CN107858344A - A kind of method of bio-mimetic syntheses nano zirconium dioxide immobilization laccase - Google Patents

A kind of method of bio-mimetic syntheses nano zirconium dioxide immobilization laccase Download PDF

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CN107858344A
CN107858344A CN201711259138.3A CN201711259138A CN107858344A CN 107858344 A CN107858344 A CN 107858344A CN 201711259138 A CN201711259138 A CN 201711259138A CN 107858344 A CN107858344 A CN 107858344A
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laccase
zirconium dioxide
bio
immobilization
nano
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张新颖
王美银
吴慧敏
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Fuzhou University
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    • 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
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
    • C12N9/0061—Laccase (1.10.3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
    • C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
    • C12Y110/03002—Laccase (1.10.3.2)

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  • Biotechnology (AREA)
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  • Molecular Biology (AREA)
  • Inorganic Chemistry (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

The invention discloses a kind of method of bio-mimetic syntheses nano zirconium dioxide immobilization laccase, it is after mixing certain density lysozyme soln and laccase solution in proportion, it is added in zirconium precursor liquid solution, white precipitate is obtained through vibration, again through centrifuging, washing, being freeze-dried, being fixed laccase particle.Operating process of the present invention is simple and easy to do, reaction condition is gentle, and gained immobilization laccase stability it is high, it is repeatable utilize, overcome the defects of immobilization process existing for traditional process for fixation is complicated, the effect of be combineding with each other of reaction condition acutely, between enzyme and carrier is weak, immobilised enzymes stability difference.

Description

A kind of method of bio-mimetic syntheses nano zirconium dioxide immobilization laccase
Technical field
The invention belongs to immobilised enzymes preparation field, and in particular to a kind of bio-mimetic syntheses nano zirconium dioxide immobilization laccase Method.
Background technology
Laccase is a kind of polyphenol oxidase containing four copper ions, and its course of reaction being catalyzed is produced using molecular oxygen The only accessory substance of water, it is " ecology is friendly " enzyme.Due to not producing secondary dirt with extensive substrate-function, oxidation substrates The features such as dye, laccase are respectively provided with wide application value in fields such as industry, food, environmental protection and cosmetics, and it is developed and application The study hotspot of always domestic and international experts and scholars.However, the laccase under free state is water miscible, and it is quick to external environment condition Sense, stability is poor, can not reuse, and this is greatly limited its application during living things catalysis.Enzyme is consolidated Surely problems above can be efficiently solved by changing technology.
It is to realize that the effective measures of enzyme stability are reused and improved to enzyme to being fixed of laccase using carrier.In recent years Come, increasing carrier is used for enzyme immobilizatio, so as to promote the industrial applications of immobilization laccase.But traditional fixation Change carrier and still suffer from some shortcomings, such as active force between expensive, carrier and enzyme is weaker so that enzyme is easy to run off, carrier is to enzyme activity There is certain inhibitory action etc., therefore, while traditional fixation support is improved, be directed to having developed new fixation support As one of focus studied now.
Nano material because its distinctive physicochemical property and high catalytic efficiency increasingly attract attention in enzyme fixed network, It is widely used as the carrier of immobilised enzymes if the nano materials such as silica, titanium dioxide, being provided for enzyme immobilizatio more has The approach of effect.
But the preparation condition of traditional nano material is complicated harsh, enzyme activity is had a great influence, by bionic structure and process Bionical thought is incorporated into the important directions for turning into the research of present immobilised enzymes in the design and preparation of carrier.
Biomimetic mineralization is to simulate the mechanism that organic matter regulation inorganic matter is formed in biomineralization, designs and synthesizes and be special inorganic Mineral material, make it have and structure and property as natural biological mineral facies.It is bionical compared with material prepared by conventional method Material has more preferable mechanical performance and physicochemical property.The example of earliest biomimetic mineralization is that organic matter is urged in simulation frustule Change the formation of regulation and control silica, on this basis, proposition regulates and controls silicon to researcher by the use of organic matters such as protamine as derivant again Hydrolytie polycondensation forms silica and titanium oxide and is used successfully to enzyme by electrostatic adsorption respectively for presoma and titanium precursors Immobilization.RSC Advance, 2016,6,38117-38123 disclose a kind of bio-mimetic syntheses titanium oxide immobilization horseradish peroxidating The method of thing enzyme, its process include：Horseradish peroxidase is dissolved in the phosphate buffer of pH=7.0 containing lactose, Butyl titanate stirring 1h is added to after being mixed with lecithin, ethanol, particle uses TritonX-100 and ultrapure washing two after being formed Secondary to be freeze-dried obtained titanium oxide immobilized HRP particle again, particle obtained by this method is that nanoscale is irregularly spherical Particle.
Principle based on bionical silication and bionical titanizing, patent of invention CN 103451176A provide one kind and are based on luring certainly The method for leading bionic zirconium immobilization proteinase, it utilizes the induction effect to zirconium precursor body such as papain, bromelain Should, albumen enzyme immobilizatio is realized during bionic zirconium is induced, obtains the immobilization proteinase using zirconium oxide as carrier. But the difficulty using biomimetic method synthesis zirconium oxide is larger, and relevant report is still less.
The content of the invention
Present invention aims at a kind of method of bio-mimetic syntheses nano zirconium dioxide immobilization laccase is provided, it is based on bionical Principle, using lysozyme as derivant, synthesize zirconium oxide immobilization laccase, its easy to operate, mild condition, be swift in response, and Avoid conventional oxidation zirconium and prepare the harsh conditions for needing acid base catalysator, it is cheap, environment-friendly.
To achieve the above object, the present invention uses following technical scheme：
A kind of method of bio-mimetic syntheses nano zirconium dioxide immobilization laccase, it is to be adjusted using lysozyme as derivant, catalysis The formation of zirconium dioxide is controlled, and for painting enzyme immobilizatio；It specifically includes following steps：
（1）Lysozyme is added in phosphate buffer, being slowly stirred makes it be uniformly dispersed, and it is 5 ~ 20mg/mL to obtain concentration Lysozyme soln（Preferably 5mg/mL lysozyme soln）；
（2）Laccase is first diluted to 0.02 ~ 0.15U/mL with phosphate buffer, then is added to step in equal volume（1）It is obtained In lysozyme soln；
（3）By potassium fluorozirconate（K2ZrF6）Before the zirconium for being 0.05 ~ 0.2mmo/L with distilled water or phosphate buffered saline concentration Drive liquid solution（Preferably 0.1mmol/L zirconium precursor liquid solution）；
（4）According to volume ratio 1:8~1:10, by step（2）Obtained mixed liquor is added to step（3）Obtained zirconium precursor body is molten In liquid, after gently vibrating 5 ~ 10min, 4000rpm centrifugation 5min, supernatant, precipitation distilled water or phosphate buffer are removed It is freeze-dried after washing 3 times, obtains the laccase of zirconium dioxide immobilization.
The pH value of the phosphate buffer is 6 ~ 7.5, and concentration is 30 ~ 60mmol/L（Preferably 50mmol/L）, it is Na2HPO4And NaH2PO4Mixed solution.
The present invention has nucleophilic group and hydrogen bond acceptor group both active groups using lysozyme, as induction Agent is played a part of adjusting mineralising, and rapid catalysis induction zirconium precursor body forms the accurate zirconium oxide of structure height；And lured with other Agent is led to compare, using lysozyme as derivant also have antibiotic property and it is cheap and easy to get the advantages of.
The present invention relies on electrostatic adsorption, and the activity for making to be fixed on the laccase on nano zirconium dioxide is effectively protected Stay, make its heat-resisting and acidproof alkali ability is strong, storage stability is good, biocompatibility preferably and possesses certain cyclical stability.
The advantage of the invention is that：
1. the immobilization operating process of the present invention is simple, mild condition, it is swift in response；
2. the method that the present invention prepares immobilization laccase is unique novel, for the enzyme activity rate of recovery up to 59%, it passes through bionical principle The problem of laccase present in traditional immobilization process is lost in is avoided, and improves the utilization rate of laccase；
3. the even particle size of nano zirconium dioxide immobilization laccase prepared by the present invention, particle diameter are about 170nm or so；
4. the heat-resisting and acidproof alkali ability of immobilization laccase prepared by the present invention is high, storage stability is good, favorable repeatability.
Brief description of the drawings
Fig. 1 is the nano zirconium dioxide immobilization laccase prepared in the embodiment of the present invention 1 using various concentrations buffer solution Enzyme activity rate of recovery comparison diagram.
Fig. 2 is the nano zirconium dioxide immobilization paint prepared in the embodiment of the present invention 2 using various concentrations lysozyme soln The enzyme activity rate of recovery comparison diagram of enzyme.
Fig. 3 is the nano zirconium dioxide immobilization prepared in the embodiment of the present invention 3 using various concentrations zirconium precursor liquid solution The enzyme activity rate of recovery comparison diagram of laccase.
Fig. 4 is the SEM figures for the laccase particle that nano zirconium dioxide prepared by the embodiment of the present invention 4 is fixed.
Fig. 5 is the storage stability curve map for the laccase particle that nano zirconium dioxide prepared by the embodiment of the present invention 4 is fixed.
Fig. 6 is that the reuse stability for the laccase particle that nano zirconium dioxide prepared by the embodiment of the present invention 4 is fixed is bent Line chart.
Fig. 7 is nano titanium oxide immobilization laccase particle and nano zirconium dioxide immobilization paint in the embodiment of the present invention 5 The enzyme activity rate of recovery comparison diagram of enzyme granulate.
Embodiment
In order that content of the present invention easily facilitates understanding, with reference to embodiment to of the present invention Technical scheme is described further, but the present invention is not limited only to this.
Embodiment 1：
At room temperature, it is respectively 30mmol/L, 40mmol/L, 50mmol/L, 60mmol/L phosphate-buffered with pH=7.0, concentration Liquid（PBS）5mg/mL lysozyme soln and 0.1U/mL laccase solution are prepared, respectively takes 1mL to be well mixed both solution Afterwards, it is added to 8mL 0.1mmol/L K2ZrF6In solution, 10min, 4000rpm are gently vibrated, white is obtained after centrifuging 5min Precipitation, gained precipitation wash the laccase to remove unreacted presoma and not be embedded three times with PBS, and it is heavy to collect Form sediment, nano zirconium dioxide immobilization laccase particle is obtained after freeze-drying.
The enzyme activity rate of recovery of gained immobilization laccase is as shown in Figure 1.As seen from Figure 1, when the concentration of phosphate buffer is During 50mmol/L, enzyme activity rate of recovery highest.
Embodiment 2：
At room temperature, with pH=7.0,50mmol/L phosphate buffer（PBS）5mg/mL, 10mg/mL, 15mg/ are prepared respectively ML, 20mg/mL lysozyme soln and 0.1U/mL laccase solution, take each 1mL of lysozyme soln of various concentrations, respectively and After the mixing of 1mL laccase solutions, 8mL 0.1mmol/L K is added to2ZrF6In solution, 10min, 4000rpm centrifugations are gently vibrated White precipitate is obtained after 5min, gained precipitation is washed three times with PBS to remove unreacted presoma and not be embedded Laccase, collect precipitation, nano zirconium dioxide immobilization laccase particle is obtained after freeze-drying.
The enzyme activity rate of recovery of gained immobilization laccase is as shown in Figure 2.From Figure 2 it can be seen that when the concentration of lysozyme is 5mg/mL When, enzyme activity rate of recovery highest.
Embodiment 3：
At room temperature, with pH=7.0,50mmol/L phosphate buffer（PBS）Prepare 5mg/mL lysozyme soln and 0.1U/ ML laccase solution, after respectively taking 1mL well mixed both solution, be added to 8mL concentration be respectively 0.05mmol/L, 0.1mmol/L, 0.15mmol/L, 0.2mmol/L K2ZrF6In solution, 10min is gently vibrated, is obtained after 4000rpm centrifugations 5min To white precipitate, gained precipitation washs the laccase to remove unreacted presoma and not be embedded three times with PBS, receives Collection precipitation, obtains nano zirconium dioxide immobilization laccase particle after freeze-drying.
The enzyme activity rate of recovery of gained immobilization laccase is as shown in Figure 3.As seen from Figure 3, K is worked as2ZrF6The concentration of solution is During 0.1mmol/L, enzyme activity rate of recovery highest.
Embodiment 4：
At room temperature, with pH=7.0,50mmol/L phosphate buffer（PBS）Prepare 5mg/mL lysozyme soln and 0.1U/ ML laccase solution, after respectively taking 1mL well mixed both solution, it is added to 8mL 0.1mmol/L K2ZrF6In solution, 10min is gently vibrated, white precipitate is obtained after 4000rpm centrifugations 5min, gained precipitation is washed three times with PBS to remove Unreacted presoma and the laccase not being embedded, precipitation is collected, nano zirconium dioxide immobilization laccase is obtained after freeze-drying Particle.
The scanning figure for the laccase particle that gained nano zirconium dioxide is fixed is as shown in Figure 4.As can be seen from Fig. 4, the immobilization The size of laccase particle is in 170nm or so.
Using ABTS methods, laccase activity is determined at wavelength 420nm using ultraviolet-uisible spectrophotometer.As a result show, The enzyme activity rate of recovery of gained immobilization laccase is 59%, and as seen from Figure 5, after being stored 30 days at 4 DEG C, its enzyme activity is maintained as Initial more than 95%, and as seen from Figure 6, it is 60.5% that gained immobilization laccase, which is continuously repeated using the residual enzyme activity after 5 times, Have and preferably reuse ability.
Embodiment 5：
The nano zirconium dioxide immobilization laccase prepared in embodiment 4 and nano titanium oxide immobilization laccase are carried out into enzyme activity to return Yield contrasts, as a result as shown in Figure 7.The preparation method of nano titanium oxide immobilization laccase：At room temperature, with pH=7.0, 50mmol/L Tris-HCl buffers 5mg/mL protamine sulfate solution and 0.1 U/mL laccase solution, by this After two kinds of solution respectively take 1mL well mixed, the ammonium of two (2 hydroxy propanoic acid) two hydroxide two for being added to 1mL 30mmol/L closes titanium （Ti-BALDH）In solution, 10min is gently vibrated, 4000rpm centrifugations 5min obtains white precipitate, gained precipitation Tris-HCl The laccase to remove unreacted presoma and not be embedded, collection are precipitated, received after freeze-drying three times for buffer solution washing Rice titanium dioxide immobilization laccase particle.
As seen from Figure 7, the enzyme activity rate of recovery of nano titanium oxide immobilization laccase particle is 41.6%, nano zirconium dioxide For the enzyme activity rate of recovery of immobilization laccase particle up to 59%, i.e. the enzyme activity rate of recovery of nano zirconium dioxide immobilization laccase particle is obvious Higher than nano titanium oxide immobilization laccase particle.
The foregoing is only presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with Modification, it should all belong to the covering scope of the present invention.

Claims (4)

  1. A kind of 1. method of bio-mimetic syntheses nano zirconium dioxide immobilization laccase, it is characterised in that：Using lysozyme as induction Agent, the formation of catalysis regulation and control zirconium dioxide, and for painting enzyme immobilizatio.
  2. 2. the method for bio-mimetic syntheses nano zirconium dioxide immobilization laccase according to claim 1, it is characterised in that：Including with Lower step：
    （1）Lysozyme is added in phosphate buffer, being slowly stirred makes it be uniformly dispersed, and it is 5 ~ 20mg/mL to obtain concentration Lysozyme soln；
    （2）Laccase is first diluted to 0.02 ~ 0.15U/mL with phosphate buffer, then is added to step in equal volume（1）It is obtained In lysozyme soln；
    （3）Zirconium precursor liquid solution with distilled water or phosphate buffered saline concentration for 0.05 ~ 0.2mmo/L；
    （4）According to volume ratio 1:8~1:10 ratio, by step（2）Obtained mixed liquor is added to step（3）Before obtained zirconium Drive in liquid solution, after gently vibrating 5 ~ 10min, 4000rpm centrifugation 5min, remove supernatant, precipitation distilled water or phosphate Buffer solution is freeze-dried after washing 3 times, obtains the laccase of zirconium dioxide immobilization.
  3. 3. the method for bio-mimetic syntheses nano zirconium dioxide immobilization laccase according to claim 2, it is characterised in that：The phosphorus The pH value of phthalate buffer is 6 ~ 7.5, and concentration is 30 ~ 60mmol/L.
  4. 4. the method for bio-mimetic syntheses nano zirconium dioxide immobilization laccase according to claim 2, it is characterised in that：The zirconium Presoma is potassium fluorozirconate.
CN201711259138.3A 2017-12-04 2017-12-04 A kind of method of bio-mimetic syntheses nano zirconium dioxide immobilization laccase Pending CN107858344A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111229193A (en) * 2020-01-15 2020-06-05 重庆师范大学 Application of zirconium dioxide nano particles as alkaline phosphatase nano mimics
CN115404225A (en) * 2022-10-14 2022-11-29 兰州大学 Nitrogen-doped titanium dioxide and titanium carbide co-modified PVDF (polyvinylidene fluoride) film immobilized laccase as well as method and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103451176A (en) * 2013-09-21 2013-12-18 桂林理工大学 Proteinase immobilization method based on autoinduction bionic zirconium
CN107164357A (en) * 2017-05-31 2017-09-15 桂林理工大学 A kind of method based on autoinduction bionic titanizing immobilization proteinase

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
CN103451176A (en) * 2013-09-21 2013-12-18 桂林理工大学 Proteinase immobilization method based on autoinduction bionic zirconium
CN107164357A (en) * 2017-05-31 2017-09-15 桂林理工大学 A kind of method based on autoinduction bionic titanizing immobilization proteinase

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YANJUN JIANG等: "Facile Synthesis and Novel Application of Zirconia Catalyzed and Templated by Lysozyme", 《IND.ENG.CHEM.RES》 *
和文祥等: "《环境微生物学》", 28 February 2007, 中国农业大学出版社 *
马翠丽: "基于仿生矿化固定化脂肪酶研究", 《中国优秀硕士学位论文全文数据库基础科学辑》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111229193A (en) * 2020-01-15 2020-06-05 重庆师范大学 Application of zirconium dioxide nano particles as alkaline phosphatase nano mimics
CN111229193B (en) * 2020-01-15 2022-10-18 重庆师范大学 Application of zirconium dioxide nano particles as alkaline phosphatase nano mimics
CN115404225A (en) * 2022-10-14 2022-11-29 兰州大学 Nitrogen-doped titanium dioxide and titanium carbide co-modified PVDF (polyvinylidene fluoride) film immobilized laccase as well as method and application thereof
CN115404225B (en) * 2022-10-14 2024-03-15 兰州大学 Nitrogen-doped titanium dioxide and titanium carbide co-modified PVDF membrane immobilized laccase and method and application thereof

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