CN102719423A

CN102719423A - New method for improving life and stability of glucose oxidase by modifying surface of glucose oxidase with biocompatible polymer

Info

Publication number: CN102719423A
Application number: CN2012101736155A
Authority: CN
Inventors: 刘敬权; 罗雄; 王蕊
Original assignee: Qingdao University
Current assignee: Qingdao University
Priority date: 2012-05-25
Filing date: 2012-05-25
Publication date: 2012-10-10

Abstract

The invention provides a new method for modifying surface of glucose oxidase, and aims at improving heat stability and life of glucose oxidase by modifying glucose oxidase utilizing amino group on the surface. The method comprises the steps: synthesizing alpha-(1-oxo-2-propenyl)-omega-methoxyl-poly(ethylene oxide) (PEG-A) high polymer with RAFT (reversible addition-fragmentation chain transfer) reagent with mercaptothiazoline terminal group through RAFT polymerization method; and carrying out amidation reaction between mercaptothiazoline group at the terminal of polymer chain and amino group on the surface of glucose oxidase to connect the mercaptothiazoline group to the surface of glucose oxidase. The polymer chain of the modified glucose oxidase partially wrap the activity center (flavin adenine dinucletide: FAD) and other components of the enzyme, so that stabilizing and protective action on the enzyme structure are realized. Therefore, the modified glucose oxidase has excellent heat stability and longer service life.

Description

A kind of biocompatible macromolecule modified P-FAD surface that utilizes is to improve the novel method of its life-span and stability

Technical field

This method relate to a kind of P-FAD remodeling method, a plurality of amino groups through P-FAD surface make a kind of high molecular polymer with biocompatibility be connected its surface and make it to be transformed.Improved P-FAD has better thermostability and longer work-ing life, belongs to the enzyme research field.

Background technology

The high purity P-FAD is pale yellow powder, and is soluble in water, is insoluble to ether, chloroform, butanols etc. fully.Molecular weight is about 160KDa, and the per molecule P-FAD contains bimolecular flavin adenine dinucleotide (FAD).The solid zymin was stablized 2 years 0 ℃ of following preservation at least, under-15 ℃, stablized 8 years.The stable TR of P-FAD is 30 ℃～60 ℃, and optimal pH is 5.P-FAD can generate the maltonic acid lactone by oxidizing glucose in the presence of molecular oxygen, oxygen consumed generates hydrogen peroxide simultaneously.Hydrogen peroxide is a kind of strong oxidizer, and the effect of sterilization and disinfection is arranged.Because it has advantages such as catalysis specificity, high reactivity and catalysis high efficiency, application is very extensive at aspects such as foodstuffs industry, medicine, fodder additivess, has played effects such as removal glucose, deoxidation, sterilization.Its topmost purposes is to produce glucono-and be widely used in the food fresh keeping process as inhibitor.

Since nineteen twenty-eight Muller find and the name P-FAD so far, all have every year many people that P-FAD is carried out research and discovery, especially to its prothetic group---flavin adenine dinucleotide (FAD).A large amount of bibliographical informations relevant FAD fixing or transform, for example: be used to prepare the research of the aspects such as application and enzymic activity of glucose sensor, electrochemistry aspect.Yet, about through the surface of P-FAD is transformed with regulate its life-span and stability report but seldom.The present invention is intended to the surface of P-FAD is directly transformed; Through connect macromolecular chain on the surface with biocompatibility; Thereby reach the thermostability of improving P-FAD and the purpose in life-span thereof, make improved glucose oxidase endonuclease capable better application in fields such as food-processing, food fresh keeping and rotproofings.And in the process of preparation, having very low toxicity, is a kind of preparation method of environmental protection.

Summary of the invention:

The present invention proposes a kind of novel method that the surface of P-FAD is transformed,, its thermostability and work-ing life are improved in the hope of it being transformed through the amino group on P-FAD surface.This method has been synthesized α-(1-oxo-2-propenyl)-ω-methoxyl group through RAFT (RAFT) polymerization and has been gathered (oxygen ethene) (PEG-A) high molecular polymer with the RAFT reagent that has the mercaptothiazoline terminal group.Through the terminal mercaptothiazoline group of macromolecular chain, the amino surperficial with P-FAD carries out amidate action, thereby is connected its surface.Improved P-FAD, its surperficial macromolecular chain partly surrounds the active site of enzyme components such as (FAD), and the structure of enzyme has been played firm and provide protection.Therefore, improved P-FAD has good thermostability and longer work-ing life.

Invention realizes through following manner, comprises the following steps:

(1) the synthetic RAFT reagent that has the mercaptothiazoline terminal group, polymerization PEG-A obtains having the polymer of mercaptothiazoline terminal group then, and its polymer chain is connected on the P-FAD.(accompanying drawing 1)

(2) mensuration of enzymic activity:, the activity of transforming the back enzyme is measured through the ultraviolet spectrophotometer analytical method.

(3) mensuration of thermostability: be dissolved in enzyme in the deionized water, be incubated 3 hours down at 70 ℃, at set intervals, sample thief carries out (4) active testing.Mensuration is reached a conclusion, and P-FAD loses activity under 70 ℃ very soon, and improved P-FAD still had activity after 3 hours.(accompanying drawing 2)

(4) mensuration in work-ing life: be put in room temperature to the aqueous solution of enzyme following 60 days, at set intervals, sample thief carries out (4) active testing.Mensuration is reached a conclusion, and P-FAD at room temperature can store 29 days, and improved P-FAD still had activity (accompanying drawing 3) after 60 days

Description of drawings:

Fig. 1 is the synthetic of polymer chain and transforms the P-FAD synoptic diagram.

Fig. 2 is thermal stability determination figure as a result.

The mensuration that Fig. 3 is to use the life-span is figure as a result.

Embodiment:

Embodiment 1. gets RAFT reagent 0.527g, and 2-mercaptothiazoline 0.238g is dissolved in the methylene dichloride of 10ml.Add dewatering agent NSC 57182 (DCC) 0.497g and catalyzer 4-Dimethylamino pyridine (DMAP) 24.5mg again, at room temperature stirred 7 hours.After reaction finished, suction filtration was removed solid particulate, then through the silicagel column purified product.The RAFT reagent 7.6mg that will have the mercaptothiazoline terminal group; α-(1-oxo-2-propenyl)-ω-methoxyl group gathers (oxygen ethene) (PEG-A) 2g, and azo-bis-isobutyl cyanide (AIBN) 1mg is dissolved in the 4ml dioxane; Wherein, the mol ratio of RAFT reagent and PEG-A is 1: 100.Logical nitrogen deoxygenation 40min, the sealed reaction system was reacted 5 hours in 75 ℃ oil bath then.Reaction is used the ether sedimentation purifying polymer, the product that obtains expecting after finishing.The glucose oxidase solution of 5mg (pH=7) in the 5ml phosphate buffered saline buffer, add the synthetic polymkeric substance according to 1: 30 ratio of mol ratio, stirred 48 hours under the room temperature.After reaction finishes, dialyse with the dialysis tubing of molecular weight cut-off 100,000 and to remove excessive polymer, the glucose oxidase cold freeze-drying of transformation is dry.Carry out enzymic activity, thermostability and test in work-ing life then.

Embodiment 2. changes mol ratio in 1 into RAFT reagent: PEG-A=1: 50, and other are like embodiment 1.

Embodiment 3. changes mol ratio in 1 into RAFT reagent: PEG-A=1: 200, and other are like embodiment 1.

Embodiment 4. changes mass ratio in 1 into RAFT reagent: PEG-A=1: 400, and other are like embodiment 1.

Claims

1. one kind is utilized biocompatible macromolecule modified P-FAD surface to improve the novel method of its life-span and stability

(1) have RAFT reagent synthetic of mercaptothiazoline terminal group: get RAFT reagent 0.527g, 2-mercaptothiazoline 0.238g is dissolved in the 10ml methylene dichloride; Add NSC 57182 (DCC) 0.497g again; With 4-Dimethylamino pyridine (DMAP) 24.5mg, at room temperature stirred 7 hours, after reaction finishes; Suction filtration is removed solid particulate, then through the silicagel column purified product.

(2) the PEG-A polymkeric substance is synthetic: will have the RAFT reagent 7.6mg of mercaptothiazoline terminal group, α-(1-oxo-2-propenyl)-ω-methoxyl group gathers (oxygen ethene) (PEG-A) 2g, and azo-bis-isobutyl cyanide (AIBN) 1mg is dissolved in the 4ml dioxane.Wherein, the mol ratio of RAFT reagent and PEG-A is 1: 100.Logical nitrogen deoxygenation 40min, sealed reaction system then, stirring reaction is 5 hours in 75 ℃ oil bath, and reaction is used the ether sedimentation purifying polymer, the product that obtains expecting after finishing.

(3) the high molecular polymer chain is connected on the P-FAD: the glucose oxidase solution of 5mg (pH=7) in the 5ml phosphate buffered saline buffer; Add the synthetic polymkeric substance according to 1: 30 ratio of mol ratio, stirred 48 hours under the room temperature, after reaction finishes; With the purification of dialysing of the dialysis tubing of molecular weight cut-off 100,000; The purification postlyophilization obtains product, carries out the test of enzymic activity, thermostability and storage characteristics then.

2. according to the described preparation method of claim 1, it is characterized in that: polymericular weight and on-fixed in the step (2) can change the monomer consumption in proportion according to the needs of polymericular weight.

3. according to the described preparation method of claim 1, it is characterized in that: temperature of reaction can be regulated in the step (2), all can in 60～90 ℃ of scopes.

4. according to the described preparation method of claim 1, it is characterized in that: ratio and the on-fixed of RAFT reagent and AIBN in the step (2), RAFT/AIBN=2-8.