TWI419972B - A method for improving enzyme activities by biomimetic silicification and a kit thereof - Google Patents

Description

Method for enhancing enzyme activity by biochemical deuteration reaction and its set

The present invention relates to a method for enhancing enzyme activity by a biochemical deuteration reaction and a kit thereof, and more particularly to a biochemical deuteration reaction using a hydrazine solution having a hydrophobic functional group and a phthalic acid-containing precursor. .

D-amino acid oxidase (EC 1.4.3.3; DAO) is a flavin adenine dinucleotide (FAD)-dependent oxidoreductase. Optically specific, specifically catalyzed by oxidative deamination of D-type amino acids, producing corresponding α-keto acids and ammonia, and reducing O ₂ to H ₂ while the reaction proceeds O ₂ . DAO is a commercially valuable enzyme in the industry. Its main applications include the production of 7-aminocephalosporanic acid (7-ACA, as a reactant for the production of cephalosporin antibiotics), the separation of racemic amino acids, and the production of α-keto acids. And detecting the degree of microbial contamination of food.

At present, the application bottleneck of DAO in the industry is its poor stability. The main reason is that DAO often causes DAO enzymes to change due to structural changes, FAD shedding, and subunit separation due to factors such as temperature, pH and oxidation of by-product H ₂ O ₂ . This causes the DAO to lose its activity.

Enzyme immobilization technology is often used in the industry to improve enzyme thermostability, pH stability, H ₂ O ₂ tolerance, and the like. Enzyme immobilization technology can be divided into physical method and chemical method according to its principle. The chemical method forms a covalent bond between the enzyme and the carrier by a chemical reagent, and the saccharide-related enzyme enhances the stability by this method; the advantage is that the bond is stable and the enzyme is not easily detached, and the disadvantage is that the structural change of the enzyme itself may result in a decrease in activity. The carrier or enzyme must be chemically modified in advance. The physical law utilizes a porous hollow fiber, a microcapsule, a water gel, a network cross-linked polymer or the like to confine the enzyme molecule to the carrier or adsorbs the enzyme on the surface of the carrier by non-covalent bonding such as electrostatic force or hydrophobic action; The method does not change the chemical structure of the enzyme itself, so the effect on the activity is usually low, and the disadvantage is that the high quality transmission disorder and the enzyme molecule are easy to fall off.

Therefore, in 1999, Luckarift et al. used the biodeuteration method, that is, the R5 peptide (i.e., one of the silaffin proteins purified from the cell wall of the algae cell) and the tannic acid cholinesterase (butyrylcholinesterase). cover. The efficiency of coating butyl choline phosphatase in this way is as high as 90%, and the enzyme retains all the activity; compared with the uncoated succinylcholine oxidase, the stability after coating with cerium oxide Significant improvement. The use of biodegradable coated enzymes can combine the good mechanical properties of cerium oxide with a mild reaction environment, and can effectively retain the activity of the enzyme after coating.

However, the above-mentioned biodeuteration technology is an enzyme immobilization method which has many potentials and has many advantages. However, to apply this method to prepare a large number of immobilized enzymes, one of the foreseeable problems is the cost of the R5 peptide, and whether it is produced by genetic recombination or peptide synthesis is quite expensive, so there is a need for improvement.

Accordingly, it is an object of the present invention to provide a method and a kit for enhancing the activity of an enzyme by a biochemical deuteration reaction, and changing the cerium oxide particle by using a hydrazine solution having a hydrophobic functional group and a phthalic acid-containing precursor. Microenvironment to enhance enzyme activity.

Another object of the present invention is to provide a method and a kit for enhancing the activity of an enzyme by a biochemical deuteration reaction, and immobilizing the enzyme by a biochemical deuteration reaction, thereby destroying the original structure of the enzyme without adding an additional agent or step. Its physical or chemical nature.

A further object of the present invention is to provide a method and a kit for enhancing the activity of an enzyme by a bio-chemical deuteration reaction, which can be reacted by using other catalysts for bio-deuteration reaction without using a large amount of R5 peptide, thereby effectively reducing the cost. .

To achieve the above object, the present invention provides a method for enhancing enzyme activity by a biochemical oximation reaction, comprising: providing a first acidic solution comprising a silicic acid precursor; providing a first compound comprising a siloxane derivative a diacid solution, wherein the oxoxane compound comprises at least one hydrophobic functional group; mixing the first acidic solution and the second acidic solution to form a mixed solution; and adding the enzyme to be immobilized and a catalyst to the mixed solution to start the first Biomimetic reaction, wherein the catalyst is selected from the group consisting of: silaffin, a pro-protein derivative, poly-L-lysine, poly-L-lysine Derivatives, poly-L-arginine, poly-L-histidine, poly-L-glutamic acid derivatives, polypropylene A group of polyallylamine (PAA), polyallylamine hydrochloride, polyethyleneimine, and cellulose to form cerium oxide particles to immobilize the enzyme therein.

In a preferred embodiment, the catalyst may be a poly L- oleic acid derivative, a polyacrylamide or a polypropylene amine hydrochloride to reduce cost.

In one embodiment, the citric acid precursor may be an oxime compound, such as, but not limited to, tetramethoxysilane (TMOS) or tetraethoxysilane (TEOS). In one embodiment, the first acidic solution may be an aqueous hydrochloric acid solution, but is not limited thereto. In another embodiment, the second acidic solution may be an aqueous hydrochloric acid solution, but is not limited thereto. In one embodiment, wherein the hydrophobic functional group comprises, for example, any one or a combination of methyl, ethyl, propyl or vinyl, but is not limited thereto.

By virtue of the hydrophobic functional group, the substituted oxoxane compounds change the degree of crosslinking, hydrophobicity, steric hindrance, surface charge, surface water molecule content and arrangement, hydrogen bonding and other micro environmental factors, and these factors are Will affect the structure of the protein. Therefore, the activity of the enzyme can be enhanced by improving the microscopic environment of the cerium oxide particles formed by the biochemical deuteration reaction.

In one embodiment, when the hydrophobic functional group is a methyl group, the ratio of citric acid in the mixed solution is 5-15 mol%. In another embodiment, when the hydrophobic functional group is a vinyl group, the ratio of citric acid in the mixed solution is from 10 to 60 mol%. In still another embodiment, wherein the hydrophobic functional group is ethyl or propyl, the ratio of citric acid in the mixed solution is from 10 to 50 mol%.

In one embodiment, the enzymes that can be used in the above methods may include, but are not limited to, butyrylcholine esterase, catalase, horseradish peroxidase, soybean peroxidation. Soybean peroxidase, hydroxylaminobenzene mutase, β-galactosidase, nitrobenzene nitroreductase, lipase, glucose isomerism Glucose isomerase, glucose-6-phosphate dehydrogenase, glucose oxidase, organophosphate hydrolase, luciferase, green fluorescein A group consisting of green fluorescent protein (GFP), β-glucuronidase, and D-type amino acid oxidase. In a preferred embodiment, the enzyme can be a D-type amino acid oxidase.

The invention also provides a kit for enhancing enzyme activity by a bio-chemical deuteration reaction, the kit comprising: a first acidic solution comprising a phthalic acid precursor; a second acidic solution comprising a decane-based compound, wherein the oxygen is contained The alkane compound comprises at least one hydrophobic functional group; and a catalyst to initiate a biomimetic reaction, the catalyst being selected from the group consisting of: a relative protein, an R5 peptide, a poly-L-lysine, Poly-L-lysine derivatives, poly-L-arginine, poly-L-histamine, poly-L-glutamic acid derivatives, polyacrylamide (PAA), polypropylene amine hydrochloride, polyethylene Amines and cellulose.

In a preferred embodiment, the catalyst may be a poly L- oleic acid derivative, a polyacrylamide or a polypropylene amine hydrochloride to reduce cost.

This kit can be widely used to enhance the activity of various enzymes. For example, enzymes include, but are not limited to, butylcholinesterase, catalase, horseradish peroxidase, soybean peroxidase, hydroxylamine mutase, beta-galactosidase, nitrobenzene nitrate Reductase, lipase, glucose isomerase, glucose-6-phosphate dehydrogenase, grape oxidase, organophosphorus hydrolase, luciferase, green fluorescent protein, beta-glucuronidase, and D-amine A group consisting of acid oxidases. In a preferred embodiment, the enzyme can be a D-type amino acid oxidase.

In addition, the citrate precursor, the first acidic solution, the second acidic solution and the hydrophobic functional group in the above-mentioned kit are referred to the above description, and will not be described herein.

Before the narrative, it should be understood that the terms or words used in the specification and claims of the present invention should not be construed as a limitation of the general and dictionary meaning, but the invention may be appropriately defined according to the inventor. Explained in terms of technical meaning and concept. Therefore, the explanations set forth herein are merely illustrative of one preferred embodiment and are not intended to limit the scope of the invention, and therefore, it should be understood that other equivalents and modifications may be made without departing from the spirit and scope of the invention.

Embodiment 1

152 μl of TMOS was added to 848 μl of 1 mM HCl, and reacted at room temperature for 15 minutes to hydrolyze it into 1 M citric acid. The citrate will be condensed into a gel after long-term storage, and needs to be prepared before each coating. Fresh citric acid solution. Another methyl-trimethoxy silane (MTMS) (136 μl MTMS with 864 μl of 1 mM HCl), dimethyl-dimethoxy silane (DMDMS) (120 μl DMDMS with 880 μl of 1 mM HCl), and trimethyl-methoxy silane (TMMS) ( 104 μl of TMMS was reacted with 896 μl of 1 mM HCl) for 15 minutes at room temperature for acid hydrolysis to a final concentration of 1 M. Next, hydrolyzed MTMS or DMDMS or TMMS was added to 1 M citric acid after hydrolysis to prepare 5, 10, 15 mol% of mixed citric acid. The composition of the solution when coated is 50 μl of 0.1 M KH ₂ PO ₄ (containing 0.1 N NaOH), 10 μl of hydrolyzed citric acid or mixed citric acid, 10 μl of 1 mg/ml DAO (dissolved in dialysis buffer (dialysis) Buffer), which consists of 50 mM KH ₂ PO ₄ , pH 6.8, contains 2 mM EDTA, 1 mM dithiothreitol, 10% (w/v) glycerol), 10 μl of 10 mg/ml The magnetic nanoparticle suspension was finally reacted by adding 20 μl of a 1 mM aqueous solution of polyacrylamide (PAA). After reacting at room temperature for 5 minutes, the solid-liquid separation was carried out with a magnetic holder and the supernatant was withdrawn, followed by washing the cerium oxide particles three times with 100 μl of deionized water, and finally, the cerium oxide particles were suspended in 100 μl of dialysis buffer. The activity of the immobilized (polyacrylamide (PAA) catalyzed) D-type amino acid oxidase by the addition of a methyl-substituted oxonane compound is shown in Figure 1. The enzyme activity increases with the degree of substitution and the added concentration. When 15 mol% of TMMS was added, the best activity increase was obtained, which was 1.4 times higher than when it was not added.

The addition of methyl-substituted oxane compounds can change the degree of cross-linking, hydrophobicity, steric hindrance, surface charge, surface water molecule content and arrangement, hydrogen bonding and other micro-environmental factors, all of which affect protein structure. In each of the examples of the present invention, a hydroxyl group-containing compound having a hydrophobic functional group is added to affect the structure of the enzyme by a similar mechanism, thereby enhancing the activity.

Embodiment 2

The same procedure as in Example 1, except that the catalyst for forming cerium oxide particles was changed to 20 μl of a 1 mM R5 peptide aqueous solution. The activity of the immobilized (R5 peptide-catalyzed) D-type amino acid oxidase by the addition of a methyl-substituted alkane is shown in Figure 2. The activity of the enzyme increases with the degree of substitution and the concentration of addition, when 15 mol% is added. The TMMS gives the best activity boost, which is 4.5 times higher than when it is not added.

Embodiment 3

The operation of the first embodiment, but the specific substituent of the oxane compound, uses vinyl-trimethoxy silane (VTMS). 148 μl of VTMS was reacted with 852 μl of 1 mM HCl for 15 minutes at room temperature to hydrolyze it to a final concentration of 1 M. VTMS was mixed with TMOS at 0-60 mol%, and this mixed tannic acid was used as a source of tannic acid. Another catalyst for the formation of cerium oxide particles was changed to 20 μl of a 1 mM R5 peptide aqueous solution. The activity of the addition of a vinyl substituted alkane to the D-type amino acid oxidase is shown in Figure 3. When 60 mol% of VTMS is added, the enzyme activity is increased by about 5.9 times.

Embodiment 4

In the same manner as in the first embodiment, the relevant experimental data of the substituted ethyl group, the propyl group, etc. (the case where the ratio of the citric acid is added) is shown in FIG. 4 and FIG. Figure 4 is a graph showing the effect of adding ethyltrimethoxysilane (ETMS) to TMOS on the coating efficiency and activity of D-amino acid oxidase immobilized in cerium oxide particles. The histogram of Figure 4 shows the fixation. Efficiency, solid dots indicate relative activity, and the catalyst used to produce cerium oxide particles is polyacrylamide (PAA). Figure 5 is the effect of adding n-propyl trimethoxysilane (n-PTMS) to TMOS on the coating efficiency and activity of D-amino acid oxidase immobilized in cerium oxide particles, Figure 5 The bar graph indicates the fixed efficiency, the solid dot indicates the relative activity, and the catalyst for producing the cerium oxide particles is polyacrylamide (PAA).

Embodiment 5

In the same manner as in Example 1, the biomimetic encapsulation of Pseudomonas cepacia esterase (EC 3.1.1.3) with polyacrylamide (PAA) was used to enhance the activity. Adding 10 and 15 mol% trimethylmethoxysilane to the tetramethoxysilane during coating can increase the activity by a factor of two. The experimental results are shown in Fig. 6. The effect of adding a methyl substituted fluorene-based compound to TMOS on the activity of Pseudomonas cepacia esterase in the biomimetic cerium oxide. Among them, black column table MTMS, dot column table DMDMS, white column table TMMS.

Embodiment 6

The experimental data for the operation of Example 1, but using other catalysts (R5 peptide) to produce cerium oxide particles are shown in Figures 7 to 9. Figure 7 is experimental data showing the activity of Pseudomonas cepacia esterase encapsulated in biomimetic cerium oxide by addition of a methyl substituted fluorene oxide compound to TMOS. Fig. 8 is the experimental data of the efficiency and activity of the D-type amino acid oxidase immobilized on the cerium oxide particles by the addition of the ethyl-substituted fluorene oxide compound ETMS to TMOS, wherein the column chart is fixed, and the solid dot table is relatively Specific activity. Figure 9 is a graph showing the effect of n-PTMS added to nMOS on the efficiency and activity of D-type amino acid oxidase immobilized on cerium oxide particles. Column chart fixed efficiency, solid dot table relative activity.

The above-mentioned embodiments are merely examples for convenience of description, and the scope of the claims is intended to be limited to the above embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a graph showing the effect of improving the activity of D-type amino acid oxidase immobilized in cerium oxide particles in Example 1 of the present invention.

Fig. 2 is a graph showing the effect of improving the activity of D-type amino acid oxidase immobilized in cerium oxide particles in the second embodiment of the present invention.

Fig. 3 is a graph showing the effect of improving the activity of D-type amino acid oxidase immobilized in cerium oxide particles in the third embodiment of the present invention, wherein the columnar chart has a fixed efficiency and a solid dot table relative activity.

Figure 4 shows the effect of adding ethyltrimethoxysilane (ETMS) to TMOS on the coating efficiency and activity of D-amino acid oxidase immobilized in cerium oxide particles, wherein the histogram shows the fixed efficiency, solid Dots indicate relative activity and the catalyst used to produce cerium oxide particles is polyacrylamide (PAA).

Figure 5 shows the effect of adding n-propyl trimethoxysilane (n-PTMS) to tetramethoxysilane (TMOS) on the efficiency and activity of D-type amino acid oxidase immobilized in cerium oxide particles. The histogram indicates the fixed efficiency, the solid dot indicates the relative activity, and the catalyst for producing the cerium oxide particles is polyacrylamide (PAA).

Figure 6 shows the effect of the addition of a methyl substituted fluorene oxide compound to TMOS on the activity of Pseudomonas cepacia esterase in a biomimetic cerium oxide, in which black column table MTMS, gray column table DMDMS, white column table TMMS.

Figure 7 shows experimental data on the activity of Pseudomonas cepacia esterase encapsulated in biomimetic cerium oxide by addition of a methyl substituted fluorene oxide compound to TMOS.

Figure 8 is a graph showing the experimental data of the efficiency and activity of the D-type amino acid oxidase immobilized on the cerium oxide particles by adding an ethyl-substituted decane-based compound ETMS to TMOS, wherein the column chart is fixed, and the solid dot table is relatively Specific activity.

Figure 9 is a graph showing the effect of the addition of n-propyl substituted fluorene-based compound n-PTMS to TMOS on the coating efficiency and activity of D-amino acid oxidase immobilized in cerium oxide particles. Column chart fixed efficiency, solid dot table relative activity.

Figure 1 is a data chart with no components representing symbols.

Claims (15)

A method for enhancing enzyme activity by a biochemical deuteration reaction, comprising: providing a first acidic solution, the first acidic solution comprising a silicic acid precursor; providing a second acidic solution, the second acidic solution comprising a oxoxane compound comprising at least one hydrophobic functional group; mixing the first acidic solution and the second acidic solution to form a mixed solution; and adding one of the mixed solution to be fixed The enzyme and a catalyst initiate a biochemical deuteration reaction, wherein the catalyst is selected from the group consisting of: silaffin, a relative protein derivative, poly-L-lysine, poly-L- Poly-L-lysine derivatives, poly-L-arginine, poly-L-histidine, poly-L-glutamic acid (poly-) a group of L-glutamic acid derivatives, polyallylamine (PAA), polyallylamine hydrochloride, polyethyleneimine, and cellulose The cerium oxide particles fix the enzyme in it The method of claim 1, wherein the phthalic acid precursor is tetramethoxysilane (TMOS) or tetraethoxysilane (TEOS). The method of claim 1, wherein the first acidic solution is an aqueous hydrochloric acid solution. The method of claim 1, wherein the second acidic solution is an aqueous hydrochloric acid solution. The method of claim 1, wherein the hydrophobic functional group is at least one selected from the group consisting of methyl, ethyl, propyl and vinyl. The method of claim 5, wherein the hydrophobic functional group is methyl, the ratio of citric acid in the mixed solution is 5-15 mol%. The method of claim 5, wherein the ratio of citric acid in the mixed solution is 10-50 mol% when the hydrophobic functional group is ethyl or propyl. The method of claim 5, wherein the ratio of citric acid in the mixed solution is from 10 to 60 mol%, wherein the hydrophobic functional group is a vinyl group. The method of claim 1, wherein the enzyme is selected from the group consisting of: butyrylcholine esterase, catalase, horseradish peroxidase, soybean peroxidase (soybean) Peroxidase), hydroxylaminobenzene mutase, β-galactosidase, nitrobenzene nitroreductase, lipase, glucose isomerase Isomerase), glucose-6-phosphate dehydrogenase, glucose oxidase, organophosphate hydrolase, luciferase, green fluorescent protein A group consisting of green fluorescent protein, GFP), β-glucuronidase, and D-type amino acid oxidase. A kit for enhancing enzyme activity by a biochemical deuteration reaction, the kit comprising: a first acidic solution, the first acidic solution comprising a citrate precursor; a second acidic solution, the second acidic solution comprising a ruthenium An oxane compound comprising at least one hydrophobic functional group; and a catalyst for initiating a biomimetic reaction, the catalyst being selected from the group consisting of: a relative Protein, R5 peptide, poly L-lysine, poly-L-lysine derivative, poly-L-arginine, poly-L-histidine, poly-L-glutamic acid derivative, polyacrylamide, poly Propylene amine hydrochloride, polyethyleneimine, and cellulose. The kit of claim 10, wherein the enzyme is selected from the group consisting of: butylcholinesterase, catalase, horseradish peroxidase, soybean peroxidase, hydroxylamine mutase, β-half Lactosidase, nitrophenylnitroreductase, lipase, glucose isomerase, glucose-6-phosphate dehydrogenase, grape oxidase, organophosphorus hydrolase, luciferase, green fluorescent protein, β- A group consisting of glucuronidase and D-type amino acid oxidase. The kit of claim 10, wherein the phthalic acid precursor is tetramethoxy decane (TMOS) or tetraethoxy decane (TEOS). The kit of claim 10, wherein the first acidic solution is an aqueous hydrochloric acid solution. The kit of claim 10, wherein the second acidic solution is an aqueous hydrochloric acid solution. A kit for enhancing enzyme activity by a biocidal deuteration reaction according to claim 10, wherein the hydrophobic functional group is at least one selected from the group consisting of methyl, ethyl, propyl and vinyl.