CN104004699A - Method for producing lactulose through whole-cell catalysis - Google Patents
Method for producing lactulose through whole-cell catalysis Download PDFInfo
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Abstract
The invention discloses a method for producing lactulose through whole-cell catalysis, and belongs to the field of the food biotechnology. According to the method, recombinant escherichia coli E. coli BL 21 (DE3) for producing cellobiose epimerase is taken as production bacterial strains, lactose is used for replacing isopropyl-beta-D-sulfo-galactoside (IPTG) and taken as an inductive agent for fermental cultivation, and thalluses obtained through centrifugation are subjected to ethyl alcohol permeabilization and vacuum freeze drying to serve as a cell biocatalyst for directly converting the lactose to produce the lactulose. The maximum percent conversion of the lactulose can reach 65.1%, the concentration of the lactulose reaches 390.6 g/L, the production rate of the lactulose reaches 195.3 g/(L*h), and the production quantity of by-product epidepride lactulose is smaller than 2%(w/w). According to the method, microbial cells are directly used for converting the lactose to produce the lactulose, and the method is simple and easy to implement, avoids the loss of enzyme activity in the separation and purification process, greatly reduces the cost of producing the lactulose through an enzymic method and is beneficial for achieving industrialization of lactulose production through the enzymic method early.
Description
Technical field
A kind of method that the present invention relates to whole-cell catalytic galactopoiesis fructose, belongs to technical field of food biotechnology.
Background technology
In recent years, obesity, diabetes and cardiovascular disorder crowd present the trend that increases year by year and become younger, the focus that makes functional sweetener low in calories, to have greater functionality characteristic become vast food practitioner research and development and pay close attention to.There is in the world 70% people to there is lactose intolerance, after EDIBLE LACTOSE, can cause serious gastrointestinal problems, the annual lactose that has in the world millions of tons causes environmental pollution because can not get further utilizing to be wasted simultaneously, how to utilize efficiently lactose to become urgent need to solve the problem.And the lactulose obtaining by lactose isomerization is just becoming the new focus of consumers in general and food practitioner concern with its superior functional performance.
Lactulose (4-O-β-D-galactopyranose base-D-Fructose) is lactose isomerization product, is functional oligose a kind of synthetic and can not be digested.Nineteen fifty-seven Austria scholar Petuely finds, by adding lactulose, can make the baby intestinal flora of artificial feeding and baby that breast milk is fed be close, and confirmed that first lactulose is a kind of bifidus bacillus multiplicaiton factor.In Japan, lactulose is identified as " specific food for health care " additive and is widely used.At present worldwide, lactulose is widely used in the industries such as medicine, health care, foodstuff additive and animal-feed.In clinical pharmaceutical industries, lactulose is mainly used in treating hepatogenic encephalopathy, constipation, intracellular toxin and hypoglycemic, decreasing cholesterol etc., in food service industry, extensively being added in baby milk powder, soft drink and healthcare products, is mainly the effect of its bifidus bacillus multiplicaiton factor of performance.
The output of the annual lactulose in the whole world has exceeded 60000 tons at present, the minority enterprises such as Jin You Dalian Chemical Research &. Design Inst. of domestic lactulose manufacturing enterprise, Dandong rehabilitation pharmaceutical Co. Ltd, product is low-purity lactulose syrup, and the high-purity lactulose of domestic pharmaceutical grade is whole dependence on import almost.And domestic industryization is produced and is all relied on chemical method, and Production by Enzymes lactulose is unrealized industrialization also.Chemical method produce lactulose have transformation efficiency high, can realize the advantages such as scale operation, but the shortcoming of chemical isomerization method is also very outstanding, as many in byproduct of reaction, desalination complicated operation, energy consumption are high and have a security hidden trouble etc.For overcome chemical isomerization method exist various drawbacks, biological enzyme transform lactose prepare lactulose because of its product of preparing single, Nantural non-toxic, meets consumers in general and pursues the consumer psychology of natural product and become current study hotspot.
At present mainly contain beta-glycosidase, beta-galactosidase enzymes and derive from the cellobiose epimerase of Caldicellulosiruptor saccharolyticus for the production of the enzyme of lactulose.The wherein transformation efficiency of beta-glycosidase and beta-galactosidase enzymes lower (7.5-30%), obtains it and is not suitable for the suitability for industrialized production of lactulose.When the pure enzyme of use cellobiose epimerase carries out the Production by Enzymes of lactulose, in reaction system, need to add a large amount of boric acid, boric acid is a kind of harmful material, remove boric acid and need to adopt the de-boron resin of membrane separation technique in conjunction with costliness, this can increase the industrial cost of lactulose, brings food safety hidden danger simultaneously.
Efficient Production by Enzymes how effectively to avoid the problems referred to above and realize lactulose will become the new task that foodstuffs industry practitioner faces.
Summary of the invention
First technical problem that the present invention will solve is to provide the genetic engineering bacterium of a strain heterogenous expression cellobiose epimerase, be taking intestinal bacteria as host, taking pET-28a (+) as expression vector, express the cellobiose epimerase of nucleotide sequence as shown in SEQ ID NO.1.
The preferred E.coli BL21 of described intestinal bacteria (DE3).
Second technical problem that the present invention will solve is to provide a kind of method of inducing described genetic engineering bacterium to express cellobiose epimerase, is to utilize IPTG or lactose-induced restructuring E.coli BL21 (DE3) fermentation to produce cellobiose epimerase.
Described method is preferably using the recombination bacillus coli E.coli BL21 (DE3) of the product cellobiose epimerase that builds as producing bacterial strain, and activated, enlarged culturing is to logarithmic phase OD
600when=0.2-2.0, add isopropyl-β-D-thiogalactoside(IPTG) (IPTG) to final concentration 0.05-1.2mM or to add lactose to final concentration be 0.5-50g/L, 15-45 DEG C, 150-250r/min shaking table is cultivated 8-24h, centrifugal collection thalline.
The 3rd technical problem that the present invention will solve is to provide a kind of method of utilizing described genetic engineering bacterium whole-cell catalytic to produce lactulose, mainly comprises the following steps: (1) produces cellobiose epimerase with lactose-induced cultivation restructuring E.coli BL21 (DE3); (2) collect cultivate the thalline that obtains by ethanol thoroughly change, vacuum lyophilization processes as biocatalyst cell; (3) biocatalyst cell is directly used in and transforms lactose production lactulose.
Described step (2) is preferably suspended in the centrifugal thalline of collecting in 1L fermented liquid in the ethanol of 1-100mL1-90% (v/v), at 4-30 DEG C, mixing carrying out of 5-120min processes, centrifugal collecting precipitation, under the 4 DEG C of conditions of bacterium powder that obtain through vacuum lyophilization, preserve, bacterium powder is directly used in and transforms lactose production lactulose as biocatalyst cell.With the enzyme work after lactose-induced full cell permeabilization be 300-400U/g.
Preferred 40% (v/v) ethanol when describedization processed.
Described step (3) is following steps preferably:
1. lactose is mixed with to 50-800g/L with damping fluid, regulates pH to 6.0-9.5 as substrate solution;
2. biocatalyst cell is added to substrate solution, conversion condition is: substrate lactose concn 50-800g/L, biological catalyst taking the enzyme work of freeze-dried vaccine powder to the consumption of substrate solution as 1-100U/mL, initial pH6.0-10.0, temperature of reaction is 40-95 DEG C, and the reaction times is 1-24h.
The preferred 600g/L of described substrate lactose concn, the preferred 12.5U/mL of bacterium powder addition, preferably 80 DEG C of temperature of reaction, react preferred time 3h.
When described substrate solution preparation, damping fluid used is: Tris-HCl damping fluid, phosphate buffered saline buffer, PIPES damping fluid, Na
2hPO
4one in-citrate buffer solution, boric acid-borate buffer solution, Britton-Robinson damping fluid and the aqueous solution.
The present invention using build product cellobiose epimerase recombination bacillus coli E.coli BL21 (DE3) as production bacterial strain, substitute IPTG using lactose and produce cellobiose epimerase as inductor induction, the thalline of collecting by ethanol thoroughly change, vacuum lyophilization processes as biocatalyst cell, in different buffer solution systems, transform lactose using lactose as single substrate and produce lactulose.Lactulose transformation efficiency reaches as high as 65.1%, and lactulose concentration reaches 390.6g/L, and the productivity of lactulose reaches 195.3g/ (Lh), and by product is less than 2% (w/w) according to a lactose growing amount.
The present invention first utilizes lactose to substitute IPTG as producing enzyme inducer, produce reference is provided for the colibacillary industrial fermentation of recombinating, utilize the free cell that obtains through lyophilize simultaneously directly, thoroughly change cell and transform lactose as biocatalyst cell and produce lactulose, effectively having avoided the enzyme of intracellular enzyme in fragmentation, extraction and separation and purification process to live loses, effectively reduce the cost of Production by Enzymes lactulose, for industrial Production by Enzymes lactulose provides reference and reference.The method catalyzed conversion lactose in phosphate buffered liquid system is produced when lactulose, phosphoric acid salt is nontoxic and consumption is less, in phosphate system, by product is less than 2% (w/w) according to the amount of a lactose simultaneously, simplify follow-up separation and purification, for the Production by Enzymes of high-purity lactulose provides a kind of new approaches.This invention is prepared lactulose by biological enzyme, product is clean nontoxic, separation and purification process is relatively simple and energy consumption is little, is conducive to realize resource-conserving, environmentally friendly production, simultaneously for the suitability for industrialized production of the Production by Enzymes lactulose that promotes cleaning, efficient, environmental protection provides useful reference.
Embodiment
The enzyme work of the resolvase of cellobiose epimerase is defined as: at 80 DEG C, and pH7.5, in 50mM Tris-HCl damping fluid, per minute catalysis lactose generates the required enzyme amount of 1 μ mol lactulose and is defined as 1U.When mensuration enzyme is lived, reaction substrate lactose concn is 50g/L, reaction times 20min.
The mensuration that the enzyme of full cell bacterium powder is lived: get 0.01g through cryodesiccated bacterium powder, be dissolved in 1mL pH7.550mM Tris-HCl damping fluid, after mixing, getting 100 μ L bacteria suspensions joins in 900 μ L lactose solutions, final system lactose concn is 50g/L, after mixing, at 80 DEG C, react 20min, the go out amount of the lactulose generating in sampling and measuring system after enzyme 5min of boiling water bath, generate the required full cell bacterium powder amount of 1 μ mol lactulose with per minute catalysis lactose and be defined as 1U, follow-up enzyme concentration is with the enzyme activity metering of bacterium powder.
The chemical determination of lactulose: be hydrolyzed lactulose under acidic conditions, hydrolysate reacts with cysteine hydrochloride-tryptophane developer, produce and absorb at 518nm place, and under optimal conditions, lactose and by product are very faint according to the colour developing of a lactose, the qualification of lactulose is disturbed very low, and the detectability of lactulose very low be 0.58 μ g/mL (Zhang Zhong, et al.International Journal of Food Science and Technology, 2010).
The separation and purification of lactulose reaction solution: the centrifugal 10min under the rotating speed of 8000r/min of the lactulose reaction solution after conversion reaction is finished collects the reaction of full cell for next batch, lactulose solution after centrifugal removes salinity through nanofiltration operation after being diluted to certain multiple, pass through again vacuum concentration, lactulose crystals is separated out, after dry, obtain highly purified lactulose finished product, and remaining lactose joins in next batch reaction as reaction substrate continuation reaction after being diluted to finite concentration; Or by the lactulose solution after centrifugal after the desalination of industrial chromatography post again through ion exchange resin column separating lactulose and lactose.
Embodiment 1
Recombination bacillus coli E.coli BL21 (DE3) structure condition is as follows: produce cellobiose epimerase gene source in Caldicellulosiruptor saccharolyticus DSM8903, its nucleotide sequence is as shown in SEQ ID NO.1, host cell is E.coli BL21 (DE3), carrier is pET-28a (+), uses respectively IPTG and lactose as inductor induction product enzyme.
The recombination bacillus coli E.coli BL21 (DE3) of the product cellobiose epimerase building is carried out to fermentation culture as producing bacterial strain, and fermentation culture step is as follows:
(1) get-80 DEG C, the bacterial classification in 30% (v/v) glycerine, connects bacterium amount by 1%, and 37 DEG C, 200r/min shaking table 12h is as seed culture fluid;
(2) fermention medium is selected LB liquid nutrient medium, adds 50mL LB liquid nutrient medium, 121 DEG C in 250mL triangular flask, 20min sterilizing, adds seed culture fluid by 1% inoculum size after cooling, adds kalamycin to final concentration 10 μ g/mL, 37 DEG C, 200r/min shaking table 1-2h to OD
600=0.6, wherein one group adds IPTG to final concentration 0.4mM, and 200r/min continues shaking table and cultivates 15h at 25 DEG C; It is 10g/L that another group is added lactose to final concentration, and under 25 DEG C of conditions, 200r/min continues shaking table cultivation 20h.
(3) centrifugal collection thalline, thalline is resuspended in the damping fluid of pH7.5, get respectively the fermented liquid ultrasonication mensuration enzyme of equivalent and live, add the fermented liquid average enzyme alive 340U/L of IPTG as inductor, and add lactose to reach 800U/L as the average enzyme work of fermented liquid of inductor.With lactose-induced, the thalline quantity in fermented liquid is approximately 2 times of cell quantity in the fermented liquid of IPTG induction, and total enzyme is lived also more than 2 times.
Embodiment 2
The thalline that in embodiment 1, fermentation obtains using lactose as inductor carries out vacuum lyophilization and obtains bacterium powder, directly transform lactulose using the bacterium powder of freeze-drying as biocatalyst cell catalysis lactose, substrate lactose is dissolved in Tris-HCl (pH7.5) damping fluid, concentration 600g/L, bacterium powder addition is 12.5U/mL, 80 DEG C of temperature of reaction, reaction times 3h, in end reaction liquid, lactulose content is 251.4g/L, and lactulose transformation efficiency reaches 41.9%.
Embodiment 3
Under 4 DEG C of conditions of thalline 40% (v/v) ethanol that in embodiment 1, fermentation obtains using lactose as inductor, saturatingization processed 15min, vacuum lyophilization is changed bacterium powder thoroughly, thoroughly change bacterium powder using gained and directly transform lactose production lactulose as biocatalyst cell, substrate lactose concn 600g/L, damping fluid is Tris-HCl damping fluid (pH7.5), 80 DEG C of temperature of reaction, bacterium powder addition 12.5U/mL, reaction times 3h, in end reaction liquid, lactulose content is 310g/L, lactulose transformation efficiency reaches 51.8%, be 49g/L according to a lactose-content, transformation efficiency according to a lactose reaches 8.2%.
Embodiment 4
Under 4 DEG C of conditions of thalline 40% (v/v) ethanol that in embodiment 1, fermentation obtains using lactose as inductor, saturatingization processed 15min, vacuum lyophilization is changed bacterium powder thoroughly, saturatingization bacterium powder using freeze-drying transforms lactulose as biocatalyst cell catalysis lactose, substrate lactose concn 600g/L, damping fluid is phosphate buffered saline buffer (pH7.5), 80 DEG C of temperature of reaction, bacterium powder addition 12.5U/mL, reaction times 2h, in end reaction liquid, lactulose content is 390.6g/L, lactulose transformation efficiency reaches 65.1%, the production efficiency of lactulose reaches 195.3g/ (Lh), be 11.6g/L according to a lactose-content, transformation efficiency according to a lactose only has 1.9%.In phosphate system, by product is less than 2% (w/w) according to the amount of a lactose, has simplified follow-up separation and purification, is conducive to the Production by Enzymes of high-purity lactulose.
Although the present invention with preferred embodiment openly as above; but it is not in order to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, therefore protection scope of the present invention should be with being as the criterion that claims were defined.
Claims (10)
1. the genetic engineering bacterium of a strain heterogenous expression cellobiose epimerase, is taking intestinal bacteria as host, expresses the cellobiose epimerase of nucleotide sequence as shown in SEQ ID NO.1.
2. genetic engineering bacterium according to claim 1, is characterized in that, described intestinal bacteria are E.coli BL21 (DE3), taking pET-28a (+) as expression vector.
3. inducing genetic engineering bacterium described in claim 1 to express a method for cellobiose epimerase, is to utilize IPTG or the fermentation of lactose-induced recombination bacillus coli to produce cellobiose epimerase.
4. method according to claim 3, is characterized in that, using the recombination bacillus coli E.coli BL21 (DE3) of the product cellobiose epimerase that builds as producing bacterial strain, activated, enlarged culturing is to logarithmic phase OD
600when=0.2-2.0, add isopropyl-β-D-thiogalactoside(IPTG) to final concentration 0.05-1.2mM or to add lactose to final concentration be 0.5-50g/L, 15-45 DEG C, 150-250r/min shaking table is cultivated 8-24h, centrifugal collection thalline, obtains the recombination bacillus coli of having expressed cellobiose epimerase.
5. the method for genetic engineering bacterium whole-cell catalytic galactopoiesis fructose described in an application rights requirement 1, it is characterized in that, mainly comprise the following steps: (1) produces cellobiose epimerase with lactose-induced cultivation restructuring E.coli BL21 (DE3); (2) collect cultivate the thalline that obtains by ethanol thoroughly change, vacuum lyophilization processes as biocatalyst cell; (3) biocatalyst cell is directly used in and transforms lactose production lactulose.
6. method according to claim 5, it is characterized in that, described step (2) is suspended in the centrifugal thalline of collecting in 1L fermented liquid in the ethanol of 1-100mL1-90%, at 4-30 DEG C, mix 5-120min, centrifugal collecting precipitation, under the 4 DEG C of conditions of bacterium powder that obtain through vacuum lyophilization, preserve, bacterium powder is directly used in and transforms lactose production lactulose as biocatalyst cell.
7. method according to claim 5, is characterized in that, described step (3) specifically comprises the following steps:
1. lactose is mixed with to 50-800g/L with damping fluid, regulates pH to 6.0-9.5 as substrate solution;
2. biocatalyst cell is added to substrate solution, conversion condition is: substrate lactose concn 50-800g/L, biological catalyst taking the enzyme work of freeze-dried vaccine powder to the consumption of substrate solution as 1-100U/mL, initial pH6.0-10.0, temperature of reaction is 40-95 DEG C, and the reaction times is 1-24h.
8. method according to claim 5, is characterized in that, substrate solution is with Tris-HCl damping fluid, phosphate buffered saline buffer, PIPES damping fluid, Na
2hPO
4one in-citrate buffer solution, boric acid-borate buffer solution, Britton-Robinson damping fluid or water is as solvent.
9. method according to claim 5, is characterized in that, step (3) is at Tris-HCl damping fluid, phosphate buffered saline buffer, PIPES damping fluid, Na
2hPO
4in-citrate buffer solution, boric acid-borate buffer solution, Britton-Robinson damping fluid or water, carry out.
10. method according to claim 7, is characterized in that, substrate lactose concn is 600g/L, and bacterium powder addition is 12.5U/mL, and temperature of reaction is 80 DEG C, reaction times 3h, pH7.5.
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Cited By (3)
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| CN104313009A (en) * | 2014-10-21 | 2015-01-28 | 江南大学 | Method for immobilizing cellobiose epimerase whole cells |
| CN104357423A (en) * | 2014-11-26 | 2015-02-18 | 吉林农业大学 | Method capable of implementing efficient induction expression of recombinant protein by D-lactitol |
| CN113699087A (en) * | 2021-08-23 | 2021-11-26 | 齐鲁工业大学 | Lactobacillus plantarum engineering strain for converting lactose to generate lactulose and construction method and application thereof |
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| CN104313009A (en) * | 2014-10-21 | 2015-01-28 | 江南大学 | Method for immobilizing cellobiose epimerase whole cells |
| CN104357423A (en) * | 2014-11-26 | 2015-02-18 | 吉林农业大学 | Method capable of implementing efficient induction expression of recombinant protein by D-lactitol |
| CN113699087A (en) * | 2021-08-23 | 2021-11-26 | 齐鲁工业大学 | Lactobacillus plantarum engineering strain for converting lactose to generate lactulose and construction method and application thereof |
| CN113699087B (en) * | 2021-08-23 | 2023-08-15 | 齐鲁工业大学 | Lactobacillus plantarum engineering strain for converting lactose to generate lactulose, construction method and application thereof |
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Application publication date: 20140827 |