CN111607584A - Method for immobilizing marine cyclodextrin glucosyltransferase by resin - Google Patents

Method for immobilizing marine cyclodextrin glucosyltransferase by resin Download PDF

Info

Publication number
CN111607584A
CN111607584A CN202010498247.6A CN202010498247A CN111607584A CN 111607584 A CN111607584 A CN 111607584A CN 202010498247 A CN202010498247 A CN 202010498247A CN 111607584 A CN111607584 A CN 111607584A
Authority
CN
China
Prior art keywords
enzyme
resin
bsi
carrier
immobilized
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.)
Pending
Application number
CN202010498247.6A
Other languages
Chinese (zh)
Inventor
郝建华
王伟
孙晶晶
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
Original Assignee
Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences filed Critical Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
Priority to CN202010498247.6A priority Critical patent/CN111607584A/en
Publication of CN111607584A publication Critical patent/CN111607584A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • C12N11/091—Phenol resins; Amino resins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C12N9/1074—Cyclomaltodextrin glucanotransferase (2.4.1.19)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01019—Cyclomaltodextrin glucanotransferase (2.4.1.19)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

The invention relates to a method for immobilizing marine cyclodextrin glucosyltransferase by resin, which belongs to the technical field of biology and comprises the following steps: weighing activated resin MI-BSI, adding the activated resin MI-BSI into CGTase enzyme solution with the concentration of 0.067mg/mL, preparing the enzyme solution by glycine-sodium hydroxide buffer solution with the pH value of 9, and immobilizing the activated resin MI-BSI and the CGTase enzyme solution for 8 hours in a shaking table with the temperature of 35 ℃ and the rpm of 200, wherein the mass-volume ratio of the activated resin MI-BSI to the CGTase enzyme solution is 10:1 (mg/mL). The invention adopts the immobilization technology of adsorption and covalent bonding method, the reaction condition is mild, the operation is simple, the cost is lower, the thermal stability of the immobilized cyclodextrin glucosyltransferase is better than that of free enzyme, the pH stability is better than that of the free enzyme, the tolerance to acid and alkali is enhanced, the reusability of the immobilized enzyme is good, the immobilized enzyme can be reused for 10 times, and the retention rate of the relative enzyme activity is more than 65%.

Description

Method for immobilizing marine cyclodextrin glucosyltransferase by resin
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for immobilizing marine cyclodextrin glucosyltransferase by resin.
Background
Cyclodextrin glucosyltransferases (CGTase, EC2.4.1.19), which are important members of the alpha-amylase family 13, catalyze four reactions, hydrolysis, cyclization, coupling and disproportionation. Among the four reactions, the cyclization reaction, the disproportionation reaction and the coupling reaction are transglycosylation reactions, and have high activity and low hydrolysis activity, and the control of the hydrolysis activity is favorable for the production of cyclodextrin, wherein the cyclization reaction is a characteristic reaction for producing cyclodextrin, and CGTase is used as a biocatalyst mainly for catalyzing the conversion of starch into cyclodextrin in industry. Cyclodextrins (CD) include primarily alpha-CD, beta-CD, and gamma-CD. The cyclodextrin is a molecule with a cylindrical structure, and based on the special structure that the interior of the cyclodextrin is hydrophobic and the exterior of the cyclodextrin is hydrophilic, the cyclodextrin and the derivatives thereof have great application prospects in the food industry, the biotechnology, the pharmaceutical industry, the cosmetics industry, the chemical industry and other aspects. Therefore, there is also an increasing research on CGTase required for the production of cyclodextrins.
The immobilized enzyme is a new technology developed in the sixties of the twentieth century. The localization and restriction of immobilized enzymes, i.e., enzymes, means that free enzymes are bound or restricted in a certain region by binding the free enzymes to a carrier, and the free enzymes cannot move freely but can still perform their specific catalytic reactions. After the enzyme is immobilized, the enzyme is easy to separate from a product and a substrate, can be effectively recovered and reused, and has enhanced thermal stability and operation stability compared with free enzyme. The traditional immobilization method mainly comprises an adsorption method, an embedding method, a cross-linking method, a covalent bonding method and the like, wherein the binding force between an enzyme and a carrier in the adsorption method is not strong, and the enzyme molecules are easy to separate from the carrier; the enzyme liquid in the embedding method is easy to run off and is not easy to catalyze macromolecular substrates; cross-linking methods are generally used in combination with other methods; in the covalent binding method, the enzyme is firmly bound with the carrier, but the enzyme activity is influenced due to the fierce immobilization condition.
Disclosure of Invention
The invention aims to provide a method for immobilizing marine cyclodextrin glucosyltransferase by a covalent bonding method, which has the characteristics of simple operation, low cost, good immobilized enzyme stability, reusability and the like.
The invention is realized by the following technical scheme:
a method for immobilizing marine cyclodextrin glucosyltransferase by resin comprises the following specific steps:
(1) the carrier activation selects amino functional resin MI-BSI as a carrier, and the carrier is activated by the following steps before immobilization: adding 0.1M PBS buffer solution with pH8.0 into the carrier, soaking and cleaning for 1h, filtering and draining, wherein the mass-volume ratio of the carrier to the PBS buffer solution is 1:4(g/ml), adding equal volume of 0.1M PBS buffer solution with pH8.0 and containing 2% glutaraldehyde, slowly stirring for 1h at 25 ℃, filtering, washing the carrier for 3-5 times by deionized water, and freeze-drying to obtain an activated carrier;
(2) accurately weighing activated resin MI-BSI, adding the resin MI-BSI into CGTase enzyme solution with the concentration of 0.067mg/mL, preparing the enzyme solution by glycine-sodium hydroxide buffer solution with the pH value of 9, and immobilizing the resin MI-BSI and the CGTase enzyme solution for 8 hours in a shaking table at the temperature of 35 ℃ and the rpm of 200, wherein the mass-to-volume ratio of the activated resin MI-BSI to the CGTase enzyme solution is 10:1 (mg/mL).
The marine microorganism cyclodextrin glucosyltransferase is prepared from marine product resources of yellow sea aquatic research institute and enzyme laboratories.
Compared with the prior art, the invention has the beneficial effects that:
1. the amino functional resin provided by the invention has the advantages of amino functional groups and the like, and is widely applied to the research in the field of biotechnology.
2. The invention adopts the immobilization technology of adsorption and covalent bonding method, has mild reaction condition, simple operation and lower cost
3. The heat stability of the immobilized cyclodextrin glucosyltransferase is superior to that of free enzyme, the pH stability is superior to that of the free enzyme, the tolerance to acid and alkali is enhanced, the reusability of the immobilized enzyme is good, the immobilized enzyme is reused for 10 times, and the retention rate of the relative enzyme activity is over 65 percent.
Drawings
FIG. 1: pH stability of immobilized enzyme
FIG. 2: metal ion tolerance of immobilized enzyme
FIG. 3: stability of operation of immobilized enzyme
Detailed Description
The technical solutions of the present invention are further illustrated by the following examples, but the scope of the present invention is not limited by the examples in any way. And are not limited to the following examples:
example 1
A method for immobilizing marine cyclodextrin glucosyltransferase by resin specifically comprises the following steps:
(1) screening immobilized carriers, respectively weighing certain resins, treating the resins before immobilization, adding CGTase enzyme solution with certain enzyme protein concentration, and shaking and fixing for a certain time. After the immobilization is finished, separating the resin of the immobilized enzyme from the supernatant, washing the resin and the supernatant for multiple times by using a buffer solution until the content of the enzyme protein in the supernatant is zero, and measuring the influence of different resins on the recovery rate of the enzyme activity and the protein immobilization rate. Screening resin with highest enzyme activity recovery rate and protein immobilization rate as immobilized carrier (resin including MA-WP8, MA-P9, MI-BSI, MC-300EP, MI-BN4, LX-1000EP and LX-1000 HA);
(2) activation of the carrier: adding 40ml of 0.1M PBS (pH 8.0) buffer solution into 10g of carrier, soaking and washing for 1h, filtering and draining, adding 0.1M PBS (pH 8.0) buffer solution containing 40ml of 2% glutaraldehyde, slowly stirring for 1h at 25 ℃, filtering, washing the carrier with deionized water for 3-5 times, and freeze-drying to obtain the activated carrier. (3) 10mg of activated amino functional resin was weighed out accurately, 1mL of CGTase enzyme solution with an enzyme protein concentration of 0.067mg/mL was added, and the mixture was fixed in a shaker at 35 ℃ and 200rpm for 8 hours. After the immobilization is finished, the immobilized CGTase is separated from the supernatant, the CGTase is repeatedly washed by glycine-sodium hydroxide buffer solution until the content of enzyme protein in the supernatant is zero, and the enzyme activity recovery rate and the protein immobilization rate of the immobilized enzyme are analyzed.
The marine microorganism cyclodextrin glucosyltransferase is prepared from marine product resources of yellow sea aquatic research institute and enzyme laboratory laboratories
Example 2 pH stability of immobilized enzyme
Respectively placing free enzyme and immobilized enzyme in buffer solutions with pH of 4, 5, 6, 7, 8, 9, 10, 11, and 12, treating for 2h, measuring enzyme activity, and exploring influence of pH on immobilized enzyme and free enzyme activity with highest enzyme activity of 100%
And (3) placing the free enzyme and the immobilized enzyme under the condition of pH 4-12 to act for 2h, measuring the enzyme activity and researching the influence of pH on the enzyme activity. As shown in FIG. 1, the results of the catalytic performance showed that the enzyme activities of the immobilized enzyme and the free enzyme increased with increasing pH at pH 4-9, and decreased with increasing pH at pH 9-12. However, under the same pH condition, the relative enzyme activity of the immobilized enzyme is higher than that of the free enzyme, which shows that after the CGTase is immobilized by a resin MI-BSI carrier, the immobilized enzyme has higher tolerance to acid and alkali than the free enzyme.
Example 3 tolerance of immobilized enzymes to Metal ions
Adding K into free enzyme and immobilized enzyme respectively+、Ca2+、Mn2+、Ba2+、Fe2+、Fe3+、Al3+、Cu2+、Mg2+Waiting 9 metal ions to ensure that the final concentration of the solution is 5mmol/L, then processing for 2h, measuring the enzyme activity, and exploring the influence of the metal ions on the activity of the immobilized enzyme and the free enzyme by taking the highest enzyme activity as 100 percent
Placing the free enzyme and the immobilized enzyme in solutions of different metal ions for acting for 2h, measuring the enzyme activity and researching the influence of different metal ions on the enzyme activity. As shown in FIG. 2, after different metal ions are treated, the enzyme activities of free enzyme and immobilized enzyme are changed, except for Mg2+In addition, the immobilized enzymes have stronger tolerance to metal ions than free enzymes. By metal ions Al3+、Fe2 +、Fe3+After treatment, the activity of the CGTase is obviously inhibited.
Example 4 operational stability of immobilized enzymes
After a certain amount of immobilized enzyme is reacted, the immobilized enzyme is magnetically adsorbed, the immobilized enzyme is washed for a plurality of times by using buffer solution, the enzyme activity of the immobilized enzyme is continuously measured, the enzyme activity measured for the first time is 100 percent, the immobilized enzyme is washed and used for the catalytic reaction of the next batch, the reaction is continuously carried out for 5 times, the residual enzyme activity of the immobilized enzyme is measured, and the operation stability of the immobilized enzyme is researched.
After the resin MI-BSI immobilized marine cyclodextrin glucosyltransferase is repeatedly used for 10 times, as shown in figure 3, the relative enzyme activity of the amino functional resin MI-BSI immobilized marine cyclodextrin glucosyltransferase is still kept at 68.2 percent, which shows that the resin MI-BSI carrier immobilized CGTase has a good combination effect, and the immobilized enzyme has good operation stability.

Claims (1)

1. A method for immobilizing marine cyclodextrin glucosyltransferase is characterized by comprising the following steps:
(1) the carrier activation selects amino functional resin MI-BSI as a carrier, and the carrier is activated by the following steps before immobilization: adding 0.1M PBS buffer solution with the pH value of 8.0 into the carrier, soaking and cleaning for 1h, filtering and draining, wherein the mass-volume ratio of the carrier to the PBS buffer solution is 1:4, the unit of the proportion is g/ml, adding equal volume of 0.1M PBS buffer solution with the pH value of 8.0 and containing 2% glutaraldehyde, slowly stirring for 1h at 25 ℃, filtering, washing the carrier for 3-5 times by using deionized water, and freeze-drying to obtain an activated carrier;
(2) accurately weighing activated resin MI-BSI, adding the resin MI-BSI into CGTase enzyme solution with the concentration of 0.067mg/mL, preparing the enzyme solution by glycine-sodium hydroxide buffer solution with the pH value of 9, fixing the activated resin MI-BSI and the CGTase enzyme solution for 8 hours in a shaking table with the temperature of 35 ℃ and the rpm of 10: 1.
CN202010498247.6A 2020-06-04 2020-06-04 Method for immobilizing marine cyclodextrin glucosyltransferase by resin Pending CN111607584A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010498247.6A CN111607584A (en) 2020-06-04 2020-06-04 Method for immobilizing marine cyclodextrin glucosyltransferase by resin

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010498247.6A CN111607584A (en) 2020-06-04 2020-06-04 Method for immobilizing marine cyclodextrin glucosyltransferase by resin

Publications (1)

Publication Number Publication Date
CN111607584A true CN111607584A (en) 2020-09-01

Family

ID=72198976

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010498247.6A Pending CN111607584A (en) 2020-06-04 2020-06-04 Method for immobilizing marine cyclodextrin glucosyltransferase by resin

Country Status (1)

Country Link
CN (1) CN111607584A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112048498A (en) * 2020-09-14 2020-12-08 江苏省奥谷生物科技有限公司 Preparation method for improving yield of beta-cyclodextrin

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108977430A (en) * 2018-09-14 2018-12-11 中国水产科学研究院黄海水产研究所 A kind of process for fixation of marine microorganism cyclodextrin glycosyltransferase

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108977430A (en) * 2018-09-14 2018-12-11 中国水产科学研究院黄海水产研究所 A kind of process for fixation of marine microorganism cyclodextrin glycosyltransferase

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
JÉSSIE DA NATIVIDADE SCHÖFFER等: "Directed immobilization of CGTase: The effect of the enzyme orientation on the enzyme activity and its use in packed-bed reactor for continuous production of cyclodextrins", 《PROCESS BIOCHEMISTRY》 *
刘桂林: "《生物技术概论》", 30 September 2010, 中国农业大学出版社 *
姜峻颖等: "海洋脂肪酶YS2071的固定化及酶学性质研究", 《食品与发酵工业》 *
朱伯儒等: "环糊精葡萄糖基转移酶的固定化及其应用──Ⅲ．固定化环糊精葡萄糖基转移酶制备环糊精的研究", 《应用化学》 *
郭姣梅: "海洋环糊精葡萄糖基转移酶的固定化及其性能研究", 《硕士中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112048498A (en) * 2020-09-14 2020-12-08 江苏省奥谷生物科技有限公司 Preparation method for improving yield of beta-cyclodextrin
CN112048498B (en) * 2020-09-14 2023-10-03 江苏省奥谷生物科技有限公司 Preparation method for improving beta-cyclodextrin yield

Similar Documents

Publication Publication Date Title
Mardani et al. Immobilization of α-amylase on chitosan-montmorillonite nanocomposite beads
Rekuć et al. Very stable silica-gel-bound laccase biocatalysts for the selective oxidation in continuous systems
Ureta et al. Recent advances in β-galactosidase and fructosyltransferase immobilization technology
Noreen et al. Performance improvement of Ca-alginate bead cross-linked laccase from Trametes versicolor IBL-04
de Segura et al. Immobilization of Dextransucrase from Leuconostocmesenteroides NRRL B‐512F on Eupergit C Supports
Gargouri et al. Fungus β-glycosidases: immobilization and use in alkyl-β-glycoside synthesis
EP0158909A2 (en) Immobilized enzymes, processes for preparing same and use thereof
Güleç et al. Immobilization of Aspergillus oryzae β-galactosidase on low-pressure plasma-modified cellulose acetate membrane using polyethyleneimine for production of galactooligosaccharide
CN109576256B (en) Method for encapsulating double enzymes by magnetic DNA hydrogel
Zhang et al. Progress and perspective of enzyme immobilization on zeolite crystal materials
Wu et al. Preparation and characterization of tannase immobilized onto carboxyl-functionalized superparamagnetic ferroferric oxide nanoparticles
JPS5978687A (en) Immobilization of catalyst on granular carbon
Wang et al. Improving the stability and reusability of dextranase by immobilization on polyethylenimine modified magnetic particles
Charoenpol et al. Marine chitin upcycling with immobilized chitinolytic enzymes: current state and prospects
CN111607584A (en) Method for immobilizing marine cyclodextrin glucosyltransferase by resin
Ozyilmaz et al. Preparation of regenerable magnetic nanoparticles for cellulase immobilization: Improvement of enzymatic activity and stability
Fatima Use of nanomaterials for the immobilization of industrially important enzymes
Safarik et al. Development of advanced biorefinery concepts using magnetically responsive materials
Graebin et al. Preparation and characterization of cross-linked enzyme aggregates of dextransucrase from Leuconostoc mesenteroides B-512F
Svensson et al. Immobilisation of CGTase for continuous production of long-carbohydrate-chain alkyl glycosides: Control of product distribution by flow rate adjustment
Kovaleva et al. Inulinase immobilization on macroporous anion-exchange resins by different methods
Rai et al. Development of recyclable magnetic cross-linked enzyme aggregates for the synthesis of high value rare sugar d-tagatose in aqueous phase catalysis
Mahmoud Immobilization of invertase by a new economical method using wood sawdust waste
Ahmad et al. Methods of enzyme immobilization on various supports
CN107760666B (en) Reversible double-enzyme co-immobilization method capable of regulating enzyme ratio

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20200901

WD01 Invention patent application deemed withdrawn after publication