain
    [60] Bacillus coagulans KCTC 1823 strain was shaken in LB liquid medium and chromosomal DNA was isolated from the recovered cells by Saito and Miura's method ( Biochim. Biophys. Acta ., 72 : 619-629 (1963)). And the N-terminal starting material based on the nucleotide sequence of the Bacillus subtilis 168 yzxA gene to amplify the phytase gene on the chromosome of Bacillus coagulans KCTC 1823 strain by polymerase chain reaction and to clone it in E. coli. (5'-GCTCTAGAGTGATAAAAGAGGAGGG-3 ') and C-terminal starting material (5'-CGGGATCCGCTGCACAAGCTGCTTTC-3') were prepared. Using these two primers, the 1.2 kb DNA fragments generated by the polymerase chain reaction were isolated, and these DNA fragments were completely cleaved with Xba l and Bam HI and dephosphorylated with the same restriction enzyme and CIP (pBluescriptII SK +). Strategene) plasmid. E. coli JM83 was transformed by electroporation with the conjugated DNA, and then transformed into MacConkey agar medium containing ampicillin antibiotic at a concentration of 50 µg / ml.
    [61] Recombinant plasmids isolated from white colonies from MacConkey plated agar plates were analyzed for Southern hybrdization and restriction enzyme patterns. 1.2 kb of plasmid was isolated from recombinant E. coli. The presence of DNA from Bacillus coagulans of size was confirmed and this recombinant plasmid was named pBSK / phyJ.
    [62] Example 4. Base Sequence Determination and Analysis of Bacillus Coagulans Phytase Gene
    [63] The sequence of Bacillus coagulans-derived DNA contained in the recombinant plasmid pBSK / phyJ was analyzed by Sanger's method ( Proc. Natl. Acad. Sci ., 74 : 5463 (1977)) using an ABI PRISM Dye Terminator Cycle Sequencing kit. It was determined using a sequence analyzer (Automatic Sequencer ABI310). The nucleotide sequence of the gene encoding the phytase derived from Bacillus coagulans found by nucleotide sequence determination is as shown in FIG. In addition, the amino acid sequence of the protein translated from the gene base sequence is as shown in FIG.
    [64] As shown in FIG. 7, the Bacillus coagulans phytase gene named phyJ was composed of 1,149 bases, which encodes 382 amino acids as shown in FIG. 8. And the phytase of the present invention can be confirmed that the amino acid sequence starting from amino acid No. 31 as shown underlined in Figure 8 exactly matches the N-terminal amino acid sequence of the phytase purified in the culture of Bacillus coagurans strain there was. In addition, when comparing the sequence homology with other well-known genes, the phytase gene sequence of the present invention is 98 and the base sequence of the yzxA gene (GenBank AF015775) derived from Bacillus subtilis 168, a putative gene whose function is not known. Showed a very high homology of%, and 72% homology between the phyC gene derived from Bacillus subtilis VTT E-68013 (GenBank AF029053) and the nucleotide sequence of Bacillus amyloliquifaciens DS11 derived phy gene (GenBank U85968), respectively. Showed. In addition, it was confirmed that the gene of the present invention is a novel phytase gene because it has little homology with the nucleotide sequence of other known genes.
    [65] Example 5 Expression of Escherichia coli and Bacillus subtilis of the Bacillus coagulans phytase gene
    [66] Recombinant plasmid pBSK / phyJ (FIG. 9 (A)) was introduced into E. coli JM83 and E. coli XL1-blue, and these recombinant E. coli were inoculated in LB medium containing 100 μg / ml of ampicillin antibiotics at 37 ° C. The shaker was incubated overnight at. After crushing the cells obtained by centrifugation and using the coenzyme solution obtained to examine the phytase activity, the recombinant E. coli containing the vector DNA pBluescriptII SK + was not measured phytase activity but contains the recombinant plasmid pBSK / phyJ Recombinant E. coli JM83 and recombinant E. coli XL1-blue showed 0.27 U / mg and 0.05 U / mg, respectively. In addition, the reaction characteristics of the phytase produced in Escherichia coli by partial purification of the coenzyme solution obtained from the recombinant E. coli JM83 strain containing pBSK / phyJ were identical to those of the phytase purified from Bacillus coagulans culture supernatant. It was.
    [67] To express the phyJ gene in Bacillus subtilis strains, the phyJ gene fragment contained in the pBSK / phyJ plasmid was introduced into the pJH27 vector ( Biotechnol. Lett ., 15 : 133-138 (1993)), an E. coli-Bacillus shuttle vector . The recombinant plasmid pJH27 / phyJ (FIG. 9 (B)) was prepared and introduced into Bacillus subtilis DB431 strain. The recombinant strain was inoculated in LB medium containing 5 ㎍ / ml kanamycin and shaken at 30 ° C to investigate the cell growth and phytase activity according to growth time. As shown in FIG. 10, the recombinant Bacillus strain exhibited the highest activity of 0.2 unit / ml in 8 hours of incubation, which is 5 times higher in productivity compared to the Bacillus coagulans strain. However, the cell mass decreased slowly after 18 hours, while the phytase activity dropped sharply after 8 hours. It is assumed that phytase is degraded by the protease produced by the Bacillus subtilis host bacterium, which may be overcome by the use of protease-deficient mutants or changes in the composition of the medium.
    [68] As described in detail above, the phytase gene derived from the Bacillus coagulans KCTC 1823 strain of the present invention is not only a novel gene that differs from the known phytase gene and its nucleotide sequence and amino acid sequence, but also of other Bacillus-derived pita. By encoding a phytase enzyme having a higher ability to degrade phytic acid in soy flour, phytase prepared from the strain or recombinant microorganism may be used in the form of a phosphorus containing indigestible phytic acid in food and feed. There is a very good effect of increasing the nutritional value of animal feed, lowering feed costs and reducing the environmental burden of undigested and secreted phosphorus.
    权利要求:
    Claims (8)
    [1" claim-type="Currently amended] Phytase gene or functional equivalent thereof having the same nucleotide sequence as SEQ ID NO: 1 isolated from Bacillus coagulans KCTC 1823 strain.
    [2" claim-type="Currently amended] A phytase having an amino acid sequence equal to or SEQ ID NO: 2 isolated from Bacillus coagulans KCTC 1823 strain.
    [3" claim-type="Currently amended] Recombinant expression vector comprising the phytase gene of claim 1.
    [4" claim-type="Currently amended] A host cell transformed with the expression vector of claim 3.
    [5" claim-type="Currently amended] A method for producing a phytase, wherein the host cell of claim 4 is cultivated under suitable culture conditions and the phytase is recovered from the host cell or the culture medium by a method known in the art.
    [6" claim-type="Currently amended] A food or feed composition comprising the phytase prepared according to claim 2 or 5 or the host cell culture according to claim 5.
    [7" claim-type="Currently amended] A method for producing phytase, comprising culturing Bacillus coagulans KCTC 1823 strain under suitable culture conditions and recovering phytase from the strain or culture medium by methods known in the art.
    [8" claim-type="Currently amended] Food or feed composition of the phytase or strain culture according to claim 7.
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    同族专利:
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    2001-12-18|Application filed by 아미코젠주식회사
    2001-12-18|Priority to KR1020010080964A
    2003-06-25|Publication of KR20030050524A
    优先权:
    申请号 | 申请日 | 专利标题
    KR1020010080964A|KR20030050524A|2001-12-18|2001-12-18|A novel phytase gene from Bacillus coagulans KCTC 1823|
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