CN101270378B - Method for preparing miglitol midbody N-substituted-1-deoxidization nojirimycin derivative - Google Patents
Method for preparing miglitol midbody N-substituted-1-deoxidization nojirimycin derivative Download PDFInfo
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Abstract
The invention relates to a preparation method of miglitol intermediate, in particular to a preparation method of a derivative of N-substitution-1-deoxynojirimycin which is the key intermediate of miglitol. Gluoconobacter oxydans or the Gluoconobacter oxydans treated by immobilized cell technology are used repeatedly for transforming the substrate to the derivative of N-substitution-1-deoxynojirimycin which is the miglitol intermediate, the transfer temperature is controlled at 5 to 25 DEG C, pH value is controlled at 4.0 to 6.5 and dissolved oxygen is controlled at 5 to 80 percent (in volume). Adopting the invention can decrease the biotransformation cost, reduce the inter-assay of cell, and be suitable for the large scale industrial production, increase the mechanical strength of the wall of the strain prepared, make the strain not to dissolve during the cultivation in long-term liquid environment, lengthen the service life and have outstanding slow release performance.
Description
Technical field
The present invention relates to a kind of preparation method of miglitol intermediate, especially, the preparation method who relates to miglitol key intermediate N replacement-1-DNJ derivative, specifically, adopt time to prepare the method for miglitol intermediate 6-(2-hydroxyethyl)-amino-6-deoxidation-α-L-sorb furanose with Gluconobacter oxydans or immobilized Gluconobacter oxydans.
Background technology
Miglitol can effectively be treated type ii diabetes as a kind of Novel alpha-alpha-glucosidase inhibitors, and its function mainly is to reduce patient's level of postprandial blood sugar, reduces the generation of diabetic complication, and becomes rapidly choice drug.
Preparation method as the synthetic important intermediate N-replacement-1-DNJ derivative of miglitol mainly contains following several at present: the people (KinastG such as (1) Kinast; Schedel M.USP4 246 345. (1981)) utilized the method for microbial transformation in 1981; successfully obtained N-replacement-1-DNJ derivative; before carrying out bio-transformation, to need to introduce protective material as shown in Equation 1; and needing a large amount of catalyzer during hydrogenation, these have increased cost and the difficulty of technique widely.(2) people (KinastG, Schedel M.USP4 405 714. (1983)) such as Kinast has carried out improving (seeing formula 2) on the original basis, but still needs to add protective material in synthetic.(3) people (GtabnerRW such as Gtabner, Landis BH.Wang PT et al.EP0 477 160. (1991) Gtabner RW, Landis BH.Wang PT USP2001/0019837 (2001)) invented another kind of more easy biosynthetic means (seeing formula 3).
The synthetic method one of formula 1N-replacement-1-DNJ
(R-substituting group P-protecting group)
The synthetic method two of formula 2N-replacement-1-DNJ
The synthetic method three of formula 3N-replacement-1-DNJ
The method has lot of advantages:
(1) conversion fluid need not separation and purification and goes out intermediate through the centrifugal thalline (being microorganism) of removing, and just can carry out next step and synthesize.
(2) need not radical protection, cost is reduced greatly, and avoid descending because removing the rate of recovery that blocking group causes.
(3) intermediate 6-(substituted amido)-6-deoxidation-o-L-furans sorbose has higher solubleness and stability, is difficult for being degraded.
But the method yeast culture cost and bio-transformation cost are higher, collect thalline with centrifugal method, and the thalline loss is large, can't adapt to industrialized production.
Immobilized cell technique is to utilize physics or chemical means with free microorganism cells or enzyme, the area of space that is positioned to limit, and make it keep a technology active and that can recycle.Initial people are used for this technology to produce acetic acid in the filter reactive system.Afterwards, people were used for food, medicine, fermentation industry with immobilized cell technique.In the last few years, in field of waste water treatment, this technology had also obtained good development, demonstrated its unique superiority.Developed country adopts immobilized cell technique to make microbial inoculant inoculation soil in the eighties in 20th century, and demonstrates good application prospect.The domestic research that also has the scholar to be devoted to this field has obtained preferably achievement equally.In US Patent No. 5602013, once mentioned and to adopt immobilized cell technique, but used carrier is uncommon, and be prone to the phenomenon that carrier breaks and cell leaks.
Entrapping method is rapidly one of emerging immobilized cell technique of development in recent years.That this method has is simple to operate, on the less and efficient high of cytoactive impact, be present immobilized cell technique research and most widely used method.Using embedding immobilization cell carrier commonly used has agar, gelatin, sodium alginate (SA), polyvinyl alcohol (PVA) and polyacrylamide (PACRM) etc., wherein sodium alginate (SA) is common using embedding immobilization cell carrier, it is few and prepare simple advantage that it has loss of enzyme activity, but different system especially phosphate system is easily gone out the phenomenon that expression vector breaks and cell leaks.
Summary of the invention
The objective of the invention is to overcome in the present prior art yeast culture cost and bio-transformation cost higher, difference is large between thalline batch, the deficiency that can't adapt to industrialized production, a kind of method that adopts time to prepare miglitol key intermediate N replacement-1-DNJ derivative with Gluconobacter oxydans or immobilized Gluconobacter oxydans is provided, to reduce the bio-transformation production cost, reduce the thalline differences between batches, be fit to industrial mass production.
The method of the Gluconobacter oxydans that time of the present invention is processed with Gluconobacter oxydans or through immobilized cell technique, that conversion of substrate is transformed preparation miglitol key intermediate N replacement-1-DNJ derivative [6-(2-hydroxyethyl)-amino-6-deoxidation-α-L-sorb furanose], described conversion of substrate is the glucose of amination, the glucose of described amination is N-(2-hydroxyethyl)-glycosamine, and described method comprises the steps:
The first step: with the conversion of substrate water dissolution, making the conversion of substrate ultimate density is 2%～10% (weight), and regulating the pH value is 4.0～6.5, adds 0.3%～0.6% (weight) MgSO
4Add bacterium liquid or immobilization Gluconobacter oxydans, the weight ratio of thalline is 1:1～3 in conversion of substrate and bacterium liquid or the immobilization Gluconobacter oxydans, the control invert point is 5 ℃～25 ℃, it is 4.0～6.5 that control transforms the pH value, and the control oxyty is in 5%～80% (relative saturation volumetric concentration);
Second step: with used in the first step once bacterium liquid or the immobilization Gluconobacter oxydans with the centrifugal thalline that obtains, or remove conversion fluid, water or 0.3%～0.6% (weight) MgSO with membrane filtration
4The solution top is washed till total reducing sugar≤1.0% (weight), amino nitrogen≤50mg/100ml, and omnidistance control temperature is at 5 ℃～30 ℃, and pressure is at 0.01MPa～1MPa, and the pH value obtains bacterium liquid 5.0～7.0.
Wherein, repeat the described the first step and apply mechanically conversion production, the add-on of whenever applying mechanically a conversion of substrate reduces by 5% (weight), and recirculation is applied mechanically 2～15 times.
Wherein, the method for preparing described bacterium liquid comprises the steps: to get the Gluconobacter oxydans fermented liquid that is cultured to logarithmic phase or stationary phase, removes fermented liquid with membrane filtration, water or 0.3～0.6% (weight) MgSO
4The solution top is washed till total reducing sugar≤1.0% (weight), amino nitrogen≤50mg/100ml, and omnidistance control temperature is at 10 ℃～30 ℃, and pressure is at 0～0.06MPa, and the pH value obtains bacterium liquid 5.0～7.0.
Wherein, prepare described immobilization Gluconobacter oxydans and undertaken by the immobilized cell technique facture, its concrete steps are as follows:
1) preparation of the mixed solution of embedding medium and bacterium liquid: prepare first weight percent concentration and be 2%～6% embedding medium solution, be cooled to 30～50 ℃ through 120 ± 1 ℃ of sterilizations, again by bacterium liquid: 2%～6% embedding medium solution=1:2～6 (weight ratio) are mixed with the mixed solution of embedding medium and bacterium liquid;
2) preparation of linking agent: 1. be 5%～10% cross-linking agent solution CaCl with weight percent concentration
2Solution, regulating the pH value is 4.0～6.0, sterilizes stand-by; 2. be 0.5%～1% cross-linking agent solution with weight percent concentration, chitosan solution, and 5% CaCl
2Solution, regulating the pH value is 5.0～6.0, sterilizes stand-by; 3. weight percent concentration is 0.5%～1% cross-linking agent solution, and glutaraldehyde solution is sterilized stand-by;
3) preparation of immobilization Gluconobacter oxydans: with the mixed solution pump delivery of embedding medium and bacterium liquid, water dropper by 0.5mm～1.0mm, splash among the cross-linking agent solution, and stir, regulate transfer rate and stirring velocity with drip as quickly as possible dual intensity Cheng Zhu be advisable, with the immobilization Gluconobacter oxydans that makes after in linking agent, hardening under 4～5 ℃ 2～10 hours with aseptic washing 3～4 times, again the immobilization Gluconobacter oxydans is soaked in 0.5%～1% chitosan and 5% CaCl
2In the purification of aqueous solutions 1～2 hour, with immobilization Gluconobacter oxydans with 0.5%～1% glutaraldehyde purification of aqueous solutions crosslinked 5～10 minutes, prepare the immobilization Gluconobacter oxydans at last.
Wherein, described conversion of substrate is the glucose of amination, and the glucose of described amination is N-(2-hydroxyethyl)-glycosamine, and the automatic or manual that is controlled to be of invert point and pH value is controlled.Described film is inorganic micro filtering or ultra-filtration membrane or organic micro-filtration or ultra-filtration membrane, and its aperture is 0.03 μ m～1.0 μ m.
Wherein, described water is tap water or deionized water or purified water.
Wherein, described total sugar content is≤1.0% (weight), and described amino nitrogen content is≤50mg/100ml.
Wherein, the solvent of described embedding medium solution is purified water; The solvent of described cross-linking agent solution is purified water.
Wherein, described bacterium liquid be preserved in≤20 ℃ of environment in 120 days with interior for subsequent use.
Wherein, described immobilization Gluconobacter oxydans is pearl type immobilized cell particle.
Wherein, described fixedly Gluconobacter oxydans be preserved in≤20 ℃ of environment in 120 days with interior for subsequent use.
Wherein, described embedding medium is natural macromolecule amylose class and synthetic macromolecular compound.Described natural macromolecule amylose class comprises agar, alginates, carrageenin, gelatin; Described synthetic macromolecular compound comprises polyacrylamide, polyvinyl alcohol, light-hardening resin; Described alginates comprises alginate calcium, sodium alginate.Described linking agent comprises chitosan, CaCl
2, glutaraldehyde; Can also be borax, boric acid.
The present invention has following advantage:
1, be that 0.03 μ m～inorganic, organic micro-filtration of 1.0 μ m or the loss of ultra-filtration membrane collection thalline are little with the aperture, treatment capacity is large, and the treatment time is short.
2, can decline to a great extent with transforming production cost by time, transform cost and can descend 5/6 at most, reduce again the generation of fermented waste fluid and waste gas simultaneously, reduce environmental pollution.
3, the bacterial strain wall physical strength that makes is high, and long-term liquid environment is cultivated, and dissolution phenomena does not occur bacterial strain.
4, the bacterial strain long service life that makes, and have excellent sustained release performance.
Embodiment
Embodiment 1
A, bacterium solution preparation: getting and be cultured to the logarithmic growth oxidizing glucose fermented liquid 10000L in latter stage, is the microfiltration of ceramic membrane filtration removal fermented liquid of 0.2 μ m with the aperture, uses 0.3%MgSO
4It is 0.065% that the solution top is washed till total reducing sugar, amino nitrogen 2.8mg/100ml, and whole process is controlled temperature at 25 ± 1 ℃, pressure 0.03MPa, pH5.0 obtains bacterium liquid 100L.
B, conversion are produced: drop into conversion of substrate N-(2-hydroxyethyl)-glycosamine 100kg and dissolve with purified water in the 5000L fermentor tank, making the conversion of substrate ultimate density is 10%, and regulating the pH value is 6.0, adds 0.32%MgSO
4Add bacterium liquid, so that conversion of substrate and thalline weight ratio are 1: 1, automatically the control invert point is 24 ± 1 ℃, automatically control conversion pH value is 6.0, and dissolved oxygen is controlled at 5%, when transforming 24h, adopt tlc (TLC) and carbonyl color reaction method to the detection of converted product, transformation efficiency reaches 99%.
C, repeat to apply mechanically production: the B step being used 1 time bacterium liquid is that the microfiltration of ceramic membrane of 0.2 μ m filters and removes conversion fluid with the aperture, uses 0.3%MgSO
4It is 0.065% that the solution top is washed till total reducing sugar, amino nitrogen 2.8mg/100ml, and whole process is controlled temperature at 20 ± 1 ℃, pressure 0.03MPa, pH value=5.0.Press the B step and transform production, whenever apply mechanically a N-(2-hydroxyethyl)-glycosamine dosage and reduce 5%, so recirculation is applied mechanically 6 times.Transformation efficiency sees the following form 1:
Table 1
| Apply mechanically number of times | 1 | 2 | 3 | 4 | 5 | 6 |
| Transformation efficiency (%) | 99 | 99 | 98 | 97 | 95 | 93 |
Embodiment 2
A, bacterium solution preparation: getting and be cultured to the logarithmic growth oxidizing glucose fermented liquid 10000L in latter stage, is the microfiltration of ceramic membrane filtration removal fermented liquid of 0.05 μ m with the aperture, uses 0.6%MgSO
4The solution top is washed till total reducing sugar 0.134%, amino nitrogen 5.6mg/100ml, and whole process is controlled temperature at 20 ℃, pressure 0.04MPa, pH7.0 obtains bacterium liquid 100L.
B, conversion are produced: drop into conversion of substrate N-(2-hydroxyethyl)-glycosamine 100kg and dissolve with purified water in the 5000L fermentor tank, making the conversion of substrate ultimate density is 10%, and regulating the pH value is 6.5, adds 0.6%MgSO
4Add bacterium liquid, conversion of substrate and thalline weight ratio are 1: 3, manually the control invert point is 6 ± 1 ℃, manually control conversion pH value is 6.5, and dissolved oxygen is controlled at 80%, when transforming 34h, adopt tlc (TLC) and carbonyl color reaction method to the detection of converted product, transformation efficiency reaches 99%.
C, repeat to apply mechanically production: the B step being used 1 time bacterium liquid is that the microfiltration of ceramic membrane of 0.05 μ m filters and removes conversion fluid with the aperture, uses 0.6%MgSO
4It is 0.134% that the solution top is washed till total reducing sugar, and amino nitrogen is 5.6mg/100ml, and whole process is controlled temperature at 10 ℃, pressure 0.04MPa, pH7.0.Press the B step and transform production, whenever apply mechanically a conversion of substrate dosage and reduce 5%, so recirculation is applied mechanically 6 times.Transformation efficiency sees the following form 2:
Table 2
| Apply mechanically number of times | 1 | 2 | 3 | 4 | 5 | 6 |
| Transformation efficiency (%) | 99 | 99 | 98 | 97 | 95 | 93 |
Embodiment 3
A, bacterium solution preparation: get and be cultured to the logarithmic growth oxidizing glucose fermented liquid 10000L in latter stage, be that the tetrafluoroethylene membrane filtration of 1.0 μ m is removed fermented liquid with the aperture, being washed till total reducing sugar with the deionized water top is 0.98%, amino nitrogen is 47.6mg/100ml, omnidistance control temperature is at 15 ℃, pressure 0.03Mpa, pH6.0 obtains bacterium liquid 100L.
B, conversion are produced: drop into conversion of substrate N-(2-hydroxyethyl)-glycosamine 100kg and dissolve with purified water in the 5000L fermentor tank, making the conversion of substrate ultimate density is 10%, and regulating the pH value is 4.0, adds 0.32%MgSO
4Add bacterium liquid, conversion of substrate and thalline weight ratio are 1: 2, automatically the control invert point is 10 ± 1 ℃, automatically control conversion pH value is 4.0, and dissolved oxygen is controlled at 45%, when transforming 24h, adopt tlc (TLC) and carbonyl color reaction method to the detection of converted product, transformation efficiency reaches 99%.
C, repeat to apply mechanically production: the B step being used 1 time bacterium liquid is that the tetrafluoroethylene membrane microfiltration of 1.0 μ m filters and removes conversion fluid with the aperture, be washed till total reducing sugar 0.98% with the purified water top, amino nitrogen 47.6mg/100ml, omnidistance control temperature is at 6 ℃, pressure 0.1MPa, pH6.0.Press the B step and transform production, whenever apply mechanically a conversion of substrate dosage and reduce 5%, so recirculation is applied mechanically 6 times.Transformation efficiency sees the following form 3:
Table 3
| Apply mechanically number of times | 1 | 2 | 3 | 4 | 5 | 6 |
| Transformation efficiency (%) | 99 | 99 | 98 | 97 | 95 | 93 |
Embodiment 4
A, bacterium solution preparation: getting and be cultured to the logarithmic growth oxidizing glucose fermented liquid 10000L in latter stage, is the microfiltration of ceramic membrane filtration removal fermented liquid of 0.2 μ m with the aperture, uses 0.6%MgSO
4The solution top is washed till total reducing sugar 0.59%, amino nitrogen 25.2mg/100ml, and whole process is controlled temperature at 25 ℃, pressure 0.03MPa, pH5.0 obtains bacterium liquid 100L.
The preparation of the mixed solution of B, embedding medium and bacterium liquid: in the 1000L tank, prepare first weight percent concentration and be 5% sodium alginate soln 400L, be cooled to 30 ℃ through 120 ℃ of sterilizations, open stirrer, add bacterium liquid so that bacterium liquid: 5% sodium alginate=1: 4 (weight ratio) is mixed with the mixed solution 500L of embedding medium and bacterium liquid, and solvent for use is purified water.
The preparation of C, linking agent: 1, the preparation weight percent concentration is 6% CaCl
2Purification of aqueous solutions 1000L, regulating the pH value is 6.0, sterilizes stand-by.2, the preparation weight percent concentration is 0.6% chitosan and 5% CaCl
2Purification of aqueous solutions 1000L, regulating the pH value is 6.0, sterilizes stand-by.3, the preparation weight percent concentration is 0.5% glutaraldehyde purification of aqueous solutions 1000L.
The preparation of D, pearl type immobilized cell particle: with the mixed solution pump delivery of above-mentioned embedding medium and bacterium liquid, by the water dropper of 1.0mm, splash into weight percent concentration and be 6% CaCl
2Among the purification of aqueous solutions, and stir with magnetic stirrer, regulate transfer rate and stirring velocity with drip as quickly as possible dual intensity Cheng Zhu be advisable, the immobilization Gluconobacter oxydans strain for preparing under 4 ℃ at the CaCl of linking agent 6%
2Sclerosis was filtered after 10 hours in the purification of aqueous solutions, with aseptic washing 3 times, the strain of immobilization Gluconobacter oxydans was soaked in 0.6% chitosan and 5% CaCl again
2In the purification of aqueous solutions 2 hours, at last with immobilization Gluconobacter oxydans strain with 0.5% glutaraldehyde purification of aqueous solutions crosslinked 10 minutes.Filter stand-by.
E, conversion are produced: drop into conversion of substrate N-(2-hydroxyethyl)-glycosamine 100kg and dissolve with purified water in the 5000L fermentor tank, making the conversion of substrate ultimate density is 10%, and regulating the pH value is 6.3, adds 0.32%MgSO
4Add fixedly Gluconobacter oxydans bacterial strain, conversion of substrate and thalline weight ratio are 1: 1, automatically the control invert point is 24 ± 1 ℃, automatically control conversion pH value is 6.5, and dissolved oxygen is controlled at 50%, when transforming 48h, adopt tlc (TLC) and carbonyl color reaction method to the detection of converted product, transformation efficiency reaches 99.5%.
F, repeat to apply mechanically production: the E step being used 1 time fixedly Gluconobacter oxydans bacterial strain is that the microfiltration of ceramic membrane of 0.2 μ m filters and removes conversion fluid with the aperture, uses 0.6%MgSO
4The solution top is washed till total reducing sugar 0.59%, amino nitrogen 25.2mg/100ml, and whole process is controlled temperature at 22 ℃, pressure 0.04MPa, pH7.0.Press the E step and transform production, whenever apply mechanically a conversion of substrate dosage and reduce 5%, so recirculation is applied mechanically 15 times.Transformation efficiency sees the following form 4:
Table 4
| Apply mechanically number of times | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 |
| Transformation efficiency (%) | 99.5 | 99.5 | 99.5 | 99 | 99 | 99 | 97 | 97 | 95 | 95 | 93 | 93 | 93 | 91 | 91 |
Embodiment 5
A, bacterium solution preparation: getting and be cultured to the logarithmic growth oxidizing glucose fermented liquid 10000L in latter stage, is the polysulfone membrane filtration removal fermented liquid of 0.22 μ m with the aperture, uses 0.6%MgSO
4The solution top is washed till total reducing sugar 0.26%, amino nitrogen 16.8mg/100ml, and whole process is controlled temperature at 25 ℃, pressure 0.04MPa, pH7.0 obtains bacterium liquid 100L.
The preparation of the mixed solution of B, embedding medium and bacterium liquid: in the 1000L tank, prepare first weight percent concentration and be 2% sodium alginate soln 400L, be cooled to 30 ℃ through 120 ℃ of sterilizations, open stirrer, add bacterium liquid so that bacterium liquid: 2% sodium alginate=1: 6 (weight ratio) is mixed with the mixed solution 500L of embedding medium and bacterium liquid, and solvent for use is purified water.
The preparation of C, linking agent: 1, the preparation weight percent concentration is 5% CaCl
2Purification of aqueous solutions 1000L, regulating the pH value is 4.0, sterilizes stand-by.2, the preparation weight percent concentration is 0.8% chitosan and 5% CaCl
2Purification of aqueous solutions 1000L, regulating the pH value is 5.0, sterilizes stand-by.3, the preparation weight percent concentration is 1% glutaraldehyde purification of aqueous solutions 1000L.
The preparation of D, pearl type immobilized cell particle: with the mixed solution pump delivery of embedding medium and bacterium liquid, by the water dropper of 1.0mm, splash into weight percent concentration and be 5% CaCl
2Among the purification of aqueous solutions, and stir with magnetic stirrer, regulate transfer rate and stirring velocity with drip as quickly as possible dual intensity Cheng Zhu be advisable, the immobilization Gluconobacter oxydans strain for preparing under 4 ℃ is 5% CaCl in the linking agent weight percent concentration
2Sclerosis was filtered after 5 hours in the purification of aqueous solutions, with aseptic washing 3 times, the strain of immobilization Gluconobacter oxydans was soaked in 1% chitosan and 5% CaCl again
2In the purification of aqueous solutions 1 hour, at last with immobilization Gluconobacter oxydans strain with 1% glutaraldehyde purification of aqueous solutions crosslinked 5 minutes.Filter stand-by.
E, conversion are produced: drop into conversion of substrate N-(2-hydroxyethyl)-glycosamine 100kg deionized water dissolving in the 5000L fermentor tank, making the conversion of substrate ultimate density is 2%, and regulating the pH value is 6.5, adds 0.6%MgSO
4Add fixedly Gluconobacter oxydans bacterial strain, conversion of substrate and thalline weight ratio are 1: 3, manually the control invert point is 6 ± 1 ℃, manually control conversion pH value is 6.5, and dissolved oxygen is controlled at 50%, when transforming 48h, adopt tlc (TLC) and carbonyl color reaction method to the detection of converted product, transformation efficiency reaches 99.5%.
F, repeat to apply mechanically production: the E step being used 1 time fixedly Gluconobacter oxydans bacterial strain is that the polysulfone membrane micro-filtration of 0.22 μ m filters and removes conversion fluid with the aperture, uses 0.6%MgSO
4The solution top is washed till total reducing sugar 0.26%, amino nitrogen 16.8mg/100ml, and whole process is controlled temperature at 25 ℃, pressure 0.04MPa, pH7.0.Press the E step and transform production, whenever apply mechanically a conversion of substrate dosage and reduce 5%, so recirculation is applied mechanically 10 times.Transformation efficiency sees the following form 5:
Table 5
| Apply mechanically number of times | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Transformation efficiency (%) | 99.5 | 99.5 | 99.5 | 99 | 99 | 98 | 97 | 97 | 95 | 95 |
Embodiment 6
A, bacterium solution preparation: getting and be cultured to the logarithmic growth oxidizing glucose fermented liquid 10000L in latter stage, is the microfiltration of ceramic membrane filtration removal fermented liquid of 0.45 μ m with the aperture, uses 0.5%MgSO
4The solution top is washed till total reducing sugar 0.65%, amino nitrogen 36.4mg/100ml, and whole process is controlled temperature at 15 ℃, pressure 0MPa, pH6.0 obtains bacterium liquid 100L.
The preparation of the mixed solution of B, embedding medium and bacterium liquid: in the 1000L tank, prepare first weight percent concentration and be 6% sodium alginate soln 400L, be cooled to 30 ℃ through 120 ℃ of sterilizations, open stirrer, add bacterium liquid so that bacterium liquid: 6% sodium alginate=1: 2 (weight ratio) is mixed with the mixed solution 500L of embedding medium and bacterium liquid, and solvent for use is purified water.
The preparation of C, linking agent: 1, the preparation weight percent concentration is 10% CaCl
2Purification of aqueous solutions 1000L, regulating the pH value is 5.0, sterilizes stand-by.2, the preparation weight percent concentration is 0.5% chitosan and 5% CaCl
2Purification of aqueous solutions 1000L, regulating the pH value is 6.0, sterilizes stand-by.3, the preparation weight percent concentration is 0.8% glutaraldehyde purification of aqueous solutions 1000L.
The preparation of D, pearl type immobilized cell particle: with the mixed solution pump delivery of embedding medium and bacterium liquid, by the water dropper of 0.8mm, splash into weight percent concentration and be 10% CaCl
2Among the purification of aqueous solutions, and stir with magnetic stirrer, regulate transfer rate and stirring velocity with drip as quickly as possible dual intensity Cheng Zhu be advisable, the immobilization Gluconobacter oxydans strain for preparing under 4 ℃ is 10% CaCl in the linking agent weight percent concentration
2Sclerosis was filtered after 6 hours in the purification of aqueous solutions, with aseptic washing 4 times, the strain of immobilization Gluconobacter oxydans was soaked in 0.8% chitosan and 5% CaCl again
2In the purification of aqueous solutions 2 hours, at last with immobilization Gluconobacter oxydans strain with 0.6% glutaraldehyde purification of aqueous solutions crosslinked 8 minutes.Filter stand-by.
E, conversion are produced: drop into conversion of substrate N-(2-hydroxyethyl)-glycosamine 100kg and dissolve with tap water in the 5000L fermentor tank, making the conversion of substrate ultimate density is 5%, and regulating the pH value is 6.0, adds 0.4%MgSO
4Add fixedly Gluconobacter oxydans bacterial strain, so that conversion of substrate and thalline weight ratio are 1: 2, automatically the control invert point is 10 ± 1 ℃, automatically control conversion pH value is 6.0, and dissolved oxygen is controlled at 80%, when transforming 48h, adopt tlc (TLC) and carbonyl color reaction method to the detection of converted product, transformation efficiency reaches 99.5%.
F, repeat to apply mechanically production: the E step being used 1 time fixedly Gluconobacter oxydans bacterial strain is that the ceramic membrane filter of 0.45 μ m is removed conversion fluid with the aperture, uses 0.4%MgSO
4The solution top is washed till total reducing sugar 0.65%, amino nitrogen 36.4mg/100ml, and whole process is controlled temperature at 10 ℃, pressure 0.08MPa, pH6.0.Press the E step and transform production, whenever apply mechanically a conversion of substrate dosage and reduce 5%, so recirculation is applied mechanically 15 times.Transformation efficiency sees the following form 6:
Table 6
| Apply mechanically number of times | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 |
| Transformation efficiency (%) | 99.5 | 99.5 | 99.5 | 99 | 99 | 99 | 97 | 97 | 95 | 95 | 93 | 93 | 93 | 91 | 91 |
Embodiment 7
A, bacterium solution preparation: get and be cultured to the logarithmic growth oxidizing glucose fermented liquid 50ml in latter stage, the centrifugal thalline 2.5g that obtains.
B, immobilized cell preparation: the preparation weight percent concentration is 3% agar-agar soln 100ml, be cooled to 50 ℃ through 120 ℃ of sterilizations, add thalline 2.5g, agar-agar soln and thalline mixed pour in the sterilized culture dish, make it cooled and solidified 2h, the dice that gel is cut into 3mm * 3mm * 3mm is for subsequent use, and solvent for use is purified water.
C, shaking flask transform: get conversion of substrate N-(2-hydroxyethyl)-glycosamine 1.25g and dissolve with tap water, making the substrate ultimate density is 5%, and regulating the pH value is 6.0, in the 250ml shaking flask of packing into, adds 0.4%MgSO
4, adding fixedly Gluconobacter oxydans bacterial strain, conversion of substrate and thalline weight ratio are 1: 2, shaking speed 220rpm, automatically controlling invert point is 15 ± 1 ℃, when transforming 58h, adopt tlc (TLC) and carbonyl color reaction method to the detection of converted product, transformation efficiency reaches 98.5%.
Embodiment 8
A, bacterium solution preparation: get and be cultured to the logarithmic growth oxidizing glucose fermented liquid 50ml in latter stage, the centrifugal thalline 2.5g that obtains.
B, immobilized cell preparation: the preparation weight percent concentration is 10% gelatin solution 100ml, be cooled to 50 ℃ through 120 ℃ of sterilizations, add thalline 2.5g, gelatin solution and thalline mixed pour in the sterilized culture dish, make it cooled and solidified 2h, gel is cut into the dice of 3mm * 3mm * 3mm, soak 4h with 0.5% glutaraldehyde solution, for subsequent use, solvent for use is purified water.
C, shaking flask transform: get conversion of substrate N-(2-hydroxyethyl)-glycosamine 1.25g and dissolve with tap water, making the conversion of substrate ultimate density is 5%, and regulating the pH value is 6.0, in the 250ml shaking flask of packing into, adds 0.4%MgSO
4, adding fixedly Gluconobacter oxydans bacterial strain, substrate and thalline weight ratio are 1: 2, shaking speed 220rpm, automatically controlling invert point is 15 ± 1 ℃, when transforming 58h, adopt tlc (TLC) and carbonyl color reaction method to the detection of converted product, transformation efficiency reaches 97.5%.
Embodiment 9
A, bacterium solution preparation: get and be cultured to the logarithmic growth oxidizing glucose fermented liquid 50ml in latter stage, the centrifugal thalline 2.5g that obtains.
B, immobilized cell preparation: the preparation weight percent concentration is 10% PVA (polyvinyl alcohol) solution 100ml, be cooled to 50 ℃ through 120 ℃ of sterilizations, add thalline 2.5g, PVA solution and thalline are mixed, pour in the sterilized culture dish, slowly add saturated boric acid solution, soak 10h, gel is cut into the dice of 3mm * 3mm * 3mm, for subsequent use, solvent for use is purified water.
C, shaking flask transform: get conversion of substrate N-(2-hydroxyethyl)-glycosamine 1.25g and dissolve with tap water, making the conversion of substrate ultimate density is 5%, and regulating the pH value is 6.0, in the 250ml shaking flask of packing into, adds 0.4%MgSO
4, adding fixedly Gluconobacter oxydans bacterial strain, substrate and thalline weight ratio are 1: 2, shaking speed 220rpm, automatically controlling invert point is 15 ± 1 ℃, when transforming 48h, adopt tlc (TLC) and carbonyl color reaction method to the detection of converted product, transformation efficiency reaches 98.5%.
Claims (15)
1. the method for the intermediate 6-(2-hydroxyethyl) for preparing miglitol-amino-6-deoxidation-α-L-sorb furanose, described method comprises the steps:
The first step: with the conversion of substrate water dissolution, making the conversion of substrate ultimate density is 2 % by weight～10 % by weight, and regulating the pH value is 4.0～6.5, adds 0.3 % by weight～0.6 % by weight MgSO
4, adding bacterium liquid, the weight ratio of thalline is 1: 1～3 in conversion of substrate and the bacterium liquid, and the control invert point is 5 ℃～25 ℃, and it is 4.0～6.5 that control transforms the pH value, and the control oxyty is at 5 volume %～80 volume %;
Wherein, described conversion of substrate is N-(2-hydroxyethyl)-glycosamine;
The method for preparing described bacterium liquid comprises the steps: to get the Gluconobacter oxydans fermented liquid that is cultured to logarithmic phase or stationary phase, removes fermented liquid with membrane filtration, water or 0.3 % by weight～0.6 % by weight MgSO
4The solution top is washed till total reducing sugar≤1.0 % by weight, amino nitrogen≤50mg/100ml, and omnidistance control temperature is at 10 ℃～30 ℃, and pressure is at 0～0.06MPa, and the pH value obtains bacterium liquid 5.0～7.0;
Second step: will use once bacterium liquid with the centrifugal thalline that obtains in the first step, or remove conversion fluid, water or 0.3 % by weight～0.6 % by weight MgSO with membrane filtration
4The solution item is washed till total reducing sugar≤1.0 % by weight, amino nitrogen≤50mg/100ml, and omnidistance control temperature is at 5 ℃～30 ℃, and pressure is at 0.01Mpa～1MPa, and the pH value obtains bacterium liquid 5.0～7.0;
The 3rd step: the bacterium liquid that second step is obtained repeats the described the first step and applies mechanically to transform and produce, and the add-on of whenever applying mechanically a conversion of substrate reduces by 5% weight.
2. method according to claim 1 is characterized in that: produce applying mechanically to transform, recirculation is applied mechanically 2～15 times.
3. method according to claim 1, it is characterized in that: described film is inorganic micro filtering membrane or ultra-filtration membrane or organic microfiltration membrane or ultra-filtration membrane, its aperture is 0.03 μ m～1.0 μ m.
4. method according to claim 1, it is characterized in that: described water is tap water or deionized water or purified water.
5. method according to claim 1 is characterized in that: described bacterium liquid is preserved in≤20 ℃ of environment in 120 days with interior for subsequent use.
6. the method for the intermediate 6-(2-hydroxyethyl) for preparing miglitol-amino-6-deoxidation-α-L-sorb furanose, described method comprises the steps:
The first step: with the conversion of substrate water dissolution, making the conversion of substrate ultimate density is 2 % by weight～10 % by weight, and regulating the pH value is 4.0～6.5, adds 0.3 % by weight～0.6 % by weight MgSO
4Add the immobilization Gluconobacter oxydans, the weight ratio of thalline is 1: 1～3 in conversion of substrate and the immobilization Gluconobacter oxydans, and the control invert point is 5 ℃～25 ℃, it is 4.0～6.5 that control transforms the pH value, and the control oxyty is at 5 volume %～80 volume %;
Wherein, described conversion of substrate is N-(2-hydroxyethyl)-glycosamine;
The preparation method who is used for preparing the bacterium liquid of described immobilization Gluconobacter oxydans comprises the steps: to get the Gluconobacter oxydans fermented liquid that is cultured to logarithmic phase or stationary phase, remove fermented liquid with membrane filtration, water or 0.3 % by weight～0.6 % by weight MgSO
4The solution top is washed till total reducing sugar≤1.0 % by weight, amino nitrogen≤50mg/100ml, and omnidistance control temperature is at 10 ℃～30 ℃, and pressure is at 0～0.06MPa, and the pH value obtains bacterium liquid 5.0～7.0;
Prepare described immobilization Gluconobacter oxydans and undertaken by the immobilized cell technique facture, its concrete steps are as follows:
1) preparation of the mixed solution of embedding medium and bacterium liquid: prepare first weight percent concentration and be 2%～6% embedding medium solution, be cooled to 30～50 ℃ through 120 ± 1 ℃ of sterilizations, again by bacterium liquid: 2%～6% embedding medium solution=weight ratio of 1: 2～6 is mixed with the mixed solution of embedding medium and bacterium liquid;
2) preparation of linking agent: 1. be 5%～10% CaCl with weight percent concentration
2Solution, regulating the pH value is 4.0～6.0, sterilizes stand-by; 2. be 0.5%～1% chitosan solution with weight percent concentration, and weight percent concentration is 5% CaCl
2Solution, regulating the pH value is 5.0～6.0, sterilizes stand-by; 3. weight percent concentration is 0.5%～1% glutaraldehyde solution, sterilizes stand-by;
3) cross-linking agent solution the preparation of immobilization Gluconobacter oxydans: carry the mixed solution of embedding medium and bacterium liquid, splash into step 2) 1. among and stirring; Regulating transfer rate and stirring velocity and can be advisable by Cheng Zhu fast with drip as far as possible, under 4～5 ℃ is 5%～10% CaCl in weight percent concentration with the immobilization Gluconobacter oxydans that makes
2With aseptic washing 3～4 times, again the immobilization Gluconobacter oxydans was soaked in weight percent concentration and is 0.5%～1% chitosan and weight percent concentration and be 5% CaCl after hardening in the solution 2～10 hours
2In the purification of aqueous solutions 1～2 hour is crosslinked 5～10 minutes of 0.5%～1% glutaraldehyde purification of aqueous solutions at last with immobilization Gluconobacter oxydans weight percent concentration, prepares the immobilization Gluconobacter oxydans;
Second step: will use once immobilization Gluconobacter oxydans with the centrifugal thalline that obtains in the first step, or remove conversion fluid, water or 0.3 % by weight～0.6 % by weight MgSO with membrane filtration
4The solution item is washed till total reducing sugar≤1.0 % by weight, amino nitrogen≤50mg/100ml, and omnidistance control temperature is at 5 ℃～30 ℃, and pressure is at 0.01Mpa～1MPa, and the pH value is 5.0～7.0, being fixed Gluconobacter oxydans bacterium liquid;
The 3rd step: the immobilization Gluconobacter oxydans bacterium liquid that second step is obtained repeats the described the first step and applies mechanically to transform and produce, and the add-on of whenever applying mechanically a conversion of substrate reduces by 5% weight.
7. method according to claim 6 is characterized in that: produce applying mechanically to transform, recirculation is applied mechanically 2～15 times.
8. method according to claim 6, it is characterized in that: described film is inorganic micro filtering membrane or ultra-filtration membrane or organic microfiltration membrane or ultra-filtration membrane, its aperture is 0.03 μ m～1.0 μ m.
9. method according to claim 6, it is characterized in that: described water is tap water or deionized water or purified water.
10. method according to claim 6, it is characterized in that: the solvent of described embedding medium solution is purified water; The solvent of described cross-linking agent solution is purified water.
11. method according to claim 6 is characterized in that: described bacterium liquid is preserved in≤20 ℃ of environment in 120 days with interior for subsequent use.
12. method according to claim 6 is characterized in that: described immobilization Gluconobacter oxydans is pearl type immobilized cell particle, it is preserved in≤20 ℃ of environment in 120 days with interior for subsequent use.
13. method according to claim 6 is characterized in that: described embedding medium is natural macromolecule amylose class or synthetic macromolecular compound.
14. method according to claim 13 is characterized in that: described natural macromolecule amylose class comprises agar, alginates, carrageenin, gelatin; Described synthetic macromolecular compound comprises polyacrylamide, polyvinyl alcohol, light-hardening resin; Wherein, described alginates comprises alginate calcium, sodium alginate.
15. method according to claim 6 is characterized in that: described linking agent comprises chitosan, CaCl
2, glutaraldehyde.
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