CN102517276A - Method for preparing magnetic nano carrier immobilized aldolase with high substrate tolerance - Google Patents
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Abstract
The invention discloses a method for preparing magnetic nano carrier immobilized aldolase with high substrate tolerance. The method comprises the following steps of: 1) preparing super-paramagnetic Fe3O4 nano particles by co-precipitation of a mixture of ferrous and ferric iron salts, modifying the particles by using a silane coupling agent, and activating the outer surfaces of the modified particles by using glutaraldehyde to obtain surface activated magnetic nano particles; 2) stirring a purified and desalted aldolase buffer solution and a magnetic carrier at a low temperature, washing, performing freeze drying, and thus obtaining the magnetic nano carrier immobilized aldolase; and 3) catalyzing 2-deoxy-D-ribose-5-phosphoric acid by using the magnetic nano carrier immobilized aldolase as a catalyst to obtain 3-glyceraldehyde phosphate and acetaldehyde. In the reaction of catalyzing the 2-deoxy-D-ribose-5-phosphoric acid by using the immobilized aldolase, the tolerance of the acetaldehyde substrate is remarkably improved, and the recycling operation of enzyme is greatly simplified.
Description
Technical field
The present invention relates to improve the method for the substrate tolerance of enzyme, relate in particular to a kind of preparation method of magnetic nano-carrier immobilization zymohexase of high substrate tolerance through immobilization.
Background technology
2-deoxy-D-ribose-5-phosphoric acid zymohexase can be unique to other three kinds of zymohexases by two aldehyde molecular complex of catalysis because of it; And the loose specificity of this enzyme allows the common replacement of two substrates; Some chain lengths are that the aldehyde below four Wasserstoffatomss can be used as acceptor, and when first acceptor was substituted acetaldehyde, it can carry out twice successive condensation with acetaldehyde; Product obtains stable cyclic acetal after resetting, and can continue on for more synthetic useful pharmaceutical intermediates.The aldol reaction that can be used for catalysis two molecules of acetaldehyde and another acceptor aldehyde such as 2-deoxy-D-ribose-5-phosphoric acid zymohexase obtains six hydrogen pyran structure, and then synthetic statins drug midbody.
But several major issues have limited its practical application in scale operation.At first, catalyst amounts is very big, and every gram separated product needs 2-deoxy-D-ribose-5-phosphoric acid zymohexase, i.e. mass percent of 20% of about 200 mg.So high enzyme concn makes synthesizes expensive and has increased the isolating difficulty of product; The second, the reaction times needs several days; The 3rd, reactant concn is limited.Various factors is comprehensive, has only the volumetric production of 2g/L/d, makes the actual application value of 2-deoxy-D-ribose-5-phosphoric acid zymohexase reduce greatly.
Summary of the invention
The objective of the invention is to overcome the deficiency of prior art, a kind of preparation method of magnetic nano-carrier immobilization zymohexase of high substrate tolerance is provided.
The preparing method's of the magnetic nano-carrier immobilization zymohexase of high substrate tolerance step is following:
1) in the 250mL oxygen-free water, adds 0.25 ~ 0.35mol trivalent iron salt and 0.15 ~ 0.2mol divalent iron salt, be adjusted to pH=1 ~ 2 with hydrochloric acid, supersound process 30-40 min; Be warming up to 70 ~ 85 ℃, under 1 200 ~ 1 400 rpm stir, N
2Bubbling 25 ~ 30 min, ammoniacal liquor or 10 ~ 15mL 10mol/L NaOH of adding 9 ~ 10 mL mass percents 25 ~ 28% make pH value of solution=8-10; Behind 20 ~ 30 min; Magnetic separates, and oxygen-free water washing 5 ~ 10 times is again with 0.010 ~ 0.015mol/L aqueous ethanolic solution washing 3 ~ 4 times;
2) adding 80 ~ 100 mL percent by volumes is 50% aqueous ethanolic solution, and 6 ~ 8 mL silane crosslinkers, 50 ~ 60 ℃, 180 ~ 220 rpm stir 5 ~ 6 h, washs 3 ~ 5 times with the phosphoric acid buffer of pH=7;
3) add the phosphoric acid buffer of 18 ~ 20 mL pH=7 and the glutaraldehyde water solution of 18 ~ 20 mL mass percents 5%; 200 rpm stir 2 ~ 3 h, and magnetic separates, and wash 5 times with the phosphoric acid buffer of pH=7; Vacuum-drying, the magnetic nano-particle that obtains surface active is microsphere supported;
4) the zymohexase protein solution of preparation 1mg/ml, the mass ratio of zymohexase albumen and carrier is: 1:1 ~ 5, in ice bath, pH is adjusted to 5-10; 200rpm stirs 3-12h, and magnetic separates; The washing immobilized enzyme, freeze-drying obtains the magnetic nano-carrier immobilization zymohexase of high substrate tolerance.Detect the protein concentration of immobilized reactant residue supernatant with the Bradford method;
5) preserve 2 ~ 12h in the acetaldehyde solution with the immobilized zymohexase 300mM of 10mg; Join the triethanolamine hydrochloride solution of the 50mM of 1ml again, its pH is 7.5, contains the Reduced nicotinamide-adenine dinucleotide of 0.1mmol; 0.4mmol substrate 2-deoxy-D-ribose-5-phosphoric acid; The 4U/ml triosephosphate isomerase, the 11U/ml glycerolphos phate dehydrogenase reacts the variation that 340 nm place absorbancys are detected in the back under 50 ℃ of conditions
Described divalent iron salt is FeSO
47H
2O or FeCl
24H
2O.Described trivalent iron salt is FeCl
36H
2O or Fe
2(SO
4)
37H
2O.Described silane crosslinker is aminopropyl triethoxysilane or aminopropyl trimethoxysilane.Described zymohexase is Escherichia coli K12 AB200068.
The present invention is used for catalysis 2-deoxy-D-ribose-5-phosphoric acid (DRP) with magnetic Nano material immobilization zymohexase; Obtain in glyceraldehyde 3-phosphate and the acetaldehyde; Its substrate tolerance is significantly increased, and makes things convenient for the recovery and the recycling of enzyme greatly, has great industrial application value.
Description of drawings
Fig. 1 hysteresis curve;
The microsphere supported immobilization zymohexase of the magnetic nano-particle of Fig. 2 surface active enzyme electron scanning micrograph.
Embodiment
The preparing method's of the magnetic nano-carrier immobilization zymohexase of high substrate tolerance step is following:
1) in the 250mL oxygen-free water, adds 0.25 ~ 0.35mol trivalent iron salt and 0.15 ~ 0.2mol divalent iron salt, be adjusted to pH=1 ~ 2 with hydrochloric acid, supersound process 30-40 min; Be warming up to 70 ~ 85 ℃, under 1 200 ~ 1 400 rpm stir, N
2Bubbling 25 ~ 30 min, ammoniacal liquor or 10 ~ 15mL 10mol/L NaOH of adding 9 ~ 10 mL mass percents 25 ~ 28% make pH value of solution=8-10; Behind 20 ~ 30 min; Magnetic separates, and oxygen-free water washing 5 ~ 10 times is again with 0.010 ~ 0.015mol/L aqueous ethanolic solution washing 3 ~ 4 times;
2) adding 80 ~ 100 mL percent by volumes is 50% aqueous ethanolic solution, and 6 ~ 8 mL silane crosslinkers, 50 ~ 60 ℃, 180 ~ 220 rpm stir 5 ~ 6 h, washs 3 ~ 5 times with the phosphoric acid buffer of pH=7;
3) add the phosphoric acid buffer of 18 ~ 20 mL pH=7 and the glutaraldehyde water solution of 18 ~ 20 mL mass percents 5%; 200 rpm stir 2 ~ 3 h, and magnetic separates, and wash 5 times with the phosphoric acid buffer of pH=7; Vacuum-drying, the magnetic nano-particle that obtains surface active is microsphere supported;
4) the zymohexase protein solution of preparation 1mg/ml, the mass ratio of zymohexase albumen and carrier is: 1:1 ~ 5, in ice bath, pH is adjusted to 5-10; 200rpm stirs 3-12h, and magnetic separates; The washing immobilized enzyme, freeze-drying obtains the magnetic nano-carrier immobilization zymohexase of high substrate tolerance.Detect the protein concentration of immobilized reactant residue supernatant with the Bradford method;
5) preserve 2 ~ 12h in the acetaldehyde solution with the immobilized zymohexase 300mM of 10mg; Join the triethanolamine hydrochloride solution of the 50mM of 1ml again, its pH is 7.5, contains the Reduced nicotinamide-adenine dinucleotide of 0.1mmol; 0.4mmol substrate 2-deoxy-D-ribose-5-phosphoric acid; The 4U/ml triosephosphate isomerase, the 11U/ml glycerolphos phate dehydrogenase reacts the variation that 340 nm place absorbancys are detected in the back under 50 ℃ of conditions
Described divalent iron salt is FeSO
47H
2O or FeCl
24H
2O.Described trivalent iron salt is FeCl
36H
2O or Fe
2(SO
4)
37H
2O.Described silane crosslinker is aminopropyl triethoxysilane or aminopropyl trimethoxysilane.Described zymohexase is Escherichia coli K12 AB200068.
Embodiment 1
1) in the 250mL oxygen-free water, adds 0.25mol trivalent iron salt and 0.15mol divalent iron salt, be adjusted to pH=1 with hydrochloric acid, supersound process 30-40 min; Be warming up to 70 ℃, under 1 200 rpm stir, N
2Bubbling 25 min add the ammoniacal liquor or the 10 mL 10mol/L NaOH of 9mL mass percent 25%, make pH value of solution=8,20 min after, magnetic separates, oxygen-free water washing 5 times is again with 0.010mol/L aqueous ethanolic solution washing 3 times;
2) adding the 80mL percent by volume is 50% aqueous ethanolic solution, and 6 mL silane crosslinkers, 50 ℃, 180 rpm stir 5 h, washs 3 times with the phosphoric acid buffer of pH=7;
3) add the phosphoric acid buffer of 18 mL pH=7 and the glutaraldehyde water solution of 18 mL mass percents 5%; 200 rpm stir 2 h, and magnetic separates, and wash 5 times with the phosphoric acid buffer of pH=7; Vacuum-drying, the magnetic nano-particle that obtains surface active is microsphere supported;
4) the zymohexase protein solution of preparation 1mg/ml, the mass ratio of zymohexase albumen and carrier is: 1:1, in ice bath, pH=5; 200rpm stirs 2h, and magnetic separates; The washing immobilized enzyme, freeze-drying obtains the magnetic nano-carrier immobilization zymohexase of high substrate tolerance.Detect the protein concentration of residue supernatant in the immobilized reactant with the Bradford method.The adsorptive capacity of carrier is 37.1%;
5) preserve 2h in the acetaldehyde solution with the immobilized zymohexase 300mM of 10mg, join the triethanolamine hydrochloride solution of the 50mM of 1ml again, its pH is 7.5; The Reduced nicotinamide-adenine dinucleotide that contains 0.1mmol; 0.4mmol substrate 2-deoxy-D-ribose-5-phosphoric acid, 4U/ml triosephosphate isomerase, 11U/ml glycerolphos phate dehydrogenase; React the variation that 340 nm place absorbancys are detected in the back under 50 ℃ of conditions, enzyme is 1.55U/mg than living.
Embodiment 2
1) in the 250mL oxygen-free water, adds 0.35mol trivalent iron salt and 0.2mol divalent iron salt, be adjusted to pH=2 with hydrochloric acid, supersound process 340 min; Be warming up to 85 ℃, under 1 400 rpm stir, N
2Bubbling 30 min add the ammoniacal liquor or the 15mL 10mol/L NaOH of 10 mL mass percents 28%, make pH value of solution=10,30 min after, magnetic separates, oxygen-free water washing 10 times is again with 0.015mol/L aqueous ethanolic solution washing 4 times;
2) adding 100 mL percent by volumes is 50% aqueous ethanolic solution, and 8 mL silane crosslinkers, 60 ℃, 220 rpm stir 6 h, washs 5 times with the phosphoric acid buffer of pH=7;
3) add the phosphoric acid buffer of 20 mL pH=7 and the glutaraldehyde water solution of 20 mL mass percents 5%; 200 rpm stir 3 h, and magnetic separates, and wash 5 times with the phosphoric acid buffer of pH=7; Vacuum-drying, the magnetic nano-particle that obtains surface active is microsphere supported;
4) the zymohexase protein solution of preparation 1mg/ml, the mass ratio of zymohexase albumen and carrier is: 1:5, in ice bath, pH=10; 200rpm stirs 4h, and magnetic separates; The washing immobilized enzyme, freeze-drying obtains the magnetic nano-carrier immobilization zymohexase of high substrate tolerance.Detect the protein concentration of residue supernatant in the immobilized reactant with the Bradford method.The adsorptive capacity of carrier is 56.1%;
5) preserve 4h in the acetaldehyde solution with the immobilized zymohexase 300mM of 10mg, join the triethanolamine hydrochloride solution of the 50mM of 1ml again, its pH is 7.5; The Reduced nicotinamide-adenine dinucleotide that contains 0.1mmol; 0.4mmol substrate 2-deoxy-D-ribose-5-phosphoric acid, 4U/ml triosephosphate isomerase, 11U/ml glycerolphos phate dehydrogenase; React the variation that 340 nm place absorbancys are detected in the back under 50 ℃ of conditions, enzyme is 1.71U/mg than living.
Embodiment 3
1) in the 250mL oxygen-free water, adds 0.3mol trivalent iron salt and 0.2mol divalent iron salt, be adjusted to pH=1.5 with hydrochloric acid, supersound process 340 min; Be warming up to 85 ℃, under 1 400 rpm stir, N
2Bubbling 30 min add the ammoniacal liquor or the 15mL 10mol/L NaOH of 10 mL mass percents 28%, make pH value of solution=10,30 min after, magnetic separates, oxygen-free water washing 10 times is again with 0.015mol/L aqueous ethanolic solution washing 4 times;
2) adding 100 mL percent by volumes is 50% aqueous ethanolic solution, and 8 mL silane crosslinkers, 60 ℃, 220 rpm stir 6 h, washs 5 times with the phosphoric acid buffer of pH=7;
3) add the phosphoric acid buffer of 20 mL pH=7 and the glutaraldehyde water solution of 20 mL mass percents 5%; 200 rpm stir 3 h, and magnetic separates, and wash 5 times with the phosphoric acid buffer of pH=7; Vacuum-drying, the magnetic nano-particle that obtains surface active is microsphere supported;
4) the zymohexase protein solution of preparation 1mg/ml, the mass ratio of zymohexase albumen and carrier is: 1:5, in ice bath, pH=7.5; 200rpm stirs 6h, and magnetic separates; The washing immobilized enzyme, freeze-drying obtains the magnetic nano-carrier immobilization zymohexase of high substrate tolerance.Detect the protein concentration of residue supernatant in the immobilized reactant with the Bradford method.The adsorptive capacity of carrier is 76.8%;
5) preserve 8h in the acetaldehyde solution with the immobilized zymohexase 300mM of 10mg, join the triethanolamine hydrochloride solution of the 50mM of 1ml again, its pH is 7.5; The Reduced nicotinamide-adenine dinucleotide that contains 0.1mmol; 0.4mmol substrate 2-deoxy-D-ribose-5-phosphoric acid, 4U/ml triosephosphate isomerase, 11U/ml glycerolphos phate dehydrogenase; React the variation that 340 nm place absorbancys are detected in the back under 50 ℃ of conditions, enzyme is 1.92U/mg than living.
Embodiment 4
1) in the 250mL oxygen-free water, adds 0.3mol trivalent iron salt and 0.2mol divalent iron salt, be adjusted to pH=1.7 with hydrochloric acid, supersound process 340 min; Be warming up to 85 ℃, under 1 400 rpm stir, N
2Bubbling 30 min add the ammoniacal liquor or the 15mL 10mol/L NaOH of 10 mL mass percents 28%, make pH value of solution=9.5,30 min after, magnetic separates, oxygen-free water washing 10 times is again with 0.015mol/L aqueous ethanolic solution washing 4 times;
2) adding 100 mL percent by volumes is 50% aqueous ethanolic solution, and 8 mL silane crosslinkers, 60 ℃, 220 rpm stir 6 h, washs 5 times with the phosphoric acid buffer of pH=7;
3) add the phosphoric acid buffer of 20 mL pH=7 and the glutaraldehyde water solution of 20 mL mass percents 5%; 200 rpm stir 3 h, and magnetic separates, and wash 5 times with the phosphoric acid buffer of pH=7; Vacuum-drying, the magnetic nano-particle that obtains surface active is microsphere supported;
4) the zymohexase protein solution of preparation 1mg/ml, the mass ratio of zymohexase albumen and carrier is: 1:5, in ice bath, pH=7.5; 200rpm stirs 8h, and magnetic separates; The washing immobilized enzyme, freeze-drying obtains the magnetic nano-carrier immobilization zymohexase of high substrate tolerance.Detect the protein concentration of residue supernatant in the immobilized reactant with the Bradford method.The adsorptive capacity of carrier is 78.1%;
5) preserve 2h in the acetaldehyde solution with the immobilized zymohexase 300mM of 10mg, join the triethanolamine hydrochloride solution of the 50mM of 1ml again, its pH is 7.5; The Reduced nicotinamide-adenine dinucleotide that contains 0.1mmol; 0.4mmol substrate 2-deoxy-D-ribose-5-phosphoric acid, 4U/ml triosephosphate isomerase, 11U/ml glycerolphos phate dehydrogenase; React the variation that 340 nm place absorbancys are detected in the back under 50 ℃ of conditions, enzyme is 2.47U/mg than living.
Embodiment 5
1) in the 250mL oxygen-free water, adds 0.3mol trivalent iron salt and 0.2mol divalent iron salt, be adjusted to pH=1.5 with hydrochloric acid, supersound process 340 min; Be warming up to 80 ℃, under 1 400 rpm stir, N
2Bubbling 30 min add the ammoniacal liquor or the 15mL 10mol/L NaOH of 10 mL mass percents 28%, make pH value of solution=9.5,30 min after, magnetic separates, oxygen-free water washing 10 times is again with 0.015mol/L aqueous ethanolic solution washing 4 times;
2) adding 100 mL percent by volumes is 50% aqueous ethanolic solution, and 8 mL silane crosslinkers, 60 ℃, 220 rpm stir 6 h, washs 5 times with the phosphoric acid buffer of pH=7;
3) add the phosphoric acid buffer of 20 mL pH=7 and the glutaraldehyde water solution of 20 mL mass percents 5%; 200 rpm stir 3 h, and magnetic separates, and wash 5 times with the phosphoric acid buffer of pH=7; Vacuum-drying, the magnetic nano-particle that obtains surface active is microsphere supported;
4) the zymohexase protein solution of preparation 1mg/ml, the mass ratio of zymohexase albumen and carrier is: 1:2, in ice bath, pH=7.5; 200rpm stirs 8h, and magnetic separates; The washing immobilized enzyme, freeze-drying obtains the magnetic nano-carrier immobilization zymohexase of high substrate tolerance.Detect the protein concentration of residue supernatant in the immobilized reactant with the Bradford method.The adsorptive capacity of carrier is 83.1%;
5) preserve 2h in the acetaldehyde solution with the immobilized zymohexase 300mM of 10mg, join the triethanolamine hydrochloride solution of the 50mM of 1ml again, its pH is 7.5; The Reduced nicotinamide-adenine dinucleotide that contains 0.1mmol; 0.4mmol substrate 2-deoxy-D-ribose-5-phosphoric acid, 4U/ml triosephosphate isomerase, 11U/ml glycerolphos phate dehydrogenase; React the variation that 340 nm place absorbancys are detected in the back under 50 ℃ of conditions, enzyme is 2.57U/mg than living.
Embodiment 6
1) in the 250mL oxygen-free water, adds 0.3mol trivalent iron salt and 0.2mol divalent iron salt, be adjusted to pH=1.7 with hydrochloric acid, supersound process 340 min; Be warming up to 85 ℃, under 1 400 rpm stir, N
2Bubbling 30 min add the ammoniacal liquor or the 15mL 10mol/L NaOH of 10 mL mass percents 28%, make pH value of solution=9.5,30 min after, magnetic separates, oxygen-free water washing 10 times is again with 0.015mol/L aqueous ethanolic solution washing 4 times;
2) adding 100 mL percent by volumes is 50% aqueous ethanolic solution, and 8 mL silane crosslinkers, 60 ℃, 220 rpm stir 6 h, washs 5 times with the phosphoric acid buffer of pH=7;
3) add the phosphoric acid buffer of 20 mL pH=7 and the glutaraldehyde water solution of 20 mL mass percents 5%; 200 rpm stir 3 h, and magnetic separates, and wash 5 times with the phosphoric acid buffer of pH=7; Vacuum-drying, the magnetic nano-particle that obtains surface active is microsphere supported;
4) the zymohexase protein solution of preparation 1mg/ml, the mass ratio of zymohexase albumen and carrier is: 1:1, in ice bath, pH=7.5; 200rpm stirs 8h, and magnetic separates; The washing immobilized enzyme, freeze-drying obtains the magnetic nano-carrier immobilization zymohexase of high substrate tolerance.Detect the protein concentration of residue supernatant in the immobilized reactant with the Bradford method.The adsorptive capacity of carrier is 78.1%;
5) preserve 12h in the acetaldehyde solution with the immobilized zymohexase 300mM of 10mg, join the triethanolamine hydrochloride solution of the 50mM of 1ml again, its pH is 7.5; The Reduced nicotinamide-adenine dinucleotide that contains 0.1mmol; 0.4mmol substrate 2-deoxy-D-ribose-5-phosphoric acid, 4U/ml triosephosphate isomerase, 11U/ml glycerolphos phate dehydrogenase; React the variation that 340 nm place absorbancys are detected in the back under 50 ℃ of conditions, enzyme is 2.13U/mg than living.
Embodiment 7
1) in the 250mL oxygen-free water, adds 0.3mol trivalent iron salt and 0.2mol divalent iron salt, be adjusted to pH=1.7 with hydrochloric acid, supersound process 340 min; Be warming up to 85 ℃, under 1 400 rpm stir, N
2Bubbling 30 min add the ammoniacal liquor or the 15mL 10mol/L NaOH of 10 mL mass percents 25%, make pH value of solution=9.5,30 min after, magnetic separates, oxygen-free water washing 10 times is again with 0.015mol/L aqueous ethanolic solution washing 4 times;
2) adding 100 mL percent by volumes is 50% aqueous ethanolic solution, and 8 mL silane crosslinkers, 60 ℃, 220 rpm stir 6 h, washs 5 times with the phosphoric acid buffer of pH=7;
3) add the phosphoric acid buffer of 20 mL pH=7 and the glutaraldehyde water solution of 20 mL mass percents 5%; 200 rpm stir 3 h, and magnetic separates, and wash 5 times with the phosphoric acid buffer of pH=7; Vacuum-drying, the magnetic nano-particle that obtains surface active is microsphere supported;
4) the zymohexase protein solution of preparation 1mg/ml, the mass ratio of zymohexase albumen and carrier is: 1:5, in ice bath, pH=12; 200rpm stirs 8h, and magnetic separates; The washing immobilized enzyme, freeze-drying obtains the magnetic nano-carrier immobilization zymohexase of high substrate tolerance.Detect the protein concentration of residue supernatant in the immobilized reactant with the Bradford method.The adsorptive capacity of carrier is 19.1%;
5) preserve 2h in the acetaldehyde solution with the immobilized zymohexase 300mM of 10mg, join the triethanolamine hydrochloride solution of the 50mM of 1ml again, its pH is 7.5; The Reduced nicotinamide-adenine dinucleotide that contains 0.1mmol; 0.4mmol substrate 2-deoxy-D-ribose-5-phosphoric acid, 4U/ml triosephosphate isomerase, 11U/ml glycerolphos phate dehydrogenase; React the variation that 340 nm place absorbancys are detected in the back under 50 ℃ of conditions, enzyme is 1.63U/mg than living.
Claims (5)
1. the preparation method of the magnetic nano-carrier immobilization zymohexase of a high substrate tolerance is characterized in that its step is following:
1) in the 250mL oxygen-free water, adds 0.25 ~ 0.35mol trivalent iron salt and 0.15 ~ 0.2mol divalent iron salt, be adjusted to pH=1 ~ 2 with hydrochloric acid, supersound process 30-40 min; Be warming up to 70 ~ 85 ℃, under 1 200 ~ 1 400 rpm stir, N
2Bubbling 25 ~ 30 min, ammoniacal liquor or 10 ~ 15mL 10mol/L NaOH of adding 9 ~ 10 mL mass percents 25 ~ 28% make pH value of solution=8-10; Behind 20 ~ 30 min; Magnetic separates, and oxygen-free water washing 5 ~ 10 times is again with 0.010 ~ 0.015mol/L aqueous ethanolic solution washing 3 ~ 4 times;
2) adding 80 ~ 100 mL percent by volumes is 50% aqueous ethanolic solution, and 6 ~ 8 mL silane crosslinkers, 50 ~ 60 ℃, 180 ~ 220 rpm stir 5 ~ 6 h, washs 3 ~ 5 times with the phosphoric acid buffer of pH=7;
3) add the phosphoric acid buffer of 18 ~ 20 mL pH=7 and the glutaraldehyde water solution of 18 ~ 20 mL mass percents 5%; 200 rpm stir 2 ~ 3 h, and magnetic separates, and wash 5 times with the phosphoric acid buffer of pH=7; Vacuum-drying, the magnetic nano-particle that obtains surface active is microsphere supported;
4) the zymohexase protein solution of preparation 1mg/ml, the mass ratio of zymohexase albumen and carrier is: 1:1 ~ 5, in ice bath; PH is adjusted to 5-10, and 200rpm stirs 3-12h; Magnetic separates, washing immobilized enzyme, freeze-drying; Obtain the magnetic nano-carrier immobilization zymohexase of high substrate tolerance, detect the protein concentration of immobilized reactant residue supernatant with the Bradford method;
5) preserve 2 ~ 12h in the acetaldehyde solution with the immobilized zymohexase 300mM of 10mg; Join the triethanolamine hydrochloride solution of the 50mM of 1ml again, its pH is 7.5, contains the Reduced nicotinamide-adenine dinucleotide of 0.1mmol; 0.4mmol substrate 2-deoxy-D-ribose-5-phosphoric acid; The 4U/ml triosephosphate isomerase, the 11U/ml glycerolphos phate dehydrogenase reacts the variation that 340 nm place absorbancys are detected in the back under 50 ℃ of conditions.
2. according to the preparation method of the magnetic nano-carrier immobilization zymohexase of the described a kind of high substrate tolerance of claim 1, it is characterized in that described divalent iron salt is FeSO
47H
2O or FeCl
24H
2O.
3. according to the preparation method of the magnetic nano-carrier immobilization zymohexase of the described a kind of high substrate tolerance of claim 1, it is characterized in that described trivalent iron salt is FeCl
36H
2O or Fe
2(SO
4)
37H
2O.
4. according to the preparation method of the magnetic nano-carrier immobilization zymohexase of the described a kind of high substrate tolerance of claim 1, it is characterized in that described silane crosslinker is aminopropyl triethoxysilane or aminopropyl trimethoxysilane.
According to the magnetic nano-carrier immobilization zymohexase of the described a kind of high substrate tolerance of claim 1, the preparation method, it is characterized in that described zymohexase is Escherichia coli K12 AB200068.
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| CN108410852A (en) * | 2018-03-16 | 2018-08-17 | 浙江理工大学 | A kind of preparation method of the chitosan-immobilized aldolase of high acetaldehyde tolerance and application |
| CN108559742A (en) * | 2018-05-11 | 2018-09-21 | 浙江理工大学 | A kind of preparation method of modification infusorial earth immobilization aldolase and application |
| CN113980951A (en) * | 2021-11-23 | 2022-01-28 | 东北农业大学 | Immobilized CALB preparation method based on nano dialdehyde starch carrier |
| CN115786290A (en) * | 2022-11-30 | 2023-03-14 | 郑州尼采生物科技有限公司 | Method for converting S adenosylmethionine into ACC (ACC) by using magnetic nano immobilized enzyme |
| CN116272702A (en) * | 2022-11-22 | 2023-06-23 | 广州蔚捷生物医药科技有限公司 | Biological nanometer microsphere and preparation method and application thereof |
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| CN108410852A (en) * | 2018-03-16 | 2018-08-17 | 浙江理工大学 | A kind of preparation method of the chitosan-immobilized aldolase of high acetaldehyde tolerance and application |
| CN108559742A (en) * | 2018-05-11 | 2018-09-21 | 浙江理工大学 | A kind of preparation method of modification infusorial earth immobilization aldolase and application |
| CN113980951A (en) * | 2021-11-23 | 2022-01-28 | 东北农业大学 | Immobilized CALB preparation method based on nano dialdehyde starch carrier |
| CN116272702A (en) * | 2022-11-22 | 2023-06-23 | 广州蔚捷生物医药科技有限公司 | Biological nanometer microsphere and preparation method and application thereof |
| CN116272702B (en) * | 2022-11-22 | 2023-09-26 | 广州蔚捷生物医药科技有限公司 | Biological nanometer microsphere and preparation method and application thereof |
| CN115786290A (en) * | 2022-11-30 | 2023-03-14 | 郑州尼采生物科技有限公司 | Method for converting S adenosylmethionine into ACC (ACC) by using magnetic nano immobilized enzyme |
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