JPS6057829B2 - Manufacturing method of modified protein - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Proteins are biologically synthesized macromolecules that have various roles in biological systems.

Enzymes are a special class of biologically active proteins that catalyze specific reactions. Currently, enzyme technology is used in many fields in industry and research, such as medical research, food processing and preservation,
It is used in the production of fermented beverages, in the production of pharmaceuticals and in the analytical determination of the concentration of various metabolites and food components by analytical enzyme technology. Enzymes are highly specific in their biological activity and generally catalyze individual reactions at much higher rates than corresponding reactions that would occur at room temperature without biological catalysis.

A single enzyme can exhibit catalytic activity with respect to many substrates on which it can act. Thus, a given enzyme can catalyze the synthesis or degradation of more than one substrate. Some proteins that are not considered canonical enzymes, such as bovine serum albumin, exhibit very limited catalytic activity with respect to one or more substrates. Many enzymes are found in nature in extremely small amounts.

Therefore, their isolation, purification and utilization is limited to small scale operations due to the expense and time required to isolate them in useful form. Some enzymes occur in relatively large quantities in nature, and are relatively easy to isolate, purify, and utilize. Unfortunately, however, due to the strict catalytic behavior of these enzymes, enzymes available in large quantities are only able to catalyze certain selected reactions. Recently, much effort has been devoted to the synthesis of synthetic biological catalysts that exhibit enzymatic behavior similar to that exhibited by native enzymes that are difficult or expensive to isolate. Further. In recent years, efforts have been made to modify Nutib enzymes to change their enzyme specificity so that they act to catalyze reactions that they were previously unable to catalyze. One known technique for achieving enzyme behavior that catalyzes specific desired reactions is the synthesis of so-called enzyme model molecules.

For example, a low molecular weight compound can be covalently attached to an active functional group in the active site of an enzyme. Examples of such preparation methods are described in the publications listed below. 1R ● Breslow (
R. BreslOw) C6 Advances in Chemistry Series 3566Advances1nCh
emistr'YSeries-Edited by R,F,Gould, A. Merican.
Chemical Society, DC 21, Washington
=43 (1971) J and 1 C.C. Tongue (C
．． C. Tang), D.Davalin (D.Dava)
lian), P. Haung and R. R. BreslOw, 66 J.A. a. Mel. Chem●Soku''C4J.Amer.Chem
．． SOc. 5, 100, 3918 (1978). J and 1R. Breslow (R. BreslOw), J.A. J.B. Doherty, G. Guillot and C. C. Lipsey, 66 J.A.M.C.
SOc'' (J.Amer.Chem.SOc!tsu100,
3277 (1978) JO et al. technique involves the use of a synthetic polymer matrix that is modified along its backbone to provide functional groups indicative of the active site function of a given enzyme.

Examples of such techniques can be found in the following papers: 1 G.wu Ff and 1.Schu1za, 66 Isreal G. Chem'1 (゜゜1srea1J. Chem.), 1
7.291 (1978) J.S.
uh) and I●M Kron (1.M.K10tz)
, '6BioOrgani
c゛), 6, 165 (1977). K. Another technique involves adding new chemical moieties to native enzymes near the enzyme's active site in an attempt to cause such enzymes to react with different catalytic activity. An example of this is illustrated in the paper below:
It converts the proteolytic enzyme papain into an oxidase-type enzyme by covalently adding a flavin near the active site of the original papain enzyme: H.L. Le Vine and E. tea. Kaiser (ET. Kaiser), 66 Jie●Amel●Chem●Soku55C4J. Amer. Ch
em. S0c) 3, 100, 7670 (1978) and T. Otsuki, Y●Nakagawa and E●T●Kaiser, 6' G●C●S●Chem●Com゛
J. C. S. Chem. COmm.゛) 11, 457 (
1978) JO Other examples of such enzymatic modifications can be found in the following papers: M.E. Wilson and G.M. Whitesides. )、J●
Amel●Chem Sog (゜゜J.Anler.Chem.
S0c. ), 100, 306 (1978). Yet another attempt to alter the specificity of the JO enzyme is to immobilize the native enzyme in a gel matrix.

For example, the trypsin enzyme has been immobilized in polyacrylamide gels. Polyacrylamide gels will allow amino acid esters to diffuse through the gel matrix and react with the enzyme, but will not allow larger proteins to diffuse; thus, the specificity of the enzyme depends on the accessibility of one of the substrate molecules to the enzyme. can be changed by eliminating. An example of such changes in specificity can be found in Kirkoosmer's R46 Encyclopedia of Chemical Technology 31, published by Willi & Son, Inc.
C6Encyc10pedia0fChemica1T
echn010gy 3, 3rd edition, 9, 148, (198
0) Described in J. It is also known that the sulfhydryl groups on this enzyme can be blocked by reacting nativridin monooxygenase.

When this particular enzyme, lysine monooxygenase, is treated in this way, it exhibits new catalytic activity towards amino acids, catalyzing an oxidative deamination reaction instead of its natural enzymatic decarboxylation reaction. However, the above-mentioned reporters cannot explain the above-mentioned reforming behavior. 1 “゜The Journal Biological Chemistry 1”
C4TheJournaIOfBiOlOgicalC
hemjstry゛), 24&. 1013750-37
52 (1973) Tee in middle school. See the paper by Yamauchi, S.Yamamoto, and Oh Hayaishi.

It has also been reported that a native enzyme, such as trypsin, can be modified in its activity with respect to its natural substrate by reacting with its natural inhibitor and then cross-linking the enzyme. 1゜“India J. Peptide Res.゛), 5, 215
-18 (1973) G●H●Beven (G
．． H. See the paper by W. B. Beaven and W. B. Gratzzer.

Although these techniques are suitable for many applications, they generally result in modified natural enzymes or total synthetic enzyme analogs that are not highly catalytically active.

Therefore, inexpensive commercially available native enzymes can be chemically modified to perform certain desired chemical reactions that have hitherto had no commercially useful reactions catalyzed by native enzymes, and which are novel. For such reactions, where the reactions can be systematically predetermined, there is a need for a simple, efficient, and economical method to produce active modifying enzymes. The methods described in the above-mentioned documents simply subject an enzyme to a set of conditions and attempt to elucidate its behavior. Those methods do not present a systematic way to modify the behavior of proteins and enzymes. The present invention achieves a modified enzyme by partially denaturing a native protein, typically an enzymatically active protein, by exposing it to a denaturing agent and partially opening (UrlfOld) the conformational structure of the protein. do.

Next, this partially denatured protein is brought into contact with an inhibitor of the model enzyme. This protein is then cross-linked to define a new conformation defined by the inhibitor, ie the shape of the modified enzyme. The inhibitor and excess crosslinker are then removed from the newly generated modified enzyme, yielding a functional analog to the model enzyme. The modified enzyme thus produced exhibits the activity characteristics of the above-mentioned model enzyme. In achieving the objects and advantages of the present invention, it has now been found that a protein can be modified from its native conformation to a modified conformation by practicing the method of the present invention.

The new conformational state defines the shape of the reforming enzyme that exhibits catalytic activity. As used herein, the term "enzyme" is defined as a protein that has a known catalytic activity toward a specific substrate.
Also, the term "protein" as used herein is defined as generally accepted in the art, ie, a polypeptide produced from amino acids to yield a biological molecule. The present invention modifies a protein from one conformation to a second conformation, thus creating a new enzymatic activity for the selected protein or limiting enzyme activity present in the native protein. to a commercially useful level.
It typically involves subjecting the enzyme to conditions and/or reagents that partially denature, usually reversibly, the structure of the protein.

Next, an inhibitor of the model enzyme is brought into contact with the partially denatured native protein. Without wishing to be bound by any theory, by partially denaturing the protein, the protein binds the inhibitor of the model enzyme according to this method,
It is believed that it forms an active site that is very similar to that of the model enzyme. The binding of the above inhibitors preserves and defines the new conformation containing at least one site capable of performing the catalytic function of the model enzyme until the new conformation can be cross-linked. I can believe it. Therefore,
After contacting the inhibitor with the partially denatured protein, a crosslinking step chemically stabilizes the new conformation of the protein. Thus, new modifying enzymes are prepared from the model protein. As defined herein:
“Partial denaturation” perturbs the shape or conformation of a protein without causing irreversible, gross denaturation of the protein (Pe
rtrb). Conformation, as generally accepted in the art, refers to the secondary and tertiary structure of a protein that has biological activity in vivo. It is defined as a combination of Partial denaturation of proteins is well known and discussed in detail in the literature listed below. Worth Publisher
sllnc. ), New York (N.Y.), A.L.?Hninger, “
゜Biochemistry゛C'BiOchemistry
3), Sekipeyo, R64 Advances in Protein Chemistry (AdvancesinPrOt)
einChemist-y'tsu, vol. 33, 167-19
2 pages, inside '6 Stability of Proteins 55
P.L.Privu Alov (P.L.Priv) titled 5 C6StabiIity0fPr0teins.
vaIOv)'s paper Ato Banshees In Protein Chemistry'5C6AdvanceinPr0
teinChemistry゛), Part C, 2 volumes, 2-9
Top, middle “゜Protein Denaturation 35C6
Pr0teinDer! C.Sanford's paper entitled 3tUrati0n3, 1 Advances in Protein Chemistry 33C6AdvancesinPr0teinCh
emistry 3 volumes, pages 74-166, inside, MOtiOni Proteins 5' (MOtiOni
F.R.N. Gur'd et al.'s paper titled nPrOtairls', 1 BBN (゜゜BBN゛), vol. 621,
Pages 227-232, inside Oh Yardetsky (0.Za
rdetzky), 1 Day●I●B●Es (゛゜TIBS゛), 197@. December, 271 pages,
R.Huber's paper and 1Moleku I
YarnayaBlOlOgiya'tsu, Volume 8, No.
6, pp. 857-863, in the article by D. S. Mark Ovich et al., The term "denaturing agent" used herein causes partial denaturation of proteins. Refers to process conditions or reagents.

For example, partial denaturation of a protein involves raising the temperature of the protein, e.g.
This can be achieved by immersion in an aqueous solution at a temperature within the range of 0.degree. C. to 6 cr.C. . 25 for most proteins
℃ to 6 (generations) will perturb the structure of the protein so as to produce changes in one protein. However, as is well known in the art, some proteins from thermophilic bacterial sources are stable near the boiling point of water and therefore generally require higher temperatures than those disclosed above. It will be decided that In addition, partial denaturation of proteins can be done by converting proteins into inorganic acids,
This can also be achieved by immersion in an aqueous solution containing an inorganic or organic or water-miscible solvent. Suitable inorganic salts to help destabilize protein structures include: NaFl(NH4)2S0, (CH3), NCIl.
(CH3)4NBr1KCH3C00. ．． N.H., C.I.
．． RbCI,. KC■, NaCIlCsC■, LiCI
．． ．． KBr. ．． NaBr,. KNO3, MgCI2, N
aNO3, CacI2, KSCN, . NaSCNlBa
CI2, NaI and Lil.

Suitable inorganic acids include: hydrochloric acid, nitric acid,
Sulfuric acid, phosphoric acid and similar strong inorganic acids that donate protons.

Suitable organic acids include acetic acid, formic acid, propionic acid and citric acid.

Suitable water-miscible solvents believed to dissolve hydrophobic groups on proteins and thus destabilize their structure include t-
Butanol, acetonitrile, dioxane, acetone,
Includes methanol, ethanol and dimethyl sulfoxide.

As used herein, the term "inhibitor" refers to a compound that has sufficient structural similarity to the natural substrate of the modifying protein to serve as a template for the active site of the modifying enzyme. .

Inhibitors, like substrates, are generally not degraded by enzymes. An example of structural similarity between an enzyme inhibitor and the enzyme's natural substrate is that of glucose oxidase. Glucose is the natural substrate of glucose oxidase, while D-
Glucal is an inhibitor of glucose oxidase. Glucose and D-glucal are structurally very similar. As defined herein, the term "crosslinking" refers to the creation of inter- or intramolecular covalent bonds between reactive sites on a protein.

For intramolecular crosslinking, the process is usually accomplished through the use of polyfunctional reagents, such as glutaraldehyde. Other examples of cross-linking reagents suitable for achieving cross-linking of proteins are: 2-amino-4,6-dichloro-S-
Triazine diasonium salt; N-hydroxysuccinamide; p-benzoyl azide and crosslinking reagents disclosed in the following documents: F.Wold
), 66 Mesotzus. Enzimol 55C6Meth0ds
Edited by C.H.W.HIRS, Academic Press, 1967, 617. JSR H●
H. FasOld et al.'6 Augen Chem India Ed Engle 5'C4Augen. Ch
em. lnt. Ed. Engl? 1, 10, 795, 1
97J and 1M.H.Key
s), Kirk-αHer, “゜Encyclopedia of Chemical Technology 8C
6Encyc10pedia0fChemica1Te
chn010gy゛tsu, 9, 3rd edition, 1980, G.I.
Willy & Sons Inc., 14
8-172. Many natural enzymes can be modeled according to this method to create modified analogs thereof, such as hydrolases, redox enzymes and transferases.

For example: the first group of hydrolytic enzymes are proteolytic enzymes that hydrolyze proteins, such as papain, fuicin, pepsin, trypsin, chymotrypsin, promelin, keratinase; carbohydrases that hydrolyze hydrocarbons, such as cellulase, amylase, maltase, pectinase. , chitinase; esterase that hydrolyzes esters;
For example, lipase, cholinesterase, lecithinase,
Alkaline and acid phosphatases; nucleases that hydrolyze nucleic acids, such as ribonucleases, deoxyribonucleases; and amidases that hydrolyze amines, such as arginase, asparaginase, glutaminase, histidase, and urease. The second group are redox enzymes that catalyze oxidation or reduction reactions. These include glucose oxidase, xanthioxidase, catalase, peroxidase, lipoxidase and cytochrome reductase; a third group includes transferases that transfer groups from one molecule to another. Examples of these are glutamine pyrpine transferaminase, glutamine-oxalacetic transaminase, transmethylase, phosphopyrpintotransphosphorylase. In normal practice, a model or first protein;
Typically enzymes are chosen.

Next, a second protein to be imitated to the first protein is selected, and a modifying enzyme is produced. By practicing the present invention, the second protein can be tailored to a desired, different, modified protein. This provides significant benefits in a wide range of clinical and industrial situations where the enzymes desired to be used are in short supply, very expensive, or difficult to purify. Thus, native proteins or enzymes that are available in large quantities and/or at low cost can be modified by the method of the present invention to exhibit the desired catalytic activity of native enzymes, but are less available than the former.

and/or can be replaced with more expensive modifying enzymes. There are many uses for such enzyme-modified products.
Examples include industrial and research applications, especially in fermentation, pharmaceutical and medical research applications, and food processing needs. In the typical practice of the invention, the native protein is purified and dissolved in a suitable buffer.

The solution is then mixed with a denaturing agent to partially denature the proteins dissolved therein. Partial denaturation of proteins typically involves adding inorganic salts to alter the iozo strength of the solution, altering the pH of the solution with inorganic or organic proton-donating acids, or
Alternatively, it may be carried out by modifying the solution by introducing a water-miscible organic solvent. The contact time between the denaturant and the protein can be from one minute to several days. In addition, the solution temperature can be determined from the above-mentioned documents,
It can be raised as a one-step modification of conditions that results in partial denaturation of the protein, as disclosed, for example, in the Privalov article. In some cases, the processed protein contains multiple disulfide bridges. In some cases, for example in the case of bovine serum albumin or urease, partial denaturation can be achieved by exposing the protein to mercapto-ethanol to break disulfide bonds within the protein. It is believed that partial denaturation of a protein results in a relaxation of the protein structure, so that the protein is able to accept and bind inhibitors that are later mixed with a solution containing the partially denatured protein. After partial denaturation, an inhibitor of the model enzyme is mixed and left in contact with the partially denatured enzyme at a temperature sufficient for a sufficient time to form a population of inhibitor-partially denatured enzyme complexes.

For example, if the native enzyme trypsin is to be converted to a modified enzyme that mimics the activity of the native enzyme chymotrypsin, the native enzyme trypsin is contacted with an inhibitor of chymotrypsin, such as indole or benzoic acid. This contacting can be carried out in an aqueous solution or in an aqueous solution containing an added amount of organic solvent sufficient to aid solubilization of the inhibitor. After contacting the inhibitor with the partially denatured enzyme, the new form of the modified enzyme becomes stable due to extensive cross-linking of the protein structure.

Typically such crosslinking is accomplished using a glutaraldehyde crosslinking agent. This is because this product is relatively inexpensive and easily available. However, any of the above crosslinking agents can be used successfully. After crosslinking the protein into a new structure to create the modifying enzyme, the inhibitor and excess crosslinker are removed from the newly formed modifying enzyme by any suitable method.

Liquid chromatography and extensive dialysis are suitable methods. Typically, the most active modifying enzyme is obtained by purifying the newly formed modifying enzyme by gel column chromatography and collecting the most active protein fraction from the eluent. For convenience of disclosure, only a portion of all patents and publications mentioned herein are presented.

The following examples illustrate the method of the invention. Example 1 Purification of Part A Enzyme Purified trypsin, twice crystallized, unsalted, and lyophilized, from bovine pancreas was purified using the procedure of Kostka and Carpenter (Kostka and Carpenter). Carpenter, 1 The Journal of Biological Chemistry (TheJO
umalOfBiOlOgicaIChemiStr'
No native chymotrypsin contaminants are detected when tested according to YJl 239, 6, 1799 (1964).

The first analysis of the specificity of trypsin substrates was performed by Walsh and Wilcox.
) as taught by the potentiometer PH stat method (K.A.
Walsh and P●E●Wilcox, 7MethodsinEnZ
ynlOgy). JS G●E●Bahlmann (G.E
．． Perlmann) and L.LOr
Edited by arKi), Academic Breath, 31-
41 (1970). Trypsin to PH3
．． Prepared in 0.001M HCI of O. absorbance at 28
Quantitated at 0r1m, absorbance 14 for 1% solution
．． 3 to create a concentration of Mg/ml. Four initial potentiometer PH-stat analyzes are performed to quantify the U/Mg activity for each substrate of native trypsin.

The esterase substrates used were: 1. Acetyltyrosine ethyl ester (ATEE) (a). 01M) 2 Benzoylarginine ethyl ester (BAEE) (0.01M) 3 Acetyltryptophan ethyl ester (AT
rEE) (4). 01M) 4 acetylphenylalanine ethyl ester (APEE) (0.01M)B
Add enough purified trypsin to the denatured A part of the enzyme at 25°C, pH 3, 0.0
When dissolved in 1100ml of 01MHC, 0 at 280nm.
．． Gives an absorbance of 98.

When trypsin is left for 3 minutes, partial denaturation occurs. Part C Addition of Inhibitor To 40 ml of the denatured enzyme solution of Part B, 30 rng of purified, dry indole powder is added and the mixture is gently shaken for 1 hour.

After 1 hour, the trypsin-chymotrypsin inhibitor complex is analyzed to ensure inhibition and thus binding of the inhibitor to the enzyme.

Crosslinking part D Add 100μ of 8% glutaraldehyde crosslinking agent to the solution of part C.
Add e.

The resulting solution is shaken at PH3, 0-5°C for 1 hour. 1
After an hour, the pH of the solution is raised to 5 by addition of 0.01M NaOH.

Part E Purification 5 ml of solution of Part D was added to 0.001M HCl1CaCI2
Sephadex (
Chromatographic analysis on a SePhadex™ G-10 gel filtration column.

Separation of indole and excess glutaraldehyde was carried out in 1
It is run in about 1 hour using a 2 x 1 inch column and an eluent flow rate of 60 ml/Hr. The protein peak is 254r
1 m and collected and analyzed as described below. Section F Results The following activity increases for substrates for chymotrypsin have been recorded from samples from section E of the chymotrypsin-like modified protein prepared according to the invention.

The results indicate that the chymotrypsin-like modified protein exhibits increased activity toward the chymotrypsin substrate (ATEB) and decreased activity toward the trypsin substrate (BAEE).

This example also shows that activity is substantially increased with respect to the substrate when using the method of the invention.

This example further demonstrates that when processed according to the present invention to produce chymotrypsin-like modified proteins, the activity of one species of peptidyl-peptide hydrolase, trypsin (with respect to native ester hydrolysis catalytic activity), is greater than that of the other. For the substrate of one peptidyl-peptide hydrolase, chymotrypsin (with respect to the activity of the native esterase type of chymotrypsin), it has been shown to increase. Example 2 Part A Purification of Enzyme Sigma Chemical CO. is type ■-A from
A salt-free, protease-free, pure form ribonuclease enzyme was purchased as bovine pancreatic ribonuclease.

Modification of Part B Enzyme When 60rr1g of purified ribonuclease from Part A is dissolved in 100ml of deionized distilled water, 0.3
It shows an absorbance of 9.

Add 30% of 0.2M mercaptoethanol denaturant to this solution.
Add 0 Pe. Gently stir at 25°C and heat to 0.
Raise the pH of the solution to 7 by adding 01M (7) NaOH dropwise and maintain at that pH for 2 hours.

Part C Addition of Inhibitor To 100 Tne of the solution from Part B add 40 rI 1 g of dry powder indole inhibitor.

Stir the solution at 25°C and add 0.01M NaOH 1-1.
After adding dropwise and keeping the pH at 7 for a period of time, all the indole will eventually dissolve. Cross-linking of part D The solution of part C at 25°C is raised to pH 9.45 while adding 0.1 Mf) NaOH dropwise, and stirred gently for 3 hours.

The solution is then cooled to O-5°C in a cold water bath. When the solution reaches 5°C, typically about 3 ml, 400 μe of 8% glutaraldehyde crosslinker is added and the solution is brought to 1'Nli! Gently shake for f. Part E Purification 5 ml of Part D solution is subjected to chromatographic analysis on a Sephadex G-15 column using 0.001M HCI eluent.

Separation of indole and excess glutaraldehyde 12×1
This is done using an inch column. Protein fraction 206nn
Collected through monitoring in step 1. Section F Results The following activity increases for substrates for esterases have been recorded from samples from Section E of the esterase-like modified proteins prepared according to the invention.

This result indicates that the esterase-like modified protein exhibits enzymatic activity with respect to esterase substrates for which no activity was detected in native ribonuclease.

This indicates that one genus of enzymes, nucleases, is changing to another genus of enzymes, esterases. Example 3 Purification of Part A Enzyme Purified trypsin, twice crystallized, unsalted and lyophilized, from bovine pancreas was purified by the procedure of Koszka and Carpenter (F. Koszka and F.H. Carpenter, 7.
Journal of Biological Chemistry (T
TleJOumaIOfBiOlOgicaIChem
istry Jl 239, 61799 (1964)), no native chymotrypsin contaminants were detected.

The first analysis of the specificity of trypsin substrates was based on the teachings of K. A. Walsh and P. E. Wilcox, J. Methods in Engineering.
Edited by Bahlmann and L. Ogy), Academics Breath, 31-41 (1
970)). Trypsin at pH 3. O
of 0.01M HCI. Absorbance at 280nm
The absorbance of 14.3 for a 1% solution is used to create a concentration of Mg/ml. 4 original potential variable P
H-stat analysis was performed to quantify the U/Mg activity for each substrate of native trypsin.

The substrates used were as follows. 1 Acetyltyronsin ethyl ester (ATEE) (4). 01M) 2 Benzoylarginine ethyl ester (BAEE)
(stomach). 01M) 3 Acetyl tryptophan ethyl ester (AT
rEE) (0.01M) 4 Acetylphenylalanine ethyl ester (APEE) (a). 01M)
Modification of Part B Enzyme Add enough purified trypsin of Part A to 0.0 at pH 3, 25°C.
When dissolved in 100 ml of 01MHC, it gives an absorbance of 1.4 at 280 m.

When this trypsin is left for 3 minutes, partial denaturation occurs. Part C Addition of Inhibitor To 40 ml of the denatured enzyme solution of Part B, add 2 ml of 1% indole solution (in 0.001 MHCI) and shake the solution gently for 2 hours.

After 2 hours, the trypsin-chymotrypsin inhibitor complex is analyzed to ensure inhibition and thus binding of the inhibitor to the enzyme.

Crosslinking part D Add 300μ of 8% glitaraldehyde crosslinking agent to the solution of part C.
Add l.

The resulting solution is shaken for 1 m at 0-5°C and PH3. 1′
IVI! After a period of f, the pH of the solution is increased to 5 by adding 0.01 M NaOH.

Part E Purification 5ml of the solution of Part D was mixed with 0.001MHCI, . CaCI2
Chromatographed on a Sephadex™ G-10 gel filtration column using 0.001M as eluent.

12 x 1 inch column and eluent flow rate 60ml/H
r. Separation of indole and superagent glutaraldehyde is carried out in about 1 hour. Protein peak is 254nn
1, collected and analyzed as described below. F
Section Results The following activity increases for substrates for chymotrypsin-like modified proteins have been recorded from samples from section E of chymotrypsin-like modified proteins prepared according to the present invention.

This result indicates that the chymotrypsin-like modified protein exhibits increased activity with respect to the chymotrypsin substrate (AT) (CE) and decreased activity with respect to the trypsin substrate (BAEE).

This example also shows that activity is substantially increased with respect to the substrate when using the method of the invention.

This example further demonstrates that the activity (with respect to native ester hydrolysis catalytic activity) of one species of peptidyl-peptide hydrolase, namely trypsin, when treated according to the present invention.
However, one other peptidyl-peptidide hydrolase,
That is, it has been shown to increase with respect to the substrate of chymotrypsin (with respect to the native esterase activity of chymotrypsin). Example 4 Purification of part A protein Sigma Chemical CO. Bovine serum albumin (BSA) was purchased in purified form as a crystalline, lyophilized protein containing 1-3% globulin from Rod A4.
Purified BSAlOOm9 from the denatured A part of the 378OB part enzyme was dissolved in deionized distilled water 10
When dissolved in 0ml, it exhibits an absorbance of 0.58 at 280nm.

The pH of the solution is maintained at PH3 for 2 hours with gentle stirring at 25°C by adding 0.01M HCI dropwise. Part C Addition of Inhibitor To 100 ml of solution from Part B add 40 ml of dry powder indole inhibitor.

Stir the solution at 25°C and add 0.01M HCI 1-1.
After dropping for 5 hours and keeping the pH at 3, all of the indole is finally dissolved. Crosslinking of part D 25°C (7) Raise the pH of the solution of part C to 7 while adding 0.1M NaOH dropwise, and stir gently for 3 hours.

The solution is then cooled to 0-5°C in a cold water bath. When the solution reaches 5°C, usually about 3 ml, 400 μe of 8% glutaraldehyde crosslinker is added and the solution is gently shaken for 17 hours. Part E Purification 5 ml of Part D solution is chromatographed on a column of Sephadex® G-15 gel using an eluent of 0.001 M HCI.

Separation of indole and excess glutaraldehyde was carried out 12×
Performed using a 1 inch column. Protein fraction, 206
Monitor and collect using r1m. Part F results in the increased activity of the sterase-like modified protein prepared according to the present invention on the substrate for esterase.
Recorded from samples from section E.

This result indicates that the esterase-like modified protein
This shows that the enzyme exhibits enzymatic activity with respect to the esterase substrate, with no activity detected in BSA.

This indicates that one genus of nonenzymatic proteins, albumin, is converted to another genus of proteins, enzymatically active esterases. Example 5 Purification of Part A Enzyme Purified twice-crystallized, salt-free, and lyophilized trypsin from bovine pancreas was purified using the procedure of Kostska and Carpenter (F.Kostska and F.H. Carpenter, 1996).

The Journal of Biological Chemistry (TlleJOuOnal″0f131010gica
1ChemistryJ1239, 6, 1799 (19
No native chymotrypsin contaminants were detected when tested by 64)). The original analysis of the specificity of trypsin substrates was based on the teachings of Walsh and Wilcox (K.A. Walsh and P.E. Wilcox, 1 Methods in Engineering).
Trypsin is PH
3. Prepared in 0.001M HCI of O. absorbance 2
Quantitated at 80 nrn and determined absorbance for 1% solution at 28
Absorbance 14.3 for 1% solution determined at 0r1m
to create a concentration of Mg/ml. Four initial potentiometer PH-stat assays are performed to quantify U/M9 activity for each substrate of native trypsin.

The esterase substrates used were: 1 Acetyltyronsine ethyl ester (ATEE) (
4). 01M) 2 Benzoylarginine ethyl ester (BAE
E) (4). 01M) 3 Acetyl tryptophan ethyl ester (AT
rEE) (0.01M)4 Acetylphenylalanine ethyl ester (,APEE) (0.01
M) Modification of Part B Enzyme Add enough purified trypsin of Part A to 0.0% at pH 3, 25°C.
When dissolved in 1100ml of 01MHC it gives an absorbance of 0.98 at 280r1m.

If this trypsin is applied for 3 minutes, it will partially denature. C
Addition of Part Inhibitor Add 2 ml of 1% indole (in 0.001 MHCI) to 40 ml of the denatured enzyme solution of Part B and shake the solution gently for 1 hour.

Crosslinking part D Add 100μ of 8% glutaraldehyde crosslinking agent to the solution of part C.
Add e.

The resulting solution was diluted with 2CB at 0-5℃ and PH3! Shake for f. After 2 hours, add 0.01M NaOH to adjust the pH of the solution.
Raise it to 5.

Part E Purification 5 mt of the solution of Part D was added to 0.001 MHCI1CaCIz
Chromatographed on a Sephadex® G-10 Gel P percolumn using 0.001M as eluent.

Separation of indole and excess glutaraldehyde was carried out in 12
×1 inch column and 60 m1/Hr. using an eluent flow rate of about 1 hour. Protein peak at 254nm
Detected, collected, and analyzed as described below. Part F Results The following activity increases for substrates for chymotrypsin have been recorded from samples from part E of the chymotrypsin-like modified protein prepared according to the invention.

The results indicate that the chymotrypsin-like modified protein exhibits increased activity with respect to the chymotrypsin substrate (ATEE) and decreased activity with respect to the trypsin substrate (BAEE).

This example also shows that activity is substantially increased with respect to the substrate when using the method of the invention.

This example further demonstrates that the activity (with respect to native ester hydrolysis catalytic activity) of one species of peptidyl-peptide hydrolase, namely trypsin, when treated according to the present invention.
has been shown to increase with respect to the substrate of another peptidyl-peptide hydrolase, namely chymotrypsin (with respect to the activity of the native esterase type of chymotrypsin). Example 6 Purification of Part A Enzyme Purified trypsin, twice crystallized, unsalted, and lyophilized, from bovine pancreas was purified using the procedure of Kostska and Carpenter (F.Kostska and F.H. Carpenter, "
The Journal of Biological Chemistry
Chemistry), 239, 61799 (196
4)) No native chymotrypsin contaminants were detected.

The original analysis of the specificity of tryptic substrates was based on the teachings of Walsh and Wilcox (K.A. Walsh and P.E. Wilcox, "Methods in Engineering").
1Ogy), edited by G●E●Bahlman and Elle Kuland, Academics●Bress, 31-41.
(1970)) by the potentiometer PH-stat method. Trypsin at pH 3. Adjust in 0.001 MHC1 of O. The absorbance is determined at 280rlm and an absorbance of 14.3 for a 1% solution is used to create a concentration of M9/ml. Four initial potentiometer PH-stat assays are performed to quantify U/M9 activity for each substrate of native trypsin.

The esterase substrates used were: 1 acetyl tyrosine ethyl ester (ATEE) (0
．． 01M) 2 Benzoylarginine ethyl ester (BAE
E) (a). 01M) 3 Acetyl tryptophan ethyl ester (AT
rEE) (a). 01M)4 Acetylphenylalanine ethyl ester (APEE) (a). 01
M) Modification of Part B Enzyme Add enough purified trypsin of Part A to 0.0% at pH 3, 25°C.
When dissolved in 1100ml of 01MHC, 280nTrL
gives an absorbance of 1.35.

When this trypsin is left for 3 minutes, partial denaturation occurs. Addition of Part C Inhibitor To 10 mL of the denatured enzyme solution of Part B, add 5 m of 1% benzoic acid in water.
1 and shake the solution gently for 1 hour.

After 1 hour, the trypsin-chymotrypsin complex is analyzed to ensure inhibition and thus binding of the inhibitor to the enzyme.

D part crosslinking 8% glutaraldehyde crosslinking agent 100P in solution of C part
Add e.

The resulting solution is shaken for 17 hours at 0-5°C and PH3. 1
After 7 hours, raise the pH of the solution to 5 by adding 0.01M NaOH.

Part E Purification 5 ml of solution of Part D was added to 0.001MHC11CaCI2
Chromatographed on a Sephadex® G-10 gel filtration column using 0.001M as eluent.

Separation of benzoic acid and excess glutaraldehyde was performed 12×
1 inch column and 60 m1/Hr. using an eluent flow rate of about 1 hour. Protein peak at 254nm
Detected, collected, and analyzed as described below. Part F Results The following activity increases for substrates for chymotrypsin have been recorded from samples from part E of the chymotrypsin-like modified protein prepared according to the invention.

- The results show that the chymotrypsin-like modified protein exhibits an increased activity with respect to the chymotrypsin substrate (ATEE) and a decreased activity with respect to the trypsin substrate (BAEE).

This example also shows that activity is substantially increased with respect to the substrate when using the method of the invention.

This example further demonstrates that the activity (with respect to native ester hydrolysis catalytic activity) of one species of peptidyl-peptide hydrolase, namely trypsin, when treated according to the present invention.
has been shown to increase with respect to the substrate of another peptidyl-peptide hydrolase, namely chymotrypsin (with respect to the activity of the native esterase type of chymotrypsin). Example 7 Purification of part A enzyme Sigma Chemical CO. is type ■-A from
The ribonuclease enzyme was purchased in purified form as salt-free, protease-free, bovine pancreatic ribonuclease.

Modification of Part B Enzyme When 60m9 of purified ribonuclease from Part A is dissolved in 100mL of deionized distilled water, 280nn1 is 0.411.
shows the absorbance of

0.01M HCl was added dropwise to lower the pH of the solution to 3.
Maintain the pH at 25°C for 2 hours with gentle stirring.

Part C Addition of Inhibitor To 100 mt of solution from Part B add dry, powdered indole inhibitor 40WL9.

The solution is stirred at 25° C., 0.01M HCl is added dropwise and kept at pH 3 for 1-1.5 hours, until all of the indole is dissolved. Crosslinking of Part D: While adding 0.1 M NaOH dropwise to the solution of Part C at 25°C, the pH thereof is raised to 7, and the solution is gently stirred for 3 hours.

The solution is then cooled to 0-5°C in a cold water bath. When the solution reaches 5°C, usually about 3 ml, 8% glutaraldehyde crosslinker 400 Pe is added and the solution is gently shaken for 3 CRs. Part E Purification The solution from Part D was purified for 2011 V against 0.01 M Tris buffer at pH 7 using SpectrapOre brand tubing with a molecular weight cut-off of approximately 3500. Dialyze at °C.

Section F Results The following activity increases for substrates for esterases have been recorded from samples from Section E of esterase-like modified proteins prepared according to the invention.

The buffer described in analysis procedure 2 was adjusted to an appropriate pH with 0.01 MHC1. This result indicates that the esterase-like modified protein exhibits enzymatic activity on esterase substrates for which no activity was detected in native ribonuclease.

This indicates that one genus of enzymes, nucleases, is converted into another genus of enzymes, esterases. Example 8 Purification of Part A Enzyme Purified trypsin, twice crystallized, unsalted, and lyophilized, from bovine pancreas was purified by the procedure of Kostska and Carpenter (F. Kostska and F. H. Carpenter, "
The Journal of Biological Chemistry (MleJOURrkllOfBlOlOgicalC
239, 61799 (1964
), no native chymotrypsin contaminants were detected.

The original analysis of tryptic substrate specificity was based on the teachings of Walsh and Wilcox (K.A. Waltsch and P.E. Wilcox, "Methods").
In Engimology (MethOdsinE former war 01
Edited by G●E●Bahlman and L●Kuland, Akademitsuku●Breath 31-41 (197
Edited by 0)), Academia Tsuku●Breath◆31-41
(1970)) by the potentiometric PH-stat method. Trypsin at pH 3. Prepared in 0.001M HCI of O. Absorbance was quantified at 280nm1 and 7F! using an absorbance of 14.3 for a 1% solution! Create a concentration of 9/Mt. Four initial potentiometer PH-stat analyzes were performed to determine the U/V for each substrate of native trypsin.
Quantify Tn9 activity.

The esterase substrates used were: 1 acetyl tyrosine ethyl ester (ATEE) (4
). 01M) 2benzoyl arginine ethyl ester (BAEE) (a). 01M) 3 Acetyl tryptophan ethyl ester (AT
rEE) (4). 01M)4 Acetylphenylalanine ethyl ester (APEE) (a). 01
M) Modification of Part B Enzyme Add enough purified trypsin of Part A to 0.0% at pH 3, 25°C.
When dissolved in 1100ml of 01MHC it gives an absorbance of 1.56 at 280r1m.

When this trypsin is left for 3C@, it partially denatures. C
Addition of Part Inhibitor Add 2 ml of pure phenyl acetate to 40 ml of the denatured enzyme solution of Part B and readjust the solution to pH 3 with dilute HCl.

The solution is then heated to 40° C. to dissolve all the phenyl acetate inhibitor into the solution and stirred for 2 hours. Cross-linking part D Add 600μ of 8% glutaraldehyde cross-linking agent to the solution of part C.
Add e.

The resulting solution is shaken for 2 ctf at O-5°C and PH3.
Part E Purification 5 ml of solution of Part D was added to 0.001MHC1, CaCI2
O. Chromatography is performed on a Sephadex trademark G-10 p-filter column using OOlM as eluent.

Separation of phenyl acetate and excess glutaraldehyde was performed in 12
×1 inch column and 60 m1/Hr. using an eluent flow rate of about 1 hour. protein peak at 254r
1rn, collected and analyzed as described below. Section F Results The following activity increases for substrates for chymotrypsin have been recorded from samples from section E of the chymotrypsin-like modified proteins prepared according to the invention.

The results indicate that the chymotrypsin-like modified protein exhibits increased activity with respect to the chymotrypsin substrate (ATEE) and decreased activity with respect to the trypsin substrate (BAEE).

This example also shows that activity is substantially increased with respect to the substrate when using the method of the invention.

This example further demonstrates that the activity (with respect to native ester hydrolysis catalytic activity) of one species of peptidyl-peptide hydrolase, namely trypsin, when treated according to the present invention.
has been shown to increase with respect to the substrate of another peptidyl-peptide hydrolase, namely chymotrypsin (with respect to the activity of the native esterase type of chymotrypsin). Example 9 Purification of part A enzyme Bacillus subtilis
Bacterial alpha amylase was purchased as a purified enzyme, type ■-A from Sigma Chemical Co., isolated from is), material of four crystallizations.

Purified bacterial alpha amylase 1 y in deionized distilled water 1 y
Dissolve 0-5 in C for 2 ml, pH 7 for 1 m
Dialyze against M phosphate buffer.

The preparation is then frozen until use. Modification of Part B Enzyme 10 ml of frozen 1% alpha-amylase from Part A is brought to room temperature and filtered through a 0.20 μm pore size filter.

The concentration was determined to be 0.67% after storage. Then, 6.5ml of this alpha amylase solution was added to 0.0ml of this alpha amylase solution.
Titrate with 0.1M NaOH to bring the pH to 10.7, and stir gently between 1α and 1α. Addition of Part C Inhibitor The solution of Part B is mixed with 0.017f of cellobiose inhibitor and stirred at 25°C for 4 minutes.

D part crosslinking The solution from C part at 25°C was mixed with glutaraldehyde crosslinking agent 1.
Mix with 0μ' and stir for 1 minute.

Upon addition of glutaraldehyde, the pH decreased to 9.9,
The solution turned from colorless to yellow. PH 0.01MHC1
Add dropwise to adjust to 9 and stir for an additional 15 minutes. Next, the pH was adjusted to 7 with 0.01MHC1 and stirred for another hour. Part E Purification 5 ml of the solution from Part D was passed through a Sephadex trademark G-10 gel filtration column 1.25 x 47 Cr! On l, flow velocity/m
l/Min. Chromatography using 0.01 MPH 7 Tris buffer.

Protein peaks are detected at 206 nm and collected. Section F Results The following increases in activity for the substrates for glycoside hydrolase have been recorded from samples from Section E of the glycoside hydrolase-like modified protein prepared according to the invention.

The substrates for the glycoside hydrolases used are: ρ-nitrophenyl-β-D-galactopyranoside (ρNPGA) and ρ-nitrophenyl-α-D monoglycoside (PNaGL). This result indicates that the glycoside hydrolase-like modified protein has no activity detected in native bacterial alpha-amylase, which is itself a species of glycoside hydrolase. It shows that it shows activity.

This indicates that one glycoside hydrolase is converted into another glycoside hydrolase. Example 1
Purification of 0A part enzyme Bacillus subtilis
Bacterial alpha-amylase was purchased as a purified enzyme of quadruple crystallized material, type ■-A from Sigma Chemical Co., isolated from is).

Purified bacterial alpha amylase 15/100y was dissolved in 15ml of deionized distilled water and heated to 0-5°C for 24JJ. Dialyze against 1 mM phosphate buffer, pH 7, for f. B
1% alpha-amylase solution from Part A denaturation of the enzyme 10T1Lt
brought to room temperature and centrifuged at 20,000 g.

The concentration was quantified and found to be 0.65%. Then, 10 ml of alpha amylase solution was added dropwise with 0.01M NaOH, and PHLO. Make it 6 and stir it slowly for 1 minute. Addition of Part C Inhibitor The solution of Part B is mixed with 0.051y of cellobiose inhibitor and stirred at 25°C for 4 minutes.

Part D Crosslinking At 25°C, mix the solution from Part C with 86μe of glutaraldehyde crosslinker.

Immediately thereafter, PHLO. O of NaCO3-NaHCO
A 0.1M solution of 3 is added until the PH is kept at 10 for 15 minutes. Approximately 0.7 ml of carbonate solution was added. E
1 ml of the solution from Part Purification Part D was added to Sephadex trademark G-10.
On gel filtration column 1.25 x 47C71, flow rate 0.3
4e/Min(7)0.01M,. Chromatography is performed using a pH 7 Tris buffer.

Protein peaks are detected at 254 nm and collected. Section F Results The following activity increases with respect to substrates for glycoside hydrolases have been recorded from samples from Section E of the glycoside hydrolase-like modified proteins prepared according to the invention.

The substrate for the glycoside hydrolase used is: ρ-nitrophenyl-β-D monoglycoside (PNβGL
) ρ-nitrophenyl-α-D monoglycoside (ρNαGL)
As a result, the glycoside hydrolase-like modified protein was found to be an enzyme with respect to the glycoside hydrolase substrate, for which no activity was detected in native bacterial alpha-amylase, which is itself a species of glycoside hydrolase. It shows that it shows activity.

This indicates that one glycoside hydrolase is converted into another glycoside hydrolase. Example 1
Purification of Part 1A enzyme Bacillus Subtilis
Bacterial alpha-amylase was purchased as a purified enzyme of quadruple crystallized material, type ■-A from Sigma Chemical CO., isolated from is).

Purified bacterial alpha-amylase 15/100' was dissolved in 15 ml of deionized distilled water and incubated at 0-5°C for 2 hours at pH 7.
Dialyze against 1rrL,M phosphate buffer. Modification of Part B Enzyme 10 ml of 1% alpha amylase from Part A is brought to room temperature and centrifuged at 20,000 g.

When the concentration was quantified, it was 0.57%. Then, 10ml of alpha amylase solution was added to 0.01N NaOH.
Titrate to pH 10.6 and stir slowly. Addition of Part C Inhibitor The solution of Part B is mixed with 0.051f of cellobiose inhibitor and stirred at 25°C for 4 minutes.

D part crosslinking The solution from C part at 25°C is mixed with glutaraldehyde crosslinking agent 7
Mix with 2μ'.

Shortly thereafter, PHLO. 0.1M NaC03-Na
Add HCO3 solution until the pH remains at 10.0. 4/10 ml of carbonate solution was used. Part E Purification Add solution 1TILI from Part D to Sephadex trademark G-10.
0.34 m1 on gel filtration column 1.25 x 47 cm
/Min. flow rate of 0.01M. ．． Chromatography is performed using Tris buffer at pH 7.

Protein peaks are detected with 254nn1 and collected. Section F Results The following activity increases with respect to substrates for glycoside hydrolases have been recorded from samples from Section E of the glycoside hydrolase-like modified proteins prepared according to the invention.

The substrates for the glycoside hydrolases used are as follows. ρ-nitrophenyl-β-D monoglycoside (PNβGL
) This result indicates that the glycoside hydrolase-like modified protein is a glycoside hydrolase substrate for which no activity was detected in native bacterial alpha-amylase, which is itself a species of glycoside hydrolase. It has been shown that it exhibits enzymatic activity.

This indicates that one glycoside hydrolase is converted into another glycoside hydrolase. Analytical Procedures The samples analyzed in Examples 1-8 can be analyzed in one of two ways.

Analysis method 1 measures proton release from reacting substrates. Analysis method 2 measures changes in the spectrum due to changes in electronic structure induced by hydrolysis of the substrate. In all cases of Examples 1-8 above, both methods yielded positive activity change measurements for the activity desired to model, and two unrelated assays previously It was confirmed that activity occurs when no activity exists at all. Example reagent for analysis method 1: 0.1M KCL 10.05MCa with pH 7.75
C12, 0.01M Tris buffer substrate: Alpha N
-benzoyl-L-arginine ethyl ester HCl
(BAEE) 343m9 is dissolved in 100 ml of buffer.

Procedure: Sargent-Welch
ch) Using the PH-Stat model PHR, fill the titration buret with 0.1M NaOH.

Add 5 ml of the substrate solution to the PH-stat beaker while stirring rapidly. Adjust the titration device to reach a pH of 7.
．． Raise it to 8. Set a fixed baseline. Enzyme solution 2m
Add 1. The recorder records the volume of base consumed per unit time as a direct measure of micromoles of substrate consumed per minute. Example reagent for analysis method 2: PH8. 0.1MKC1, 0.05MCaC of O
12, 0.5M Squirrel buffer substrate: Alpha N-
Bensoyl-L-arginine ethyl ester HCl
(BAEE) 34.3 is dissolved in 100 ml of buffer.

Procedure: Using a Beckman ACTA spectrometer, set the wavelength adjuster to 255 nm with a slit width of 1.25 nm.

Prepare zero buffer in controls and samples. Empty the sample chamber and rinse the cuvette with acetone and then water. Add 2.5 ml of BAEE substrate solution and 1 minute baseline.

Add 0.5 mL of solution enzyme and record the rate of increase in absorbance as BAEE is hydrolyzed to alpha-N-benzoyl-L-arginine. Plot absorbance versus time (delta A/min) for at least 5 minutes.
For delta extinction coefficient substrate-to-product at 808M-1 (1-1), 1 unit is equal to BAEE/micromole hydrolysis per minute at 25°C and pH 8.0.

G*Double Schubert and T*Takenake,
“BIOCHEMICA・BIOCHEMICA・BIOCHEMICA
aetBiOphisica), ACTAll6, 57
0, 1955”.

Example of Analysis Procedure 3 The sample analyzed in Example 9 can be analyzed by the following procedure.

Reagents: Sodium citrate buffer with pH 4.6.

0. ? of sodium carbonate.

Substrate: 25mM p-nitrophenyl-α-D monoglycoside in 0.05M sodium citrate buffer at pH 4.6
solution.

ρ-Nitrophenyl-α-D galacto pyranoside 25rnM solution in 0.05r11V4 sodium citrate buffer at pH 4.6.

Procedure: 1 of 25n111!4 solution of either substrate in 0.05mM sodium citrate buffer at pH 4.6
00μ' sample with the same buffer 350μ' at 30°C.
Incubate for 5 minutes.

Five such solutions were prepared.

Add 50 μe of enzyme to three of these solutions.
After adding 50 μe of citrate buffer to the remaining two test solutions, the solutions are incubated at 30°C.

In one sample, one enzyme-containing solution and one control are selected.

The reaction is stopped by adding 0.2M sodium carbonate 700Pe.

The absorbance is measured at 420r1m. In 3 cases, the other 1
Analyze one enzyme solution, and analyze the last enzyme solution and the remaining target in six samples.

1Methods●in●engineology (MethOdsi
nEnzymOlOgy), 2? , pp. 720-21.

Activity is calculated using an extinction coefficient of 1.83 x 10+4 M-Cm-1. “J●Biol●Chem (J
．． BiOl. Chem. ), 233s1113 (195
8)”.

Calculate the amount of enzyme present using the absorbance at 280r1m of 25.2 for the α-amylase 1% solution.

Example of Analytical Procedure 4 The sample analyzed in High Pressure Liquid Chromatography Analysis Example 9 is analyzed by the following procedure.

Reagent: Galactose, 0.1% solution.

Galactose, 1.0% solution.

ρ-nitrophenyl-β-D-galactopyranoside (ρNf3GL), 12 mM solution.

ρ-Nitrophenol, 0.5% solution. Glucosidase, 0.5% solution.

MicrO-Pak trademark
VarianAssOciat
es) Column 30x4cm0 absorbance detector 206rI
Set it to 1M.

Water eluent at 25°C.

At flow rate -2000PSi, 25rr1e/Mi
rlO procedure: A mixture of semisynthetic glycoside hydrolase and ρNPGL was incubated and then added to the column.

In specific time, the peak at the position set for galactose was recorded. Peak heights and concentrations were determined from standard galactose solutions. The activity of the semisynthetic enzyme was calculated to be 3 x 10-3 U/ml. Example of Analytical Procedure 5 The samples analyzed in Examples 10 and 11 are analyzed by the following procedure.

Reagent: PH5. O sodium citrate buffer 0.05
M00.2M sodium carbonate.

Substrate: ρ-nitrophenyl-β-D-glucopyranoside 25n1M solution in deionized distilled water.

Dissolved in deionized distilled water, ρ-nitrophenyl α
-D-glucopyranoside 25mM solution.

Procedure: pH5 sodium citrate buffer 700p'
Add aliquots of to 5 tubes (2 of which are controls).

). 100 Pe of semisynthetic enzyme is added to three of the above tubes, while 100 μe of sodium citrate at PH5 is added to the two controls.

These tubes are incubated in a shaker for 3 cycles. After this one-pour period, 200 Pe of the appropriate substrate is added to all five tubes and left to incubate in a shaker at 30C. After incubation of the powder, remove one control tube and one enzyme-containing tube. The solution in the control tube is immediately mixed with 1.4 me of 0.2 M sodium carbonate. Separate enzyme-containing tubes for 2 minutes. After a period of 2 minutes, pour the liquid in the tube into the marked tube.
and discard the precipitate.

Next, 500 μe of the above solution was pipetted into another marked tube, and 700 μe of the 0.2M sodium carbonate buffer was added.
The reaction is stopped by adding μe. absorbance at 420r1m
Measure with. At 30 minutes, remove one bottle of enzyme content and centrifuge for approximately 2 minutes.

The solution is then injected into the marked tube, and 500 μe of the solution is pipetted into another marked tube. In this tube, 0.2
The reaction is stopped by adding 700 μe of M sodium carbonate solution. At 4 doses, remove the last two tubes (one containing enzyme, the other control) and repeat the procedure described above for the 1 tube.

U using the extinction coefficient for ρ-nitrophenol at 420 nm, 1.38 x 10 M
/7F! Calculate 9.

Claims (1)

[Scope of Claims] 1. Selecting a model enzyme; Selecting a native protein to be modified to imitate the enzyme; a native protein is mixed with a denaturing agent; the resulting partially denatured native protein is mixed with a competitive inhibitor of the model enzyme; and between the reactive sites of the partially denatured native protein in the presence of the inhibitor of the model enzyme. A modification characterized in that the partially denatured native protein and the inhibitor are mixed with a cross-linking agent at a temperature sufficient to cross-link the partially denatured native protein by forming covalent bonds. Protein manufacturing method. 2. Partially denaturing the native protein by forming an aqueous solution of the native protein and holding the aqueous solution at a temperature sufficient for a sufficient time to partially denature the native protein. Manufacturing method. 3. The time is about 15 minutes to 24 hours, and the temperature is about 25°C to 60°C. The manufacturing method according to claim 2. 4. The manufacturing method according to claim 1, wherein the native protein is partially denatured by mixing the native protein with water to form an aqueous solution and mixing the resulting solution with a denaturing agent. 5. The manufacturing method according to claim 4, wherein the modifier is an inorganic acid. 6. The method of claim 4, wherein the modifier is an organic acid. 7. The method of claim 4, wherein the modifier is a water-miscible organic solvent. 8. The method of claim 4, wherein the modifier is an inorganic acid.