CN1030530C - Preparation of parakin sugar by solidified alpha-glucosyl group transferase - Google Patents
Preparation of parakin sugar by solidified alpha-glucosyl group transferase Download PDFInfo
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- CN1030530C CN1030530C CN89100684A CN89100684A CN1030530C CN 1030530 C CN1030530 C CN 1030530C CN 89100684 A CN89100684 A CN 89100684A CN 89100684 A CN89100684 A CN 89100684A CN 1030530 C CN1030530 C CN 1030530C
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- palatinose
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
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Abstract
The present invention relates to a method for preparing palatinose from immobilized alpha-glucose group transferases, which belongs to enzyme engineering in biotechnology. The method comprises the following steps: the strains of alpha-glucose group transferases are fermented and immobilized, so that a cane sugar solution is converted, concentrated and crystallized to be prepared into the palatinose. In the method, a flow addition nutrient source is used for fermenting and culturing mycelium cells; in the cell immobilization technology, clay using kaolin as the main component is used as an additive for absorbing cells, etc. and providing a set of optimized technological conditions, and a cell immobilization technique by a nontraditional inner gelling method is also proposed; the method has the advantages of simple technique, low material cost, easy material acquisition and high enzyme activity, is favourable for enhancing the conversion rate and the yield of the palatinose, and is convenient for wide popularization and application.
Description
The invention belongs to the enzyme engineering in the biotechnology, relate to the industrial utilization of microorganism cells curing technology and curing enzyme.
Palatinose, formal name used at school is that Palatinose (Isomaltulose) is a kind of carious tooth that do not produce, wholesome sweeting agent, this sugar is the isomers of sucrose, can utilize alpha-glucosyl transferring enzyme (α-glucosyltransferase) transform sucrose to obtain palatinose, this enzyme is present in the cell pericentral siphon of certain micro-organisms bacterial classification, industrial common employing enzyme immobilization technology is produced palatinose, its technology comprises, select certain to contain the bacterial classification of alpha-glucosyl transferring enzyme, through fermentation propagation somatic cells, will be through the immobilization of centrifugal somatic cells, sucrose solution stream is added to the reaction column that immobilized enzyme is housed, and making sucrose inversion is palatinose, makes its crystallization after the palatinose conversion fluid being concentrated again and obtains the palatinose product.Various countries have formed a plurality of concrete technical schemes in the production of palatinose at present.United States Patent (USP) (U.Spat.No.435931) discloses a kind of method with the whole cells produce palatinose of immobilization, this patent is used E.rhapontici NCPPB1578 bacterial strain, its substratum is sucrose 4%, peptone 1%, beef extract 0.4%, cell was cultivated in substratum 70 hours, and at harvested cell stationary phase, cell yield is 1%(WWC%), curing technology is: sodium alginate consumption 5%, 0.1MCaCl
2Stirred 1 hour down in 30 ℃, to solidify cell is contained in φ 5 * 30cm post with dissection, flow velocity is the 0.01(V-0.01ecv/h of free column volume, empty column Volumn/h) sucrose inversion is that palatinose and other sugar reach balance, and its enzymic activity is 0.325g product/g wet cell hour.The method that a kind of immobilization that proposed United States Patent (USP) (U.S.Pat.NO.4390627) contains the whole cell of P.rubrum bacterium of α-grape-transferase enzyme comprises use tannin (Tannic Acid) absorption, add polymine (polyethylemine) flocculation, add epihalohydrins/polyamines polymkeric substance (epihalohydrin/polyamine Copolymer) and glutaraldehyde (glutaraldehyde) again in nutrient solution, obtain reaction product, filter or the wet bacterium cake of centrifugal concentrated collection, be drying to obtain the curing cell under 55 ℃, the polymkeric substance of this patent requires to use the product of the commercially available trade mark of the U.S. as BETZ1180.English Patent (U.K.Fat.Appl.GB.NO.2082591) proposes a kind of method of improved immobilization alpha-glucosyl transferring enzyme again, the microorganism cells that will have the alpha-glucosyl transferring enzyme is embedded in back polymine and glutaraldehyde processing in the alginate calcium, this patent uses S.Plymuthica bacterium, substratum to be sucrose 5%, corn steep liquor 3%, Na
2HPO
40.3%, NaCl 0.2%, with 30 liters of canned 15 liters of fermention mediums, cultivates 16 hours down at 28 ℃, through centrifugal concentrated harvested cell slurry, uses calcium alginate embedded curing again.Above-mentioned technology ubiquity following problems: (1) once supplies with nutrition source when cell fermentation, cause cell yield low, generally has only 1～3%(WWC%); (2) the curing material therefor has particular requirement, price expensive (U.S.Pat.NO4390627), and the general curing enzyme enzyme that obtains is alive lower, causes reality palatinose conversion fluid flow velocity in reaction column low, transformation time is long, is difficult to improve the output of palatinose.
The present invention is directed to above-mentioned prior art problems, propose a kind of improved novel method of utilizing immobilized enzyme to transform and prepare palatinose.The technology of this method to simplify, the material that cheaply is easy to get, preparation has higher yield and enzyme curing enzyme alive, and the transformation efficiency and the output that improve palatinose also reduce cost, and make it easy to apply.
Method of the present invention, comprising ferments to the bacterial classification that contains the alpha-glucosyl transferring enzyme makes its proliferative cell, behind the centrifugal concentrated harvested cell, the cell fixation of collecting is made immobilized enzyme (or claiming to solidify cell), to solidify the enzyme reaction column of packing into, stream adds sucrose solution, make it change into palatinose liquid, again the palatinose conversion fluid is concentrated, crystallization or directly solidify, dry and make the palatinose product, one of characteristics of the present invention are that bacterial classification adopts during fermentation culture the method in the stream Ensure Liquid source while ferment to promote bacterium cell to breed in substratum, with preparation alpha-glucosyl transferring enzyme (α-glucosyltanferase), employed bacterial classification is that (this bacterial classification is stored in American Type Culture Collection (ATCC) (ATCC) to the Serratiaplymuthica bacterial classification, its standard model number is ATCC15928) culture, the strain fermentation nutrient media components that adopts contains 1～6%(W/V) sucrose, 0.5～5%(W/V) corn steep liquor (removing by filter throw out), 0.01～0.4%(W/V) KCl, the nutrition source that stream adds is that concentration is 1～7% the yeast extract paste or the corn steep liquor (removing by filter throw out) (nitrogenous source) of concentration 1～8%, and the sucrose solution of concentration 1～15%, fed-batch mode can be that Continuous Flow adds or at interval stream add.Following is one group of parameter index preferably, nutrient media components: 2%(W/V) sucrose, 2%(W/V) corn steep liquor (removing by filter throw out), 0.2%(W/V) KCl, Continuous Flow add or at interval stream add corn steep liquor (removing by filter throw out) that concentration is 2～3% yeast extract paste or 2% and 2% sucrose solution.
Its processing condition are: available any volume fermentation culture bacterial classification is with proliferative cell, and the fermenting process temperature is 25～35 ℃, pH5.0～8.5, stir, in stream Ensure Liquid source, 1～20 hour inner edge fermentation limit, fermentation time is 8～24 hours, in logarithmic phase results somatic cells.
Two of characteristics of the present invention are that the solidification process of alpha-glucosyl transferring enzyme or somatic cells comprises: use with kaolin to the additive of the clay class of major ingredient as upholder and adsorb somatic cells.With the whole somatic cells of sodium alginate to embed, use CaCl again
2Solution is handled the material cured moulding of institute's embedding again with glutaraldehyde, crosslinked again, kills the curing enzyme of viable cell with the preparation Transglucosylase simultaneously.
The said additive that is used to adsorb somatic cells can be potter's clay, clay, kaolin etc.
Additive amount can be 2～30% of fermented liquid, and is good with 6～9%, and granularity can be 100～300 orders, and is good with 150～200 orders.
The specific embodiment that alpha-glucosyl transferring enzyme or somatic cells immobilization are solidified enzyme with preparation is: (1), with additive add fully mix adherent cell in the fermented liquid after, centrifugal results additive and adsorbed cell thereof, perhaps earlier with the centrifugal cytoplasm that gets of fermented liquid, again additive is added wherein, fully mix adherent cell; (2) material (cell that comprises additive and absorption) and the sodium alginate with (1) results mixes, and forms gel, fully stirs.The consumption of sodium alginate is 1～6%(W/V), and the cell embedding amount is 5～50(WWC); (3), with the particle squeezer embedded material being clamp-oned concentration is 0.1～0.8% CaCl
2Middle curing molding, the limit squish lip stirs more than 2 hours; (4), suction filtration blots the immobilized enzyme that collection has been cured, with physiological saline or phosphoric acid buffer (pH7.0) washing; (5), this solidified enzyme handle with glutaraldehyde, exchange again, the killing living cell is washed with physiological saline or phosphoric acid buffer more simultaneously, cryodrying promptly gets solidifies enzyme.
The present invention is the recovery technology of palatinose and palatinose product with sucrose inversion, is the curing enzyme is contained in the filling bed type column type reactor, and the volume of post is not limit, any footpath height, and volume all can.This reactor enzyme post way flow adds or the circulation Continuous Flow adds sucrose solution, and sucrose solution concentration can be 5～75%, and sucrose solution flows through the enzyme post and promptly is converted into the palatinose conversion fluid, and by adjusting flow velocity, the control transformation efficiency is at 60～100%.
Collect the palatinose conversion fluid, after directly it being concentrated, can or add seeded crystallization or conversion fluid is directly solidified drying by organic solvent such as alcohol extractive crystallization and can obtain the palatinose product.
The present invention also can adopt gelling process immobilization alpha-glucosyl transferring enzyme (or cell) in the unconventional alginates, uses this method except that the cell fixation process, and all the other technological processs are identical with aforesaid method.
The gelling process processing method comprises and will add a CaCO behind a somatic cells slurry and a sodium alginate mixing in the said non-traditional alginates
3Solution stirs, and adds a Gluconolactone solution then, leaves standstill more than 2 hours after stirring fast, and solid good material is made particle, immerses CaCl again
2Solution, filter do or low temperature dry down.
The concrete processing condition of gelling process are in adopting: the sodium alginate consumption is 1～5%(W/V), 2% being good, and CaCO
3Consumption be 0.5～8.0%, 3% to be good, the consumption of gluconic acid lactone is 0.5～4.0%, is good with 0.5～1%, cell embedding concentration can be 1～30%(WWC%), is good with 11～14%, with CaCl
2Strength of solution is 0.1～0.6M, is good with 0.2M.
The present invention adopts continuously or flows at interval the method in Ensure Liquid source in the strain fermentation process, make the nutrition of adding fully be used for the cells physiological metabolism and proliferative cell, thereby cell proliferation is fast, the present invention was fermentation 6～10 hours, and cell yield can reach 3～8%, is much higher than prior art.Cell curing method of the present invention, comprise traditional outer gelling process and unconventional interior gelling process, its outer gelling process has adopted performance good, the additive that material cheaply is easy to get, the adsorption rate of this additive pair cell can reach 98～100%, the enzyme that solidifies enzyme is lived to be improved greatly, can reach 0.42～0.49g handkerchief sugar/g wet cell hour, thereby the efficient that makes this curing enzyme be used for the palatinose conversion is improved, be reflected in the actual production, the flow through stream scooter 0.1ecv/h～0.14ecv/h of enzyme post of sucrose, transformation time is 5～8 hours, and index all is better than prior art, helps improving the output of palatinose, the interior gelling process that the present invention proposes, it is alive stable also to have the enzyme that makes the curing enzyme, and enzyme activity descends and waits advantage slowly, and the prospect of applying is arranged greatly.The transformation efficiency of using palatinose of the present invention can reach 80～100%, and the rate of recovery is more than 80%, and product purity 85～100% meets edible demand, can match in excellence or beauty with external product fully.
Accompanying drawing is a process flow diagram of the present invention.
Embodiment one
Use MSJ-N
220L type fermentor tank, 12～14 liters of fermented liquids of dress, bacterial classification is Serratia Plymuthica(ATCC15928), the substratum composition: sucrose 2%(W/V), corn steep liquor 2%(W/V), KCl0.2%, ventilated stirring velocity 300rpm, 28～31 ℃ of temperature 1: 1; PH7.0, add 3% yeast extract paste and 2% sucrose solution at cell log stream in vegetative period, stir fermentation 8～16 hours, at the logarithmic phase harvested cell, kaolin with 8% adds in the fermented liquid, fully mix, with the cytoplasm of whizzer in the centrifugal results additive absorption of 3000～4000r/min, this material is washed 2～3 times with physiological saline, make pulpous state with deionized water again, add sodium alginate to embed, its consumption is 3% to stir, and with squeezer embedded material is injected 0.2MCaCl
2In the liquid, stirred 2 hours, filter, blot and promptly get the curing enzyme that is cured, this is solidified enzyme handle with penta-aldehyde, crosslinked again, while killing living cell, forming granularity is the curing enzyme granulate of 2～5mm.With this moisture curing enzyme 25 liters of filling bed type enzyme reaction posts of packing into, moisture curing enzyme dress post amount is 52.6%(W/V), adding concentration to enzyme post way flow is 50% sucrose solution, the adjustment flow velocity is 0.12ecv/h, enzyme post effluent liquid is collected, vacuum and low temperature (60～70 ℃) directly is concentrated into 50～70% of original volume, add concentration and be 85～95% alcohol, stir, leave standstill, crystallization, suction filtration, washing 1～2 time, in 50 ℃ of dryings or vacuum-drying, promptly get 90～100% the pure and mild chromatographically pure palatinose white crystals of reagent.
Embodiment two
Cell fixation adopts unconventional interior gelling process, and other technical process are with embodiment one.
Attached used unit explanation: W/V represents: weight/volume, and WWC% represents: the heavy % of wet cell, ecv/h represents: free column volume/hour.
Claims (6)
1, a kind of method of utilizing immobilized enzyme to make sucrose inversion and preparing palatinose, comprise the strain fermentation propagation that contains the alpha-glucosyl transferring enzyme, concentrate, immobilization, conversion sucrose solution are that palatinose, palatinose conversion fluid concentrate, crystallization or directly solidify, drying step, it is characterized in that: (1) bacterial classification is a Seratiaplymuthica ATCC15928 culture of strains thing; (2) adopt the method fermentation propagation somatic cells that in substratum, flows the Ensure Liquid source while fermenting, with preparation alpha-glucosyl transferring enzyme (α-glucogyltrangferase), its fermention medium component contains 1-6% (W/V) sucrose, 0.5-5% (W/V) removes by filter sedimentary corn steep liquor, 0.01-0.4% (W/V) KCl, the nutrition source that stream adds is yeast extract paste or the corn steep liquor of 1-8% and the sucrose solution of 1-15% of concentration 1-7%, the fermenting process temperature is 25-35 ℃, pH5.0-8.5, stir, fermentation time is 8-24 hour, stream Ensure Liquid source, the limit of inner edge fermentation during fermentation, the time in stream Ensure Liquid source was controlled in 1-20 hour, in logarithmic phase results somatic cells; (3) use kaolin or clay as additive as upholder and adsorb somatic cells, with sodium alginate to embed, use CaCl again
2Solution is fixed-type with institute's embedded material, handle crosslinked with glutaraldehyde again, kill the immobilized enzyme of viable cell simultaneously with preparation alpha-glucosyl transferring enzyme, being used to the kaolin that adsorbs somatic cells and make upholder or the consumption of clay is the 2-30% (W/V) of fermented liquid, granularity is the 100-300 order, the consumption of said marine alga ester sodium is 1-6% (W/V), and the cell embedding amount is 5-50% (WWC%), used CaCl
2The concentration of solution is 0.1-0.8%; (4) immobilized enzyme is contained in the filling type column type reactor, stream adds the sucrose solution that concentration is 5-75%, makes sucrose solution be converted into the palatinose conversion fluid, adjusts the flow rate control transformation efficiency between 60-100%.
2, by the described method for preparing palatinose of claim 1, it is characterized in that said fermention medium component contains 2%(W/V) sucrose, 2%(W/V) remove by filter sedimentary corn steep liquor, 0.2%(W/V) KCl, the nutrition source that stream adds is the yeast extract paste of concentration 2% and 2% sucrose solution, but Continuous Flow adds or interval stream adds.
3, by the described method for preparing palatinose of claim 1, it is characterized in that as the kaolin of additive or the consumption of clay be the 6-9% of fermented liquid, granularity is the 150-200 order.
4, by the described method for preparing palatinose of claim 1, the preparation that it is characterized in that alpha-glucosyl transferring enzyme immobilized enzyme comprises (1), additive is added in the fermented liquid fully the whip attachment cell after, the cell of centrifugal results additive and absorption, perhaps earlier with the centrifugal cytoplasm that gets of fermented liquid, again additive is added wherein, fully mix adherent cell; (2) material (additive and adsorbed cell) and the sodium alginate with (1) results mixes, and forms gelling, fully stirs, and (3) clamp-on CaCl with the particle squeezer with embedded material
2Middle curing molding, the limit squish lip stirred more than 2 hours, and (4), suction filtration blot, and collect the immobilized enzyme that has been cured, with physiological saline or phosphoric acid buffer (pH7.0) washing; (5), will solidify enzyme and handle crosslinkedly with glutaraldehyde, killing living cell simultaneously is again with physiological saline or phosphoric acid buffer washing.
5, by the described method for preparing palatinose of claim 1, it is characterized in that with the palatinose conversion fluid directly concentrate the back by the alcohol extractive crystallization add seeded crystallization or with conversion fluid directly solidify, dry preparation palatinose product.
6, a kind of method of utilizing immobilized enzyme to make sucrose inversion and preparing palatinose, comprise the strain fermentation propagation that contains the alpha-glucosyl transferring enzyme, concentrate, immobilization, conversion sucrose solution are that palatinose, palatinose conversion fluid concentrate, crystallization or directly solidify, drying and other steps, it is characterized in that: (1) bacterial classification is a Seratia plymuthica ATCC15928 culture of strains thing; (2) adopt the method fermentation propagation somatic cells that in substratum, flows the Ensure Liquid source while fermenting, with preparation alpha-glucosyl transferring enzyme (α-glucosyltransferase), its fermention medium component contains 1-6%(W/V) sucrose, 0.5-5%(W/V) remove by filter sedimentary corn steep liquor, 0.01-0.4%(W/V) KCl, the nutrition source that stream adds is yeast extract paste or the corn steep liquor of 1-8% and the sucrose solution of 1-15% of concentration 1-7%, the fermenting process temperature is 25-35 ℃, pH5.0-8.5, stir, fermentation time is 8-24 hour, stream Ensure Liquid source, the limit of inner edge fermentation during fermentation, the time in stream Ensure Liquid source was controlled in 1-20 hour, in logarithmic phase results somatic cells; (3) by the immobilized enzyme of unconventional interior gelling process immobilized bacterium somatocyte, soon add CaCO behind the somatic cells of results slurry and the sodium alginate mixing with preparation alpha-glucosyl transferring enzyme
3Solution stirs, and adds Gluconolactone solution then, stirs fast to leave standstill more than 2 hours, and the material that is cured is made particle, immerses CaCl again
2Solidify in the solution, filter and do or cryodrying, said sodium alginate consumption is 1-5%(W/V), CaCO
3Consumption be 0.5-8.0%(W/V), the consumption of Gluconolactone is 0.5-4.0%(W/V), cell embedding concentration is 1-30%(WWC%), used CaCl
2The concentration of solution is 0.1-0.6M; (4) immobilized enzyme is contained in the filling type column type reactor, stream adds the sucrose solution that concentration is 5-75%, makes sucrose solution be converted into palatinose, adjusts the flow rate control transformation efficiency between 60-100%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN89100684A CN1030530C (en) | 1989-02-03 | 1989-02-03 | Preparation of parakin sugar by solidified alpha-glucosyl group transferase |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN89100684A CN1030530C (en) | 1989-02-03 | 1989-02-03 | Preparation of parakin sugar by solidified alpha-glucosyl group transferase |
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| CN1044676A CN1044676A (en) | 1990-08-15 |
| CN1030530C true CN1030530C (en) | 1995-12-20 |
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| CN89100684A Expired - Fee Related CN1030530C (en) | 1989-02-03 | 1989-02-03 | Preparation of parakin sugar by solidified alpha-glucosyl group transferase |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100396780C (en) * | 2005-06-22 | 2008-06-25 | 万代生物技术（深圳）有限公司 | Process for preparing isomaltitol |
Families Citing this family (6)
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| AU783125B2 (en) * | 2000-10-31 | 2005-09-29 | Dsm Ip Assets B.V. | Optimisation of fermentation processes |
| DE102011100772A1 (en) * | 2011-05-05 | 2012-11-08 | Evonik Degussa Gmbh | Process for the preparation of isomaltulose from plant juices |
| CN102286454B (en) * | 2011-07-01 | 2013-04-17 | 广西科学院生物研究所 | Method for preparing isomaltulose by polyvinyl alcohol immobilized sucrose isomerase producing bacteria |
| CN106591275A (en) * | 2016-11-30 | 2017-04-26 | 温县兴发生物科技有限公司 | Method for preparing calcium gluconate through co-immobilization of glucose oxidase and catalase |
| CN107557351A (en) * | 2017-09-07 | 2018-01-09 | 苏州艾科飞材料科技有限公司 | One kind processing mthod of white water from paper making embedded particles immobilization mixed enzyme and preparation method thereof |
| CN116179631B (en) * | 2023-04-27 | 2023-11-28 | 山东百龙创园生物科技股份有限公司 | Isomaltulose crystal and preparation method thereof |
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1989
- 1989-02-03 CN CN89100684A patent/CN1030530C/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100396780C (en) * | 2005-06-22 | 2008-06-25 | 万代生物技术（深圳）有限公司 | Process for preparing isomaltitol |
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| CN1044676A (en) | 1990-08-15 |
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