CN1292064C - Microorganism strain and method for converting cephalosporin C into deacetylate cephalosporin C by using same - Google Patents
Microorganism strain and method for converting cephalosporin C into deacetylate cephalosporin C by using same Download PDFInfo
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Abstract
The present invention relates to novel Rhodotorula. sp for producing and preparing deacetyl cephalosporin C (DCPC) by using a biologic conversion method and a method for preparing the deacetyl cephalosporin C by using Rhodotorula. sp. The microbe strain is characterized in that the strain is classified in rhodotorula and named as Rhodotorula R-92, and the accession serial number of the strain in the preservation center is CGMCC NO. 1277. In the conversion method, the strain is used as a biologic conversion agent; the water solution of a cephalosporin salt is used as a biologic conversion substrate; the conversion agent is added to the substrate for conversion or mixed fermentation, and off-white or yellowish deacetyl cephalosporin C powder is obtained by resin treatment, concentration and solvent crystallization. The present invention aims to solve the problems of low yield, high cost, non adaptability for industrial production on a large scale, etc. existing in the production of the deacetyl cephalosporin C (DCPC).
Description
One, technical field: the present invention relates to a kind of new biotransformation method production that is used for and prepare the rhodotorula bacterium (Rhodotorula.sp) of deacetyl cephalosporin C (DCPC) and utilize this bacterial strain to prepare the method for deacetylation cephalosporin.
Two, background technology:
Cephalosporin (Cephalosporin C is called for short CPC) is a leavened prod, and its structural formula is:
Behind 3 last deacetylates of CPC, promptly get deacetyl cephalosporin C (DeactylCephalosporin C is called for short DCPC), its structural formula is:
DCPC is a kind of impurity in the CPC fermentative production, because amount is little, the recovery trouble is unworthy as byproduct.But it is as the intermediate of semi-synthetic cephalo medicine, has suitable value, can be used for the chemosynthesis of multiple cephalo medicine, as cefotaxime sodium, rocephin, ceftazime, Cefixime Micronized, Cefdinir, cefaclor, a cephalo Lip river, a cephalo nurse, cefuroxime etc.; And be used for some cephalo medicine synthetic yield as raw material with DCPC, it is high when ratio is made raw material with CPC or 7-ACA, for example: pharmaceutical factory, Japanese military field uses DCPC to compare with CPC, synthetic BMY28142 yield exceeds 29.4%, synthetic FK027 yield exceeds 58.99% (medicine industry development program basic data special edition P265 in 2000, technical situation institute of medicine management general bureau of country, 1988), Ge Lansu company is 97% with the yield that DCPC makes the synthetic rocephin of raw material, being about domestic at that time is 1.5 times that raw material synthesizes rocephin with 7-ACA, Hoffmann-La Roche DCPC is the synthetic ceftazime of raw material, it is 2 times (Recl.Trav.Chim.Pay.Bas 112,066-081 (1993) P78) of the synthetic ceftazime of raw material with 7-ACA that yield is about domestic at that time.
In view of the These characteristics of DCPC, a large amount of research has been carried out in its preparation abroad, the preparation method of the DCPC that has reported has two classes:
1. chemical preparation method:
(B.P 1289814 as CPC alkali hydrolysis method, lactone compound open loop method etc.; Heymes, Rene etal.Bull.Soc.Chim, Fr, 563 (1974); USP 4148995), owing to beta-lactam nucleus to reasons such as the unstable of alkali and yield are low, reduced this The Application of Technology and be worth.
2. microbial method:
Many microorganisms all can be used for the preparation of DCPC, produce the negative mutant strain of bacterium etc. as bacterium, actinomycetes, yeast, CPC, and the technological line of the microbe transformation method of having reported has multiple.As:
(USP 3926729 (1973), adopt CPC to produce the mutagenic fungi fermentative preparation DCPC of bacterium, and final DCPC fermentation unit is 1900r/ml for the patent of Teng Ze good fortune husband in 1973 application.
Alam Smith prepares DCPC and has applied for patent (USP 3912589) with the esterase hydrolyzed CPC that cultivation rhodotorula (Rhodotorula) or other fungi obtain.Alam Smith has proposed to produce the idea (day clear 56-42596 of disclosure special permission communique) of DCPC replaced C PC again in 1981, be that DCPC is taken as impurity and discards because 15% beta-lactam thing is arranged in the CPC tunning in the past.Smith adds the CPC acetylase during the fermentation, and DCPC increases by 88% than CPC after 189 hours, however, and the fermentation level of this patent still lower (3000-4000r/ml).
Nineteen eighty-two, Japanese Fujisawa company has developed to use and has produced esterase microorganism Aureobasidium pullulans and prepare the processing method of DCPC and declared European patent (EPA0044736).The used microorganism Aureobasidium pullulans of this patent also is the yeast quasi-microorganism, in this patent, be substrate with the CPC sodium salt, in the 300ml CPC aqueous solution, (contain CPC sodium salt 7.5g) and add the wet thallus of 10g Aureobasidium pullulans, under PH4.5,30 ℃, transform, steps such as warp concentrates, 75% alcohol crystal get DCPC crystallization 3.8g, yield 50.7%; Adopt this microorganism and the cephalosporium acremonium that produces CPC to carry out mixed fermentation, total fermentation period is 5 days, and CPC can be converted into DCPC well after testing, but the concentration of fermentation termination DCPC only has 500r/ml for lower.
It is in recent years a focus that the genetic engineering bacterium fermentative production prepares DCPC, and big drugmaker of external some such as LILLY CO ELI, SQUIBB BRISTOL MYERS CO etc. all carried out research in this respect, and applied for a plurality of patents (EP1263969; US6180361; EP0465189; WO016767).Though fermentation level significantly improves, have that engineering bacteria throughput is easily degenerated, a fermentation condition requirement height, and potential environmental safety problem.
Three, summary of the invention:
1, goal of the invention: the invention provides and a kind ofly can effectively transform the new bacterial strain of microorganism that CPC is DCPC, be rhodotorula bacterium (Rhodotorula.sp), its purpose is solve to produce and has in the deacetyl cephalosporin C (DCPC) that yield is low, cost is high, is unsuitable for the problem that aspect such as industrial-scale production exists.
2, technical scheme: the present invention is achieved through the following technical solutions:
A kind of microorganism strains is characterized in that: this strain classification is a Rhodotorula, and called after RhodotorulaR-92, this bacterial strain register on the books at the preservation center and be numbered CGMCC NO.1277; This microorganism strains obtains through screening, challenge by oneself to separate in CPC Study on Fermentation process, and its vegetative cell in glucose-protein culture medium is rounded, has more bud; On glucose-peptone-nutrient agar, form red circular bacterium colony, soft, moistening, have reflective, the bacterium colony surface smoothing, the edge is smooth.
A kind of to transform cephalosporin with microorganism strains is the method for deacetyl cephalosporin C, it is characterized in that this method carries out according to the following steps:
(1). the microorganism strains Rhodotorula R-92 described in the claim 1 is carried out routine cultivate, ferment, the conventional cultivation comprises slant culture or liquid culture, the nutrient solution of acquisition or medium centrifugal filtered after the wet mycelium that obtains as biological catalyst;
(2). produce the cephalosporin nutrient solution of bacterial strain preparation as the bio-transformation substrate with the aqueous solution of cephalosporin salt or with cephalosporin;
(3). with the fermented liquid of (1), or the wet mycelium of centrifugal collection, transform or mixed fermentation in the substrate solution of adding (2), obtain the conversion fluid of deacetyl cephalosporin C;
(4). with the conversion fluid of (3), remove mycelium, through plastic resin treatment, concentrate and step such as solvent crystal obtains off-white color or faint yellow deacetyl cephalosporin C powder.
In the step (1), when carrying out microorganism strains Rhodotorula R-92 slant culture, substratum consists of: glucose 0.5-2%, peptone 0.2-1.2%, extractum carnis 0.1-1.0%, agar 2-2.5%, all the other are distilled water, each component concentration is volume percent meter by weight all, i.e. g/100ml, and the pH value of substratum transfers to 5.0-7.0, sterilized 30 minutes for culture condition: 110-120 ℃, sterilization postcooling, bevel, inoculation was cultivated 2-6 days for 25-35 ℃.
In the step (1), when carrying out the liquid culture of microorganism strains Rhodotorula R-92, substratum consists of: glucose 0.5-2.5%, peptone 0.2-1.2%, extractum carnis 0.1-1.0%, sal epsom 0.0075-0.05%, all the other are distilled water, each component concentration is volume percent meter by weight all, i.e. g/100ml, and the pH value of substratum transfers to 5.0-7.0; Sterilized 30 minutes for culture condition: 110-120 ℃, sterilization postcooling, inoculation, inoculum size is per-cent meter 2-15% by volume, culture temperature 25-35 ℃, shaking speed 100-260r/min, 5L fermentor tank, stirring velocity 250-300r/min, ventilate 1: 0.5～1: 1V/V.M, incubation time 1-4 days, respectively as seed or fermentation culture.
Wet thallus method for transformation in the step (3): concentration of substrate 1-5%, volume percent meter by weight; The wet thallus add-on is 1-8g/100ml; Wet thallus can repeatedly reclaim use; Invert point 25-35 ℃; Conversion fluid pH transfers to 4-7.
Composting fermentation in the step (3): microorganism strains Rhodotorula R-92 kind liquid measure 2-15%V/V; The microorganism strains Rhodotorula R-92 joining day is cephalosporin fermentation back 48-96 hour; 25-35 ℃ of mixed fermentation temperature; Stir 300-1000r/min; Air flow 1: 0.5～1: 1V/V.M; Control fermented liquid pH5.0-7.0; Total fermentation period 140-172 hour.
The method that this strain classification name is contrast " fungi identification handbook " (the super posthumous work of Wei Jing, Shanghai science tech publishing house, 1979, Shanghai p103-115) is carried out.
3, advantage and effect:
(1). microbial performance is stable:
The Rhodotorula R-92 that research is used is the wild strain that natural separation obtains, and the ability that its thalline catalysis CPC is converted into DCPC is stable, under the condition of the inventive method, is not subjected to the influence of passage number.
(2). the cultivation of microorganism used therefor Rhodotorula R-92, the preparation method of thalline are simple.
(3). thalline can repeatedly be used for conversion reaction repeatedly, and active nothing obviously reduces.
(4). the concentration height that feeds intake of CPC in the conversion fluid reaches as high as 5%.
(5) .CPC is converted into the transformation efficiency height of DCPC, reaches as high as 100%, far above the bibliographical information level.
(6) .R-92 can produce DCPC with cephalosporium acremonium collocation carrying out mixed fermentation, compare with the CPC fermentation, under the same terms, the beta-lactam total amount of mixed fermentation is higher than the CPC fermentation, and the ratio of fermentation termination DCPC is up to 90-100%, and the concentration of DCPC can reach 1.5-3 ten thousand γ/ml.Factory with CPC fermentation technique can easily produce DCPC, and production cost is low, and is equally matched with CPC.
Four, description of drawings:
Accompanying drawing 1 is IR (infrared) collection of illustrative plates of product DCPC of the present invention;
Accompanying drawing 2 is NMR (nuclear-magnetism) collection of illustrative plates of product DCPC of the present invention;
Accompanying drawing 3 transforms the process flow sheet that CPC prepares DCPC for the present invention uses R-92;
Five, embodiment: the present invention adopts following scheme implementation:
The present invention by the principle that CPC prepares DCPC is:
The bioconversion method that the present invention prepares DCPC comprises:
(1) bacterial strain Rhodotorula R-92 is carried out routine is cultivated, fermentation, the nutrient solution of acquisition or medium centrifugal filtered after the wet mycelium that obtains as biological catalyst, the conventional cultivation comprises: inclined-plane or liquid;
(2) produce the CPC nutrient solution of bacterial strain preparation as the bio-transformation substrate with the aqueous solution of CPC or with CPC
(3) wet mycelium of the fermented liquid of (1) or centrifugal collection is added in the substrate solution of (2) and transform, generate DCPC
(4) with the conversion fluid of (3), remove thalline, handle, concentrate, reach the solvent crystal step through resin or film and obtain off-white color or faint yellow DCPC powder.
Transform cultivation and fermentation with bacterial strain:
Slant culture: preparation contains glucose 0.5-2%, peptone 0.2-1.2%, and extractum carnis 0.1-1.0%, agar 2-2.5%, all the other are distilled water, cultivate keynote pH5.0-7.0, and each components contents is percent weight in volume, and promptly g/100ml is together following.Sterilized 30 minutes for 110-120 ℃, sterilization postcooling, bevel, inoculation was cultivated 2-6 days for 25-35 ℃.
Seed culture and fermentation: preparation contains glucose 0.5-2.5%, peptone 0.2-1.2%, extractum carnis 0.1-1.0%, sal epsom 0.0075-0.05%, all the other are distilled water, cultivate keynote pH5.0-7.0, liquid amount is 10-60ml/250ml triangular flask or 100-250ml/1000ml triangular flask, or the 2.5-3.5L/5L fermentor tank, sterilized 30 minutes the sterilization postcooling for 110-120 ℃, inoculation, inoculum size 2-15% (V/V), culture temperature 25-35 ℃, shaking speed 100-260r/min, 5L fermentor tank, stirring velocity 250-300r/min, ventilate 1: 0.5～1: 1V/V.M, incubation time 1-4 days, respectively as seed or fermentation culture.
The preparation of substrate:
The preparation of the CPC aqueous solution is dissolved in CPC sodium salt or sylvite in the damping fluid of PH4.5-7.0 by 1-5% (volume percent by weight) concentration.
The substratum of preparation cephalosporium acremonium inserts the cephalosporium acremonium bacterial classification, and aerated culture 50-100 hour nutrient solution is as the substrate that transforms.
The microbial transformation reaction:
With cultivating the wet thallus that the back obtains, be substrate with the aqueous solution of CPC salt, the amount that adds wet thallus is the 1-8g/100ml conversion fluid, invert point 25-35 ℃, measures the terminal point of conversion reaction by high pressure liquid chromatographic analysis.Mycelium is reuse repeatedly, carries out repeatedly conversion reaction
Perhaps, kind liquid with Rhodotorula R-92, be added in the cephalosporium acremonium fermented liquid of cultivating suitable time (48-96 hour), co-cultivation is carried out mixed fermentation with it, the add-on 2-15% (V/V) of Rhodotorula R-92 kind liquid, culture temperature 25-35 ℃, stirring velocity 300-1000r/min, air flow 1: 0.5～1: 1.5V/V.M, PH 5.0-7.0, total incubation time 140-180 hour.
The HPLC of CPC and DCPC (high performance liquid chromatography) measures:
Waters 600 pumps, the Waters486 UV-detector detects wavelength 254nm; UBondapakC183.9 * 150mm chromatographic column; Moving phase: 3% methyl alcohol, 97%0.02M NaH
2PO
4The aqueous solution; Flow velocity: 1ml/min
The extraction of DCPC:
The conversion situation decision that HPLC detects CPC transforms end.Conversion fluid 4000rpm is centrifugal, collects centrifugate, and with 0.8 μ membrane filtration or use resin decolorization, being evaporated to volume is 1/3 of conversion fluid, adds 3-4 times of volume of ethanol, stirred crystallization, leaves standstill 2hr, and suction filtration, drying obtain micro-yellow powder.
Example 1:R-92 yeast culture base: glucose 0.5%, peptone 1.2%, extractum carnis 0.1%, sal epsom 0.05%, all the other are distilled water, transfer pH5.0, the every 250ml triangular flask 10ml prepared culture medium of packing into, 115 ℃ of steam sterilizings 30 minutes, insert the R-92 bacterial classification, 25-27 ℃, 100r/min shaking culture 72 hours, with nutrient solution in 4000r/min centrifugal the R-92 wet thallus.
CPCNa0.5g is dissolved among the damping fluid 50ml of pH5.8, the wet thallus 0.5g that adds above-mentioned preparation, transform 38 hours in 27 ℃, 200r/min, HPLC measures transformation efficiency 80%, conversion fluid is centrifugal in 4000r/min, and centrifugate is with the membrane filtration of 0.8 μ m, and 40 ℃ are evaporated to 1/5 of original volume, the ethanol that adds 4 times of amounts, stirred crystallization, suction filtration, the dry micro-yellow powder 0.22g that gets.
Example 2: yeast culture base: glucose 1%, peptone 1%, extractum carnis 0.3%, sal epsom 0.02%, all the other are distilled water, transfer medium pH 6.0, every 250ml triangular flask 50ml substratum of packing into, 115 ℃ of steam sterilizings 30 minutes insert R-92, in 240r/min, 30 ℃ of shaking culture 48 hours, centrifugal thalline.
CPCNa1.5g is dissolved in the 50mlpH5.8 phosphoric acid buffer, adds the 2g wet thallus, and 28-30 ℃, 200r/min conversions of vibrate, HPLC measures conversion end in 28 hours, transformation efficiency 100%.The centrifugal thalline that goes of conversion fluid 4000r/min, centrifugate 0.8 μ m membrane filtration, 40 ℃ are evaporated to 1/3 of original volume, add 4 times of volume ethanol, stirred crystallization, suction filtration, the dry micro-yellow powder 1.1g that gets.
Example 3: with example 2, difference is that the PH of conversion fluid is 4.5, and transformation time is 50 hours, it is 49% that HPLC measures transformation efficiency, conversion fluid is removed unconverted CPC through centrifugal removal thalline through the resin anion(R.A) post, gets DCPC solid 0.36g through steps such as condensing crystals again.
Example 4: with example 2, difference is that the PH of conversion fluid is 7.0, transformation time 40 hours, and it is 85% that HPLC measures transformation efficiency, and conversion fluid is undertaken by the treatment process of example 2, and the result gets faint yellow DCPC crystallization 0.8g.
Example 5: yeast culture base: glucose 2.5%, peptone 0.2%, extractum carnis 1.0%, sal epsom 0.0075%, all the other are distilled water, medium pH is transferred to about 7.0, the 200ml substratum of packing in the 1000ml triangular flask, 115 ℃ of sterilizations 30 minutes insert the R-92 bacterial classification, cultivated 30 hours centrifugal wet thallus in 260r/min, 33 ℃.
CPCNa 9g is dissolved in the damping fluid of 200ml pH5.8, add above-mentioned wet thallus 16g, 32 ℃, 200r/min transform 32 hours, it is 82% that HPLC measures transformation efficiency, centrifugal removal thalline is concentrated into 1/3 of conversion fluid volume after the plastic resin treatment, add 4 times of volume of ethanol, stirred crystallization gets DCPC solid 5.0g after suction filtration, the drying.
Example 6: the substratum 3000ml that presses the proportioning preparation R-92 of example 2, the 5L fermentor tank of packing into, sterilized 30 fens for 115 ℃, ratio in 10% inserts R-92 kind liquid, air flow: 1: 0.75V/V.M, stirring velocity 300r/min, pH control 6.0, cultivated 48 hours for 30 ℃, get wet thallus after nutrient solution 4000r/min is centrifugal.
6g CPCNa is dissolved in the phosphate buffered saline buffer of 200ml pH5.8, add wet thallus 10g, in the conversion of vibrating of 28.5 ℃, 200r/min, detect 30 hours through HPLC and transform end, transformation efficiency 99%, conversion fluid is in the centrifugal collection supernatant liquor of 4000r/min, through resin decolorization, be concentrated into 1/3 of conversion fluid volume, add 4 times of volume ethanol crystallizations, get white DCPCNa solid 4.5g after filtration, the drying.
Example 7: wet thallus prepares with example 6, CPCNa 8g is dissolved among the phosphate buffered saline buffer 200ml of pH5.8, add the wet thallus 12g for preparing then, carry out conversion reaction, after the HPLC inspection transformed and finishes, conversion fluid is centrifugal, and the wet thallus of collecting was added to the CPC solution for continuous for preparing and carries out conversion reaction, according to this repeatedly, test-results sees the following form
Wet thallus is conversion results in batches repeatedly
| Transform number of | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| Transformation efficiency | 89％ | 93％ | 98％ | 100％ | 100％ | 100％ | 100％ |
| DCPC must measure | 6.5g | 8.4g | 8.6g | 9.0g | 9.1g | 9.5g | 9.8g |
Cephalosporium acremonium substratum 2700ml packs in the example 8:5L fermentor tank, its compound method is as follows: corn steep liquor 8%, dextrin 6%, starch 3%, α-Dian Fenmei 0.02%, ammonium sulfate 0.5%, potassium primary phosphate 0.9%, sal epsom 0.3%, DL-methionine 0.6%, manganous sulfate 0.01%, zinc sulfate 0.01%, phosphorous acid 0.06%, lime carbonate 0.75%, all the other are distilled water, sterilize 30 minutes for 115 ℃, insert the cephalosporium acremonium bacterial classification, 27 ℃ of culture temperature, according to the metabolism situation, control stirring velocity 300-1000r/min, air flow 1: 0.5～1: 1V/V.M in the culturing process, ammoniacal liquor control fermented liquid PH6.0, cultivate and insert R-92 kind liquid 300ml after 72 hours, continue to cultivate 90 hours, the concentration that HPLC detects DCPC in the fermented liquid is 22,936 γ/ml, the ratio of DCPC (DCPC/DCPC+CPC) is 95.5%, and the simple CPC fermentation of carrying out simultaneously, the total concn of DCPC+CPC has only 16897 γ/ml.
Example 9: with example 8, difference is that the volume of cephalosporium acremonium fermented liquid is 2550ml, the add-on of R-92 kind liquid is 450ml, joining day is that cephalosporium acremonium was cultivated 96 hours, and the time of continuing to cultivate is 80 hours, and the concentration that HPLC measures DCPC in the fermented liquid is 202,36 γ/ml, the ratio of DCPC (DCPC/DCPC+CPC) is 90.5%, and the simple CPC fermentation of carrying out simultaneously, the total concn of DCPC+CPC has only 18762 γ/ml.
Example 10: with example 8, difference is that the add-on of R-92 is 100ml, joining day is that cephalosporium acremonium was cultivated 48 hours, the time of continuing to cultivate is 96 hours, and the concentration that HPLC measures DCPC in the fermented liquid is 160,04 γ/ml, the ratio of DCPC (DCPC/DCPC+CPC) is 89.5%, and the simple CPC fermentation of carrying out simultaneously, the total concn of DCPC+CPC has only 149,68 γ/ml.
The Rhodotorula R-92 that the present invention is claimed; on December 21st, 2004; through China Committee for Culture Collection of Microorganisms's common micro-organisms preservation center preservation, deposit number is CGMCC NO.1277, and the address is the No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City.
Claims (6)
1, a kind of microorganism strains is characterized in that: this strain classification is a Rhodotorula, and called after Rhodotorula R-92, this bacterial strain register on the books at the preservation center and be numbered CGMCC NO.1277; This microorganism strains obtains through screening, challenge by oneself to separate in CPC Study on Fermentation process, and its vegetative cell in glucose-protein culture medium is rounded, has more bud; On glucose-peptone-nutrient agar, form red circular bacterium colony, soft, moistening, have reflective, the bacterium colony surface smoothing, the edge is smooth.
2, a kind of to transform cephalosporin with microorganism strains is the method for deacetyl cephalosporin C, it is characterized in that this method carries out according to the following steps:
(1). the microorganism strains Rhodotorula R-92 described in the claim 1 is carried out inclined-plane or liquid culture, fermentation, the nutrient solution of acquisition or medium centrifugal filtered after the wet mycelium that obtains as biological catalyst;
(2). produce the cephalosporin nutrient solution of bacterial strain preparation as the bio-transformation substrate with the aqueous solution of cephalosporin salt or with cephalosporin;
(3). with the fermented liquid of (1), or the wet mycelium of centrifugal collection, transform or mixed fermentation in the substrate solution of adding (2), obtain the conversion fluid of deacetyl cephalosporin C;
(4). with the conversion fluid of (3), remove mycelium, obtain off-white color or faint yellow deacetyl cephalosporin C powder through plastic resin treatment, the concentrated solvent crystal step that reaches.
3, described a kind of to transform cephalosporin with microorganism strains be the method for deacetyl cephalosporin C according to claim 2, it is characterized in that: in the step (1), when carrying out microorganism strains Rhodotorula R-92 slant culture, substratum consists of: glucose 0.5-2%, peptone 0.2-1.2%, extractum carnis 0.1-1.0%, agar 2-2.5%, all the other are distilled water, each component concentration is volume percent meter by weight all, be g/100ml, the pH value of substratum transfers to 5.0-7.0, culture condition: sterilized 30 minutes the sterilization postcooling for 110-120 ℃, bevel, inoculation was cultivated 2-6 days for 25-35 ℃.
4, described a kind of to transform cephalosporin with microorganism strains be the method for deacetyl cephalosporin C according to claim 2, it is characterized in that: in the step (1), when carrying out the liquid culture of microorganism strains Rhodotorula R-92, substratum consists of: glucose 0.5-2.5%, peptone 0.2-1.2%, extractum carnis 0.1-1.0%, sal epsom 0.0075-0.05%, all the other are distilled water, and each component concentration is volume percent meter by weight all; Be g/100ml, the pH value of substratum transfers to 5.0-7.0, culture condition: sterilized 30 minutes sterilization postcooling, inoculation for 110-120 ℃, inoculum size is per-cent meter 2-15% by volume, culture temperature 25-35 ℃, shaking speed 100-260r/min, 5L fermentor tank, stirring velocity 250-300r/min, ventilate 1: 0.5～1: 1V/V.M, incubation time 1-4 days, respectively as seed or fermentation culture.
5, described a kind of to transform cephalosporin with microorganism strains be the method for deacetyl cephalosporin C according to claim 2, it is characterized in that: wet thallus method for transformation in the step (3): concentration of substrate 1-5%, volume percent meter by weight; The wet thallus add-on is 1-8g/100ml; Wet thallus can repeatedly reclaim use; Invert point 25-35 ℃; Conversion fluid pH transfers to 4-7.
6, described a kind of to transform cephalosporin with microorganism strains be the method for deacetyl cephalosporin C according to claim 2, it is characterized in that: composting fermentation in the step (3): microorganism strains Rhodotorula R-92 kind liquid measure 2-15%V/V; The microorganism strains Rhodotorula R-92 joining day is cephalosporin fermentation back 48-96 hour; 25-35 ℃ of mixed fermentation temperature; Stir 300-1000r/min; Air flow 1: 0.5～1: 1V/V.M; Control fermented liquid pH5.0-7.0; Total fermentation period 140-172 hour.
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