WO2010058590A1 - Protein substance hydrolysis method, hydrolysis device, and hydrolytic product analysis device - Google Patents

Abstract

With the objective of providing a protein substance hydrolysis method, a hydrolysis device, and a hydrolytic product analysis device which are simple, fast, and can be automated, disclosed is a protein substance hydrolysis method to hydrolyze the protein substance whereby a protein substance is brought into contact with a solid acid catalyst, with the solid acid catalyst being arranged on a protein substance holding base which has multiple fine pores, and is heated in the presence of water. In addition, the protein substance hydrolysis device/hydrolytic product analysis device (21) is equipped with: an auto sampler (5) that supplies a solution which includes a protein substance; hydrolysis columns (8) used in the execution of the hydrolysis method; a flow path that connects the auto sampler (5) and the hydrolysis columns (8); an electric oven (9) that heats the hydrolysis columns (8); and a hydrolysis product analysis unit (11).

Description

Protein hydrolysis method, hydrolysis apparatus, and hydrolysis product analysis apparatus

The present invention relates to a protein hydrolysis method, a hydrolysis apparatus, and a protein hydrolyzate analysis apparatus.

By analyzing peptides and amino acids that are protein hydrolysates, information on amino acid composition, protein amount, post-translational modification of amino acids, and the like, which are important properties of the protein, can be obtained.

As a method for hydrolyzing proteins, for example, many hydrolysis methods using an enzyme as a catalyst have been developed. However, since enzymatic degradation has substrate specificity and cannot completely degrade amino acids, this method has a problem that its application is limited.

In general, biopolymers such as proteins are organic or inorganic strong acids (hydrochloric acid, sulfuric acid, trifluoroacetic acid, p-toluenesulfonic acid, methanesulfonic acid, etc.) with addition of water or strong alkali ( Hydrolysis by mixing with sodium hydroxide, potassium hydroxide, etc.) is known to produce oligomers and monomers (peptides and amino acids in proteins, oligosaccharides and monosaccharides in polysaccharides) (non-patent literature) 1). Based on this finding, a number of protein hydrolysis methods using strong acid or strong alkaline aqueous solutions have also been developed.

The above-mentioned hydrolysis with a strong acid or strong alkaline aqueous solution has a problem that amino acids as degradation products are further degraded, and at present, a complete protein hydrolysis method has not been established. However, among these methods, (1) a hydrolysis method in which a constant boiling point hydrochloric acid (about 6N hydrochloric acid) is used and heated to 110 degrees C. for 20 to 72 hours in a test tube sealed under reduced pressure (non-patent document) 2) And (2) A modified method (gas phase hydrolysis method) in which constant boiling point hydrochloric acid is directly distilled in a sealed tube in order to prevent contamination from hydrochloric acid and handle a small amount of sample is the best. It is used.

By the way, the methods (1) and (2) described above require complicated and skilled techniques, and in particular, in the microanalysis of proteins, the influence of proteins mixed during human operations on the analysis results becomes large. For this reason, attempts have been made to automate the method for the purpose of realizing a simpler operation and preventing protein contamination during the operation.

For example, as an example of the above attempt, an apparatus was developed that automated the hydrolysis process using constant boiling hydrochloric acid and the derivatization process (prelabel PTC (phenylthiocarbamyl) amino acid derivatization method) (Patent Document 1). However, in this apparatus, a failure due to corrosion of the valve by hydrochloric acid has been reported.

US Pat. No. 5,106,583 (patent date April 21, 1992)

That is, in the above conventional protein hydrolysis method using constant boiling hydrochloric acid, 1) the amino acid that is a protein hydrolyzate is more easily decomposed by oxidation, and a very complicated operation such as a vacuum sealed tube is required. 2) There are problems such as being unsuitable for automation of the apparatus.

The present invention has been made in view of the above-mentioned problems, and its purpose is to apply a novel method for hydrolyzing proteins more easily than the method using conventional constant boiling hydrochloric acid, and to which the method is applied. And a hydrolyzate analyzer. Moreover, it is providing the hydrolysis method of the protein which can automate reaction as needed.

In order to solve the above-mentioned problems, the inventors of the present application have intensively studied a new hydrolysis method not using constant boiling hydrochloric acid. As a result, prior to the present invention, it was found for the first time that a protein can be hydrolyzed to an amino acid by heating in a state where a solid acid catalyst such as a cation exchange resin is in contact with the protein. The inventors of the present application have further studied diligently, and found that the catalytic activity of the solid acid catalyst is significantly derived by combining the protein holding substrate having a plurality of pores and the solid acid catalyst, The present invention has been completed.

That is, in the protein hydrolysis method according to the present invention, in order to solve the above-mentioned problem, a solid acid catalyst is disposed on at least a part of the inner surface of the protein holding substrate having a plurality of pores. In this state, the protein is brought into contact with the solid acid catalyst, and the protein is hydrolyzed by heating in the presence of water.

According to the above method, the catalytic activity of the solid acid catalyst with respect to the hydrolysis reaction of the protein is significantly extracted, and the protein can be hydrolyzed better than the case where the solid acid catalyst is used alone. There is an effect. As a result, it becomes possible to hydrolyze proteins more easily than the conventional method using constant boiling hydrochloric acid. Further, automation of the protein hydrolysis method becomes easier as compared with the conventional method.

In order to solve the above problems, a protein hydrolyzing apparatus according to the present invention includes a sample supply means for supplying a solution containing a protein, and at least a part of inner surfaces of a plurality of pores of the protein holding substrate. Hydrolysis means comprising a composite substrate on which a solid acid catalyst is arranged, a flow path connecting the sample supply means and the hydrolysis means, and a heating means for heating the hydrolysis means It is said.

In addition, in order to solve the above problems, an apparatus for analyzing a protein hydrolyzate according to the present invention further comprises an analysis means for analyzing a protein hydrolyzate after the hydrolysis means in the above hydrolysis apparatus. It is characterized by having.

According to the present invention, a novel protein hydrolysis using a solid acid catalyst can be provided. Furthermore, the hydrolysis apparatus which employ | adopted the said hydrolysis method, and the analysis apparatus of a hydrolysis product can be provided.

It is the schematic explaining an example of the protein hydrolysis apparatus / hydrolyzate analysis apparatus which concerns on this invention.

Hereinafter, embodiments of the present invention will be described in detail.

[Summary of protein hydrolysis method according to the present invention]
The method according to the present invention comprises contacting a protein with a solid acid catalyst in a state where a solid acid catalyst is disposed on at least a part of the inner surface of the protein holding substrate having a plurality of pores, The protein is hydrolyzed by heating in the presence. The inventors of the present application have already tried to hydrolyze proteins using a solid acid catalyst such as a strong cation exchange resin alone, but by adopting the method of the present invention, the hydrolysis of proteins It is believed that the catalytic activity of the solid acid catalyst for the reaction is significantly extracted. As a result, in the method according to the present invention, the protein can be hydrolyzed more satisfactorily as compared with the case where the solid acid catalyst is used alone (see also Examples and Comparative Examples described later).

In addition, the method according to the present invention does not damage the hydrolysis apparatus as compared with the conventional hydrolysis method using constant boiling hydrochloric acid or the like. Further, it is easy to recover the solid acid catalyst and the protein-holding base material used for the hydrolysis reaction, and the environmental burden is reduced as compared with the conventional method.

In the present invention, the “protein holding substrate” is not particularly limited as long as it is a substrate having a plurality of pores (that is, a porous substrate) and can be held by adsorbing or adhering proteins. The material is not particularly limited, but porous nitrocellulose carrier, porous nylon carrier, porous polyvinylidene difluoride (PVDF) carrier, porous silica carrier, porous polyethersulfone carrier, porous polycarbonate carrier, porous Polytetrafluoroethylene (PTFE) carrier, porous cellulose mixed ester carrier, or composite carrier composed of a plurality of these materials, etc., and hydrophobization treatment or hydrophilization treatment to improve protein retention Etc. may be given. Among these, a porous polyvinylidene difluoride (PVDF) carrier is more preferable from the viewpoint of excellent protein retention and heat resistance. The amount of protein retained in the protein retaining substrate is not particularly limited, but the amount retained for bovine serum albumin (BSA) is preferably in the range of 50 μg / cm ² to 250 μg / cm ² , and is preferably 100 μg / cm 2. More preferably, it is in the range of ² to 250 μg / cm ² . Here, the “protein retention amount” is measured by, for example, a radioisotope labeling method.

The shape of the protein-holding substrate is not particularly limited, and examples thereof include a film shape, a rod shape, and a granular shape. In the case of a film, the film thickness is not particularly limited, but is preferably in the range of 20 to 1000 μm, and more preferably in the range of 80 to 200 μm. Examples of the membrane-like protein holding substrate include a protein blotting membrane, an ultrafiltration membrane, and the like, and a plurality of membranes may be laminated.

The average pore diameter of the pores possessed by the protein-holding substrate is not particularly limited, but for the majority of proteins, in order to retain the whole or a part of the protein in the pores, 0.01 μm to It is preferably in the range of 0.75 μm, and more preferably in the range of 0.05 μm to 0.6 μm. The “average pore diameter” can be measured by observation with an electron microscope.

In addition, when the protein-holding substrate is in the form of a membrane, it is preferable that at least a part of the pores is a through-hole penetrating from one side of the membrane to the other side. If it has a through-hole, it can hydrolyze by supplying the solution containing a protein from the one side of the membrane of the protein holding substrate, and a hydrolyzate such as an amino acid can be recovered from the other side of the membrane.

In the present invention, the “solid acid catalyst” is not particularly limited as long as it is a solid catalyst that can function as an acid, but from the viewpoint of hydrolysis efficiency, saturated carbonization such as a methanesulfonic acid group and a vinylsulfonic acid group. Hydrogen sulfonic acid group, unsaturated hydrocarbon sulfonic acid group, halogenated saturated hydrocarbon sulfonic acid group such as difluoromethane sulfonic acid group, halogenated unsaturated hydrocarbon sulfonic acid group, (these hydrocarbon sulfonic acid groups are (Including cyclic hydrocarbon sulfonic acid groups) or sulfonic acid type cation exchangers including aromatic sulfonic acid groups such as toluene sulfonic acid groups; phenolic cation exchangers including picric acid groups; mono and dichloroacetic acid groups Halogenated acetic acid groups such as mono- and difluoroacetic acid groups, salicylic acid groups, nitrobenzoic acid groups, chlorobenzoic acid groups, phthalic acid groups, hemi Carboxylic acid type ion exchangers containing aromatic carboxylic acid groups such as littic acid groups and pyromellitic acid groups, pyruvic acid groups, acetopyruvic acid groups, malonic acid groups and aconitic acid groups; fluorine-containing or chlorine Sulfinic acid type ion exchangers containing sulfinic acid groups, toluene sulfinic acid groups, nitrobenzene sulfinic acid groups, halogenated benzene sulfinic acid groups, benzene sulfinic acid groups, etc .; sulfenic acid type ion exchangers containing fluorine-containing or chlorine sulfenic acid groups A saturated hydrocarbon phosphoric acid group, an unsaturated hydrocarbon phosphoric acid group, a halogenated saturated hydrocarbon phosphoric acid group, a halogenated unsaturated hydrocarbon phosphoric acid group, (these hydrocarbon phosphoric acid groups include heterocyclic hydrocarbon phosphorus Acid group), glycerophosphate group, hexose phosphate group, aromatic phosphate group, aromatic allyl phosphate group, allyl Is preferably at least one selected from the group consisting of; phosphoric acid type cation exchanger containing a phosphate group such as phospho groups and halogenated Arirurin groups. One kind of solid acid catalyst may be used, or two or more kinds may be used in combination.

The sulfonic acid type cation exchanger is an ion exchanger having a cation exchange capacity containing a sulfonic acid group (—SO ₃ H) as described above, and a cation exchange resin containing a sulfonic acid group as a preferred embodiment. Can be mentioned. For example, when the sulfonic acid type cation exchanger has counter ions other than protons (-SO ₃ X (X represents a counter ion)), the sulfonic acid type cation exchanger is used after being replaced with a proton type. The cation exchange resin is not particularly limited, but for the purpose of rapidly hydrolyzing the protein described later at a relatively high temperature and quickly, a polytetrafluoroethylene having a sulfonic acid type ion exchange group having excellent heat resistance (introduced). A fluoroethylene (co) polymer is more preferred. An example of a polytetrafluoroethylene (co) polymer having a sulfonic acid type ion exchange group is Nafion ((Nafion) registered trademark) as an example of a perfluorosulfonic acid / polytetrafluoroethylene copolymer. . In the present invention, the term “(co) polymer” is a concept encompassing both a homopolymer obtained by polymerizing one kind of monomer and a copolymer obtained by polymerizing two or more kinds of monomers.

Furthermore, for the purpose of rapidly hydrolyzing proteins at a relatively high temperature, a particularly suitable combination of “protein holding substrate” and “solid acid catalyst” includes, for example, PVDF and Nafion. The combination with the polytetrafluoroethylene (co) polymer which has a sulfonic acid type ion exchange group is mentioned.

Nafion has long been known as a solid polymer electrolyte and has been used for various purposes as a state-of-the-art material, particularly a fuel cell material, due to its durability and chemical stability. Nafion is available in the form of membranes, powders, pellets, and suspensions, and there are reports on the properties of cast membranes using suspensions (“Densityensand Solubility of Nafion: Recast, Annealed, and Commercial Films ”, Zook, L. A., LeddyedJ., Anal. Chem.,. 68, 3793-37963-3 (1996)). Also, examples of protein mass spectrometry using matrix-assisted laser desorption / ionization (MALDI) (“Diffusive transfer to membranes as an effective interface between gel electrophoresis and mass spectrometry”, gorOgorzalek Loo, R. R., Mitchell, C ., Stevenson, T. I., Loo, J. A., Andrews, P. C., Int. J. Mass Spectrom. IonesProcesses, 169/170, 273-290 (1997)) As for the Nafion membrane, not a cast membrane but a composite membrane in which Nafion is developed on a PVDF membrane is produced.

However, the present inventors were the first to use the above composite membrane for protein hydrolysis, 1) the hydrolysis can be performed at a relatively high temperature and quickly, and 2) the composite. By using the membrane, it is possible to realize an extremely excellent amount of recovered amino acid as compared with a Nafion cast membrane (see also Examples and Comparative Examples described later).

Needless to say, hydrolysis can always be performed more rapidly even if a combination of a relatively solid heat-resistant "solid acid catalyst" and a "protein holding substrate" is arbitrarily selected. Is not limited. Indeed, the rate of hydrolysis reaction increases with increasing temperature. However, the higher the temperature, the greater the risk of unwanted side reactions between the proteins in the reaction system, degradation products (amino acids, etc.), the solid acid catalyst, and the protein holding substrate. May adversely affect quantity and composition. Therefore, in order to perform protein hydrolysis more rapidly, factors other than heat resistance in the combination of the “solid acid catalyst” and the “protein holding substrate” are also important. As a result of trial and error, the present inventors have found that a combination of PVDF and a polytetrafluoroethylene (co) polymer having a sulfonic acid-type ion exchange group such as Nafion can hydrolyze proteins more rapidly. I found it very suitable.

In the present invention, “the solid acid catalyst is arranged on at least a part of the inner surface of the pore of the protein-holding substrate” means that at least a part or the whole of the inner surface of the pore of the protein-holding substrate is solid. It means that an acid catalyst is attached (including a state infiltrating the surface), and more preferably, a part or the whole of the inner surface is coated with a solid acid catalyst. In addition, as long as the solid acid catalyst is arranged on the inner surface of the pore, the solid acid catalyst may be attached to a part or the whole of the surface (surface other than the inner surface) of the protein holding substrate.

In addition, the method of arranging the solid acid catalyst on the protein holding substrate is not particularly limited. For example, 1) A solution obtained by dissolving or suspending the solid acid catalyst in an appropriate solvent is used for the protein holding substrate (coating, etc.). 2) A method for immersing the protein-holding substrate in the solution, and 3) evaporating a solid acid catalyst on the protein-holding substrate. Among these examples, the method 1) or 2) is preferable from the viewpoint of easy and reliable operation.

The amount of the solid acid catalyst arranged on the protein holding substrate is not particularly limited, but 0.1 mg per surface area calculated from the outer dimensions of the protein holding substrate on which the solid acid catalyst is arranged, excluding the inner surface. / Cm ² to 100 mg / cm ² is preferable, and 0.4 mg / cm ² to 2 mg / cm ² is more preferable.

In the present invention, the protein is hydrolyzed by being brought into contact with the solid acid catalyst disposed on the protein holding substrate and heated in the presence of water. Here, as a method of bringing the protein into contact with the solid acid catalyst, (A) the protein holding substrate and the solid acid catalyst are brought into contact with each other, and the solid acid catalyst is formed on at least a part of the inner surface of the pore of the protein holding substrate. Is obtained, and then the protein is brought into contact with the composite substrate, or (B) the protein holding substrate and the protein are brought into contact with each other to hold the protein on the protein holding substrate. Next, the protein-holding substrate holding the protein and the solution containing the solid acid catalyst are brought into contact with each other, and the protein is placed in a state where the solid acid catalyst is arranged on at least a part of the inner surface of the pore. And a method of contacting with a solid acid catalyst.

Here, if a film-like substrate is used as the “composite substrate” by the method (A), for example, the present invention can be applied to the following applications. (1) Since this composite substrate can be incorporated into a column, automatic protein hydrolysis using an automatic hydrolysis apparatus becomes possible. (2) Affinity with existing formats such as a commercially available 96-well dot blot apparatus is high, and it is easy to process a large amount of samples (protein samples) in parallel by using them together. (3) Usually, in addition to various types of chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is frequently used for protein separation because of its relatively good resolution. However, when amino acid composition analysis is performed, a method of directly hydrolyzing a gel containing a protein to be analyzed to obtain an amino acid generates a large amount of ammonia, which hinders subsequent analysis. Moreover, even if the protein is eluted using SDS or formic acid, it is difficult to remove substances that interfere with hydrolysis. Therefore, the conventional method is to electrically transfer the gel to a blotting membrane (protein holding substrate), stain and remove the background, check the protein band, cut out, wash and hydrolyze. Have taken. However, the more complicated such operations, the greater the risk that contamination from the operating human will interfere with the analysis. On the other hand, if a column incorporating the membrane-like composite base material is used according to the present invention, the protein solution eluted from the gel after electrophoresis can be added, and after washing, heating can be performed from hydrolysis to amino acid recovery. Even if SDS or formic acid is added for protein elution, it is possible to suppress contamination from the environment that inhibits microanalysis as much as possible.

In addition, as a specific example of the method (B), although not particularly limited, the protein after gel electrophoresis is transferred from the gel to the protein holding substrate (membrane for blotting), and then the protein holding substrate And a method of bringing the solid acid catalyst into contact with each other. Usually, since a protein sample to be subjected to hydrolysis is a mixture containing undesired proteins and other contaminants, a separation operation is performed by gel electrophoresis or the like before hydrolysis. According to the method (B), the protein transferred from the gel after electrophoresis to the protein holding substrate can be directly hydrolyzed by acting a solid acid catalyst.

Furthermore, as another specific example of the method (B), although not particularly limited, a solution containing protein is filtered through an ultrafiltration membrane (protein holding base material) or the like, and then the ultrafiltration membrane and the solid acid catalyst are filtered. Can be mentioned. By using a commercially available ultrafiltration membrane or a filter plate plate such as 96-hole or 384-hole with a PVDF membrane, it is possible to hydrolyze multiple samples.

In the present invention, the “protein” to be hydrolyzed is not particularly limited in terms of its type, origin, size, etc., as long as it is formed by connecting a plurality of amino acids by peptide bonds. However, in general, a protein consisting of 100 or more and 2000 or less amino acid residues is preferred.

In the present invention, “protein hydrolysis” refers to a reaction in which a peptide bond is hydrolyzed to a shorter protein (peptide) and / or a constituent amino acid in the presence of water. The reaction conditions for the hydrolysis are not particularly limited as long as the decomposition reaction proceeds and the solid acid catalyst and the protein-holding substrate are not substantially decomposed. The reaction temperature is preferably within a temperature range of 100 ° C. or higher and 220 ° C. or lower, and within a temperature range of 110 ° C. or higher and 160 ° C. or lower, as long as the solid acid catalyst and the protein holding substrate are not substantially decomposed. Is more preferable. Particularly preferred is a temperature range of 140 ° C. or higher and 160 ° C. or lower. Note that the combination of PVDF and a polytetrafluoroethylene (co) polymer having a sulfonic acid type ion exchange group does not cause a remarkable deterioration in hydrolytic resolution within the temperature range exemplified here.

The hydrolysis reaction time depends on the reaction temperature, but is preferably about 5 minutes to 1 week, more preferably within the range of 10 minutes to 24 hours. Particularly preferably, it is in the range of about 10 minutes to 2 hours.

Furthermore, in the hydrolysis reaction according to the present invention, the amount ratio of the protein weight serving as the reaction substrate and the solid acid catalyst weight is not particularly limited as long as the reaction proceeds smoothly, but the protein amount and the solid acid catalyst amount are: It is preferably in the range of 1: 1000 to 1: 1.

The use of the protein hydrolyzate obtained by the method according to the present invention is not particularly limited. For example, it can also be used as a raw material for various industrial products including food additives and supplements, and can also be used for analysis of protein constituent amino acids.

[Protein hydrolysis apparatus and hydrolysis product analysis apparatus]
The protein hydrolysis apparatus according to the present invention includes: 1) a sample supply means for supplying a solution containing protein; 2) a hydrolysis means comprising a composite base material in which a solid acid catalyst is arranged on a protein holding base material; 3) a flow path connecting the sample supply means and the hydrolysis means, 4) a liquid feed means for feeding the washing liquid and an eluate for eluting the hydrolyzed product after hydrolysis, and 5) the hydrolysis means. Heating means for heating. Further, as necessary, 6) a hydrolyzate recovery unit and a flow path for connecting the hydrolysis unit and the recovery unit are provided on the downstream side of the hydrolysis unit.

The sample supply means supplies a protein to be hydrolyzed to the hydrolysis means, and can be constituted by, for example, a known sample injector. Moreover, it is good also as a structure which supplies the sample containing protein automatically using a well-known autosampler.

The above-mentioned hydrolysis means is means for executing the protein hydrolysis method according to the present invention, and specifically comprises a composite substrate in which a solid acid catalyst is arranged on a protein holding substrate. Although not particularly limited, the hydrolysis means is preferably either a column packed with a granular composite substrate or a column in which a membrane-shaped composite substrate is arranged in a flow path. If the column is provided, the protein to be hydrolyzed supplied from the sample supply means is retained in the column, while the hydrolyzate can be recovered on the downstream side of the column. Therefore, the hydrolysis apparatus can be easily automated, and the hydrolysis product and undegraded protein can be separated relatively easily.

The method for sending the hydrolyzate to the downstream side of the column is not particularly limited, and examples include a method of eluting the hydrolyzate by feeding an appropriate eluate to the column after the hydrolysis reaction. . In addition, as eluent, basic buffer solution such as boric acid solution and triethylamine aqueous solution; high salt concentration solution such as sodium chloride (for example, concentration of 0.1M to 2M); acidic solution such as trifluoroacetic acid aqueous solution; etc. Any known eluate for protein hydrolysates can be used as appropriate.

The above heating means is a means for heating the hydrolysis means to a desired temperature to promote the protein hydrolysis reaction, and can be constituted by, for example, a known electric furnace. Moreover, said collection | recovery means is a means to collect | recover protein hydrolysates (amino acid etc.), and is provided in the back | latter stage side of a hydrolysis means.

Although not particularly limited, the hydrolysis apparatus is preferably a closed system including the flow path in order to prevent contamination other than the hydrolysis target. Furthermore, it is preferable to provide a hydrolysis apparatus capable of automatic operation by providing a control means for automatically controlling the operation of each means described above.

The apparatus for analyzing a protein hydrolyzate according to the present invention further comprises an analysis means for analyzing peptides and / or amino acids which are protein hydrolysates after the hydrolysis means in the hydrolysis apparatus. is there. In addition, what is necessary is just to use a well-known peptide and / or amino acid analyzer for an analysis means. In addition, amino acids may be collected by a known fraction collector and analyzed separately after derivatization.

Here, as shown in FIG. 1, a specific example of the protein hydrolyzing apparatus / hydrolysate analyzing apparatus 21 according to the present invention is as follows: a cleaning liquid tank 1 for storing a cleaning liquid; an elution liquid tank for storing an elution liquid 2; Liquid feed pump (liquid feed means) 3 connected to the washing liquid tank 1 and the eluate liquid tank 2 through a flow path; any sample pipe 4 is selected from a plurality of sample pipes 4 each holding a different protein solution And an autosampler (sample supply means) 5 for supplying the protein solution to a hydrolysis column (hydrolysis means) 8 described later; a plurality of hydrolysis columns 8 connected to the autosampler 5 via a flow path; An electric furnace (heating means) 9 that houses a plurality of hydrolysis columns 8 and heats these hydrolysis columns 8; a decomposition product recovery unit (decomposition product recovery) connected to the hydrolysis column 8 through a flow path hand ) 10; a decomposition product analysis unit (analysis unit) 11 connected to the hydrolysis column 8 through a flow path; and a control unit (control unit) 12 for controlling the operation of the entire apparatus. .

The plurality of hydrolysis columns 8 include a membrane-like composite base material in which a solid acid catalyst is disposed on at least a part of the inner surfaces of the plurality of pores of the protein holding base material on the flow path (examples described later) Equivalent to the Nafion-PVDF membrane). The plurality of hydrolysis columns 8 are connected to an autosampler 5 disposed on the front side, a degradation product recovery unit 10 disposed on the rear side, and a flow path switching valve (flow path switching means) 7. The degradation product analysis unit 11 is connected. Thereby, protein hydrolysis is performed while appropriately switching the hydrolysis column 8 to be used.

The control unit 12 automatically controls the operations of the flow path switching valve 7, the decomposition product recovery unit 10, and the decomposition product analysis unit 11 via the liquid feed pump 3, the autosampler 5, and the valve controller 6.

For example, protein hydrolysis / hydrolysis product analysis using the hydrolysis apparatus / hydrolysis product analysis apparatus shown in FIG. 1 is performed as follows.
(1) First, MilliQ water stored in the washing liquid tank 1 is passed through the entire apparatus to equilibrate the hydrolysis column 8.
(2) Next, the protein solution in the sample tube 4 to be subjected to hydrolysis and the like is passed through the hydrolysis column 8 through the autosampler 5. As a result, the protein introduced into the hydrolysis column 8 is held on the composite substrate disposed in the column 8.
(3) Next, MilliQ water stored in the washing solution tank 1 is passed through the hydrolysis column 8 for washing, and then the hydrolysis column 8 is heated by the electric furnace 9. Thereby, the protein hold | maintained at the said composite base material is hydrolyzed to a peptide or an amino acid according to the method concerning this invention.
(4) Next, heating by the electric furnace 9 is stopped, and after cooling, the eluate stored in the eluate tank 2 is passed through the hydrolysis column 8. Thereby, the peptide or amino acid which is a hydrolysis product elutes to the back | latter stage side of the said column 8 for hydrolysis.
(5) Next, the eluted peptide or amino acid is recovered by the degradation product recovery unit 10 and is used for applications such as analysis in the degradation product analysis unit 11. Alternatively, the eluted peptide or amino acid is directly subjected to analysis in the degradation product analysis unit 11 (that is, not via the degradation product recovery unit 10).

As described above, in the protein hydrolysis method according to the present invention, the solid acid catalyst is a polytetrafluoroethylene (co) polymer having a sulfonic acid type ion exchange group, and the protein holding substrate is Polyvinylidene difluoride is preferred. Further, in this case, the hydrolysis of the protein is preferably performed within a temperature range of 100 ° C. or higher and 220 ° C. or lower.

The combination of polytetrafluoroethylene (co) polymer having sulfonic acid type ion exchange groups and polyvinylidene difluoride is not only excellent in heat resistance but also excellent in the property of catalyzing protein hydrolysis reaction. ing. Therefore, according to the above method, for example, protein is rapidly and satisfactorily hydrolyzed at a high temperature of 100 ° C. or higher and 220 ° C. or lower, more preferably 110 ° C. or higher and 160 ° C. or lower. I can do it.

The method for hydrolyzing a protein according to the present invention includes a composite group in which a protein holding substrate and a solid acid catalyst are brought into contact with each other, and the solid acid catalyst is arranged on at least a part of the inner surface of the pores of the protein holding substrate. It may include a step of obtaining a material and a step of bringing the protein into contact with the composite base material.

Alternatively, in the method for hydrolyzing a protein according to the present invention, the step (1) of bringing the protein holding base material into contact with the protein and holding the protein in the protein holding base material, and then the above protein holding holding the protein The substrate and the solution containing the solid acid catalyst are contacted, and the protein and the solid acid catalyst are contacted with the solid acid catalyst disposed on at least a part of the inner surface of the pores of the protein holding substrate. Step (2) to be performed. Further, in this case, the step (1) may be a step of transferring the protein after gel electrophoresis from the gel to the protein holding substrate.

According to the above method, for example, the protein transferred from the gel after electrophoresis to the protein holding substrate can be directly hydrolyzed by acting a solid acid catalyst.

The present invention is not limited to the above-described embodiment, and various modifications can be made within the scope indicated in the claims. That is, embodiments obtained by combining technical means appropriately modified within the scope of the claims are also included in the technical scope of the present invention.

Next, the present invention will be specifically described by way of examples and comparative examples, but the present invention is not limited thereto. First, the method of “amino acid analysis by fluorescence derivatization” used in Examples and Comparative Examples will be described below.

(1) Amino acid analysis by fluorescent derivatization Synthesis of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and fluorescent derivatization (prelabeling) of amino acids using AQC are well-known (SA Cohen et al., Anal) Biochem. 211. p279-287 (1993)).

The fluorescent derivatives of amino acids thus prepared were separated by HPLC. Separation by HPLC was performed by a gradient elution method using a reversed phase column and a buffer solution system containing an ion pair reagent. The following conditions were used to separate a standard amino acid mixture or protein hydrolyzate containing 17 amino acids and ammonium fluorescently derivatized by AQC.
<Conditions>
a) HPLC apparatus: Agilent 1100 series (including vacuum degasser G1379A, binary pump G1312A, autosampler G1367A, column thermostat G1330B / G1316A, diode array detector G1315B, fluorescence detector G1321A)
b) Column: Inertsil ODS-3 (4.6 × 150 mm, 3 μm), manufactured by GL Sciences
c) Mobile phase: A: 95% 5 mM tetrabutylammonium bromide, 30 mM phosphate buffer (pH 7.3), 5% acetonitrile; B: 50% acetonitrile / 30 mM phosphate buffer (pH 7.3)
d) Concentration gradient: The mobile phase B concentration was changed to 2-7.3-72.3% between 0-3 and 40 minutes.
e) Flow rate: 0.5ml per minute f) Temperature: 40 ° C
g) Fluorescence excitation wavelength (Ex) and monitor wavelength (Em): Ex = 250nm, Em = 395nm
A 10 pmol / μl solution prepared from Pierce Amino Acid Standard H (2.5 μmol / ml) was used as the standard amino acid solution. 5 μl (50 pmol) of the standard amino acid solution was fluorescently derivatized with AQC, and the protein hydrolyzate was quantified based on the analysis value when 1/10 volume was injected.

[Reference Example 1: Preparation of Nafion membrane]
About 1 ml of 5 wt% Nafion resin solution in IPA (isopropanol) -water mixture (15-20% water, Aldrich 274704) (hereinafter referred to as “Nafion suspension”) is developed on a Weinboat and dried in a draft I let you. Thereafter, it was left in a vacuum desiccator containing silica gel for 2 days. The obtained film was peeled off from the Weinboat, heated in a muffle furnace at 140 ° C. for 1 hour (annealed), allowed to cool, and immersed in about 10 ml of concentrated nitric acid. After 24 hours, the membrane was washed with water until the supernatant became neutral, and then the membrane was immersed in distilled water for 24 hours. Thereafter, it was sufficiently dried in a vacuum desiccator to obtain a Nafion membrane for hydrolysis.

[Example 1: Preparation of Nafion-PVDF membrane and hydrolysis of protein]
(Creation of Nafion-PVDF membrane)
Polyvinylidene difluoride (PVDF) membrane (ProBlott PVDF membrane, Applied Biosystems 400994) is cut into a 5 mm square in a glove box with a scalpel, and the amount of Nafion suspension described in Reference Example 1 is 10 μl / cm ² . The catalyst was dropped onto the PVDF membrane at a catalyst weight of about 0.4 mg / cm ² . After drying, Nafion suspension was similarly dropped on the back surface of the PVDF membrane and dried again to obtain a Nafion-PVDF membrane (membrane of composite substrate) as a membrane for hydrolysis.

(Protein hydrolysis using Nafion-PVDF membrane)
After the obtained Nafion-PVDF membrane was moistened with acetonitrile, 5 μl of 0.2 μg / μl bovine serum albumin (BSA, Sigma A-7638) aqueous solution was added to the membrane. The membrane was placed in a 6 × 32 mm small test tube and 10 μl of MilliQ water was added. The small test tube was placed in a 25 ml screw-cap vial, 25 μl of MilliQ water was added, and the tube was sealed. This vial was heated at 110 ° C. for 20 hours to hydrolyze BSA. After the vial is allowed to cool, the small test tube is taken out, 10 μl of acetonitrile is added to the small test tube to wet the membrane, 20 μl of 20 mM hydrochloric acid is added and stirred, and then 0.2 M borate buffer (pH 8.8) is added. 50 μl and 20 μl of AQC solution were added, and fluorescence derivatization was performed according to the method described in “(1) Amino acid analysis by fluorescence derivatization”, and amino acids were quantified.

Table 1 shows the total amount of recovered amino acids (the amount of recovered amino acids (pmol)) and the ratio of each amino acid to the total amount of recovered amino acids (the amount of recovered amino acids (%)) as a result of hydrolysis. For reference, the results of hydrolytic hydrolysis of the same amount of BSA aqueous solution at constant boiling hydrochloric acid are also shown in Table 1 (shown as “gas-phase” in the table).

Here, the constant-boiling-point hydrochloric acid gas phase hydrolysis was carried out as follows. Specifically, 5 μl of 0.2 μg / μl BSA aqueous solution was put into a 6 × 32 mm small test tube and dried. Next, this small test tube was put into a 25 ml screw mouth vial in which phenol crystal was added to 200 μl of constant boiling hydrochloric acid, and after deaeration using a Mininert Valve, it was sealed. And this vial was heated at 110 degreeC for 20 hours, and the hydrolysis of BSA was performed. Next, 20 μl of 20 mM hydrochloric acid was added to the small test tube after the gas phase hydrolysis, and after stirring, 60 μl of 0.2 M borate buffer (pH 8.8) and 20 μl of the AQC solution were added. According to the method shown in “Amino acid analysis by derivatization”, fluorescent derivatization was performed, and amino acids were determined.

[Comparative Example 1: Protein hydrolysis using Nafion membrane]
Instead of the Nafion-PVDF membrane, hydrolysis of BSA was carried out according to the method described in Example 1 above, except that the Nafion membrane obtained in Reference Example 1 was cut in a glove box with a knife to about 5 mm square. And amino acid quantification. Table 1 shows the total amount of recovered amino acids (the amount of recovered amino acids (pmol)) and the ratio of each amino acid to the total amount of recovered amino acids (the amount of recovered amino acids (%)).

As shown in Table 1, when the hydrolysis method of the present invention was compared with the hydrolysis method by the gas phase method, almost the same results were obtained with respect to the total amount of recovered amino acids. On the other hand, when the Nafion membrane was used, the total amount of recovered amino acids was reduced (Comparative Example 1). From this, it can be seen that the Nafion-PVDF membrane in which Nafion and PVDF are combined significantly extracts the catalytic activity of Nafion for the protein hydrolysis reaction. The recovered amino acid amount (%) was almost the same between the case where the Nafion-PVDF membrane was used and the gas phase method, and a very good result was obtained.

[Example 2: Rapid hydrolysis of various proteins using Nafion-PVDF membrane]
A 1 cm square Nafion-PVDF membrane was prepared in the same manner as in Example 1, and the membrane was cut into a 1 mm wide strip with a knife. After moistening with acetonitrile, 1 μl of 1 μg / μl of BSA aqueous solution, ovalbumin (OVA, Sigma A-5503) aqueous solution, or histone H3 (H3, Roche 1034758) aqueous solution was added to the membrane. Each was put in a small test tube of 32 mm. The small test tube was placed in a 25 ml screw-mouth vial in which phenol crystal was added to 200 μl of MilliQ water, and after deaeration using a Mininert Valve, the tube was sealed. This vial was heated at 150 ° C. for 2 hours to hydrolyze each protein.

Next, 10 μl of acetonitrile is added to each small test tube after hydrolysis, the membrane is moistened, 20 μl of 20 mM hydrochloric acid is added and stirred, 50 μl of 0.2 M borate buffer (pH 8.8), and 20 μl of AQC solution are added. And derivatized with fluorescence according to the method described in “(1) Amino acid analysis by fluorescence derivatization”, and amino acids were quantified.

Tables 2 to 4 show, in order, the total amount of recovered amino acids (the amount of recovered amino acids (pmol)) as a result of hydrolysis of BSA, OVA, and H3, and the ratio of each amino acid to the total amount of recovered amino acids (the amount of recovered amino acids (%)). ). For reference, the results of vapor-phase hydrolysis of the same amount of protein aqueous solution with constant boiling hydrochloric acid are also shown in Tables 2 to 4 (shown as “gas phase method” in the table).

Here, the constant-boiling-point hydrochloric acid gas phase hydrolysis was carried out as follows. That is, 1 μl of a 1 μg / μl BSA aqueous solution, an OVA aqueous solution, or an H3 aqueous solution was placed in a 6 × 32 mm small test tube and dried. Next, this small test tube was put into a 25 ml screw mouth vial in which phenol crystal was added to 200 μl of constant boiling hydrochloric acid, and after deaeration using a Mininert Valve, it was sealed. And this vial was heated at 110 degreeC for 20 hours, and each protein was hydrolyzed. Next, 20 μl of 20 mM hydrochloric acid was added to the small test tube after the gas phase hydrolysis, and after stirring, 60 μl of 0.2 M borate buffer (pH 8.8) and 20 μl of the AQC solution were added. According to the method shown in “Amino acid analysis by derivatization”, fluorescent derivatization was performed, and amino acids were determined.

When the hydrolysis method of the present invention is compared with the hydrolysis method by the gas phase method, the total amount of recovered amino acids is 96% of the gas phase method for BSA, 74% for OVA, and 77% for H3. It was. Also, the recovered amino acid amount (%) was almost the same as the gas phase method, and very good results were obtained.

That is, a Nafion-PVDF membrane (a pore having a size sufficient for protein and pores) coated with Nafion with heat resistance and acidic residue on a porous PVDF membrane with heat resistance and excellent protein retention It was possible to hydrolyze it into an amino acid that is a component of protein by sealing and heating the protein aqueous solution and the acid aqueous solution. Furthermore, it is possible to recover an amino acid equivalent to the amino acid obtained at the conventional hydrolysis time of 110 ° C. for 20 hours in a short time of 2 hours at 150 ° C., and the throughput is dramatically improved.

[Example 3: Hydrolysis on electroblotted membrane]
A standard molecular weight marker (GE Healthcare Bioscience, LMW Marker Kit, 17-0446-01) containing 0.87 μg of BSA was applied to a polyacrylamide gel, and BSA gel electrophoresis was performed. After SDS-PAGE, electroblotting was performed on a PVDF membrane (ProBlott PVDF membrane, Applied Biosystems 400994). This membrane was stained with Coomassie blue, and the detected BSA band was cut out. Next, a Nafion suspension was applied to the PVDF membrane corresponding to this band and dried. As a result, the surface of the PVDF membrane and the inner surface of the pores were in a state where Nafion was added. For reference, a Nafion-PVDF membrane prepared by the same method as in Example 1 was cut into the same size as the cut out band, and 1 μl of 1 μg / μl BSA aqueous solution was added thereto.

Next, these were placed in small test tubes each having a size of 6 × 32 mm, placed in a 25 ml screw mouth vial in which phenol crystal was added to 200 μl of MilliQ water, and after deaeration using a MininertValve, they were sealed. This vial was heated at 150 ° C. for 2 hours to hydrolyze BSA. After allowing the vial to cool, take out the small test tube, add 10 μl of acetonitrile to the small test tube to wet the membrane, add 20 μl of 20 mM hydrochloric acid and stir, and then add 50 μl of 0.2 M borate buffer (pH 8.8). Then, 20 μl of AQC solution was added, and the resulting amino acid was fluorescently derivatized according to the method described in “(1) Amino acid analysis by fluorescent derivatization”, and the amino acid was quantified.

Table 5 shows the ratio of each amino acid to the total amount of recovered amino acids (the amount of recovered amino acids (%)) as a result of hydrolysis. In the table, “blotted PVDF-Nafion” is the result when Nafion is applied to the electroblotted PVDF membrane, which is almost the same as the result using the Nafion-PVDF membrane according to Example 1. I understand.

[Example 4: Hydrolysis by incorporating a Nafion-PVDF membrane into a column]
A Nafion-PVDF membrane coated with BSA was prepared by adding 2 µl (10 µg in terms of BSA) of 5 µg / µl BSA aqueous solution to the Nafion-PVDF membrane. Then, a precolumn filter assembly A-318 manufactured by Upchurch Scientific was attached with the Nafion-PVDF membrane sandwiched between frit A-700 and frit A-701, and a precolumn was assembled. After MilliQ water was passed through the precolumn at 0.2 ml / min for 1 minute, both ends of the precolumn were sealed with PEEK end plugs (Tosoh 0017791). This precolumn was heated at 150 ° C. for 2 hours to hydrolyze BSA on the Nafion-PVDF membrane. Extraction of the hydrolyzate from the Nafion-PVDF membrane is performed by the following two methods (a) and (b), and the obtained amino acid is fluorescent according to the method described in the above “(1) Amino acid analysis by fluorescent derivatization”. Derivatization and quantification of amino acids.
(A) The Nafion-PVDF membrane is taken out from the precolumn, moistened with acetonitrile, and then the hydrolyzate is extracted with 0.2 M borate buffer. Table 6 shows the ratio of each amino acid to the total recovered amino acid amount (recovered amino acid amount (%)) as a result of hydrolysis.
(B) A 0.2 M borate buffer solution is passed through the precolumn at a flow rate of 0.05 ml / min, and the hydrolyzate is recovered at 1 min / tube with a fraction collector. Table 7 shows the ratio of each amino acid to the total amount of recovered amino acids (the amount of recovered amino acids (%)) as a result of hydrolysis.

As shown in Tables 6 and 7, it can be seen that BSA was hydrolyzed even when a Nafion-PVDF membrane was incorporated in the column. In addition, some recovered amino acid amounts (%) of some amino acids are out of the theoretical value, but this does not indicate that BSA is not hydrolyzed. That is, it is considered that the recovered amino acid amount (%) closer to the theoretical value can be realized by optimizing column design, elution conditions of amino acids as degradation products, and the like.

[Example 5: Hydrolysis by incorporating a Nafion-PVDF membrane into a column (automatic protein introduction)]
A frit made only of PEEK material is created by combining the outer frame of Frit A-100 made by Upchurch Scientific and the filter part of A-702, and a Nafion-PVDF membrane sandwiched between the created frit is a pre-column filter A precolumn was assembled by attaching to assembly A-318. First, acetonitrile is introduced into the precolumn to wet the membrane, then 5 μl of 2 μg / μl BSA aqueous solution (10 μg in BSA amount) is automatically introduced using an autosampler, and MilliQ water is added at 0.05 ml / min. The solution was passed for 10 minutes. This precolumn was heated at 150 ° C. for 2 hours to hydrolyze BSA on the Nafion-PVDF membrane. Extraction of the hydrolyzate from the Nafion-PVDF membrane is performed by the following two methods (a) and (b), and the obtained amino acid is fluorescent according to the method described in the above “(1) Amino acid analysis by fluorescent derivatization”. Derivatization and quantification of amino acids.
(A) The Nafion-PVDF membrane is taken out from the precolumn, moistened with acetonitrile, and then the hydrolyzate is extracted with 0.2 M borate buffer. The ratio of each amino acid to the total amount of recovered amino acids (recovered amino acid amount (%)) is shown in the item “BSA manual elution” in Table 8 as a result of hydrolysis.
(B) A 0.2 M borate buffer solution is passed through the precolumn at a flow rate of 0.05 ml / min, and the hydrolyzate is recovered at 1 min / tube with a fraction collector. That is, (b) corresponds to an embodiment in which all steps (column pretreatment, sample injection, washing, heating, elution, and recovery) are automated. In the item of “BSA automatic elution” in Table 8, the ratio of each amino acid to the total amount of recovered amino acids (the amount of recovered amino acids (%)) is shown as a result of hydrolysis.

As shown in Table 8, it can be seen that BSA is hydrolyzed even when Nafion-PVDF membrane is incorporated in the column and BSA is automatically introduced. In addition, some recovered amino acid amounts (%) of some amino acids are out of the theoretical value, but this does not indicate that BSA is not hydrolyzed. That is, it is considered that the recovered amino acid amount (%) closer to the theoretical value can be realized by optimizing column design, elution conditions of amino acids as degradation products, and the like.

According to the present invention, it is possible to provide a protein hydrolysis method, hydrolysis apparatus, and hydrolysis product analysis apparatus that are simpler, faster, and more automated than conventional methods.

5 Autosampler (sample supply means)
8 Column for hydrolysis (hydrolysis means)
9 Electric furnace (heating means)
11 Decomposition product analysis section (analysis means)
21 Hydrolysis equipment / hydrolysis product analysis equipment

Claims (8)

1. By contacting the protein with the solid acid catalyst in a state where the solid acid catalyst is disposed on at least a part of the inner surface of the pore of the protein holding substrate having a plurality of pores, and heating in the presence of water, the protein is retained. A method for hydrolyzing a protein, comprising hydrolyzing the protein.
2. The solid acid catalyst is a polytetrafluoroethylene (co) polymer having a sulfonic acid type ion exchange group, and the protein-holding substrate is polyvinylidene difluoride according to claim 1, A method for hydrolyzing the protein described.
3. The method for hydrolyzing a protein according to claim 2, wherein the protein is hydrolyzed within a temperature range of 100 ° C or higher and 220 ° C or lower.
4. Contacting the protein-holding substrate with a solid acid catalyst to obtain a composite substrate in which the solid acid catalyst is disposed on at least a part of the inner surface of the pore;
   The method for hydrolyzing a protein according to any one of claims 1 to 3, further comprising a step of bringing the protein into contact with the composite substrate.
5. A step (1) of bringing the protein holding substrate and the protein into contact with each other and allowing the protein holding substrate to hold the protein;
   Next, the protein-holding base material holding the protein and the solution containing the solid acid catalyst are brought into contact with each other, and the protein and the solid acid are placed in a state where the solid acid catalyst is disposed on at least a part of the inner surface of the pore. The method for hydrolyzing a protein according to any one of claims 1 to 3, comprising a step (2) of contacting the catalyst.
6. The method for hydrolyzing a protein according to claim 5, wherein the step (1) is a step of transferring the protein after gel electrophoresis from the gel to the protein holding substrate.
7. Sample supply means for supplying a solution containing protein;
   A hydrolysis means comprising a composite substrate in which a solid acid catalyst is disposed on at least a part of the inner surfaces of the plurality of pores of the protein-holding substrate;
   A flow path connecting the sample supply means and the hydrolysis means;
   And a heating means for heating the hydrolysis means.
8. Sample supply means for supplying a solution containing protein;
   A hydrolysis means comprising a composite substrate in which a solid acid catalyst is disposed on at least a part of the inner surfaces of the plurality of pores of the protein-holding substrate;
   A flow path connecting the sample supply means and the hydrolysis means;
   Heating means for heating the hydrolysis means;
   An apparatus for analyzing a protein hydrolyzate, comprising an analysis unit for analyzing a protein hydrolyzate after the hydrolysis unit.

Images