CN100478451C - Method for catalyzed synthesizing alpha arbutin from free cells or immobilized cells - Google Patents
Method for catalyzed synthesizing alpha arbutin from free cells or immobilized cells Download PDFInfo
- Publication number
- CN100478451C CN100478451C CNB2005100803646A CN200510080364A CN100478451C CN 100478451 C CN100478451 C CN 100478451C CN B2005100803646 A CNB2005100803646 A CN B2005100803646A CN 200510080364 A CN200510080364 A CN 200510080364A CN 100478451 C CN100478451 C CN 100478451C
- Authority
- CN
- China
- Prior art keywords
- arbutin
- alpha
- cell
- xanthomonas campestris
- immobilized
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
A process for catalytically synthesizing alpha-arbutoside from the free or immobilized cells of Xanthomonas campestris (CGMCC No.1243) includes such steps as preparing somatic cells, choosing reaction system, choosing immobilizing carrier, preparing immobilized cells, optimizing reaction condition, and extracting product. Said alpha-arbuto side can be used for beautifying skin.
Description
Technical field
The invention belongs to biological technical field, relate to the preparation method who utilizes xanthomonas campestris (Xanthomonas campestris) CGMCCNO.1243 free cell or immobilized cell catalyzed synthesizing alpha-arbutin.
Background technology
Alpha-arbutin has significant restraint of tyrosinase activity, reduces the deposition of tyrosine oxidase in skin, skin is had the effect of bleaching, anti-look change and removing beverage; Be internationally recognized efficient, the safe whitening agent promoted of doing one's utmost, be whiten, removing beverage, sun care preparations ideal add composition.
Fixation of microbial cell (immobilized cells) technology is an emerging biometric technology that grows up the sixties in 20th century, and in industries such as chemical industry, fermentative production, the energy, medicine, practical application effect is remarkable.It is to utilize the means of physics or chemistry with the area of space of free microorganism cellular localization in qualification, and makes it keep the active method of recycling.The fixation of microbial cell technology is compared with traditional suspended biological facture, and immobilized cell has the nectar degree that improves unit volume, and thalline easily reclaims, to the tolerance enhancing advantages such as (as pH value, temperature, organic solvent, toxic substances etc.) of environment.
At present, owing to utilize chemical synthesis process to be difficult to synthetic a-arbutin, and mainly be by being catalyzer with the α Glycosylase about the preparation method of a-arbutin, synthetic Resorcinol of catalysis and glucose are alpha-arbutin.Because the α Glycosylase costs an arm and a leg, and adopt method for preparing alpha-arbutin productive rate not high, this certainly will cause production cost can not to be in any more, has brought very big difficulty to actual applying.So alpha-arbutin is generally synthetic by microbial fermentation catalysis, but also there are following problems in this method:
One, utilize fermented liquid production alpha-arbutin product subsequent treatment process complexity, facility investment and process cost all bigger;
Two, utilize the cycle of fermented liquid production alpha-arbutin longer;
Three, utilize the output of fermented liquid production alpha-arbutin unit volume alpha-arbutin lower.
In order to overcome these problems, free cell or immobilized cell are best terms of settlement.The alginates gel is as a kind of cell fixation medium of widespread use, has that the immobilization temperature is low, intensity is high, chemical stability is good, nontoxic, embedding efficiency is high and advantage such as cheap.
Summary of the invention
The objective of the invention is to overcome the defective of prior art, utilize xanthomonas campestris free cell or immobilized cell to be repeatedly used, shorten the production cycle, improved the productive rate of alpha-arbutin, reduce the production cost of alpha-arbutin, make the scale operation alpha-arbutin become possibility.Wherein immobilized cell promptly adopts entrapping method that xanthomonas campestris CGMCC NO.1243 is carried out immobilization, and with this immobilized cell catalyzed synthesizing alpha-arbutin.This preparation method carries out immobilization by selecting suitable fixing condition for use to xanthomonas campestris CGMCC NO.1243, again with this immobilized cell as catalyzer, add reactant Resorcinol and sucrose, catalyzed synthesizing alpha-arbutin under optimum reaction condition.This method has fixation cell cytoactive height, and good stability is easy to recovery, regeneration and reusable advantage; Advantages such as its technological operation is simple, production cost is low, operational safety.
Overall technology design of the present invention is:
The method of free cell catalyzed synthesizing alpha-arbutin, this method is made up of following process steps:
A. the preparation of xanthomonas campestris (Xanthomonas campestris) CGMCC NO.1243 cell is comprising seed culture and fermentor cultivation;
B. free cell catalyzed synthesizing alpha-arbutin;
C. the recovery of free cell and being repeatedly used;
D. the separation and purification of free cell catalysis synthetic alpha-arbutin.
The method of immobilized cell catalyzed synthesizing alpha-arbutin, this method is made up of following process steps:
A. the preparation of xanthomonas campestris (Xanthomonas campestris) CGMCC NO.1243 cell is comprising seed culture and fermentor cultivation;
B. the preparation of immobilization xanthomonas campestris (Xanthomonas campestris) CGMCC NO.1243;
C. immobilized cell catalyzed synthesizing alpha-arbutin;
D. the recovery of immobilized cell and being repeatedly used;
E. the separation and purification of immobilized cell catalysis synthetic alpha-arbutin.
Concrete processing step of the present invention and processing condition are:
The preparation of xanthomonas campestris in step a and the steps A (Xanthomonas campestris) CGMCC NO.1243 cell:
Xanthomonas campestris (Xanthomonas campestris) the CGMCC NO.1243 bacterial strain that picking 1 ring inclined-plane is preserved joins in the liquid nutrient medium that volume is 50ml, and at 25-40 ℃, the 120r/min shaking table gets primary seed solution after cultivating 24-48h; Primary seed solution 10-15% is inoculated in the 500ml Erlenmeyer flask that contains the above-mentioned substratum of 200ml, at 25-40 ℃, the 140r/min shaking table is cultivated 24-48h and is got secondary seed solution again; With 10-15% secondary seed solution inoculation fermentation jar, fermented liquid is at 6000r/min, and 4 ℃ of centrifugal 15min get xanthomonas campestris (Xanthomonas campestris) CGMCC NO.1243 cell.
Seed liquor and fermentation tank culture medium are made up of following parts by weight of component:
60 parts of sucrose, (NH
4)
2SO
42 parts, 0.5 part in sal epsom, 2 parts in sodium-chlor, CaCl
22H
20.05 part of O, ZnCl
20.01 part, FeCl
24H
20.1 part of O, Na
2MoO
42H
20.05 part of O, 0.002 part of VITMAIN B1,0.005 part of vitamin B12,0.004 part of calcium pantothenate, 0.001 part in nicotinic acid, para-amino benzoic acid are received 1000 parts in 0.002 part and water, pH7.0;
The processing condition of fermentation are 10-15% for the seed liquor inoculum size, and the pH value is 7.0, and culture temperature is 25-40 ℃, and ventilation is 0.4-0.6vvm, and mixing speed is 120-160r/min, and fermentation time is 48-72h.
The reaction conditions of the synthetic alpha-arbutin of free cell catalysis is among the step b:
In the 10mmol/L phosphate buffer solution, cell concn is 100-200g/L, and reactant Resorcinol amount concentration is 45-70mmol/l, sucrose amount concentration is 100-200mmol/l, temperature of reaction is 30-55 ℃, and the reaction times is 36-72 hour, and mixing speed is 160-200 rev/min.Under this reaction conditions, the transformation efficiency that Resorcinol is converted into alpha-arbutin reaches 75-92% (calculating with Resorcinol, down together), contains alpha-arbutin 10-15g in every liter of reaction solution.
The cellifugal recovery in step c middle reaches is reused again:
After the free cell catalyzed synthesizing alpha-the arbutin reaction finishes, at 6000r/min, 4 ℃ of centrifugal 15min can reclaim free cell, the cell that reclaims is with 10mmol/L phosphate buffer solution washing 2-3 time, and input contains in the phosphate buffer solution of 10mmol/L of reactant Resorcinol and sucrose and reacts once more; Recirculation like this, realize repeatedly catalyzed synthesizing alpha-arbutin of xanthomonas campestris (Xanthomonas campestris) CGMCC NO.1243 cell, with this understanding, the thalline free cell is transforming 5-8 during the cycle, cell still keeps 75% vigor, thereby reduced fermentation cell and product subsequent disposal expense, saved production cost greatly.
The separation and purification of free cell catalysis synthetic alpha-arbutin comprises following four steps in the steps d;
1. the phosphate buffer solution after reaction being finished was removed thalline in centrifugal 15 minutes under 6000 rev/mins of conditions;
2. carrying out separation and purification through the 1. phosphate buffer solution of step by polar macroporous adsorbent resin column, polar macroporous adsorbent resin column is a macroporous adsorbent resin S-8 post;
3. through the 2. effluent liquid vacuum-drying of step macroporous adsorptive resins, can obtain purity and be the alpha-arbutin more than 98%, vacuum-drying vacuum tightness is 0.1Mpa, and temperature is 65 degree, and dried finished products is a white powder;
4. after water-soluble, carry out crystallization through the alpha-arbutin crude product of 3. step, promptly obtain purity and be more than 99.5% the pure product of alpha-arbutin, Tc is 4 degree.
Xanthomonas campestris among the step B (Xanthomonas campestris) CGMCC NO.1243 cell fixation:
With mass percent concentration is that the sodium alginate adding distil water of 2-7% boils dissolving, the cooling back is the bacteria suspension mixing of 150-300g/L with the equal-volume cell concn, get the emulsion tube of the suitable 4mm * 6mm of length, at one end adopt the water dropper of the about 1.0mm-4.0mm of internal diameter.With the emulsion tube peristaltic pump of packing into, regulate peristaltic pump speed and drip/min to 50-100.Under agitation condition, mixed solution is dropwise splashed into 0.1-1.0mol/L boric acid and 0.5-2.0mol/L CaCl
2In the mixing solutions, form immobilization gel beads of uniform size, diameter maximum difference≤0.5mm takes out behind the curing 5-15h, and making diameter is the 1-5mm gel beads.
The reaction conditions of immobilized cell catalyzed synthesizing alpha-arbutin is among the step C:
In the 10mmol/L borate buffer solution, immobilized cell is that the gel beads concentration behind the cell fixation is 150-300g/L, reactant Resorcinol amount concentration is 50-80mmol/l, sucrose amount concentration is 200-350mmol/l, temperature of reaction is 30-60 ℃, reaction times is 36-72 hour, and mixing speed is 150-200 rev/min.Under this reaction conditions, the transformation efficiency that Resorcinol is converted into alpha-arbutin reaches 75-92%, contains alpha-arbutin 10-16g in every liter of reaction solution.
The recovery of immobilized cell is reused again among the step D:
After the immobilized cell catalyzed synthesizing alpha-the arbutin reaction finishes, utilize and filter and to reclaim immobilized cell, the immobilized cell that reclaims drops in the 10mmol/L borate buffer solution that contains reactant Resorcinol and sucrose once more and reacts with 10mmol/L borate buffer solution washing 2-3 time; Recirculation like this, realize repeatedly catalyzed synthesizing alpha-arbutin of sodium alginate immobilization xanthomonas campestris (Xanthomonas campestris) CGMCC NO.1243 cell, with this understanding, the thalline immobilized cell has used 5-9 during the cycle, immobilized cell still keeps the vigor more than 75%, for the suitability for industrialized production that realizes alpha-arbutin is laid a good foundation.
The separation and purification of immobilized cell catalysis synthetic alpha-arbutin comprises following four steps in the step e;
1. the borate buffer solution after reaction being finished is removed the immobilized cell gel beads with filter paper filtering;
2. carrying out separation and purification through the 1. borate buffer solution of step by polar macroporous adsorbent resin column, polar macroporous adsorbent resin column is a macroporous adsorbent resin S-8 post;
3. through the 2. effluent liquid vacuum-drying of step macroporous adsorptive resins, can obtain purity and be the alpha-arbutin more than 98%, vacuum-drying vacuum tightness is 0.1Mpa, and temperature is 65 degree, and dried finished products is a white powder;
4. after water-soluble, carry out crystallization through the alpha-arbutin crude product of 3. step, promptly obtain purity and be more than 99.5% the pure product of alpha-arbutin, Tc is 4 degree.
The method of utilizing xanthomonas campestris (Xanthomonas campestris) CGMCC NO.1243 immobilized cell catalyzed synthesizing alpha-arbutin provided by the invention is applicable to that with sodium alginate, alginate calcium, polyvinyl alcohol (PVA) or silica gel be the synthetic alpha-arbutin of entrapment media immobilization xanthomonas campestris (Xanthomonas campestris) CGMCC NO.1243 cell catalysis.
Used xanthomonas campestris (Xanthomonas campestris) the CGMCC NO.1243 of the present invention is in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, and preserving number is xanthomonas campestris CGMCCNO.1243.
The obtained technical progress of the present invention is:
The present invention is by xanthomonas campestris (Xanthomonas campestris) CGMCC NO.1243 free cell or immobilized cell catalyzed synthesizing alpha-arbutin, solved the direct product later stage separating technology complexity that is caused with fermented liquid catalyzed synthesizing alpha-arbutin, catalyst recovery is utilized difficulty again, the difficult problem under production cost is in not; Xanthomonas campestris (Xanthomonas campestris) CGMCC NO.1243 free cell or immobilized cell have improved the nectar degree of unit volume, make thalline strengthen (as pH value, temperature, organic solvent, toxic substance etc.) to the tolerance of environment, this has not only shortened fermentation period greatly, reduced production cost, and the separation purifying technique condition of reaction after product is simpler, the safer property of production operation.The alpha-arbutin output height that adopts the present invention to produce contains the 10-16g alpha-arbutin in every liter of reaction solution, cell survival is the 5-9 cycle, has great industrialization meaning.
Embodiment: the present invention is described further below in conjunction with embodiment.
Embodiment 1
(a) preparation of somatic cells:
Xanthomonas campestris (Xanthomonas campestris) the CGMCC NO.1243 bacterial strain that picking 1 ring inclined-plane is preserved joins in the liquid nutrient medium that volume is 50ml, and at 35 ℃, the 130r/min shaking table gets primary seed solution after cultivating 48h; Again 10% primary seed solution is inoculated in the 500ml Erlenmeyer flask that contains the above-mentioned substratum of 200ml, at 35 ℃, the 140r/min shaking table cultivate behind the 36h secondary seed solution; With 10% secondary seed solution inoculation fermentation jar, fermented liquid is at 6000r/min, and 4 ℃ of centrifugal 15min get xanthomonas campestris (Xanthomonas campestris) CGMCC NO.1243 cell.
Seed liquor and fermentation tank culture medium are made up of following parts by weight of component:
60 parts of sucrose, (NH
4)
2SO
42 parts, 0.5 part in sal epsom, 2 parts in sodium-chlor, CaCl
22H
20.05 part of O, ZnCl
20.01 part, FeCl
24H
20.1 part of O, Na
2MoO
42H
20.05 part of O, 0.002 part of VITMAIN B1,0.005 part of vitamin B12,0.004 part of calcium pantothenate, 0.001 part in nicotinic acid, para-amino benzoic acid are received 1000 parts in 0.002 part and water, pH7.0; The processing condition of fermentation are 10% for the seed liquor inoculum size, and the pH value is 7.0, and culture temperature is 35 ℃, and ventilation is 0.5vvm, and mixing speed is 150r/min, and fermentation time is 60h.
(b) reaction conditions of free cell catalyzed synthesizing alpha-arbutin:
In the 10mmol/L phosphate buffer solution, free cell concentration is 150g/L, and reactant Resorcinol amount concentration is 45mmol/l, sucrose amount concentration is 100mmol/l, temperature of reaction is 35 ℃, and the reaction times is 72 hours, and mixing speed is 200 rev/mins.Under this reaction conditions, the transformation efficiency that Resorcinol is converted into alpha-arbutin reaches 91.5%, contains alpha-arbutin 11.2g in every liter of reaction solution.
(c) recovery of free cell and being repeatedly used:
After the free cell catalyzed synthesizing alpha-the arbutin reaction finishes, at 6000r/min, 4 ℃ of centrifugal 15min can reclaim free cell, the cell that reclaims is with 10mmol/L phosphate buffer solution washing 2-3 time, and input contains in the phosphate buffer solution of 10mmol/L of reactant Resorcinol and sucrose and reacts once more; Recirculation like this, realize repeatedly catalyzed synthesizing alpha-arbutin of xanthomonas campestris (Xanthomonas campestris) CGMCC NO.1243 cell, with this understanding, the thalline free cell transformed for 8 cycles, the transformation efficiency of alpha-arbutin is respectively: 91.5%, 91.5%, 90.7%, 90.2%, 89%, 88%, 85%, 82%, and corresponding alpha-arbutin mass concentration is: 11.2g/L, 11.2g/L, 11.1g/L, 11g/L, 10.9g/L, 10.8g/L, 10.4g/L, 10g/L.So free cell is after using 8 times, cell viability still remains on more than 80%.
(d) separation and purification of alpha-arbutin comprises following four steps;
1. the phosphate buffer solution after reaction being finished was removed thalline in centrifugal 15 minutes under 6000 rev/mins of conditions;
2. carrying out separation and purification through the 1. phosphate buffer solution of step by polar macroporous adsorbent resin column, polar macroporous adsorbent resin column is a macroporous adsorbent resin S-8 post;
3. through the 2. effluent liquid vacuum-drying of step macroporous adsorptive resins, can obtain purity and be the alpha-arbutin more than 98%, vacuum-drying vacuum tightness is 0.1Mpa, and temperature is 65 degree, and dried finished products is a white powder;
4. after water-soluble, carry out crystallization through the alpha-arbutin crude product of 3. step, promptly obtain purity and be more than 99.5% the pure product of alpha-arbutin, Tc is 4 degree.
Embodiment 2
(a) preparation of somatic cells:
(a) step that repeats among the embodiment 1 prepares somatic cells;
(b) reaction conditions of free cell catalyzed synthesizing alpha-arbutin:
In the 10mmol/L phosphate buffer solution, free cell concentration is 200g/L, and reactant Resorcinol amount concentration is 60mmol/l, sucrose amount concentration is 200mmol/l, temperature of reaction is 45 ℃, and the reaction times is 72 hours, and mixing speed is 200 rev/mins.Under this reaction conditions, the transformation efficiency that Resorcinol is converted into alpha-arbutin reaches 92%, contains alpha-arbutin 15g in every liter of reaction solution.
(c) recovery of free cell and being repeatedly used:
After the free cell catalyzed synthesizing alpha-the arbutin reaction finishes, at 6000r/min, 4 ℃ of centrifugal 15min can reclaim free cell, the cell that reclaims is with 10mmol/L phosphate buffer solution washing 2-3 time, and input contains in the phosphate buffer solution of 10mmol/L of reactant Resorcinol and sucrose and reacts once more; Recirculation like this, realize repeatedly catalyzed synthesizing alpha-arbutin of xanthomonas campestris (Xanthomonas campestris) CGMCC NO.1243 free cell, with this understanding, the thalline free cell transformed for 6 cycles, the transformation efficiency of alpha-arbutin is respectively: 92%, 92%, 91.5%, 87%, 80%, 75%, and corresponding alpha-arbutin mass concentration is: 15g/L, 15g/L, 14.9g/L, 14.2g/L, 13.1g/L, 12.2g/L.So free cell is after using 5 times, cell viability still remains on 75%.
(d) separation and purification of alpha-arbutin comprises following four steps;
1. the phosphate buffer solution after reaction being finished was removed thalline in centrifugal 15 minutes under 6000 rev/mins of conditions;
2. carrying out separation and purification through the 1. phosphate buffer solution of step by polar macroporous adsorbent resin column, polar macroporous adsorbent resin column is a macroporous adsorbent resin S-8 post;
3. through the 2. effluent liquid vacuum-drying of step macroporous adsorptive resins, can obtain purity and be the alpha-arbutin more than 98%, vacuum-drying vacuum tightness is 0.1Mpa, and temperature is 65 degree, and dried finished products is a white powder;
4. after water-soluble, carry out crystallization through the alpha-arbutin crude product of 3. step, promptly obtain purity and be more than 99.5% the pure product of alpha-arbutin, Tc is 4 degree.
Embodiment 3
(A) preparation of somatic cells:
(a) step that repeats among the embodiment 1 prepares somatic cells;
(B) immobilization of somatic cells:
With mass percent concentration is that 2% sodium alginate adding distil water boils dissolving, and cooling back and equal-volume cell concn are the bacteria suspension mixing of 150g/L, get the emulsion tube of the suitable 4mm * 6mm of length, at one end adopt the water dropper of the about 4.0mm of internal diameter.With the emulsion tube peristaltic pump of packing into, regulate peristaltic pump speed to 100 droplet/min.Under agitation condition, mixed solution is dropwise splashed into 0.1mol/L boric acid and 1.5mol/L CaCl
2In the mixing solutions, form immobilization gel beads of uniform size, diameter maximum difference≤0.5mm takes out behind the curing 10h, makes the gel beads that diameter is 3mm.
(C) immobilized cell catalyzed synthesizing alpha-arbutin reaction conditions is:
In the 10mmol/L borate buffer solution, immobilized cell is that the gel beads concentration behind the cell fixation is 150g/L, reactant Resorcinol amount concentration is 50mmol/l, sucrose amount concentration is 200mmol/l, temperature of reaction is 40 ℃, reaction times is 48 hours, and mixing speed is 180 rev/mins.Under this reaction conditions, the transformation efficiency that Resorcinol is converted into alpha-arbutin reaches 91.5%, contains alpha-arbutin 12.4g in every liter of reaction solution.
(D) recovery of immobilized cell is reused again:
After the immobilized cell catalyzed synthesizing alpha-the arbutin reaction finishes, utilize and filter and to reclaim immobilized cell, the immobilized cell that reclaims drops in the 10mmol/L borate buffer solution that contains reactant Resorcinol and sucrose once more and reacts with 10mmol/L borate buffer solution washing 2-3 time; So recirculation realizes repeatedly catalyzed synthesizing alpha-arbutin of sodium alginate immobilization xanthomonas campestris (Xanthomonas campestris) CGMCC NO.1243 cell.With this understanding, the thalline free cell transformed for 9 cycles, the transformation efficiency of alpha-arbutin is respectively: 91.5%, 91.5%, 91%, 89%, 88%, 85%, 81%, 79%, 75%, corresponding alpha-arbutin mass concentration is: 12.4g/L, 12.4g/L, 12.3/L, 12.1g/L, 12g/L, 11.6g/L, 11g/L, 10.7g/L, 10.2g/L.So free cell is after using 9 times, cell viability still remains on more than 75%.
(E) separation and purification of alpha-arbutin comprises following four steps;
1. the borate buffer solution after reaction being finished is removed the immobilized cell gel beads with filter paper filtering;
2. carrying out separation and purification through the 1. borate buffer solution of step by polar macroporous adsorbent resin column, polar macroporous adsorbent resin column is a macroporous adsorbent resin S-8 post;
3. through the 2. effluent liquid vacuum-drying of step macroporous adsorptive resins, can obtain purity and be the alpha-arbutin more than 98%, vacuum-drying vacuum tightness is 0.1Mpa, and temperature is 65 degree, and dried finished products is a white powder;
4. after water-soluble, carry out crystallization through the alpha-arbutin crude product of 3. step, promptly obtain purity and be more than 99.5% the pure product of alpha-arbutin, Tc is 4 degree.
Embodiment 4
(A) preparation of somatic cells:
(a) step that repeats among the embodiment 1 prepares somatic cells;
(B) immobilization of somatic cells:
With mass percent concentration is that 5% sodium alginate adding distil water boils dissolving, and cooling back and equal-volume cell concn are the bacteria suspension mixing of 200g/L, get the emulsion tube of the suitable 4mm * 6mm of length, at one end adopt the water dropper of the about 2.0mm of internal diameter.With the emulsion tube peristaltic pump of packing into, regulate peristaltic pump speed to 75 droplet/min.Under agitation condition, mixed solution is dropwise splashed into 0.1mol/L boric acid and 1.5mol/L CaCl
2In the mixing solutions, form immobilization gel beads of uniform size, diameter maximum difference≤0.5mm takes out behind the curing 15h, makes the gel beads that diameter is 5mm.
(C) immobilized cell catalyzed synthesizing alpha-arbutin reaction conditions is:
In the 10mmol/L borate buffer solution, immobilized cell is that the gel beads concentration behind the cell fixation is 250g/L, reactant Resorcinol amount concentration is 65mmol/l, sucrose amount concentration is 250mmol/l, temperature of reaction is 50 ℃, reaction times is 48 hours, and mixing speed is 180 rev/mins.Under this reaction conditions, the transformation efficiency that Resorcinol is converted into alpha-arbutin reaches 92%, contains alpha-arbutin 16g in every liter of reaction solution.
(D) recovery of immobilized cell is reused again:
After the immobilized cell catalyzed synthesizing alpha-the arbutin reaction finishes, utilize and filter and to reclaim immobilized cell, the immobilized cell that reclaims drops in the 10mmol/L borate buffer solution that contains reactant Resorcinol and sucrose once more and reacts with 10mmol/L borate buffer solution washing 2-3 time; So recirculation realizes repeatedly catalyzed synthesizing alpha-arbutin of sodium alginate immobilization xanthomonas campestris (Xanthomonas campestris) CGMCC NO.1243 cell.With this understanding, the thalline free cell transformed for 6 cycles, the transformation efficiency of alpha-arbutin is respectively: 92%, 92%, 89%, 85%, 78%, 75%, and corresponding alpha-arbutin mass concentration is: 16g/L, 16g/L, 15.7g/L, 15g/L, 13.8g/L, 13.3g/L.So free cell is after using 6 times, cell viability still remains on more than 75%.
(E) separation and purification of alpha-arbutin comprises following four steps;
1. the borate buffer solution after reaction being finished is removed the immobilized cell gel beads with filter paper filtering;
2. carrying out separation and purification through the 1. borate buffer solution of step by polar macroporous adsorbent resin column, polar macroporous adsorbent resin column is a macroporous adsorbent resin S-8 post;
3. through the 2. effluent liquid vacuum-drying of step macroporous adsorptive resins, can obtain purity and be the alpha-arbutin more than 98%, vacuum-drying vacuum tightness is 0.1Mpa, and temperature is 65 degree, and dried finished products is a white powder;
4. after water-soluble, carry out crystallization through the alpha-arbutin crude product of 3. step, promptly obtain purity and be more than 99.5% the pure product of alpha-arbutin, Tc is 4 degree.
Embodiment 5
(A) preparation of somatic cells:
(a) step that repeats among the embodiment 1 prepares somatic cells;
(B) immobilization of somatic cells:
With mass percent concentration is that 7% sodium alginate adding distil water boils dissolving, and cooling back and equal-volume cell concn are the bacteria suspension mixing of 250g/L, get the emulsion tube of the suitable 4mm * 6mm of length, at one end adopt the water dropper of the about 1.0mm of internal diameter.With the emulsion tube peristaltic pump of packing into, regulate peristaltic pump speed to 50 droplet/min.Under agitation condition, mixed solution is dropwise splashed into 0.1mol/L boric acid and 1.5mol/L CaCl
2In the mixing solutions, form immobilization gel beads of uniform size, diameter maximum difference≤0.5mm takes out behind the curing 15h, makes the gel beads that diameter is 1.5mm.
(C) immobilized cell catalyzed synthesizing alpha-arbutin reaction conditions is:
In the 10mmol/L borate buffer solution, immobilized cell is that the gel beads concentration behind the cell fixation is 300g/L, reactant Resorcinol amount concentration is 70mmol/l, sucrose amount concentration is 200mmol/l, temperature of reaction is 35 ℃, reaction times is 60 hours, and mixing speed is 200 rev/mins.Under this reaction conditions, the transformation efficiency that Resorcinol is converted into alpha-arbutin reaches 81%, contains alpha-arbutin 15.4g in every liter of reaction solution.
(D) recovery of immobilized cell is reused again:
After the immobilized cell catalyzed synthesizing alpha-the arbutin reaction finishes, utilize and filter and to reclaim immobilized cell, the immobilized cell that reclaims drops in the 10mmol/L borate buffer solution that contains reactant Resorcinol and sucrose once more and reacts with 10mmol/L borate buffer solution washing 2-3 time; So recirculation realizes repeatedly catalyzed synthesizing alpha-arbutin of sodium alginate immobilization xanthomonas campestris (Xanthomonas campestris) CGMCC NO.1243 cell.With this understanding, the thalline free cell transformed for 5 cycles, and the transformation efficiency of alpha-arbutin is respectively: 81%, 79%, 78%, 76%, 75%, and corresponding alpha-arbutin mass concentration is: 15.4g/L, 15g/L, 14.9g/L, 14.5g/L, 14.3g/L.So free cell is after using 5 times, cell viability still remains on more than 75%.
(E) separation and purification of alpha-arbutin comprises following four steps;
1. the borate buffer solution after reaction being finished is removed the immobilized cell gel beads with filter paper filtering;
2. carrying out separation and purification through the 1. borate buffer solution of step by polar macroporous adsorbent resin column, polar macroporous adsorbent resin column is a macroporous adsorbent resin S-8 post;
3. through the 2. effluent liquid vacuum-drying of step macroporous adsorptive resins, can obtain purity and be the alpha-arbutin more than 98%, vacuum-drying vacuum tightness is 0.1Mpa, and temperature is 65 degree, and dried finished products is a white powder;
4. after water-soluble, carry out crystallization through the alpha-arbutin crude product of 3. step, promptly obtain purity and be more than 99.5% the pure product of alpha-arbutin, Tc is 4 degree.
Claims (10)
1, the method for free cell or immobilized cell catalyzed synthesizing alpha-arbutin, it is characterized in that: with free cell or immobilized cell catalysis Resorcinol and the synthetic alpha-arbutin of sucrose in buffer system of xanthomonas campestris (Xanthomonas campestris) CGMCC NO.1243, wherein:
The method of free cell catalyzed synthesizing alpha-arbutin, this method is made up of following process steps:
A. the preparation of xanthomonas campestris (Xanthomonas campestris) CGMCC NO.1243 cell is comprising seed culture and fermentor cultivation;
B. free cell catalyzed synthesizing alpha-arbutin;
C. the recovery of free cell and being repeatedly used;
D. the separation and purification of free cell catalysis synthetic alpha-arbutin;
The method of immobilized cell catalyzed synthesizing alpha-arbutin, this method is made up of following process steps:
A. the preparation of xanthomonas campestris (Xanthomonas campestris) CGMCC NO.1243 cell is comprising seed culture and fermentor cultivation;
B. the preparation of immobilization xanthomonas campestris (Xanthomonas campestris) CGMCC NO.1243;
C. immobilized cell catalyzed synthesizing alpha-arbutin;
D. the recovery of immobilized cell and being repeatedly used;
E. the separation and purification of immobilized cell catalysis synthetic alpha-arbutin.
2, the method for free cell according to claim 1 or immobilized cell catalyzed synthesizing alpha-arbutin is characterized in that: seed liquor and fermentation tank culture medium in described a and the A step are made up of following parts by weight of component:
60 parts of sucrose, (NH
4)
2SO
42 parts, 0.5 part in sal epsom, 2 parts in sodium-chlor, CaCl
22H
20.05 part of O, ZnCl
20.01 part, FeCl
24H
20.1 part of O, Na
2MoO
42H
20.05 part of O, 0.002 part of VITMAIN B1,0.005 part of vitamin B12,0.004 part of calcium pantothenate, 0.001 part in nicotinic acid, para-amino benzoic acid are received 1000 parts in 0.002 part and water, pH7.0.
3, according to the method for claim 1 or 2 described free cells or immobilized cell catalyzed synthesizing alpha-arbutin, it is characterized in that: the processing condition of fermentation are 10-15% for the seed liquor inoculum size, the pH value is 7.0, culture temperature is 25-40 ℃, ventilation is 0.4-0.6vvm, mixing speed is 120-160r/min, and fermentation time is 48-72h.
4, the method for free cell according to claim 1 or immobilized cell catalyzed synthesizing alpha-arbutin, it is characterized in that: the reaction conditions in the described b step is: in the 10mmol/L phosphate buffer solution, cell concn is 100-200g/L, reactant Resorcinol amount concentration is 45-70mmol/l, sucrose amount concentration is 100-200mmol/l, temperature of reaction is 30-55 ℃, and the reaction times is 36-72 hour, and mixing speed is 160-200 rev/min.
5, the method for free cell according to claim 1 or immobilized cell catalysis synthetic alpha-arbutin is characterized in that: the separation and purification of the free cell catalysis synthetic alpha-arbutin in the described d step comprises following four steps:
1. the phosphate buffer solution after reaction being finished was removed thalline in centrifugal 15 minutes under 6000 rev/mins of conditions;
2. carrying out separation and purification through the 1. phosphate buffer solution of step by polar macroporous adsorbent resin column, polar macroporous adsorbent resin column is a macroporous adsorbent resin S-8 post;
3. through the 2. effluent liquid vacuum-drying of step macroporous adsorptive resins, can obtain purity and be the alpha-arbutin more than 98%, vacuum-drying vacuum tightness is 0.1Mpa, and temperature is 65 degree, and dried finished products is a white powder;
4. carry out crystallization after water-soluble through the alpha-arbutin crude product of 3. step, promptly obtain purity and be the pure product of alpha-arbutin more than 99.5%, Tc is 4 degree.
6, the method for free cell according to claim 1 or immobilized cell catalyzed synthesizing alpha-arbutin is characterized in that:
The entrapment media of xanthomonas campestris (Xanthomonas campestris) CGMCC NO.1243 cell is selected from sodium alginate, alginate calcium, polyvinyl alcohol (PVA) or silica gel.
7, the method for free cell according to claim 1 or immobilized cell catalyzed synthesizing alpha-arbutin, it is characterized in that: the preparation of immobilization xanthomonas campestris in the described B step (Xanthomonas campestris) CGMCC NO.1243 is to utilize the preparation of sodium alginate to embed method, the steps include: to make earlier the sodium alginate aqueous solution that mass percent concentration is 2-7%, the cooling back is the bacteria suspension mixing of 150-300g/L with the equal-volume cell concn, get the emulsion tube of the suitable 4mm * 6mm of length, at one end adopt the water dropper of the about 1.0mm-4.0mm of internal diameter; With the emulsion tube peristaltic pump of packing into, regulate peristaltic pump speed and drip/min to 50-100; Under agitation condition, mixed solution is dropwise splashed into 0.1-1.0mol/L boric acid and 0.5-2.0mol/L CaCl
2In the mixing solutions, form immobilization gel beads of uniform size, diameter maximum difference≤0.5mm takes out behind the curing 5-15h, and making diameter is the 1-5mm gel beads.
8, the method for free cell according to claim 1 or immobilized cell catalyzed synthesizing alpha-arbutin, it is characterized in that: the reaction conditions in the described C step is: in the 10mmol/L borate buffer solution, immobilized cell is that the gel beads concentration after the immobilization is 150-300g/L, reactant Resorcinol amount concentration is 50-80mmol/l, sucrose amount concentration is 200-350mmol/l, temperature of reaction is 30-60 ℃, reaction times is 36-72 hour, and mixing speed is 150-200 rev/min.
9, the method for free cell according to claim 1 or immobilized cell catalyzed synthesizing alpha-arbutin is characterized in that: be the 5-8 cycle work-ing life of described free cell, and be the 5-9 cycle work-ing life of immobilized cell.
10, the method for free cell according to claim 1 or immobilized cell catalyzed synthesizing alpha-arbutin is characterized in that: the separation and purification of the immobilized cell catalysis synthetic alpha-arbutin in the described E step comprises following four steps:
1. the borate buffer solution after reaction being finished is removed the immobilized cell gel beads with filter paper filtering;
2. carrying out separation and purification through the 1. borate buffer solution of step by polar macroporous adsorbent resin column, polar macroporous adsorbent resin column is a macroporous adsorbent resin S-8 post;
3. through the 2. effluent liquid vacuum-drying of step macroporous adsorptive resins, can obtain purity and be the alpha-arbutin more than 98%, vacuum-drying vacuum tightness is 0.1Mpa, and temperature is 65 degree, and dried finished products is a white powder;
4. carry out crystallization after water-soluble through the alpha-arbutin crude product of 3. step, promptly obtain purity and be the pure product of alpha-arbutin more than 99.5%, Tc is 4 degree.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100803646A CN100478451C (en) | 2005-07-04 | 2005-07-04 | Method for catalyzed synthesizing alpha arbutin from free cells or immobilized cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100803646A CN100478451C (en) | 2005-07-04 | 2005-07-04 | Method for catalyzed synthesizing alpha arbutin from free cells or immobilized cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1727493A CN1727493A (en) | 2006-02-01 |
| CN100478451C true CN100478451C (en) | 2009-04-15 |
Family
ID=35927015
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005100803646A Expired - Fee Related CN100478451C (en) | 2005-07-04 | 2005-07-04 | Method for catalyzed synthesizing alpha arbutin from free cells or immobilized cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100478451C (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101701237B (en) * | 2009-11-05 | 2011-08-31 | 北京化工大学 | Method for producing alpha-glucosyl eugenol by fermentation |
| CN102978265A (en) * | 2012-12-03 | 2013-03-20 | 韦慧芳 | Method for synthesizing alpha-arbutin by enzymic method through catalysis |
| CN104711248A (en) * | 2013-12-16 | 2015-06-17 | 北京化工大学 | Substrate toxicity eliminating method |
| CN104498565A (en) * | 2014-09-11 | 2015-04-08 | 北京化工大学 | Method for co-production of alpha-arbutin and xanthan gum by xanthomonas |
| CN107446844A (en) * | 2017-08-03 | 2017-12-08 | 浙江工业大学 | A kind of xanthomonas campestris and its application |
| CN107653282A (en) * | 2017-10-25 | 2018-02-02 | 北京化工大学 | A kind of method of substrate and the double immobilization production glucoside compounds of catalyst |
| CN111979282A (en) * | 2019-05-21 | 2020-11-24 | 北京化工大学 | Method for producing alpha-arbutin by substrate and cell double-immobilization fermentation |
| CN113073061B (en) * | 2021-04-06 | 2022-10-18 | 浙江美迪生物科技有限公司 | Method for efficiently producing alpha-arbutin by immobilized cells |
| CN114058660B (en) * | 2021-10-15 | 2023-06-27 | 安徽华恒生物科技股份有限公司 | Immobilized enzyme conversion synthesis method of alpha-arbutin and application |
-
2005
- 2005-07-04 CN CNB2005100803646A patent/CN100478451C/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| Enzymatic synthesis of α-arbutin by α-anomer-selectiveglucosylation of hydroquinone using lyophilized cells ofXanthomonas campestris WU-9701. JUN KUROSU et al.JOURNAL OF BIOSCIENCE AND BIOENGINEERING,Vol.93 No.3. 2002 |
| Enzymatic synthesis of α-arbutin by α-anomer-selectiveglucosylation of hydroquinone using lyophilized cells ofXanthomonas campestris WU-9701. JUN KUROSU et al.JOURNAL OF BIOSCIENCE AND BIOENGINEERING,Vol.93 No.3. 2002 * |
| 熊果苷的制备方法研究. 周烽，卢定强，韦萍.精细与专用化学品，第2005卷第01期. 2005 |
| 熊果苷的制备方法研究. 周烽，卢定强，韦萍.精细与专用化学品，第2005卷第01期. 2005 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1727493A (en) | 2006-02-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100478451C (en) | Method for catalyzed synthesizing alpha arbutin from free cells or immobilized cells | |
| CN100562581C (en) | A kind of method and special-purpose reaction column thereof of producing γ-An Jidingsuan | |
| CN101838672B (en) | Method for producing gamma-amino butyric acid by using immobilized lactobacillus plantarum | |
| CN107354188A (en) | The technique of ETEC JL GlcN fermenting and producing N acetylglucosamines | |
| CN1165624C (en) | Production process of cane-fruit oligosaccharide with immobilized fructose-base transferase | |
| CN101235394B (en) | Method for separating and extracting fumaric acid | |
| CN102943069A (en) | Co-immobilization glucose oxidase/catalase microspheres and application thereof in production of gluconic acid or gluconic salt | |
| CN101591628B (en) | Acinetobacter juni. X8 and application thereof in preparing algin lyase | |
| CN101555503A (en) | Method for separating and extracting L-arabinose from waste wood sugar mother liquid from wood sugar production | |
| CN104341535B (en) | A kind of high-valued extracting method of Enteromorpha | |
| CN100523209C (en) | Method of transforming enzyme to prepare L-ornithine | |
| CN102363797B (en) | Method for producing epsilon-poly-L-lysine | |
| CN103864954B (en) | A kind of extracting method of peanut meal polysaccharides | |
| CN103805657A (en) | Method of synthesizing alpha-arbutin by catalyzing immobilized hydroquinone by free cells | |
| CN102925418B (en) | Method for recycling sucrose phospholylase in alpha-arbutin production process | |
| CN100478440C (en) | Method of immobilized cell continuously producing L-carbamyl ornithine | |
| CN103773827A (en) | Method for improving output of alpha-arbutin | |
| CN101974151A (en) | Method for preparing bio-based nylon polybutyrolactam | |
| CN101701243B (en) | Method for producing R-mandelic acid and derivates thereof by biocatalysis | |
| CN101701237B (en) | Method for producing alpha-glucosyl eugenol by fermentation | |
| CN104711248A (en) | Substrate toxicity eliminating method | |
| CN101691555B (en) | Method for producing L-ornithine by transforming free cell or immobilized cell micro-organism | |
| CN101709323A (en) | Method for producing R-mandelic acid with biocatalysis and separating and coupling method | |
| CN102071235B (en) | Method for promoting enzymic preparation to convert isomaltulose by utilizing ultrasonic wave | |
| CN103194374B (en) | Column type immobilization reactor for fermentation production of gamma-polyglutamic acid and process thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20160826 Address after: Haicang District of Xiamen City, Fujian province 361028 Weng Kok Road No. 289 Building No. 3 Building 4 branch on the west side of building Patentee after: Xiamen Qiyuan Green Biotechnology Co. Ltd. Address before: 100029, box 53, Beijing University of Chemical Technology, 15 East Third Ring Road, Beijing Patentee before: Zhang Peng Patentee before: Zhang Shurong Patentee before: Liu Chunqiao |
|
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20090415 Termination date: 20190704 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |