JPH11206368A - Secretory production of phytase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for the secretory production of phytase capable of continuing the culture essentially without increasing the amount of the cells of the microorganism in the extracellular secretion of phytase by the culture of a recombinant Candida boidinii in a liquid medium. SOLUTION: A recombinant Candida boidinii is produced by transforming Candida boidinii with a recombination plasmid having a DNA containing a phytase-coding gene bonded to the downstream of the promoter of AOD1 gene of Candida boidinii. The recombinant Candida boidinii is cultured in a liquid medium containing 0.3-3.0 g/L of phosphoric acid or phosphoric acid compound in terms of phosphate radical for >=10 days while adding methanol to the medium for secretory production of phytase from the cell of the recombinant Candida boidinii.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for culturing recombinant Candida boidinii in a liquid medium to secrete and produce phytase outside the cells of the recombinant Candida boidinii. Recombinant Candida voidinii obtained by transforming Candida voidiny with a recombinant plasmid having a DNA in which a gene encoding phytase is linked downstream of the promoter of the AOD1 (primary alcohol oxidase) gene of Davidinii is phosphoric acid or The present invention relates to a method of culturing in a liquid medium containing a phosphate compound while adding methanol to the medium to secrete and produce the phytase out of the recombinant Candida voidinii cells.

[0002]

2. Description of the Related Art Phytase is an enzyme that catalyzes a reaction to produce phosphoric acid by hydrolyzing phytin. In recent years, phytase has been added to livestock feed as a feed additive to decompose phytin that can not be digested by monogastric animals such as chickens and pigs, thereby reducing the concentration of phosphoric acid in livestock excrement and reducing phosphorus in feed. Attempts have been made to improve the use of acids.

[0003] EP-A-0 699 762 describes Schwaniomyces oxydentalis ( Schwann).
niomycs occidentalis ) and its genes have been described. The phytase is capable of hydrolyzing phytic acid to myo-inositol and has excellent heat stability. Therefore, the phytase is industrially useful as a feed additive because the feed is hardly deactivated even when exposed to high temperatures in the drying step of feed production.

[0004] EP-A-0697762 describes Saccharomyce.
Phytase production method using the transformed yeast of s cerevisiae) have been disclosed. Nevertheless, the method has room for improvement in secretory productivity.

By the way, among the methods for obtaining a recombinant protein using a recombinant microorganism, the construction of a purification process in which feedback regulation is easily released, the recombinant protein is not degraded, and it is highly likely that the recombinant protein exists as an active form. For example, a method of secreting and producing the recombinant protein out of the cells of the recombinant microorganism is desirable.

[0006] On the other hand, using yeast as a recombinant microorganism is
It is preferable for stably producing a recombinant protein because it is easy to culture and the gene encoding the introduced recombinant protein can be integrated into the chromosome by homologous recombination. For such reasons, secretory production of recombinant proteins using recombinant yeast has been studied. However, compared to secretory production using bacteria represented by Bacillus subtilis, the secretory production of yeast was at a lower level especially in the secretory production of proteins derived from heterologous organisms outside the cells.

[0007] Examples of highly secretory production of recombinant proteins using recombinant yeasts which have been known so far are described in EP-A-0558024, EP-A-0794256, Sakai. Et al. (Y. Sakai et a
l., Biochim. Biophys. Acta vol. 1308, pp. 81-87, (199
6)).

[0008] EP-A-0555824 discloses an expression cassette having a promoter and a terminator for the alcohol oxidase (AOD) gene of Candida voidinii, an expression vector having the expression cassette, and a transformed yeast having the expression vector. Is disclosed. The specification describes expression of adenylate kinase, cytochrome C552, and peroxidase using the expression cassette.

[0009] In EP-A-0 794 256, Kex of Saccharomyces cerviche is described.
A transformed yeast (Candida voidinii) having a recombinant plasmid in which a polypeptide derived from 2-prosthesis is incorporated into the above expression vector is prepared, and the transformed yeast is cultured while adding methanol to a liquid medium, thereby obtaining a Kex2 prosthesis. Producing polypeptides of interest.

Sakai et al. Used a transformant strain of Candida voidinii, a methanol-assimilating yeast, and cultured it while adding a concentrated solution of methanol, glycerol and a medium component to obtain Rhizopus oryza ( Rhizopus oryza).
e ) 3.4 g / l of glucoamylase from
Has accumulated.

[0011] In these techniques, the target protein or polypeptide is produced in large quantities, but the amount of microbial cells is increasing as the target product is produced. This is from the viewpoint of the productivity of the target substance per cell mass,
This means that productivity has decreased over time, and it is not always an efficient production method.

Further, as a result of the study by the present inventors, it has been found that Candida voidinii secretes certain viscous polysaccharides outside the cells during the culture. The increase in the bacterial cell concentration during the culture causes the accumulation of polysaccharides secreted and produced by the bacterial cells in the medium, resulting in an increase in the viscosity of the culture solution, which hinders the oxygen supply of the medium, and is the target Production of recombinant proteins becomes difficult. Furthermore, generally, microorganism cells that have grown excessively are not always preferable in an industrial process because they make the purification of the target substance difficult and increase the cost of treating waste cells.

For the above reasons, it is desirable to develop a fed-batch cultivation method that can suppress the growth of microorganisms to a certain level and efficiently and stably produce the desired product for a long period of time. However, in secretory production of recombinant proteins using yeast, the above-mentioned fed-batch culture method is not known,
Furthermore, no secretion production method is known when the recombinant protein is phytase.

[0014]

Accordingly, an object of the present invention is to provide a method of culturing recombinant Candida voidinii in a liquid medium to secrete and produce phytase outside the cells, thereby substantially increasing the amount of cells. It is an object of the present invention to provide a novel and efficient phytase secretion production method capable of continuously producing phytase without phytase.

[0015]

Means for Solving the Problems The present inventors have developed a DNA in which a phytase-encoding gene is linked downstream of the promoter of the AOD1 gene of Candida voidinii.
Recombinant Candida voidinii obtained by transforming Candida voidinii with a recombinant plasmid having a, in a liquid medium containing a limited amount of phosphoric acid or a phosphate compound, for a certain period of time while adding methanol to the medium By culturing as described above, the present inventors have found that phytase can be continuously secreted and produced outside the cells of the recombinant Candida voidinii without proliferating the cells, thereby completing the present invention.

That is, the present invention provides a recombinant plasmid having a DNA in which a phytase-encoding gene is linked downstream of the AOD1 gene promoter of Candida boidinii.
Recombinant Candida voidinii obtained by transforming Voidinii is cultured in a liquid medium while adding methanol to the medium, and the phytase is secreted and produced outside the cells of the recombinant Candida voidinii.
A phytase secretion production method characterized in that secretory production of the phytase continues without substantially increasing the amount of bacterial cells by containing a limited amount of phosphoric acid or a phosphate compound in the liquid medium, and It is a recombinant Candida voidinii used in the method.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION The promoter used in the present invention is a promoter inducible by methanol, and the AOD1 (primary alcohol oxidase) gene which is a methanol oxidase of Candida voidinii, a methanol-assimilating bacterium. Is a promoter. And as such, a promoter containing the sequence of SEQ ID NO: 1 can be exemplified.

A DNA containing a part of the nucleotide sequence of SEQ ID NO: 1 can be used in the present invention as a promoter as long as it has the activity of the promoter and is induced by methanol. Such DNA is specifically DNA having 70%, preferably 90%, homology to the sequence set forth in SEQ ID NO: 1. Further, when hybridization is performed using a DNA having the base sequence of SEQ ID NO: 1 as a probe, a DNA that hybridizes
Is also a category of the promoter used in the present invention as long as it has the activity of the promoter and is induced by methanol.
However, in the above, hybridization is performed under the following conditions. That is, hybridization was carried out at 42 ° C. for 12 hours in 5 × SSC containing 50% by weight of formamide and 0.1% by weight, and then 68 ° C., 15 ° C. in 0.1 × SSC containing 0.1% by weight. Wash for minutes.
SSC means an aqueous solution containing 3M NaCl and 0.3M citric acid.

The phytase-encoding gene used in the present invention is a gene encoding an enzyme having an activity of hydrolyzing phytic acid, which can be expressed downstream of the promoter of the AOD1 gene of Candida voidinii. When bound, it refers to one that is expressed in the host Candida voidinii and can be secreted out of the cells of the cell. As such, SEQ ID NO: 2 in the sequence listing
And a phytase derived from Schwaniomyces oxydentalis described in EP-A-0 699 972. In addition, 70%, preferably 90%, of the DNA of another base sequence encoding the amino acid sequence of the phytase shown in SEQ ID NO: 2 in the sequence listing and the base sequence of the phytase gene shown in SEQ ID NO: 2 in the sequence listing DNA having homology is also included in the phytase-encoding gene of the present invention as long as it has phytase activity.

When a DNA having the nucleotide sequence of SEQ ID NO: 2 is used as a probe for hybridization, the hybridizing DNA encodes the phytase used in the present invention as long as the DNA has the activity of the phytase. This is the category of genes that do. However, in the above, hybridization is performed under the following conditions. That is, after hybridization at 42 ° C. for 12 hours in 5 × SSC containing 50% by weight formamide and 0.1% by weight, 0.1 × SSC containing 0.1% by weight was used.
Washing is performed at 68 ° C. for 15 minutes. SSC means an aqueous solution containing 3M NaCl and 0.3M citric acid.

The recombinant plasmid used in the present invention comprises:
A DNA having a gene encoding phytase bound downstream (3 ') of the promoter of the AOD1 gene of Candida voidinii;
NA is inserted into a vector that is replicable in the host Candida voidinii. Examples of vectors that can be used for this purpose include known vectors used for transformation of Escherichia coli, such as pUC18 and pBR322.

It is preferable that an appropriate selection marker is linked to the recombinant plasmid of the present invention in order to determine the transformed host cell. Examples of the selectable marker include a gene related to nucleic acid synthesis, a gene related to amino acid synthesis, a gene related to growth, and a gene imparting drug resistance.
In addition, since the recombinant plasmid of the present invention is retained in the transformed host cell, it contains a sequence that enables autonomous replication in the cell, or contains the sequence of the chromosome of the host cell for integration into the chromosome by homologous recombination. It is desirable to include a sequence highly homologous to the sequence. Furthermore, in order to improve expression efficiency,
It is desirable to add a terminator downstream of the gene encoding the polypeptide of interest. As such a terminator, the terminator of the AOD1 gene of Candida voidinii is exemplified.
As an example of such a recombinant plasmid, pNNPH60 in which a gene encoding a phytase derived from Schwaniomyces oxydentalis is linked downstream of an AOD1 promoter derived from Candida voidinii (FIG. 2)
Can be exemplified.

The host transformed with the recombinant plasmid of the present invention is Candida voidinii. In order to transform the host Candida voidinii with the above-described recombinant plasmid, the recombinant plasmid may be introduced into the host by a known transformation method.

In order to secrete and produce phytase using the recombinant Candida voidinii obtained as described above, the recombinant Candida voidinii is cultured in a special liquid medium for a certain period of time while adding methanol. do it. Hereinafter, the culture conditions for secretory production of phytase will be described in detail.

In the present invention, the liquid medium refers to a medium in which the medium is water. Basically, various types of carbon sources, nitrogen sources, inorganic salts, and trace amounts of organic and A synthetic medium or a natural medium containing an inorganic component.

Although methanol is an inducer and an important medium component as a carbon source, the carbon source added to the liquid medium at the start of culture does not need to be limited to methanol. There is no particular limitation as long as it can grow. Examples of such carbon sources include glycerol, glucose, sucrose, organic acids, and other carbon sources, as well as molasses, peptone, yeast extract, corn steep liquor, and nitrogen simultaneously with carbon sources such as meat extract. It may be a source or a phosphate source. It is also possible to use a plurality of these. Further, these carbon sources and methanol can be used in combination.

Examples of the nitrogen source include ammonia and urea, and ammonium salts such as ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate and ammonium nitrate.

As the inorganic salts, it is convenient to use monopotassium phosphate, dipotassium phosphate, sodium chloride, magnesium sulfate, calcium chloride, zinc sulfate, copper sulfate, ferrous sulfate and the like, if necessary. . further,
Addition of vitamins such as thiamine hydrochloride and biotin as a trace component may be effective in some cases.

In the present invention, it is important to limit the amount of phosphoric acid or a phosphoric acid compound in the medium components. That is, the amount of phosphoric acid or phosphate compound is added so as to be a limiting factor for the growth of recombinant Candida voidinii. In that sense, the amount of the above-mentioned medium components other than the phosphoric acid or the phosphate compound must be an amount that does not become insufficient throughout the culture. Whether the amount of phosphate or phosphate compound is the limiting factor for growth is determined by the fact that the growth of the recombinant yeast reaches a steady state, and the addition of phosphate or phosphate compound increases the growth amount. It can be easily determined based on whether or not.

Here, the term "phosphate compound" means an organic phosphoric acid derived from a biological component contained in a natural medium, in addition to inorganic phosphates such as ammonium salt, potassium salt, sodium salt and calcium salt of phosphoric acid. . The concentration of phosphoric acid or a phosphate compound in the medium varies depending on the cell concentration to be finally obtained, but it shows a certain amount of secretory production and, from the viewpoint of suppressing excessive production of cells, is usually used as a phosphate group. 0.3 ~
3.0 g / l, preferably 0.5 to 2.0 g / l
It is.

In the present invention, the above liquid medium is inoculated with recombinant Candida voidinii and cultured.
The culture temperature, aeration and stirring conditions, the pH of the medium, and other conditions for the culture may be in accordance with the usual culture conditions suitable for Candida Boydinii used as a host. Specifically, the culture temperature is usually 20 to 40 ° C, and the pH of the medium is 3.0 to 7.0.
It is desirable to keep it at zero. In addition, for efficient production of phytase, at least 10 days or more, preferably 15
Continue culturing for at least one day.

Shortly after the cultivation, the carbon source is depleted and the concentration of dissolved oxygen in the medium increases as the recombinant Candida voidinii grows. After this stage, culture is continued by adding only methanol and an alkali as a pH adjuster in the presence of a nitrogen source. Unless the carbon source added at the start of the culture is particularly difficult to assimilate, the time point at which the addition of methanol is started by depletion of the carbon source is usually 24 to 96 hours after the start of the culture. At this time, if a sufficient amount of nitrogen source is added at the start of the culture, p
The H adjuster may be a caustic solution such as sodium hydroxide or potassium hydroxide, but usually the pH is adjusted by adding ammonia water or ammonia gas, also serving as a nitrogen source supply.

As soon as methanol is added in the presence of a nitrogen source, the amount of phosphoric acid becomes a limiting factor for growth, and the increase in the bacterial cell concentration stops, so that there is no substantial increase in the amount of bacterial cells. The state where there is no substantial increase in the amount of cells means a state where the number of cells does not increase more than twice in one week.

When the addition rate of methanol is kept constant, the secretory production of phytase continues linearly with time.
In consideration of the productivity of practical phytase, the cell concentration at this time is 5 g / l to 120 g / l, preferably 10 g / l to 80 g / l in terms of dry cell weight. 5g cell concentration
If it is less than / l, the productivity of phytase will be poor. If the amount of the cells is more than 120 g / l, the medium components are consumed for the growth of the cells and are wasted. On the other hand, if the amount of cells is too large, the load in the purification step of phytase produced by culturing increases, and the cost of treating waste cells also increases.

Further, as described above, Candida voidinii secretes certain viscous polysaccharides outside the cells during culture, so that if the cell concentration during culture is too high,
Polysaccharides accumulate in the medium, resulting in an increase in the viscosity of the culture solution, which hinders the oxygen supply of the medium and makes it difficult to produce the target recombinant protein.

The method of adding methanol to the liquid medium may be intermittent or continuous. At that time, if a large amount of methanol is added in a short period of time, the productivity of phytase will decrease, and in some cases, the bacteria will die and the production of phytase will stop. Therefore, the cultivation is performed while controlling the amount of methanol in the medium as an inducer / carbon source usually to less than 1.0% (v / v).

The methanol concentration in the culture can be determined by, for example, analyzing a small amount of the culture by gas chromatography. Practically, a method of adding methanol while continuously monitoring with a methanol sensor can be employed. Further, under conditions in which carbon sources other than methanol are depleted, it is also possible to continuously monitor the presence or absence of methanol depletion by measuring the concentration of dissolved oxygen at a dissolved oxygen electrode.

When the cultivation is continued, the amount of recombinant Candida voidinii cells does not substantially increase and does not extremely decrease. Thus, the finding that the recombinant candy davoydinii maintains the cell concentration for a long period of time is completely unexpected, and was first found by the present inventors.

The end point of the culture may be determined from the viewpoint of phytase productivity and cost, but is usually about 10 minutes from the start of the culture.
It may end on day 0, preferably about day 50.

After removing the cells from the resulting culture by centrifugation or the like, phytase may be recovered according to a commonly used protein recovery method. The recovered phytase may be further purified. Further, considering that the phytase produced by the present invention is useful as an additive for livestock feed, a crude phytase recovered from a culture solution can be used as a final product, and furthermore, a livestock feed. Phytase containing a medium component can also be used as a final product as long as there is no problem in growth.

[0041]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but these Examples do not limit the present invention in any way.

Example 1 (1) Preparation of Expression Plasmid A plasmid containing a cDNA encoding a phytase derived from Schwaniomyces oxydentalis cloned with a ZAP-cDNA synthesis kit (Strategene) was replaced with MT-1.
[0743] This strain is designated as deposit number FERM BP-5108 in 1994.
1-3, Higashi 1-3-1 Tsukuba, Ibaraki Prefecture, Japan
(Deposited under the Budapest Treaty on International Recognition of Microbial Deposits in Patent Procedures with the National Institute of Bioscience and Biotechnology, National Institute of Advanced Industrial Science and Technology). A restriction map of pPHY36 is shown in FIG. 1 (FIG. 1). This is the vector region of pPHY36.
pBluescript SK- (Strategene) is 2958 bp, and a cDNA of about 1800 bp encoding phytase is cloned into its EcoRI and XhoI sites, and contains a terminator and Poly (A). This plasmid
After making EcoRI and NotI sites blunt-ended with T4 polymerase, a linker having a NotI site (Takara Shuzo Co., Ltd.) was added and ligated to create a NotI site. Xho
After making the I site blunt-ended with T4 polymerase, NotI linker was added and ligated, and phytase cDNA was added.
A plasmid having NotI sites at both 5 ′ and 3 ′ ends was prepared. CDNA prepared by cutting this plasmid with NotI
Was ligated to the NotI site of pNote I (European Patent Application Publication No. 0558024), a plasmid for expressing a heterologous protein of Candida voydinii, with T4 ligase to prepare pNNPH60. pNNP
The restriction enzyme map of H60 is shown in FIG. 2 (FIG. 2).

(2) Transformation Candida voidinii TK62 strain described in European Patent Application Publication No.
(1% yeast extract, 2% polypeptone, 2% glucose) The medium was inoculated into a medium, and shake-cultured at 28 ° C. for 30 hours to obtain a preculture liquid. Using the obtained preculture, 100
The cells were inoculated into ml of YPD medium so that OD ₆₁₀ became 0.2, and cultured at 28 ° C. for about 12 hours until OD ₆₁₀ was around 1.0. The culture was collected, and 25 ml of a lithium solution (10 mM Tris-HCl (pH 8.0), 1 ml) was collected.
mM EDTA, 3 mM lithium acetate).
The cells were collected by centrifugation, suspended again in 5 ml of a lithium solution, and incubated at 30 ° C. for 1 hour with shaking to obtain competent cells. 1.5m
10 μg / μl calf heat-denatured in 1 tube
thymus, 10 μl, 1 μg / μ cut with BamHI
After adding 5 μl of pNNPH60 and 100 μl of competent cells and mixing gently,
Let stand for minutes. After standing, 700 μl of a 45% PEG solution (45% (w / v) polyethylene glycol # 4000)
(PEG), 10 mM Tris-HCl (pH 8.
0), 1 mM EDTA, and 3 mM lithium acetate) were added, mixed gently, and allowed to stand at 30 ° C. for 1 hour. Then, after heat shock at 42 ° C for 5 minutes,
It was left still at 0 ° C. for 5 minutes. After collecting cells by centrifugation, 1 ml
200 μl of each suspension in sterilized water (0.67% Yeast Nitrogen base)
ew / o Amino acid (manufactured by Difco),
2% glucose, 2% agar) and cultured at 28 ° C for 3 days. Only the cells transformed by this operation formed colonies.

(3) Screening of phytase-high-producing strain From the Candida voidinii transformant obtained in the above section (2), the following culture was selected to select a strain that highly expresses phytase derived from Schwaniomyces oxydentalis. And phytase activity in the culture supernatant was compared.

Each single colony was pre-cultured in 5 ml of YPD medium at 28 ° C. for 24 hours. Using this preculture, 20 ml of a YPGM medium (1% yeast extract, 2% polypeptone, 3% glycerol, 1.5% methanol) was inoculated to an OD ₆₁₀ of 0.2, and incubated at 28 ° C. for 3 hours. After culturing for one day, the culture was centrifuged to obtain a supernatant containing the enzyme. After concentrating the supernatant 10 times by ultrafiltration,
The medium components were replaced with ion exchanged water. Next, the enzyme solution is further concentrated or diluted to a concentration suitable for the reaction.
Add 900 μl of 60 mM sodium acetate buffer (pH 4.2) containing 5 mM sodium phytate to μl,
The reaction was performed at 70 ° C. for 30 minutes. After the reaction is completed, add 5N
The reaction was stopped by adding 100 μl of sulfuric acid. The control group was prepared by adding 100 μl of 5N sulfuric acid to a buffer solution containing a substrate in advance and adding an enzyme solution. After the reaction was stopped, the reaction solution was diluted appropriately, and the method of Heinonen and Lahti (Anal. Bioche
m., Vol. 113, PP. 313-317, (1981)).

The phytase production was calculated assuming that 1 mg of phytase catalyzes a reaction that releases 320 μmol of phosphoric acid per minute under the above-mentioned activity measurement conditions.
By this operation, it was found that there were various phytase-producing strains in the transformed strain, and MT-40544 was obtained as a strain having high phytase-producing ability. This strain has the accession number FERM BP-5936 on May 3, 1996.
As of 1st, it has been deposited at the Institute of Biotechnology and Industrial Technology, 1-3-1 Higashi, Tsukuba, Ibaraki Prefecture, based on the Budapest Treaty on the International Recognition of Microbial Deposits for Patent Procedures.

Example 2 Secretory production of phytase (1) The transformed strain MT-40544 obtained in Example 1 was cultured by the following method. 100 ml of a medium consisting of 1% corn steep liquor, 0.5% ammonium sulfate, 0.1% dipotassium phosphate and 2% glucose was placed in a 500 ml baffled Erlenmeyer flask, sterilized at 121 ° C. for 20 minutes, and then added thereto.
One platinum loop of MT-40544 was inoculated and cultured with shaking at 28 ° C. for 24 hours. Next, 1200 ml of a medium (0.5% yeast extract (phosphate content 3.3%), 3% glycerol, 0.1%
5% ammonium sulfate, 0.2% monopotassium phosphate,
(0.1% magnesium sulfate heptahydrate) in a 2 liter jar fermenter and sterilized in the same manner, and then 18 ml of methanol was added.
28 ° C, aeration volume 1.0vvm, stirring blade rotation speed 700rpm
m, pH of the culture solution was adjusted to 5.5 with 5% aqueous ammonia, and culture was performed.

After the start of the cultivation, the dissolved oxygen concentration in the culture solution once decreased to 0%, and thereafter, the addition of methanol was started 60 days after the start of the culture when the dissolved oxygen concentration began to increase. Only the pH adjuster aqueous ammonia was continued to be added. Methanol was added when the dissolved oxygen concentration reached 30% of the saturated concentration while monitoring the dissolved oxygen concentration. Methanol continued to be added at a rate of about 1 g / l / h, but the water volume was kept almost constant throughout the culture period because no water was recovered from the exhaust of the jar fermenter. The progress of the culture is shown in FIG. 3 (FIG. 3). The secretory production of phytase was continued even after 50 days from the start of the cultivation, while substantially maintaining the state where the amount of cells (dry cell weight) did not increase.

Example 3 Secretory Production of Phytase (2) The transformant MT-40544 was cultured in the same manner as in Example 1 except that the amount of phosphate in the culture solution in the jar fermenter was changed. That is, the addition amount of monopotassium phosphate was set to 0.1% or 0.05%. As shown in FIG. 3 (FIG. 3), the amount of bacterial cells and the amount of phytase changed with time but were similar to those in Example 2 but differed in strength. Therefore, it was shown that the secretory production of phytase can be continued while maintaining the state in which the amount of bacterial cells does not substantially increase by using the amount of phosphate groups in the culture solution as the growth limiting factor.

[0050]

According to the method of the present invention, it is possible to produce phytase in large quantities and efficiently. According to the method of the present invention, the bacterial cell concentration in the liquid medium does not increase beyond a certain level, so that the phytase production per bacterial cell is extremely good. In addition, since phytase production is continued for a long period of time without a substantial increase in the amount of cells, the phytase production rate, effective use of medium components, and reduction of the load on the purification step are achieved. The method of the present invention is effective as a method for industrially producing phytase.

[0051]

[Sequence list]

SEQ ID NO: 1 Sequence length: 734 Sequence type: Number of nucleic acid strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin Organism: Candida boidinii Differentiation degree: Wild strain Sequence: TCTAGAATTT CATTATTTAT TTTTTATTGA CTGGAAATTT TCAATCAATT TTATTTATTT 60 TTATTTATTT ATTTTCATAT TCTTAGATTT AAACTTTTTA GATGACCGCT ATTTTACTTA 120 CTTACTTACT TACTTACTTA CTTACTTACT TACATACCTA CTTACTGTGA TTTTATAATA 180 TGATAAGAAT TAATTTTCAT ATTTATGATG ATGTAAATTT AACCTAGTAT ACTATTTTAA 240 AGTTATCACT ATCTTTTAGT GCTGGCATTT TTTATTCTAT TTTCATATAT GTATATAAGT 300 AAATTAAGTA TCATCACGCT GCTTACTGTA CGTTTAAAAT GTGGAGATGG AAATAGAGAT 360 GGGGATGAAG ATGAAGATGA TGAGAATTAT AAACCATTCA TTCATTAATC AATCAATATA 420 ACTTATAAAA AAATTTATAT TTAAATGAAT TAATTTCCTT TATTTTAATA ATATCGTTAA 480 TTCTTTTAAA TTCTATTTTA TTTTAATTCT TTCTTTATCA TAGTTATCATCAT ATAACAATTA 540 TATAACATAG ATACACAATT ATTATTATAT TATCATATTA TTTTTTAATA TATTTATATATATATATATATATTAATTAATTAATTA TTTCT 660 TTTTTTAACG AATTTTAACG AACTTTTAAA AAAACAAAAA AAAAAAAACA AAATTATTTT720 TCAATAGCGG CCGC 734

SEQ ID NO: 2 Sequence length: 1631 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA origin Organism: Schwanniomyces occidentalis Degree of differentiation: wild-type sequence GTCAATC ATG GTC TCG ATC TCA AAA TTA ATT AAT AAC GGT TTA CTC TTA 49 Met Val Ser Ile Ser Lys Leu Ile Asn Asn Gly Leu Leu Leu 5 10 GCT GGT CAA AGT GTT TAC CAA GAT TTA GCT ACT CCA CAA CAA TCT TCC 97 Ala Gly Gln Ser Val Tyr Gln Asp Leu Ala Thr Pro Gln Gln Ser Ser 15 20 25 30 GTC GAG CAG TAT AAT ATT ATT AGG TTT TTA GGT GGT TCG GGT CCT TAC 145 Val Glu Gln Tyr Asn Ile Ile Arg Phe Leu Gly Gly Ser Gly Pro Tyr 35 40 45 ATT CAA CGC AGT GGT TAT GGT ATT TCC ACT GAT ATT CCT GAT CAG TGC 193 Ile Gln Arg Ser Gly Tyr Gly Ile Ser Thr Asp Ile Pro Asp Gln Cys 50 55 60 ACA ATT AAG CAA GTT CAG TTG ATG TCA AGG CAT GGG GAA AGA TAC CCT 241 Thr Ile Lys Gln Val Gln Leu Met Ser Arg His Gly Glu Arg Tyr Pro 65 70 75 TCA AAA AAC TCT GGT AAG AAG TTA AAA ACA ATA TAT GGT AAA TTA A AG 289 Ser Lys Asn Ser Gly Lys Lys Leu Lys Thr Ile Tyr Gly Lys Leu Lys 80 85 90 AGC TAC AAT GGC ACT TTC ACA GGT AGC TTA GCT TTT TTG AAT GAC TAT 337 Ser Tyr Asn Gly Thr Phe Thr Gly Ser Leu Ala Phe Leu Asn Asp Tyr 95 100 105 110 GAA TAT TTT GTT CCG GAT GAT AGT TTG TAC GAA AAG GAA ACA AGT GCA 385 Glu Tyr Phe Val Pro Asp Asp Ser Leu Tyr Glu Lys Glu Thr Ser Ala 115 120 125 CTG AAC TCG CAA GGT TTA TTT GCA GGT ACT ACA GAT GCC TTA AGA CAT 433 Leu Asn Ser Gln Gly Leu Phe Ala Gly Thr Thr Asp Ala Leu Arg His 130 135 140 GGT GCT GCT TTT AGA GCT AAA TAT GGA TCA TTG TAT AAA CAA AAT TCT 481 Gly Ala Ala Phe Arg Ala Lys Tyr Gly Ser Leu Tyr Lys Gln Asn Ser 145 150 155 ACC TTG CCA GTT TTC ACT TCA AAT TCC AAC AGA GTC TAC CAG ACT TCT 529 Thr Leu Pro Val Phe Thr Ser Asn Ser Asn Arg Val Tyr Gln Thr Ser 160 165 170 GAA TAC TTT GCC AGA GGT TTC TTA GGT GAT GAA TTT TCT GAT GCT ACT 577 Glu Tyr Phe Ala Arg Gly Phe Leu Gly Asp Glu Phe Ser Asp Ala Thr 175 180 185 190 GTT CAC TTT GCT ATC ATT GAT GAA GAC CCT AAA ATG G GT GTT AAT TCA 625 Val His Phe Ala Ile Ile Asp Glu Asp Pro Lys Met Gly Val Asn Ser 195 200 205 TTA ACA CCA AGA GCC GCT TGT GAC AAT TAT AAT GAG GAT GTG AAT GAC 673 Leu Thr Pro Arg Ala Ala Cys Asp Asn Tyr Asn Glu Asp Val Asn Asp 210 215 220 GGC ATT GTC AAT CAA TAT AGC ACT GAC TAT TTG GAT GAA GCC CTT AAA 721 Gly Ile Val Asn Gln Tyr Ser Thr Asp Tyr Leu Asp Glu Ala Leu Lys 225 230 235 AGA TTC CAA TCA TCA AAT CCA GGA TTG AAT TTG ACC TCG GAA GAC GTT 769 Arg Phe Gln Ser Ser Asn Pro Gly Leu Asn Leu Thr Ser Glu Asp Val 240 245 250 TAC CAA CTT TTC GCT TAC TGT GCA TAT GAG ACT AAT GTT AAG GGT GCA 817 Tyr Gln Leu Phe Ala Tyr Cys Ala Tyr Glu Thr Asn Val Lys Gly Ala 255 260 265 270 TCC CCA TTC TGT GAC TTA TTT ACT AAT GAA GAA TAC ATT CAA TAT TCC 865 Ser Pro Phe Cys Asp Leu Phe Thr Asn Glu Glu Tyr Ile Gln Tyr Ser 275 280 285 TAC AGC GTT GAT CTT TCT AAT TAT TAT TCT CAC GGG GCA GGT CAT AAT 913 Tyr Ser Val Asp Leu Ser Asn Tyr Tyr Ser His Gly Ala Gly His Asn 290 295 300 CTA ACT AAA ACC ATT GGT TCT ACT TTA T TA AAT GCC TCA TTA ACC TTA 961 Leu Thr Lys Thr Ile Gly Ser Thr Leu Leu Asn Ala Ser Leu Thr Leu 305 310 315 TTA AAA GAT GGC ACC AAT GAC AAT AAA ATC TGG TTA TCT TTT TCA CAC 1009 Leu Lys Asp Gly Thr Asn Asp Asn Lys Ile Trp Leu Ser Phe Ser His 320 320 325 330 GAT ACT GAT TTG GAA ATC TTC CAT AGT GCC TTA GGA ATT GTT GAG CCA 1057 Asp Thr Asp Leu Glu Ile Phe His Ser Ala Leu Gly Ile Val Glu Pro 335 340 345 345 350 GCT GAA GAT TTA CCA GTT GAT TAC ATT CCT TTT CCA TCG CCA TAT ATT 1105 Ala Glu Asp Leu Pro Val Asp Tyr Ile Pro Phe Pro Ser Pro Tyr Ile 355 360 365 CAC TCA CAA ATT GTT CCA CAA GGT GCT AGA ATT TAT ACT GAG AAA TAT 1153 His Ser Gln Ile Val Pro Gln Gly Ala Arg Ile Tyr Thr Glu Lys Tyr 370 375 380 TCA TGT GGC AAC GAA ACC TAT GTT AGA TAT ATA CTT AAT GAT GCA GTT 1201 Ser Cys Gly Asn Glu Thr Tyr Val Arg Tyr Ile Leu Asn Asp Ala Val 385 390 395 GTT CCA ATT CCA AAA TGC TCT TCT GGT CCA GGG TTC TCA TGT GAG CTT 1249 Val Pro Ile Pro Lys Cys Ser Ser Gly Pro Gly Phe Ser Cys Glu Leu 400 405 410 AGT AAA TTC GAA GAA TAT ATT AAT AAA AGA CTT AGG GAT GTT GAC TTT 1297 Ser Lys Phe Glu Glu Tyr Ile Asn Lys Arg Leu Arg Asp Val Asp Phe 415 420 425 430 GTT GAA CAA TGT GAT TTA AAA GAT GCT CCA ACT GAA GTT ACT TTT TAC 1345 Val Glu Gln Cys Asp Leu Lys Asp Ala Pro Thr Glu Val Thr Phe Tyr 435 440 445 TGG GAT TAC ACG TCG GTG AAC TAT AGT GCG TCC CTT ATT AAT GGT TAA 1393 Trp Asp Tyr Thr Ser Val Asn Tyr Ser Ala Ser Leu Ile Asn Gly 450 455 460 ATTGAGTATA GGAGAATATC TTATTTCTAG TTTGATCACT ATCTGAATCC ACTTTGCTCT 1433 TTCTCCTTGT TTTGATTGCT TATCCATTGT TTAGAAATAC GTTATAAAGC AATCATTTTT 1463 ACAACTATGT CGTCTAATAC TTTGTTTCTA GAATTAAAAA AATAAATAGT ATACCTGTGT 1493 AAAGTCTTAC TTGATAGCTA ACTAGTCACT TCTTAATCAT ACACCTAATC TATCCTAA 1631

[Brief description of the drawings]

FIG. 1 is a restriction map of a plasmid PHY36 containing a phytase cDNA derived from Schwaniomyces oxydentalis. In the figure, Ampr indicates the ampicillin resistance gene, and ori indicates the replication origin.

FIG. 2 is a restriction map of a recombinant plasmid pNNPH60 for expressing a phytase gene derived from Schwaniomyces oxydentalis in Candida voidinii. In the figure, Ampr indicates an ampicillin resistance gene, and C.boidiniiURA3 indicates a URA3 gene derived from Candida voidinii.

FIG. 3 is a graph showing the change over time in the phytase production amount and the increase in the amount of bacterial cells when the transformed yeast MT-40544 is cultured by addition of methanol while changing the phosphate concentration in the culture solution. In the figure, a square symbol indicates a test in which the amount of monopotassium phosphate added to the liquid medium was 0.5 g / l, a triangle symbol indicates that it was 1.0 g / l, The circle symbol indicates that it is 1.0 g / l. Among the symbols, the filled symbols indicate the bacterial cell concentration, and the hollow symbols indicate the phytase activity.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12N 1/19 C12R 1:72) (C12N 15/09 ZNA C12R 1: 645) (72) Inventor Nobuo Kato Kameoka-shi, Kyoto 2-3-18 Nishi-Atsujigaoka Miyamadai 2-72 (72) Inventor Yasunobu Sakai 2-40-8 Motomiya, Otsu-shi, Shiga Prefecture (72) Inventor Daisuke Mochizuki 1144 Togo, Mobara-shi, Chiba Prefecture Mitsui Chemicals Co., Ltd. Person Hitoshi Takahashi 1 Toonuma-cho, Sunagawa-shi, Hokkaido Mitsui Chemicals, Inc.

Claims (7)

[Claims] 1. Candida Voidiny
boidinii ) The recombinant Candida voidinii obtained by transforming Candida voidinii with a recombinant plasmid having a DNA in which a gene encoding phytase is linked downstream of the promoter of the AOD1 gene is prepared by adding methanol to a medium in a liquid medium. When the phytase is secreted and produced outside the cells of the recombinant Candida voidinii, the phytase is substantially reduced in amount by including a limited amount of phosphoric acid or a phosphate compound in the liquid medium. A method for secretory production of phytase, characterized in that secretory production of said phytase continues without increase. 2. The liquid medium according to claim 1, wherein said liquid medium contains 0.3 to 3.
2. The method for secretory production of phytase according to claim 1, comprising 0 g / L of phosphoric acid or a phosphoric acid compound. 3. The method for secretory production of phytase according to claim 1, wherein the culture time is 10 days or more. 4. The method according to claim 1, wherein the phytase is a phytase derived from Schwanniomyces occidentalis . 5. The method according to claim 5, wherein the recombinant Candida voidinii is MT.
The method for secretory production of phytase according to claim 4, which is -40544 (FERM BP-5936). 6. A recombinant Candida voidinii for use in the method of claim 4. 7. The recombinant Candida Voidinii MT-40544 (FERM BP-593) used in the method according to claim 5.
6).