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/1, 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/1, 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 the water dropper of-about 2.0mm of end employing 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 it is corresponding that (the 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.