CN103451176A

CN103451176A - Proteinase immobilization method based on autoinduction bionic zirconium

Info

Publication number: CN103451176A
Application number: CN2013104278825A
Authority: CN
Inventors: 李海云; 余艳葵; 阮贵华; 李子院
Original assignee: Guilin University of Technology
Current assignee: Guilin University of Technology
Priority date: 2013-09-21
Filing date: 2013-09-21
Publication date: 2013-12-18

Abstract

The invention discloses a proteinase immobilization method based on autoinduction bionic zirconium. By adopting the bionic zirconium induction effect of bromelain, papain or trypsin on a K2ZrF6 zirconium precursor, proteinase immobilization is realized in the induction bionic zirconium process so as to obtain the immobilized proteinase taking zirconium oxide as a carrier. The method overcomes the defects of the traditional immobilization method that the immobilization process is complex, biocompatibility between the proteinase and the carrier is bad, the combination effect of the proteinase and the carrier is relatively weak, stability of the immobilized proteinase is poor, the service life is short and the proteinase is inactive due to commonly severe immobilization reaction conditions, and the like, and has the advantages of simple immobilization process, no need of additional inducer, mild conditions, high efficiency and reusability, etc.

Description

A kind of method based on the bionical zirconium immobilization proteinase of self-induction

Technical field

The invention belongs to the immobilized enzyme preparing technical field, particularly a kind of method based on the bionical zirconium immobilization proteinase of self-induction.

Background technology

Proteolytic enzyme has the advantages such as highly selective, high catalytic activity, reaction conditions gentleness, environmental protection.In fields such as food, medicine, environment, be used widely.Yet under state of nature, most of enzymes are water miscible, make the application of enzyme in the biocatalysis process under unbound state be very restricted, be mainly manifested in the poor stability of resolvase, the product that can not recycle, generate is difficult to separation, purifying etc. from reaction mixture.The immobilization of carrying out enzyme is the effective ways that address the above problem, so explore fixation support and the process for fixation of enzyme, the immobilized enzyme for preparing high reactivity, high stability, high mechanical strength will have important theoretical and practical significance.

Existing enzyme immobilization method commonly used is broadly divided into 4 classes: absorption method, crosslinking, covalent coupling method, entrapping method.Absorption method is easy and simple to handle, and little to the destruction of enzyme conformation, but, a little less than the bonding force of enzyme and carrier, after enzyme immobilization, easily comes off; The reaction conditions of crosslinking is fiercer, and in the enzyme molecule, some group is crosslinked, and can cause the enzyme rate of recovery alive to reduce larger; The covalent attachment rule require enzyme have special can with the functional group of carrier generation linked reaction, but violent chemical reaction often can cause the inactivation of enzyme, generally enzymatic activity recovery only has 30% left and right; With absorption method, crosslinking, covalent coupling method, compare, the preparation method of the carrier that entrapping method is used has that required reaction conditions is gentle, the enzyme molecule is difficult for leaking, the advantage such as little and biological activity fixedly high density unit to biological activity unit deactivation.Enzyme is enclosed in the grid of superpolymer or is surrounded by semipermeable polymeric film and become two kinds of lattice type and microcapsule-types, due to carrier, enzyme and external environment can be separated again, so also contribute to improve the stability of enzyme.The suitability that entrapping method has more generally, make it obtain in recent years paying close attention to widely.

Fixed enzyme vector is expensive at present, and the vigor thermostability of most immobilized enzyme is high not enough, and the operation transformation period also falls short of, and is difficult to realize industrial serialization production.When improving the conventional carriers material, the research and development type material becomes the main direction of fixed enzyme vector research from now on.Along with the bionic development of material, the researchist in immobilized enzyme field is by bionical thought, particularly the bionical thought of bionic structure and process is incorporated in the design and preparation of this exotic materials of fixed enzyme vector, has developed novel fixed enzyme vector.

The biomimetic mineralization of simulation biomineralization is exactly that the mechanism of biomineralization is introduced to the synthetic of inorganic materials, take the organism assembly as template, removing to control inorganics forms, the organic mineral complex that preparation has special assembling mode and multilevel hierarchy characteristics, make material have excellent physical and chemical performance.The basic skills of inducing biomimetic mineralization is generally to utilize inductor to induce inorganic precursor (as tetraethoxy, potassium fluozirconate, two hydroxide two ammoniums close titanium etc.) hydrolysis polycondensation with negative electricity to generate oxide compound, enzyme is embedded among oxide particle simultaneously, form immobilized enzyme, at aspects such as materialogy, industrial production, chemical analysis and medicine, tempting application prospect is arranged.

The general method that utilizes at present biomimetic mineralization to carry out enzyme immobilization is to utilize the basic proteins such as protamine as inductor.Owing to having added extra protein induce agent in system, the method is only applicable to the immobilization of nonprotein hydrolase and should not carries out the immobilization of proteolysis enzyme.The present invention utilizes bromeline, papoid and trypsinase to realize the biomimetic mineralization immobilization to the inducing action of bionical zirconium, in system, does not need additionally to add protein induced dose, has overcome the deficiency of aforesaid method.Utilize at present the method for the bionical zirconium immobilization proteinase of self-induction to there is not yet report.

Summary of the invention

The object of the invention is to overcome the defect of traditional process for fixation, a kind of method based on the bionical zirconium immobilization proteinase of self-induction is provided.

Concrete steps are:

Under room temperature, add the zirconium precursor liquid solution that 8 ~ 12 mL concentration are 0.01 ~ 0.05 mol/L in immobilization reactor, then adding the concentration of the damping fluid preparation of pH 6.0 ~ 8.0 for 8 ~ 24 mL, concentration 0.02 mol/L ~ 0.12 mol/L is 4 mg/mL ~ 12 mg/mL floating preteins enzyme solution, and the volume ratio that makes zirconium precursor liquid solution and floating preteins enzyme solution is 1:1 ~ 2; Stir gently 5 min, 5000 rpm frozen centrifugation 10 min, collecting precipitation; Precipitate with after distilled water wash 3 times lyophilize, being fixed enzyme.

Described floating preteins enzyme is a kind of in bromeline, trypsinase and papoid.

Described zirconium precursor liquid solution is K ₂zrF ₆solution.

Described damping fluid is phosphoric acid salt (SODIUM PHOSPHATE, MONOBASIC and Sodium phosphate dibasic mix) damping fluid.

Advantage of the present invention and effect are: the immobilization process of resolvase is simple, mild condition, time shorten, good biocompatibility, Energy Intensity Reduction; The enzyme of the immobilized enzyme rate of recovery alive and embedding rate are higher; Immobilized enzyme stability is good.

The accompanying drawing explanation

Fig. 1 is the bionical zirconium immobilization of the embodiment of the present invention 1 bromeline, zirconium dioxide standard reference and K ₂zrF ₆infrared spectrogram.

In figure: the A-zirconium dioxide; B-immobilization bromeline; C-K ₂zrF ₆.

Embodiment

The damping fluid that following phosphate buffered saline buffer is SODIUM PHOSPHATE, MONOBASIC and Sodium phosphate dibasic mixed preparing.

Embodiment 1:

Under room temperature, add 10 mL 0.02 mol/L K in immobilization reactor ₂zrF ₆solution, then be 6 mg/mL bromelain enzyme solution to the concentration that adds in immobilization reactor pH 7.5 for 20 mL, concentration to be 0.10 mol/L phosphate buffered saline buffer preparation, make K ₂zrF ₆the volume ratio of solution and bromelain enzyme solution is 1:2; Stir gently 5 min, 5000 rpm frozen centrifugation 10 min, collecting precipitation.Distilled water wash precipitation 3 times, collect washing lotion, merge supernatant liquor, then will precipitate lyophilize 24 hours, the immobilized enzyme enzyme of the gained rate of recovery alive is 59.52%, the bromeline embedding rate is 63.03%, and after same batch immobilized enzyme is reused 6 times, enzyme is lived and still can be kept more than 60%.

The immobilization bromeline obtained, after the sample porphyrize, pressing potassium bromide troche, adopt Fourier infrared spectrograph to measure its infrared spectra, as shown in Figure 1.Contrast immobilization bromeline (Fig. 1-B) and zirconium precursor body K ₂zrF ₆(Fig. 1-C) is known, through bromelain enzyme induction zirconium precursor body K ₂zrF ₆after zirconium, at 3360 cm ^-1, 2977 cm ^-1, 1667 cm ^-1, 1557 cm ^-1, 537 cm ^-1etc. the wave number place, new spectrum peak appears, wherein 3360 cm ^-1belong to the amine groups in the amide group in the bromeline molecule, 2977 cm ^-1belong to alkyl, 1667 cm ^-1with 1557 cm ^-1be that carbonylic stretching vibration in amide group causes, show bromelain enzyme induction K ₂zrF ₆really be embedded with bromeline in the precipitation of zirconium generation.Contrast immobilization bromeline (Fig. 1-B) is known with zirconium dioxide (Fig. 1-A), at 500 ~ 600 cm ^-1obvious absorption is all arranged in scope, and this is caused by the Zr-O-Zr stretching vibration, shows under the inducing of bromeline K ₂zrF ₆zirconiumization has generated zirconium dioxide really.Result shows, bromeline can be used as template-mediated K ₂zrF ₆thereby itself being embedded in precipitation, zirconium generation zirconium dioxide and bromeline molecule realize immobilization.

Embodiment 2:

Under room temperature, add 10 mL 0.035 mol/L K in immobilization reactor ₂zrF ₆solution, then be 8 mg/mL trypsin solutions to the concentration that adds in immobilization reactor pH 8.0 for 10 mL, concentration to be 0.08 mol/L phosphate buffered saline buffer preparation, make K ₂zrF ₆the volume ratio of solution and trypsin solution is 1:1; Stir gently 5 min, 5000 rpm frozen centrifugation 10 min, collecting precipitation; Distilled water wash precipitation 3 times, collect washing lotion, merge supernatant liquor, then will precipitate lyophilize 24 hours, the immobilized enzyme enzyme of the gained rate of recovery alive is 46.69%, the trypsinase embedding rate is 63.03%, and after same batch immobilized enzyme is reused 5 times, enzyme is lived and still can be kept more than 60%.

Embodiment 3:

Under room temperature, add 10 mL 0.02 mol/L K in immobilization reactor ₂zrF ₆solution, then be 8 mg/mL papoid solution to the concentration that adds in immobilization reactor pH 6.5 for 20 mL, concentration to be 0.08 mol/L phosphate buffered saline buffer preparation, make K ₂zrF ₆the volume ratio of solution and papoid solution is 1:2; Stir gently 5 min, 5000 rpm frozen centrifugation 10 min, collecting precipitation; Distilled water wash precipitation 3 times, collect washing lotion, merge supernatant liquor, then will precipitate lyophilize 24 hours, the immobilized enzyme enzyme of the gained rate of recovery alive is 54.45%, the papoid embedding rate is 89.15%, and after same batch immobilized enzyme is reused 6 times, enzyme is lived and still can be kept more than 60%.

Claims

1. the method based on the bionical zirconium immobilization proteinase of self-induction is characterized in that concrete steps are:

Under room temperature, add the zirconium precursor liquid solution that 8 ~ 12 mL concentration are 0.01 ~ 0.05 mol/L in immobilization reactor, then adding the concentration of the damping fluid preparation of pH 6.0 ~ 8.0 for 8 ~ 24 mL, concentration 0.02 mol/L ~ 0.12 mol/L is 4 mg/mL ~ 12 mg/mL floating preteins enzyme solution, and the volume ratio that makes zirconium precursor liquid solution and floating preteins enzyme solution is 1:1 ~ 2; Stir gently 5 min, 5000 rpm frozen centrifugation 10 min, collecting precipitation; Precipitate with after distilled water wash 3 times lyophilize, being fixed enzyme;

Described floating preteins enzyme is a kind of in bromeline, trypsinase and papoid;

Described zirconium precursor liquid solution is K ₂zrF ₆solution;

Described damping fluid is phosphate buffered saline buffer, for SODIUM PHOSPHATE, MONOBASIC and Sodium phosphate dibasic are mixed.