JPS58158183A - Production of semi-synthetic enzymes - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Proteins are biologically synthesized macromolecules that have various roles in biological systems. Enzymes are a variety of biologically active proteins, among others, that catalyze specific reactions. Currently, enzyme technology is used in many fields in industry and research, such as medical research, food processing and preservation, fermented beverage production, pharmaceutical production, and analytical determination of various metabolites and food constituents using analytical enzyme technology. , used in quantity.

Enzymes are highly specific in their biological activity and generally carry out individual reactions at much higher rates than corresponding reactions that 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 quantities. Therefore, their isolation, purification and utilization is limited to small scale operations σζ due to the expense and time required to isolate them in their normal form.

Some enzymes occur in relatively large quantities in nature, and are relatively easy to isolate, purify, and utilize. Unfortunately, due to the strict catalytic behavior of these enzymes, enzymes available in large quantities are only capable of catalyzing selected reactions.

Recently, much effort has been devoted to the synthesis of synthetic biological catalysts that exhibit enzymatic behavior similar to that exhibited by natural enzymes that are difficult or expensive to isolate.

Additionally, some attempts have been made to modify natural enzymes to change their enzymatic specificity so that they serve to access previously inaccessible reactions.

One technique known to achieve enzyme behavior that contacts specific, desired reactions is the synthesis of so-called enzyme model molecules. For example, a low molecular weight compound can be covalently attached to the active functional group of the active locus of the enzyme. Examples of such preparation methods are described in the following publications: Advances In Chemistry 5er
ies), R6F, Gould Publishing, American Chemical Society, Washington, D.
, C6,2/-g3 (/ri7/) and c, c, Tang: D, daguaria.

(Davalian); P, i□Ung (Hau
ng) and R, Pre et al.'s technique involves the use of synthetic polymer matrices that are modified along their backbones to provide functional groups that are indicative of the ability of a given enzyme to act as an active site. Examples of such techniques can be found in the following papers: 2G, WUTFF and 1. Schulz 7 (5chu1
．． za), J, wildebeest (Suh), 10M, black (
Klotz), Bioorganic % Otsu, / Otsuj (/977).

Another technique involves adding new chemical moieties to natural enzymes near the active seat of the enzyme in an attempt to cause such enzymes to react with different catalytic activity. An example of this is the conversion of the proteolytic enzyme papain into an oxidase-type enzyme by the covalent addition of funbin near all active sites of the native papain enzyme, as illustrated in the paper below. That is: H, L, Levine and E, T,
Kaiser, J, Amer, Che
m, Soc,, 100.7 Otsu 70 (/P7g) and T,
Otsuki: Y., Nakagawa and E.T., Kaiser, J.C.
,S,Chem,Comm1,//,'l-37CI'
? 7g). Other examples of such enzymatic modifications can be found in the following papers: M, E, Virso7 (W
i l son ) and G, M, white size (Wh
) % J+Amer, Chem,
Soc, 100.30 (797K).

Yet another attempt to alter the specificity of the enzyme is to immobilize the native enzyme in a cell matrix. For example, tridicine enzymes have been immobilized in polyacrylamide gels. Polyacrylamide glue will allow amino acid esters to diffuse through the glue matrix and react with the enzymes, but will not allow the diffusion of more sensitive proteins. In other words, the specificity of an enzyme is altered by eliminating the access of one of the substrate molecules to the enzyme. An example of such a change in specificity is found in Kirk-Othmer's Encyclopedia of Chemicals, 3rd edition, published by Willi and Hung, Inc.
/llt♂, CI'? IO).

It is also known that the sulfhydryl groups on this enzyme can be blocked by reacting with natural linone monooxidanase. When this particular enzyme, lysine monooxycanase, is treated in this way, it exhibits new catalytic activity towards amino acids, catalyzing an oxidative membrane amine reaction in place of its natural enzymatic decarboxylation reaction. Of course,
See the paper by T. Yamauchi; S. Yamamoto and O. Hayaishi in the above reporter/θ, 37!0-37!2 (/973). It has also been reported that the activity of a natural enzyme, especially tridicine, with respect to its natural substrate can be modified by reacting it with its natural inhibitor and then cross-linking the enzyme. Int, J, Peptide Re58
, j12/, 3--/1fc7973), H,
Beaven and W. B. Gratz 7-
(Gratzer).

Although these techniques are suitable for many applications, they generally result in modified natural enzymes or total synthetic enzyme homologs that are not highly catalytically active. Therefore, it is possible to chemically modify commercially available natural enzymes to perform certain desired chemical reactions that have hitherto not been commercially useful reactions catalyzed by natural enzymes, and which There is a need for a simple, efficient and economical method for producing active semisynthetic enzymes for such reactions in which novel reactions can be systematically predetermined. The method described in the above-mentioned documents simply subjects the enzyme to a set of conditions, and exposes the protein whose behavior is being attempted to be partially denatured to a denaturing agent, thereby partially changing the conformational structure of the protein. A semi-synthetic enzyme is achieved by unfolding. Next, this partially denatured protein is brought into contact with a model enzyme inhibitor. This protein is then cross-linked,
The new conformation defined by the inhibitor defines the shape of the semisynthetic enzyme. The inhibitor and excess crosslinker are then removed from the newly generated semisynthetic enzyme, yielding a functional homologue to the model enzyme. The semisynthetic 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 proteins can be modified from their native conformation to a semi-synthetic conformation by practicing the method of the present invention. This new conformational state defines the shape of the semisynthetic enzyme that exhibits catalytic activity.

As used herein, the term ``enzyme'' is specifically defined as a polypeptide that is produced from amino acids to yield a biological molecule.

The present invention modifies a protein from one conformation to a second conformation, thus producing new enzymatic activity for the selected protein, or limiting enzymes present in the naturally occurring protein. including methods of increasing activity to commercially useful levels.

The method involves subjecting a native protein, typically an enzyme, to conditions and/or reagents that partially denature, usually reversibly, the structure of the protein. Then, the model enzyme inhibitor is brought into contact with the partially denatured natural protein. Without wishing to be bound by any theory, it is believed that by partially denaturing the protein described above, the protein binds the inhibitor of the model enzyme according to this method and forms an active whole that is very similar to the active whole of the model enzyme. I believe that you will. To this end, the constraint of one drug preserves the new conformation containing at least one total seat capable of performing the catalytic function of the model enzyme and preserves its shape until the new conformation can be cross-linked. It is believed that it is established. Therefore, after contacting the inhibitor with the partially denatured protein, a crosslinking step chemically stabilizes the new conformation of the protein. After sniffing, a new semi-synthetic enzyme is prepared from the model protein.

As defined herein, "partial modification"
without causing irreversible and severe denaturation of proteins.
It means to change the conformation of a protein in a way that perturbs the shape or conformation of the protein. As generally accepted in the art, the
It is defined as the combination of secondary and tertiary structure of a protein that has biological activity in vivo. Partial denaturation of proteins is well known and discussed in detail in the following literature: Worth Publishers N Inc.
, ' ), N, Y, , A, L, Lehninger (Le
hnhnger), Book, Biochemistry, sg page: “Advances in Proteochemistry”
einChemistry), Volume 33, /Otsu7-/9
．． Page 2, entitled “Protein Stability”,
An article by C. Sanford titled “1 Protein Denaturation” in “Advances in Protein Chemistry”, Part 0, Vol. 21I, pp. 2-27, by L. Zolivalov ) °゛Motion in Proteins
) titled, F, RoN, Guard (Gurd)
et al.; BBA, Vol. 627, pp. 1.227-232,
0 inside. Zardetzky's paper; TlB5. / 5;' 72/72 1.27I
Page, in R. Huber's paper; and M.
o1ekulyarnaya Biologiya,
Volume 1, 1, g37-,! i>63 pages, Nakanori, S.
The paper by Markovich et al.

As used herein, the term "modifier" means
Refers to process conditions or reagents that result in partial denaturation of proteins. For example, partial denaturation of a protein can be achieved by immersing the protein in an aqueous solution at an elevated temperature, for example, within the range of 2 parts C. to 20 degrees Celsius. For most proteins, temperatures between 23°C and 60°C will perturb the structure of the protein, causing changes in the 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 temperature increases than those disclosed above. It will be decided that Partial denaturation of a protein can also be achieved by immersing the protein in an aqueous solution containing an inorganic salt, an inorganic or organic acid, or a water-miscible solvent.

Examples of suitable inorganic salts that serve to destabilize protein structures include: NaF, (NH4)2s04,
(CH3)4NC11 (CH3)4NBr1KCH3c
o01NH4c1, RbC1, Kct X NaC1-
, ' CsCl X LICI-, KBr, NaBr5
KNO3, MgCl2, NaNo3, CaCl2,
KSCN, Na5CN 1B a Cl2, Na
1 and Li10 Suitable inorganic acids include: hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid and similar strong proton-donating inorganic acids.

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

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

As used herein, the term "inhibitor" refers to a compound that has sufficient structural similarity to the natural substrate of a semi-synthetic protein to serve as a template for all activities of the semi-synthetic enzyme. Inhibitors, like substrates, are generally not degraded by enzymes. An example of the structural similarity between an enzyme inhibitor and the enzyme's natural substrate is that of glucose oxidase.

Glucose is the natural substrate for glucose oxidase, while D-glucal is an inhibitor for glucose oxidase. Glucose and D-glucal are structurally very similar.

As defined herein, the term cross-linking refers to the creation of inter- or intra-molecular 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-aminogu, o-nochloro-3-) riazine diazonium salt; N-hydroxysuccinamide; p-benzoyl azide and Crosslinking reagents 2F, wota, disclosed in the following documents:
, Methods Enzymol % //, HIR8
, C0H, W, edition, Academic Press, 15;) Otsu 7, Otsu/7; H, Fasold
et al., and KH, Keyes, Kirk-Othmer: Encyclopenia of Chemical Technology, 3ci ed, 10. J. Willie
& Nones, Inc., /≠了-/7.2゜Many natural enzymes can be modeled using this method.
ng) and will create their semi-synthetic analogues, such as hydrolases, redox enzymes and transferases. For example: Group 1 hydrolytic enzymes are proteolytic enzymes that hydrolyze proteins, such as Evazoguin, ficin, pezocin, tridicin, chymotrigicin, promelin, and keratinase; cargohydrase that hydrolyzes hydrocarbons, such as Evacellulase. , amylase, maltase, pectinase, chitinase: esterases that hydrolyze esters, e.g. -ases, cholinesterases, lecithinases, alkaline and phosphatases: include nucleases that hydrolyze nucleic acids, such as ribonucleases, deoxyribonucleases; and amidases that hydrolyze amines, such as arginase, Asi9 laginase, glutaminase, histidase and urease. The second group are redox enzymes that carry out oxidation or reduction reactions.

These include glucose oxidase, xanthine oxidase, catalase, peroxidase, lipoxidase and cytochrome reductase.

The third group includes transferases that transfer groups from one molecule to another. Examples of these are glutamine pyruvate transaminase, glutamine-oxalacetate transmethylase, transmethylase, phosphopyruvate transphosphorylase.

In normal Zolactis, the model or first protein,
Typically enzymes are chosen. Next, a second protein is selected to imitate the first protein and serve as a model, and a semisynthetic enzyme is produced. By practicing the present invention, the second protein can be tailored to a desired, different, semi-synthetic protein.

This provides great benefits in a wide range of clinical and industrial situations where the enzymes that are desired to be used are in short supply, very expensive, or difficult to purify.

Natural 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 natural enzymes, the former of which are not available elsewhere, and/or Alternatively, one can switch to a more expensive semi-synthetic enzyme.

There are many uses for such enzymatically modified products,
For example, there are many industrial and research applications, particularly in fermentation, pharmaceutical and medical research applications, and food processing needs.

In the conventional Zolactis of the present invention, the natural protein is purified and dissolved in an appropriate buffer. The solution is then mixed with a denaturing agent to partially denature the proteins dissolved therein. Partial denaturation of proteins is typically accomplished by changing the ionic strength of the solution by adding inorganic salts, by changing the ionic strength of the solution with inorganic or organic proton-donating acids, or by introducing water-miscible organic solvents. This is done by modifying the solution. The contact time between the denaturant and the protein can be from 75 minutes to several days.

Also, the solution temperature can be increased as a one-step condition modification that results in partial denaturation of the protein, as disclosed in the above-mentioned publications, such as the Prigualov paper. In some cases where the protein to be treated contains multiple disulfide bridges, for example in the case of bovine serum albumin or urease, exposing the protein to mercapto-ethanol can destroy the nosulfide bonds within the protein. Good partial modification can be achieved.

It is believed that partial denaturation of the 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 partially denaturing the protein, a model enzyme inhibitor is mixed,
The inhibitor is kept in contact with the partially denatured enzyme for a time and at a temperature sufficient to create a population of complexes of the partially denatured enzyme.

For example, when converting natural trypsin into a semisynthetic enzyme that models the activity of the natural enzyme chymotrypsin, the natural enzyme trypsin is contacted with an inhibitor of chymotrypsin, such as indole or benzoic acid. This contacting may occur 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 partially denatured enzyme with an inhibitor, the new form of the semisynthetic 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 cross-linking the protein into a new structure to create the semi-synthetic enzyme, the inhibitor and excess cross-linking agent are removed from the newly formed bovine synthase by any suitable method. Liquid chromatography and extensive dialysis are suitable methods. Typically, new bovine synthase is produced by glucolumn chromatography and the most active protein fraction from the eluent is collected to yield the most active semi-synthetic enzyme.

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.

Examples in the margin below / Part A Purification of the enzyme Purified trypsin, twice crystallized, unsalted, and lyophilized, from bovine pancreas was purified using the Kos tka and Carpenter procedure (V,:I).
Sutsuka and F, H, Karinter, The Tsuyaru Ono Gomeioro:,! Gopishi-+S,--Tori-, 237, Otsu, /7 Tata (/9 Otsu 4'),)
No natural chymotrinosin contaminants were detected. The first analysis of the specificity of trinosine substrates was performed using the potentiometer-stat method taught by Walsh and Wilcox.
K., A., Wolsey and P.

E. Wilcox, Methods of Enzymology
in5Hz zymology), G, E, no e-
Le Mans (Per 1mann) and Roland (
Edited by David Lorand, Academic Press, 3
7-7/C7970)). Trypsin is prepared in 0.00/MHCI of PI-13,0.

The absorbance is determined at 1f, 't-, 20 nm, and a concentration of ~/ml is made using the absorbance/'A, 3 for the solution.

Two initial potentiometer-stat analyzes are performed to quantify U/In9 activity for each substrate of native trypsin. The substrates used were: /, 7 cetyl tyrosine ethyl ester (ATEE) <o, ciM) 2, pensoyl arginine ethyl ester (BAEE)
(0,0 fM) 3. Acetyl tryptophan ethyl ester (ATr
EE) (0,0/M) Ku Acetylphenylalanine ethyl ester (APE)
E)<0°0 fM) Sufficient purified trypsin of denatured part A of part B enzyme was added to 0.0 ml of trypsin at 23°C, pH 3.
When dissolved in 00/M HCI/00 ml, -
Gives an absorbance of 0.7 g at 2f(1) nm. Trisian f: Partially denatures when left for 30 minutes.

Addition of Part 0 Inhibitor To 4tOml of the denatured enzyme solution of Part B, add 30rn9f of purified, dry indole powder and shake the mixture slowly for 7 hours. After 7 hours, the trypsin-chymotrigsin inhibitor complex is analyzed (inhibited) thus ensuring binding of the inhibitor to the enzyme.

0 parts crosslinking 700μ of glutaraldehyde crosslinking agent in 0 parts solution
Add l. Product solution f: PI-13, shaken at 0-3°C for 7 hours. After 7 hours, the solution is raised in the evening by the addition of o.i.M. NaOH.

0.00/M HCI 1C 0.00/M HCI 1C
Chromatographic analysis is performed on a Sep, hadex G-10 Glu filtration column using aC12θ, 00 fM as eluent. Separation of indole and excess glutaraldehyde was carried out using a column of /2×/2 parts and an eluent flow rate of approximately 7 ml/hr.
Do it in time. Protein peaks are detected at 123'4' nm and collected and analyzed as described below.

Part E Results The following increased activity on the substrate for chymotrypsin of the semi-formed chymotrinosin prepared according to the present invention
Recorded from samples from section E.

Substrate ATEE (U/In/RI BAEE (U/ml) Initial activity Left 2/!;!;, 3 Final activity analysis procedure/ and 37 30.2/Changes
+/Otsu0 -≠2 This result shows that semi-synthetic chymotrypsin 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 when processed according to the present invention to produce semi-synthetic chymotrypsin, the activity of one species of butynol-peptide hydrolase, i.e., IJ EXAMPLE 2 Purification of Part A Enzyme Type [1-A, unsalted, norotease from Sigma Chemical Co. No bovine pancreatic IJ g
Pure form glue as a nuclease? nuclease enzyme i
I bought it.

Modification of Part B Enzyme When the purified ribonuclease from Part A is dissolved in 100ml of deionized distilled water, it yields -21! It shows an absorbance of 0.37 at i'' Onm. Add 300 μl of 0.2M mercazotoethanol denaturant to this solution.
Raise the temperature to 7, add 0.0 fM NaOH dropwise, and maintain the temperature above - while stirring slowly at t°C.

Addition of Part 0 Inhibitor To 700 ml of solution from Part B add dry powder indole inhibitor a o my. Stir the solution t-jj℃, 0
．． 0/M NaOH was added dropwise for 7,3 hours.
If kept at 7, all the indole will eventually dissolve.

Part D Crosslinking 0 parts of the solution at 2I°C was added to 0. /M NaOH was added dropwise to raise the pH to Z4Lj and stirred slowly for 3 hours. The solution is then cooled to 0-SC in a cold water bath. When the solution reaches t°C, usually in about 30 minutes, add 00 μl of glutaraldehyde crosslinker and shake the solution slowly for 77 hours. Chromatographic analysis is carried out on a column of Itsephadex G-/3 for M HCI eluent. Separation of indole and excess glutaraldehyde is carried out using a /2x/2 part column. Protein fractions are collected by control at 20 nm.

Part F Results The following activity increases for substrates for esterases have been recorded from samples from part E of semisynthetic esterases prepared according to the invention.

Substrate BAEE (U/my) Initial Activity 0.00 Final Activity Assay Procedure 70.3 Assay Procedure 20. % N/A This result indicates that the semisynthetic esterase exhibits enzymatic activity with respect to esterase substrates for which no activity was detected in the native IJ & nuclease. This indicates that the seven genus of enzymes, nucleases, are transformed into another genus of enzymes, esterases.

Example 3 Purification of Part A Enzyme Purified twice-crystallized, salt-free and lyophilized trypsin from bovine pancreas was purified by Koskka and Carter.
in (V, Koska and F, H, Carventer, the.
Shiny Ossi Biorozocal Chemistry, 2
39, Otsu, /7ri9 (/9otsugu)), natural chymotrigisin contaminants were not detected. The first analysis of the specificity of trigsin substrates was based on the teachings of Walshy and Wilcox (K., A., Walsh and P.
E. Wilcox, Methods of Enzymology, edited by G. E.-''-Leman and Roland, Academic Press, 3/-1I-/(7970)).

Trypsin is prepared in 3.0 0.00/M HCI. The absorbance is quantified in 20 Onm and the absorbance for the relevant solution 11A3f: is used to create a concentration of my/ml.

Two initial potentiometer-one-stat analyzes were performed to quantify the U/my activity for each substrate of native trypsin. The substrates used were: /, acetyl tyrosine ethyl ester (ATEE)
(0,0IM), 2. Hair soil arginine ethyl ester (BA
EEX O,0/M > 3, acetyltryptophan ethyl ester (ATrE
E) (0,0/M) ≠ Acetylphenylalanine ethyl ester (APE
EXO,0/M) Enough purified trypsin of the denatured A part of the B part enzyme was added to PI-131,2, t°C.
Dissolving in 0.00 IM HCI/00 ml gives an absorbance of 1.2 f Onm/, ≠. When this trypsin is left for 30 minutes, it partially denatures.

Addition of 0 part inhibitor to part B of the denatured enzyme solution pQml (0,001M HCI
Add 2 ml of the indole solution and shake the solution slowly for an hour. After 2 hours, the complex of the IJ inhibitor is analyzed to ensure inhibition and thus binding of the inhibitor to the enzyme.

Crosslinking part D: Add 300 μg of glutaraldehyde crosslinking agent to 0 parts of crosslinking solution.
Add l. The resulting solution is shaken at O-S°C, PH3 for 77 hours. After 77 hours, bring the solution to -j by adding 0.0/M NaOH.

E part purification part solution jml 0.00/M HCI XCa
Chromatography is performed on a Sephadex G-10R filtration column using Cl 20.007M as eluent.

/, 2X/inch column and eluent flow rate Oml/
Separation of indole and excess glutaraldehyde is carried out in about 7 hours using hr. The protein peak is 2j
Detected at 4'nm, collected and analyzed as described below.

Results The following increases in activity for the substrate for IJ psin have been recorded from 6 samples from part E of the semi-synthetic chymotrypsin prepared according to the invention.

Substrate ATEE (U/mJ) BAEE (U/i/) Initial Activity 3.2 32.0 Final Activity Analysis Procedure / /, 2. I3; lI-yo7j
Change factor 10≠0/-/4' This result shows that semi-synthetic chymotrypsin is a chymotrypsin substrate (ATEE).
) shows an increase in activity with respect to the tridicine substrate (BAEE), and a decrease in activity with respect to the tridicine 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 shows that when treated according to the present invention, the activity of one species of penotinolupegutide hydrolase, namely tridicine, is
For another vegutinorhenotide hydrolase, a substrate for chymotrypsin, it has been shown to increase.

Example ≠ Part A Bovine serum albumin 4 (BSA) in purified form as a crystalline, lyophilized protein containing /-3% globulin was purchased from Sigma Chemical Co., Lot A.
≠37g.

Denaturation of Part B Enzyme Purified BSA/00 m9 from Part A is dissolved in 100'ml of deionized distilled water to give 1.2% and 0.6 at θr1m.
It shows the absorbance of g. 0.0j MHC1 was added dropwise to the solution, and the pH was maintained at 2j℃ for 2 hours with slow stirring.
Keep it at 3.

Addition of 0 part inhibitor Add dry powder I:/Dole inhibitor 1ltOrn9 to 00 ml of solution from part B. The solution is stirred at 2j°C and 0.0/M HCl is added dropwise for /-/j hours and maintained at P)I3 until all of the indole is dissolved.

D part crosslinking, 0 parts of solution at 2j°C was mixed with 0. / M NaOH was added dropwise at 17, and the - level was increased to 7, and the mixture was stirred slowly for 3 hours. The solution is then cooled to O-S°C in a cold water bath.

When the solution reaches t°C, usually in about 30 minutes, add 4t00 till t of glutaraldehyde crosslinker of J'[
The solution is gently shaken for 17 hours.

Part E is subjected to purification chromatography. Separation of indole and excess glutaraldehyde is performed using a 2X/inch column. Protein fractions are collected under control at 20 nm.

Part F Results The following increases in activity for the substrates of the esterase were recorded from samples from part E of the semi-synthetic esterase prepared according to the invention.

Substrate BAEE (u1m&) Initial activity 0.00 Final activity analyzer Jlliii/ 0.0 Otsu analysis procedure, 2
0.022 Variation Chi N/A This result indicates that the semi-synthetic esterase exhibits enzymatic activity with respect to the esterase substrate, with no activity detected in natural BSA. This means that albumin, which is one of the seven genera of non-enzymatic proteins, is one other genus of proteins.
This indicates that it is converted to an enzymatically active esterase.

Example S Part A Purified trypsin, twice crystallized, unsalted, and lyophilized, from bovine pancreas was purified using the Kostka and Carpenter procedure (v1 Kostka and F.H. Carpenter, The Journal of・Nokiorosocal Chemistry, λ3
No natural chymotrypsin contaminants were detected when tested using RI, Otsu, /799 (/RIOtsu≠)). The original analysis of the specificity of trinonone substrates was based on the teachings of Walsh and Wilcox (K., A., Walsh and P.
, E. Wilcox, Methods of Enzymology, G.E. /?
- Le Mans, edited by Rolando, Academic
Potentiometer PH by press, 37-≠/(/970))
It is done by the stat method. Trypsin was added at pH 3.0. Prepare in OO/MHCI. Absorbance at 210nm
and use absorbance/4t, 3 for a 7% solution to create a concentration of ~/ml.

Perform t initial potential variable PH stat analysis,
The U/mg activity for each substrate of native trypsin is quantified. The substrates used were as follows: Acetyl tyrosine ethyl ester (ATEE) (0.0/M) Yu Pensoyl arginine ethyl ester (BAEE)
(0.0/M) 3. Acetyl trithophan ethyl ester (AT
rEE) (0.0/M) ≠ Acetylphenylalanine ethyl ester (APE
EX o. 0/M ) PHJ sufficient purified trypsin of the denatured A part of the B part enzyme, 2! ;℃ 0,
When dissolved in 00/M HCI/00 ml,
Gives an absorbance of 0,5;'f at 2fOnm. If this trypsin is allowed to stand for 30 minutes, it will partially denature.

Addition of 0 part inhibitor Part B of denatured enzyme solution 41 to Qrnl (0.0)0
2 ml of )/% indole in M HCI are added and the solution is shaken slowly for 7 hours.

Add 700μ of horse chestnut glutaraldehyde crosslinking agent to a solution of 0 parts of D-part crosslinking.
Add j. The resulting solution is shaken at O-SC, P) (3) for 20 hours. After 20 hours, 0.0/M NaOH is added to bring the solution to -5.

0.00/M HCI
, C aC 1 20,007M as eluent on a Sephadex® G-10 gel permeation column. Separation of indole and excess glutaraldehyde was carried out using a /2×/2/h column and a 10 m
l/hr. using an eluent flow rate of about 7 hours.

Protein peak. 2 J” detected at 41 nm, collected and analyzed as described below.

Part F Results The following increased activity on the substrate for chymotrypsin of semi-synthetic chymotryzosin prepared according to the present invention
Recorded from samples from section E.

Initial activity of substrate 3. 32. O final activity analysis procedure / l1lt! ; Female τ change section
+, 2≠ -g This result shows that semi-synthetic chymotrypsin exhibits increased activity with respect to the chymotrypsin substrate (ATEE) and decreased activity with respect to the trypsin substrate (BAEE).

The examples also demonstrate that activity is substantially increased with respect to the substrate when using the method of the invention. This example further shows that when treated according to the present invention, the activity of one species of 4-zotinol-zotide hydrolase, namely trypsin, is
For another peptinolupephrotide hydrolase, a substrate for chymotrypsin, it has been shown to increase.

Example B Part A Purification of Enzyme Purified trypsin, twice crystallized, unsalted, and lyophilized, from bovine pancreas was purified by the Koszka and Carpenter procedure (
V. Koszka and F.H. Carpenter, The Universal Op Biological Chemistry, 23.
When tested by Otsu, /7ri9 (/riOtsu4')),
No natural chymotrypsin contaminants were detected. The original analysis of the specificity of trypsin substrates was based on the teachings of Walsh and Wilcox (K., A., Walsh and P.
It is carried out by the potentiometric-one-stat method according to E. Wilcox, Methods of Enzymology, edited by Lemans and Roland, Academic Press, 3/-11, / (/70)). Trypsin at pH 3.0
．． Prepared in 00/MHC1. The absorbance was determined at 2 gOnm, and the absorbance at /443 for the /di solution was used to determine m
Make a concentration of 9/ml.

Two initial potentiometer-stat analyzes are performed to quantify U 1m9 activity for each substrate of native trypsin. The substrates used were as follows.

/, acetyl tyrosine ethyl ester (ATEEX
O,0/M) ノ, pensoyl arginine ethyl ester (BAEE)
(0,0/M) 3, acetyl) IJ near') fan ethyl ester (ATrEE) (0,0/M) acetylphenylalanine ethyl ester (APEE)
) (0,0/M) Part B Add enough purified trypsin from Part A to 0.0 at pH 3,2/°C.
When dissolved in 0/M HCI/00 ml, 2I
! For i>Onm/, gives an absorbance of 3j. When this trypsin is left for 30 minutes, it partially denatures.

Part C Part B denatured enzyme solution/Qml in water/3 ml of benzoic acid
1 and shake the solution gently for 7 hours. After 7 hours, the tricytosine-chymotrypsin complex is analyzed to ensure inhibition and thus binding of the inhibitor to the enzyme.

Cross-linking of part D Add 100 μl of glutaraldehyde cross-linking agent to the solution of part C.
Add g. The resulting solution is shaken at os° C. and PH3 for 77 hours. After 77 hours, the pH of the solution is raised in the evening by adding 0.0/M NaOH.

3 ml of the solution from the E section purification section was mixed with 0-00/M HCl 1C
Chromatography is performed on a Sephadex G-10 Glu filtration column using aCl 20.00/M as eluent. Separation of benzoic acid and excess glutaraldehyde was carried out using a column of /2×/2/h and Qml/h.
r.

using an eluent flow rate of about 7 hours. 2 protein peaks! ; detected at 4Lnm, collected and analyzed as described below.

Part F Results The following increased activity for the substrate of chymotrinosin was observed in the semi-synthetic chymotrinosin prepared according to the present invention.
Recorded from samples from section E.

Substrate ATrEE (U/mA) Initial activity /3 Final activity analysis procedure / O, 3j Change +3 Za2 This result shows that semi-synthetic chymotrigisin increases the activity with respect to the chymotrigin substrate (ATEE), and increases the activity with respect to the tricyan substrate (BAEE). It has been shown that the activity decreases with respect to

This example also shows that activity is substantially increased with respect to the substrate when using the method of the invention. This example further shows that when treated according to the present invention, the activity of one species of penotinolupenotide hydrolase, namely trypun, is
For one other penotino-hesotide hydrolase, a substrate for chymotrigisin, it has been shown to increase.

Example 7 Purification of Part A Enzyme A purified form of glue bonuclease enzyme, Tights It-A, salt-free, protease-free, bovine pancreatic glue bonuclease from Sigma Chemical Co., was purchased.

Modification of Part B Enzyme When purified lysonuclease O■ from Part A is dissolved in 100 nll of deionized distilled water, ? 0.11 in I Onm
The absorbance at t// is shown. Add 0.0j MHC1 dropwise,
The pH of the solution was lowered to 3 and kept there for 2 hours with slow stirring at Kota°C.

Addition of Part 0 Inhibitor To the solution ioomt from Part B, add dry, powdered indole inhibitor lI-om9. The solution was stirred at jJ-°C and 0.0/M HCI was added dropwise at /-/ for j hours.
If kept at a constant temperature, all of the indole will eventually dissolve.

D part crosslinking, 0 parts of solution at 2j°C was mixed with 0. / M Raise the - to 7 while adding NaOH dropwise and stir slowly for 3 hours. The solution is then cooled to O-S°C in a cold water bath. When the solution reaches 5° C., usually in about 30 minutes, g% glutaraldehyde crosslinker 00μl is added and the solution is shaken slowly for 30 hours.

E part The solution from the purification part is about 3! Dialyze against 0.0 jM Tris buffer, pH 7, for 20 hours at O-S°C using Spectrapore brand tubing with a molecular weight cutoff of ;00.

Part F Results The following increases in activity for the substrates of the esterase were recorded from samples from part E of the semi-synthetic esterase prepared according to the invention.

The buffer described in analysis procedure 2 was adjusted to an appropriate concentration of 0.0jMHC1.

Substrate BAEE (for U) Initial activity 0.00 Final activity analysis procedure 2P) 'Iri, ogri PH1, /j pH 7,, 2ri PH Otsu, 7ri pH 3;, gu/ Change N/A This result show that semisynthetic esterases exhibit enzymatic activity with respect to esterase substrates for which no activity was detected in the native IJ yf nuclease. This indicates that one genus of enzymes, nucleases, is converted into another genus of enzymes, esterases.

EXAMPLES Part A Purification of the Enzyme Purified trypsin, twice crystallized, unsalted, and lyophilized, from bovine pancreas was prepared using the procedure of Kostska and Carventer (V. Kostka and F.H., Carventer, The. No natural chymotrypsin contaminants are detected when tested by the Journal of Pyronochal Chemistry, 237, O, /79 (/L? Otsull).The original analysis of tryptic substrate specificity was carried out by Walsh and Will. Cox's teachings (K.

A. Walsh and P. E. Wilcox, Methods of Enzymology, edited by G. E. Gullman and R. Roland, Academic Press, 3/-! / (/ri7
It is carried out by the potentiometer PH-stat method using 0. Trypsin is prepared in 0.001M HCl at pH 3.0. The absorbance is determined at 210 nm and the absorbance of /4t, 3 for a 7% solution is used to create a concentration of my/ml.

≠ One initial potentiometer...Stat analysis is performed to quantify 01m9 activity for each substrate of native trypsin. The substrates used were: /, acetyl tyrosine ethyl ester (ATEE) (0,07M) λ, benzoyl arginine ethyl ester (BAEE)
) (0,0/M) 3. Acetyl tryptophan ethyl ester (ATr
EE) C0,07M) ≠ Acetylphenylalanine ethyl ester (APE)
E) (0,0/M) Enough purified trypsin of the denatured A part of the B part enzyme was added to PI-13,2/0 °C.
．． When dissolved in 00/M HCI/00ml, 2
At 10 nm/, it gives an absorbance of j. When this trypsin is left for 30 minutes, it partially denatures.

Addition of 0 part inhibitor Part B denatured enzyme solution ≠ Q 2 ml pure phenyl acetate
3 and the solution is readjusted to pi-I3 with dilute HCI.

The solution is then heated to ≠θ°C to dissolve all the phenyl acetate inhibitor into the solution and stirred for 2 hours.

■ g% glutaraldehyde crosslinking agent OOOμ in 0 parts crosslinking solution
Add t. The resulting solution was expressed at 0-j'c, PH3 for 20 hours.

3 ml of the solution from the E section purification section was mixed with 0.00/M HCl 1
Chromatography on a Sephadex brand G-10 filtration column using CaCl 20.00/M as eluent. Separation of phenyl acetate and excess glutaraldehyde was carried out using a /2×/2/ch column and OtsuQ me
/hr.

using an eluent flow rate of about 7 hours. The protein t' peak is detected at 234'nm, collected and analyzed as described below.

(Results in Part F) The following increase in activity for the substrate of IJ desine was observed in the semi-synthetic chymotrypsin prepared according to the present invention.
Recorded from samples from section E.

Initial activity of substrate 3.2 j≠ Final activity analysis procedure / Otsu, 23 3.2. Otsu change chi
+/rij −,22 This result indicates that semi-synthetic chymotrypsin is a chymotrypsin substrate (ATEE).
increased activity for and trypsin substrate (BAEE)
It has been shown that the activity decreases with respect to

This example also shows that activity is substantially increased with respect to the substrate when using the method of the invention. This example further shows that when treated according to the present invention, the activity of one species of vedetinol-detide hydrolase, namely trypsin, is
For one other peptidyl-peptide hydrolase, the substrate of chymotrypsin, it has been shown to increase.

EXAMPLE Purification of N-part Enzyme Bacterial alpha amylase, isolated from Bacillus subtilis, was purchased as a purified enzyme, type II-A from Sigma Chemical CO., material for ≠ crystallization.

Purified bacterial alpha amylase/g in deionized distilled water 1
00ml, at O-S°C, 2/h, pH 7/
Dialyze against mM IJ phosphate buffer.

The preparation is then frozen until use.

Modification of Part B Enzyme Frozen 10 ml of Thialpha amylase from Part A are brought to room temperature and filtered through a 0.20 μm pore size filter. When the concentration was determined, it was 0.67 after storage.
It is Chi. Next, this alpha amylase solution.

Titrate jml with 0.0/M NaOH to pH 7.
0.7 and stir slowly for 70 minutes.

Addition of Inhibitor The solution of Part B is mixed with 0.0/7 g of cellobiose inhibitor and stirred for ≠j minutes at 2/°C.

Part D Crosslinking, 2/°C solution from 6 parts is mixed with 10 μt of glutaraldehyde crosslinker and stirred for 75 minutes. Upon addition of daltaraldehyde, the - value decreased to 2 and the solution turned from colorless to yellow. - Adjust by adding 0.07M HCl dropwise and stir for another 5 minutes.

Then, the pH was slowly adjusted to 7 with 0.0/MHCl and stirred for an additional 7 hours.

ml of the solution from the E section purification section was passed through a SEFRYODEX trademark G-10 filtration column/. Chromatography is carried out at 177 cm at a flow rate of 0, θ/M, PI (7 cm) at a flow rate/mV min-. The protein peak is detected at 20 mV and collected.

Part F Results The following activity increases for the substrates for glycoside hydrolases have been recorded from samples from Part E of the semisynthetic glycoside hydrolases prepared according to the invention. The substrates for the glycoside hydrolases used are: ρnitrophenyl-β-D- is lactopyranoside (ρ
NβGA) and ρ-nitrophenyl-α-D-glycoside (ρNαGL) substrates ρNβGA (U/ml) ρNαGL (U/m/l') Initial activity o. oo o. o.

Final activity analysis procedure 3/. f×10-3/. ,! ;×10-'
Analytical procedure ≠ 3 × 10 = This result indicates that the semi-synthetic glycoside hydrolase is itself a glycoside hydrolase whose activity was not detected in natural bacterial alpha amylase, which is a species of glycoside hydrolase. It has been shown to exhibit enzymatic activity with respect to body hydrolase substrates. This indicates that one glycoside hydrolase is converted into another glycoside hydrolase.

Example 10 Purification of part A enzyme isolated from Bacillus subtilis produced by Sigma Chemical Co. Bacterial allele-amylase was purchased as a purified enzyme of type II-A from ≠ times crystallized material.

Purified bacterial alpha-amylase/v10011 was dissolved in deionized distilled water/ml and incubated for 2/hr at 0°C.
Dialyze against H7/mM phosphate buffer.

Denaturation of Part B Enzyme 10 ml of thialpha amylase solution from Part A was brought to room temperature, 2 o. With the gravity of ooo. Centrifuge for 20 minutes. Quantify the concentration and find that it is 0.0%. Then, add the alpha amylase solution/Qml to 0.000%.
Titrated with 0/M NaOH and PHio. to t,
Stir slowly for 10 minutes.

Addition of Part 6 Inhibitor The solution of Part B is mixed with the cellobiose inhibitor o, osig and stirred at 25° C. for ≠ evening.

D part crosslinking 2! ℃, mix the solution from 6 parts with the glutaraldehyde crosslinker and the μt. Immediately thereafter, N with a pH of 10.0
a 0.1M solution of C03-Na HCO5, - is 7
Add until held at 10 for 5 minutes. carbonate solution 9
．． 7 mli was added.

The solution/ml from the E section purification section was added to Sephadex trademark α-1.
0 glu offshore column/, on 23x4t7cm, flow rate 0,
Chromatography using 34' mVmin 0.0/M, PH7, Lith buffer. Protein peaks are detected at 251 A nm and collected.

The following activity increases with respect to the substrates of the F-part glycoside hydrolase have been recorded from samples from the E-part of the semisynthetic glycoside hydrolase prepared according to the invention. The substrates for the glycoside hydrolase used are: ρ-nitrophenyl-β-monoglycoside (ρNβGL) and ρ-nitrophenyl,-α-monoglycoside (ρNctGL).
) Substrate ρNβGL (U/ff19) ρNαGL (goods ζ) Initial activity o, oo o, o.

Final activity analysis procedure! ; 3. /×/θ−' /, lI−×
10-' As a result, the semi-synthetic glycoside hydrolase itself has no activity detected in natural bacterial alpha amylase, which is a type of glycoside hydrolase. This indicates that the enzyme exhibits enzymatic activity regarding the enzyme substrate. This indicates that one glycoside hydrolase is converted into another glycoside hydrolase.

EXAMPLE // Purification of Part A Enzyme Bacterial alpha amylase isolated from Bacillus subtilis, type II-A from Sigma Chemical Co., was purchased as a purification enzyme of group crystallized material.

Purified bacterial alpha amylase/! /100 g in deionized distilled water/ml and at O-S°C 2 parts for 2 hours P
) (7/'mM'J phosphate buffer).

Denaturation of Part B Enzyme 101 nl of amylase from Part A are brought to room temperature and centrifuged at 20,000 gravity for 20 minutes. When the concentration was quantified, it was found to be 0. j 7%. Then, 10 ml of alpha amylase solution was added to 0.0
/N Titrated with NaOH to 10. Stir slowly for 70 minutes.

Addition of Inhibitor Part B solution is mixed with cellobiose inhibitor 0.0/9 and 2! Stir for 4 minutes at °C.

Part D crosslinking, mix the solution from 6 parts of 25u with 72 μl of glutaraldehyde crosslinker. Immediately after pH/0.0,
/M NaC0-NaHCO3 solution is added until - is kept at 10 ρ for 75 min. carbonate solution'l/10
ml was used.

Sephadex trademark α-1 solution/rul from E part purification part
0dal filtration column/, m? 0.3 on jX4'7tM
Chromatograph using 0007M, pH 7 Tris buffer at a flow rate of 11-mV min. 2 protein peaks! ; Detect at II nm and collect.

Part F Results The following increases in activity with respect to the substrates of the glycoside hydrolases have been recorded from samples from part E of the semi-synthetic glycoside hydrolases prepared according to the invention. The substrates of the glycoside hydrolase used are as follows: ρ-nitrophenyl-β-D-glycoside (ρNβGL) substrate ρNβGL (U/m9) Initial activity 0.00 Final activity analysis procedure j 2 ．． I×70-' This result indicates that the semisynthetic glycoside hydrolase itself is a type of glycoside hydrolase, and no activity was detected in natural bacterial alpha-amylase. This indicates that the enzyme exhibits enzymatic activity regarding the degrading enzyme substrate. This indicates that one glycoside hydrolase is converted into another glycoside hydrolase.

The sample analyzed in Analytical Procedure Perform/-g can be analyzed by one of two methods. The analytical method/measures proton release from reacting substrates. Analysis method 2 measures the change in sheath due to the change in electronic structure induced by hydrolysis of the substrate.

In all cases of Example/-g above, either method yields a positive activity change measurement with respect to the activity desired to model, and two, unrelated assays indicate that the previously It was confirmed that activity occurs in the absence of any.

Reagent: pj-17,73; O,/MKCI, 0.03
; M CaCl2.0, 0/M Tris Buffer Substrate: Alpha N-benzoyl-thi-arginine ethyl ester HCI (BAEE) 34t3 m9 is dissolved in 100 ml of buffer.

Procedure: Sargent-Welch
lch) PH-stat model pHRf: using the titration vinylet at 0. /M Fill with NaOH. Substrate Solution 3; Place the ml into a PH-stat beaker with rapid stirring. Adjust the titrator to increase - to 7 g. Set a fixed base line. Add 2 ml of enzyme solution.

The recorder records the volume of base consumed per unit time as a direct measure of micromoles of substrate consumed per minute.

Analysis method! Example reagent: pHf, O of Q, /MKC110, Oj
M Ca Cl 2.0.3 M Tris Buffer Substrate: Flufur N-benzoyl-mono-arginine ethyl ester HCl (BAEE) 34t, 3rn9 is dissolved in 100 ml of buffer.

Procedure: Using a Beckman ACTA spectrometer, adjust the wavelength adjuster with a slit width of /, 23 nm at 2! ;3;n
Set to m. Prepare zero buffer in controls and samples. Empty the sample chamber and wash with Cubela (Kubera) acetone and then water. .: Add 3 ml of BAEE substrate solution and/needle base line. Add 0.5 ml of solution enzyme and record the rate of increase in absorbance as BAEE is hydrolyzed to alpha-N-benzoyl-di-arginine. Plot absorbance versus time (delta A/min) for at least 5 minutes. For a delta extinction coefficient substrate-product in I O + S'M-''cm-', one unit is the fractionation si BAEE /
Equivalent to micromolar hydrolysis.

G, W, Schwert and T, Takenake, Biochemistry.
Biochemica et Bi
ophisica), ACTA, /Otsu, j70, /9!
;! reference.

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

Reagent: pHIA Otsu sodium citrate buffer.

0,. 2M sodium carbonate.

Substrate: pop, Otsu's 0. ρ-nitrophenyl-α-D-glycoside 23 mM bath solution PHI/l in Oj M sodium citrate buffer, 0. ρ-nitrophenyl ~ α-D-galactopyranoside 2 in Oj M sodium citrate buffer! rmM solution.

Procedure: PI (4t, 0.0!; 700μl samples of a 26mM bath solution of both substrates in mM sodium citrate soaking solution with 3JOμg of the same buffer at 30°C for 5 minutes. A solution was prepared.

After adding jOμl of enzyme to three of these solutions and jOμl of citrate buffer to the remaining two control solutions, the solutions are placed in a greenhouse at 30°C. At 75 minutes, one enzyme-containing solution and one control are selected. The reaction is stopped by adding 0.2M sodium carbonate 7θOμl. The absorbance is measured at ≠ 20 nm. At 30 minutes, one other enzyme solution is analyzed, and at 60 minutes, the last enzyme solution and the remaining controls are analyzed.

See Methods of Enzymology, Vol. 2g, pp. 7.20-27.

Activity is calculated using an extinction coefficient of /, g3×10 M cm.

J%Bio1. Chem, , 233, ///3C/
See 93g').

Calculate the amount of enzyme present using the absorbance at 210 nm for the α-amylase/thi solution, 2×2.

The samples analyzed in Examples can be analyzed by the following procedure.

Reagent: Galactose, 0.1% solution.

is lactose, 7.0% solution.

ρ-nitrophenyl-β-D-galactopyranoside (ρ
NβGL), / ,! mM bath solution ρ-nitrophenol, O, SS solution.

Glucosidase, 0. −jchi solution.

Micro-Pak Trademark (Varian Asso-ciat)
eg)) Column 3QXlILcm.

Set the absorbance detector at 20°C.

2! °C water elution cutting.

Flow rate - 2,000 pSi odor'''cQj; ml!/
Min.

Step 2 A mixture of semi-synthetic glycoside hydrolase and ρN/lGL was added to the column in the greenhouse.

/g latency, the peak at the position set for galactose was recorded.

Peak heights and concentrations were determined from standard galactose solutions. When the activity of the semisynthetic enzyme was calculated, it was 3 × 10
It was U/me.

The samples analyzed in Example 10 and/or are analyzed by the following procedure.

Reagent 2 PHvo sodium citrate buffer 0.0!
;M.

0.2M sodium carbonate.

Substrate: ρ-nitrophenyl-β-D-1'rucohyranoside 23 dissolved in deionized distilled water; mM bath solution ρ-nitrophenyl-α-D dissolved in deionized distilled water
-1'Rukoeu/Sito, 2! ;mM bath solution procedure: pH, 5-700μ of sodium citrate buffer
Add 1 aliquot to 5 tubes, 2 of which are controls. 100 μg of semi-synthetic enzyme was added to the three above tubes, while 100 μlf of sodium citrate at pH 3-
Add to the two controls. The tubes are incubated in a shaker at 30° C. for 70 minutes. After this 70 minute period, 200 μl of the appropriate substrate is added to all 5 tubes and left in the greenhouse in a shaker at 30°C. After 75 minutes in the greenhouse,
Remove 7 control tubes and 7 enzyme-containing tubes. Immediately add the solution in the control tube to 10.2M sodium carbonate, rum/, ≠ml
Mix with. Centrifuge the enzyme-containing tube for 2 minutes.

After the 2 minute period, pour the liquid in the tube into the marked tube and discard the precipitate. Next, the solution is placed in the tube and 700 μl of 1.2M sodium carbonate buffer is added to stop the reaction.

The absorbance is measured at ≠20 nm. At 30 minutes, remove the 7 enzyme-containing tubes and centrifuge for approximately 2 minutes. Then,
Pour the solution into the marked tube and pipette 500 μl of the solution into another marked tube. The reaction is stopped by adding 700 μl of 1.2M sodium carbonate solution to the tube. At 4'J-min, remove the last two tubes (7 containing enzyme, others controls) and repeat the procedure described for the 75 minute tubes above.

≠) Extinction coefficient for ρ-nitrophenol at θnm/, U/ using 3g×IO'M'col-'
Calculate m9.

Absorbance (A'%) for α-amylase is also used in dividing the enzyme abundance during analysis.

Name of the agent Kawahara 1) - Director of the Patent Office for amendments to the proceedings Kazuo Wakasugi Tono (1982-36) Indication patent application for the case 36 Otsu No. Postal code: 105 6 Number of inventions increased by amendment 7, subject of amendment Detailed explanation of the invention in the specification column 8
Contents of the amendment As shown in Attachment 9 Contents of amendment to the list of attached documents The description that can be submitted to the recipient will be amended as follows.

(1) On page 70, line 6, “2 is” is corrected to “or”.

(2) On page 2j, first line, "E section" is corrected to "F section."

(3) From the bottom of page 57, in the second line, ``Implementation'' is corrected to ``Example''.

(4) Page 2j, line g (2 locations), page 32, line g (,
2 places), page 39, line ≠ (2 places), page 4t, page 3, g
i U, written in row ≠ row (, 2 places) on page O,
/ mt J is corrected to rU'm9-1, respectively.

that's all

Claims (1)

[Scope of Claims] (1) Protein substrate specificity, which comprises: (1) partially denaturing the protein; and crosslinking the partially denatured protein in situ in the presence of a predetermined inhibitor for semi-synthetic proteins. A method of creating predetermined semisynthetic proteins by chemically changing their sex. (2) forming an aqueous solution of the protein and maintaining the aqueous solution at a temperature and for a period of time sufficient to partially denature the protein; ) method. (3) The method of claim (2), wherein said time is about 75 minutes to 2≠ hours and said temperature is about 2j°C to 60°C. (4) Mix the above protein with water to dissolve it in water. A method according to claim 1, characterized in that the protein is produced by preparing a protein and mixing the resulting solution with a denaturing agent. (5) Claim No. 1, wherein the modifier is an inorganic acid.
4) Method. (6) Claim No. 1, wherein the modifier is an organic acid.
4) Method. (7) The method of claim (4), wherein the modifier is a water-miscible organic solvent. (8) Claim No. 1, wherein the modifier is an inorganic salt.
4) Method. (9) Select a model enzyme protein; select a second protein to be modified to imitate the enzyme protein; select the second protein to partially denature the second protein; A method for making a semi-synthetic enzyme comprising mixing with a denaturing agent for a sufficient time and at a temperature; and crosslinking said second protein in situ in the presence of an inhibitor for said enzyme protein. αO Make an aqueous solution of the above second protein, and add this aqueous solution to
10. The method of claim 9, characterized in that said second protein is maintained at a temperature and for a period of time sufficient to partially denature said protein. 11. The method of claim 4, wherein said time is about 75 minutes to 21 hours and said temperature is 23°C to 60°C. 0bu Mix the above second protein with water to make an aqueous solution,
By mixing this produced solution with a modifier, the second
The method according to claim (9), characterized by the protein. 03. The method of claim alpha, wherein the modifier is an inorganic acid. α◆ Claim 0, wherein the modifier is an organic acid
■Section Method. q→ The method of claim 92, wherein the modifier is a water-miscible organic solvent. 0→ Claim 0, wherein the modifier is an inorganic salt
Method in Section 4.