CN103695409A - Preparation method of immobilized enzyme and application of immobilized enzyme in geniposide conversion - Google Patents
Preparation method of immobilized enzyme and application of immobilized enzyme in geniposide conversion Download PDFInfo
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- CN103695409A CN103695409A CN201310737625.1A CN201310737625A CN103695409A CN 103695409 A CN103695409 A CN 103695409A CN 201310737625 A CN201310737625 A CN 201310737625A CN 103695409 A CN103695409 A CN 103695409A
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Abstract
The invention discloses a preparation method of an immobilized enzyme. The preparation method comprises the following steps: performing cross-linking on polysaccharide sodium alginate with genipin; adding beta-glucosidase, and polymerizing to obtain a polymerized solution; dropwise adding the polymerized solution into a CaCl2 solution to form immobilized beads; performing metal ion substitution on the immobilized beads in a MnSO4 solution to form hybrid manganese alginate immobilized particles. The invention further discloses a geniposide conversion method which comprises the following steps: performing continuous bioconversion on the prepared hybrid manganese alginate immobilized particles in a packed bed reactor (PBR), and hydrolyzing geniposide to prepare the genipin. The preparation method has the benefits that through an immobilization method, the immobilized enzyme is better in stability and hydrolysis performance in comparison with a free enzyme, the time for achieving the same hydrolysis effect is shortened by more than half, and the hydrolysis process is more complete; the duration time is more than 500 hours and the conversion rate is kept about 90% during the continuous conversion process in the PBR.
Description
Technical field
The present invention relates to a kind of preparation method and the application in Geniposide transforms thereof of immobilized enzyme, belong to biological technical field.
Background technology
Since the second half in last century, along with the innovation of technology of preparing, the immobilization of enzyme and microorganism cells obtains tremendous development, impels immobilized enzyme to be able to widespread use.Immobilized enzyme is compared with free state enzyme, the advantage such as have that enzyme and product are easily separated, stability is high, operate continuously, reaction process are easy to control, can reuse and can significantly reduce production costs, has been widely used in industrial circle and has carried out enhanced biological catalytic process.
According to the difference of immobilization carrier used, working method, and immobilized dominant mechanism, immobilization technology can be divided into entrapping method, absorption method, covalent method, crosslinking and microencapsulation, wherein with entrapping method application at most.Although basic process for fixation can be summed up as five above classes, but increasing fixing means is not limited to the use of single method, but come as required two kinds of compound uses and two or more fixation method to reach the object of the good immobilization material of preparation.Adopt compound method, can overcome the limitation of single method.For example, enzyme is carried out to physically trapping immobilization with gel, the easy leakage loss of embedded particles, mass transfer are poor, poor stability, and these deficiencies can solve by compound other different process for fixation.
In entrapping method, with macromolecular organic or inorganic polymkeric substance, enzyme or microorganism cells are wrapped in gel, these carriers are generally the materials such as alginate calcium, silica gel, gelatin, collagen, agar and polyacrylamide, wherein with alginate calcium, are most widely used again.Calcium-alginate-immobilized particle has the advantages such as grid-gap is little, resistance to mass transfer is little, preparation method is simple, cheap, but shortcoming is bad mechanical strength, is subject in system phosphatic impact and causes and break and disintegrate and stability is significantly reduced.Use linking agent to be fixed the physical strength that front modification can significantly improve calcium alginate gel.The most frequently used linking agent is glutaraldehyde, but has certain bio-toxicity, and biocompatibility is poor, has the security risk that enters transformation system and final product, thus be crosslinked gradually reaction temperature and, the natural biological linking agent of good biocompatibility replaces.Genipin (Genipin) is exactly a kind of good natural biological linking agent, can with the crosslinked biomaterials of preparing such as collagen, gelatin, chitosan, protein, for the immobilization of enzyme, the feature such as have that cytotoxicity is little, good biocompatibility, safe and anti-degradation capability are strong.On the other hand, genipin has important prospect in medicine, it is content seldom (for example content is only about 0.01% in cape jasmine dry fruit) in Gardenia Ellis fruit and the bark of eucommia, but its glycoside Geniposide (Geniposide) content abundant (for example content is about 4% in cape jasmine dry fruit) relatively.By screening high biological activity and Geniposide is there is to the beta-glucosidase of high substrate avidity, method with enzymolysis, can be under gentle condition, Efficient Conversion Geniposide is prepared genipin, and then can also synthesis of natural food dye---gardenia blue pigment (Gardenia blue pigment).In the preparation process of medicine material and food raw material, should select safe material and reagent, the safety of guarantee final product as far as possible.Select genipin as the crosslinked material of immobilization, can meet the requirement of target product to security.
Summary of the invention
One of the technical problem to be solved in the present invention is to provide a kind of preparation method of immobilized enzyme,
A preparation method for immobilized enzyme, comprises step: polysaccharide sodium alginate and genipin are crosslinked; Add beta-glucoside enzymatic polymerization to obtain polymer fluid; Above-mentioned polymer fluid is splashed into CaCl
2solution becomes immobilized spherule; By above-mentioned immobilized spherule at MnSO
4in solution, carry out metal ion displacement, form hydridization Manganese Alginate immobilization particle.
Further, above-mentioned beta-glucosidase is to utilize aspergillus niger, and obtain by microbe fermentation method.
Aspergillus niger Aspergillus niger bacterial strain, be through low energy ion implantttion technique modify the beta-glucosidase superior strain select (radiation research and radiation process journal, 2010,28(6): 345-351).
Above-mentioned beta-glucosidase also can be used commercially available zymin, does not affect immobilization effect.
Further, the preparation method of above-mentioned beta-glucosidase, comprises step: seed culture medium sterilizing, and after inoculation 2 shovel aspergillus niger spores, at 30 ℃, 200rpm cultivates and within 18 hours, forms seed liquor; The bottled 100mL fermention medium of 500mL triangle for fermentation shake flask, inoculates the above-mentioned seed liquor of 10mL after sterilizing, 33 ℃, 200rpm cultivates and obtains beta-glucosidase crude enzyme liquid; Above-mentioned beta-glucosidase crude enzyme liquid is obtained to throw out with salting-out process preliminary purification; Above-mentioned throw out is dissolved in to damping fluid, and dialysis desalting obtains beta-glucosidase.
Further, above-mentioned seed culture medium comprises 12g/L glucose, 10g/L CaCO
3, 3.0g/L KH
2pO
4, 4.0g/L NaNO
3, 2.0g/L (NH
4)
2sO
4, 0.5g/L MgSO
47H
2o, 0.5g/L CaCl
2, 0.0075g/L FeSO
47H
2o, 0.0025g/L MnSO
4h
2o, 0.002g/L ZnSO
4, 0.003g/L CoCl
2, pH5.5 – 6.0.
Further, above-mentioned fermention medium comprises 5% corn cob meal, 4% wheat bran, 0.6% (NH4)
2sO
4, 0.6% yeast extract, 0.6% glucose.
Further, above-mentioned beta-glucosidase crude enzyme liquid is with (NH
4)
2sO
4salting-out process carries out preliminary purification.
(NH
4)
2sO
4saturation ratio be 80%.
Further, above-mentioned throw out is dissolved in 20mM phosphate buffered saline buffer (pH7.0, Buffer A), is placed in semi-permeable membranes dialysis desalting and obtains beta-glucosidase.
The enzyme activity determination of above-mentioned beta-glucosidase.
P-nitrophenyl-β-D-the glycopyranoside (pNPG) of take is substrate, measures free and immobilized β-glucosidase enzyme activity.Per minute discharges 1 μ mol p-nitrophenyl and is defined as 1 Ge Meihuo unit (U).Concrete grammar is as follows: the pNPG that is 5mM with 0.2M acetic acid-sodium-acetate buffer (pH4.8, Buffer B) compound concentration.Get resolvase liquid 1mL, or immobilized enzyme 40 μ L, add 1mL Buffer B, mix with the 5mM pNPG solution of 1mL, 50 ℃ of water-baths 2 minutes, add the 1%Na of 2mL
2cO
3solution termination reaction, ultraviolet spectrophotometer 400nm measures absorption value, and reference standard curve determination calculates enzyme and lives.
Scanning electron microscope (SEM, Scanning Electron Microscopy) is analyzed.
Adopt surface topography and the inner structural features of 4 kinds of immobilized spherules of the above-mentioned preparation of scanning electron microscope analysis.Immobilized spherule, after lyophilize, is observed microstructure characteristic with scanning electron microscope (model: Sirion200, FEI, USA) under 5kV condition.
High-efficient liquid phase chromatogram HPLC testing conditions
The content of the gentle Geniposide of transformation system Zhong jing Buddhist nun adopts Waters high performance liquid phase system, is equipped with Kromasil C18 reversed-phase column (200mm * 4.6mm, 5 μ).Moving phase is methanol/water (45:55, v/v), flow velocity 1.0mL/min, and 25 ℃ of column temperatures, sample size 20 μ L, detect wavelength 238nm.The standard substance of genipin and Geniposide (purity 98%) are purchased from Delta natural organic-compound information center.
Two of the technical problem to be solved in the present invention is to provide a kind of application of this immobilized enzyme in Geniposide transforms that utilize.
A Geniposide method for transformation, carries out continuous bio-transformation by the hydridization Manganese Alginate immobilization particle of above-mentioned preparation with packed bed reactor, and hydrolysis Geniposide is prepared genipin.
Further, above-mentioned invert point is 40-50 ℃, concentration of substrate 1%-3%, and flow velocity 0.5-1 column volume/hour.
Transformation efficiency reaches 90% left and right, can transform continuously more than 500 hours.
Beneficial effect of the present invention:
By before fixing, modify, in Lalgine gel, add natural linking agent genipin fixing for enzyme, realize the interaction of enzyme-linking agent-carrier, effectively increase the physical strength of immobilization particle, and be fixed rear modification with the calcium ion in mn ion displacement immobilization particle, increase, to phosphatic resistance in system, strengthens stability and the work-ing life of immobilization particle.And prepare immobilized β-glucosidase with this method, safe and efficient conversion Geniposide is with preparation high purity genipin and high-quality gardenia blue pigment.
Accompanying drawing explanation
Fig. 1 is the HPLC collection of illustrative plates (A: Geniposide standard substance of Geniposide and genipin; B: genipin standard substance; C: the Geniposide in conversion fluid and genipin);
Fig. 2 is the schematic diagram of immobilized enzyme and resolvase conversion process;
Fig. 3 is the schematic diagram (A: bead 1 of immobilized spherule internal microstructure sem analysis; B: bead 2; C: bead 3; D: bead 4);
Fig. 4 is the rectangular coordinates schematic diagram of the selection of invert point;
Fig. 5 is the rectangular coordinates schematic diagram of concentration of substrate on the impact transforming;
Fig. 6 is for transforming the rectangular coordinates schematic diagram of the selection of flow velocity;
Fig. 7 is the rectangular coordinates schematic diagram that packed bed reactor transforms continuously.
Embodiment
According to drawings and embodiments the present invention is described in further detail below.
Embodiment 1
The preparation of immobilized enzyme bead
Prepare 100mL2% sodium alginate soln and 1000mL1%CaCl
2solution, prepares the immobilization particle of 4 types according to the following steps.
Bead 1: above-mentioned sodium alginate soln dropwise splashes into CaCl
2solution, and stirring gently, forms calcium alginate gel beads, harden after 12 hours with Buffer B wash 2 times standby.
Bead 2: get the above-mentioned sodium alginate soln of 20mL and add 50-125mg genipin, stir crosslinked 5-8 hour, then dropwise splash into CaCl
2solution balling-up, subsequent step is with bead 1.
Bead 3: get the above-mentioned sodium alginate soln of 80mL and add 200-500mg genipin, stir crosslinked 2-4 hour, then add the beta-glucosidase enzyme liquid of the above-mentioned preparation of 5-8mL, stir crosslinked 3-5 hour, then dropwise splash into CaCl
2solution balling-up, subsequent step is with bead 1.
Bead 4: by the bead of above-mentioned preparation 3, after Buffer B washing 2 times, move into 1%MnSO
4solution solution is replaced 24 hours under 4 ℃ of conditions, makes Mn
2+fully replace Ca
2+, form hydridization Manganese Alginate immobilization particle.
The immobilized spherule volume averaging of above-mentioned preparation is 40 μ L.
Investigate immobilized β-glucosidase changing effect prepared by different methods
According to above-mentioned enzyme activity determination method, measure the calcium-alginate-immobilized enzyme of hydridization (bead 3) of above-mentioned preparation and the enzyme work of hydridization Manganese Alginate immobilized enzyme (bead 4) and be respectively 3.1 and 5.7U/ml gel.Immobilized enzyme and the resolvase of getting same enzyme (34U) alive carry out transformation experiment, and wherein two kinds of immobilized spherules are got same volume.Transformation system is: substrate is 1% Geniposide (dissolving with Buffer B); According to different situations, add respectively CaCl
2or MnCl
2solution, CaCl
2or MnCl
2concentration in system reaches 0.1%; In 150mL triangular flask, pack conversion fluid 40mL into, water-bath concussion, 40 ℃ of temperature, with reference to figure 1, timing sampling is measured the content of Geniposide and genipin, calculates transformation efficiency.Conversion results as shown in Figure 2.Transformation efficiency and the conversion rate of immobilized enzyme are better than resolvase, react Geniposide after 8-10 hour and reach complete hydrolysis, and resolvase now Geniposide percent hydrolysis only have 70%, after 24 hours, just reach 90%, also be that immobilized enzyme is compared with resolvase, reach same hydrolysis effect time shorten over half, and hydrolysis is more thorough.In the immobilized enzyme of preparation, hydridization Manganese Alginate (bead 3) immobilization effect is better than hydridization alginate calcium (bead 4), and transformation efficiency is high by 5%.
Embodiment 3
The SEM image analysis of immobilized spherule
Different immobilized spherule internal microstructure SEM images as shown in Figure 3.Along with adding of linking agent genipin and beta-glucosidase enzyme liquid, the internal structure of bead 1 to 4 presents the trend becoming increasingly complex, also given their physical strengths of becoming better and better, and good physical strength contributes to increase physically impaired resistibilitys such as stirrings.And with Mn
2+replace the Ca in calcium alginate gel
2+, can resist phosphoric acid salt in enzyme liquid or in the transformation system splitting action to gel, the work-ing life that strengthens saccharase.
Invert point preferably
Under 40,50 and 60 ℃ of conditions, investigate the changing effect of immobilized enzyme to 1% Geniposide, result is as shown in Figure 4.Conversion rate increases along with the raising of invert point, transforms after 12 hours, and at above-mentioned three kinds of temperature, Geniposide is all transformed completely.Although consider that higher temperature is favourable to enzyme reaction, the unstable of enzyme be can increase and the coefficient of expansion of immobilization particle and the risk of unfavorable chemical reaction increased, therefore select 40-50 ℃ of temperature, be suitable invert point.
Concentration of substrate preferably
Under the conversion condition of 50 ℃, investigate the changing effect of immobilized enzyme to 1%, 3%, 5% and 8% Geniposide in the above-described embodiments, the results are shown in Figure shown in 5.1% Geniposide is that hydrolyzable is complete after transforming 4 hours, and along with the raising of concentration of substrate, percent hydrolysis reduces gradually, and high concentration substrate hydrolysis completely just needs to extend the reaction times.Consider the water-soluble little characteristic of hydrolysis rate and genipin, concentration of substrate selects 1%-3% proper.
The study on the stability of enzyme
Packed bed reactor (PBR) with jacketed water-bath carries out serialization transformation experiment.PBR reactor specification is 1.6cm * 20cm, fills the immobilized enzyme bead of 30mL volume in chamber, and substrate is 1% Geniposide and 0.1%MnCl
2(with Buffer B solution preparation).When flow velocity be 0.5 column volume/hour, the percent hydrolysis of Geniposide is greater than 90%(Fig. 6).With this PBR reactor, flow velocity is 0.5 column volume/while hour transforming continuously, can continue to transform more than 500 hours, and transformation efficiency maintains 90% left and right, and immobilized enzyme shows quite high catalytic stability (Fig. 7).
Embodiment 7
The separation and purification of genipin in conversion fluid
The conversion fluid of the above-mentioned PBR reactor of flowing through, with macroporous resin adsorption genipin.Macroporous adsorbent resin can be selected the models such as HPD100, HPD100A, HPD400, HZ801, D301, D151, selects HPD100 macroporous resin wet method dress post in this example, and on conversion fluid, column equilibration is 1 hour.With 20%-100% ethanolic soln stepwise elution, to collect each elutriant and detect genipin purity with HPLC method, in 40% alcohol elutriant, genipin purity is 90%, concentrated by crystallization and recrystallization, purity to 98%; In 100% alcohol elutriant, genipin purity reaches 98%, concentrates and obtains 98% purity genipin solid by One-step crystallization.The crystallization obtaining is analyzed through LC-MS, FTIR, and feature is consistent with genipin standard substance.
The high purity genipin of above-mentioned acquisition can further be prepared high-quality natural food colour---gardenia blue pigment in 80 ℃ of water-bath 0.5-5 hour with a-amino acids such as L-Ala, Methionin, Histidine, glycine, tryptophane, L-glutamic acid, glutamine, phenylalanines.In this example, select glycine to react and produce gardenia blue pigment with genipin.Genipin and amino acid whose molar concentration rate are 1:10-10:1.The gardenia blue pigment that adsorbs above-mentioned generation with HPD100 macroporous resin column, with 80% ethanolic soln wash-out, collects elutriant after deionization washing, concentrating under reduced pressure, and lyophilize obtains gardenia blue pigment, its OD value (absorbance ratio when 238nm and 590nm, A
238/ A
590) be 0.20-0.24, reach good refining purification effect.
Above-described embodiment is only explanation technical conceive of the present invention and feature, and its object is to allow the personage who is familiar with this art can understand content of the present invention and be implemented, and can not limit the scope of the invention with this.All equivalences that spirit is done according to the present invention change or modify, and all should be encompassed in protection scope of the present invention.
Claims (9)
1. a preparation method for immobilized enzyme, is characterized in that, comprises step: polysaccharide sodium alginate and genipin are crosslinked; Add beta-glucoside enzymatic polymerization to obtain polymer fluid; Described polymer fluid is splashed into CaCl
2solution becomes immobilized spherule; By described immobilized spherule at MnSO
4in solution, carry out metal ion displacement, form hydridization Manganese Alginate immobilization particle.
2. the preparation method of immobilized enzyme according to claim 1, is characterized in that, described beta-glucosidase is to utilize aspergillus niger, and obtain by microbe fermentation method.
3. the preparation method of immobilized enzyme according to claim 2, is characterized in that, the preparation method of described beta-glucosidase, comprises step: seed culture medium sterilizing, and inoculated aspergillus niger spore is cultivated and is formed seed liquor; After fermention medium sterilizing, inoculate described seed liquor, cultivate and obtain beta-glucosidase crude enzyme liquid; Described beta-glucosidase crude enzyme liquid is obtained to throw out with salting-out process preliminary purification; Described throw out is dissolved in to damping fluid, and dialysis desalting obtains beta-glucosidase.
4. the preparation method of immobilized enzyme according to claim 3, is characterized in that, described seed culture medium comprises 12g/L glucose, 10g/L CaCO
3, 3.0g/L KH
2pO
4, 4.0g/L NaNO
3, 2.0g/L (NH
4)
2sO
4, 0.5g/L MgSO
47H
2o, 0.5g/L CaCl
2, 0.0075g/L FeSO
47H
2o, 0.0025g/L MnSO
4h
2o, 0.002g/L ZnSO
4, 0.003g/L CoCl
2, pH5.5 – 6.0.
5. the preparation method of immobilized enzyme according to claim 3, is characterized in that, described fermention medium comprises 5% corn cob meal, 4% wheat bran, 0.6% (NH4)
2sO
4, 0.6% yeast extract, 0.6% glucose.
6. the preparation method of immobilized enzyme according to claim 3, is characterized in that, described beta-glucosidase crude enzyme liquid is with (NH
4)
2sO
4salting-out process carries out preliminary purification.
7. the preparation method of immobilized enzyme according to claim 3, is characterized in that, described throw out is dissolved in phosphate buffered saline buffer, is placed in semi-permeable membranes dialysis desalting and obtains beta-glucosidase.
8. the immobilized enzyme as prepared in claim 1-7 Arbitrary Term of a utilization is at Geniposide method for transformation, it is characterized in that, the hydridization Manganese Alginate immobilization particle of described preparation is carried out to continuous bio-transformation with packed bed reactor, and hydrolysis Geniposide is prepared genipin.
9. Geniposide method for transformation according to claim 8, is characterized in that, described invert point is 40-50 ℃, concentration of substrate 1%-3%, and flow velocity 0.5-1 column volume/hour.
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| CN112251430A (en) * | 2020-10-14 | 2021-01-22 | 浙江农林大学 | Method for extracting gardenia blue pigment |
| CN114164238A (en) * | 2021-10-25 | 2022-03-11 | 福州一诺维生物科技有限公司 | Enzymatic synthesis method of L-tyrosine |
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| CN112251430A (en) * | 2020-10-14 | 2021-01-22 | 浙江农林大学 | Method for extracting gardenia blue pigment |
| CN114164238A (en) * | 2021-10-25 | 2022-03-11 | 福州一诺维生物科技有限公司 | Enzymatic synthesis method of L-tyrosine |
| CN114164238B (en) * | 2021-10-25 | 2024-03-26 | 福州一诺维生物科技有限公司 | Enzymatic synthesis method of L-tyrosine |
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Application publication date: 20140402 |