CN105176963B - The bio-microcapsule and preparation method of a kind of calcium alginate-sodium carboxymethylcellulose co-immobilization whiterot fungi and its ectoenzyme - Google Patents
The bio-microcapsule and preparation method of a kind of calcium alginate-sodium carboxymethylcellulose co-immobilization whiterot fungi and its ectoenzyme Download PDFInfo
- Publication number
- CN105176963B CN105176963B CN201510588127.4A CN201510588127A CN105176963B CN 105176963 B CN105176963 B CN 105176963B CN 201510588127 A CN201510588127 A CN 201510588127A CN 105176963 B CN105176963 B CN 105176963B
- Authority
- CN
- China
- Prior art keywords
- solution
- concentration
- white rot
- whiterot fungi
- calcium chloride
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to calcium alginate-sodium carboxymethylcellulose co-immobilization whiterot fungi and its bio-microcapsule and preparation method of ectoenzyme, the bio-microcapsule includes shell and enclosure interior core material；Case material is calcium alginate gel, and white rot enzyme cross-linked aggregates are embedded inside calcium alginate gel；Enclosure interior core material is calcium chloride-sodium carboxymethyl cellulose solution, and whiterot fungi cell and extracellular cross-linked enzyme aggregate are dispersed with inside calcium chloride-sodium carboxymethyl cellulose solution.In preparation process directly cross-linked enzyme aggregate is made in white rot enzyme by bio-microcapsule of the present invention, eliminates the step of purifying Ligninolytic Enzymes, can effectively be reduced production cost, and form the close relations of dependence with whiterot fungi, be improved degradation efficiency.
Description
Technical field
The invention belongs to microbes and field of environment engineering technology, are related to a kind of whiterot fungi common immobilization method, tool
Body is related to bio-microcapsule and its preparation of a kind of calcium alginate-sodium carboxymethylcellulose co-immobilization whiterot fungi and its ectoenzyme
Method.
Background technology
Whiterot fungi can realize the degradation to heteroplasia matter under aerobic condition, have in wastewater treatment, environment remediation etc.
Wide application prospect.Whiterot fungi starts from the secondary metabolism stage to the degradation of heteroplasia matter, relies primarily on the lignin of its secretion
Degradation enzyme system.The extracellular enzyme includes mainly lignin peroxidase, manganese peroxidase and laccase, they are by causing one
The radical reaction of series realizes the indirect degradation of heteroplasia matter.
However, whiterot fungi, which is directly used in wastewater treatment, has some inevitable disadvantages.First, whiterot fungi is filiform
Fungi, anti-shearing force difference, in reactor shearing force too conference destroy its mycelial structure, influence its producing enzyme；It is too small to influence water body
Mass transfer and dissolved oxygen to influence growth and the degradation capability of bacterium.In addition, whiterot fungi growth and producing enzyme by aquatic environment influenced compared with
Greatly, it means that very important person was in practical applications controls waste component, limits the range of its application, increases and answer
Use cost.
Simple immobilized white rot fungus can effectively improve adaptability of the whiterot fungi to environment, but the producing enzyme of whiterot fungi is still
It is limited by own metabolism.Simple immobilization Ligninolytic Enzymes can break away from whiterot fungi metabolic effect, improve the place of waste water
Efficiency is managed, but in Ligninolytic Enzymes in addition to laccase, other two kinds of enzymes are all anti-to start for direct substrate with hydrogen peroxide
It answers, and the amount of hydrogen peroxide is difficult control in practical operation, which has limited manganese peroxidases and lignin peroxidase
Application.
Invention content
It is an object of the invention to be directed to the deficiencies in the prior art, provide it is a kind of can be applied to wastewater treatment,
The bio-microcapsule of calcium alginate-sodium carboxymethylcellulose co-immobilization whiterot fungi and its ectoenzyme.Meanwhile the present invention also provides
Preparation method.
Technical solution of the present invention is：
A kind of bio-microcapsule of calcium alginate-sodium carboxymethylcellulose co-immobilization whiterot fungi and its ectoenzyme, it is special
Sign is：The bio-microcapsule includes shell and enclosure interior core material；Case material is calcium alginate gel, alginic acid
White rot enzyme cross-linked aggregates are embedded inside calcium gel；Enclosure interior core material is that calcium chloride-sodium carboxymethylcellulose is water-soluble
Liquid is dispersed with whiterot fungi cell and white rot enzyme cross-linked aggregates inside calcium chloride-sodium carboxymethyl cellulose solution；
The lignin-degrading enzymes total enzyme activity concentration of the white rot enzyme cross-linked aggregates is more than 60U/L.
The whiterot fungi cell is whiterot fungi mycelia or spore；When whiterot fungi cell is mycelia, concentration 10-300g/L,
Weight in wet base；When whiterot fungi cell is spore, 500-10000/mL of concentration.
In calcium chloride-sodium carboxymethyl cellulose solution, calcium chloride and sodium carboxymethylcellulose concentration are all higher than 0.005
g/mL。
The bio-microcapsule granule size is：3-15mm；Thickness of shell is：0.2-2mm.
The preparation of the bio-microcapsule of above-mentioned calcium alginate-sodium carboxymethylcellulose co-immobilization whiterot fungi and its ectoenzyme
Method includes the following steps：
1）The preparation of white rot enzyme cross-linked aggregates：Whiterot fungi is inoculated in fluid nutrient medium, constant temperature incubation 6-
10 days, mycelia was collected in filtering, spare；Crude enzyme liquid is made through dialysis, concentration in filtered filtrate；Saturation is added into crude enzyme liquid
Ammonium sulfate and phosphate buffer stir 15-20min, obtain reaction solution；A concentration of 25% glutaraldehyde is added dropwise into reaction solution,
Crosslinking, centrifugation, is washed with acetate buffer solution, obtains white rot enzyme cross-linked aggregates；
2）The preparation of white rot bacteria suspension selects either method to prepare white rot bacteria suspension from following a or b：
A is by step 1）Collected mycelia is inoculated in after being cultivated 5-8 days in solid medium, scrapes white in culture medium
Rotten bacterium spore is scattered in calcium chloride-sodium carboxymethyl cellulose solution, and white rot bacteria suspension is made；
B is by step 1）In the mycelia that is collected into break up, be directly scattered in calcium chloride-sodium carboxymethyl cellulose solution,
White rot bacteria suspension is made；
3）The preparation of co-immobilization microcapsules：Take step 1）In ectoenzyme cross-linked aggregates obtained be scattered in sodium alginate
In aqueous solution, solution A is obtained；Then step 1 is taken again）In ectoenzyme cross-linked aggregates obtained be scattered in step 2）In whiterot fungi
In suspension, solution B is obtained；Solution B is added drop-wise in solution A under stirring, stirs encystation, then adds sterile water dilution, filtering obtains micro-
Then microcapsules are placed in calcium chloride water fixed, filtering by capsule, with sterile water washing 3-4 times, obtain calcium alginate-
The bio-microcapsule of sodium carboxymethylcellulose co-immobilization whiterot fungi and its ectoenzyme.
Described, step 1）In, the group of fluid nutrient medium becomes：2 g/L KH2PO4、0.25 g/L MgSO4、0.1 g/L
CaCl2、5 mg/L MnSO4, 5 mg/L VB1,0.2 g/L ammonium tartrates, 20 g/L glucose and 150 mL/L micro member
Plain storing solution；
The group of the micro- storing solution becomes：NaCl 1.0g/L,MgSO4•7H2O 3.0g/L、FeSO4•7H2O
100mg/L、CoSO4•7H2O 100mg/L、CaCl2 100mg/L、ZnSO4•7H2O 100mg/L、CuSO4•5H2O 10mg/L、
KAl(SO4)2 100mg/L、H3BO310mg/L and NaMoO4 10mg/L。
Described, step 1）In, the volume ratio of crude enzyme liquid, saturated ammonium sulfate solution and phosphate buffer is 1：5-9：0-1；
The phosphate buffer pH is 7.4, a concentration of 0.2-0.5M.
Described, step 1）In volumetric concentration of 25% glutaraldehyde in reaction solution be 0.2-0.4%.
Described, step 1）Middle crosslinking is that isothermal vibration is crosslinked, and crosslinking temperature is 30 DEG C, crosslinking time 1.5-9h, is handed over
The concussion frequency of connection is 60-100rpm, centrifugal speed 10000r/min, centrifugation time 20min.
Described, step 1）Middle acetate buffer solution pH is 4.5, a concentration of 20mM.
Described, step 1）Middle crude enzyme liquid includes one kind in lignin peroxidase, manganese peroxidase and laccase
Or it is a variety of.
Described, step 2）In, in white rot bacteria suspension obtained, the mycelia containing a concentration of 10-300g/L of weight in wet base or dense
Degree is the spore of 500-10000/mL.
Described, step 2）In, in calcium chloride-sodium carboxymethyl cellulose solution, calcium chloride and sodium carboxymethylcellulose
Concentration be all higher than 0.01g/mL.
Described, step 2）In, the group of solid medium becomes：200g/L potato leachates, 20g/L glucose sugar, 3g/L
Potassium dihydrogen phosphate, 1.5g/L magnesium sulfate and 20g/L agar.
Described, step 3）In, the lignin-degrading enzymes total enzyme activity concentration of ectoenzyme cross-linked aggregates is more than 60U/L；
Described, step 3）In, a concentration of 0.005-0.04g/mL of sodium alginate in sodium alginate aqueous solution；
Described, step 3）In, the stirring encystation time is 1-5min, when solution B is added drop-wise in solution A, in solution B
Calcium chloride can react into rapidly Nang with the sodium alginate in solution A, and the purpose of stirring is two microcapsules adhesions in order to prevent, micro-
Capsule wall thickness can change with time and thicken, with sterile water dilute filtration also for the adhesion for preventing microcapsules after encystation.
Described, step 3）In, the set time is 30-60min in calcium chloride water, fixed calcium chloride water used
Middle CaCl2A concentration of 0.02-0.03g/mL, the volume of calcium chloride water are 1 times or more that gained fixes microcapsules volume altogether.
Described, step 3）In, dropwise operation can be can be used for injector for medical purpose, peristaltic pump etc. be added dropwise equipment；Stirring
Operation can use the mixing plants such as magnetic stirring apparatus.
The present invention prepares the bio-microcapsule of calcium alginate-sodium carboxymethylcellulose co-immobilization whiterot fungi and its ectoenzyme
Principle be：Bio-microcapsule is a kind of large biological molecule or microorganism, animal and plant cells to be encapsulated in one layer hydrophilic half
It is formed by pearl microcapsules in permeable membrane, microenvironment can be provided to be fixed on internal microorganism or enzyme, promote the life of microorganism
Long and enzyme catalytic action.
White rot enzyme cross-linked aggregates, which are embedded in calcium alginate gel, is used as wall material, is scattered in calcium chloride-carboxylic first
Whiterot fungi spore or mycelia and cross-linked enzyme aggregate in base sodium cellulosate solution form co-immobilization microcapsules as core material.
In degradation process, whiterot fungi cross-linked enzyme aggregate first engages in the degradation of heteroplasia matter, and at the same time, whiterot fungi is in metabolic process
The H of secretion2O2H can be provided for cross-linked enzyme aggregate by generating enzyme system2O2, promote the degradation of heteroplasia matter.
Technical solution of the present invention advantage is：
1, white rot enzyme is directly directly prepared into cross-linked enzyme aggregate by the present invention, is eliminated Ligninolytic Enzymes
The step of purification, can effectively reduce production cost.
2, ectoenzyme cross-linked aggregates are contacted with heteroplasia matter first in the outside of microcapsules in the present invention, can effectively reduce drop
The startup time of solution improves degradation efficiency, and can play the protective effect to capsule whiterot fungi.Meanwhile capsule
Production lactoperoxidase system secreted by whiterot fungi can provide direct substrate for cross-linked enzyme aggregate, promote degradation.
3, capsule of the present invention is hollow-core construction, and a microenvironment can be provided for cell growth, is conducive to the life of thalline
It is long.
Description of the drawings
Fig. 1 is the micro- glue of biology of calcium alginate of the present invention-sodium carboxymethylcellulose co-immobilization whiterot fungi and its ectoenzyme
The structural schematic diagram of capsule；
Fig. 2 is the micro- glue of biology of calcium alginate of the present invention-sodium carboxymethylcellulose co-immobilization whiterot fungi and its ectoenzyme
The preparation process flow of capsule.
Specific implementation mode
It is further illustrated the present invention below by specific embodiment, it should be understood that the embodiment of the present invention is only used for explaining
The bright present invention, rather than limiting the invention；Under the premise of present inventive concept, simple modifications to the present invention program and replace
Change belong to the present invention claims protection domain.
Experimental method used in following examples is conventional method unless otherwise specified；Material used in following examples
Material, reagent unless otherwise specified, can directly be bought or be obtained by conventional method.
1, fluid nutrient medium component：2 g/L KH2PO4、0.25 g/L MgSO4、0.1 g/L CaCl2、5 mg/L
MnSO4, 5 mg/L VB1,0.2 g/L ammonium tartrates, 20 g/L glucose and 150 mL/L micro- storing solution.
2, the component of micro- storing solution：NaCl 1.0g/L,MgSO4•7H2O 3.0g/L、FeSO4•7H2O 100mg/
L、CoSO4•7H2O 100mg/L、CaCl2 100mg/L、ZnSO4•7H2O 100mg/L、CuSO4•5H2O 10mg/L、KAl
(SO4)2 100mg/L、H3BO310mg/L and NaMoO4 10mg/L。
3, the group of solid medium becomes：200g/L potato leachates, 20g/L glucose sugar, 3g/L potassium dihydrogen phosphates,
1.5g/L magnesium sulfate and 20g/L agar.
4, the structure for system of degrading：The fluid nutrient medium of the dyestuff containing 40mg/L.
Embodiment 1
It choosesPhanerochaete chrysosporium（Purchased from Guangdong Culture Collection, bacterial strain preservation is compiled
Number be GIM3.383）For whiterot fungi strain.
1）The preparation of white rot enzyme cross-linked aggregates：By whiterot fungiPhanerochaete chrysosporiumIt connects
Kind in fluid nutrient medium, mycelia is collected by filtration, and mycelia is spare, and filtrate is dialysed, and concentrates, system in 30 DEG C of constant temperature incubations 10 days
Obtain crude enzyme liquid；Saturated ammonium sulfate solution and phosphate buffer are added into crude enzyme liquid（0.2M, pH7.4）, crude enzyme liquid, saturation sulphur
Acid ammonium solution and phosphate buffer volume ratio are 1：6：1, assemble 15min under magnetic stirrer, obtains reaction solution；To reaction solution
25% glutaraldehyde of middle dropwise addition makes glutaraldehyde final volume a concentration of 0.25%, isothermal vibration be crosslinked 3h, and crosslinking temperature is 30 DEG C, is handed over
Connection concussion frequency is 60rpm, and centrifugal speed 10000r/min, centrifugation time 10min use acetate buffer solution（20mM, pH4.5）
Washing, obtains white rot enzyme cross-linked aggregates；
2）The preparation of white rot bacteria suspension：By step 1）Collected mycelia is inoculated in after being cultivated 7 days in solid medium,
Whiterot fungi spore in scraping culture medium is scattered in calcium chloride-sodium carboxymethyl cellulose solution, and white rot bacteria suspension is made；Chlorine
Change in calcium-sodium carboxymethyl cellulose solution, calcium chloride concentration 0.02g/mL, a concentration of 0.03 g/ of sodium carboxymethylcellulose
mL；In white rot bacteria suspension obtained, spore concentration is 1623/mL.
3）The preparation of co-immobilization microcapsules：Take step 1）In ectoenzyme cross-linked aggregates obtained be scattered in it is a concentration of
In the sodium alginate soln of 0.02g/mL, solution A is obtained, ectoenzyme cross-linked aggregates dispersion concentration is weight in wet base 3g/L, wood in solution A
Lignin-degrading enzymes total enzyme activity is 102U/L；Then step 1 is taken again）In ectoenzyme cross-linked aggregates obtained be scattered in step 2）In
White rot bacteria suspension in, obtain solution B, ectoenzyme cross-linked aggregates dispersion concentration is weight in wet base 2g/L, lignin-degrading enzymes in solution B
Total enzyme activity is more than 102U/L；Solution B is added drop-wise to injector for medical purpose in the solution A under magnetic stirrer, encystation is stirred,
The stirring encystation time is 3min, then adds sterile water dilution, and filtering obtains microcapsules, it is a concentration of that microcapsules are then placed in 50mL
60min is fixed in the calcium chloride water of 0.02g/mL, is filtered, and with sterile water washing 3 times, it is fine to obtain calcium alginate-carboxymethyl
The bio-microcapsule of the plain sodium co-immobilization whiterot fungi of dimension and its ectoenzyme.
The biology of calcium alginate made from embodiment 1-sodium carboxymethylcellulose co-immobilization whiterot fungi and its ectoenzyme is micro-
In capsule, granule size is：5 mm；Calcium alginate gel thickness of shell is：0.3 mm；White rot enzyme cross-linked aggregates exist
It is a concentration of in calcium alginate gel：3g/L, weight in wet base；Lignin-degrading enzymes total enzyme activity is 150U/L in ectoenzyme cross-linked aggregates.
It is dispersed with whiterot fungi mycelia or spore in enclosure interior core material calcium chloride-sodium carboxymethyl cellulose solution；Whiterot fungi spore is dense
Spend 1623/mL.In calcium chloride-sodium carboxymethyl cellulose solution, calcium chloride concentration 0.02g/mL, sodium carboxymethylcellulose
A concentration of 0.03 g/mL..
4）Degradation capability compares：The fluid nutrient medium for preparing the Acid Brilliant Scarlet GR containing 40mg/L is cultivated according to inoculum concentration for 1mL
The ratio that base connects 1 bio-microcapsule is inoculated with, 39 DEG C, 100rpm, three days degradation rates for measuring basic fuchsin of culture, and in
Traditional immobilization of same inoculum concentrationPhanerochaete chrysosporiumDegradation rate under gel particle the same terms into
Row comparison.
The result shows that the percent of decolourization of bio-microcapsule of the present invention and traditional immobilization gel particle is respectively 53% He after 3 days
30%, this shows that total fixed microcapsules have higher decolorizing efficiency.
Embodiment 2
It is chosen forTrametes versicolor（Purchased from Chinese industrial Culture Collection, bacterial strain deposit number
For CICC 50001）Whiterot fungi strain.
1）The preparation of white rot enzyme cross-linked aggregates：By whiterot fungiTrametesversicolorIt is inoculated in liquid training
It supports in base, mycelia is collected by filtration in 30 DEG C of constant temperature incubations 10 days, and mycelia is spare, and filtrate is dialysed, and crude enzyme liquid is made in concentration；To
Saturated ammonium sulfate solution and phosphate buffer are added in crude enzyme liquid（0.2M, pH7.4）, crude enzyme liquid, saturated ammonium sulfate solution and phosphorus
Acid buffer volume ratio is 1：9, assemble 15min under magnetic stirrer, obtains reaction solution；25% penta 2 is added dropwise into reaction solution
Aldehyde makes glutaraldehyde final volume a concentration of 0.3%, isothermal vibration be crosslinked 3h, and crosslinking temperature is 30 DEG C, and crosslinking concussion frequency is
100rpm, centrifugal speed 10000r/min, centrifugation time 10min use acetate buffer solution（20mM, pH4.5）Washing, obtains white rot
Enzyme cross-linked aggregates；
2）The preparation of white rot bacteria suspension：By step 1）Collected mycelia is broken up and is scattered in calcium chloride-carboxymethyl cellulose
In sodium water solution, white rot bacteria suspension is made；In calcium chloride-sodium carboxymethyl cellulose solution, calcium chloride concentration is 0.02 g/
ML, a concentration of 0.06 g/mL of sodium carboxymethylcellulose；In white rot bacteria suspension obtained, mycelial concentration 200g/L.
3）The preparation of co-immobilization microcapsules：Take step 1）In ectoenzyme cross-linked aggregates obtained be scattered in it is a concentration of
In the sodium alginate soln of 0.015 g/mL, solution A is obtained, ectoenzyme cross-linked aggregates dispersion concentration is weight in wet base 2g/L in solution A,
Lignin-degrading enzymes total enzyme activity is 150U/L；；Then step 1 is taken again）In ectoenzyme cross-linked aggregates obtained be scattered in step
2）In white rot bacteria suspension in, obtain solution B, ectoenzyme cross-linked aggregates dispersion concentration is weight in wet base 2g/L, lignin drop in solution B
It solves enzyme total enzyme activity and is more than 150U/L；Solution B is added drop-wise to injector for medical purpose in the solution A under magnetic stirrer, is stirred
Encystation, stirring encystation time are 5min, then add sterile water dilution, and filtering obtains microcapsules, microcapsules are then placed in 100mL
60min is fixed in the calcium chloride water of a concentration of 0.03 g/mL, is filtered, with sterile water washing 3 times, obtains calcium alginate-carboxylic
The bio-microcapsule of sodium carboxymethylcellulose pyce co-immobilization whiterot fungi and its ectoenzyme.
The biology of calcium alginate made from embodiment 2-sodium carboxymethylcellulose co-immobilization whiterot fungi and its ectoenzyme is micro-
In capsule, granule size is：7 mm；Calcium alginate gel thickness of shell is：0.25 mm；White rot enzyme cross-linked aggregates
Lignin-degrading enzymes total enzyme activity is 150U/L in a concentration of 2g/L exoenzymes cross-linked aggregates in calcium alginate gel.Enclosure interior
It is dispersed with whiterot fungi mycelia or spore in core material calcium chloride-sodium carboxymethyl cellulose solution；Whiterot fungi mycelial concentration 200g/L.
In calcium chloride-sodium carboxymethyl cellulose solution, calcium chloride concentration be 0.02 g/mL, sodium carboxymethylcellulose it is a concentration of
0.06 g/mL。。
4）Degradation capability compares：The fluid nutrient medium for preparing the Acid Brilliant Scarlet GR containing 40mg/L is cultivated according to inoculum concentration for 1mL
The ratio that base connects 1 bio-microcapsule is inoculated with, 39 DEG C, 100rpm, three days degradation rates for measuring basic fuchsin of culture, and in
Traditional immobilization of same inoculum concentrationTrametes versicolorDegradation rate under gel particle the same terms is compared.
The result shows that the percent of decolourization of co-immobilization microcapsules and traditional immobilization gel particle is respectively 70% He after 3 days
20%, this shows that total fixed microcapsules have higher decolorizing efficiency.
Claims (9)
1. the bio-microcapsule of a kind of calcium alginate-sodium carboxymethylcellulose co-immobilization whiterot fungi and its ectoenzyme, feature
It is that the bio-microcapsule includes shell and enclosure interior core material；Case material is calcium alginate gel, calcium alginate
White rot enzyme cross-linked aggregates are embedded inside gel；Enclosure interior core material is calcium chloride-sodium carboxymethyl cellulose solution,
Whiterot fungi cell and white rot enzyme cross-linked aggregates are dispersed with inside calcium chloride-sodium carboxymethyl cellulose solution；
The bio-microcapsule granule size is：3-15mm；Thickness of shell is：0.2-2mm；
It is made of following methods:
1）The preparation of white rot enzyme cross-linked aggregates：Whiterot fungi is inoculated in fluid nutrient medium, constant temperature incubation 6-10 days,
Mycelia is collected in filtering, spare；Crude enzyme liquid is made through dialysis, concentration in filtered filtrate；Saturation sulfuric acid is added into crude enzyme liquid
Ammonium salt solution and phosphate buffer stir 15-20min, obtain reaction solution；A concentration of 25% glutaraldehyde is added dropwise into reaction solution, hands over
Connection, centrifugation, is washed with acetate buffer solution, obtains white rot enzyme cross-linked aggregates；
2）The preparation of white rot bacteria suspension selects either method to prepare white rot bacteria suspension from following a or b：
A is by step 1）Collected mycelia is inoculated in after being cultivated 5-8 days in solid medium, scrapes the whiterot fungi in culture medium
Spore is scattered in calcium chloride-sodium carboxymethyl cellulose solution, and white rot bacteria suspension is made；
B is by step 1）In the mycelia that is collected into break up, be directly scattered in calcium chloride-sodium carboxymethyl cellulose solution, be made
White rot bacteria suspension；
3）The preparation of co-immobilization microcapsules：Take step 1）In ectoenzyme cross-linked aggregates obtained to be scattered in sodium alginate water-soluble
In liquid, solution A is obtained；Then step 1 is taken again）In ectoenzyme cross-linked aggregates obtained be scattered in step 2）In white rot bacteria suspension
In, obtain solution B；Solution B is added drop-wise in solution A under stirring, stirs encystation, then adds sterile water dilution, filtering obtains micro- glue
Then microcapsules are placed in calcium chloride water fixed, filtering by capsule, with sterile water washing 3-4 times, obtain calcium alginate-carboxylic
The bio-microcapsule of sodium carboxymethylcellulose pyce co-immobilization whiterot fungi and its ectoenzyme.
2. bio-microcapsule according to claim 1, it is characterised in that：White rot enzyme in the bio-microcapsule
The lignin-degrading enzymes total enzyme activity of cross-linked aggregates is more than 60U/L.
3. bio-microcapsule according to claim 2, it is characterised in that：The whiterot fungi cell be whiterot fungi mycelia or
Spore；When whiterot fungi cell is mycelia, concentration 10-300g/L, weight in wet base, when whiterot fungi cell is spore, concentration 500-10000
A/mL；In the calcium chloride-sodium carboxymethyl cellulose solution, calcium chloride and sodium carboxymethylcellulose concentration are all higher than
0.005g/mL。
4. the biology of a kind of calcium alginate described in claim 1-sodium carboxymethylcellulose co-immobilization whiterot fungi and its ectoenzyme
The preparation method of microcapsules, which is characterized in that include the following steps：
1）The preparation of white rot enzyme cross-linked aggregates：Whiterot fungi is inoculated in fluid nutrient medium, constant temperature incubation 6-10 days,
Mycelia is collected in filtering, spare；Crude enzyme liquid is made through dialysis, concentration in filtered filtrate；Saturation sulfuric acid is added into crude enzyme liquid
Ammonium salt solution and phosphate buffer stir 15-20min, obtain reaction solution；A concentration of 25% glutaraldehyde is added dropwise into reaction solution, hands over
Connection, centrifugation, is washed with acetate buffer solution, obtains white rot enzyme cross-linked aggregates；
2）The preparation of white rot bacteria suspension selects either method to prepare white rot bacteria suspension from following a or b：
A is by step 1）Collected mycelia is inoculated in after being cultivated 5-8 days in solid medium, scrapes the whiterot fungi in culture medium
Spore is scattered in calcium chloride-sodium carboxymethyl cellulose solution, and white rot bacteria suspension is made；
B is by step 1）In the mycelia that is collected into break up, be directly scattered in calcium chloride-sodium carboxymethyl cellulose solution, be made
White rot bacteria suspension；
3）The preparation of co-immobilization microcapsules：Take step 1）In ectoenzyme cross-linked aggregates obtained to be scattered in sodium alginate water-soluble
In liquid, solution A is obtained；Then step 1 is taken again）In ectoenzyme cross-linked aggregates obtained be scattered in step 2）In white rot bacteria suspension
In, obtain solution B；Solution B is added drop-wise in solution A under stirring, stirs encystation, then adds sterile water dilution, filtering obtains micro- glue
Then microcapsules are placed in calcium chloride water fixed, filtering by capsule, with sterile water washing 3-4 times, obtain calcium alginate-carboxylic
The bio-microcapsule of sodium carboxymethylcellulose pyce co-immobilization whiterot fungi and its ectoenzyme.
5. preparation method according to claim 4, it is characterised in that：Described, step 1）In, the composition of fluid nutrient medium
For：2g/L KH2PO4、0.25g/L MgSO4、0.1g/L CaCl2、5mg/L MnSO4、5mg/L VB1, 0.2g/L ammonium tartrates,
The micro- storing solution of 20g/L glucose and 150mL/L；
The group of the micro- storing solution becomes：NaCl1.0g/L,MgSO4·7H2O3.0g/L、FeSO4·7H2O
100mg/L、CoSO4·7H2O 100mg/L、CaCl2 100mg/L、ZnSO4·7H2O 100mg/L、CuSO4·5H2O 10mg/
L、KAl(SO4)2 100mg/L、H3BO3 10mg/L and NaMoO4 10mg/L。
6. preparation method according to claim 4, it is characterised in that：Described, step 1）In, crude enzyme liquid, saturation sulfuric acid
The volume ratio of ammonium salt solution and phosphate buffer is 1：5-9：0-1；The phosphate buffer pH is 7.4, a concentration of 0.2-0.5M；
Described, volumetric concentration of 25% glutaraldehyde in reaction solution is 0.2-0.4%；The acetate buffer solution pH is 4.5, a concentration of
20mM；Described, crude enzyme liquid includes one or more in lignin peroxidase, manganese peroxidase and laccase.
7. preparation method according to claim 4, it is characterised in that：Described, step 1）Middle crosslinking is that isothermal vibration is handed over
Connection, crosslinking temperature are 30 DEG C, crosslinking time 1.5-9h, and crosslinked concussion frequency is 60-100rpm, and centrifugal speed is
10000r/min, centrifugation time 20min.
8. preparation method according to claim 4, it is characterised in that：Step 2）In,
Described, in white rot bacteria suspension obtained, the mycelia containing a concentration of 10-300g/L of weight in wet base or a concentration of 500-10000
The spore of a/mL；
Described, in calcium chloride-sodium carboxymethyl cellulose solution, calcium chloride and sodium carboxymethylcellulose concentration are all higher than
0.01g/mL；
Described, the group of solid medium becomes：200g/L potato leachates, 20g/L glucose, 3g/L potassium dihydrogen phosphates,
1.5g/L magnesium sulfate and 20g/L agar.
9. preparation method according to claim 4, it is characterised in that：Step 3）In, described, ectoenzyme cross-linked aggregates
Middle lignin-degrading enzymes total enzyme activity is more than 60U/L；A concentration of 0.005-0.04g/mL of sodium alginate in sodium alginate aqueous solution；
The stirring encystation time is 1-5min；The set time is 30-60min in calcium chloride water, in fixed calcium chloride water used
CaCl2A concentration of 0.02-0.03g/mL, the volume of calcium chloride water are 1 times or more that gained fixes microcapsules volume altogether；Drop
Add operation can use injector for medical purpose, peristaltic pump；Stirring operation can use magnetic stirring apparatus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510588127.4A CN105176963B (en) | 2015-09-16 | 2015-09-16 | The bio-microcapsule and preparation method of a kind of calcium alginate-sodium carboxymethylcellulose co-immobilization whiterot fungi and its ectoenzyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510588127.4A CN105176963B (en) | 2015-09-16 | 2015-09-16 | The bio-microcapsule and preparation method of a kind of calcium alginate-sodium carboxymethylcellulose co-immobilization whiterot fungi and its ectoenzyme |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105176963A CN105176963A (en) | 2015-12-23 |
| CN105176963B true CN105176963B (en) | 2018-10-26 |
Family
ID=54899394
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510588127.4A Active CN105176963B (en) | 2015-09-16 | 2015-09-16 | The bio-microcapsule and preparation method of a kind of calcium alginate-sodium carboxymethylcellulose co-immobilization whiterot fungi and its ectoenzyme |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105176963B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107619825A (en) * | 2016-07-21 | 2018-01-23 | 济南航晨生物科技有限公司 | A kind of agricultural probiotics method for preparing microsphere based on biological polyoses |
| CN106011124B (en) * | 2016-08-05 | 2019-03-26 | 齐鲁工业大学 | A kind of whiterot fungi biological microsphere and preparation method |
| CN106669839A (en) * | 2016-12-26 | 2017-05-17 | 华侨大学 | Iron carbon and bentonite sodium alginate gel catalyst and preparation method and application thereof |
| CN107162367B (en) * | 2017-06-19 | 2020-05-22 | 河海大学 | Polluted river sediment purification capsule |
| CN109136213A (en) * | 2018-08-22 | 2019-01-04 | 贵州大学 | A method of utilizing the black dyestuff of immobilized white rot fungus degrading activity |
| CN114032253B (en) * | 2021-11-11 | 2024-04-05 | 哈尔滨工业大学 | Hydrogen production method based on hydrogel-coated bacteria aggregation |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1986787A (en) * | 2006-12-21 | 2007-06-27 | 天津大学 | Bionic preparing process of silica-alginic acid microcapsule for immobilized beta-glucurosidase |
| CN104498470A (en) * | 2015-01-16 | 2015-04-08 | 齐鲁工业大学 | New type co-immobilization white rot fungi gel particle |
-
2015
- 2015-09-16 CN CN201510588127.4A patent/CN105176963B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1986787A (en) * | 2006-12-21 | 2007-06-27 | 天津大学 | Bionic preparing process of silica-alginic acid microcapsule for immobilized beta-glucurosidase |
| CN104498470A (en) * | 2015-01-16 | 2015-04-08 | 齐鲁工业大学 | New type co-immobilization white rot fungi gel particle |
Non-Patent Citations (1)
| Title |
|---|
| 中空海藻酸钙微胶囊的性质及其固定化枯草杆菌生产纳豆激酶的研究;柴燚;《中国优秀硕士学位论文全文数据库（电子期刊）工程科技I辑》;20040315;第17-20页2.2.2.1/2.3.1，第四章4.3，第五章，第23-24页，表2.2-2.5，图2.6-2.7，第23页2.3.3，28页，17页2.2.2.1.1页,21页2.3.2 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105176963A (en) | 2015-12-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105176963B (en) | The bio-microcapsule and preparation method of a kind of calcium alginate-sodium carboxymethylcellulose co-immobilization whiterot fungi and its ectoenzyme | |
| CN101144097B (en) | Method for preparing chitin and its chitosan and chitosan oligosaccharide | |
| CN103025668B (en) | A kind of method of quick processing wastewater and combinations thereof thing | |
| CN108996709B (en) | A kind of round-the-clock composite photocatalyst collaboration system and preparation method thereof | |
| CN103896407B (en) | A kind of quick startup, biofilm carbon antimicrobial composition process for purifying water | |
| CN101875928A (en) | Embedding immobilization method for microbial preparation | |
| CN106006950B (en) | A kind of method that mycelium pellet promotes aerobic sludge granulation | |
| Pallerla et al. | Characterization of a Ca-alginate-immobilized Trametes versicolor bioreactor for decolorization and AOX reduction of paper mill effluents | |
| CN108017793A (en) | A kind of application being sustained in the preparation method and its chemical wastewater treatment of polyurethane mesh carrier | |
| CN107285480A (en) | Efficient water-purifying agent based on biology enzyme | |
| CN107937382A (en) | A kind of preparation method of immobilized microalgae | |
| Adlercreutz et al. | Oxygen supply to immobilized cells: 1. Oxygen production by immobilized Chlorella pyrenoidosa | |
| CN106045010A (en) | Speed separation ball filter material | |
| Sharari et al. | Treatment of bagasse preparation effluent by Phanerochaete chrysosporium immobilized on polyurethane foam: Enzyme production versus pollution removal | |
| Chen et al. | Encapsulation technology to improve biological resource recovery: recent advancements and research opportunities | |
| CN104498470A (en) | New type co-immobilization white rot fungi gel particle | |
| CN108439750A (en) | A method of it is reduced using Fenton oxidation realization sludge recessive growth and strengthens wastewater treatment | |
| CN107445289A (en) | PVA/ chitosan sphere bio-carriers and preparation method thereof | |
| CN109019874A (en) | A kind of biological growth promoter and preparation method thereof for paper waste | |
| CN110964651A (en) | Preparation method of microbial agent for papermaking wastewater | |
| CN103508564A (en) | Embedded immobilized microorganism deep denitrification reactor and method | |
| Geng et al. | Fast granulation of flocculent activated sludge by mycelium pellet and wastewater biological treatment performance | |
| CN107629962A (en) | A kind of method using starch wastewater culture microalgae | |
| CN106754532A (en) | A kind of composite bacteria agent that surface active agent wastewater is rich in for degrading and preparation method thereof | |
| CN106430831A (en) | Bio-microcapsule-ultrafiltration membrane combined process for treating heavy metals and ammonia nitrogen in water |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20221215 Address after: 250306 Room 3115, No. 135, Ward Avenue, Ping'an Street, Changqing District, Jinan City, Shandong Province Patentee after: Shandong Jiqing Technology Service Co.,Ltd. Address before: 250399 No. 3501 University Road, Changqing District, Jinan City, Shandong Province Patentee before: Qilu University of Technology |
|
| TR01 | Transfer of patent right |