CN104988196A - Fermentation production method for N-acetylglucosamine - Google Patents
Fermentation production method for N-acetylglucosamine Download PDFInfo
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- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 title claims abstract description 80
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 title claims abstract description 80
- 238000000855 fermentation Methods 0.000 title claims abstract description 57
- 230000004151 fermentation Effects 0.000 title claims abstract description 57
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 11
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 title abstract 4
- 229950006780 n-acetylglucosamine Drugs 0.000 title abstract 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims abstract description 95
- 239000004202 carbamide Substances 0.000 claims abstract description 51
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 43
- 239000008103 glucose Substances 0.000 claims abstract description 43
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- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims abstract description 10
- 239000007788 liquid Substances 0.000 claims description 43
- 238000000034 method Methods 0.000 claims description 40
- 230000008569 process Effects 0.000 claims description 18
- 238000011218 seed culture Methods 0.000 claims description 15
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- 239000001963 growth medium Substances 0.000 claims description 10
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 6
- 229910019142 PO4 Inorganic materials 0.000 claims description 6
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 6
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 6
- 239000004473 Threonine Substances 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 6
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 6
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 claims description 6
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 6
- RUTXIHLAWFEWGM-UHFFFAOYSA-H iron(3+) sulfate Chemical compound [Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O RUTXIHLAWFEWGM-UHFFFAOYSA-H 0.000 claims description 6
- 229910000360 iron(III) sulfate Inorganic materials 0.000 claims description 6
- 238000002386 leaching Methods 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 6
- 229930182817 methionine Natural products 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 6
- 239000010452 phosphate Substances 0.000 claims description 6
- 239000011591 potassium Substances 0.000 claims description 6
- 229910052700 potassium Inorganic materials 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 6
- 229960001763 zinc sulfate Drugs 0.000 claims description 6
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 6
- 239000002609 medium Substances 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 4
- 241000894006 Bacteria Species 0.000 claims description 3
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 3
- 238000009413 insulation Methods 0.000 claims description 3
- 230000000968 intestinal effect Effects 0.000 claims description 3
- 230000035800 maturation Effects 0.000 claims description 3
- 239000001301 oxygen Substances 0.000 claims description 3
- 229910052760 oxygen Inorganic materials 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 230000006698 induction Effects 0.000 abstract description 4
- 238000006243 chemical reaction Methods 0.000 abstract description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 51
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 19
- 229960002989 glutamic acid Drugs 0.000 description 17
- 230000009466 transformation Effects 0.000 description 17
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 16
- 235000013922 glutamic acid Nutrition 0.000 description 16
- 239000004220 glutamic acid Substances 0.000 description 16
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- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 6
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 6
- 229960002442 glucosamine Drugs 0.000 description 6
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
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- 238000003556 assay Methods 0.000 description 1
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
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Abstract
The invention provides a fermentation production method for N-acetylglucosamine. In the fermentation process, when the residual glucose content of fermentation liquor falls below 0.5 g/L, a glucose solution is flowingly added into the fermentation liquor at the flow acceleration of 5-8 g/L.h, when OD660nm of the fermentation liquor is equal to 28-31, IPTG is added for induction, and then a urea solution is flowingly added into the fermentation liquor at the flow acceleration of 0.5-0.8 g/L.h. According to the fermentation production method for the N-acetylglucosamine, in the fermentation process, induction is performed through the IPTG, the flow adding speed of the glucose solution and the urea solution is optimized when induction is performed, and the N-acetylglucosamine content and the conversion rate at the fermentation end point are significantly improved.
Description
Technical field
The present invention relates to field of fermentation engineering, specifically, relate to a kind of fermentation method for producing of 2-Acetamido-2-deoxy-D-glucose.
Background technology
The purposes of glucosamine is very extensive, is mainly used in the fields such as food, medical treatment, makeup, husbandry.At present, what glucosamine was maximum is used to arthritic prevention and therapy.Glucosamine is the main component of reticular tissue and chondrocyte, if pathology occurs in joint, supplementary glucosamine can repair impaired cartilage, increases interarticular lubrication.
Now, the glucosamine of China is raw material production mainly with chitosan greatly, but there is raw material sources instability, and some crowds exist the shortcomings such as anaphylaxis.Although Production by Microorganism Fermentation 2-Acetamido-2-deoxy-D-glucose is (containing in the fermented liquid of 2-Acetamido-2-deoxy-D-glucose, add the hydrochloric acid of 0.1mol/L, 3h is reacted at 100 DEG C, the 2-Acetamido-2-deoxy-D-glucose of more than 90% can be converted into glucosamine) above shortcoming can be overcome, but fermentable is produced 2-Acetamido-2-deoxy-D-glucose existence and is yielded poorly, low conversion rate and by-product acetic acid, the phenomenon that content of glutamic acid is high, therefore, need the new process for fermenting developing a kind of 2-Acetamido-2-deoxy-D-glucose, to improve fermentation yield and the transformation efficiency of 2-Acetamido-2-deoxy-D-glucose, reduce fermentation byproduct acetic acid simultaneously, the content of L-glutamic acid.
Summary of the invention
In order to solve problems of the prior art, the object of this invention is to provide a kind of fermentation method for producing of 2-Acetamido-2-deoxy-D-glucose.
In order to realize the object of the invention, technical scheme of the present invention is as follows:
A kind of fermentation method for producing of 2-Acetamido-2-deoxy-D-glucose, during the fermentation, when the residual sugar content of fermented liquid is down to below 0.5g/L, glucose solution is added to stream in fermented liquid according to the stream rate of acceleration of 5 ~ 8g/Lh, when OD660nm=28 ~ 31 of fermented liquid, add IPTG to induce, then add urea soln according to the stream rate of acceleration of 0.5 ~ 0.8g/Lh to stream in fermented liquid.When stream adds urea, glucose still simultaneously stream add.
Further, the stream rate of acceleration of described urea soln is 1:10 with the ratio of the stream rate of acceleration of glucose solution.
Further, the add-on of described IPTG is 0.03mM.
Further, the mass concentration of described glucose solution is 55%.
Further, the mass concentration of described urea soln is 15%.
Further, said method comprising the steps of:
(1) seed culture:
Preparation seed culture medium, seed culture medium pH to 7.3 is adjusted with the ammoniacal liquor that concentration is 25% (m/m), logical saturation steam to 121 DEG C in seed culture medium, insulation 30min, setting seed culture condition: temperature 37 DEG C, tank pressure 0.05mpa, ventilating ratio 1:0.5, rotating speed 300rpm, pH6.8 ~ 7.0; Access is produced 2-Acetamido-2-deoxy-D-glucose intestinal bacteria and is cultivated, when seed OD660nm=3.5 ~ 5 range intervals, as seed maturity standard;
(2) fermentation culture:
Preparation fermention medium, 121 DEG C, sterilizing 30min, fermentation culture conditions is set: temperature 37 DEG C, tank pressure 0.03 ~ 0.05mpa, ventilating ratio 1:0.5 ~ 1:1 after cooling, rotating speed 300 ~ 500rpm, pH are omnidistance is 6.9 ~ 7.0 with the automatic control of 25% (m/m) ammoniacal liquor; The 2-Acetamido-2-deoxy-D-glucose seed liquor of maturation is inoculated into fermentor tank cultivates according to 10% (V/V) inoculum size, by regulating tank pressure, air quantity, rotating speed to control dissolved oxygen 30-40% in fermenting process;
When the residual sugar content of fermented liquid is down to below 0.5g/L, glucose solution is added to stream in fermented liquid according to the stream rate of acceleration of 5 ~ 8g/Lh, when OD660nm=28 ~ 31 of fermented liquid, add IPTG and induce, then add urea soln according to the stream rate of acceleration of 0.5 ~ 0.8g/Lh to stream in fermented liquid; Cultivate 52 ~ 56 hours.
Further, described seed culture medium is: glucose 15g/L (singly disappearing), dipotassium hydrogen phosphate 11g/L, potassium primary phosphate 16g/L, yeast leaching powder 1g/L, Threonine 0.2g/L, methionine(Met) 0.15g/L, magnesium sulfate 0.5g/L, ammonium sulfate 5g/L, manganous sulfate 0.013mg/L, ferric sulfate 0.024mg/L, zinc sulfate 0.047mg/L, cobalt chloride 0.01mg/L.
Further, described fermention medium is: glucose 5g/L (singly disappearing), dipotassium hydrogen phosphate 16g/L, potassium primary phosphate 10g/L, yeast leaching powder 0.3g/L, yeast extract paste 0.1g/L, Threonine 0.1g/L, methionine(Met) 0.15g/L, magnesium sulfate 0.25g/L, ammonium sulfate 3g/L, Repone K 0.1g/L, manganous sulfate 0.015mg/L, ferric sulfate 0.35mg/L, zinc sulfate 0.41mg/L, cobalt chloride 0.02mg/L.
As preferably, after stream adds urea soln, cultivate 54 hours.
Beneficial effect of the present invention is:
The fermentation method for producing of 2-Acetamido-2-deoxy-D-glucose provided by the invention, induced by IPTG at fermenting process, and the stream rate of acceleration of glucose solution and urea soln when optimizing induction, significantly improve fermentation termination 2-Acetamido-2-deoxy-D-glucose content and transformation efficiency.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
In following embodiment and comparative example:
OD pH-value determination pH: adopt 7230G visible spectrophotometer, measure light absorption value under wavelength 660nm visible ray.
The concentration of reducing sugar is measured according to the method for GB/T5009.7-2008.
2-Acetamido-2-deoxy-D-glucose assay: high performance liquid chromatography.
Acetic acid content measures: high performance liquid chromatography.
Content of glutamic acid measure: get fermented liquid centrifugal after supernatant liquor, be diluted to residual aminoglutaric acid concentration within 1g/L, use bio-sensing analysis-e/or determining.
Transformation efficiency (%)=2-Acetamido-2-deoxy-D-glucose amount/total consumption sugar amount × 100%.
Embodiment 1
(1) seed culture medium is prepared: glucose 15g/L, dipotassium hydrogen phosphate 11g/L, potassium primary phosphate 16g/L, yeast leaching powder 1g/L, Threonine 0.2g/L, methionine(Met) 0.15g/L, magnesium sulfate 0.5g/L, ammonium sulfate 5g/L, manganous sulfate 0.013mg/L, ferric sulfate 0.024mg/L, zinc sulfate 0.047mg/L, cobalt chloride 0.01mg/L.
Put in 50L fermentor tank, seed culture medium pH to 7.3 is adjusted with the ammoniacal liquor that concentration is 25% (m/m), logical saturation steam to 121 DEG C in seed culture medium, insulation 30min, setting seed culture condition: temperature 37 DEG C, tank pressure 0.05mpa, ventilating ratio 1:0.5, rotating speed 300rpm, pH6.8-7.0.Access is produced 2-Acetamido-2-deoxy-D-glucose intestinal bacteria and is cultivated, when seed OD660nm=3.5-5 range intervals, as seed maturity standard.
(2) fermention medium is prepared: glucose 5g/L (singly disappearing), dipotassium hydrogen phosphate 16g/L, potassium primary phosphate 10g/L, yeast leaching powder 0.3g/L, yeast extract paste 0.1g/L, Threonine 0.1g/L, methionine(Met) 0.15g/L, magnesium sulfate 0.25g/L, ammonium sulfate 3g/L, Repone K 0.1g/L, manganous sulfate 0.015mg/L, ferric sulfate 0.35mg/L, zinc sulfate 0.41mg/L, cobalt chloride 0.02mg/L.
Put in 50L fermentor tank, 121 DEG C, sterilizing 30min, sets fermentation culture conditions after cooling: temperature 37 DEG C, tank pressure 0.03-0.05mpa, ventilating ratio 1:0.5-1:1, and rotating speed 300-500rpm, pH are omnidistance is 7.0 with 25% (m/m) automatic control.The 2-Acetamido-2-deoxy-D-glucose seed liquor of maturation is inoculated into fermentor tank cultivates according to 10% (V/V) inoculum size, by regulating tank pressure, air quantity, rotating speed to control dissolved oxygen 30-40% in fermenting process.
When residual sugar is down to below 0.5g/L in 2-Acetamido-2-deoxy-D-glucose fermentation culture process, add 55% (m/m) glucose solution according to the stream rate of acceleration of 5g/Lh to stream in 2-Acetamido-2-deoxy-D-glucose fermented liquid.
When 2-Acetamido-2-deoxy-D-glucose fermented liquid OD660nm=28-31 is interval, the disposable 0.03mM of adding IPTG induces, now add urea soln according to glucose solution and urea solution mass than the data rate stream of 10:1, namely add 15% (m/m) urea soln according to stream rate of acceleration 0.5g/Lh to stream in 2-Acetamido-2-deoxy-D-glucose fermented liquid.
The present invention does not produce 2-Acetamido-2-deoxy-D-glucose according to the fermentation later stage or produces 2-Acetamido-2-deoxy-D-glucose and judges fermentation termination more slowly, in 54 hours fermentation culture cycles, 2-Acetamido-2-deoxy-D-glucose content is 107g/L, and transformation efficiency is 38.3%, acetic acid content is 3.9g/L, and content of glutamic acid is 5.7g/L.
Embodiment 2
When residual sugar is down to below 0.5g/L in 2-Acetamido-2-deoxy-D-glucose fermentation culture process, add 55% (m/m) glucose solution according to the stream rate of acceleration of 6g/Lh to stream in 2-Acetamido-2-deoxy-D-glucose fermented liquid.Now add urea soln according to glucose solution and urea solution mass than the data rate stream of 10:1, namely add 15% (m/m) urea soln according to stream rate of acceleration 0.6g/Lh to stream in 2-Acetamido-2-deoxy-D-glucose fermented liquid.Other conditions and cultural method are with embodiment 1.During fermentation culture 54h, 2-Acetamido-2-deoxy-D-glucose content is 110g/L, and transformation efficiency is 40.1%, and acetic acid content is 1.9g/L, and content of glutamic acid is 2.1g/L.
Embodiment 3
When residual sugar is down to below 0.5g/L in 2-Acetamido-2-deoxy-D-glucose fermentation culture process, add 55% (m/m) glucose solution according to the stream rate of acceleration of 7g/Lh to stream in 2-Acetamido-2-deoxy-D-glucose fermented liquid.Now add urea soln according to glucose solution and urea solution mass than the data rate stream of 10:1, namely add 15% (m/m) urea soln according to stream rate of acceleration 0.7g/Lh to stream in 2-Acetamido-2-deoxy-D-glucose fermented liquid.Other conditions and cultural method are with embodiment 1.During fermentation culture 54h, 2-Acetamido-2-deoxy-D-glucose content is 98g/L, and transformation efficiency is 38.5%, and acetic acid content is 3.2g/L, and content of glutamic acid is 3.5g/L.
Embodiment 4
When residual sugar is down to below 0.5g/L in 2-Acetamido-2-deoxy-D-glucose fermentation culture process, add 55% (m/m) glucose solution according to the stream rate of acceleration of 8g/Lh to stream in 2-Acetamido-2-deoxy-D-glucose fermented liquid.Now add urea soln according to glucose solution and urea solution mass than the data rate stream of 10:1, namely add 15% (m/m) urea soln according to stream rate of acceleration 0.8g/Lh to stream in 2-Acetamido-2-deoxy-D-glucose fermented liquid.Other conditions and cultural method are with embodiment 1.During fermentation culture 54h, 2-Acetamido-2-deoxy-D-glucose content is 101g/L, and transformation efficiency is 38.7%, and acetic acid content is 4.4g/L, and content of glutamic acid is 4.7g/L.
Embodiment 5
Embodiment 5 difference from Example 1 is to adopt 500L fermentor tank to carry out the fermentation culture of 2-Acetamido-2-deoxy-D-glucose, during fermentation culture 54h, 2-Acetamido-2-deoxy-D-glucose content is 103g/L, and transformation efficiency is 38.4%, acetic acid content is 3.5g/L, and content of glutamic acid is 4.9g/L.
Comparative example 1
When residual sugar is down to below 0.5g/L in 2-Acetamido-2-deoxy-D-glucose fermentation culture process, add 55% (m/m) glucose solution according to the stream rate of acceleration of 3g/Lh to stream in 2-Acetamido-2-deoxy-D-glucose fermented liquid.Now add urea soln according to glucose solution and urea solution mass than the data rate stream of 10:1, namely add 15% (m/m) urea soln according to stream rate of acceleration 0.3g/Lh to stream in 2-Acetamido-2-deoxy-D-glucose fermented liquid.Other conditions and cultural method are with embodiment 1.During fermentation culture 54h, 2-Acetamido-2-deoxy-D-glucose content is 80.3g/L, and transformation efficiency is 31.1%, and acetic acid content is 2.1g/L, and content of glutamic acid is 2.5g/L.
Comparative example 2
When residual sugar is down to below 0.5g/L in 2-Acetamido-2-deoxy-D-glucose fermentation culture process, add 55% (m/m) glucose solution according to the stream rate of acceleration of 4g/Lh to stream in 2-Acetamido-2-deoxy-D-glucose fermented liquid.Now add urea soln according to glucose solution and urea solution mass than the data rate stream of 10:1, namely add 15% (m/m) urea soln according to stream rate of acceleration 0.4g/Lh to stream in 2-Acetamido-2-deoxy-D-glucose fermented liquid.Other conditions and cultural method are with embodiment 1.During fermentation culture 54h, 2-Acetamido-2-deoxy-D-glucose content is 85.7g/L, and transformation efficiency is 36.1%, and acetic acid content is 1.8g/L, and content of glutamic acid is 2.7g/L.
Comparative example 3
When residual sugar is down to below 0.5g/L in 2-Acetamido-2-deoxy-D-glucose fermentation culture process, add 55% (m/m) glucose solution according to the stream rate of acceleration of 9g/Lh to stream in 2-Acetamido-2-deoxy-D-glucose fermented liquid.Now add urea soln according to glucose solution and urea solution mass than the data rate stream of 10:1, namely add 15% (m/m) urea soln according to stream rate of acceleration 0.9g/Lh to stream in 2-Acetamido-2-deoxy-D-glucose fermented liquid.Other conditions and cultural method are with embodiment 1.During fermentation culture 54h, 2-Acetamido-2-deoxy-D-glucose content is 90.5g/L, and transformation efficiency is 35.2%, and acetic acid content is 4.7g/L, and content of glutamic acid is 5.8g/L.
Comparative example 4
When residual sugar is down to below 0.5g/L in 2-Acetamido-2-deoxy-D-glucose fermentation culture process, add 55% (m/m) glucose solution according to the stream rate of acceleration of 10g/Lh to stream in 2-Acetamido-2-deoxy-D-glucose fermented liquid.Now add urea soln according to glucose solution and urea solution mass than the data rate stream of 10:1, namely add 15% (m/m) urea soln according to stream rate of acceleration 1g/Lh to stream in 2-Acetamido-2-deoxy-D-glucose fermented liquid.Other conditions and cultural method are with embodiment 1.During fermentation culture 54h, 2-Acetamido-2-deoxy-D-glucose content is 75.3g/L, and transformation efficiency is 32.1%, and acetic acid content is 7.5g/L, and content of glutamic acid is 8.5g/L.
Comparative example 5
When residual sugar is down to below 0.5g/L in 2-Acetamido-2-deoxy-D-glucose fermentation culture process, add 55% (m/m) glucose solution according to the stream rate of acceleration of 5g/Lh to stream in 2-Acetamido-2-deoxy-D-glucose fermented liquid.Now add urea soln according to glucose solution and urea solution mass than the data rate stream of 8:1, namely add 15% (m/m) urea soln according to stream rate of acceleration 0.625g/Lh to stream in 2-Acetamido-2-deoxy-D-glucose fermented liquid.Other conditions and cultural method are with embodiment 1.During fermentation culture 54h, 2-Acetamido-2-deoxy-D-glucose content is 81.5g/L, and transformation efficiency is 34.6%, and acetic acid content is 3.2g/L, and content of glutamic acid is 5.1g/L.
Comparative example 6
When residual sugar is down to below 0.5g/L in 2-Acetamido-2-deoxy-D-glucose fermentation culture process, add 55% (m/m) glucose solution according to the stream rate of acceleration of 5g/Lh to stream in 2-Acetamido-2-deoxy-D-glucose fermented liquid.Now add urea soln according to glucose solution and urea solution mass than the data rate stream of 9:1, namely add 15% (m/m) urea soln according to stream rate of acceleration 0.56g/Lh to stream in 2-Acetamido-2-deoxy-D-glucose fermented liquid.Other conditions and cultural method are with embodiment 1.During fermentation culture 54h, 2-Acetamido-2-deoxy-D-glucose content is 85.5g/L, and transformation efficiency is 35.5%, and acetic acid content is 4.5g/L, and content of glutamic acid is 6.3g/L.
Comparative example 7
When residual sugar is down to below 0.5g/L in 2-Acetamido-2-deoxy-D-glucose fermentation culture process, add 55% (m/m) glucose solution according to the stream rate of acceleration of 5g/Lh to stream in 2-Acetamido-2-deoxy-D-glucose fermented liquid.Now add urea soln according to glucose solution and urea solution mass than the data rate stream of 11:1, namely add 15% (m/m) urea soln according to stream rate of acceleration 0.45g/Lh to stream in 2-Acetamido-2-deoxy-D-glucose fermented liquid.Other conditions and cultural method are with embodiment 1.During fermentation culture 54h, 2-Acetamido-2-deoxy-D-glucose content is 87.5g/L, and transformation efficiency is 33.5%, and acetic acid content is 4.7g/L, and content of glutamic acid is 6.2g/L.
Comparative example 8
When residual sugar is down to below 0.5g/L in 2-Acetamido-2-deoxy-D-glucose fermentation culture process, add 55% (m/m) glucose solution according to the stream rate of acceleration of 5g/Lh to stream in 2-Acetamido-2-deoxy-D-glucose fermented liquid.Now add urea soln according to glucose solution and urea solution mass than the data rate stream of 12:1, namely add 15% (m/m) urea soln according to stream rate of acceleration 0.42g/Lh to stream in 2-Acetamido-2-deoxy-D-glucose fermented liquid.Other conditions and cultural method are with embodiment 1.During fermentation culture 54h, 2-Acetamido-2-deoxy-D-glucose content is 86.5g/L, and transformation efficiency is 32.9%, and acetic acid content is 2.8g/L, and content of glutamic acid is 4.7g/L.
Comparative example 9
Comparative example 9 difference from Example 1 is that in 2-Acetamido-2-deoxy-D-glucose fermentation culture process, stream does not add urea soln, during fermentation culture 54h, 2-Acetamido-2-deoxy-D-glucose content is 90.2g/L, and transformation efficiency is 32.3%, acetic acid content is 4.1g/L, and content of glutamic acid is 5.2g/L.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (9)
1. the fermentation method for producing of a 2-Acetamido-2-deoxy-D-glucose, it is characterized in that, in fermenting process, when the residual sugar content of fermented liquid is down to below 0.5g/L, glucose solution is added to stream in fermented liquid according to the stream rate of acceleration of 5 ~ 8g/Lh, when OD660nm=28 ~ 31 of fermented liquid, add IPTG and induce, then add urea soln according to the stream rate of acceleration of 0.5 ~ 0.8g/Lh to stream in fermented liquid.
2. method according to claim 1, is characterized in that, the stream rate of acceleration of described urea soln is 1:10 with the ratio of the stream rate of acceleration of glucose solution.
3. method according to claim 1 and 2, is characterized in that, the add-on of described IPTG is 0.03mM.
4. method according to claim 3, is characterized in that, the mass concentration of described glucose solution is 55%.
5. method according to claim 3, is characterized in that, the mass concentration of described urea soln is 15%.
6. the method according to claim 4 or 5, is characterized in that, said method comprising the steps of:
(1) seed culture:
Preparation seed culture medium, regulates pH to 7.3, logical saturation steam to 121 DEG C in seed culture medium, insulation 30min, setting seed culture condition: temperature 37 DEG C, tank pressure 0.05mpa, ventilating ratio 1:0.5, rotating speed 300rpm, pH6.8 ~ 7.0; Access is produced 2-Acetamido-2-deoxy-D-glucose intestinal bacteria and is cultivated, when seed OD660nm=3.5 ~ 5 range intervals, as seed maturity standard;
(2) fermentation culture:
Preparation fermention medium, 121 DEG C, sterilizing 30min, sets fermentation culture conditions after cooling: temperature 37 DEG C, tank pressure 0.03 ~ 0.05mpa, ventilating ratio 1:0.5 ~ 1:1, rotating speed 300 ~ 500rpm, pH 6.9 ~ 7.0; The 2-Acetamido-2-deoxy-D-glucose seed liquor of maturation is inoculated into fermentor tank according to 10% inoculum size cultivate, controls dissolved oxygen 30 ~ 40%;
When the residual sugar content of fermented liquid is down to below 0.5g/L, glucose solution is added to stream in fermented liquid according to the stream rate of acceleration of 5 ~ 8g/Lh, when OD660nm=28 ~ 31 of fermented liquid, add IPTG and induce, then add urea soln according to the stream rate of acceleration of 0.5 ~ 0.8g/Lh to stream in fermented liquid; Cultivate 52 ~ 56 hours.
7. method according to claim 6, is characterized in that, described seed culture medium is: glucose 15g/L, dipotassium hydrogen phosphate 11g/L, potassium primary phosphate 16g/L, yeast leaching powder 1g/L, Threonine 0.2g/L, methionine(Met) 0.15g/L, magnesium sulfate 0.5g/L, ammonium sulfate 5g/L, manganous sulfate 0.013mg/L, ferric sulfate 0.024mg/L, zinc sulfate 0.047mg/L, cobalt chloride 0.01mg/L.
8. method according to claim 6, is characterized in that, described fermention medium is: glucose 5g/L, dipotassium hydrogen phosphate 16g/L, potassium primary phosphate 10g/L, yeast leaching powder 0.3g/L, yeast extract paste 0.1g/L, Threonine 0.1g/L, methionine(Met) 0.15g/L, magnesium sulfate 0.25g/L, ammonium sulfate 3g/L, Repone K 0.1g/L, manganous sulfate 0.015mg/L, ferric sulfate 0.35mg/L, zinc sulfate 0.41mg/L, cobalt chloride 0.02mg/L.
9. method according to claim 6, is characterized in that, after stream adds urea soln, cultivates 54 hours.
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