CN100370032C - Producing dihydroxy acetone by microbe method - Google Patents
Producing dihydroxy acetone by microbe method Download PDFInfo
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- CN100370032C CN100370032C CNB2005100619690A CN200510061969A CN100370032C CN 100370032 C CN100370032 C CN 100370032C CN B2005100619690 A CNB2005100619690 A CN B2005100619690A CN 200510061969 A CN200510061969 A CN 200510061969A CN 100370032 C CN100370032 C CN 100370032C
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Abstract
The present invention discloses a method for producing dihydroxy acetone by using new microorganisms, which relates to a Clavispora lusitaniae in Clavispora of which the preservation number is CCTCC No. M205116 or Pichia membranifaciens in Pichia of which the preservation number is CCTCC No. M205117. The microorganism is used for producing dihydroxy acetone, the transformation efficiency of the glycerol reaches 50 to 90 percent under the good process condition, and the microorganism of the present invention is suitable for commercially producing dihydroxy acetone.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to from soil screening and separating to the new microorganism of two strains, also relate to and utilize these two kinds new microbial transformation glycerine to produce the method for otans.
Background technology
Otan is called 1 again, and the 3-otan (1,3-Dihydroxyacetone), be called for short DHA, be the most single ketose, have the white powder crystallization of sweet taste, organic solvents such as soluble in water, ethanol, acetone and ether.The molecular weight of this chemical substance is 90.08.
DHA is a kind of important chemical material, medicine intermediate and foodstuff additive, and is of many uses:
(1) contains three functional groups in the dna molecular, chemical property is active, and wide participation is such as various chemical reactions such as polymerization, condensations, is a kind of intermediate of important chemosynthesis, also be an important poly functional reagent, if can participate in effectively from formaldehyde to the aldol, the condensation reaction of hydroxyl ketone.(2) DHA is widely used in the makeup, can stop the moisture content excessive vaporization of skin, skin is played preserve moisture and sunscreen effect, and in tanning industry, DHA can be used as the protective material of leatherware.(3) DHA is a kind of important medicine intermediate and medicine, now has been applied to hypoglycemia and diabetes and some treating for skin disease.(4) DHA can be used as a kind of foodstuff additive, is widely used in foodstuff production.
Because DHA is of many uses, market capacity is big, and the research of producing otan with microbial method glycerine has great importance.
Abroad with the microbial method glycerine converting be otan oneself a lot of patents and bibliographical information are arranged, if the microbial host Gluconobacter (Gluconobacter) that is used to produce otan (US5770411) and genus acetobacter (Acetobacter) (US 4076589) microorganism, especially the report of gluconobacter oxydans (Gluconobacter oxydans) and weak oxide acetobacter (AcebobacterSuboxydans) is in the majority, studied the Bacillus subtilus (Bacillus subtilis FERM P-10524) (JP 2286089) of bacillus (Bacillus) in addition, Micremonospora (Micromonospora) (JP 62210994), Pseudomonas (Pseudomonas), serratia (Serratia), restrain mould uncle Bordetella (Klebsiella), the Irving program Bordetella (Erwinia) of planting, Aspergillus (Aspergillus), Penicillium (Penicillium), Rhizopus (Rhizopus) (US4576913).
The used bacterial classification of the producing dihydroxy acetone by microbe method of domestic report is weak oxidized acetic acid bacteria (Acebobacter Suboxydans) (food and fermentation industries, 2005,31 (6): 22-26) and gluconobacter suboxydans (Gluconobacter suboxydans) (CN1687434).
Microbe transformation method production DHA compares with chemical synthesis has many advantages: advantages such as specificity is strong, reaction conditions is gentle, substrate utilization ratio is high, transformation efficiency is high, production technique is simple.From the economy of technology, the angle of the friendly of environment, microbial transformation is produced DHA and is had more economic benefit and social benefit.And conversion technology reaches its maturity, and this technology has been widely used in the production of all kinds of materials, as microbiotic, steroid hormone, amino acid etc.
Summary of the invention
The invention provides a kind of screening from soil the glycerine bio-transformation is the new microorganism strains of otan; Next provides a kind of method of utilizing this new microbe cell biological glycerine converting to produce otan.
The new microorganism strains of two strains provided by the invention belongs to yeast, and bacterial strain 1 feature is as follows:
Colonial morphology: the bacterium colony surface wettability, oily, color is yellowish-white, and bacterium colony is rounded, and is smooth all around, and central authorities are projection slightly, and the bacterium colony quality is even, the edge rounding.
Cellular form: circle, ellipse.
18s rDNA sequential analysis: with the total DNA of the cell that extracts is template, utilize the 18S rDNA gene of primer P1:5 '-TCC GTA GGT GAA CCT GCG G-3 ' and P2:5 '-TCC TCC GCT TAT TGATAT GC-3 ' amplification bacterial strain, gene product is connected with the T carrier, confirm that through order-checking this fragment physical length is 382bp, (submitted this sequence to GenBank, the GenBank registration number is DQ223426) with GenBank in related data carry out similarity analysis and find, the highest (the homology of homology of this bacterium and Clavispora lusitaniae yeast (Clavispora lusitaniae), 98.43%/382bps, based on 18s rDNA).Physiology and biochemistry is identified the binding molecule evaluation, can confirm that this bacterial strain belongs to the Clavispora lusitaniae yeast (Clavisporalusitaniae) of excellent spore yeast belong (Clavispora).Oneself is preserved in Chinese typical culture collection center this bacterial strain on October 12nd, 2005, be called for short CCTCC, and deposit number is CCTCC No.M 205116.
New bacterial strain 2 features that the present invention screens from soil are as follows:
Colonial morphology: the bacterium colony surface wettability, oily, color is yellowish-white, and bacterium colony is rounded, and is smooth all around, and central authorities are projection slightly, and the bacterium colony quality is even, the edge rounding.
Cellular form: circle, ellipse.
18s rDNA sequential analysis: with the total DNA of the cell that extracts is template, utilize the 18S rDNA gene of primer P1:5 '-TCC GTA GGT GAA CCT GCG G-3 ' and P2:5 '-TCC TCC GCT TAT TGATAT GC-3 ' amplification bacterial strain, gene product is confirmed that through order-checking this fragment physical length is 447bp with the T carrier, (submitted this sequence to GenBank, the GenBank registration number is DQ223427) with GenBank in related data carry out similarity analysis and find, the highest (the homology of homology of this bacterium and Pichia membranaefaciens (Pichia membranifaciens), 98.54%/477bps, based on 18srDNA).Physiology and biochemistry is identified the binding molecule evaluation, can confirm that this bacterial strain belongs to the Pichia membranaefaciens of Pichia (Pichia) (Pichia membranifaciens).Oneself is preserved in Chinese typical culture collection center this bacterial strain on October 12nd, 2005, be called for short CCTCC, and deposit number is CCTCC No.M 205117.
According to above microbial characteristic, these two new bacterial strains are belonged to Clavispora lusitaniae yeast (Clavispora lusitaniae) by evaluation, and Pichia membranaefaciens (Pichia membranifaciens) bacterial strain, these two two strains do not belong to any of above-mentioned own publication bacterial classification, it is the ability of otan that this two strains microorganism strains has transformation of glycerol, can be used for the production of otan.
The used raw material of the present invention is a kind of widespread use industrial chemical---glycerine (glycerol), 2 hydroxyls of glycerine (2-OH) generate ketone group through glycerol dehydrogenase enzymic catalytic reaction in the microorganism cells, obtain otan.
Method with this microorganisms producing otan is as follows:
The substratum of new microorganism Clavispora lusitaniae yeast (Clavispora lusitaniae) CCTCCNo.M 205116 of the present invention or Pichia membranaefaciens (Pichia membranifaciens) CCTCC No.M205117 and carbonaceous sources, nitrogenous source, inorganic salt, substrate is a glycerine, ferment, otan in the separate fermentation, and purify.
Substratum of the present invention consist of (w/v, %): glycerine: 0.5%～12%, yeast powder: 0.1%～2.0%, peptone 0.1%～2.0%, (NH
4)
2SO
4: 0%～1.5%, NaH
2PO
42H
2O:0.1%～0.8%, MgSO
4: 0.01%～0.1%, CaCO
3: 0.1%～0.4%, with tap water preparation, pH value 4.0～7.0.
Glycerine in this substratum also can substitute with N.F,USP MANNITOL.
Produce DHA with new bacterial strain Clavispora lusitaniae yeast (Clavispora lusitaniae) of the present invention or Pichia membranaefaciens (Pichia membranifaciens), its method has:
(1) substratum of above-mentioned preparation is through sterilization, the bacterial classification Clavispora lusitaniae yeast (Clavispora lusitaniae) of access after cultivating, CCTCC No.M 205116 or Pichia membranaefaciens (Pichia membranifaciens), CCTCC No.M205117, inclined-plane or inoculate with seed liquor, cultivate, usually select temperature at 20 ℃～40 ℃, with 28 ℃～35 ℃ better, initial pH is 4.0～7.0, with near better neutral, incubation time is that 24h～72h is better, wherein to be best about 48h, reaction can be carried out under leaving standstill in addition, but is stirring, ventilate, carry out to good under the oscillating condition.During the fermentation, the pH value is regulated control with mineral acid or organic acid, bases.Glycerine during substratum is formed in this method is the carbon source that microorganism utilizes, again by the substrate of microbial transformation; Substrate glycerine generates DHA through the dehydrogenation reaction of microorganism.
(2) substratum of above-mentioned preparation is through sterilization, access is bacterial classification Clavispora lusitaniae yeast (Clavispora lusitaniae) after cultivating, CCTCC No.M 205116 or Pichia membranaefaciens (Pichiamembranifaciens), CCTCC No.M 205117 with inclined-plane or seed liquor inoculation, cultivates thalline, culture condition: select temperature usually at 20 ℃～40 ℃, with 28 ℃～35 ℃ better, initial pH is 4.0～7.0, with near better neutral; Stream adds substrate---the glycerine of microbial transformation reaction in culturing process, can when just having begun to grow, thalline add by stream, also can be at thalli growth for some time of after, promptly carrying out stream after the thalli growth logarithmic phase again adds, the speed that stream adds can be regulated according to the concentration of glycerine in the fermented liquid, and incubation time is that 48h～120h is better, wherein being best about 72h, reaction can be carried out under leaving standstill in addition, but carries out under stirring, ventilation, oscillating condition to good; During the fermentation, the variation of pH value is regulated control with mineral acid or organic acid, bases, makes tunning.
(3) substratum of above-mentioned preparation is through sterilization, access is bacterial classification Clavispora lusitaniae yeast (Clavispora lusitaniae) after cultivating, CCTCC No.M 205116 or Pichia membranaefaciens (Pichiamembranifaciens), CCTCC No.M 205117 with inclined-plane or seed liquor inoculation, cultivates thalline, culture condition: select temperature usually at 20 ℃～40 ℃, with 28 ℃～35 ℃ better, initial pH is 4.0～7.0, with near better neutral; Cultivate thalline to logarithmic phase, carry out centrifugation, obtain thalline; The thalline that obtains is added substrate glycerine, and the substrate glycerol concentration is 0.5%～15.0%, and the reaction times is that 1h～100h is better, and wherein to be best about 24h, reaction can be carried out under leaving standstill in addition, but carries out under stirring, ventilation, oscillating condition to good; In the bioconversion reaction process, the variation of pH value is regulated control with mineral acid or organic acid, bases.
During from the broth extraction target product, can adopt conventional tunning extraction method.As: filter, centrifugal, precipitation, crystallization, recrystallization, concentrate, dry, lyophilize, absorption, ion-exchange, chromatography or the like.
Concrete extracting method is as follows: the employing volume ratio is that 95: 5 ethyl acetate-ethanol mixed solvent is a moving phase, use sherwood oil wet method dress post, at aspect ratio 10～40: 1, elution flow rate 1mL/min～10mL/min, the application of sample amount is column volume 3%～10% o'clock, and glycerine and otan separating effect are better in its enriched material.Collection contains otan section sample, carries out vacuum concentration, obtains white otan crystallization in 95: 5 ethyl acetate-ethanol solution.
The analytical procedure of otan adopts gas-chromatography and thin-layer chromatography side to carry out: otan thin-layer chromatography method.G type silica-gel plate is adopted in experiment, and (20mm * 10mm), developping agent: chloroform-methanol-distilled water mixed solvent (volume ratio is 80: 19: 1), developer consists of: phospho-molybdic acid 3g, methyl alcohol 45mL, distilled water 45mL and vitriol oil 10mL.Concrete operations step: the silica gel thin sheet behind the point sample is placed on downwards in the chromatography cylinder that developping agent is housed with reference line launches 10～15min, diffuse to apart from 1～2cm place, thin plate top until developping agent.Take out thin plate, dry up, stifling 2～5min in the iodine cylinder, baking oven is put in colour developing, toasts about 10min down at 110 ℃, can present the otan spot, by comparing otan content in the judgement sample with the spot size of different concns gradient standard substance demonstration.
DHA gas chromatography analysis method: used instrument: Varian varian cp-3800 gas chromatograph, band flame ionization ditector; PY-2020iD type cracker (Frontier LaboratoriesLtd.Japan).Agents useful for same: TMAH (25% aqueous solution), otan (chromatographically pure), glycerine, anhydrous methanol (analytical pure), distilled water.Chromatographic condition: Ultra ALLOY-5 Stainless Steel Capillary tubing string (30m * 0.25mm * 0.25 μ m); 400 ℃ of vaporization temperatures; 280 ℃ of detector temperatures; 50 ℃ of beginnings of column temperature rise to 60 ℃ with 1 ℃/min, rise to 280 ℃ with 10 ℃/min again; Splitting ratio 30: 1; Carrier gas, nitrogen; Post flow 1mL/min.Application of sample: 0.2 μ L fermentation supernatant and 0.4 μ LTMAH (25% aqueous solution).
Microorganism strains Clavispora lusitaniae yeast of the present invention (Clavispora lusitaniae) or Pichia membranaefaciens (Pichia membranifaciens), can not only on the substratum that with glycerine is sole carbon source, grow, and it is stronger that glycerine is carried out the ability of dehydrogenation reaction; Utilize Clavispora lusitaniae yeast (Clavispora lusitania) or Pichia membranaefaciens (Pichia membranifaciens) to produce otan, under the preferred process condition, the transformation efficiency of glycerine reaches 50%～90%, (promptly 1 mole glycerine can be converted into 0.50～0.90 mole otan), microorganism strains Clavispora lusitaniae yeast of the present invention (Clavispora lusitaniae) or Pichia membranaefaciens (Pichiamembranifaciens) are applicable to the suitability for industrialized production of otan.
Specific embodiments
Following embodiment is intended to illustrate rather than limit the scope of the invention.
The microorganism of adopting in following examples is Clavispora lusitaniae yeast (Clavispora lusitania) ZHB-05571 bacterial strain, CCTCC No.M 205116.
Embodiment 1:
Culture medium prescription (weight kg/ volume L per-cent, as follows): glycerine: 1.0%, yeast powder: 0.8%, peptone: 0.1%, NaH
2PO
42H
2O:0.1%, (NH
4)
2SO
4: 0.1%, MgSO
4: 0.01%, with the tap water preparation, pH is transferred to 4.0 with HCl solution, add CaCO again
3: 0.2%, it is standby to sterilize.
Get the above-mentioned substratum of 100mL, average mark is contained in 2 250mL triangular flasks, sterilization.Insert slant strains CCTCC No.M 205116, cultivate thalline, shaking speed 150r/min cultivates 24h as seed liquor at 28 ℃ of shaking tables, and is standby.
Get the above-mentioned substratum of 2L, average mark 40 500mL that pack into shake in the bottle, sterilization.Insert seed liquor, inoculum size is 1.5% (v/v, promptly commensurability's volume ratio is as follows), cultivates, and culture temperature is 20 ℃, and incubation time is 72h, shaking speed 150r/min.
Collect above-mentioned fermented liquid 1.5L, through centrifugal (10000 * g, 10min) afterwards, supernatant concentration is to 500mL, at 55 ℃ of following activated carbon decolorizing 30min, vacuum concentration is to pulpous state then, add an amount of dehydrated alcohol mixing, vacuum concentration removes ethanol, adds ethanol repeatedly three times, be concentrated into till the no ethanol, enriched material is standby.
Above-mentioned enriched material is by otan and glycerine in the adsorption chromatography post separation enriched material that 100～200 order silica gel are housed, the ethyl acetate-ethanol mixed solvent that adopted 95: 5 is a moving phase, use sherwood oil wet method dress post, aspect ratio 35: 1, elution flow rate 2mL/min, when the application of sample amount was column volume 5%, glycerine and otan separating effect were better in its enriched material.Collection contains otan section sample, carries out vacuum concentration, obtains white otan crystallization in 95: 5 ethyl acetate-ethanol solution, obtains the 0.95g otan after the lyophilize.
Embodiment 2:
Culture medium prescription: glycerine: 8.0%, yeast powder: 2.0%, peptone: 0.5%, (NH
4)
2SO
4: 0.1%, NaH
2PO
42H
2O:0.8%, MgSO
4: 0.1%, CaCO
3: 0.4%, with the tap water preparation, pH is transferred to 7.0 with NaOH solution, it is standby to sterilize.
Get the 500mL substratum, average mark is contained in 10 500mL triangular flasks, sterilization.Insert slant strains Clavispora lusitaniae yeast (Clavispora lusitaniae), CCTCC No.M205116 cultivates, and culture temperature is 28 ℃, and incubation time is 50h, is reflected under ventilation, the oscillating condition to carry out shaking speed 200r/min.
Collect above-mentioned fermented liquid 400mL, separate, purifying, step obtains 1.26 gram DHA with embodiment 1.
Embodiment 3:
Fermentative medium formula: glycerine: 12%, yeast powder: 2%, peptone: 2%, (NH
4)
2SO
4: 0.5%, NaH
2PO
42H
2O:0.1%, MgSO
4: 0.02%, CaCO
3: 0.1%, with the tap water preparation, pH is transferred to 7.0 with NaOH solution, it is standby to sterilize.
Seed culture based formulas: glycerine: 1.0%, yeast extract powder: 1%, peptone 0.1%, MgSO
4: 0.05%, with the tap water preparation, pH is transferred to 7.0 with NaOH solution, it is standby to sterilize.
Get the 100mL seed culture medium, average mark is contained in 2 500mL triangular flasks, sterilization.Insert slant strains CCTCC No.M 205116, cultivate thalline, shaking speed 200r/min cultivates 24h as seed liquor at 30 ℃ of shaking tables, and is standby.
Get the 4L fermention medium, average mark is contained in 80 500mL triangular flasks, sterilization.Insert seed liquor, inoculum size is 2% (v/v), cultivates, and culture temperature is 30 ℃, and incubation time is 72h, is reflected under ventilation, the oscillating condition to carry out shaking speed 200r/min.
Collect above-mentioned fermented liquid 3.5L, separate, purifying, step obtains 13.4 gram DHA with embodiment 1.
Embodiment 4
Culture medium prescription: glycerine: 1.0%, yeast powder: 0.5%, peptone 1%, (NH
4)
2SO
4: 0.2%, NaH
2PO
42H
2O:0.5%, MgSO
4: 0.01%, CaCO
3: 0.2%, with the tap water preparation, pH7.0, it is standby to sterilize.
Get the above-mentioned substratum of 200mL, average mark is contained in 4 250mL triangular flasks, sterilization.Insert slant strains CCTCCNo.M 205116, cultivate thalline, shaking speed 150r/min cultivates 24h as seed liquor at 28 ℃ of shaking tables, and is standby.
Add above-mentioned substratum 6.0L in the 10L fermentor tank, sterilization inserts seed liquor 100mL, ferments.
After inoculation, begin stream and add the glycerine 1.0L that concentration is 50% (V/V), flow acceleration 0.2mL/min; 30 ℃ of culture temperature, incubation time are about 84h, air flow 5L/min, stirring velocity 300r/min.
Collect above-mentioned fermented liquid 6L, separate, purifying, step obtains 11.32 gram DHA with embodiment 1.
Embodiment 5
Culture medium prescription: glycerine: 2.0%, yeast extract powder: 2%, peptone: 1%, (NH
4)
2SO
4: 1%, NaH
2PO
42H
2O:0.8%, MgSO
4: 0.01%, with the tap water preparation, pH6.5 adds CaCO again
3: 0.2%, it is standby to sterilize.
Get the above-mentioned substratum of 200mL, average mark is contained in 4 500mL triangular flasks, sterilization.Insert slant strains CCTCC No.M 205116, cultivate thalline, shaking speed 150r/min cultivates 72h as seed liquor at 28 ℃ of shaking tables, and is standby.
Add above-mentioned substratum 7L in the 10L fermentor tank, sterilization inserts seed liquor 100mL, ferments.
Thalli growth during to logarithmic phase (behind the fermentation 20h) carry out stream to add concentration be 50% glycerine 1L, flow acceleration 0.3mL/min; 28 ℃ of culture temperature, incubation time are about 96h, air flow 5L/min, stirring velocity 300r/min.
Collect above-mentioned fermented liquid 7L, separate, purifying, step obtains 34.51 gram otans with embodiment 1.
Embodiment 6
Culture medium prescription: glycerine: 2.0%, yeast powder: 2.0%, peptone: 0.2%, (NH
4)
2SO
4: 0.1%, NaH
2PO
42H
2O:0.1%, MgSO
4: 0.01%, with the tap water preparation, regulating pH with HCl is 6.0, adds CaCO again
3: 0.1%, it is standby to sterilize.
Get the 1000mL substratum, average mark is contained in 20 500mL triangular flasks, sterilization.Insert slant strains CCTCC No.M 205116, cultivate thalline to logarithmic phase, carry out centrifugation then, obtain thalline, it is in 0.5% the glycerine solution that these thalline are joined 300mL concentration, utilizes thalline that glycerine is carried out dehydrogenation reaction; Reaction conditions: 28 ℃ of temperature, initial pH is 6.5, the reaction times is about 24h, shaking speed 100r/min.
Collect above-mentioned reaction solution 250mL, separate, purifying, step obtains 0.35 gram DHA with embodiment 1.
Embodiment 7
Culture medium prescription glycerine: 2.0%, yeast powder: 0.5%, peptone 0.2%, (NH
4)
2SO
4: 0.1%, NaH
2PO
42H
2O:0.1%, MgSO
4: 0.01%, CaCO
3: 0.1%, with the tap water preparation, regulating pH with NaOH solution is 7.0, it is standby to sterilize.
Get the 1000mL substratum, average mark is contained in 20 500mL triangular flasks, sterilization, insert slant strains CCTCC No.M 205116, cultivate thalline to logarithmic phase, carry out centrifugation then, obtain thalline, it is in 12% the glycerine solution that these thalline are joined 300mL concentration, utilizes the enzymes biocatalysis glycerine in the thalline to produce otan; Reaction conditions: 32 ℃ of temperature, initial pH is 5.0, the reaction times is about 48h, shaking speed 100r/min.
Collect above-mentioned reaction solution 250mL, separate, purifying, step obtains 2.77 gram DHA with embodiment 1.
The microorganism of adopting in following examples is Pichia membranaefaciens (Pichiamembranifaciens) bacterial strain ZJB-05389, CCTCC No.M 205117
Embodiment 1:
Culture medium prescription (weight/volume percent, as follows): glycerine: 1.0%, yeast powder: 0.8%, peptone: 0.1%, KH
2PO
4: 0.1%, MgSO
4: 0.01%, with the tap water preparation, pH is transferred to 4.0 with HCl solution, add CaCO again
3: 0.2%, it is standby to sterilize.
Get the above-mentioned substratum of 100mL, average mark is contained in 2 250mL triangular flasks, sterilization.Insert slant strains CCTCC No.M 205117, cultivate thalline, shaking speed 150r/min cultivates 24h as seed liquor at 28 ℃ of shaking tables, and is standby.
Get the above-mentioned substratum of 2L, average mark 40 500mL that pack into shake in the bottle, sterilization.Insert seed liquor, inoculum size is 1.5% (v/v), cultivates, and culture temperature is 30 ℃, and incubation time is 50h, shaking speed 150r/min.
Collect above-mentioned fermented liquid 1.5L, through centrifugal (10000 * g, 10min) afterwards, supernatant concentration is to 500mL, at 55 ℃ of following activated carbon decolorizing 30min, vacuum concentration is to pulpous state then, add an amount of dehydrated alcohol mixing, vacuum concentration removes ethanol, adds ethanol repeatedly three times, be concentrated into till the no ethanol, enriched material is standby.
Above-mentioned enriched material is by otan and glycerine in the adsorption chromatography post separation enriched material that 100～200 order silica gel are housed, the ethyl acetate-ethanol mixed solvent that adopted 95: 5 is a moving phase, use sherwood oil wet method dress post, aspect ratio 35: 1, elution flow rate 2mL/min, when the application of sample amount was column volume 5%, glycerine and otan separating effect were better in its enriched material.Collection contains otan section sample, carries out vacuum concentration, obtains white otan crystallization in 95: 5 ethyl acetate-ethanol solution, obtains the 1.15g otan after the lyophilize.
Embodiment 2:
Culture medium prescription: glycerine: 10%, yeast extract powder: 2.0%, peptone: 0.5%, KH
2PO
4: 0.2%, MgSO
4: 0.05%, CaCO
3: 0.4%, with the tap water preparation, pH is transferred to 7.0 with NaOH solution, it is standby to sterilize.
Get the 500mL substratum, average mark is contained in 10 500mL triangular flasks, sterilization.Insert slant strains CCTCC No.M205117, cultivate, culture temperature is 28 ℃, and incubation time is 50h, is reflected under stirring, ventilation, the oscillating condition to carry out shaking speed 200r/min.
Collect above-mentioned fermented liquid 400mL, separate, purifying, step obtains 1.42 gram DHA with embodiment 1.
Embodiment 3
Culture medium prescription: glycerine: 2.0%, yeast powder: 0.5%, peptone 1%, kH
2PO
4: 0.1%, MgSO
4: 0.01%, CaCO
3: 0.2%, with the tap water preparation, natural pH, it is standby to sterilize.
Get the above-mentioned substratum of 200mL, average mark is contained in 4 250mL triangular flasks, sterilization.Insert slant strains CCTCC No.M 205117, cultivate thalline, shaking speed 150r/min cultivates 24h as seed liquor at 28 ℃ of shaking tables, and is standby.
Add above-mentioned substratum 6.0L in the 10L fermentor tank, sterilization inserts seed liquor 100mL, ferments.
After inoculation, begin stream and add the glycerine 1.0L that concentration is 50% (V/V), flow acceleration 0.2mL/min; 30 ℃ of culture temperature, incubation time are about 84h, air flow 5L/min, stirring velocity 300r/min.
Collect above-mentioned fermented liquid 6L, separate, purifying, step obtains 11.32 gram DHA with embodiment 1.
Embodiment 4
Culture medium prescription glycerine: 2.0%, yeast extract powder: 2%, peptone: 1%, kH
2PO
4: 0.3%, MgSO
4: 0.01%, with the tap water preparation, pH6.5, it is standby to sterilize.
Get the above-mentioned substratum of 200mL, average mark is contained in 4 500mL triangular flasks, sterilization.Insert slant strains CCTCC No.M 205117, cultivate thalline, shaking speed 150r/min cultivates 72h as seed liquor at 28 ℃ of shaking tables, and is standby.
Add above-mentioned substratum 7L in the 10L fermentor tank, sterilization inserts seed liquor 100mL, ferments.
Thalli growth during to logarithmic phase (behind the fermentation 20h) carry out stream to add concentration be 50% glycerine 1L, flow acceleration 0.3mL/min; 28 ℃ of culture temperature, incubation time are about 96h, air flow 5L/min, and stirring velocity 300r/min, the pH value is controlled at 6.0, regulates with HCl and NaOH.
Collect above-mentioned fermented liquid 7L, separate, purifying, step obtains 38.26 gram DHA with embodiment 1.
Embodiment 5
Culture medium prescription: N.F,USP MANNITOL: 4%, yeast powder: 2.0%, peptone: 0.2%, KH
2PO
4: 0.1%, MgSO
4: 0.01%, with the tap water preparation, regulating pH with HCl is 6.0, it is standby to sterilize.
Get the 1000mL substratum, average mark is contained in 20 500mL triangular flasks, sterilization.Insert slant strains CCTCC No.M 205117, cultivate thalline (24h) to the logarithmic phase later stage, carry out centrifugation then, obtain thalline, it is in 0.5% the glycerine solution that these thalline are joined 300mL concentration, utilizes thalline that glycerine is carried out dehydrogenation reaction; Reaction conditions: 28 ℃ of temperature, initial pH is 6.5, the reaction times is about 24h, shaking speed 100r/min.
Collect above-mentioned reaction solution 250mL, separate, purifying, step obtains 0.39 gram DHA with embodiment 1.
Embodiment 6
Culture medium prescription N.F,USP MANNITOL: 4%, yeast powder: 0.5%, peptone 0.2%, KH
2PO
4: 0.1%, MgSO
4: 0.01%, with the tap water preparation, regulating pH with HCl is 6.0, it is standby to sterilize.
Get the 1000mL substratum, average mark is contained in 20 500mL triangular flasks, sterilization, insert slant strains CCTCC No.M 205117, cultivate thalline to logarithmic phase, carry out centrifugation then, obtain thalline, it is in 12% the glycerine solution that these thalline are joined 300mL concentration, utilizes the enzymes biocatalysis glycerine in the thalline to produce otan; Reaction conditions: 32 ℃ of temperature, initial pH is 5.0, the reaction times is about 48h, shaking speed 100r/min.
Collect above-mentioned reaction solution 250mL, separate, purifying, step obtains 2.85 gram DHA with embodiment 1.
Claims (9)
1. the utilization method that screening and separating is produced otan to new microbial transformation glycerine from soil, it is characterized in that described microorganism is the Clavispora lusitaniae yeast (Clavispora lusitaniae) of excellent spore yeast belong (Clavispora), the Pichia membranaefaciens of CCTCC No.M 205116 or Pichia (Pichia) (Pichia membranifaciens), CCTCC No.M205117, Clavispora lusitaniae yeast or Pichia membranaefaciens and carbonaceous sources, nitrogenous source, the substratum of inorganic salt, substrate is a glycerine, ferment, otan in the separate fermentation then, and purify.
2. method according to claim 1 is characterized in that described substratum forms, and the per-cent of weight kg/ volume L is glycerine or N.F,USP MANNITOL: O.5%～12%, yeast powder: O.1%～2.0%, peptone O.1%～2.O%, (NH
4)
2SO
4: O%～1.5%, NaH
2PO
42H
2O:O.1%～O.8%, MgSO
4: 0.01%～O.1%, CaCO
3: O.1%～0.4%, prepare with tap water.
3. method according to claim 1, the separation, the purification step that it is characterized in that described fermented liquid are: the employing volume ratio is that 95: 5 ethyl acetate-ethanol mixed solvent is a moving phase, use sherwood oil wet method dress post, at aspect ratio 10～40: 1, elution flow rate 1mL/min～10mL/min, the application of sample amount is column volume 3%～10% o'clock, collects to contain otan section sample, carry out vacuum concentration, in 95: 5 ethyl acetate-ethanol solution, obtain white otan crystallization.
4. method according to claim 1 and 2, it is characterized in that in substratum, inserting inclined-plane or the seed liquor inoculation of CCTCCNo.M 205116 or CCTCC No.M 205117, ferment culture condition: 20 ℃～40 ℃ of temperature, initial pH is 4.0～7.0, and incubation time is 24h～72h.
5. method according to claim 4 is characterized in that culture condition: 28 ℃～35 ℃ of temperature, and initial pH is 7.0, incubation time is 48 h.
6. method according to claim 1 and 2, it is characterized in that in substratum, inserting inclined-plane or the seed liquor inoculation of bacterial classification CCTCC No.M 205116 or CCTCC No.M 205117, cultivate thalline, culture condition: temperature is at 20 ℃～40 ℃, initial pH is 4.0～7.0, incubation time is 48h～120h, in the culturing process, begins to flow the substrate glycerine that adds microbial reaction when thalline has just begun to grow; Or after the thalli growth logarithmic phase, flow the substrate glycerine that adds microbial reaction again.
7. method according to claim 6 is characterized in that culture condition: 28 ℃～35 ℃ of temperature, and initial pH is 7.0, incubation time is 72h.
8. method according to claim 1 and 2, it is characterized in that in substratum, inserting bacterial classification CCTCC No.M 205116 or CCTCC No.M 205117, with inclined-plane or seed liquor inoculation, cultivate thalline, culture condition: temperature is at 20 ℃～40 ℃, initial pH is 4.0～7.0, cultivate thalline to logarithmic phase, carry out centrifugation, obtain thalline, the thalline that obtains is joined in the substrate glycerine solution, utilize thalline conversion of substrate glycerine, the substrate glycerol concentration is 0.5%～15.0%, reaction times 1h～100h.
9. method according to claim 8 is characterized in that culture condition: 28 ℃～35 ℃ of temperature, and initial pH is 7.0, the reaction times is 24h.
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| CN100494347C (en) * | 2006-09-22 | 2009-06-03 | 浙江工业大学 | Bacillus lichenformis B-05571 and application in preparation of 1,3-dihydroxy acetone |
| CN101367783A (en) | 2008-10-10 | 2009-02-18 | 中国科学技术大学 | Preparation method of 5-hydroxymethyl furfural |
| CN101591681B (en) * | 2009-04-13 | 2011-11-23 | 浙江工业大学 | Method for producing dihydroxyacetone through microbial transformation |
| EP2495239B1 (en) | 2009-07-09 | 2016-11-16 | University of Science and Technology of China | Method for preparing 4-hydroxymethylfurfural |
| CN103667369A (en) * | 2012-09-26 | 2014-03-26 | 北京贯虹科技集团有限公司 | Method for producing dihydroxyacetone by converting glycerin by virtue of biological fermentation process |
| CN109196090A (en) * | 2016-05-12 | 2019-01-11 | 三得利控股株式会社 | The manufacturing method of thallus or thalline culture or their extract of the yeast containing L- hydroxyproline and application thereof and L- hydroxyproline |
| CN112831537B (en) * | 2021-01-24 | 2022-11-15 | 广东石油化工学院 | Preparation method of bio-based end-capped polyether |
| CN113621658B (en) * | 2021-08-09 | 2023-05-02 | 长兴制药股份有限公司 | Preparation method for continuously producing 1,3-dihydroxyacetone and erythrulose |
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| US5770411A (en) * | 1994-12-14 | 1998-06-23 | Merck Patent Gesellschaft Mit Beschrankter Haftung | Microbial process for the preparation of dihydroxyacetone with recycling of biomass |
| CN1687434A (en) * | 2005-03-30 | 2005-10-26 | 赵金红 | Method for preparing dihydroxy acetone |
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| US5770411A (en) * | 1994-12-14 | 1998-06-23 | Merck Patent Gesellschaft Mit Beschrankter Haftung | Microbial process for the preparation of dihydroxyacetone with recycling of biomass |
| CN1687434A (en) * | 2005-03-30 | 2005-10-26 | 赵金红 | Method for preparing dihydroxy acetone |
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