CN1821418A - Producing dihydroxy acetone by microbe method - Google Patents
Producing dihydroxy acetone by microbe method Download PDFInfo
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- CN1821418A CN1821418A CN 200510061969 CN200510061969A CN1821418A CN 1821418 A CN1821418 A CN 1821418A CN 200510061969 CN200510061969 CN 200510061969 CN 200510061969 A CN200510061969 A CN 200510061969A CN 1821418 A CN1821418 A CN 1821418A
- Authority
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- Prior art keywords
- glycerine
- cctcc
- thalline
- otan
- substratum
- Prior art date
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- Granted
Links
- RXKJFZQQPQGTFL-UHFFFAOYSA-N dihydroxyacetone Chemical compound OCC(=O)CO RXKJFZQQPQGTFL-UHFFFAOYSA-N 0.000 title claims abstract description 51
- 238000000034 method Methods 0.000 title claims abstract description 30
- 229940120503 dihydroxyacetone Drugs 0.000 title abstract description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 124
- 235000011187 glycerol Nutrition 0.000 claims abstract description 60
- 241001508813 Clavispora lusitaniae Species 0.000 claims abstract description 28
- 241000235062 Pichia membranifaciens Species 0.000 claims abstract description 28
- 241001508811 Clavispora Species 0.000 claims abstract description 5
- 241000235648 Pichia Species 0.000 claims abstract description 5
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 36
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 30
- 230000001580 bacterial effect Effects 0.000 claims description 20
- 238000006243 chemical reaction Methods 0.000 claims description 19
- 239000000843 powder Substances 0.000 claims description 17
- 239000001888 Peptone Substances 0.000 claims description 16
- 108010080698 Peptones Proteins 0.000 claims description 16
- 235000019319 peptone Nutrition 0.000 claims description 16
- 239000008399 tap water Substances 0.000 claims description 16
- 235000020679 tap water Nutrition 0.000 claims description 16
- 238000011534 incubation Methods 0.000 claims description 15
- 244000005700 microbiome Species 0.000 claims description 15
- 230000000813 microbial effect Effects 0.000 claims description 12
- 239000000758 substrate Substances 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 11
- LJQKCYFTNDAAPC-UHFFFAOYSA-N ethanol;ethyl acetate Chemical compound CCO.CCOC(C)=O LJQKCYFTNDAAPC-UHFFFAOYSA-N 0.000 claims description 8
- 238000000855 fermentation Methods 0.000 claims description 8
- 230000004151 fermentation Effects 0.000 claims description 8
- 230000009466 transformation Effects 0.000 claims description 8
- 238000011081 inoculation Methods 0.000 claims description 7
- 230000035484 reaction time Effects 0.000 claims description 7
- 238000005119 centrifugation Methods 0.000 claims description 6
- 238000002425 crystallisation Methods 0.000 claims description 6
- 230000008025 crystallization Effects 0.000 claims description 6
- 241001052560 Thallis Species 0.000 claims description 5
- 239000012046 mixed solvent Substances 0.000 claims description 5
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 4
- 229930195725 Mannitol Natural products 0.000 claims description 4
- 238000010828 elution Methods 0.000 claims description 4
- 239000000594 mannitol Substances 0.000 claims description 4
- 235000010355 mannitol Nutrition 0.000 claims description 4
- 239000002689 soil Substances 0.000 claims description 4
- -1 ferment Chemical compound 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 2
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 2
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims 1
- 238000000746 purification Methods 0.000 claims 1
- 238000009776 industrial production Methods 0.000 abstract 1
- 230000001954 sterilising effect Effects 0.000 description 23
- 238000004659 sterilization and disinfection Methods 0.000 description 23
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 18
- 238000002360 preparation method Methods 0.000 description 18
- 239000000243 solution Substances 0.000 description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 16
- 239000001963 growth medium Substances 0.000 description 13
- 239000000463 material Substances 0.000 description 12
- 241000894006 Bacteria Species 0.000 description 9
- 230000001105 regulatory effect Effects 0.000 description 8
- 238000003756 stirring Methods 0.000 description 8
- 229960004756 ethanol Drugs 0.000 description 7
- 108020004463 18S ribosomal RNA Proteins 0.000 description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
- 238000009423 ventilation Methods 0.000 description 5
- 241000589232 Gluconobacter oxydans Species 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 230000001133 acceleration Effects 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 238000006356 dehydrogenation reaction Methods 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000000741 silica gel Substances 0.000 description 4
- 229910002027 silica gel Inorganic materials 0.000 description 4
- 229960001866 silicon dioxide Drugs 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241000589220 Acetobacter Species 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 239000002054 inoculum Substances 0.000 description 3
- 229910052500 inorganic mineral Inorganic materials 0.000 description 3
- 239000011707 mineral Substances 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 150000007524 organic acids Chemical class 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- HSJKGGMUJITCBW-UHFFFAOYSA-N 3-hydroxybutanal Chemical compound CC(O)CC=O HSJKGGMUJITCBW-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 241000228212 Aspergillus Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 2
- 241000588807 Bordetella Species 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 241000589236 Gluconobacter Species 0.000 description 2
- 241000228143 Penicillium Species 0.000 description 2
- 241000589516 Pseudomonas Species 0.000 description 2
- 241000235527 Rhizopus Species 0.000 description 2
- 241000607720 Serratia Species 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 238000005377 adsorption chromatography Methods 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 229960000935 dehydrated alcohol Drugs 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000004817 gas chromatography Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000035479 physiological effects, processes and functions Effects 0.000 description 2
- 238000011218 seed culture Methods 0.000 description 2
- 238000012882 sequential analysis Methods 0.000 description 2
- WGTYBPLFGIVFAS-UHFFFAOYSA-M tetramethylammonium hydroxide Chemical compound [OH-].C[N+](C)(C)C WGTYBPLFGIVFAS-UHFFFAOYSA-M 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 238000009834 vaporization Methods 0.000 description 2
- 230000008016 vaporization Effects 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000588698 Erwinia Species 0.000 description 1
- 101000892220 Geobacillus thermodenitrificans (strain NG80-2) Long-chain-alcohol dehydrogenase 1 Proteins 0.000 description 1
- 208000013016 Hypoglycemia Diseases 0.000 description 1
- 208000007976 Ketosis Diseases 0.000 description 1
- 241000588748 Klebsiella Species 0.000 description 1
- 241000187708 Micromonospora Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000006482 condensation reaction Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 125000000468 ketone group Chemical group 0.000 description 1
- 150000002584 ketoses Chemical class 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- DHRLEVQXOMLTIM-UHFFFAOYSA-N phosphoric acid;trioxomolybdenum Chemical compound O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.OP(O)(O)=O DHRLEVQXOMLTIM-UHFFFAOYSA-N 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 208000017520 skin disease Diseases 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 239000003270 steroid hormone Substances 0.000 description 1
- 230000000475 sunscreen effect Effects 0.000 description 1
- 235000019605 sweet taste sensations Nutrition 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention discloses one new method of producing dihydroxy acetone with microbes, and the related microbes include Clavispora lusitaniae in Clavispora, CCTCC No. M205116, and Pichia membranifaciens in Pichia, CCTCC No. M205117. Producing dihydroxy acetone with these microbes can obtain glycerin converting rate as high as 50~90 % in perfect technological conditions. The microbe strains of the present invention are suitable for industrial production of 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, of many uses: as to contain three functional groups in (1) dna molecular, chemical property is active, wide participation is such as various chemical reactions such as polymerization, condensations, it 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) (CN 1687434).
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 18th, 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 18s rDNA).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 18th, 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 the above-mentioned bacterial classification of publication, 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.M 205117, 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 25 ℃~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.M 205116 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 CCTCC No.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.M 205117, 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 composition, the per-cent of weight kg/ volume L, for: glycerine or N.F,USP MANNITOL: 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%, 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 a column volume 3%~10%, 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 48h.
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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