A kind of subtilis and the method for fermentative production D-riboseTechnical field
The invention belongs to technical field of microbial fermentation, be specifically related to a kind of subtilis and the method with this bacterium fermentative production D-ribose.
Background technology
D-ribose is a kind of epochmaking aldopentose, is the important composition composition of genetic material nucleic acid in organism, is also the important intermediate metabolites of organism composite part amino acid, base etc., has important physiological function.
D-ribose is used to semi-synthesis method at first and produces vitamins B2.Along with carrying out in a deep going way of D-ribose industrial expansion and applied research thereof, its prospect in foodstuffs industry and medicine industry etc. is also realized gradually, and D-ribose can as raw materials for production synthetic nucleosides acids flavour enhancer, as 5 '-IMP, 5 '-GMP etc.; On medicine industry, it is antiviral as synthesis, the investigation and application of the bulk drug of cancer therapy drug is also more and more extensive.D-ribose is as the natural components be present in all cells simultaneously, close relationship is had with the formation of adenylic acid (AMP) and the regeneration of ATP, it is one of the most basic energy derive of vital metabolic, play a crucial role in heart and muscle metabolism, the recovery of local organization ischemic, anoxic can being promoted, at treatment myocardial ischaemia, preventing and treating because also there is good action in kinetic sore muscle and antifatigue.In a word, D-ribose has boundless application prospect.
Since going out D-ribose from late nineteenth century human identification, the production method of D-ribose mainly experienced by enzymatic hydrolysis RNA, with other sugar or organic acid for raw material chemosynthesis with utilize fermentable to produce three classes.Wherein, the procedure of enzymatic hydrolysis RNA separating D-ribose is numerous and diverse, manufacturing cost is high, yield is low, is not suitable for carrying out scale operation; Because the technological process of mercury electrode point solution can be used in chemical synthesis production process, easily cause serious environmental pollution.Enter the seventies in last century, people start with metabolic control fermentation theoretical for instructing, and utilize microbial nutrition deficient strain to carry out the research of D-Ribose by Fermentation.Sasajima found in the research of 1971 subtilis transketolase deficient type bacterial strain with 12.5% glucose for during carbon source through 6 days fermentation, in final fermented liquid, the accumulation volume of D-ribose is 35g/L; Within 1976, carry out aerobic fermentation with the subtilis after improvement and bacillus pumilus transketolase deficient mutant strain, the accumulation volume of D-ribose reaches 72.3g/L.The bacillus pumilus transketolase deficient mutant strain such as Kishimoto in 1988, in the substratum adding die aromatischen Aminosaeuren, the output of D-ribose reaches 92.1 g/L.Within 1997, Wulf bacillus pumilus transketolase deficient mutant strain produces D-ribose, and its productive rate is 45 g/L.Within 1997, the bright subtilis transketolase deficient mutant strain waiting seed selection of Jiangsu institute of microbiology Deng Chong is that substrate carries out aerobic fermentation with glucose, and D-ribose output is up to 92 g/L.
Research through above-mentioned investigators finds that subtilis and bacillus pumilus transketolase deficient mutant strain can accumulate D-ribose in a large number.In addition, by research D-ribose biosynthesizing and relevant metabolic pathway known, the carrying out that microorganism accumulates D-ribose in a large number except blocking its downstream metabolic approach being made, also should increase the synthesis of D-ribose precursor, namely increase the vigor of Hexose phosphate dehydrogenase.Hexose phosphate dehydrogenase is the regulatory enzyme of this biosynthetic pathway, irreversible reaction, and the size of its vigor determines D-ribose output height.In genus bacillus, Hexose phosphate dehydrogenase is relevant with the formation of gemma, is induced to produce in sporulation process.But the formation of gemma can change kind and the quantity of microbe endoenzyme, affects its normal metabolic activity, is unfavorable for the accumulation of D-ribose.The D-ribose superior strain obtained is separated from subtilis, the synthesis of Hexose phosphate dehydrogenase is not suppressed, can independently be present in nourishing body cell, the research of Sasajima also confirms this point, he produces bacterial strain to D-ribose and carries out mutagenesis repeatedly, when maintaining transketolase disappearance, select the mutant strain of a strain glucose dehydro enzyme activity height and the forfeiture of gemma generative capacity, D-ribose output reaches 70 g/L.
In above-mentioned domestic and international report, D-ribose fermentation is carried out by seed selection subtilis or bacillus pumilus transketolase deficient mutant strain, the output of D-ribose reaches a higher level, but also there is the shortcoming that residual sugar is high, fermented liquid impurity is many, be unfavorable for the subsequent extracted of D-ribose; Fermenting process easily generates gemma, affects the output of D-ribose; Fermentation period is long, is generally greater than 70 hours.
Summary of the invention
The object of the present invention is to provide a kind of subtilis lost gemma generative capacity, stablize high yield D-ribose, and this strain fermentation produces the method for D-ribose, to overcome the defect that prior art residual sugar is high, easily produce gemma, fermentation period length.
Bacterial strain provided by the present invention be subtilis (bacillussubtilis) BAV20120416, so that the strains A TCC21360 of D-ribose can be accumulated on a small quantity for starting strain purchased from a strain of Beijing North Na Chuanlian Bioteknologisk Institut, adopt routine mutagenesis method, the physics such as ultraviolet, nitrosoguanidine, chemical mutagen process are carried out to ATCC21360, through repeatedly repeatedly mutagenesis screening to cultivate, final fermented liquid residual sugar can be made to reduce to 0, other impurity are few, fermenting process not easily produces gemma, fermentation period is short, and can stablize the bacterial strain carrying out D-ribose suitability for industrialized production.
Subtilis of the present invention (bacillussubtilis) BAV20120416 bacterial strain, on April 16th, 2012 in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preserving number is: CGMCC NO.6013, and preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
One, the acquisition of subtilis BAV20120416
With subtilis ATCC21360 for initial strain, on slant medium, cultivate 1 ~ 2 day under 32 ~ 38 DEG C of conditions, scraping somatic cells, in stroke-physiological saline solution, makes bacteria suspension, makes cell concn be 1 × 108~ 109individual/mL.Conveniently mutafacient system, with the physics such as ultraviolet lamp, nitrosoguanidine, chemical mutagen is alternate or process continuously.Get above-mentioned bacteria suspension in culture dish, under being placed in 15W ultraviolet lamp, distance 20cm, irradiates 0.5 ~ 5 minute; Get the nitrosoguanidine treatment solution of above-mentioned bacteria suspension 1mg/mL, 32 ~ 38 DEG C of insulation concussions 30 ~ 60 minutes, by centrifugal for the thalline of nitrosoguanidine process, with normal saline flushing several times, to remove residual nitrosoguanidine.Or the continuous bacterium liquid that processed alternate with ultraviolet lamp, nitrosoguanidine are coated on plate culture medium, cultivate 1 ~ 2 day under 32 ~ 38 DEG C of conditions, get single bacterium colony to put respectively on minimum medium and supplemental medium, screening well-grown on supplemental medium, and the bacterium colony that can not grow on minimum medium, i.e. subtilis transketolase deficient type bacterial strain.The bacterial strain of screening is sieved and further separation screening again through seed culture medium and fermention medium shake flask fermentation, select a strain stable hereditary property, not easily sprout that spore, fermentation period are short, the bacterial strain of high yield D-ribose, be subtilis transketolase deficient mutant strain that preserving number is CGMCC NO.6013 (bacillussubtilis) BAV20120416.
Above-mentioned subtilis BAV20120416 biological property: cell size 0.7 ~ 0.8 × 2 ~ 3 microns, in rod-short, Dan Sheng, in pairs or in catenation, without pod membrane, can motion, bacterium colony is circular or irregular, and bacterium colony is flat, surface irregularity is opaque, dirty white or micro-yellow.Physio-biochemical characteristics: Gram-positive, aerobic, chemoheterotrophy, liquefy gelatin, peptonized milk, reduction nitrate, catalase is positive, can assimilate carbohydrate (glucose, sorbyl alcohol, Zulkovsky starch, N.F,USP MANNITOL, fructose etc.), can utilize nitrogenous source (ammonium sulfate, yeast powder, extractum carnis, peptone, corn steep liquor etc.), growth pH 5 ~ 8, growth temperature 32 ~ 38 DEG C.
Two, the method for novel bacterial BAV20120416 fermentative production D-ribose
1, slant culture
Above-mentioned mutagenic obtained subtilis transketolase deficient mutant strain BAV20120416 is inoculated in slant medium, cultivates 1 ~ 2 day for 32 ~ 38 DEG C, obtained slant strains.
2, seed culture
Get slant strains and be inoculated in seed culture medium 32 ~ 38 DEG C of shaking tables cultivation 12 ~ 16h, obtained fermentation kind of a daughter bacteria liquid, OD value 1.2-1.5.
3, fermentation culture
By 5% ~ 10%(by volume %) inoculum size seed liquor is accessed in preprepared fermention medium, fermentation culture is carried out under 32 ~ 38 DEG C of conditions, fermentation time 40 ~ 60h, the initial pH 6.5 ~ 7.2 of fermented liquid, adding soda acid by stream in fermenting process makes fermented liquid pH control 5.6 ~ 7.0, timing sampling in fermenting process, observe growth state, measure the concentration of glucose and D-ribose in fermented liquid, when in fermented liquid, glucose concn drops to 0, maintain for some time, when D-ribose concentration no longer rises, fermentation ends.
Above-mentioned slant medium composition (%): peptone 0.5 ~ 2.0, corn steep liquor 0.5 ~ 1.0, yeast powder 0.5 ~ 1.0, sodium-chlor 0.2 ~ 0.5, manganous sulfate 0.005 ~ 0.01, agar 1.2 ~ 2.0, regulates pH 6.5 ~ 7.2.
Above-mentioned seed culture medium composition (%): glucose 1.0 ~ 3.0, corn steep liquor 0.5 ~ 1.0, dipotassium hydrogen phosphate 0.2 ~ 0.5, potassium primary phosphate 0.05 ~ 0.2, manganous sulfate 0.005 ~ 0.02, regulates pH 6.5 ~ 7.2.
Above-mentioned fermention medium composition (%): glucose 12.0 ~ 20.0, corn steep liquor 1.0 ~ 3.0, ammonium sulfate 0.3 ~ 1.0, phosphorus ammonium 0.2 ~ 1.0, manganous sulfate 0.005 ~ 0.02, regulates pH 6.5 ~ 7.2.
The measuring method of glucose in above-mentioned fermented liquid: use the SBA-40D type bio-sensing analyser that Shandong Province academy sciences Biology Research Institute produces, by glucose content in the mensuration fermented liquid that immobilized glucose oxidase film is single-minded.
D-Glucose+H2oglucose oxidasemaltonic acid+H2o2
The measuring method of above-mentioned D-ribose: adopt Dionex U3000 liquid chromatographic system to measure the content of D-ribose in fermented liquid, actual conditions is as follows:
Pump: Dionex U3000 Pump
Post: Kromasil 100-5NH2 (200 × 4.6mm)
Moving phase: acetonitrile: water=70:30(V:V)
Flow velocity: 1.0ml/mL
Column temperature: 40 DEG C
Detector: Shodex RI-101 Composition distribution
BAV20120416 of the present invention and parental plant ATCC21360 glucose fermentation comparing result are in table 1:
As can be seen from above-mentioned comparing result, the D-ribose superior strain BAV20120416 that the present invention obtains is subtilis transketolase-deficient mutant, have genetic stability good, not easily produce the feature such as gemma; More than 90g/L D-ribose can be accumulated in the fermention medium being main raw material with glucose, corn steep liquor, be about three times of its parental plant subtilis ATCC21360, fermentation period is short, general control is at 50 hours, lower than prior art about 1/3, particularly impurity is few, final fermented liquid residual sugar reduces to 0(glucose and ribulose residual quantity by existing testing method, all do not detect), be specially adapted to medical production requirement, more existing bacterial classification has greater advantage, is the strain excellent with industrial applications potentiality; And adopt the ribose that existing strain fermentation is produced, the wherein residual sugar impurity of about residual 1%, do not reach the requirement of medical production standard.
Accompanying drawing explanation
Fig. 1 represents the liquid chromatogram of bacterial strain of the present invention final fermented liquid D-ribose assay
Fig. 2 represents the liquid chromatogram of existing parental plant final fermented liquid D-ribose assay
(note: sample is sample introduction after dilution 5 times)
As seen from Figure 1, in the final fermented liquid of bacterial strain BAV20120416 of the present invention, desolventize outside peak, only have a D-ribose peak, the sample size of D-ribose is 0.0189 × 5g/ml, i.e. 94.5g/L.As seen from Figure 2, in the final fermented liquid of existing parental plant ATCC21360, desolventize peak, D-ribose peak, also have glucose peaks and ribulose peak, the sample size of D-ribose is 0.0067 × 5g/ml, i.e. 33.5 g/L; The sample size of glucose is 0.0010 × 5g/ml, i.e. 5g/L, that is there is residual sugar in final fermented liquid; Also have a ribulose peak in addition, illustrate and create other impurity at the fermenting process of existing parental plant ATCC21360.
Embodiment
embodiment 1
By subtilis ATCC21360 on slant medium, cultivate 2 days under 35 ~ 38 DEG C of conditions, scraping somatic cells, in stroke-physiological saline solution, makes bacteria suspension, be cell concn is 1 × 108~ 109individual/mL.Conveniently mutafacient system, with the physics such as ultraviolet lamp, nitrosoguanidine, chemical mutagen is alternate or process continuously.Get above-mentioned bacteria suspension in culture dish, under being placed in 15W ultraviolet lamp, distance 20cm, irradiates 0.5 ~ 5 minute; Get the nitrosoguanidine treatment solution of above-mentioned bacteria suspension 1mg/mL, 35 ~ 38 DEG C of insulation concussions 30 ~ 60 minutes, by centrifugal for the thalline of nitrosoguanidine process, with normal saline flushing several times, to remove residual nitrosoguanidine.By alternate with ultraviolet lamp, nitrosoguanidine or the continuous bacterium liquid coating processed with on plate culture medium, cultivate 1 ~ 2 day under 35 ~ 38 DEG C of conditions, get single bacterium colony to put respectively on minimum medium and supplemental medium, screening well-grown on supplemental medium, and the bacterium colony that can not grow on minimum medium, i.e. subtilis transketolase deficient type bacterial strain.The bacterial strain of screening is sieved and further separation screening again through seed culture medium and fermention medium shake flask fermentation, select a strain stable hereditary property, not easily sprout that spore, fermentation period are short, the bacterial strain of high yield D-ribose, i.e. subtilis transketolase deficient mutant strain BAV20120416.
Above-mentioned nitrosoguanidine treatment solution preparation: take 10mg nitrosoguanidine in brown bottle, add 1mL acetone and make it dissolve, then add water 10mL, be made into the nitrosoguanidine treatment solution of 1mg/mL.
Above-mentioned slant medium composition (%): peptone 0.5 ~ 2.0, corn steep liquor 0.5 ~ 1.0, yeast powder 0.5 ~ 1.0, sodium-chlor 0.2 ~ 0.5, manganous sulfate 0.005 ~ 0.01, agar 1.2 ~ 2.0, regulates pH 6.5 ~ 7.2.
Above-mentioned plate culture medium composition (%): peptone 0.5 ~ 2.0, Dried Corn Steep Liquor Powder 0.5 ~ 1.0, yeast powder 0.5 ~ 1.0, sodium-chlor 0.2 ~ 0.5, manganous sulfate 0.005 ~ 0.01, agar 1.2 ~ 2.0, regulates pH 6.5 ~ 7.2.
Above-mentioned minimum medium composition (%): glucose 0.5 ~ 1.0, ammonium sulfate 0.1 ~ 0.3, dipotassium hydrogen phosphate 0.5 ~ 1.5, potassium primary phosphate 0.3 ~ 0.8, magnesium sulfate 0.01 ~ 0.04, manganous sulfate 0.001 ~ 0.01, agar 1.2 ~ 2.0, regulates pH 6.5 ~ 7.2.
Above-mentioned supplemental medium composition (%): the shikimic acid adding 40 ~ 60mg/mL in minimum medium.
Above-mentioned seed culture medium composition (%): glucose 1.0 ~ 3.0, corn steep liquor 0.5 ~ 1.0, dipotassium hydrogen phosphate 0.2 ~ 0.5, potassium primary phosphate 0.05 ~ 0.2, manganous sulfate 0.005 ~ 0.02, regulates pH 6.5 ~ 7.2.
Above-mentioned fermention medium composition (%): glucose 14.0 ~ 20.0, corn steep liquor 1.0 ~ 3.0, ammonium sulfate 0.3 ~ 1.0, phosphorus ammonium 0.2 ~ 1.0, manganous sulfate 0.005 ~ 0.02, regulates pH 6.5 ~ 7.2.
embodiment 2
Mutagenic obtained subtilis transketolase deficient mutant strain BAV20120416 is inoculated in slant medium, cultivates 2 days for 36 DEG C, obtained slant strains.Get a ring slant strains to be inoculated in and to be equipped with in the 500mL triangular flask of 50mL seed culture medium, 36 DEG C, 14h cultivated by 150 r/min shaking tables, inoculum size access by 10% is preprepared is equipped with in the 5L shaking flask of 500mL fermention medium, at 36 DEG C, rotating speed 150r/min carries out shaker fermentation cultivation, ferments after 48 hours, in fermented liquid, glucose content drops to 0 after measured, and D-ribose content reaches 90.2g/L; And its parental plant ATCC21360 under the same conditions, need cultivate 72 hours, and in fermented liquid, glucose content no longer declines after dropping to 4 g/L, in fermented liquid, D-ribose content is only 29.7g/L.
Above-mentioned slant medium composition (%): peptone 1.0, corn steep liquor 0.5, yeast powder 0.5, sodium-chlor 0.5, manganous sulfate 0.005, agar 2.0, regulates pH 7.0 ~ 7.2.
Above-mentioned seed culture medium composition (%): glucose 2.0, corn steep liquor 0.5, dipotassium hydrogen phosphate 0.3, potassium primary phosphate 0.1, manganous sulfate 0.005, regulates pH 6.8 ~ 7.0.
Above-mentioned fermention medium composition (%): glucose 14.0, corn steep liquor 2.0, ammonium sulfate 1.0, phosphorus ammonium 0.4, manganous sulfate 0.01, regulates pH 6.8 ~ 7.0.
embodiment 3
Above-mentioned mutagenic obtained subtilis transketolase deficient mutant strain BAV20120416 is inoculated on the slant medium of eggplant bottle, cultivates 2 days for 37 DEG C, obtained large slant strains.Bacterial classification under aseptic washing is added in eggplant bottle, make bacteria suspension, by this bacterial suspension inoculation in the 5L shaking flask that 800mL seed culture medium is housed, at 37 DEG C, rotating speed 150r/min shaking table cultivates 16 hours, and the inoculum size by 10% accesses in preprepared fermention medium, 50L fermentor tank liquid amount 30L, carry out ventilation stir culture at 37 DEG C, wherein stirring velocity is 200 ~ 300r/min, and air flow is 1 ~ 2Nm3/ h, tank internal pressure 0.05Mpa, initial pH6.5 ~ 7.2 of fermented liquid, add soda acid by stream in fermenting process and make fermented liquid pH control 5.4 ~ 7.0, and in fermenting process, timing sampling measures the concentration of glucose and D-ribose in fermented liquid.Ferment after 49 hours, in fermented liquid, glucose content drops to 0, fermentation ends, measures D-ribose concentration 94.5 g/L; And its parental plant is under the same conditions, fermentation time 74 hours, and in fermented liquid, glucose content no longer declines after dropping to 6 g/L, D-ribose content is only 33.5g/L.
Above-mentioned slant medium composition (%): peptone 1.0, corn steep liquor 0.5, yeast powder 0.5, sodium-chlor 0.5, manganous sulfate 0.005, agar 2.0, regulates pH 7.0 ~ 7.2.
Above-mentioned seed culture medium composition (%): glucose 2.0, corn steep liquor 0.5, dipotassium hydrogen phosphate 0.3, potassium primary phosphate 0.1, manganous sulfate 0.005, regulates pH 6.8 ~ 7.0.
Above-mentioned fermention medium composition (%): glucose 18.0, corn steep liquor 2.0, ammonium sulfate 1.0, phosphorus ammonium 0.4, manganous sulfate 0.01, regulates pH 6.8 ~ 7.0.