CA1040123A - Physiologically active peptide and its n-acyl derivatives and processes for producing thereof - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

The present invention relates to novel physiologically active N-acyl peptides of the general formula:
R'-Val-X-Ala-X
wherein R' is an acyl radical having 1 to 16 carbon atoms or said acyl radical partially substituted by one or more hydroxyl groups or phenoxy groups or halogen atoms, Val is L-valine, the X between Val and Ala is 4-amino-3-hydroxy-6-methylheptanoic acid and Ala is L-alanine, the other X is 4-amino-3-hydroxy-6-methylheptanoic acid or salt or an ester thereof and the amino group of Val being bound to said acyl radical by an amide bond, the carboxyl group of Val being bound to the amino group of the X between Val and Ala to form a peptide bond, the carboxyl group of the X between Val and Ala being bound to the amino group of Ala to form a peptide bond, the carboxyl group of Ala being bound to the amino group of the other X to form a peptide bond, and the carboxyl group of the other X being free or esterified or bound to a cation to form a salt,and a process for their preparation.

Description

lV'~lilZ~

It has been well known -that certain peptides have physiological activities and their utilization has been studied extensively. Especially peptides bearing and acyl moiety at their N-terminal, viz., N-acyl peptides have shown interesting activity. Acid proteases, for example, have been reported to be specifically inhibited by N-acyl peptides, i.e., pepstatins, of the general formula:
R-Val-Val-X-Ala-X
wherein Val is L-valine, the X between Val and Ala is 4-amino-3-hydroxy-6-methylheptanoic acid and the other X is 4-amino-3-hydroxy-6-methylheptanoic acid or a salt or an ester thereof and Ala is L-alanine, and the carboxyl group of the X between Val and Ala being bound to the amino group of Ala to form a peptide bond and the amino group of the X between Val and Ala being bound to the carboxyl group of Val to form a peptide bond, the amino group of the other X being bound to the carboxyl group of Ala to form a peptide bond and the carboxyl group of the other X being free or esterifiea or bond to a cation to form a salt, and R is an acyl radical having carbon atoms of 2 to 8 or an acyl radical partially substituted by one or more hydroxyl groups or halogen atoms or a C-terminal of an esterified carboxyl group, and the amino group of the Val adjacent to X being bound to the carboxyl group of the other Val to form a peptide bond and the carboxyl ; group of the R being bound to the amino group of the other Val to form an amlde bond.
Some of these compounds are reportedly effective on gastric ulcers due to their anti-pepsin activity. Clinical studies to this effect have been published.
These N-acyl peptides are characterized by the presence of a novel amino acid, designated in the formula as X, which is thought to be closely related to their physiological activity.
The length of the peptide chain and the character of the acyl group are also related to the activity of these peptides.
9~ ' ~4~Z3 ` It is pos~ible to presume that i a peptide bearing X but no acyl group, havi~g a shorter pep-tide chain length and still maintaining the protease inhibitory activity can be obtained, it will exhibit more varied actions and be able to be widely utilized~ because its physiochemical and biological properties such as solubility, absorption and distribution in various organs will be different.
According to the present invention, it has been found that an N-acyl peptide of the general formula : R-Val-Val-X-Ala-X
is partly decomposed by microorganisms, namely, bacteria, molds, yeasts and ray fungi, or an enzyme produced by such microorganisms.
It has been further discovered that a peptide of the formula:
Val-X-Ala-X, a novel compound, is obtained by this degradation.
This substance still has the important novel amino acid, X, in the molecule but no acyl moiety. It has been still further found that a new N-acyl peptide of the following general formula:
R'-Val-X-Ala-X
wherein R' is an acyl radical having 1 to 16 carbon atoms or an acyl radical partially substituted by one or more hydroxyl groups or halogen atoms or a C-Terminal of an esterified carboxyl group and the carboxyl group of the R' being bound to the amino group of Val to form an amide bond and is an acyl radical which is the same as or different from the R of the above R-Val-Val-X-Ala-X and Val, Ala and X are the same as previously defined is produced by chemical acylation of the N-terminal o the above-mentioned new peptide. It has been still further discovered that these novel compounds, Val-X-Ala-X and R'-Val-X-Ala-X are physiolo-gically or biochemically active.
The present invention therefore provides a process for the preparation of a novel physiologically active peptide of the formula Val-X-Ala-X and N-acylated compounds thereof in which a compound of the general formula R-Val-Val-X-Ala-X or a salt

- 2 -~34~3 thereof or an ester thereo~ is hydrolyzed by an enzyme produced by a microorganism and capable of decomposing R-Val-Val-~-Ala-X
to Val-X-Ala-X and, subsequently, the thus obtained Val-X-Ala-X
may be acyla-ted by a conventional method.
As stated above, the peptide, Val-X-Ala-X, produced from R-Val-Val-X-Ala-X is a valuable substance, and a novel compound as well~ which is obtained by the method of the present invention for the first time. ~o process for the specific decomposition of R-Val-Val-X-Ala-X to Val-X-Ala-X, whether enzymatic or not, has heretofore been known.
The N-acyl peptides fo the general formula Rl-Val-X-Ala-X are prepared from the peptide Val-X-Ala-X according to a known method of acylation, but are also novel and valuable compounds never before known.
Figure 1 is a diagram representing the effect of pH
on the activity of the enzyme which is denoted by the number of n moles of isovaleric acid produced.
Figure 2 shows the effect of pH on the enzyme, which is indicated by the ~ of residual activity, after keeping the enzyme at vaxious values of pH for 60 minutes Figure 3 shows the effect of heat on the enzyme acti-vity which is denoted by the number of n moles of isovaleric acid produced.
Figure 4 shows the thermostability of the enzyme, which is also indicated by the % of residual activity, after keeping the enzyme at various temperature for 10 minutes.
Figure 5 is the ultraviolet absorption spectrum of the new tetrapeptide, L-valyl-4-amino-3-hydroxy-6-methylhep-ta-noyl-L-alanyl-4-amino-3-hydroxy-6-methylheptanoic acid, in a methanol solution.
Figure 6 is the infrared absorption spectrum o~ the above compound when it is pelleted in potassium bromide.

- 3 -;;* ~ .

Figures 7, 8, 9, lQ, 11 and 12 show the in~ra red absorption spectra (KBr pellet) o~ N-acetyl-Val-X-Ala-X, N-isobutyryl-Val-X-Ala-X, N-isovaleryl-Val-X-Ala-X, N-benzoyl-Val-X-Ala-X, N-phenoxyacetyl-Val-X-Ala-X and N-2-phenoxypropionyl-Val-X-Ala-X, respectively.
Figures 13, 14, 15, 16 and 17 show the NMR spectra (CD30D~ of N-acetyl-Val-X-Ala-X, N-isovaleryl-Val-X-Ala-X, N-phenoxyacetyl-Val-X-Ala-X, N-2-phenoxypropionyl-Val-X-Ala-X
and N-benzoyl-Val-X-Ala-X, respectively.
Figure 18 shows the mass spectrum of N-acetyl-Val-X-Ala-X.
In carrying out the present invention, the compound of the general formula R-Val-Val-X-Ala-X or a material containing the compound is used as the starting material. R may be any acyl group but acyl groups of C2 to C8 are generally preferable.
Moreover, the acyl group may be partially substituted by one or more hydroxyl groups or halogen atoms or a C-terminal of an esterified carboxyl group. There may be employed as the starting sustrate a single compound or a mixture of compounds of the above general formula. These compounds can be used in the form of crude material or of salts with cations which will not interfere with the enzymatic reaction. Alternatively, fermentation broth or a crude extract - 3 a -~ ..

lZ3 therefrom, when said compounds were produced by fermentation, or a crude extract Prom a reaction ni.~{ture, when said compounds were synthesized ohemically, may be employed a~ the starting materi~l, The microorganisms employed in the present invention are strains producing an enzyme capable of decomposing R-Val-Val-~-Ala-X to Val-X-Ala-X and~ i.n particular, belong to acillus~ other genera of bacteria and ~ Imperfecti.
Such micro-organisms will be described hereafter, (a) ~acillus The ~ollowing are examples of bacteria belonging to Bacillus producing an enzyme of the aforementioned capacity:
~acillus meg~erium ATCC 13639, ~acillus cereus ATaC 963~
~acilus subtilis ATCC 10783, ~acillu_ circulans ATCC 13403 and acillus ~haericus ATCC 14577.
A suitable enzyme prepara~io~ for the process of the present invention may be obtained by c~ltivation of the micro-organism~on a suitable medium and under suitable conditions~
The microorganism may be grown in liquid c~lture with or without aeration and agitation, or on solid c~lture.
~he media used ~or growth of the microorganisms i~
the pre~ent in~ention are the nutrient media known as suitable for growth of the microorganisms. As the carbon source any o~
those carbohydrates may be used ~'nich are normally employed in ~ermentation, such as starch, dextrin, sucrose, maltose, glyce-rine and the like. The nitroge.n may be furnished by any of those materials which are usually used~ such as peptone, meat extract, yeast, soybean meal, corn steep liauor, glute.nJ urea, ammonium salts, n~trate salts and the like. l,.r~eat and rice bran, soybean treated with organic solvents, nutrient agar and the like may be employed for solid culture. The media 1~ 4~ ~ ~ 3 may co.ntai~ Mg~ a~, Fe~, Mg~, phosphate ions, various vitamins, amino acids and the li~e a~ inorganic ions and trace elements as ~cessary.
The cultivat~on may be conduc-ted at the temperature and the pH which are usually employed for the growth of micro-organisms~ ~he initial pH of the cultivatio.n is preferably maintained between 4.5 and 8.59 and the tem~erature between 15C
and 40C, depending upon the microorganism employed~ Por induc-tio.n of the enzyme, a small amount of the starting material may 10 . be added to the meaium before cultivation or after initial gro~l~h of the microorganism.
Any mater,~l containi.ng the acti~e enzyme may be employed as the en~yme preparation for the process of the pre-se~t in~ention, such as c~ltured broth with cells, broth fil-trate, extract from solid culture, cells, cells treated with organic solvents, lyzed cells, dried cells or extract from cells and also.concentrated preparations, or partially or hi~hly purified preparations obtained ~rom the aforementioned substances by means of salting out, precipitation with organic sol~ents, gel filtratio.n, or chromatography, Insoluble enzyme or immobilized cells which have bee.n prepc~red by recently ~ developed remarkable techni~ues may be used.
~ he speci~ic decomposition reactio.n may take place by placing the enzyme in contact with the starting material.
During the reaction the temperature is preferably maintained between 15C and 75~ and the pH between 5 and 9. ~he preferred . concentration of the starting material is from 0.01 ~0 to 5 %
by weight. The reaction sho~ld continue for 30 minutes to 48 hours. These conditions depend upon the form of the enzyme prepa~ation, the starting matericil, the microorgc~nism and the like.

lZ~

~ he following experimen-t shows the ef~ect of pH on the degradation re~ction using a cell suspension o~ Bac.
~ A~CC 13403 and'~ac. me ~ AT~C 1~639.
E3~eximent 1 A medium containing 1 ~ peptone, 0.7 % meat e~rtract, 0,5 ~0 glucose and 0.3 Clo ~aCl, all by weight, pH 7.0, was pre_ pared and 50 m,l of the medium was poured into a 500 m,l flask and sterilized a-t 120C for te~ minutes, The medium was inoc~lated with ~acO circulans A~CC 13403. Afte~ incubation at 30C for 48 hours on a reciprocal shaker (120 r.pOm.), cells were harvested by centrifugation at 10,000 r.p.m. for ten minutes.
~he cells thus obtained were washed ~th cold 0.9 ~0, by weight, ~aCl and suspended in distilled water (0D61om~=34), ' A mixture of 1 ml of 2 mg/ml isovaleryl-I-valyl-I-valyl-4-amino-3-hydroxy-6-methylheptanoyl-I-alanyl 4-~mino-3-hydroxy-6-methylheptanoic acid7 0.5 ml of 0.4M buffer solution , ~aving the pH value indicated in ~able 1; and 0.5 ml of the cell I suspension was incubated at 37C for one hour, ~he pH of the i, reaction mixture ~Jas adjusted to 2.0 with HCl and the super-1 20 natant was applled onto a small colunm (d=0.7 x 7 cm) o~
¦ cation exchange resin (Dowex 50). ~he colu~n was washed ¦ with dist~H20 to remove unreacted ~tarting material and the reaction product was elutea with 0,5N l~H~OH quantitativelyO
Pepsi~-inhibitory activity of the eluate ~Jas assayed accordin~
to the method mentioned below, ~he effect of pH on this de-gradation using a cell suspension of ~ac. megaterium A~CC 13639 was also examined in the same manner as above, .
~able 1 shows the results.

~ .

Table 1: The effect o~ pH o.n activity . ~
P~ ~ctivi-ty _ _ _ _~ __ _ __ __ . ~ac. ~ac. ~ ne- Pseudo- Colieto- Mac~o circul~n~ me~aterium ~ ~ ~- ~
A~CC1340 ATCC13639 ATCC6939 ATCC4358 ATC~20438 A~CC20441 _ _ __ __ . ,.- _ ._ _

4~ 200 > 200 >200 >200 ~ 200 >200 _ _ ~ _ __ ,_~
4.5200 180 200 180 200 200 . _ ~ .............. ........ _~ ____ _ , _ . . _ ___

5. 65 80 80 100 100 80 _ ; - _ _

6 65 65 80. 80 ~5 65 _ _ _ _ _ _ . 6.5 65 65 80 80 80 65 _ ~ r --_~_~ _

7 ~ 65 65 80 80 80 65 ~ _ . _ _ _ - 65 8d 80 80 65 . __ __ . _, . _. .
2 0 8 65 65 100 ~0 80 80 _ _ , _ . _ ~ ___ ~
. 8.5 80 65 125 80 100 80 ..,- , _~ __, ____ . l ~ _~ _ .~
9.5 125 125 200 150> 200 200 _ , ,~ ___ _____ ,. __ _ _ 10 > 200~ 200 L~ 200 ~ 200 ? 200 ~ 200 _ _ I _ .

~ote 1:
Activity in this table and hereinbelow is defined as the volume of the eluate from the ion e~change resin in terms of ~ necessary to attain ID50 of the pepsin-inhibitory acti-vity.

.

~Jote 2:
___ This t~ble also relates to the use of genera of bacteria other than _cillus, in particular C~Q~y_e~Q~E~ ATCC6939 and Pseudomonas se~nis A'~CC4358 (see Ixperiment 6 below) and to the use of a Fungus ~ rfectus, in particular Colletotricl1um sn. ~TCC20438 and Macro~homina phaseoli A'rCC20441 (see Expe-riment 12 below).
The concentration of the reaction ~roduct in the eluate is in inverse proportion to the volume ol the eluate necessary for ID50 of the pepsin-inhibitory ac~ivity. In this degrada-tion, ~ac, circulans A'~CC 13403 showed high activity between p~I 5.0 and 8.5, and ~ac. ~ ATCC 13639 between pEI
5.5 and 9Ø
Assay of pepsin-inhibitory activity in this invention was performed according to the following method: A mi~ture of 1 ~1 of 0.6 5', by weight, high~y purified casein dissolved in 0.08 M lactate buffer solution, pH 2.2, 0.7 ~1 of 0.02III~Cl-IICl buffe~r solution, pH 2.0, and 0.2 ml of solution containing the compound sample ~as incubated at 37C for three minutes.
To this mixture 4 mcg of ~epsin (SIG~ ~ 2XCRY) dissolved in Oo1 ml of 0.2M KCl-IICl buffer 9 olution were added. 'rhe mixture was lncubated a-t 37C for 30 minutes~ '~he reaction was stopped by the addition of 2.0 ~1 of 1 7I~I perchloric acid. After one - hour at ~oom tem~erature, the optical density (a) of the super-natant llquid was read at 280 m~. An inhibition rate was obtained according to:
(b) - (a) ~ 100 (b) where (b) is the optical density at 280 mju o~ the tube - without the sample solution.
Trademark in the name of Sigma Chemical Co., St-Louis, Missouri, U.S.A, -- & --~'b ' ~a~4`~123 ~ he ID50 is de~ined as the ~moun-t necessary ~or 50~o of the inhibition rate.
In carr~ing out the process of degradation in the present invention, especially using paxtially puri~ied enzyme preparation~ the reaction mixtuxe may c~ntain Mg~+ 7 Mn++
Ca~+, Zn~+, Co++ or ~e~+ as needed.
The degxadation is usuall~ per~ormed in aqueous solution9 but non-aqueous solution may also bç employed. In that case~ a suitable solvent and concentration should be selected 90 as not to inhibit the e~zyme activity.
As mentioned above, the process OI the present inven-tion is not limited by the kind o~ acyl group, designated R in the formula, which is demonstrated by the follo~ng experimental results.
Experiment 2 Various starting materials indicated in Table 2 were reacted at pH 7.0 wi-th a cell suspension of ~ac~ sphaericus A~CC 14577 prepared according to the same procedure as in ~xperiment 1~ (Table 2 also illustrates the use of a stxain of bacteria o~ a genus other than acillus, namely Micrococcus rukens A~CC 186, see Experimen-t 7 below~ and the use of a ~ungus ImPer~ectus, namely Colletotrlchum ~p A~CC 204~8 ~ee ~xperiment 13 below).
'' .

_ 9 _ ~L~Z3 Table 2: ~fect o~' acyl rad~cal on ac-tivity \ ~c-tivity R in \ micro~ Baclllus Micrococcus Col~eto ~ s~haericus rubens ~
R-Val-Va~ ga~ism ~a~ 577 h~ 86 ATCC 20438 X-Ala-X ~ _ - _- _ pèriod Q min O min O rnin 60 min O min 60 min ~ _ _. __ _ , ,._ ___ CX3-CO- >200 65 ~200 80 ~200 7 ~ ~ __ _ ~ _ CH3-cH2-co ~200 65 ~200 80 >200 7o _ ~ __ ~ __~ _ CH3-(CH2)2-C >20.0 65 >200 80~ 200 7o ~ --_ _ _ _ cx3>CH-Co- >200 65 >200 80>200 7o 3 .
,, ____ . _. _ CH3-(CH2)3-CO- >200 65 ~200 80> 200 65 _ ~ __ ~ ~ __ CH3~CH-CH2-CO- >200 65>200 80 ~200 65 CH3 .
~_ . . ~ ~ ~
cx3_(cx2) ~-co- >200 70> 200 85 > 200 7 ~ _ __ _ . _ _ 3> CH~(CH2) 2-CO- > 200 65~200 80 > 200 7o ~__ _. - __. _. _ CH3-CH~-~H-(Cx2)2 ~ >200 7o~200 80 >200 65 CH3 .
_ _ __ . . __ . ____ __ CX3-(CH2) 6 ~ > 200 65 ~200 80 > 200 5o . _ _ _ _ ~ __ _ ~ Cl-CH2-CO- >200 7o ~200 85 ~ 200 7o . _ _ l . ~ -CH3>CH-CH2-CO- ~200 7o ~20085 ¦ ~ 200 7o CH3 (methyl este~) . .
. _ __ __ ~ 2 3 As sho~n in Table 2, the degxadation actlvity on the compounds wi~h various acyl groups or an estex thereof was ~ almost the same~
A ~ew preliminary experiments easily gave the appro-priate concentration of starting material to be employed, taking degradation rate and economy into consideration. The substance o~ the general formula R-Val-Val-X-~la-X used as the starting material in the process of the present inven-tion includes some compounds having poor solubility in water but those compounds can be used in the ~orm o~ salts or suspensions of an appropria-te concentration. When the concentration of the starting mate-rial employed is higher than its solubility, a satisfactory re-sult ~or the degradation process may be obtained by such modifi-cations as extended reaction time, gentle stirring or use of additional enzyme preparation.
~xperiment 3 ` ~ixtures of the same starting material as used i~
Experimen~ 1 of the weight concentrations indicated in Table 3 and the cell suspension f I C--E~e~b~ A~CC 14577 ob ained ln Experiment 2 were incubated at 37C ~or the periods indicated in ~able 3. ~ubes 5), 6) and 7) were stirred during the incu-bation. ~ube~ 6) and 7) were supplemented with another 0.5 ml of the cell suspe~sion after 24 hours of incubation. ~Table 3 also illustrates the use of a strain of bacteria of a genus other than acillus, namely Escherichia coli A~Ca 11303, see Experiment 8 below, and the use o~ a ~ungus Imperfectus, namely Kabatiella caulivora ATCa 20439, see ~xperiment 14 below).

- .. 11 _ ~able 3: Effect oP concentration of starting material on activity Actlv~ty . .. _ __= ,~ __ Concentration reactin ¦Bac. ~scherichia Kabatiella of the startin~ period sphaericus coli ca~ivora material (%) (hr) ATCC 14577~ 11303 ~3~r~5~9 I _ _ _ . __ . _ (1) 0.005 1 2 2 2.35 7.5 (2) 0.01 5 2 35 2~5 8.0 (3) 0.05 24 2.35 2.5 8.0 (4) 0.l 24 2,35 2.85 8.0 ~5) 0.5 24 2,85 3.0 8.5 (6) 1.0 48 2.85 3.0 8~5 (7) 2.0 48 2.~5 3.0 8.5 _ ~ _ , _ , .

As shown above, the degradation reaction proceeds with any concentra~ion of the starting material by selecting a suitable reaction period and vPlume of cell suspension, but - 0.01 % to 5 ~0~ by weight, are pre~erable concentrations from the viewpoint of e~ficient consumption of the starting material and econom~. ~ower concentrations ma~ be employed when using speoial methods such as with insoluble enz~me or immobiliæed cells~
~he temperature for the degradation process is easily determined by a few preliminar~ experiments and depends upon the form of the enzyme preparation, the kind of the starting material and the economical operation of the process. ~he ~- ~ollowing experiment shows the effect of temperature upon the degradation activity of ~ac. cereus A~CC 9634.
~xperiment 4 A cell suspension of ~acO cereus A~'CC 9634 prepared by the same procedure as in ~xperiment 1, and the same starting _ 12 -~ Z 3 material as u~ed in ~xperiment 1 were incubated at pH 7.0 in the same manner as in Experi~ent 1 at various teraperat~re~ in-dicated in ~able 4 ana the degrada-tion activity ~Jas a~sayed.
~able 4 also il~ustrates the use o~ a strain o~ bacteria of a genus other -than ~acillus~ namely Arthrobacter A~CC 7562, see E~periment 9 belowy and the use of a Fungus Imperfectus, namely (Ascoch~ E~ _lorum A~CC 14728, see ~xperiment 15, below).
~able 4O E~e~t o~ temperature on activity 10 _ .. ..~
~empera- Activity ture (C.) ~ac. Arthrobacter Ascochyta cereus ureaIaciens ~haseolorum ATCC-~634A~CC 7~~ A~lCC 1472~
_ _ _~_ . _ _ _ , . . _ ._ ....... .

_ _ .___ __ .,__ __ ~0 60 40 __ ,~ _ . _ ~ ____ ___ . _ _~ __ _ _ > 200 7 200 > 200 As given in ~able 4, ~ac. cereus A~CC 96~4 showed high activity between 30C and 70C. The preferable temperature range o~ this process is between 15C and 75C because no acti-vity is lost at a lower temperature in spite of a slower reac-tion velocity.
As stated above, the process o~ the specific ~4~12~

decomposition of the present invention is co.nducted by putting the en~yme in contact with the starting material, ~his may also be performed by adding the starting material to a growin~
culture of the microorganism.
In carrying out the degradation process of the present i.nvention during cultivation of the microorganism, the starting material may be added to the medium in the foxm ol an aqueous solution or suspension or a powder depe~ding upon its properties, principally solubility in water, at the start o~ the c~ltivation or after the initial growth of the microorganism. A concentra-tion Of 0~01~o to 5%, by weight, of the starting material is preferably employed, which may be adaed all at once or in portions.
.A product of this degradation reaction, a tetrapeptide, I~valyl-4-amino-3-hydroxy-6-methylheptanoyl-I~alanyl-4-amino-3-hyaroxy-6-methylheptanoic acid, may be reco~ered by any of those methods which are usually employed for isolation and purifica tion of oligopeptide, such as adsorption on active carbon or ion e~change resin, extraction ~ith organic sol~ents, chromatography with silica gel or almina gel, ~el filtration and the likeO
~he following experiment demonstrates the degradation process uslng ~ac~ ATCC 9634 and the recovery of a crystall~ne product.
E~periment 5 ~ ac~ cereus A~C~ 9634 was grow.n in the same manner as in Experiment 1. Washed cells were suspended in 0O01M
.. phosphate buffer, pH 7.0 and disrupted with a ~rench press.
A cell-free extract was obtained by centrifugation at 10,000 r.p.m. for 20 minutes. A mixture of 50 ~l o~ 2 mg/ml n-caproyl-I-valyl~ l-4-amino-3-hydroxy-G-methylheptanoyl-I-alanyl-4-amino-3-hydro~J-6~methylh~ptanoic acid suspension, .- 50 ~l of the cell-fxee extract and 1 ~l o~ toluene (to provide . 14 1g~4~23 protec-tion from ~nicrobial contamin~tion) ~as incubated at 37 C
for 15 hours. r~he preci~itate resultin~ froT.q adjusting the pII
to 2 with ~ICl was removed by cen-tri~ugatio.n. The supernatant was applied onto a Dowe~ 50 (~4, II+ type) column (~=2 x 20 cm).
The column was ~Jashed w.ith ~/ater. Active fractions elu-ted with 0.5N ~II40lI~rere coU ected and extracted with an equal volume of n-butanol. The butanol layer was adsorbed onto a silica gel column (~=2 x 30 cm).
The elution was carried out with a solvent system of n-butanol:acetic acid: 1120 4 : 1 : 1, by volume. After concen-tration, the active fraction was collected and applied to a Sephadex -~II 20 column (~=1 x 50 cm).
Crude crystalline L-valyl-4-amino-3-hydro:~y-6-methylheptano~vl-L-ana~yl-4-amino-3-llydroxy-6-aminoheptanoic acid was obtained from the active fraction of the gel filtra-tionO Recrystalli~.ation in methano~ providea 45.2 mg of color-less crystals.
b) ~acterla Belongin~ to ~enera other than Bac.i~lus.
The following are examples of bacteria producing an enzyme of the aforementioned capabi.lity belonging to genera other t~an Bacillus: Pseudomonas se~nis Al'CC 4358, anthomonas ca pestris ~mR~ B1459 (hTCC 13951), Acetobacter rancens ~L
B65 (A'~CC 7839), Aeromonas hydrophile i~L B 909, Protam_ o-bac-ter ruber I~TRl~L B1048 (ATCC 8457), rIicrocyclus f vus ATCC
23276, A~robacterium ~ ATCC 4718, Alcaligenes faecalis ATCC 8750, Escherichia coli ATCC 11303, Citrobacter freundii ATCC ~090, ~nterobacter aero~enes ATCC 8329, Sarcina utea ATCG 9341, Micrococcus rubens Al'CC 186, Staphylococcus epidermidis ATCC 155, ~revibacterium ammoniagenes ATCC 6871, Leuconostoc mesenteroides NRRL ~1299 (ATTC 11449), I.acto-bacillus brevis ATCC 8287, Pro~ionibacterium shermanii ATCC
Registered trademark in the name of Dow Chemical Co., Midland, ~ichigan, U.S.A.
- 15 _ ~ 2 3 13673~ Dcs_l blot^,ri~ equi A~CC 6939, ~ ~ lacticum A~CC 8180, Cellulomonas fimi A~CC 8183~ Axthrobacter u ATCC 7562, and ~ butyricum ATCC 6014.
The materials ana techniques for cultivation o~ the microorganisms and production of the enzyme preparations, such as culture broths, cells, partially or highly purified enzyme preparations and the llke, which were described in the case of the microorganisms belonging to the genus acillus, may be employed in the same manner ~hen using bacteria belonging to 1Q genera other than acillus~ -The following experiment shows the effect of pH on the degradatio.n activity of cell suspensions of Cor~nebacterium equi ATCC 6939 and Pseudomonas segnis ATCC 4358.
xperiment 6 Using Corynebacterium equi ATCC 6939 and Pseudomonas se~nis ATCC 4358~ the effect ol pH on the degradation activity was examined in the same manner as in Experiment 1. The results are sho~n in the a~ove Table 1, ~s shown in Ta~le 1 the activity was high between pH
5 and 8, and between pH 5.5 and 990, in ~ ~ e~
~ATCC 6939 and in ~ A~CC 4358, respectively.
The degradation activity is independent of the kind o~ acyl group of the starting matexla,l, ~xperiment 7 Using ~crococcus rubens ATCC 186, the degradation activity on various starting materials was examined in the same manner as in Experiment 2. The results are sho~m in the above ~able 2~
As shown in Table 2, almost the same activity on all of the vario~s compo~mds was observed.

i~4~9~3 ~xperiment 8 Using ~scherichia coli ~ca 11303, concentration o~
the starti.ng material and reactio.n time in the de~radation pro~
oess were studied in the same man.ner as in ExpeYlment 6, ~he results are sho~m in the abo~e ~able 3.
~ ubes 5) 6) and 7) were gently stirred during the incubation and ~ubes 6) and 7) were supplemented t~th another 0.5 ml of the cell suspension after.24 hours of the incubationO
As shown in Table 3, the results were very similar to those obtained in ~xperiment 3.
Experiment 9 Using Axthrobacter ureafaciens A~CC 7562, the effect __ of temperature on the degradatio.n activity was examined in the same manner as in Experiment 6. ~he results are sho~n in the above ~able 4.
As sho~n i~ ~able 4? high acti~ity was obser~ed be~-wee.n 30C and 70C. ~he preferable temperature employsd in the present in~entlon i9 between 15C and 75C, as already has . bee~ mentioned, because no enzyme activity is lost at lower tem-peratures in spite o~ slow reaction v~locity.
Experiment 10 Isovaleryl-I-valyl-I-valyl-4-amino-3-nydroxy-6-methylheptanoyl-~-alanyl-4-amino-3-hydroxy-6-methylheptanoic acid was degraded with acell suspension o~ Saroina lutea ~CC
9341 and the degradation product was o~tained in the same manner as in 3xperiment 5. Colorless crystals (34 mg) were recovered.
Experiment 11 .
~y a procedure similar to ~xperiment 10, using n-caproyl-I-valyl-I-valyl-4-amino-3-hydroxy 6-methylheptanoyl-I-alanyl-4-amino-3-hydroxy-6-methylheptanoic acid and Cellulomonas fimi A~CC 818~, 38 mg of colorless crystals of the degradation product ~as obtained~

_ 17 -.

1(~40~Z3 (c) ~ ~
'~he following are examples of microorganisms belonging to the gene~a ~ a~ producing an en~yme o~ the a~orementioned capability: ~homales including Macrophomina phaseoli ATCC 20441 and Ascoch~ta ~ eolorum ATCC 14728 Melanconiales including Colletotorium ~E~ A~CC 204389 and Kabatiella caulivora ATC~ 20439,~loniliales including Stemph~lium sarcinaeforme ATCC 20442 and ~usa~ium sP0 ATCC
20440 and ~elia sterilla including Rhizoctania sp, ATCC
20433, The materials a~d techniques ~or cultivation of the microorganisms and production of the enæyme preparations, such as culture broth, cells, partially or highly purified enzyme preparations and the like are similar to the procedures des-cribed i.n the case of the bacteria belonging to the genus Baclllus eYcept that a medi~lm usually employea ~or growth o~
fungi ls used at a tempe~ature between 10C and 35C and a pH between ~ and 8. Preferabl~ incubation time ~or the degra-datio.n reaction is between 30 mi.nutes and 72 h.~urs.
The ~ollowing experime.nt ~ith Colletotrichum ~, A~CC 204~8 and ~ phaseoli ATCC 20441 i9 an example of the effect o~ pH on the decomposition.
~xpeximent 12 A medium containing Or5 ~O starcht 0~5 $ glucose, 0.5 ~ soybean meal, 0.1 ~0 KH2P04, and 0~05 /~ MgS04 , 7H20, all by weight, was prepared and 50 ~1 of the medium was poured into a 500 ~l flask and sterilized at.120C for 20 minutes. The medium ~as i~oculated with _olletotrichum sp AT~C 204~8~
After incubatio~ at 28C or 72 hours on a rotary shaker (200 r.p.m.), cells were har~ested by centri~ugation at 3000 r.p.m~ for ten minutesO Isovaleryl-~valyl-l-valyl-_ 18 -~-amino~ hydroxy-6-methylheptanoyl-l~a~anyl~4-amino-3-h~dro~y-6-methylheptanoic acid and the ce:Lls were incubated at variou~
pHs in the same manner as in Experime.~t 1, ~he same procedure was oarried out and similar results were observed rith Macrophomina ~ Ar~CC 20441 as sho~m in Table 1 : high decomposing activity was observed between pH 5,5 and 8~5 with Colletotrichum ~. ATCC 20438 and between pE 5.0 and 9.0 with Macro,,homina phaseoli ATCC 20441 ~xperiment 13 The various starting materia.~s listed in ~able 2 were reacted with a cell suspensio,n of Col~etotrich~m ~.
~TCC 20438 prepared by the same way as in Experiment 12 and the decomposing activity was assayed, Almost the same acti.vity was observed on all compounds tested notwithstanding the differe.nce o~ the acyl group. ~he res~lts shown in ~able 2 are almost the same for Experiment 13 as ~or Experiment 2.
Experiment 14 ~he ~ame starting material as used iD ~xperiment 12 and a oell suspension o~ Kabatiel~a ~ ~ A~CC 20439 prepared by the same procedure as ln ~xperiment 12 were incu-bated at the various concentrations indicated ~n ~able 3 in the same manner as in ~xperimen~ 3, ~he results given in ~able 3 for ~xperiment 1~ are similar to those obtained in Experiment 3.
Experiment 15 _ ~he effect of temperature on the degradation activity was e~amined usi~g a cell suspeDsion o~ Ascocn~ta ~seolorlum A~CC 14728 prepared by the same procedure as in ~xperiment 12 :
the res~lts were the same as in ~xperiment 4~ High activity was obtai.ned between 30C and 70C as sho~.n in the above Tab.1e 4 ~or other microorganisms. As stated above for bacteria, ~or _ 19 -~ Z 3 un~i ImPerfecti, too~ a temperature o~ fxom 15C to 75C may be employed.
Experiment 16 Stemph~lium ~ naeforme ATCC 204~2 was grown and the cells were obtained in the same manner as in Experiment 12, A cell-free extract was prepared by disrupting the cells with a ~rench press and centrifuging the lysate at 10,000 r.p~m. for 20 minutes.
Similarly to Experiment 5, n~caproyl-~-v~lyl-~-valyl-4-amino~-hydroxy-6-methylheptanoyl-I-aMalyl-4-amino-3-hydroxy-6-methylheptanoic acid was reacted with the cell-free extract to yield 45.2 mg of colorless crystalline product.
(d) En~yme ~ he microorganisms capable o~ producing an enzyme which decomposes R-Val-Val-X-Ala-X to Val-X-Ala-~ speci~ically are widely distributed within the ~ollowing genera: Pseudomonas, Xanthomonas, Acetobacter, Aeromonas, Protaminobacter, M~cro-cyclus, A~robacterium, Alcali~ , ~ , Citrobacter, Enterobacter, Micrococcus, Stapnylococ_us, ~ 0 ~ vibac-~ , StrePt~gQe~g~, ~euc~ actobac _~us, Propioni ~iam- Cor~nebacter~m, M~crob~ uGi3~ ~QllUlm~
Y ~. ~J ~_, a~ la~ S m~yllya~ ~usariuun and Rhizoc-EQ;E~-aO
~ he enzyme formed by these microorganisms may be isolated and puri~ied ~rom the cultured broths or the cells obtained by cultiva~ing the microorganisms under the above stated conditions with the media described above. A small amount of the substrate or the startin~ material of the degra-dation process may be added to the medium at the appropriate - time, ~or example, at the start of the cultivation or a~ter . .

.

~46~z3 the initial growth, for inductlon o~ the format~on o~ the enzyme, in order to obtain higher potency of the en~yme. Al~
the microorganisms stated above may be used but the microorga-nisms belonging to -the genus ~ aLL~ are advantageously employed ~or industrial production of the enzyme. The cultiva-tion of the microorganlsms may be conducted in a liquid culture or a solid one, but a submerged c~lture has industrial advan-tages. Purified enzyme is obtained from the cultured broth~
which purified enzyme is, in particular, cell-lree extract prepared by extractlon with water or a buffer solution f~om the cells after disruption with a sonic oscillator, a French press7 or other homogeni~ers, lysozyme, organic solvents and the like, by me~ns of ammonium sulfate salting out, 0-(diethylaminoethyl) cellulose (D~AE cellulose) chromatography, Sephadex G-200 gel filtration and second D~AF cell~lose chromatography. Strep-tomycin-treatment may be employed, as neèded, to remove nucleic acids. Heat treatment at 60C for ten minutes before the first DEAF cellulose chxomatography may also be possible and satisfactory. In some cases, the ammonium sulfate salting out step may be omitted. These depe~d upon the initial crude pre-paration employed ~ox the purification.
~ he following experiment is a more detailed example o~ the purification procedure.
~xperiment 17 A medium having the same composition as in Experiment 1 was prepared and 50 ml of the medium ~lS poured into a 500 ml flask. AI`ter sterilization at 120C for ten mLnutes, the medium was inoculated with Bacillus ~phaericus ATCC 14577 and incubated on a reciprocal shaker (120 r.p.m.) at 30C for 24 hours to obtain a seed culture. ~o a 20 ~ jar fermentor was added 10~ of the medium having the same com~osition, sterilized _ 21 -3iLZ3 at 120C for 15 minu-tes c~nd inoc~lated wi-th the seed cu~ture, The cu~tivation t'Ja3 p~r:Eormed at 30~ with a~itation (300 r,p,m~) and aera-tion (5(/rninute).
AIter 15 hours ~ro~r~ isovalery-~ valvy-L valyl-4-amino-3-hydro~J-6-metll~Jlheptanoyl-L-alanyl-/1-arnino-3~hydroxy-6-methylheptanoic acid was added to the culture in a concentration of 50 mc~/ml and the cultivation ~ras continued for another 33 ho~lrs. Cells were harvested by centrifu~ation with a Spharpless centrifuge, suspended in 2j of cold 0001~I phosphate buffer, pll 7~09 and disrupted by sonica-tion at 20 Kc for two minutes, These and the following procedures were carried out under 4C, A supernatant from the sonicate was heated at 60C for ten minutes at pl1 7,0, The resultin~ precipitate was removed by centrif~gation and 3,8 g of streptomycin sulfate was added to 1,7~ of the supernatant, Precipitated nucleic acid was removed b~r centrifugation, The thus obtained supernatant was dialyzed a~ainst 0~01M Tris-llCl buffer, pI1 8.0, for 16 hours, Onto a DE~E cellulose co~umn (1R), previously eauilibrated to 0,011~1 Tris-IICl buffer, pll 800, the dialyzed supernatan~ was a~sorbed and the colu1nn was washed ~ith t'ne same buffer and with 0~11I
~TaCl disso~ved in the buf~er9 successivelyO The enzyme ~ras eluted witl1 31 o 0.3~I MaCl dissolved in -the Tris buffer.
The acti~e e~uate was concentrated usin~ an ultrafiltration apparatus and subjected to gel filtration with a Sephadex G-200 column (~=5 x 100 cm) equilibrated to the s~e Tris buffer containing 0.2M NaCl, The active fractions from the column were collected and dialyzed against 0,01II ~hosphate bufferg p~I
6,5, for more than six hours. The dialyzed solution ~ras adsorbed onto a DE~E cellulose column (50 ~1) equilibrated to the same phosphate buffer, The column was washed ~rith the buffer and 150 ml of ~'ne same buffer containin~ 0.1II MaCl, Registered trademark in the name of Pharmacia Fine Chemical AB, Uppsala , Sweden.

successively. ~he enzyme wa~ eluted with a linear gradient of 600 ~1 of 0,1M NaCl to 600 ~l of 0.3~ NaCl in the same phosph~te buffer.
A summary of the puri~ication is sho~n in Table 5.
~able 5 : Purificatio~ of the enzyme . _ _ . ~ _ __ ~otal ~otal Specific activit~ Yield vo~ume Activity (unit/mg protein) (~0) (~l) (unit) _ . _ _,_ _ Supernat~nt from sonicate 1,700 10,500 0.02 100 1st D~A~ cellulose520 7,392 0.11 7o Sephadex G-200 61 5 9 491 73 52 2nd DEAE cell~lose28 1,900 225 18 _ _ l . .
The enzyme preparation obtained from an active peak of the chromatography showed a single band in disc gel electro-phoresis, gel electrofocusing and ultracentrifugation and has the following enzymatic and physicochemical properties.
(d)-1 Action and substrate specificity ~his enzyme is unique both in its action and its substxate specificity. It acts on a compound o~ the general ~ormula R-Val-Val-X-Ala-X to produce a peptide (Yal-X-Ala-X), an organic acid (RCOOH) and ~-Yaline. ~amely9 it hydrolyzes two ~- ~ bonds shown as 1 and 2 in the following formula ,.
CH3 /CH3 CH3\ ~ H3 \ o~ ---NX / \ ~ t N~I / \ O--X--Ala--X

~ ~
.

~ _ 23 -~ Z 3 '~he aotions on the ~two bonds h~ve been thought to be di~erent enzymatic actions, such as aminoacylase action ~
and peptidase action ~, but this en~yme has bo-th the actions.
A more interesting and important property is as described belo~
in detail, that is, this partic~lar en~yme does not split other C-N bonds such as the linkage between I-valine and X, X and I-alanine, or ~-alanine and X. The action on various kinds of N-acyl peptide, given i~ ~able 6, show~ a very interesting ~ubstrate specificityO ~hese results were obtained from 20 hours reaction under the conditio.ns described in the hereinbelow section e.ntitled "Assay of the activity".
Table 6 Substrate speci~icity . . ~
Products Substrate _ _ _ ~al-X-Ala-X Organic acids T-Valine .. _ __ __ ~ _ IVA-Val _ . ~
_~,ral_Val _ _ ~ IYA-Val-Val-X ~* + +
I IVA-Val-Val-X-Ser ~** ++ ++
. TvA-~al-val-x-Ala-x ~+ +~ ~
IV~-Vai-Val-X-Ala-Thr ~+*** ++ ~t IVA-Val-Val-X~Ser-X ++**** +~ ++
~ _~__ _ ______ ___ Acetyl-Val-Val-X-~la-X -~+ ++ +~
Propionyl-Val-Val-~Ala-.X ++ ~ ++
II Isovaleryl-Val-Val-lY-~la- ++ ~ ++
Caproyl-Val-Val-X-Ala-X +++ +++ +++
: Isocaproyl-V~l-Val-X-~la- - ++ ++ ++
, ~ . . _ .. _ . .
. Acetyl-Val-X-~la-X _ _ . Isovaleryl-Val-X-Ala-X _ _ III Palmitoyl-Val-.Y-Ala-X _ _ ~enzoyl-Val-X-Ala-X _ _ 2-Phenoxy propionyl-Val- _ _ X-Ala-X
~ . . ~ ~ _ . _ ,_ .

2~

wherein IVA: Isovaleryl, Ser: L-Serine, Thr: L-Threonine, * Val-X- instead of Val-X-Ala-X
** Val-X-Ser " "
*** Val-X-Ala-Thr " "
**** Val-X-Ser-X " "
Thus the N-acyl peptides having the general formula:

R-Val-Val-X-Y, wherein R, Val are as previously defined and Y is a member selected from the group consisting of amino acid, peptide and hydroxyl residues, and the carboxyl group of the X
being bound to amino group of the amino acid or the peptide to form an amide bond when Y is amino acid or peptide and being free when Y is hydroxyl residues, were all susceptible to the enzyme.
Group I of the table illustrates the susceptibility of N-isovaleryl peptides to the enzyme. IVA-Val and IVA-Val-Val, wherein IVA stands for isovaleryl, were not split by the enzyme. Hydrolysis/takes place on the N-acyl peptides having longer chain length than IVA-Val-Val, such as IVA-Val-Val-X.
The products of this reaction were isovaleric acid, L-valine and Val-X. Thus, IVA-Val-Val-X-Ser, wherein Ser stands for L~serine, IVA-Val-Val-X-Ala-X, having more amino acid residues than the above, IVA-Val-Val-X-Ala-Thr, having L-threonine at the terminal of a carboxyl radical, wherein Thr stands for L-threonine, and IVA-Val-Val-X-Ser-X, having L-serlne residue instead of L-alanine, were all susceptible to the enzyme.
In these cases, in addition to isovaleric acid and L-valine, the reaction products were Val-X, Val-X-Ser, Val-X-Ala-X, Val-X-Ala-Thr, and Val-X-Ser-X, respectively. Group II of the table gives a relationship between the kind of acyl group and the susceptibility. This enzyme is also active on N-acyl pep-tides other than isovaleryl peptides. That is, acetyl-Val-Val-~ - 25 -X-Ala-X, propionyl-Val-Val-X-Ala-X, isovaleryl-Val-Val-X-Ala-X~
n-caproyl-Val-Val-X-Ala-X, and isocaproyl-Val-Val-X-Ala-X were all split to organic acids, L-valine and Val-X-Ala-X. No N-acyl-peptide bearing an acyl group at the N-terminal of the valine adjacent to X, such as N-acyl-Val-X-Ala-, is susceptible to this enzyme, as shown in Group III. This particularly enzyme N ~-V~ ~e ~id,A -.-r~ e~

- 25 a -~ Z 3 in their molecules to produce peptides (Va~-A~ orga~ic acids and ~-va~ine. Prior to this invention, no enzyme with the above me~tio.~ed aotion and substrate speci~icity has ever been kno~m.
~d)-2 Optimum pH and plI stability ~ he optimw~ pH of the enzymatic reaction is 7.0 ~he hydrolysis reaction may a~so take place at a ~reakly acidic or a weakly alXaline pH as sho~n in ~igure 1. This en~.yme was stable between pH 6 and 9 as showm i.n ~igure 2.
(d)-3 Assay of the activity ~he enzymatic activity is ass~yed by measuring by gas chromatography the organic acid released from ~-acyl peptide.
The activity was assayed i.n the present invention according to the fol~owing method: A reaction mixture consist-ing of 5 mg/ml IVA-'~'al-Val-X-Ala-~, 0.05M phosphate buffer, pH 7.0, and e.nzyme in a total volume of 1.0 ~1 was incubated at 37C for 30 minutes. Isovaleric acid formed was extracted with ether and assayed by gas chromatography with ~-valeric acid as an interDal standard. A unit of activity corresponds to the formation of one n mole of isovaleric acid per minute.
Ihe enzyme activity may also be assayed by measuring I-valine or Val-~-Ala-X with an amino acid autoanalyzer or by densitometry on a thin-layer chromatogram treated with ninhy-drin. ~he method described in ~xperiment 1 ma~ also be employed.
(d)-4 Optimum temperature ~he optimum temperature is 63C~ IV4-Val-Val-X-Ala-X
was i.ncubated with the enæyme under the co.nditions described above in "Assay of the activity" except at various temperatures.
The resu~ts are shown in ~igure 3~ ~he tempera-ture range for optimum activity is 15C to 70C.

4~ 1 2 (d)-5 ~hermostabili-ty ~ he enzyme is markedly thermostableO After treatment at 80C. ~or ten minutes at pH 7.0, 60 ~ o~ the origina~ activi-ty still remained. Heating at 90C for 10 minutes eliminated 98% of the activity. ~igure 4 is a thermal inactivation curve.
(d)-6 I.nhibition and activation Inhibition o~ the enzyme was observed in the presence o~ 2n~l o-phe.nanthroline9 p-chloromercuriben~oate? ~-mercapto-ethanol, dithiothreitol, N-bromosuccinimide and HgC12. Addi~ion of Co~ gave an increase in activity of 34~0 at 2 m~l and 66 ~0 at 10 m~ and addition of Ca~+ gave a 29 % increase in activity at 10 mM. ~able 7 shows the effects of various reagents on the acti~ity~
~ able 7 : Ef~ect o~ various inhibitors and metal ions ~ _ . . .
i.nhibitors and metal ions Concentratio.n Relative (n~I) actiYity ,, _ . - ~
~thylenedi~minetetraacetic Acid 2 30 . o-Phenanthroline 2 0 . .p-Chloromercuriben~oate ~ 2 Mo.noiodoacetic Acid 2 84 Diisopropylphosphorofluoridate 10 100 ~-Mercaptoethanol 2 0 .
Dithlothreitol 2 0 : NaN~ 2 100 l ~-~romosuccinimide 2 0 : ~ead Acetate 2 43 Mercuric Acetate 2 0 Cobalt Chlo~ide 2 134 . 10 166 Calcium Chloride 2 110 Zinc Chloride . 66 . 10 44 Coutrol (No Addi~ion) _ 100 .

4~ 1 ~ 3 (d~-7 Physicochemical properties Visc ge~ eleotrophoresis provided a single band of a R~p~ v~lue of 0.048 at plI 9,5 in 7.5 %, acrylamide gel and 0.232 in 5 %, gel. Gel electrofocusing showed a single band of an iso~lectric point at pH 4~2. Molec~lar weight of the enzyme was calculated by the gel filtration method to give a Yalue of 345~000. ~he sedimentation coefficient was calculated as 14.5S. In SDS gel electrophoresis a single band was observed the molecular weight of ~hich was calculated as 45,500. ~his suggests the presence of subunits.
As stated above, this particular enzyme has several novel and unique characteristics~ Primarily, acid protease inhibitors such as pepstatins are restrictively and specifi-cally hydrolyzed by this en~yme. Secondarily, the enzyme ~Yhich shows a single band in disc gel electrophoresis, gel electro-focusing, and ultracentrifugation has both acylase and peptidase - activity. In addition, it has unique substrate specificity.
Only N-acyl-valyl-valyl-4-amino-3-hydroYy-6-methylheptanoyl compounds are susceptible to this enzyme. N-acyl valine, ~-acyl ~alyl-valine and N-acyl valyl-4-amino 3-hydro~y-6-, methylheptanoyl compounds are not attacked. ~he bond suscep~
i tible to this enzyme is a linkage between an acyl group and I-valine and a linkage between ~-valine and I-valine. A bond bet~leen I-vaLine and X, ana others are not splito l~o enzyme haYing the above mentioned characteristics has ever heretofore been ~ound. ~his enzyme is useful for the method of the pre-sent inven~ion, that i5, for production of the novel physio-logically active peptide and N-acyl derivatives. In addition, the possible application of the enz~me to other processes, for example, modification of antibiotics, hormones, enzyme inhibi-tors and production of novel physiologically active substances by a combination of enzymatic reaction and chemical synthesis9 ~ 2 3 ma~ be presumed.
~e) Identification o~ the hydrolysis product ~ he produc-t of the enzymatic degradation of R-Val~
Val-X-Ala-X is a novel compound as stated above. Idenkification and properties of the compound are as follo~Js:
~ he compounds was obtained as colorless crystals, w3th m.p. of 17~-172C (colored at 168 C) and L ~ 23 = _ 51.0 (1 ~0, by weight, in methanol). ~igure 5 shows the ultraviolet spectrum~ '~he infrared spectrum is shown in ~igure 6, in which the ~ollowing bands are observed: 3320, 3090, 2955, 1660, 1545, 1470, 1450, 1390, 1300, 1260, 1178, 1080~ 938, 890, 860, 760, and 650 cm 1, ~mino acids analysis of hydrolysate of the crystals in 6N HCl at 105C ~or 15-24 hours gave I-valine~
I-alanine and 4-amino-3-hydroxy-6-methylheptanoic acid in the ratio of 1 : 1 : 1.6 to 1.9~ No organic acid was detected with an ether extract of the hydrolysate by gas chromatography~
Elementary analysis showed C=57~37, H=9.16, N=11.20 and o=22,27, which provided C24H4607~49 Considering that the compound was the hydrolysi~ product of R-Val-Val~ la-X) the following s~ructure was proposed, I-~alyl-4-amino-3 hydroxy-6-methyl-hsptanoyl-I-alanyl-4-a~ino-3-hydroxy-6-methylheptanoic acid, ~he mass spectrum of an acetylated compound of the product also indicated that structure. ~herefore, the structure of the hydrolysis product of R~Val-Val-X-Ala-X by the aforementioned enzyme was considered determined.
~ he product is soluble in methanol, ethanol, pyri-dine, and acetic acid, slightly soluble in n-proponal, n-buta-nol, ~-amyl alcohol and acetone, but insoluble in ether, ethyl acetate and butyl acetate. It gives positive thionyl chloride, 3o hydroxamic acid-ferric chloride, potassium permanganate, Rydon-Smith and ninhydrin reactions but negative ~hrlich, Sakaguchi, napthoresorcinol, anisaldehyde-sulfuric acid reactions.

l~flllZ~

~ 'he following Rf vaLues for this compound are observed in thin-layer cl~romatography using a silica ~el p~ate : 0.57, 0.15 and 0.23 in the sol~ent systems of n-butanol-ace-tic acid~
water (4 : l : 1 by vol~), n-butanol-butyl ace-tate-acetic acid-~rater (4 : 1 : 1 : 1 by vol.) ~nd aqueous n-butano~, res-pectively. '~he product mi~rate9 towards the cathodes as a cation in high voltage paperelectrophoresis at 3,500 V for 15 minutes) with a buffer solution of forMic acid - acetic acid -water (25 : 75 : 900 by vol ), (f) Acylation Acylation of the peptidet Va~ -Ala-X, obtained by hydrolysis of R-VaL-VaL-X-Ala-~ witn the various enzyme prepa-rations described in (a)-(d) is carried out at the amino group o~ the valine according to conventional methods, A~ny suitable acylating agent possessing the group desired to be introduced into the amino ~roup may be used.
Suitable exa~ples include carboxylic acid halides, carboxylic acid anhydrides or mixed anhydrides, thiocarboxylic acids and esters of halogenocarbonlc acidsA ~hese agents m~y bear any group to be bound to the amino group of the peptide, and the aforementioned group of the agent may a~50 have a moiety such as hydroxy, a~koxy7 amino or halogeno group~ ~he following are examp~es of such agents : carbox~lic acid ha~ides, carbo-~ylic anhydrides or mixed anhydrides, thioc~rbo ~lic acid ana-.Logs, and esters of halogenocarbonic acids possessing a formyL, acetyl, propionyl, n-butyryl, isobutyryl, n-valeryl, isovaleryl, n-caproyl, isocaproyl, n-heptanoyl, capryloyl, capryl, lauroyl, myristoyl, palmitoyl, stearoyl, arachidoyl, oleoyl ? erucyl, linoleoyl, linolenoyl, ~-hydroxypropionyl, lactoyl, oxalyl, malonyl, benzoyl, cinnamoyl, phthaloyl, acryloyl, phenoxyacetyl or phenoxypropionyl groupO

- 3~ -~lV~3lZ3 Suitab~e conditions ~or the acylating reaction may be employed according to conven-tional methods, N-acylated peptide is ~ormed b~ the following reaction~
CH3 \ / CH3 CH~ ~ CH3 ~ acylating ~H
H2~-HC-C0-~-Ala-X Acyl-N-HC-C0-X-~la-X
(their salts or esters) H
When esters o~ the peptide are used for the reaction, free ~-acyl peptide may be ob~ained by sapo~ification o~ the esterified product. As already described, the ~T-acyl pepti-des thus obtained are novel and valuable compounds which exhi-bit various physiological acti~ities. Possible utilization of the particular substance is presumed in the fields of medicine, biochemistry and the like.
(g) Anti-protease activity and to~icity As an example of one of the physiological activities of the peptide and ~-acyl derivatives -thereof obtained according to the present invention, anti-pepsin and anti-cathepsin D acti-vities and also acute toxicity are given in ~able 8. The acti~vi~ies (ID50) were assayed according to the method described above and ~he Journal o~ ~ntibiotics 25 689 (1972). ~`he anti protease activities of the peptide and ~-acyl derivatives thereof are extremely high, Using mice to estimate the toxi-city, it was found that the toxicity was xemarkably low.

l3!LZ3 Tab~e 8: ~nti-protease activity and acute toxicity . . _ _ . -Peptide and lT-acy~ Peptide ID50 (mcg/~l) Acute Pepsin Cathepsin D tox c ty _ _ _ __ .. .
Val-X-Ala-X 9.98 605 ~Do 5000 or more Acetyl-Val-X-Ala-X 0.031 0042 ~Do 4000 or more Isobutyryl-Val-X~Ala-X 0~021 0.28 _.
Isovaleryl-Val-X-~la-X 0.01 O. 045 ~Do 3000 or more Benzoyl-Val-X-Ala-X 0.031 O. 05 ~D50 1350 Phenoxyacetyl-Val-X-Ala-X 0.02 0.008 ~D50 1500 Phenoxypropionyl-Val-X-Ala-X 0.02 0,01 ~D50 2 0 Palmitoyl-Val-X-Ala-X ~ _ Note: Acute toxicity was examined by administrating the compounds to the nuce by abdo~inal injection, .According to the present invention, the ne~ peptide o~ the formula Val-X-Ala-X and various ~acyl derivatives there-of may be prepared, for example, N-formyl, M-acetyl, N-~ropionyl, N-butyryl, N-isobutyryl, N-valeryl, N-isovaleryl, N-caproyl, N-isocaproyl, N-heptanoyl, N-isoheptanoyl, N-anteisoheptanoyl, N-octanoyl, ~T-lauroyl, N-myristoyl, N-palmitoyl, N-stearoyl, M-eleoyl, N-benzoyl, N-phthaloyl, N-acryloy.l 7 N-phenoxyacetyl.
N-phenoxypropionyl, N-ox~lyl, M-malonyl9 and N-~-hydroxypro-pionyl peptide.
BIO~OGICA~ PROP~R~Y AND APP~ICATTON
~he peptide VaL-Y~ La-X and its N-acyl derivatives ~(~4V~L23 according to this invention ha~e low toxiclty, and mice, dogs, rats and rabbits tolerated oral ad~inistration of more than 2,000 mg/kg o~ the compounds and ~50 o~ t~e compound~ to an~-mal by the intraperitoneal injections are sho~ in ~able 8.
Daily 250 mg/kg of oral administration each of the compounds to rats for 90 days caused no toxicity and rats grew with the Dormal growth rate.
A strong pepsin inhibitor has never been kno~m before the discovery of pepstatin in USP 3740319 ana USP 3840516 and the analogous compounds of the present invention. Though s~l-furic esters of polysaccharide have been known to inhibit pep-sin, its effect is ver~ weak and it also inhibits blood caV~u~a-tion. ~he present cornpounds are strong inhibitor of pepsin as sho~m by 50~0 inhibition concentration described above and it has no effect on blood coagu~ation. The strong effect inhi-biting pepsin indicates that these compounds are effective on stomach ulcer. Really it has been confirmed by the clinical ~tudies as described in USP 3740319 and USP 38405160 Stomach ~lcer of rats made by the method described by ~akagi et al. in the Japanese Journal of Pharmacology, 18, 9-18p, 1968, that is, placing male rats in a cage at 23 C for 22 hours, are protected or therapeutically treated by the compounds. ~hen 50 mg/kg of pepstatin and the compounds according to the invention were orally given 30 min.'before the stress, and rats were sacrificed 48 hours after the stress.
~he curative ratio of the ~lcer was 60 - 75%. When the dose was 10 mg/kg, the inhibition percent of the ulcer was 50 - 60~.
When 50 - 200 mg/kg of the compound was given immediately after the stress and one daily for 4 days, and rats were sacrificed.
~he rapid cure of ulcers were proved in rats treated with pe,-statin and the analogous compound.

~ - ~3 -The effect against stomach ulcers of rats caused by pylorus ligation(shay rats) is also sho~m. ~he method de-scribed by 1,latanabe and Kasuya in Chemical Pharmaceutic~l ~lletin~ 11, 1282, 1963 was employed. In rats administered with 2 - 10 m ~ kg or the larger dose 30 minutes and 14 hours after pylorus ligation no ulcer was found~ ~ifty ~0 inhibition dose was 0.5 - 3 mg/kg.
Pepsin acti~ity in gastric juice was none or less than 10% of that of the control. Thus, they showed strong protective and curative effect against ~hay rats ulcer~
The following examples illustrate methods of carrying out the present in~Tention, but it is to be understood that they are given for the purpose of illustration and not of limitation.
Example 1 A medium consisting of 1~o peptone, 0.7 ~0 meat extract, 0.5~0 glucose and 0.3 % NaCl, all by wei~nt, pH 7.0, was pre-pared. ~he medium ( 50 ml/500 ~1 flask ) was ~teriliæed at 120a for ten minutes and inoculated with a c~lture of ~
sphae_icus A~CC 14577 After incubation on a reciprocal shaker (120 r.p~m.) at 30C for 48 hours, cells were harvested by centrifugation at 10,000 r.p.m. and suspended in 0~01M phos-phate bufferg pX 7.0 (OD610 = 35). A mixture of 50 ~1 of the cell suspension, 50 ~ of 2 mg/mL insovaleryl-~-valyl-~-valyl-4-amino-3-.hydroxy-6-methylheptanoyl-~-alanyl-4-~mino-3-hydroxy-6-methylheptanoic acid and 0.5 ml of toluene was incubated at 37C for 24 hours. A supernatant fluid obtained by centrifu-gating the incubated mixture aftsr pH adjustment to pH 2 with HCl, was applied onto a Dowex 50 (H+) co~umn (~ = 3 x 5 cm).
~he column ~7as washed with X20. Active frac-tions eluted with o.5N ~H40H were combined and extracted with an equal volume of n-butanol, Chromatography of the extract was performed . . .

using a s~lica ~el colu~n (b = 2 x 30) with a. solvent system of n-butanol-acetio acid-~I20 (4 : 1 : 1 by ~ol.), The active fraction was applied onto a Sephaclex ~I-20 co~umn (,~ - 1 x 50 cm~ for gel ~iltrationO Grystals were obtained by concentra-tion of the active fraction from the Sephadex column, Recrys-tallization in me-thanol pro~ided 45.3 mg of crystalline I~valyl-4-amino-3-hydroxy-6-methylheptanoyl- L alanyl-4-amino-3-hydroxy-6-methylheptanoic acid ~Val-X-Ala-X)~ Using scherichia coli ATCC 11303 in exactly the same manner, the same starting mate-rial was hydroly~ed to give 36.5 mg of crystalline Val-X-~la-~r.
~2 ._ acillus me~aterium ~ 13938 (ATCG 13639) was grown in a manner similar to Example 1 and the cells were removed.
To 200 ml of the broth filtrate was added 100 mg of n-caproyl-~-yalyl-I~alyl-4-amino-3-hydroxy-6-methylhep-tanoyl-I~alanyl-4-amino-3-hydroxy-6-methylheptanoic acid. The mixture was incubated at 37C with gentle stirring for 24 hours. In a way similar to Example 1, 42.8 mg of crystalline Val X-Ala-~
was obtained.
Using Sta~99e9~d~ A~CC 155 by the same procedure as above, 29 mg oî crystalline Val-X-~la-X was obtained.
Example 3 13acillus s~haericus ATGG 14577 was grown in the same manner as in Example 1 except it was incubated for 20 hours.
~he culture was added aseptically, in a ratio of 5 ml per 100 ~1, to a fermentation broth of pepsta-tin, which has been cultivated for 96 hours, produced according to ,he exampLe in U.S. Patent No. 3,740,319.
~he fermentation at 27C was continued for another 35 hours. ~y the same procedure as in Example 1, 38.8 mg oî

" ~4~LZ3 crystal~ine Val~ A~a-~ was recovered ~rom 1~ o~ the broth filtrate. Using ~re_iba I~=DL_~ aC~ A~CC 68711 in the same manner, 25,7 mg of crystalline Val-X-Ala-X was obtained, Example 4 .
~acillus sphaericus A~C 1~577 was grown in the same manner as in ~xample 1. At 16 hours of growth, ~ine powder of the same starting materia~ as used in ~ample 1 was asepti-call~ added to the culture to give a final concentration o~
2 mg/ml. After a further 30 hours cultivation, 39.5 mg of crystalline Val-X-Ala-X wa6 recovered from 50 ml o~ the broth filtrate by the same procedure as described in ~xample 1, ~y the same procedure, 32 mg of crystalline Val-X-Ala_g was obtained using I~icrobacterium lacticu~ A~CC 8180.
Example 5 A cell suspension of Brcl rus ~ n~RL B938 (A~CC 13639) prepared by the same procedure as in Example 1 - was lyophilized. ~o 50 ml o~ the same solution of starting material as used in ~xample 1, 0~7 g o~ lyophili~ed cell pre-paration and water were added to give a total volume of 100 ml.
~he pH was adjusted to 7Ø ~he mixt~e was incubated at 37C
for 24 hours. ~y the same procedure as in ~ample 1, 45.4 mg of crystalline Val-X-~la-X was ~ecovered.
Using nterobacter~ _s A~CC 8329 in the same procedure as above, 30.2 mg of crystalline Val-X-Ala-X was obtained.
Example 6 A cell suspension of ~acillus ci cul~næ A~CC 1~403 was prepared by the same procedure as in ~xample 1, The sus-~0 pension was treated with a ~rench press and a cell-free extract was obtained by centrifugation at 10,000 r.p.m. ~or 20 minutes.

~4~ 3 Ammonium s~lfate (30 g) was added to 50 ml of the extract~ ~he preoipitate was dissolved in a small ~uantitY of 0,01 M phos-phate buf~er? pX 7.2, and dia.lyzed against the same buf~er.
~he dialyzed so~ution was adsorbed onto a D~AE cellulose column (~ = 3 x 30 cm) equilibrated to the sa~me buffer~ ~he column was washed ~ith the same buffer and the en~yme was eluted by the same buffer containing 0.3 M ~aCl~ A mixture of 50 ~1 of the partially purified en~yme solution (OD28o=25) and 50 ~ of the same so.lution of starti~g material as used in Example 1 was incubated at 37C for 5 hours. ~y the same procedure as in Example 1, 49 mg of crystalline Val-X-Ala-X was obtained from the reaction mixture. Similarly, by using ~lc~ enes faecalis A~CC 8750, 36 mg of crystalline was obtained.
Val-X-~la-X
~xample 7 A medium consisting of 1 part wheat bran and 1 part 0.2 % by weight, yeast extract solution ~^ras sterilized at 120C
for ten minutes and inoculated ~rith a culture of acillus ¦ 20 ` subtilis ~RR~ ~543 (A~C 10783)o Cultivatio.n was per~ormed at30C ~or 96 hours, ~he enzyme was extracted with a 5-fold . volume of 0.01~;I p.hosphate buffer, pH 7~Q~ A mixture of 200 ~l of the e~xtrac-t and 100 mg of fine po~der of the same starting material as used in ~xample 1 was incubated with gentle stir ring at 37C for 20 hours, ~y the same procedure as described in ~x~mple 1, the incubation mixture provided 43.2 mg of crys-talline Val-X-Ala-X.
. Example 8 ~ medium containing 200 ~l potato extract solution9 5 g glucose, 15 g glycerine, 30 g yeast extract, 5 g beef extract, 2 ml ethanol, and 20 g CaC03 in a tota~ volume of 1~ , ~ 2 3 pH 7.0, t~ras prepaxed, The medium was introduced into a Roux flas~ to give a 5 mm depth, ster~liæed at 120C ~or 15 minutes.
and lnoculated with a culture of cetobacte~
NRR~ ~65 (ATCC 7839). C~ltivation was carried out at 30C
for 72 hours and the cells were harvested, ~y using the c~ells in the same manner as in Example 1, 27 mg of crystalline Val-X-~la-X ~as obtained, Example 9 A medium containing 200 ~1 potato extract solutio.n, 20 g glucose, 1 g yea~t extract, and 20 g agar in a total volume of lQ, pH 605 - 7.00, was prepared. The medium was introduced into a Roux flask to give a 5 mm dep-tn, sterilized at 120C for 15 minutes, and i.ncoulated with Xanthomonas cam-Pestris ~UR~ ~1459 (A~CC 13951), The flask was incubated at 30C for 72 hours. ~he cells were harvested ~rom the agar culture, ~y using the cells in the same manner as in Example 1, 37 mg of crystalline Val-,r~-Ala-X was obtained.
Exampls 10 A medium consistiDg of the same composition as that in Example 1 and 2 %, by weight, agar was prepared and in~ro-duced into a Roux.flask to give a 5 mm depth. The medium was sterilizad at 120C for 10 minutes and inoc~Lated with A~ro-bacterium radiobacter ATC~ 4718~ After incubation at 30C
for 72 hours~ the cells were harvested from the agar c~lture.
~y using the cells in the same procedure as in Example 1, 32.5 mg o~ crystalline Val-X-Ala-X was obtained, Example 11 - . Using Aeromonas h~drophila ~URL B909 (I~I 1018), in the same procedure as in ~xample 1, the ~ydrolysis gave 25 mg o~ crystalline Val~ Ala-~, ' ~xample 12 ~ 3 Using Protc~mlnobacter rub~r N1~ 1048 (ATaa 8457) in the same procedux0 as in ~xample 1, the degradation provided 24.~ mg o~ crystalline T~al-~-Ala-~.
Example 1~
Using Microc~clus flavus ATCC 23276 in the same procedure as in ~xample 1, the hydrolysis gave 22.5 mg of crystalline Yal-X-Ala-X.
Example 14 Using Citroba ~ reundii ATCC 8090 in the same procedure as in Example 1, the hydrolysis gave 28 mg of crys-talline Val-X-~la-X.
~xample 15 - A medium containing 2 % glucose, 1 % peptone, 0.001 NaCl, 0.05 ~0 ~H2P04~ 0.05 ~0 K2HP04, 0.02 yO MgS0~. 7~2~ 0.001 MnS04 , 5H20 and 0.001 ~p ~eO4 . 7H20, all by weight, pH 6.8, was prepared and sterilized at 120C for 10 minutes. The sterilized medium was aseptically intro~uced into a sterilized 500 ml flask up to the neck of the flask and inoc~lated ~ritn a seed culture of Clostxidium butyricum ATW 6014 prepared by cultivation in 5 ~0~ b~ we~ght, corn medium. The flask was incubated at 37C for 48 hours. ~y the same procedure as in ~x~mple 1, using the cells harvested from ~he culture, the hydrolysis gave 32,2 mg of crys~alline ~al~ Ala-X.
Example 16 A medium containing 0.55 ~0 yeast extract, 1.25 c,~
peptone, 1,1 ~ glucose, 1 ~0 CH3COO~a.3II20, 0.01 % MgS04 . 7H20, 0,005 ,~OIiinS04 . 5H20, 0.0005 ~O FeS04.7H20, 0.025 ~O -~2P04, 0.025 % K2HP04, 20 ~ liver extract solution and 0.5 ~ CaC03, all by weight, pH 7.0, was prepared and sterili~ed at 120C
for 10 minutes. The medium was introduced into a sterilized . . ~
. .
~ ~ 39 ~

500 ml flasl~ up to the neck of the ~lask and inoculated with Stre ~ococcu~ ~aeca1is A~CC 8043, ~he flask was incubated at _~ .
37C for 48 hours.
~ y the same procedure as ~n ~xam~le 1, using the cells harvested ~rom the culture, the hydrolysis gave 17.5 mg of crystalline Val-X-Ala-X.
~xample 17 Using euc~ Q~ enteroides I~URL ~1299 ~ATCC
11449) in the same procedure as in Example 16, the degradation yielded 15~8 mg of crystalline Val-X-Ala-V.
Example 18 ~ Using ~a~tobacillus brevis ~TCC 8287 in the same method as in 3xample 16, the degradation provided 15.9 mg of crystalline Val-~Y-Ala-lY.
3xample 19 Using Pro~ionibac-teriu~ shel~manii ATCC 13673 in the sa~e procedure as in ~xam le 1~, the hydrolysis gave 14 mg of crystalline Val-X-Ala-X.
3xample 20 A medium of the same composi-tion as in ~xample 1 was prepared and sterili3ed at 120C for 10 minutes. An amount of n-caproyl-I-valyl-l-valyl-4-amino-3-nydrox~-5-methylheptanoyl-I-alanyl-~-amino-3-hydro~y-6-methylheptanoic acid was asepti-cally added to the medium to give a concentration of 5 mg/ml.
The medium containing n-caproyl-Val-Val-X-Ala-X was inoculated ~ith Ba llus me~aterium ~ 938 (ATCC 136~9). The cultiva-tion was conclucted in the same manner as in Exampls 1 for 48 hours. The cultured broth was diluted 5-lold with water and filtered after the pH was adjusted to 2.00 ~0 Crystalline Val-X-~La-X (70.1 mg) was obtained from 50 ~1 of the filtrate in the s~me man-ner as in 3xample 1.

- , .. . ..

Example 21 A medium containing 0,5 7' glucose, 0,5 ~0 starch, 0.5 ,' soybean meal, 0.1 c,~ ~2P0~ and 0.05 /~ MgS04.7~0, a~l by weight, was prepared. The medium (50 mg in a 500 r~l flask) was sterilized at 120C fcr 10 minutes and inoculated with Kaba-- tiella eau~ivora ATCC 20439. ~he flask was incubated on a rotary shaker (220 r.p.m~) at 28C for 72 hours. The cells were harvested by centrifugation at 3000 r.p.m. and suspended in 0.01M phosphate buPfer (OD61o=55). Using the cells in a similar procedure to that described in Example 1, the hydro-lysis gave 43 mg of crystalline Val-X-Ala-X.
Example 22 ~usarium s~. AT~C 20440 was OE own in a way similar to ~xample 21 and a broth filtrate was prepared. To 200 ml of the filtrate was added 100 mg of finely powdered n-caproyl-~-valyl-~-valyl-4-amino-3-hydroxy-6-methylheptanoyl-l-alanyl-4-amino-3-hydroxy-6-methylheptanoic acid. ~he reaction mixture was incubated at 37C with gentle stirring for 24 hours. By the same procedure as in Example 21, 44 mg of crystalline Val-X-Ala-X was obtalned.
~xample 23 ~ ATCC 20442 was grown for 48 hours inthe same manner as in Example 22. ~he cul-ture was aseptically added to a fermentation ~roth of pepstatin, which has been cultivated for 96 hours, obtained according to the same procedure as that of ~xample 3, in a volumetric ratio of 1 to 5, and fermentation was continued at 28C for another 3~
hours, ~rom 1~ of the broth filtrate from the fermentation process 36 mg of crystalline Val-X-A~a-X was recovered by a procedure similar to that described in Examp~e 21.

_ 41 -~xample 2~
.
Rhizoct~ CC 20~43 was c~tivated for ~8 hours in the same manner as in ~ample 21. The same starting material as used in ~xample 21 was aseptically added to the culture to give a final concentration of 2 mg~ he c~ltiva-tion was continued for another 48 hours. ~y a similar proce-dure to that described in ~xample 1, 30.5 mg of crystalline Val-X-~la-X was recovered from 50 ~1 of the culture ~iltrate.
Example 25 A cell suspension of Colletotrichum sP. ATCC 20438 prepared bythe same procedure as in ~xample 21 was lyophilized.
A reaction mixture containing 1.1 g of the lyophilizedcells and 50 ~l of the same solution of starting material as used in Example 1 in a total volume of 100 ml, pX 7.0, was incubated at 37C ~or 24 hours. ~y a similar procedure to that described in ~xample 17 43.0 mg of crystalline Va1-X-Ala-X was obtained~
3xample 26 A cell suspension of Ascoh~rta ~haseoloru~ AT~C 14728 prepared by the same procedure as that in ~xample 21 was treated with a ~rench press and a cell-free extract was ob-tainéd by cen~rifugation at 10,000 r.p.m. for 20 minutes~ By the same procedure as described in Example 16, using -the cell-free ext~act3 the degradation gave 50.8 mg of crystalline Val-X-Ala-X.
Example 27 ,~
A medium consisting of 1 part ~rheat bran and 1 part0,2 ~0, by weight, yeast extract solution was prepared and ster-sterilized at 120C ~or 10 minutesO ~he medium was inoculated with Kabatiella caulivora A~CC 20439, and in¢ubated at 28C
3~ for 120 hours. The enzyme was extracted with a 5-~old volume ~ . , ~ V9~
o~ 0.01M phosphate buffer~ pH 7 0. r~he same ~ub~trate as used in ~x~mple I was incubated with ~entle stirring at 37C for 20 hours. ~y a similar procedure to that described in ~xample 1, 45 mg of crystalline Val-X-Ala-X was recovered ~rom the incubation mixture.
Example 28 -A meaium containing 0.5 % glucose, 0.7 % meat extract, 1 ~0 peptone and~0,3 ~ NaClt all by weight, pH 7,0, was prepared.
~he medium (10`~ ) was introduced into a 20~ jar ~ermentor and sterilized at 120 ~or 10 minutes~ ~or Pxeparation of a seed culture, 50 ~l of the same medium was introduced into a 500 ~l flask and inoculatt~ with Bacillus circ~lars A~CC 13403.
The flask was incu~ated on a reciprocal shaker (120 r.p.m.) at 30C for 24 hours. ~o the jar fermentor 100 ml of the seed culture was added and the cultivation ~as accomplished by stirring at 300 r.p.mQ with aeration of 5~/min at 30C for 48 hours. ~oluene was added to give a final concentration of 2 ~J~
by volume to the cultured broth at pH 7Ø ~he mixture was incubated with gentle ~tirring at 30~ for two hours and then filtered. ~nzyme activity of the clear filtrate (8.7~) was 0.8 unit/~1. ~o the liltrate 5.3 kg of (~)2S04 was added with cooling and stirring to obtain a precipitate~ A
half o~ the precipitate was l~ophilized. ~he activity o~ the lyophilized powder (5.45 g) was 520 units/g. ~he o-ther ha1f of the precipitate wa~ dissolved in 1,~ of H20 and dialyzed to remove ammonium s~lfate and then, lyophilized. A crude enzyme preparation (3.78 g) with an activity of 625 units/g was obtained. Yields were 81.4 ~0 and 6708 ,'o re~pectively.
Example 29 A reaction mixture containing 100 mg of isovaleryl-l-valyl-~-valyl-4-amino-3-hydroxy-6-methylheptanoyl-I.-alanyl-4-. .
.
_ ~3 -4~)~23 amino-3-hydroxy-6-methylheptanoic acid~ 250 units of purified enzyme prepared by the procedure described in Experiment 17 and 0.05N phosphate buffer, pH 7.2, in a total volume of 100 ml was incubated at 37C for 20 hours. By a procedure similar to - that described in Example 1, 61.8 mg of crystall~ne Val-X-Ala-X
was obtained.
Example 30 To 51 m8 of Val-X-Ala-X dissolved in 15 ml of H2O at pH 8.5, 1 ml of acetyl chloride was added dropwise with stir-ring at room temperature while maintaining the pH of the reac-tion mixture at 8.5 with lN ~aOH by using a pH-stat (C0MBI-TITRATOR~30, METROHM HERISAU, SWITZERLAND). After the reaction was oompleted~ the pH was ad~usted to 1.8 with 6N HCl. The resulting solution was extracted with 4 x 5 ml of n-butanol.
The butanol layer was washed with 3 ml of water three times and then evaporated. The resulting residue was dissolved in 0.5 ml of methanol and 30 ml of H2O was added.
The solution was passed through a Dowex 50 x 8 column (50-100 mesh, H+, 10 ml) and the column was washed with 20 ml of H2O. The effluent and the wash water were collected, com-bined and evaporated to give, after drying, 45 mg of white powder which was N-acetyl-Val-X-Ala-X. The field was 81 /~.
Anal. Calcd for C26H48N4O8 : C~57.33; ~,8.88; N~ 10-29; 0~23-Found: C,57.29; H,8.91; N~ 10.05; 0,23.83Example 31 By a procedure similar to Example 30, using isobuty-ryl choride, acylation of 50 mg of Val-X-Ala-X gave 32 mg of white powxer of N-isobutyryl-Val-X-Ala-X in a S0 % yield.

Anal. Calcd for C28H52N4O8 : C,58.72; H,9.15; N~9-78; 0~22-35 Found: C,58.95; H,9,16; N~9.56; 0,22.12 Trademark ~44_ Example 32 By a procedure similar to Example 30, using iso-valeryl chloride, acylation of 50 mg of Val-X-Ala-X gave 28 mg of white powder which was N-isovaleryl-Val-X-Ala- in a ~8 %
yield.
Example 33 To 100 mg of Val-X-Ala-X dissolved in 5 ml of metha-nol 54.2 mg of benzoic anhydride was added dropwise with stir-ring and cooling in an ice bath. The solution was stirred overnight at 5 - 7 C and 5 ml of H2O was added. After evapo-ration to remove methanol, the resulting solution was extracted with ether to give a white precipitate between the ether and aqueous layers. The precipitate was collected and dried. The residue was washed with ether and dissolved in a small volume of methanol and, then, the solution was applied to a Sephadex LH-20 column (~ = 1.5 x 80 cm) packed in methanol. The effluent was collected and evaporated to give 32 mg of N-benzoyl-Val-X-Ala-X as a white powder in a 32 % yield.
Figure 10 shows the infra red absorption spectrum (KBr pellet) of 20 N-benzoyl-Val-X-Ala-X and Figure 17 shows the NMR spectrum of the same.
EXA~PLE _ To 50 mg of Val-X-Ala-X suspended in 3 ml of dioxane 42.9 mg of phenoxyacetic anhydride dissolved in 3 ml of dioxane was added dropwise with stirring at room temperature. The solution was stirred overnight and 10 ml of H2O was added.
Dioxane was removed by evaporation. The resulting solution was washed with ben~ene twice and extracted with ethylacetate. The ethylacetate layer was evaporated and the residue was dissolved 30 in a small volu~e of methanol. Gel filtration with a Sephadex LH-20 column provided 22 mg of white powder which was N-phenoxy-acetyl-Val-Ala-X in a 44 % yield.

t Figure 11 shows the infra red absorption spectrum (KBr pellet) of N-phenoxyacetyl-Val-X-Ala-X and Figure 15 shows the NMR speckrum of the same.
Example 35 To 50 mg of Val-X-Ala-X dissolved in 4 ml of M2O a-t pH 8.5, 20.2 mg of 2-phenoxypropionyl chloride dissolved in 1 ml of acetone was added dropwise at roorn temperature with stirring while maintaining the pII at 8.5 with 1 N NaOH by using a pH-stat. The solution was stirred Eor an hour at room tempe-rature and evaporated. The residue was extracted with a small volume of methanol. Sephadex LH-20 gel filtration gave 49 mg of N-2-phenoxypropionyl-Val-X-Ala-X as a white powder in 75 g6 yield.
Figure 12 shows the infra red absorption spectrum (KBr pellet) of N-2-phenoxypropionyl-Val-X-Ala-X and Figure 16 shows the NMR
spectrum of the same.
Example 36 By a procedure similar to Example 35, using oxalyl chloride, acylation of 50 mg of Val-X-Ala-X gave 39 mg of N-oxalyl-Val-X-Ala-X in a 68 % yield.
Example 37 By a procedure similar to Example 35, using malonyl chloride r acylation of 50 mg of Val-X-Ala-X gave 25.1 mg of N-malonyl-Val-X-Ala-X in a 43 % yield.
Example 38 By a procedure similar to Example 35, using palmitoyl chloride, acylation of 50 mg of Val-X-Ala-X gave 57.5 mg of N-palmitoyl-Val-X-Ala-X in a 78 % yield.
Example 39 To a solution of 50.2 mg of Val-X-Ala-X in 2.5 ml of H2O was added 50.4 mg of NaHCO3 at 0C. Then, 20.6 mg of monobromopropionyl chloride dissolved in 2 ml of chloroform lZ3 was added dropwise with vigorous stirring. The stirring was continued for another 60 minutes after the addition of the acylating agent. An aqueous layer was collected and 1 ml of lN NaOH was added. The resulting solution was refluxed at 100C for two hours and allowed to cool to room temperature.
The reaction mixture was extracted with 3 x 2 ml of n-butanol after acidifying with HCl. The organic layer was washed with 2 x 2 ml of H2O and evaporated. The residue was dissolved in methanol. Gel filtration with Sephadex LH-20 gave 27.3 ml of N-~-hydroxypropionyl-Val-X-Ala-X as a white powder in a 43 %
yield.
Example 40 Reflux of 100 mg of Val-X-Ala-X dissolved in 10 ml of HCl-methanol at 60 C for two hours and evaporation of the mixture provided methyl ester of Val-X-Ala-X. The ester (residue) was dissolved in 10 ml of methanol and neutralized with triethylamine.
To the solution 5-fold moles of acetic anhydride was added dropwise with stirrin~. The reaction was continued with stirring overnight and monitored by thin layer chromatography and ninhydrin reaction.
The resulting mixture was evaporated and the residue was dissolved in 5 ml of H2O and then extracted with 3 x 5 ml ethylacetate. The organic layer was evaporated to give methyl ester of N-acetyl-Val-X-Ala-X. The material was dissolved in 3 ml of methanol and 3 ml of lN NaOH was added. Saponification of the ester was accomplished by refluxing the mixture at 60C for three hours.
The resulting solution was neutralized with HCl. Gel filtration with Sephadex LH-20 gave 70.4 mg of N-acetyl-Val-X-Ala-X as a white powder in a 65 % yield.
Some physical properties of the N-acyl tetrapeptides in accordance with the present invention are summerized in Table 9.

- ~7 -~ .

Table 9: Physical properties of the N-acyl tetrapeptides name Rf value m.p.
(I) (II) ( C) Val-X-Ala-X (material) 0.01 0.01 171 - 172 Acetyl-Val-X-Ala-X 0.09 0.05 122 - 124 Isobutyryl-Val-X-Ala-X 0.17 0.12 125 - 128 Isovaleryl-Val-X-Ala-X 0.19 0.15 128 - 132 Benzoyl-Val-X-Ala-X 0.24 0.21 123 - 125 Phenoxypropionyl-Val-X-Ala-X 0.19 0.18 110 - 113 2-Phenoxypropionyl-Val-X-Ala-X 0.24 0.21 103 - 105 Palmitoyl-Val-X-Ala-X 0.34 0.17 207 - 210 Oxalyl-Val-X-Ala-X 0.04 0.02 Malonyl-Val-X-Ala-X 0.43 0.23 ~-Hydroxypropionyl-Val-X-Ala-X 0.27 0.21 Note: thin layer chromatograph with silica gel GF (Merck Co., Inc.) and solvent: (I) chloroform : methanol : acetic acid 100 : 10 : 2 (II) n-buthanol : n-butylacetate : acetic acid : H2O
: 100 : 4 f~3 SUPPI.EM~NTARY DISCLOSUR~

The present Supplementary Disclosure relates to the preparation of lactoyl-Val-X~Ala-X.
Example 41 To a mixture of 0.08 ml of lactic acid and 5 ml of dried dimethylformamide, 162 mg of N,N'-carbonyldiimidazole was added and stirred at room temperature for 1.5 hours. A solution consis-ting of 502 mg of Val-X-Ala-X, 20 ml of dried dimethylformamide and 0.14 ml of triethylamine was added to this mixture dropwise with stirring. The stirring was continued further 24 hours at room temperature. A residue obtained by evaporation of the reaction mixture _ vacuo was dissolved in 40 ml of water and applied onto a Dowex 50 column (x8, Httype, 50-100 mesh, 20 ml).
The column was washed with water. The effluent and washings were combined (200 ml) and washed with 20 ml of ethylether three times.
Evaporation ln vacuo of the aqueous layer gave 429 m~ of white powder. The powder was dissolved in a small volume of methanol and applied onto a silica gel column ~Mallinckrodt Co., 100 mesh, 200 ml). Chromatography was performed with a solvent system of n-butanol - n-butyl acetate acetic acid - water (8:8:1:1, by volume) and 2~6 mg of white powder was obtained by evaporation of the active fractions. The powder was suspended in 10 ml of 0.lN ~laOH and the suspension was stirred at room temperature for 20 hours. After the pH of the solution was adjusted to 2.0 with 0.lN HCl, the aimed product was extracted by 20 ml of _-butanol three times. Evaporation ln vacuo of the butanol layer, after washing with water, provided 229 mg of lactoyl-Val-X-Ala-X.
The yield was 40%.
Anal. Calcd. for C27H50N4Og : C 56.45, H 8.71, N 9.76j 0 25.09 Found: C 56.68, H 8.90, N 9.59, 0 24.71 M.W.: 575 mp : 113-5C

~-~' .

UV spectrum: End absorption Organic analysis: lactic acid The infra red absorption spectrum (KBr pellet) and the mass spectrum of lactoyl-Val-X-Ala-X are shown in the appended Figures 19 and 20, respectively.
The above compound has been tested under the same conditions as for the peptides give~ in Table 8 of page 41, and showed an ID50 (mcg/ml) of 0.02 (pepsin) and 0.08 (cathepsin D), and an LDo (acute toxicity, mg/kg) of 6,000 or more and LDo (intra-venous injection) of 550 or more.
Lactoyl-Val-X-Ala-X is much more water soluble than other derivatives and known anti-acid protease inhibitors as shown in the following Table 10.
Table 10 Solubility in water Solubility(mc /ml,20C~
~ g N-lactoyl derivative50,000 or more N-acetyl derivative 10,000 N-isovaleryl derivative 5,000 N-phenoxyacetyl derivative20,000 N-2-phenoxypropionyl derivative 15,000 Protease inhibitors Pepstatin A 200 Pepstatin B 100 Pepstanon A , 50

Claims (16)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for the preparation of N-acyl peptides of the general formula:

R'-Val-X-Ala-X

wherein R' is an acyl radical having 1 to 16 carbon atoms or said acyl radical partially substituted by one or more hydroxyl groups or phenoxy groups or halogen atoms, Val is L-valine, the X between Val and Ala is 4-amino-3-hydroxy-6-methylheptanoic acid and Ala is L-alanine, the other X is 4-amino-3-hydroxy-6-methylheptanoic acid or salt or an ester thereof and the amino group of Val being bound to said acyl radical by an amide bond, the carboxyl group of Val being bound to the amino group of the X between Val and Ala to form a peptide bond, the carboxyl group of the X between Val and Ala being bound to the amino group of Ala to form a peptide bond, the carboxyl group of Ala being bound to the amino group of the other X to form a peptide bond, and the carboxyl group of the other X being free or esterified or bound to a cation to form a salt, which process comprises decomposing an N-acyl peptide of the formula:
R-Val-Val-X-Ala-X

wherein R is an acyl radical having 2 to 8 carbon atoms or said acyl radical partially substituted by one or more hydroxyl groups or halogen atoms and Val, X and Ala are as defined above, the acyl radical being bound to the amino group of Val by an amide bond, the carboxyl group of the Val which is bound to R
being bound to the terminal amino group of Val-X-Ala-X to form a peptide bond and Val-X-Ala-X is the same as in the R'-Val-X-Ala-X
defined above, specifically to a tetrapeptide of the formula:
Val-X-Ala-X
wherein the meaning of the formula is as defined above, with a microbial enzyme having both aminoacylase and peptidase activities and characterized by a substrate specificity for N-acyl peptides of the general formula:

R-Val-Val-X-Y

wherein R, Val and X are as defined above and Y is a member selected from the group consisting of amino acid, peptide and hydroxyl residues, and the carboxyl group of the X being bound to amino group of the amino acid or the peptide to form an amide bond when Y is amino acid or peptide and being free when Y is hydroxyl residues, and by the capability of decomposing said N-acyl peptide specifically to carboxylic acid, L-valine and Val-X-Y, acylating said tetrapeptide with an acylating agent possessing an R' group defined above to provide the desired said N-acyl peptide of the general formula:

R'-Val-X-Ala-X

wherein R', Val, X and Ala are as defined above and recovering the resulting N-acyl peptide.

2. A process according to claim 1, in which an N-acyl peptide of the formula:

R-Val-Val-X-Ala-X

wherein R, Val, X and Ala are as defined in claim 1, is decomposed specifically to a tetrapeptide of the formula:

Val-X-Ala-X

wherein Val, X and Ala are as defined in claim 1, with a microbial enzyme having both aminoacylase and peptidase activities and characterized by a substrate specificity for N-acyl peptides of the general formula:

R-Val-Val-X-Y
wherein R, Val, and X are as defined in claim 1 and Y is a member selected from the group consisting of amino acid, peptide and hydroxyl residues, and the carboxyl group of the X being bound to amino group of the amino acid or the peptide to form an amide bond when Y is amino acid or peptide and being free when Y is hydroxyl residues, and by the capability of decomposing said N-acyl peptide specifically to carboxylic acid, L-valine and Val-X-Y and the resulting tetrapeptide is recovered.

3. A process according to claim 1, in which a tetrapeptide of the formula:

Val-X-Ala-X

wherein Val, X and Ala are as defined in claim 1, is acylated with an acylating agent possessing an R' group defined in claim 1 to provide an N-acyl peptide of the formula:

R'-Val-X-Ala-X

wherein R', Val, X and Ala are as defined in claim 1, and the resulting N-acyl peptide is recovered.

4. A process according to claims 1 or 3, in which the acylation is effected with an acylating agent of the group consisting of carboxylic acid halides, carboxylic anhydrides or mixed anhydrides, thiocarboxylic acid analogues, and esters of halogenocarbonic acid, possessing formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, caproyl, isocaproyl, heptanoyl, capryloyl, capryl, lauroyl, myristoyl, palmitoyl, stearoyl, arachidoyl, oleoyl, erucyl, linoleoyl, linolenoyl, lactoyl, .beta.-hydroxypropionyl , oxalyl, malonyl, benzoyl, cinnamoyl, phthaloyl, acryloyl, phenoxyacetyl and phenoxypropionyl group.

5. A process according to claims 1 or 2, in which the enzyme is obtained by cultivating a microorganism selected from the group consisting of Bacillus megaterium NRRL B938 (ATCC 13639), Bac. circulans ATCC 13403, Bac. sphaericus ATCC 14577, Bac. cereus ATCC 9634, Bac. subtilis NRRL B543 (ATCC 10783), Clostridium butyricum ATCC 6014, Pseudomonas Acetobacter rancens NRRL B65 (ATCC 7839), Aeromonas hydrophila NRRL B909, Protaminobacter ruber NRRL B1048 (ATCC 8457), Microcyclus flavus ATCC 23276, Agrobacterium radiobacter ATCC 4718, Alcaligenes faecalis ATCC 8750, Escherichia coli ATCC 11303, Cictrobacter freundii ATCC 8090, Enterobacter aerogenes ATCC 8329, Micrococcus rubens ATCC 186, Staphylococcus epidermidis ATCC 155, Sarcina lutea ATCC 9341, Brevibacterium ammoniagenes ATCC 6871, Streptococcus faecalis ATCC 8043, Leuco-nostoc mesenterodides NRRL B1299 (ATCC 11449), Lactobacillus brevis ATCC 8287, Propionibacterium shermanii ATCC 13673, Corynebacterium equi ATCC 6939, Microbacterium Lacticum ATCC 8180, Cellulomonas fimi ATCC 8183, Arthrobacter urea-faciens ATCC 7562, Macrophomina phaseoli ATCC 20441 Ascochyta phaseolorum ATCC 14728, Colletotrichum sp. ATCC
20438, Kabatiella caulivora ATCC 20439, Stemphylium sarcinaeforme ATCC 20442, Rhizoctonia sp. ATCC 20443 and Fusarium sp. ATCC 20440 in a medium whereby said enzyme is formed and recovering said enzyme from said medium.

6. A process according to claim 1 or 2, in which the microorganism is one selected from the genus Bacillus.

7. A process according to claim 1 or 3, wherein the acylation is effected with an ester of halogenocarbonic acid possessing a lactoyl group.

8. A process according to claim 1, which further comprises reacting an N-acyl peptide of the formula:

R'-Val-X-Ala-X

wherein the meaning of the formula is as defined in claim 1, with a cation to provide the corresponding pharmaceutically acceptable salt thereof.

9. A process according to claim 1, which further comprises reacting an N-acyl peptide of the formula:

R'-Val-X-Ala-X

wherein the meaning of the formula is as defined in claim 1, with an esterifying agent to provide the corresponding pharma-ceutically acceptable ester thereof.

10. An N-acyl peptide of the formula:
R'-Val-X-Ala-X

wherein the meaning of the formula is as defined in claim 1, whenever obtained by a process according to claim 1 or its obvious chemical equivalents.

11. An N-acyl peptide according to claim 10, in which R' is a member selected from the group consisting of formyl, acetyl, propionyl, n-butyryl, isobutyryl, n-valeryl, isovaleryl, n-caproyl, isocaproyl, n-heptanoyl, capryloyl, capryl, lauroyl, myristoyl, palmitoyl, stearoyl, arachidoyl, oleoyl, erucyl, linoleoyl, linolenoyl, lactoyl , .beta.-hydroxy-propionyl, oxalyl, malonyl, benzoyl, cinnamoyl, phthaloyl, acryloyl, phenoxyacetyl and phenoxypropionyl residues, whenever obtained by a process according to claim 1 or 3 or its obvious chemical equivalents.

12. An N-acyl peptide according to claim 10, in which R' is lactoyl radical, whenever obtained by a process according to claim l or 3 or its obvious chemical equivalents.

13. A pharmaceutically acceptable salt of an N-acyl peptide whenever obtained by a process according to claim 8 or its obvious chemical equivalents.

14. A pharmaceutically acceptable salt of an N-acyl peptide whenever obtained by a process according to claim 9 or its obvious chemical equivalents.

CLAIMS SUPPORTED BY THE SUPPLEMENTARY DISCLOSURE

15. Process according to claim 3, wherein the acylation is effected with an ester of halogenocarbonic acid possessing a lactoyl group.

16. An N-acyl peptide according to claim 8, in which R' is lactoyl radical, whenever obtained by a process according to claim 11 or its obvious chemical equivalents.