CN101705222A - Method for preparing immobilized enzyme by using spherical bacterial cellulose as carrier - Google Patents

Method for preparing immobilized enzyme by using spherical bacterial cellulose as carrier Download PDF

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Publication number
CN101705222A
CN101705222A CN200910198537A CN200910198537A CN101705222A CN 101705222 A CN101705222 A CN 101705222A CN 200910198537 A CN200910198537 A CN 200910198537A CN 200910198537 A CN200910198537 A CN 200910198537A CN 101705222 A CN101705222 A CN 101705222A
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bead
enzyme
immobilized enzyme
balls
bacteria cellulose
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CN200910198537A
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Chinese (zh)
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洪枫
邹敏
杨光
杨雪霞
曹张军
杜明
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东华大学
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Abstract

The invention relates to a method for preparing immobilized enzyme by using spherical bacterial cellulose as a carrier. The method comprises the following steps of: inoculating activated slant strains into a seed culture medium for cultivation to obtain a liquid seed, shifting the liquid seed into a fermentation medium under a condition of 80 to 200 rpm at the temperature between 25 and 30 DEG C for 48 to 96 hours, and carrying out decantation to obtain bacterial cellulose balls; processing the bacterial cellulose balls in a water bath at the temperature between 80 and 90 DEG C for 30 to 120 min with 0.1 percent NaOH, rinsing the balls with deionized water, collecting the balls, neutralizing residual alkali liquor in the balls with 0.1 percent acetic acid, standing, rinsing the balls with the deionized water, taking the balls out, absorbing surface moisture, and freezing and drying the balls to obtain cellulose balls; and finally, preparing the immobilized enzyme by a physical adsorption method or an adsorption-crosslinking method. The spherical bacterial cellulose balls can effectively adsorb biological enzyme, maximally keep enzyme activity, and have safety and environmental protection. The method is simple and easy to operate and has strong controllability.

Description

A kind of is the method for preparing carriers immobilized enzyme with the spherical bacterial cellulose
Technical field
The invention belongs to the enzyme immobilization technology field, particularly relating to a kind of is the method for preparing carriers immobilized enzyme with the spherical bacterial cellulose.
Background technology
Because enzyme has Substratspezifitaet preferably, has significant application value in a lot of fields.But the free zymoprotein is subject to environmental influence and the sex change inactivation in actual application, and is difficult for separating to realize reusable purpose from reaction system, and this has limited the industrial applications of enzyme to a certain extent.Enzyme immobilization technology is to realize that enzyme repeats to use continuously and improves its stable effective means.The fixation support of enzyme comprises that mainly inorganic carrier material (as silicon-dioxide, gac, porous glass etc.), organic synthesis macromolecular material (as polyacrylamide, polyethylene, polystyrene, urethane etc.) and natural macromolecular material are (as structural albumen at present, chitosan, but still do not have and utilize the report of bacteria cellulose bead sodium alginate etc.), for fixing of carrier enzyme.The existing fixed zymotechnic mainly contains 5 kinds: (1) microencapsulation: this method is to utilize semi-permeable membranes can stop macromolecular enzyme to spread out from capsule, and the characteristic that small molecules reactant and product can freely spread realizes; (2) inert support absorption method: enzyme can pass through Van der Waals force, hydrogen bond, and hydrophobic interaction, electrostatic interactions etc. are adsorbed onto on the insoluble upholder, thereby reach immobilized purpose; (3) covalent cross-linking method: utilize bifunctional reagent can make molecule and molecule crosslinked and form macromolecular particle, thereby realize the immobilization of enzyme; (4) glue investigate things reason entrapping method: this embedding is to realize that by the polymerization in the aqueous solution of enzyme of one-tenth glue material the most frequently used is polyacrylamide gel, and other also has amylan etc.; (5) with water insoluble carrier covalent attachment method:, make enzyme and carrier produce covalent attachment by covalent coupling according to the physico-chemical property of enzyme and carrier itself.
(bacterial cellulose is a kind of by the outer biological fiber of microorganism synthetic high purity born of the same parents BC) to bacteria cellulose.The primary structure of bacteria cellulose is similar to plant cellulose, the straight-chain polysaccharide that is formed by connecting with β-1,4 glycosidic link by the Glucopyranose monomer, but the fibrous texture of BC but differs widely with vegetable fibre.BC is made up of microfibrous, and it is banded that this microfibrous is, and its thickness is about 0.1 μ m, than little two orders of magnitude of thickness (10 μ m) of vegetable fibre.In addition, this microfibrous has tangible reticulated structure.Because the characteristic of BC microstructure, it has the character of many uniquenesses, excellent mechanical intensity for example, superfine nano fibre network, biodegradability, bigger specific surface area, electroconductibility and high characteristics such as degree of crystallinity.In recent years, there is research report to utilize bacteria cellulose film after the chemical modification to be used for the immobilization of microorganism cells for carrier, microorganism cells after its immobilization is good to the degradation effect of malachite green dyestuff, still can keep stable percent of decolourization under the constant situation of nutrient solution initial pH value and dyestuff starting point concentration.Bacteria cellulose has become the focus of domestic and international research as a kind of novel nanofiber biomaterial.Bacteria cellulose has nanofiber network structure, so this filamentary material has the very high specific surface and the gap structure of One's name is legion, helps absorption and contains zymoprotein.
Summary of the invention
It is the method for preparing carriers immobilized enzyme with the bacteria cellulose bead that technical problem to be solved by this invention provides a kind of, and it is easy to have technology, workable, and enzyme is lived and lost advantages such as little.
The invention provides a kind of is the method for preparing carriers immobilized enzyme with the spherical bacterial cellulose, comprising:
(1) preparation of bacteria cellulose bead
The activatory slant strains is inserted the seed culture medium of pH4.8～5.2, under 25～30 ℃, 100～200rpm condition, cultivate 12～16h and make liquid seeds, with volume percent is that 4～10% inoculum sizes are forwarded to liquid seeds in the fermention medium of pH4.8～5.2, contain 200～300mL fermentation culture in every 500mL Erlenmeyer flask, 25～30 ℃, cultivate 48～96h under 80～200rpm condition, decantation gets the bacteria cellulose bead;
(2) processing of bacteria cellulose bead
It is that 0.1% NaOH handles 30～120min in 80～90 ℃ of water-baths that the bacteria cellulose bead massfraction obtain is collected in fermentation, with rinsed with deionized water 3～5 times, collect, with volume fraction residual alkali lye in 0.1% the acetic acid and in the bead, leave standstill 12～24h, with rinsed with deionized water 2～3 times, in water, keep its spherical-like morphology, bead is taken out, blot surface-moisture, quick-frozen or freezing in cryogenic refrigerator in liquid nitrogen, dry back obtains the plain bead of white fiber in freeze drier then;
(3) preparation of bacteria cellulose bead immobilized enzyme
With the bacteria cellulose bead is that carrier adopts physisorphtion or absorption～crosslinking to prepare immobilized enzyme.
Bacterial classification in the described step (1) is selected from acetobacter (Acetobacter sp.), acetobacter xylinum (A.xylinum), produce vinegar acetobacter (A.acetigenum), the acetify bacillus (line film acetic bacteria, A.aceti), Xu Shi acetic bacteria (A.schutzenbachii), stench acetic bacteria (A.rances), Ao Erlan acetobacter (A.orleanense), curved acetobacter (A.curvum), Acetobacter pasteurianus (Acetobacter pasteurianus), glucose bacillus (Gluconobacter sp.), glucose oxidation and bacillus (Gluconobacter oxydans), Agrobacterium (Agrobacterium tumefaciens), root nodule bacterium (Rhizobium trifolii), pseudomonas cepacia (Seudomonas cepacia), sarcina (Sarcina ventriculi), Pseudomonas cocovenenans (P.cocovenenans), in the campylobacter jejuni (Campylobacter jejuni) one or more.
Seed culture medium in the described step (1) by weight percentage, comprises: glucose 2.0%, yeast powder 0.5%, Tryptones 0.5%, Na 2HPO 412H 2O 0.27%, citric acid 0.115%.
Fermention medium in the described step (1) by weight percentage, comprises: glucose 2.0%, yeast powder 0.5%, Tryptones 0.3%, Na 2HPO 412H 2O 0.27%, citric acid 0.115%.
Physisorphtion in the described step (3), comprise: add bacteria cellulose bead 0.05～0.4g in 100～5000U/L enzyme liquid of 5～10ml and make its submergence, slight vibration 3～5min, under 4 ℃ of conditions, adsorb 6～24h, take out the bacteria cellulose bead, with the sodium acetate buffer solution rinsing of pH4.8～5.2 1～2 time, blot the bead surface-moisture, 4 ℃ of refrigerators are preserved.
Absorption～crosslinking in the described step (3), comprise: add bacteria cellulose bead 0.05～0.4g in 100～5000U/L enzyme liquid of 5～10ml and make its submergence, slight vibration 3～5min, under 4 ℃ of conditions, adsorb 6～24h, with 1g Mierocrystalline cellulose bead: it is 25% glutaraldehyde solution that the ratio of 25～200mL glutaraldehyde solution adds the quality volume percent, crosslinking reaction 1～3h under 20～30 ℃ of agitation conditions, take out the bacteria cellulose bead, with the sodium acetate buffer solution rinsing of pH4.8～5.2 1～2 time, blot the bead surface-moisture, 4 ℃ of refrigerators are preserved.
Beneficial effect
The present invention compared with prior art exists obvious improvement and positive effect:
(1) the invention provides a kind of novel enzyme immobilization technology.Biological enzyme can be efficiently adsorbed in the present invention, keeps the activity of enzyme to greatest extent, and safety and environmental protection, and method is simple, easy handling, and controllability is strong.Compare with the method that adopts chemically crosslinked, initial enzyme retention rate alive exceeds about 25%.
(2) Application Areas of bacteria cellulose has been widened in the cellulosic production of spherical bacteria and with its solid support material as enzyme immobilization.
(3) spherical bacteria cellulose materials purity height, biologically stable is strong.
(4) spherical bacteria cellulose materials mass-transfer performance is good, helps realizing the efficient catalytic of enzyme.
(5) the spherical bacteria cellulose materials is good in natural biodegradability, good biocompatibility.
Description of drawings
Fig. 1 is the optimal reactive temperature of free laccase (▲) and spherical bacterial cellulose immobilization laccase of the present invention (■).
Fig. 2 is free laccase (▲) and spherical bacterial cellulose immobilization laccase of the present invention (■) optimal reaction pH value.
Fig. 3 is the thermostability of free laccase (▲) and spherical bacterial cellulose immobilization laccase of the present invention (■).
Fig. 4 is the pH stability of free laccase (▲) and spherical bacterial cellulose immobilization laccase of the present invention (■).
Fig. 5 is the repeated use stability of absorption method of the present invention (◆) and absorption-crosslinking (■) immobilization laccase.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Embodiment 1
The single colony inoculation of picking 1 ring is in 100mL aseptic seed substratum from the bacillus aceticus inclined-plane, and described seed culture medium comprises (mass percent): glucose 2.0%, yeast powder 0.5%, Tryptones 0.5%, Na 2HPO 412H 2O0.27%, citric acid 0.115%, pH5.0 at 30 ℃, cultivates 12h under the 130rpm condition.Inoculum size with 6% is linked into seed liquor and 300mL is housed does not have in the 500mL Erlenmeyer flask of bacteria fermentation culture medium.Described fermention medium comprises (mass percent): glucose 2.0%, yeast powder 0.5%, Tryptones 0.3%, Na 2HPO 412H 2O 0.27%, citric acid 0.115%, pH5.0; 30 ℃, behind the cultivation 72h, in substratum, generate many bacteria cellulose beads under the 110rpm condition.The Mierocrystalline cellulose bead will be taken out after the substratum decantation, wash repeatedly with deionized water, remove bead surface medium and impurity, again bead is soaked in 0.1% the NaOH solution, handle 60min for 80 ℃, thalline and the residual substratum removed in the Mierocrystalline cellulose bead are translucent to oyster white; With deionized water rinsing and add in a small amount of 0.1% the acetic acid and residual NaOH in the bead, use deionized water rinsing bacteria cellulose bead after the standing over night more then; The bead surface liquid is blotted with filter paper, dry in freeze drier then one by one with liquid nitrogen flash freezer or place cryogenic refrigerator freezing, obtain bacteria cellulose bead carrier.
The preparation of immobilization laccase
Absorption method: the laccase enzyme liquid 10mL that in the 50mL beaker, adds the enzyme 300U/L of unit alive, add the freeze dried bacteria cellulose bead of 0.2g and make its submergence, slight vibration 3～5min, under 4 ℃ of conditions, adsorb 24h, take out the bacteria cellulose bead, with the acetic acid of 200mM, pH5.2-sodium acetate buffer solution rinsing 1～2 time, blot the bead surface-moisture, 4 ℃ of refrigerators are preserved standby.
Absorption-crosslinking: the laccase enzyme liquid 10mL that in the 50mL beaker, adds the enzyme 300U/L of unit alive, add the freeze dried bacteria cellulose bead of 0.2g and make its submergence, slight vibration 3～5min, under 4 ℃ of conditions, adsorb 24h, add the 10mL glutaraldehyde solution, crosslinking reaction 1～3h under 30 ℃, magnetic agitation condition, take out the bacteria cellulose bead, with the sodium acetate buffer solution rinsing of pH4.8～5.2 1～2 time, blot the bead surface-moisture, 4 ℃ of refrigerators are preserved.
The sign of enzyme is as follows:
(a) optimal reactive temperature of resolvase and immobilized enzyme:
With 0.4mM 2,2 '-azino-two-(3-ethyl benzo thiazoline quinoline-6-sulfonic acid) di-ammonium salts (2,2 '-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid), be called for short ABTS) be substrate, (pH=5.2) measures the laccase vigor in 50mM sodium acetate buffer solution, to determine the optimal reactive temperature of immobilization laccase and free laccase, the result as shown in Figure 1.The relatively variation of enzyme activity from figure (is 100% with high enzymatic activity in the experiment on the same group) as can be known, when temperature of reaction＜60 ℃, the vigor of immobilized enzyme increases along with the rising of temperature of reaction, when temperature of reaction＞60 ℃, the vigor of immobilized enzyme descends to some extent, and its optimal reaction temperature (60 ℃) specific ionization enzyme (50 ℃) has improved 10 ℃.
(b) the optimal reaction pH value of resolvase and immobilized enzyme:
With 0.4mM ABTS is substrate, in the citrate buffer solution of 200mM, measures the laccase vigor under 50 ℃ of conditions, to determine the optimal reaction pH of resolvase and immobilized enzyme.With every group of high enzymatic activity is 100% mapping, and the result as shown in Figure 2.As we know from the figure, the optimal reaction pH value of resolvase and immobilized enzyme is 3.5 all, and between pH3.5-5.5, the pH value is less than influence to resolvase to the influence of immobilized enzyme.
(c) thermostability of resolvase and immobilized enzyme:
With resolvase enzyme liquid and immobilized enzyme behind the maintenance 30min, is substrate with 0.4mM ABTS in 30～70 ℃ of water-baths respectively, measures the laccase vigor under 50 ℃ of conditions, determines the temperature stability of resolvase and immobilized enzyme.High enzymatic activity with every group is 100%, the results are shown in Figure 3.As seen from the figure, resolvase 40 ℃ with interior comparatively stable, and immobilized enzyme 50 ℃ with interior more stable, the specific ionization enzyme has improved 10 ℃.
(d) the pH stability of resolvase and immobilized enzyme:
Resolvase and immobilized enzyme are placed the Sodium phosphate dibasic-citric acid solution that contains the different pH of 0.2M and the reaction solution of 0.4mM ABTS substrate respectively, after being incubated 30min under 30 ℃, with 0.4mM ABTS is substrate, in 50mM sodium acetate buffer solution (pH5.2), measure the laccase vigor under 50 ℃ of conditions, to determine the pH stability of resolvase and immobilized enzyme.High enzymatic activity with every group is 100%, and it the results are shown in Figure 4.As can be seen from the figure, resolvase is more stable when pH5, and its enzyme work has kept the relative vigor more than 90% in the scope of pH4～7; Immobilized enzyme is more stable when pH4, and its enzyme work keeps the relative vigor more than 80% in the scope of pH3～6.The ph stability of immobilized enzyme has moved 1 pH unit than resolvase oxytropism direction.
(e) the repeated use stability of immobilized enzyme:
In the 50mL small beaker, (200mM, pH5.2) with 5mL ABTS (2mM), magnetic stir bar slowly stirs, and adds the 0.2g immobilized enzyme after temperature reaches 50 ℃ for adding 10mL deionized water, 5mL sodium acetate buffer solution.Measuring the immobilized enzyme enzyme according to enzyme activity determination method in (d) lives.Behind the 20min, take out immobilized enzyme,, wash thorough washing reaction substrate and product more repeatedly with deionized water with sodium acetate buffer solution flushing 1～2 time.Blot residual moisture and repeat above operation with filter paper, up to the immobilized enzyme that obtains reusing after 7 times.That measures enzyme activity the results are shown in Figure 5, and using the enzyme work of being surveyed for the first time in the absorption method immobilization laccase is 100%, and other enzyme work of surveying is that benchmark calculates relative enzyme work with it.As seen from the figure, two kinds of immobilized enzyme all have stability preferably after being repeatedly used, and after using through 4 times, its relative enzyme activity still is higher than 35%.

Claims (6)

1. one kind is the method for preparing carriers immobilized enzyme with the spherical bacterial cellulose, comprising:
(1) preparation of bacteria cellulose bead
The activatory slant strains is inserted the seed culture medium of pH4.8～5.2, under 25～30 ℃, 100～200rpm condition, cultivate 12～16h and make liquid seeds, with volume percent is that 4～10% inoculum sizes are forwarded to liquid seeds in the fermention medium of pH4.8～5.2, contain 200～300mL fermentation culture in every 500mL Erlenmeyer flask, 25～30 ℃, cultivate 48～96h under 80～200rpm condition, decantation gets the bacteria cellulose bead;
(2) processing of bacteria cellulose bead
It is that 0.1% NaOH handles 30～120min in 80～90 ℃ of water-baths that the bacteria cellulose bead massfraction obtain is collected in fermentation, with rinsed with deionized water 3～5 times, collect, with volume fraction residual alkali lye in 0.1% the acetic acid and in the bead, leave standstill 12～24h, with rinsed with deionized water 2～3 times, in water, keep its spherical-like morphology, bead is taken out, blot surface-moisture, quick-frozen or freezing in cryogenic refrigerator in liquid nitrogen, dry back obtains the plain bead of white fiber in freeze drier then;
(3) preparation of bacteria cellulose bead immobilized enzyme
With the bacteria cellulose bead is that carrier adopts physisorphtion or absorption～crosslinking to prepare immobilized enzyme.
2. according to claim 1 a kind of be the method for preparing carriers immobilized enzyme with the spherical bacterial cellulose, it is characterized in that the bacterial classification in the described step (1) is selected from one or more in acetobacter, acetobacter xylinum, product vinegar acetobacter, acetify bacillus, Xu Shi acetic bacteria, stench acetic bacteria, Ao Erlan acetobacter, curved acetobacter, Acetobacter pasteurianus, glucose bacillus, glucose oxidation and bacillus, Agrobacterium, root nodule bacterium, pseudomonas cepacia, sarcina, Pseudomonas cocovenenans, the campylobacter jejuni.
3. according to claim 1 a kind of be the method for preparing carriers immobilized enzyme with the spherical bacterial cellulose, it is characterized in that the seed culture medium in the described step (1), by weight percentage, comprise: glucose 2.0%, yeast powder 0.5%, Tryptones 0.5%, Na 2HPO 412H 2O 0.27%, citric acid 0.115%.
4. according to claim 1 a kind of be the method for preparing carriers immobilized enzyme with the spherical bacterial cellulose, it is characterized in that the fermention medium in the described step (1), by weight percentage, comprise: glucose 2.0%, yeast powder 0.5%, Tryptones 0.3%, Na 2HPO 412H 2O 0.27%, citric acid 0.115%.
5. according to claim 1 a kind of be the method for preparing carriers immobilized enzyme with the spherical bacterial cellulose, it is characterized in that, physisorphtion in the described step (3), comprise: add bacteria cellulose bead 0.05～0.4g in 100～5000U/L enzyme liquid of 5～10ml and make its submergence, slight vibration 3～5min, under 4 ℃ of conditions, adsorb 6～24h, take out the bacteria cellulose bead, with the sodium acetate buffer solution rinsing of pH4.8～5.2 1～2 time, blot the bead surface-moisture, 4 ℃ of refrigerators are preserved.
6. according to claim 1 a kind of be the method for preparing carriers immobilized enzyme with the spherical bacterial cellulose, it is characterized in that, absorption～crosslinking in the described step (3), comprise: add bacteria cellulose bead 0.05～0.4g in 100～5000U/L enzyme liquid of 5～10ml and make its submergence, slight vibration 3～5min, under 4 ℃ of conditions, adsorb 6～24h, with 1g Mierocrystalline cellulose bead: it is 25% glutaraldehyde solution that the ratio of 25～200mL glutaraldehyde solution adds the quality volume percent, crosslinking reaction 1～3h under 20～30 ℃ of agitation conditions, take out the bacteria cellulose bead, with the sodium acetate buffer solution rinsing of pH4.8～5.2 1～2 time, blot the bead surface-moisture, 4 ℃ of refrigerators are preserved.
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