CN1118006A - Process for production of inositol and microorganism used therefor - Google Patents

Abstract

A process for production of inositol comprising the steps of (1) culturing a microorganism capable of extracellularly secreting inositol and belonging to the genus Candida in a medium so as to extracellularly accumulate inositol in the medium; and optionally (2) recovering inositol from the culture. Preferably, the producer microorganism is resistant to an antibiotic such as cerulenine or D-cycloserine, or resistant to a glucose metabolism antagonist such as 2-deoxyglucose.

Description

The preparation method of inositol and microorganism used therefor

The present invention relates to the preparation method of inositol and the new production bacteria microorganism useful to described method.

Known up to now inositol preparation method has: extracting inositol (E.P.No.506289 from rice bran or each thing leach liquor, 1992) (Japanese unexamined patent publication number 61-56142) and cultivate bread yeast (yeast saccharomyces cerevisiae) and from culture, obtain inositol (E.P.No.506289,1992).

Yet extractive method is disadvantageous, because contain a large amount of different impurity in rice bran and each thing leach liquor, makes from rice bran or each thing leach liquor extract the purifying inositol difficulty and expensive that becomes.Moreover yield poorly in cultivating the process of bread yeast, so this method also is expensive, utilizes the industrialized preparing process of bread yeast still not have progress.Except bread yeast, also not sending other microorganism can be to the cell exocrine inositol up to now.

The present inventor through various research to find except bread yeast, to find that at last a certain mutant of a kind of microorganism of candidiasis subordinate can be to the cell exocrine inositol to the microorganism of cell exocrine inositol.The present inventor finds that also inositol not only can be by conventional fermentation process preparation in addition, wherein produces bacteria microorganism and grows in the substratum that contains carbon source and nitrogenous source with to the cell exocrine inositol, and inositol can obtain from culture; Can also prepare by the following method, wherein produce bacteria microorganism and cultivated obtaining culturing cell, and the cell of the minimum of growth use with the nutrient solution form, separate complete cell or modification cell with to the cell exocrine inositol.The enzyme reaction of not following cell to grow in one method of back has participated in the production of inositol.

Correspondingly, the invention provides the method for preparing inositol, may further comprise the steps: cultivate a kind of microorganism in substratum with at extracellular accumulation inositol, this microorganism belongs to the candiyeast Pseudomonas, can be to the cell exocrine inositol, and from culture, can optionally obtain inositol.

The present invention also provides the method for preparing inositol that comprises the following steps in addition: the culture cell that can contain the synthetic institute of inositol indispensable enzyme to the microorganism of cell exocrine inositol with acquisition of cultivating the candiyeast Pseudomonas in substratum, with enzyme with the precursor incubation of inositol precursor being transformed into inositol and at extracellular accumulation inositol, and reclaim inositol as required.

According to embodiment preferred, the present invention uses can be to the cell exocrine inositol, belong to the candiyeast Pseudomonas and antibioticly have the microorganism of resistance as producing bacterium to a kind of.

Therefore, the present invention further provides the method for preparing inositol that contains following steps: cultivation is a kind of in substratum can be to the cell exocrine inositol, the microorganism that belongs to candiyeast Pseudomonas and anti-a kind of antibiotic is with at extracellular accumulation inositol, and reclaims inositol as required from substratum.

The present invention also provides the method for preparing inositol that contains the following step: cultivation is a kind of in substratum can be to the cell exocrine inositol, the microorganism that belongs to candiyeast Pseudomonas and anti-a kind of antibiotic is to obtain to contain the culture cell of the synthetic institute of inositol indispensable enzyme, with enzyme with the precursor incubation of inositol so that precursor is transformed into inositol and to the cell exocrine inositol, and reclaim inositol as required.

Another specific embodiments according to the present invention be the present invention use a kind of can be to the cell exocrine inositol, the microorganism that belongs to the candiyeast Pseudomonas and the glucose metabolism antagonist is had a resistance is as producing bacteria microorganism.

Correspondingly, the invention provides the method for preparing inositol that contains the following step: cultivation is a kind of in substratum can be to the cell exocrine inositol, the microorganism that belongs to the candiyeast Pseudomonas and the glucose metabolism antagonist is had a resistance is with at extracellular accumulation inositol, and reclaims inositol as required.

The present invention also further provides the method for preparing inositol that contains the following step: cultivation is a kind of in substratum can be to the cell exocrine inositol, belong to the candiyeast Pseudomonas and the glucose metabolism antagonist is had the microorganism of resistance contain the culture cell that inositol synthesizes institute's indispensable enzyme with acquisition, with enzyme with the precursor incubation of inositol so that precursor is transformed into inositol and at extracellular accumulation inositol, and reclaim inositol as required.

According to the present invention, anyly belong to the candiyeast Pseudomonas and can be used to the microorganism of cell exocrine inositol.The microorganism that preferably belongs to the candiyeast Pseudomonas is a Candida boidinii, as the Candida boidinii mutant IP-2 that derives from Candida boidinii TR-1 is by preferred, attention: Candida boidinii TR-1 is called as TR-1, it is deposited in the industrial science technical body of national bio-science and the meeting of human technology on April 4th, 1994, number is FERM P-14261, it is transferred to international depositary institution in the budapest treaty in nineteen ninety-five, and number is FERMBP; Candida boidinii IP-2 is called as IP-2, it is deposited in the industrial science technical body of national bio-science and the meeting of human technology on April 4th, 1994, number is FERM P-14262, be transferred to international preservation mechanism in the budapest treaty in nineteen ninety-five, number is FERM BP.

Candida boidinii TR-1 and Candida boidinii IP-2 have following denominator:

(a) cultivation and morphological characteristic

(1) in malt juice liquid medium, cultivates

Cell grows wild, and color is yellow and is suspending equably to cream-colored, and individual cells is and waits ellipse, and slight curving, the cell size is 1.5-3.5 * 7～12 μ m.

(2) in the wort solid medium, cultivate

It is yellow to cream-colored, clear and smooth, soft and moistening that bacterium colony is.

(3) in the Corn Meal Agar substratum, be coated with dull and stereotyped the cultivation

Cell is interconnected to form chain as mycelium, and branch is arranged sometimes.

(b) formation of spore

On some substratum, do not form spore, be included on the wort agar substratum.

(C) Physiology and biochemistry sort feature

(1) optimum growing condition: pH3 to 7; 15 to 37 ℃ of temperature

(2) tolerance condition: pH2 to 9; 4 to 47 ℃ of temperature

(3) death point is 50 ℃

(4) the nitrate assimilation positive

(5) gelatine liquefication reaction negative

(6) abundant with ethanol as the sole carbon source growth

(7) the generation positive of carotenoid

(8) pegnin feminine gender

(9) the generation feminine gender of ester

(10) lipid acid scission reaction feminine gender

(11) Suan the generation positive

(12) the generation feminine gender of starchy material

(13) reaction negative of the blue B of diazonium

(14) carbohydrate fermentation effect glucose, semi-lactosi and seminose fermentation are good; Starch, threharose and pectinose fermentation are very slow; Gala is half congealed, maltose, sucrose, rhafinose and inositol nonfermented.

(15) assimilation of carbohydrate: glucose, fructose, lactose, seminose, wood sugar and N.F,USP MANNITOL can fully be assimilated; Semi-lactosi can assimilate or not assimilate a little; Sucrose, maltose, threharose, pectinose, dextrin, starch, O14-Methyldelectine, rhafinose and inositol are not absorbed.

(16) utilization of carbon source: methyl alcohol, ethanol and glycerine can be utilized well; Pyruvic acid, fumaric acid, α-ketoisocaproic and Virahol can be utilized; Citric acid, propyl alcohol, ethylene glycol, propylene glycol, acetaldehyde, glycine, L-glutamic acid, asparagus fern nitronic acid and L-L-Ala can be utilized slightly.

(17) utilization of nitrogenous source: peptone, ammonium sulfate, ammonium nitrate, SODIUMNITRATE, urea, thiocarbamide, L-L-glutamic acid, glycine, DL-L-Ala and l-asparagine can be utilized; Sodium Nitrite is not utilized.

(18) demand of VITAMIN: vitamin H is essential.

(19) amino acid whose demand: do not have

Correspondingly, according to J.Cen.Appl Microbial.26,133-158 (1980) (especially 156 pages) are accredited as Candida boidinii with microorganism strains TR-1 and IP-2.

Can be among the present invention to the representative strain Candida boidinii IP-the 2nd of cell exocrine inositol, the mutant of wild type strain Candida boidinii TR-1.Sudden change can be carried out according to conventional steps, for example, and by obtaining mutant with processing parent strains such as physical mutagen such as UV-irradiation or chemical mutagen such as N-methyl-N '-nitro-N-nitrosoguanidine, ethyl-methyl sulfonate such as wild type strain.Parental cell is after mutagenic compound are handled, and treated cell can be screened expeditiously to obtain and can for example press following method to the mutant of cell exocrine inositol:

Also promptly, treated cell is applied on cell can well-grown nutrient agar flat board, as on the natural nutrition nutrient agar to form bacterium colony.In addition, also need a kind of nutrient agar that does not contain inositol, can grow thereon, a kind of bacterial strain suspension liquid of inositol that needs is covered on this substratum through the cell such as the Candida boidinii cell of mutagenesis.Then the bacterium colony of above-mentioned generation is copied on the nutrient agar flat board by above-mentioned preparation, if certain bacterium colony that duplicates on the nutrient agar after several days can produce inositol and to the cell exocrine inositol, so before be coated on the nutrient agar, needs the strain cell of inositol growing to the bacterium colony (positive bacterium colony) of cell exocrine inositol on every side.Therefore, just can from corresponding to separate the primary colony of positive bacterium colony, purifying can be to the mutants which had of cell exocrine inositol.

Although producing the frequency of required bacterial strain is 1/10 ⁴To 1/10 ⁶, but still can simply and effectively select required bacterial strain according to above mentioned screening system.Although therefore described the representative of mutants which had TR-1 in this specification sheets as required bacterial strain, but as long as someone thinks that above-mentioned screening system is that effectively the common skilled person of this area just can obtain easily can be to the mutant of cell exocrine inositol for obtaining required bacterial strain.

According to the present invention, it is the sort of mutant that higher resistance is arranged than its hydrophilic bacterial strain antagonist that demonstrates that a certain antibody is had the mutant of resistance.Preferably, it is that those are in containing a kind of substratum of antibiotic that antagonist of the present invention has the mutant of resistance, demonstrate at least 60% allometry amount (cell concn), and wherein parent strain demonstrates 30% or the mutant of lower allometry amount (cell concn).In this case, " allometry amount " refers to microbial growth per-cent (cell concn) in containing a kind of substratum of antibiotic, with same but do not contain in the substratum of antibiotic with a kind of microbial growth amount (cell concn) as 100%, wherein increment (cell concn) is determined by measure light absorption under 600nm.

Used antibody among the present invention, for example, cerulenin, BrefeldinA, D-seromycin and other commercial antibiotic all can use.

Mutant of the present invention can be to the cell exocrine inositol, belongs to the candiyeast Pseudomonas and to certain antibiotic resistance that have, it can obtain to the cell preparation of cell exocrine inositol by mutagenesis is any.Be Candida boidinii IP-2 (FERM BP) preferably by an example of the parent strain of mutagenesis, mutagenesis can be carried out according to above-mentioned steps, to be coated on through the cell that mutagenic compound are handled on the nutrient agar that contains antibiotic to select the mutant of anti-this antibiotic, in this case, have only the mutant of the resistance that has obtained this antibiotic to grow and form bacterium colony, therefore, can select required mutant easily, only be about 1/10 although produce the frequency of this required mutagenesis ⁴To 1/10 ⁶

As can be to the cell exocrine inositol, and cerulenin there is the example of the microorganism of resistance, must mention the mutant Candida boidinii CER176 of Candida boidinii IP-2, mutant Candida boidinii CER176 is called as CER176, it was deposited in the industrial science technical body of national bio-science and the meeting of human technology on May 20th, 1994, number is FERM P-14318, it is transferred to international depositary institution in the budapest treaty April 6 nineteen ninety-five, and number is FERM BP5069.

As can and the example of the microorganism of resistance being arranged to D-seromycin to the cell exocrine inositol, must mention Candida boidinii DCSR0.2-59 and DCSR0.3-11, they all are the mutant of Candida boidinii IP-2, mutant Candida boidinii DCSR0.2-59 is called as DCSR0.2-59, it was preserved in the industrial science technical body of understanding as the national bio-science and the human technology of the international depositary institution in the budapest treaty April 6 nineteen ninety-five, and number is FERM BP5071.Mutant Candida boidinii DCSR 0.3-11 is called as DCSR 0.3-11, it was preserved in the industrial science technical body of understanding as the national bio-science and the human technology of the international depositary institution in the budapest treaty April 6 nineteen ninety-five, and number is FERM BP5072.

Another preferred specific examples is the resistant mutants that the present invention uses the glucose metabolism antagonist, and it belongs to the candiyeast Pseudomonas, can be to the cell exocrine inositol.The glucose metabolism antagonist is (1) a kind of material that suppresses microorganism growth, wherein restraining effect can be recovered by adding glucose, (2) a kind of inhibition and the material that synthesizes the relevant enzyme of inositol from glucose biological, wherein said restraining effect can be recovered by a kind of material that adds inositol and/or inositol metabolism mysoinositol downstream.

The analog that the anti-agent of glucose metabolism antagonism comprises glucose is as 2-deoxyglucose, 1-thioglucose, 5-thioglucose etc.

Mutant of the present invention can belong to candida bacterial strain to the cell exocrine inositol, and anti-agent has resistance to the glucose metabolism antagonism, and it can prepare to the strain cell of cell exocrine inositol by mutagenesis is any.An example that preferably is used for the parent strain of mutagenesis is Candida boidinii IP-2 (FERM BP).Mutagenesis can be carried out according to above-mentioned steps.Cell is after mutagenic compound are handled, and the step that stands again to screen is to select the mutant of the anti-agent of anti-glucose metabolism antagonism, and is wherein applied on the nutrient agar flat board that contains the anti-agent of glucose metabolism antagonism through the cell of mutagenic compound processing.The substratum of screening usefulness necessarily can not contain glucose, can contain a kind of alcohol for example glycerine, methyl alcohol, ethanol etc.Under this occasion, have only the mutant of the resistance that obtains anti-glucose metabolism antagonist to grow and form bacterium colony.Therefore desired mutant can obtain easily, only is about 1/10 although produce the frequency of required mutagenesis ⁴To 1/10 ⁶

An example of the resistant strain of the anti-agent of glucose metabolism antagonism of the present invention is Candida boidinii DGR1-14, it is called as DGR1-14, the industrial science technical body that the industrial science that it was understood by the national bio-science of preservation on May 20th, 1994 is understood, number is FERM P-14319, and be transferred to international depositary institution under the condition of Budapest on April 6th, 1985, be numbered FERM BP-5070.

According to the present invention, inositol can produce through the following steps: cultivating as mentioned above can be to the microorganism of cell exocrine inositol, contain carbon source in the substratum, nitrogenous source and mineral ion, organic trace constituent can be contained if necessary with at extracellular accumulation inositol, inositol (being called fermentation method here) can be from culture, obtained if necessary.

Another concrete scheme optionally according to the present invention is that inositol can produce through the following steps: cultivation is above-mentioned in substratum can be to the microorganism of cell exocrine inositol, microorganism can grow in substratum as mentioned above and contain the microorganism cells that participates in the biosynthetic enzyme of inositol with preparation, with enzyme with the biosynthetic precursor incubation of inositol so that precursor is transformed into inositol, from reaction culture medium, obtain inositol (being called Enzymology method here) if necessary.

Prepare inositol with fermentation method, can use as carbohydrate as carbon source: glucose, fructose, starch or cellulose hydrolysis product, syrup etc.; A kind of organic acid such as fumaric acid, citric acid, succsinic acid etc.; A kind of alcohol such as methyl alcohol, ethanol, glycerine etc., these can also can unite use separately in fermentation method.According to the characteristic of carbon source, preferred carbon source concentration is 1 to 15%.

As nitrogenous source, for example, organic ammonium salt such as ammonium acetate etc.; Inorganic ammonium salt class such as ammonium sulfate, ammonium chloride, ammonium phosphate etc.; Ammonia such as gaseous ammonia or ammoniacal liquor; Urea or analogue, separately or unite use.According to the preferred nitrogen concentration of the characteristic of nitrogenous source is 0.1 to 4.0%.

As organic trace constituent, other VITAMIN that vitamin H or microorganism need can be independent or jointly involved in fermention medium.Concentration according to the preferred organic trace constituent of characteristic of organic trace constituent is 0.000001% to 0.1%.If necessary, can add a kind of material in the substratum, its rich vitamin, amino acid and other somatomedin such as diffusion juice, peptone, Yeast diffusion juice etc.According to the characteristic of this material, preferred material concentration is 0.01 to 5%.

In addition, fermention medium can contain inorganic salt such as potassiumphosphate, sal epsom, calcium chloride, sodium-chlor, zinc sulfate, copper sulfate, ferrous sulfate and other trace elements, separately or unite and be added in the fermention medium, according to the characteristic of adding inorganic substance, preferred inorganic salt concentration is 0.0001 to 0.5%.If necessary also foam reducing composition can be added in addition so that culture condition is stable.

Cultivate and preferably carry out under aerobic conditions, this condition can be provided by ventilation, stirring and/or vibration, in cultural method, the pH value is controlled between 3 and 8, culture temperature is controlled between 20 ℃ and 35 ℃, and cultivation will be carried out 24 to 96 hours, optimizing breathing and stirring.

Prepare inositol for enzyme process, by in the used substratum of above-mentioned fermentation method, cultivate microorganism of the present invention with the used growth conditions of above-mentioned fermentation method and just can obtain required cell, it contains the biosynthetic enzyme of participation inositol.

The enzyme that uses in the enzyme process preferably uses the form of culture, isolated intact cell from culture, and the protoplasm somatocyte of step preparation routinely, the somatoblast that the biosynthetic enzyme system of inositol can be kept, and other.

As the biosynthetic precursor of inositol, for example precursor glucose of glucose-6-phosphoric acid or glucose-6-phosphoric acid etc. all can be used, reaction is preferably carried out in the presence of Reduced nicotinamide-adenine dinucleotide (NAD) and ammonium ion, when glucose is used as precursor, preferably add magnesium ion and adenosine-3-phosphoric acid again, the synthetic necessary compound of those inositols adds in the reaction culture medium discriminably, and perhaps the form with the organic substance that contains these compounds adds.In addition; other material such as SH base protective material also can be added in the reaction culture medium, and the building-up reactions of inositol is to carry out for 3 to 8 times in the pH value preferably, and temperature is 20 to 35 ℃; reacted 10 to 72 hours, and preferably ventilating and/or stirring under the aerobic conditions that is provided and carry out.

The inositol that accumulates in culture or reactant can be utilized, for example the one-tenth as feed grades, selectively, inositol can separation and purification from culture or reactant, for example, can separate and the purifying inositol according to the step of normally used isolated or purified inositol, preferably by with culture or reactant is centrifugal or filter to obtain to contain the supernatant liquor or the filtered solution of inositol, then, filtered solution or supernatant liquor are handled with the deionizing material through positively charged ion and anionresin, and reconcentration is so that the inositol crystallization.

Embodiment

The present invention comes at length further to explain by following examples.The separation of the mutant of embodiment 1. secretion inositols

N-methyl-N-nitro-N-nitrosoguanidine of using 300 μ g/ml is according to a conventional method handled Candida boidinii TR-1 cell 10 minutes under 30 ℃, pH6.0, suitably dilution, be coated with flat board on the nutrient agar that contains composition shown in the table 1, dull and stereotyped cell was cultivated two days down at 30 ℃.

Table 1

Glucose 20g/L

Yeast diffusion juice 2g/L

MgSO ₄·7H ₂O 0.5g/L

Peptone 5g/L

K ₂HPO ₄ 1g/L

(, adding 15g/L agar) for agar plate

In addition, the cell inoculation of yeast saccharomyces cerevisiae ATCC 34893 is contained in the substratum of composition shown in the table 1 in 5ml,, wash culturing cell, it is coated on the agar plate of substratum shown in the table 2 with physiological saline 30 ℃ of following shaking culture one day.

Table 2

Grape sugar 10g/L

(NH ₄) ₂SO ₄ 5g/L

KH ₂PO ₄ 1g/L

MgSO ₄·7H ₂O 0.5g/L

CaCl ₂·2H ₂O 0.1g/L

NaCl 0.1g/L

ZnSO ₄·7H ₂O 0.4mg/L

FeSO ₄·7H ₂O 0.4mg/L

CuSO ₄·5H ₂O 0.04mg/L

Vitamin H 0.04mg/L

Agar 15g/L

With INKOH PH is transferred to 5.5

Next step will copy on the nutrient agar flat board that contains composition shown in the table 1 of above-mentioned preparation at the bacterium colony that contains the Candida boidinii TR-1 that handles through mutagenic compound that forms on the above-mentioned agar plate of composition shown in the table 1, revealed the cell that has covered yeast saccharomyces cerevisiae on it, to pick out as positive bacterium colony around the bacterium colony that duplicates of yeast saccharomyces cerevisiae ATCC34893 cell growth, in corresponding to the bacterium colony of the original agar plate of the positive bacterium colony that duplicates, separate and the purified mutant body, by this method, a kind of required mutant, Candida boidinii IP-2 has just obtained.Embodiment 2 is by the fermentative preparation inositol

With Candida boidinii TR-1 in contrast, Candida boidinii IP-2 of the present invention cultivates in the substratum that contains composition shown in the table 3 separately in advance, vibrated 24 hours down at 30 ℃, the pre-culture of gained is connect in the substratum that the 50ml that is beneficial in the 500ml erlenmeyer flask contains composition shown in the table 3, and this substratum is 110 ℃ of sterilizations 10 minutes down.Cell is at 180rpm, and the oscillating condition that reciprocal span is 30cm was cultivated 48 hours down.

Table 3

Glucose 50g/L

(NH ₄) ₂SO ₄ 2.5g/L

KH ₂PO ₄ 1g/L

MgSO ₄·7H ₂O 0.5g/L

CaCl ₂·2H ₂O 0.1g/L

NaCl 0.1g/L

ZnSO ₄·7H ₂O 0.4g/L

FeSO ₄·7H ₂O 0.4g/L

CuSO ₄·5H ₂O 0.04g/L

Vitamin H 0.04g/L

With IN KOH with PH transfer to 5.5 cultivate after, remove culturing cell and residual lime carbonate, press DIFCO handbook (10th, method among the ed.p1092-1095 (1984) is measured inositol concentration in the supernatant liquor with biologic assay, end-result is: control strain Candida boidinii TR-1 can not accumulate measurable inositol (0g/L) in the extracellular, and mutant of the present invention, Candida boidinii IP-2 can accumulate the inositol of 1.5g/L in the extracellular.Embodiment 3 prepares inositol by enzyme process

Control strain is Candida boidinii TR-1, mutant Candida boidinii IP-2 of the present invention single culture in the substratum that contains composition shown in the table 1,30 ℃ vibrated 24 hours down, the 10ml that last culture is inoculated in the test tube that diameter is 25mm contains in the substratum of composition shown in table 4 and 5, and cell was 30 ℃ of following shaking culture 24 hours.

Table 4

Glucose 10g/L

(NH ₄) ₂SO ₄ 0.5g/L

KH ₂PO ₄ 2.5g/L

K ₂HPO ₄ 1g/L

NH ₄Cl ₂ 4g/L

Table 5

Methyl alcohol 2% (V/V)

(NH ₄) ₂SO ₄ 2.5g/L

KH ₂PO ₄ 0.5g/L

K ₂HPO ₄ 1g/L

MgSO ₄·7H ₂O 0.5g/L

NH ₄Cl ₂ 4g/L

By centrifugal culturing cell is separated from culture, isolated yeast cell is dispersed in the distilled water so that cell concn reaches 80g/L (equivalent dry weight), suspension tolerates 45 minutes down at 37 ℃, then, in cell suspending liquid, add 4mol/L D-sorbitol aqueous solution so that the final concentration of D-sorbyl alcohol is 1.5mol/L, whole solution is tolerated 10 minutes down at 37 ℃.

In the substratum that contains composition shown in table 6 and 7, suspension descended the vibration incubations 20 hours at 30 ℃ with treated cell suspension.

Table 6

100mM Tris-HCl damping fluid (pH7.5)

14mM NH ₄Cl

2.7mM MgCl ₂·6H ₂O

0.8mM Reduced nicotinamide-adenine dinucleotide (NAD)

6mM glucose-6-phosphoric acid (substrate)

Table 7

100mM Tris-HCl cushions dense (pH7.5)

14mM MgCl ₂·6H ₂O

0.8mM NAD

5mM glucose (substrate)

2.5mM adenosine-3-phosphoric acid

After the reaction, reactant is filtered to remove cell, with biologic assay that the inositol concentration in the filtrate is quantitative, the results are shown in table 8 and 9.

Table 8

Substrate: glucose-6-phosphoric acid bacterial strain inositol concentration (g/L)

Methyl alcohol substratum dextrose culture-medium Bo Yidingshi vacation

00 yeast TR-1 (contrast) Candida boidinii IP-2

0.3 0.5 (the present invention)

Table 9

Substrate: glucose bacterial strain inositol concentration (g/L)

The false silk of methyl alcohol substratum dextrose culture-medium Bo Yidingshi

00 yeast TR-1 (contrast) Candida boidiniis

0.3 the purifying and the crystallization of 0.5 IP-2 (the present invention) embodiment, 4 inositols

1 liter of culture supernatants of gained among the embodiment 2 is passed through Zeo-karb Diaion SKIB, to flow out in the segment set, again anionite-exchange resin Diaion PA316 is partly passed through in the outflow of being collected, concentrate and flow out part, concentrate this part with the crystallization inositol, in this way, can obtain purity is 97% 1g crystalline inositol.Embodiment 5 separates the cerulenin resistant mutants

With the cell of Candida boidinii IP-2 at 30 ℃, pH6.0 handled 10 minutes with 300 μ g/ml N-methyl-N-nitro-N-nitrosoguanidine down, the cell that the position mutagenic compound are handled suitably dilutes, be coated in the substratum that contains composition shown in the table 10, the cerulenin that in this substratum, has added 15g/L agar and 40mg/L, with flat board 30 ℃ of following incubations 4 days, bacterium colony separation and purification with the Candida boidinii IP-2 of the mutagenesis that forms on the flat board has obtained Candida boidinii CER176 in this way.

Table 10

Glycerine 10g/L

(NH ₄) ₂SO ₄ 5g/L

KH ₂PO ₄ 1g/L

MgSO ₄·7H ₂O 0.5g/L

CaCl ₂·2H ₂O 0.1g/L

NaCl 0.1g/L

ZnSO ₄·7H ₂O 0.4mg/L

FeSO ₄·7H ₂O 0.4mg/L

CuSO ₄·5H ₂O 0.04mg/L

Vitamin H 0.04mg/L

PH is transferred to the resistance level of 5.5 embodiment, 6 cerulenin resistant mutants with IN KOH

Control strain is Candida boidinii IP-2, and cerulenin resistant mutants Candida boidinii CER176 single culture in the substratum that contains composition shown in the table 11, is collected grown cell, washs with physiological saline.

Table 11

Glucose 20g/L

The female diffusion juice 2g/L of alcohol

MgSO ₄·7H ₂O 0.5g/L

Polyprotein peptone 5g/L

K ₂HPO ₄ 1g/L

The cell suspending liquid that obtains thus is inoculated in contains composition shown in the table 10, and contain respectively 0,10,20,40 and the 5ml substratum of 100mg/L cerulenin in, cultivate after 72 hours, measure the growth of cell, the results are shown in Table 12.

Table 12

Bacterial strain allometry amount (%)

Add the concentration (mg/L) of cerulenin

0 10 20 40 100IP-2 (contrast), 100 3.3 2.4 1.1 0.2CER 176 (the present invention) 100 90 82 67 11

From The above results, compare with parent strain, the growth of cerulenin resistant mutants is not subjected to the inhibition of the cerulenin of high density among the present invention, and this shows that mutant has obtained higher cerulenin resistance.Embodiment 7 utilizes the cerulenin resistant mutants by the fermentative preparation inositol

Control strain is Candida boidinii IP-2, cerulenin resistant mutants Candida boidinii CER176 of the present invention is incubated in the substratum that contains composition shown in the table 13 separately in advance, 30 ℃ vibrated 24 hours down, the pre-culture of gained is inoculated in 50ml in the 500ml erlenmeyer flask to be contained shown in the table 13 in the substratum, this substratum was sterilized 10 minutes down at 110 ℃ in advance, cultivation was carried out 48 hours under the oscillating condition that reciprocal span is 30cm at 180rpm.

Table 13

Glucose 50g/L

(NH ₄) ₂SO ₄ 2.5g/L

KH ₂PO ₄ 1g/L

MgSO ₄·7H ₂O 0.5g/L

CaCl ₂·2H ₂O 0.1g/L

NaCl 0.1g/L

ZnSO ₄·7H ₂O 0.4g/L

FeSO ₄·7H ₂O 0.4mg/L

CuSO ₄·5H ₂O 0.04mg/L

Vitamin H 0.04mg/L

With IN KOH PH is transferred to 5.5

After the cultivation, culture is filtered to remove cell and residual lime carbonate, measure the concentration of filtered solution mysoinositol with biologic assay, the result, control strain Candida boidinii IP-2 has accumulated the 1.5g/L inositol in the extracellular, and cerulenin resistant strain Candida boidinii CER176 of the present invention has accumulated the 2.7g/L inositol in the extracellular.Embodiment 8 utilizes the cerulenin resistant mutants to prepare inositol by enzyme process

Control strain is Candida boidinii IP-2, be incubated at cerulenin resistant mutants Candida boidinii CER176 of the present invention in the substratum that contains composition shown in the table 11 separately in advance, vibrated 24 hours down at 30 ℃, it is in the 10ml substratum that contains composition shown in table 14 and 15 in the 25mm test tube that the pre-culture of gained is inoculated in diameter, this substratum was sterilized 10 minutes down at 115 ℃ in advance, cultivation is carried out under 30 ℃, and vibrates 24 hours.

Table 14

Glucose 10g/L

(NH ₄) ₂SO ₄ 2.5g/L

KH ₂PO ₄ 0.5g/L

K ₂HPO ₄ 1g/L

MgSO ₄·7H ₂O 0.5g/L

NH ₄Cl 4g/L

Table 15

Methyl alcohol 2% (V/V)

(NH ₄) ₂SO ₄ 2.5g/L

KH ₂PO ₄ 0.5g/L

K ₂HPO ₄ 1g/L

MgSO ₄·7H ₂O 0.5g/L

NH ₄Cl 4g/L

From substratum, separate through cultured cells by centrifugal, isolated yeast cell is scattered in the distilled water so that cell concn is 80g/L (an equivalent dry weight), suspension liquid is put 37 ℃ tolerate 45 minutes down, D-the sorbitol aqueous solution that adds 4mol/L then so that the final concentration of D-sorbyl alcohol is 1.5mol/L, is put 37 ℃ of tolerances 10 minutes down with whole solution in cell suspension.

In the substratum that contains composition shown in table 16 and 17, vibration incubation suspension liquid is 20 hours under 30 ℃ with treated cell suspension.

Table 16

100mM Tris-HCl (damping fluid) (pH7.5)

14mM NH ₄Cl

2.7mM MgCl ₂·6H ₂O

0.8mM two nucleoside of nicotinamide adenine is got (NAD)

5mM glucose-6-phosphoric acid (substrate)

Table 17

100mM Tris-HCl (damping fluid) (pH7.5)

14mM NH ₄Cl

2.7mM MgCl ₂·6H ₂O

0.8mM NAD

5mM glucose (substrate)

2.5mM adenosine-3-phosphoric acid

Reaction later the filtering reaction thing to remove cell, by biologic assay that the inositol concentration in the filtrate is quantitative, the results are shown in table 18 and 19.

Table 18

Substrate: glucose-6-phosphoric acid

Bacterial strain inositol concentration (g/L)

Methyl alcohol substratum dextrose culture-medium Candida boidinii 0.3 0.5IP-2 (contrast) Candida boidinii 0.6 1.0CER 176 (the present invention)

Table 19

Substrate: glucose

Bacterial strain inositol concentration (g/L) methyl alcohol substratum dextrose culture-medium Candida boidinii IP-(contrast) 0.3 0.5 Candida boidinii CER176 0.5 1.0 (the present invention)

As can be seen from the above table, the cerulenin resistant mutants of the present invention output of comparing inositol with parent strain is significantly increased.The purifying and the crystallization of embodiment 9 inositols

1 liter of culture supernatants of gained among the embodiment 7 is passed through Zeo-karb Diaion SKIB, collect and flow out part, anionite-exchange resin Diaion PA316 is partly passed through in the outflow of collecting, concentrate and flow out part, concentrate this part with the crystallization inositol, can obtain 2g purity by this method is 97% inositol crystallization.Embodiment 10 separates 2-deoxyglucose resistant mutants

The cell of Candida boidinii IP-2 was handled 10 minutes under 30 ℃, pH6.0 with N-methyl-N-nitro-N '-nitrosoguanidine of 300 μ g/ml, to suitably dilute through the cell that mutagenic compound are handled, coat on the plate culture medium, this substratum contains composition shown in the table 20, and the agar and the 2-deoxyglucose (final concentration is 1mM) that have added 15g/L, with flat board 30 ℃ of following incubations 4 days, the bacterium colony through the Candida boidinii IP-2 of mutagenesis that separation and purification forms on flat board has obtained Candida boidinii DGR1-14 by this method.

Table 20

Glycerine 10g/L

(NH ₄) ₂SO ₄ 5g/L

KH ₂PO ₄ 1g/L

MgSO ₄·7H ₂O 0.5g/L

CaCl ₂·2H ₂O 0.1g/L

NaCl 0.1g/L

ZnSO ₄·7H ₂O 0.4mg/L

FeSO ₄·7H ₂O 0.4mg/L

CuSO ₄·5H ₂O 0.04mg/L

Vitamin H 0.04mg/L

PH is transferred to the resistance level of 5.5 embodiment, 11 2-deoxyglucose resistant mutants with IN KOH

Control strain is Candida boidinii IP-2, and 2-deoxyglucose resistant mutants Candida boidinii DGR1-14 single culture in the substratum that contains composition shown in the table 22, is collected grown cell, with the washing of physiology salt.

Table 21

Glucose 20g/L

Yeast diffusion juice 2g/L

MgSO ₄·7H ₂O 0.5g/L

Poly peptone 5g/L

K ₂HPO ₄ 1g/L

The cell suspension of the preparation of method thus is inoculated in 5ml contains composition shown in the table 20, and contain 0,0.25,0.5,0.75 respectively; In the substratum of 1.0mM 2-deoxyglucose, cultivate after 72 hours, measure cell yield, the results are shown in Table 22.

Table 22

Bacterial strain Xiang Dui Sheng Long measures (%)

Add the concentration (mM) of 2-deoxyglucose

0 0.25 0.5 0.75 1.0rP-2 (contrast), 100 26 21 1DGR1-14 (the present invention) 100 94 94 77 53

From The above results, 2-deoxyglucose resistant mutants of the present invention is compared with parent strain, and growth is not subjected to the inhibition of 2-deoxyglucose of high density, and this shows that this mutant has obtained the resistance of 2 higher-deoxyglucose.Embodiment 12 utilizes 2-deoxyglucose resistant mutation body and function fermentation method to prepare inositol

Control strain is Candida boidinii IP-2,2-deoxyglucose resistant mutants Candida boidinii DGR1-14 of the present invention is incubated in the substratum that contains composition shown in the table 23 separately in advance, vibrated 24 hours down at 30 ℃, the pre-culture of gained is inoculated in the 50ml substratum that contains composition shown in the table 23 in the 500ml erlenmeyer flask, substratum was sterilized 10 minutes down at 110 ℃ in advance, shaking culture 48 hours, the reciprocal span of vibrating is 30cm, rotating speed is 180rpm.

Table 23

Glucose 50g/L

(NH ₄) ₂SO ₄ 2.5g/L

KH ₂PO ₄ 1g/L

MgSO ₄·7H ₂O 0.5g/L

CaCl ₂·2H ₂O 0.1g/L

NaCl 0.1g/L

ZnSO ₄·7H ₂O 0.4g/L

FeSO ₄·7H ₂O 0.4mg/L

CuSO ₄·5H ₂O 0.04mg/L

Vitamin H 0.04mg/L

With IN KOH pH is transferred to 5.5

Cultivate later culture is filtered to remove cell and residual lime carbonate, filter inositol concentration in crossing with bioassay, the result is that control strain Candida boidinii IP-2 has accumulated the 1.5g/L inositol in the extracellular, and 2-deoxyglucose resistant strain Candida boidinii DGR1-14 of the present invention has accumulated the 3.2g/L inositol in the extracellular.Embodiment 13 utilizes 2-deoxyglucose resistant mutants to prepare inositol by enzyme process

Control strain is Candida boidinii IP-2,2-deoxyglucose resistant mutants Candida boidinii DGR1-14 of the present invention is incubated in the substratum that contains composition shown in the table 21 separately in advance, vibrated 24 hours down at 30 ℃, it is in the 10ml substratum that contains composition shown in table 24 and 25 in the 25mm test tube that the pre-culture of gained is inoculated in diameter, substratum was in advance 115 ℃ of down sterilizations 10 minutes, 30 ℃ of following shaking culture 24 hours.

Table 24

Glucose 10g/L

(NH ₄) ₂SO ₄ 2.5g/L

KH ₂PO ₄ 0.5g/L

K ₂HPO ₄ 1g/L

MgSO ₄·7H ₂O 0.5g/L

NH ₄Cl 4g/L

Table 25

Methyl alcohol 2% (V/V)

(NH ₄) ₂SO ₄ 2.5g/L

KH ₂PO ₄ 0.5g/L

K ₂HPO ₄ 1g/L

MgSO ₄·7H ₂O 0.5g/L

NH ₄Cl 4g/L

To from substratum, separate through cultured cells by centrifugal, isolated yeast cell is scattered in the distilled water so that cell concn is 80g/L (an equivalent dry weight), suspension liquid is put 37 ℃ and is tolerated 45 minutes down, D-the sorbitol aqueous solution that adds 4mol/L in cell suspension is put 37 ℃ with whole solution and is tolerated 10 minutes down so that the final concentration of D-sorbyl alcohol is 1.5mol/L again.

In the substratum that contains composition shown in table 26 and 27, vibration incubation suspension liquid is 20 hours under 30 ℃ with treated cell suspension.

Table 26

100mM Tris-HCl (damping fluid) (pH7.5)

14mM NH ₄Cl

2.7mM MgCl ₂·6H ₂O

0.8mM Reduced nicotinamide-adenine dinucleotide (NAD)

5mM glucose-6-phosphoric acid (substrate)

Table 27

100mM Tris-HCl (damping fluid) (pH7.5)

14mM NH ₄Cl

2.7mM MgCl ₂·6H ₂O

0.8mM NAD

5mM glucose (substrate)

2.5mM adenosine-3-phosphoric acid

Reaction later the filtering reaction thing to remove cell, with biologic assay that the inositol concentration in the filtrate is quantitative, the results are shown in Table 28 and 29.

Table 28

Substrate: glucose-6-phosphoric acid

Bacterial strain inositol concentration (g/L)

Methyl alcohol substratum dextrose culture-medium Candida boidinii 0.3 0.5IP-2 (contrast) Candida boidinii DGR1-14 (the present invention) 0.6 1.0

Table 29

Substrate: glucose

Bacterial strain inositol concentration (g/L)

The purifying and the crystallization of methyl alcohol substratum dextrose culture-medium Candida boidinii 0.3 0.5IP-2 (contrast) Candida boidinii DGR1-14 (the present invention) 0.6 1.0 embodiment 14 inositols

1 liter of culture supernatants of gained among the embodiment 12 is passed through Zeo-karb Diaion SKIB, concentrate and flow out part, anionite-exchange resin DianonPA316 is partly passed through in the outflow of collecting, concentrate and flow out part, concentrate this part with the crystallization inositol, can obtain 2.1g purity by this method is 97% inositol crystallization.Embodiment 15 separates D-seromycin resistant mutants

With the cell of Candida boidinii IP-2 with N-methyl-N-nitro-N '-nitrosoguanidine of 300 μ g/ml at 30 ℃, pH6.0 handled 10 minutes down, suitably dilution is through the cell of mutagenic compound processing, coat on the culture medium flat plate, this substratum contains composition shown in the table 30 and has added 15g/L agar and 40mg/L D-seromycin, with flat board 30 ℃ of following incubations 4 days, the bacterium colony through the Candida boidinii IP-2 of mutagenesis that occurs on the separation and purification flat board has obtained Candida boidinii DCSR0.2-59 and Candida boidinii DCSR0.3-17 by this method.

Table 30

Glycerine 10g/L

(NH ₄) ₂SO ₂ 5g/L

KH ₂PO ₄ 1g/L

MgSO ₄·7H ₂O 0.5g/L

CaCl ₂·2H ₂O 0.1g/L

NaCl 0.1g/L

ZnSO ₄·7H ₂O 0.4mg/L

FeSO ₄·7H ₂O 0.4mg/L

CuSO ₄·5H ₂O 0.04mg/L

Vitamin H 0.04mg/L

PH is transferred to the resistance level of 5.5 embodiment, 16 D-seromycin antibiosis mutant with IN KOH

Control strain is Candida boidinii IP-2, it and D-seromycin resistant mutants Candida boidinii DCSR0.2-59 and DCSR0.3-11 separately single culture in the substratum that contains composition shown in the table 31, collect grown cell, wash with physiological saline.

Table 31

Glucose 20g/L

Yeast diffusion juice 2g/L

MgSO ₄·7H ₂O 0.5g/L

Poly peptone 5g/L

K ₂HPO ₄ 1g/L

The cell suspension of preparation thus is inoculated in and contains composition shown in the table 30, and contain respectively 0,100,200,300 and the 5ml substratum of 400mg/L D-seromycin in, cultivate after 72 hours, the increment of mensuration cell the results are shown in Table 32.

Table 32

Bacterial strain allometry amount (%)

Add the concentration (mg/L) of D-seromycin

0 10 20 40 100

IP-2 (contrast) 100 2000

DCSR?0.2-59

(the present invention) 100 96 94 31 2

DCSR?0.3-11

(the present invention) 100 98 97 92 4

From The above results, D of the present invention-seromycin resistant mutants is compared with parent strain, and growth is not subjected to the inhibition of the D-seromycin of high density, and this shows that mutant has obtained the resistance of higher D-seromycin.Embodiment 17 utilizes D-seromycin resistant mutants by the fermentative preparation inositol

Control strain is Candida boidinii IP-2, it and D of the present invention-seromycin resistant mutants Candida boidinii DCSR0.2-59 and DCSR0.3-11 are incubated in the substratum that contains composition shown in the table 33 separately separately in advance, vibrated 24 hours down at 30 ℃, the pre-culture of gained is inoculated in the 50ml substratum that contains composition shown in the table 33 in the 500ml erlenmeyer flask, this substratum was sterilized 10 minutes down at 110 ℃ in advance, cultivation was carried out in vibration 48 hours, rotating speed is 180rpm, and back and forth span is 30cm.

Table 33

Glucose 50g/L

(NH ₄) ₂SO ₄ 2.5g/L

KH ₂PO ₄ 1g/L

MgSO ₄·7H ₂O 0.5g/L

CaCl ₂·2H ₂O 0.1g/L

NaCl 0.1g/L

ZnSO ₄·7H ₂O 0.4g/L

FeSO ₄·7H ₂O 0.4mg/L

CuSO ₄·5H ₂O 0.04mg/L

Vitamin H 0.04mg/L

With IN KOH pH is transferred to 5.5

Cultivate and to filter culture later with divided by cell and residual lime carbonate, measure the concentration of filtered solution mysoinositol with biologic assay, the result is that control strain Candida boidinii IP-2 accumulates the 1.5g/L inositol in the extracellular, and D of the present invention-seromycin resistant strain Candida boidinii DCSR0.2-59 and DCSR0.3-11 have accumulated 3.6g/L and 4.3g/L inositol respectively in the extracellular.Embodiment 18 utilizes D-seromycin resistant mutants to prepare inositol by enzyme process

Control strain is Candida boidinii IP-2, it and D of the present invention-seromycin resistant mutants Candida boidinii SR 0.2-59 and DCSR 0.3-11 are incubated in advance separately separately and contain composition shown in the table 11 and throw away in the substratum, vibrated 24 hours down at 30 ℃, the pre-culture of gained is inoculated in the 10ml substratum that contains composition shown in table 34 and 35 in the test tube that diameter is 25mm, this substratum was sterilized 10 minutes down at 115 ℃ in advance, cultivation is carried out under 30 ℃, vibrates 24 hours.

Table 34

Glucose 10g/L

(NH ₄) ₂SO ₄ 2.5g/L

KH ₂PO ₄ 0.5g/L

K ₂HPO ₄ 1g/L

MgSO ₄·7H ₂O 0.5g/L

NH ₄Cl 4g/L

Table 35

Methyl alcohol 2% (V/V)

(NH ₄) ₂SO ₄ 2.5g/L

KH ₂PO ₄ 0.5g/L

K ₂HPO ₄ 1g/L

MgSO ₄·7H ₂O 0.5g/L

NH ₄Cl 4g/L

By centrifugal cultured cell is separated from substratum, isolated yeast cell is scattered in the distilled water,, make suspension liquid tolerate 45 minutes down at 37 ℃ so that cell concn is 80g/L (an equivalent dry weight).In cell suspension, add 4mol/L D-sorbitol aqueous solution then so that the final concentration of D-sorbyl alcohol is 1.5mol/L, allow whole solution tolerate 10 minutes down at 37 ℃.

In the substratum that contains composition shown in table 36 and 37, suspension liquid was 30 ℃ of following incubation vibrations 20 hours with treated cell suspension.

Table 36

100mM Tris-HCl damping fluid (pH7.5)

14mM NH ₄Cl

2.7mM MgCl ₂·6H ₂O

0.8mM Reduced nicotinamide-adenine dinucleotide (NAD)

5mM glucose-6-phosphoric acid (substrate)

Table 37

100mM Tris-HCl damping fluid (pH7.5)

14mM NH ₄Cl

2.7mM MgCl ₂·6H ₂O

0.8mM NAD

5mM glucose (substrate)

2.5mM adenosine-3-phosphoric acid

Reaction after-filtration reactant is to remove cell, and is with bioassay method that the inositol concentration in the filtrate is quantitative, the results are shown in Table 38 and 39.

Table 38

Substrate: glucose-6-phosphoric acid

Bacterial strain inositol concentration (g/L)

Methyl alcohol substratum dextrose culture-medium Candida boidinii IP-2 (contrast) 0.3 0.5 Candida boidinii DCSR0.2-59 (the present invention) 0.8 1.2 Candida boidinii DCSR0.3-11 (the present invention) 1.0 1.6

Table 39

Substrate: glucose

Bacterial strain inositol concentration (g/L)

Methyl alcohol substratum dextrose culture-medium Candida boidinii IP-2 (contrast) 0.3 0.5 Candida boidinii DCSR0.2-59 (the present invention) 0.7 1.2 Candida boidinii DGSR0.3-11 (the present invention) 1.0 1.6

From last table, D of the present invention-seromycin mutant is compared inositol with parent strain output has raising significantly.The purifying and the crystallization of embodiment 19 inositols

The 1 liter of culture supernatants that obtains among 2 parts of embodiment 17 is passed through Zeo-karb Diaion SKIB, 2 parts that concentrate the outflow part will collect flow out part by anionite-exchange resin Diaion PA316 again, concentrate and flow out part, concentrate this 2 part with the crystallization inositol, can obtain 2.2g and 3.0g purity respectively by this method from the supernatant liquor of DCSR0.2-59 and DCSR0.3-11 is 97% inositol crystallization.

Claims (36)

1. prepare the method for inositol, may further comprise the steps:

(1) in substratum, cultivate a kind of candiyeast Pseudomonas that belongs to, can be to the microorganism of cell exocrine inositol with at extracellular accumulation inositol; With can be randomly

(2) from culture, obtain inositol.

2. according to the process of claim 1 wherein that the microorganism that belongs to the candiyeast Pseudomonas is a Candida boidinii.

3. according to the method for claim 2, wherein Candida boidinii is Candida boidinii IP-2.

4. prepare the method for inositol, may further comprise the steps:

(1) in substratum, cultivate a kind of candiyeast Pseudomonas that belongs to, can be to the derivative of cell exocrine inositol to obtain containing the culturing cell of inositol biosynthesizing institute indispensable enzyme;

(2) in reaction culture medium with enzyme with the biosynthetic precursor incubation of inositol so that precursor is transformed into inositol, finally in reaction culture medium, accumulate inositol; With can be randomly

(3) from reaction culture medium, obtain inositol.

5. according to the method for claim 4, the microorganism that wherein belongs to the candiyeast Pseudomonas is a Candida boidinii.

6. according to the method for claim 5, wherein Candida boidinii is Candida boidinii IP-2.

7. according to the method for claim 4, wherein precursor is glucose-6-phosphoric acid or glucose.

8. prepare the method for inositol, may further comprise the steps:

(1) in substratum, cultivates a kind of can belonging to the candiyeast Pseudomonas and certain antibiotic microorganism that have resistance is accumulated inositol with extracellular in substratum to the cell exocrine inositol; With can be randomly

(2) from culture, obtain inositol.

9. method according to Claim 8, the microorganism that wherein belongs to the candiyeast Pseudomonas is a Candida boidinii.

10. according to the method for claim 10, wherein Candida boidinii is Candida boidinii CFR176, and DCSR0.2-59 or DCSR0.3-11.

11. method according to Claim 8, wherein antibiotic is cerulenin, D-seromycin or BrefeldinA.

12. prepare the method for inositol, may further comprise the steps:

(1) in substratum, cultivate a kind of can be to the cell exocrine inositol, belong to the candiyeast Pseudomonas and antibiotic had the microorganism of resistance, to obtain to contain the culturing cell of the required enzyme of inositol biosynthesizing;

(2) in reaction culture medium with enzyme with the biosynthetic precursor incubation of inositol so that precursor changes inositol into, finally in reaction culture medium, accumulate inositol and can be randomly

(3) from reaction culture medium, obtain inositol.

13. according to the method for claim 12, the microorganism that wherein belongs to the candiyeast Pseudomonas is a Candida boidinii.

14. according to the method for claim 13, wherein Candida boidinii is Candida boidinii CER176, DCSR0.2-59 or DCSR0.3-11.

15. according to the method for claim 11, wherein precursor is glucose-6-phosphoric acid or glucose.

16. according to the method for claim 12, wherein antibiotic is cerulenin, D-seromycin or DrefeldinA.

17. prepare the method for inositol, may further comprise the steps:

(1) in substratum, cultivates a kind of can belonging to the candiyeast Pseudomonas and the glucose metabolism antagonist be had the microorganism of resistance accumulate inositol to the cell exocrine inositol with extracellular in substratum; With can be randomly

(2) from culture, obtain inositol.

18. according to the method for claim 17, the microorganism that wherein belongs to the candiyeast Pseudomonas is a Candida boidinii.

19. according to the method for claim 18, wherein Candida boidinii is Candida boidinii DGR1-14.

20. according to the method for claim 17, wherein the glucose metabolism antagonist is 2-deoxyglucose, 1-thioglucose or 5-thioglucose.

21. prepare the method for inositol, may further comprise the steps:

(1) in substratum, cultivate a kind of can be to the cell exocrine inositol, the microorganism that belongs to the candiyeast Pseudomonas and the glucose metabolism antagonist is had a resistance is to obtain to contain the culturing cell of inositol biosynthesizing institute indispensable enzyme;

(2) in reaction culture medium with enzyme with the biosynthetic precursor incubation of inositol so that precursor is transformed into inositol, finally in reaction culture medium, accumulate inositol; With can be randomly

(3) from reaction culture medium, obtain inositol.

22. according to the method for claim 21, the microorganism that wherein belongs to the candiyeast Pseudomonas is a Candida boidinii.

23. according to the method for claim 22, wherein Candida boidinii is Candida boidinii DGR1-14.

24. according to the method for claim 21, wherein precursor is glucose-6-phosphoric acid or glucose.

25. according to the method for claim 21, wherein the glucose metabolism antagonist is 2-deoxyglucose, 1-thioglucose or 5-thioglucose.

26. an energy belongs to the microorganism of candiyeast Pseudomonas to the cell exocrine inositol.

27. the microorganism according to claim 26, wherein candiyeast is a Candida boidinii.

28. the microorganism according to claim 27, wherein Candida boidinii is Candida boidinii IP-2 (FERM BP).

29. a microorganism can be to the cell exocrine inositol, belongs to the candiyeast Pseudomonas and to certain antibiotic resistance that have.

30. the microorganism according to claim 29, wherein the candiyeast Pseudomonas is the Candida boidinii that cerulenin is had resistance.

31. the microorganism according to claim 30, wherein cerulenin being had the Candida boidinii of resistance is Candida boidinii CER176 (FERM BP5069).

32. the microorganism according to claim 29, wherein the candiyeast Pseudomonas is the Candida boidinii that D-seromycin is had resistance.

33. the microorganism according to claim 32, wherein D-seromycin being had the Candida boidinii of resistance is Candida boidinii DCSR 0.2-59 (FERM BP5071) or DCSR0.3-11 (FERM BP5072).

34. a microorganism can belong to the candiyeast Pseudomonas and the glucose metabolism antagonist is had resistance to the cell exocrine inositol.

35. the microorganism according to claim 32, wherein candiyeast is the Candida boidinii that 2-deoxyglucose is had resistance.

36. the microorganism according to claim 33, wherein Candida boidinii is Candida boidinii DGR1-14 (FERM BP5070).