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July 9, 1963 HIRAo sHlMAzoNo ETAI- 3,097,145 ACID PROTEASE AND THE PRODUCTION THEREOF 3 Sheets-Sheet 1 Filed March 30, 1962 FIG I ha)2Y|DOE7(PNT5ISECAL ACTIVITY AGAINST CASEIN EFFECT OF pH O c» ON FIG 2 ^ 30 l l l I 2 3 4 è 5 6 7 8 Q 25 pH N ACIlvlTx m E20 AGAlNsT HAEMOGLOBIN cn l5 EFFECT oF pH Z ä 1o _1 FIG 3 3 5I: 5 D. ACTIVITY )2DlENO7/SPIT5LYC)AE. O | 2 3 4 5 5 AGAlNsT ALBUMEN EFFEcToF pH N PH FIG 4 $6 Èloo > E) 75 §TAB|L|TY <x ^ _j 50 è E ~ ë g 25 w ä ll-I C» fr ì AQIlVlIY g4 '-U O I: 5 l l | | l | 2 s 4 5 e J 7 EFFECT oF TEMPERATURE I 3 pH AGAINST CA§E|N a’ 2 INVENTORS H/'rao ôfhl'mazano 9 E EFFECT OF pH Kafsum/ Tomada l i . o by WMM ¿M/¿??awgä 2o 3o 4o 5o so 7o TEMPERATURE (oC) Aìtornays July 9, 1963 HIRAo sHlMAzoNo ETAL 3,097,145 Acïn PROTEASE AND THE PRODUCTION THEREOF Filed March 50, 1962 3 Sheets-Sheet 2 FIG 6 ë loo > egaal-_IH eo g wlIHQLE SUESTRATE E eo EFFECT 0F TEMPERATURE <1 _J <1 :> 40 g zo m Lu Ir 4o 5o eo 7o TEMPERATURE (°C) FIG 7 ULTRAVIOLET ABSOR PTION DOEPNTSICAYL SPECTRUM 5 0.5 230 240 250 260 270 280 290 300 3IO WAVE LENGTH (MILLIMICRONS) _ . /N VENTORS H/rao .S‘h/mazono Kafsum/ Tomada Afforneys July 9, 1963 3,097,145A HIRAO SHIMAZONO ETAL ACID PROTEASE AND THE PRODUCTION THEREOF 3 Sheets-Sheet 5 Filed March 50, 1962 70 | I4 750 B0 850 9|0H500 (cnFR/E1QU"N)CY | |20 IO (MILCWERNOAGVST)HE 8 FIG |3450 0 20 0 23540IO0 O /N VEA/70H5 TRANSMITTANCE (°/°TRANSMIS 0N) H/'roo S@ im ozono Kofsum/ Tomado by «JWM/W »L WHMMÉ, Altar/lays United States ant ‘ ice 3,097,145 Patented July> 9, 1963> 2 l As the classification of fungi is rather complicated and a microorganism is sometimes called by two or more 3,097,145 ACID PROTEASE AND THE PRODUCTION THEREOF Hirao Shimazono, Suita, and Katsumi Tomoda, Toyo naka, Japan, assignors to Takeda Chemical Industries, Ltd., Osaka, Japan Filed'Mar. 30, 1962, Ser. No. 183,919 5 Claims. (Cl. 19E-_62) names which may be quite different from each other, the descriptions of the microorganisms in this specifica tion is based on the system set fort-h in “Mycological Flora of Japan” by Seiya I-to, vol. Il, published :by Yokendo, Tokyo, Japan, in 1955. Thus, the principal object of the present invention is to provide a process for producing novel protease, espe cially the acid protease, which has stable and strong ac This invention relates to a method for producing pro 10 tivity fio-r catalyzing the hydrolysis of peptider bonds in tease, and more particularly, to a method for the produc proteins at an acid pH. »tion .of acid protease, which is useful in the -food industry Another object of this invention is to provide a method as well as for medicinal purposes such as a digestive. for the production of acid protease which can be etil It is known that proteases, which have a catalyzing activity on the hydrolysis of peptide bonds in various 15 ciently carried out on an industrial scale with very good yield. proteins, are contained in animal viscera or in the cells It is `a further object of the invention to provide -an acid and culture broth of microorganisms such as molds and protease which is novel and useful in the `food industries, bacteria, and some of these proteases can be industrially and for medicinal purposes such as a digestive, etc. ow produced and are commercially available. Proteases may ing to its stability and strong activity to degrade proteins be divided on the basis of the optimal pH at which they 20 at an acidic pH. exhibit the said catalytic activity into three groups, i.e. The first object is realized by incubating a fungus neutral protease, -alkaline protease and acid protease. selected from those belonging to the family Polypcraceae commercially available proteases are almost exclusively in an appropriate liquid medium containing assimilable neutral proteases; carbon sources, digestible nitrogen sources and other 25 One of the shortcomings of known protease .production necessary nutrients for the growth of the fungus under is the low yield «of protease inevitably caused by the poor aerobic conditions, and by recovering from the resulting capacity of the microorganisms employed for producing broth the protease produced. the protease. Another defect is that neutral protease The microorganism used in the method of this inven shows neither desirable catalytic action nor stability under 30 tion can be selected from those belonging to the family acidic conditions of pH lower than 4. Polyporaceae. Among tho-se microorganisms of this In sharp' contrast, the microorganisms employed in the family, Tramezes sanguínea (L. ex Fr.) Lloyd (=P0lypresent Iinvention can produce and accumulate acid pro stictus sanguìneus Fr.), Trametes cinnabarìno (Jacq.) Fr. tease in a high concentration in the culture broth. More (=P0lyslictus cinnalbarinus Sacc.), Poria vaporaria (Fr. over, the acid protease shows maximum catalytic activity Pers.) Cooke, etc., for example, have been found at an acidic pH ranging about from l to 4, and this char 35 non to be especially suitable for the process of this invention acteristic of the acid protease Iis especially suitable lfor a because of their :properties not only to produce respec digestive which acts against dietary protein in the stomach tively a stable acid protease having very strong activity where gastric fluid provides strongly acid conditions of but also to accumulate a large amount of the -acid pro about pH l to 3. . tease du-ring the cultivation in a liquid medium in a tank. It is known that acid protease is produced by Asper 40 That is to say, .an industrial production of 'acid protease gilli, and that industrial production has -been tried, em has become possible by the use of these microorganisms. ploying one of these microorganisms, for rendering the For the purpose of industrial production, it is in gen useful acid protease commercially available. However, eral lpreferable to use liquid culture media. Thus, the these attempts have not yet yielded any desirable result because of the fact that the cultivation of the micro 45 present invention now makes it possible to produce acid protease industrially. organism has been carried out lby solid culture, for ex As the assimilable carbon sources, one or more of ample according to the so-«called koji-process, and such glucose, dextrin, soluble starch, sucrose, maltose, lactose, a process cannot readily be scaled up to industrial magni etc., for example, may be used, and various organic corn tude. The present invention is based on the use of micro organisms belonging to the family Polyporaceae, these being widely, capable of producing protease in good yield; 50 pounds or organic materials such as organic ammonium salts, organic nitrates, urea, various amino acids, corn steep liquor, peptone, polypeptone, casein, meat extract, soybean cake, soybean flour, potato juice, etc. may be more especially, the protease thus produced is usually used not only as carbon source but also as digestible acid protease showing a remarkably strong and stable activity to catalyze Ithe hydrolysis of peptide bonds in 55 nitrogen source in the same way as the use of inorganic nitrogen sources, for example, inorganic ammonium salts, proteins. such as ammonium sulfate, ammonium carbonate, am Based upon the widely acknowledged classification of monium phosphate, ammonium nit-rate, etc. or inorganic microorganisms, the family Polyporaceae is placed in the nitrates such as sodium nitrate, potassium nitrate, etc. following situation: 60 In addition, mineral salts, phosphates, vitamins or growth factors may desirably be used for the culture media as accessory nutrients, e.g. water-soluble salts of vitamin B1. The initial pH of the culture medium is adjusted to Order _______________________ _- Agaricales Family _______________ __ Polyporaceae about 3 to 7. When Trametes sanguínea (L. ex Fr.) Lloyd is used for the production of the acid protease, it It is a novel feature of this invention that any of the 65 is preferred to incubate the microorganism in -the liquid microorganisms belonging to even thewide scope of the medium adjusted to and kept at a pH of about 3, espe class Basidiomycetes, not to speak of those of the family `cially Ifrom 3.0 to 3.2, because the resulting culture filtrate Polyporaceae, can produce acid protease; more espe contains more than two -times as much acid protease as cially, it is a novel 'aspect of this invention that the pro 70 in the case where the ¿medium is adjusted to labout pH 5. tease production is carried out by the use of said micro Subdivision Class ________________________ __ Eumycetales ________________ ..1 _____ _.- Basidiomycetes organisms belonging to the family Polyporaceae. (The preparation' of acid protease is also possible even 3 3,097,145 by the means `so-‘called koji-process where solid materials such a-s sawdust, rice fbuan, wheat bran, etc. are used for the culture.) The incubation is carried out desirably at a tempera ture of Iabout 25-35° C. Iand the accumulated protease in the culture broth reaches «the maximum usually be tween the second and fthe fifteenth day of incubation. 4 ments was »acetone powder of enzymes extracted from the iiltered broth obtained «by the cultivation of Trametes sanguínea (L. ex Fr.) Lloyd. (a) Action on beef-Ln 20 milliliters of an aqueous suspension of 0.1 gram of chopped beef in a bulîer solu tion of pH 2.8, «the protease preparation in a quantity cor responding to 10 milligrams of protein was allowed to Under such conditions, »the protease produced by the mi react with the chopped beef at 55° C. After 20 minutes, crooragnisrn is not accumulated within mycelia, but pene no precipitate formed on the addition of trichloroacetic trates out to the environment and is accumulated in the l0 acid to the mix-ture. The absorption of the suspension at culture medium. Therefore, in the method of the pres the wave length of 275 millimicrons-which corresponds ent invention, the accumulated protease is mainly recov to the characteristic absorption band of tyrosine--re ered ffrom the liquid part of the culture broth, usually markably increased and reached the maximum 80 min from the filtered broth. utes after the start of the reaction. After 100 minutes, Generally-known means for recovering enzymes from 15 the chopped beef lost its intrinsic stickiness as meat and their solutions can ‘bepapplied to the recovery of the pro vbecame brittle. tease by utilizing -the properties of the same. The pro On the other hand, another chopped beef suspension tease obtained by the present invention can be 'adsorbed having the same composition as above was allowed to on various «adsorbents, or can be precipitated by precipi stand `at 55° C. without addition thereto of the protease tants. Moreover, generalmeans for recovery such as preparation, whereupon' no change was observed during precipitation near the ‘isoelectric point, saltin-g out, di alysis, and combinations of these expedients may be far longer period than 100 minutes. Reaction time, minutes: Absorption 1 effected for the purpose of recovery and purilication. For example, an aqueous solution containing the pro ___________________________________ __ 0.000 20 __________________________________ __ 0.455 tease is subjected to salting out Iby the addition of :a salt 25 40 __________________________________ __ 0.460 such as sodium sulfate, ammonium sulfate, sodium chlo 60 __________________________________ __ 0.540 ride, or the like to the solution, or to fractional precipi 80 __________________________________ __ 0.680 tation by the addition of an appropriate hydrophilic or ganic solvent such as methanol, ethanol, normal-pro panol, acetone, dioxane, ttetrohydrofurane, or the like to 30 the solution. As the acid protease obtained by the present invention 100 _________________________________ __ 0.680 1(E-value) of beef suspension (1 cm. layer) at the wave length of 275 mu. (b) Action on "waste yeast.”---“Waste Yeast,” which is obtained as waste material from the extraction of ribo nu-cleic acids from yeast and of course is not soluble due concomitantly with its susbtrates, i.e. proteins-as well 35 to cell walls, is solubilized by the action of the acid pro as at fairly strong acid pH, the filtered «broth wherein con tease. In each of 5 milliliters of the enzyme solutions is stable at a rather high tempera-ture such as 40 to 50° C.--or 60 to 70° C. when the acid protease is placed centration of the protease is still low, may `directly be listed below in the concentration of 300 milligrams per concentrated under reduced pressure. I-f desired, it may cent, 100 milligrams of Waste yeast was suspended, and be freeze-dried. the suspensions were kept at 37° C. for 24 hours to give When it is desired to employ lan «adsorption process, ad 40 the result shown Ibelow: sorbents such as calcium phosphate, alumina, bentonite _a colloidal native hydrated aluminium silicate (clay) solubilized _magnesium silicate, carboxymethylcellulose, weak acid carboxylic acid-type cation exchange resin, and -the like, may advantageously be employed. Enzyrnes - Acîdîty of the medium As the cation ex 45 yeast (percent) change resins, Ifor example, “Duolite CS~-10l”-manufactured and sold by Chemical Process Co., U.S.A. “Amberlite lRC-50”-manufactured and sold by Rohm & Haas Co., U.S.A.-etc., are especially suitable for this purpose. These resins 'are commercially available and can 'generally be produced by such methods as described, for example, in “Ion Exchange Resins,” second edition, pages 85-87, by Robert Kunin, published by John Wiley & Sons, Inc., U.S.A., in 1958. Adsorption and elution solid part rel ative to the original solid part in waste N/lOO-HCI acidic _______ __ 11. 9 Trgpsin _________________ __ pH 7 (phosphate buüer)... 26.4 Acid protease preparation. N /100-HC1 acidic _______ ._ Pe sin __________________ __ 61. 0 The following examples set forth presently-preferred exemplary embodiments of the present invention; they >are intended to be `solely illustrative, however, and not at rall limitative of the invention. In the examples, per may be carried out 'by column chromatography or in 55 centages are on the weight basis unless` otherwise noted. batch process. “ATCC” refers to American Type Culture Collection, The protease may be precipitated from its aqueous so Washington, D.C., U.S.A. lution by the addition of `a proteín-precipitant such as Example I nucleic acids, tannic acid, phosphotungstic acid, or the like. Tnametes sanguínea (L. ex Fr.) Lloyd (IPO-7045) was 60 inoculatedin 5 liters of the culture medium of pH about Precipitation of the protease is facilitated by adjust 5.2 which consisted of 5 percent of dext-rin, 2 percent of ing the pH of the solution to .fthe isoelectric point. Elec cornsteep liquor, 0.15 percent of potassium dihydrogen trodialysis may also .be employed for the purpose of puri phosphate, 0.05 percent of magnesium sulfate (7 hy iication o-f the acid protease. The ‘acid protease thus produced and recovered has 65 drate), 2 milligrams per liter of thiamine hydrochloride, and water, and then incubated for 6 days under shaking the outstanding properties of strongly catalyzing the hy at la temperature between 25 and 30° C. At the end of dolysis of peptide-bonds in proteins, and the catalytic the incubation, mycelia were plentifully grown. The action is independent of the kind of protein involved. culture ibroth was centrifuged to separate a clear aqueous Therefore, the protease can remarkably degrade, a Vari layer from the mycelia. Ethanol was added to` the aque 70 ety of proteins such as casein, hemoglobin, albumin, etc. ous layer to obtain 25 »grams of white enzyme powder. to the same degree. The enzyrnic activity as protease of the powder at pH 'I'he following illustrations show the activity of the 3.0 was: acid protease and indicate the practical utilization of the same. The protease preparation used in these experi 75 3,097,145 il The Iabbreviation to show protease activity, i.e. PUâiìâlyî" 2"’ m“ means that casein as substrate was exposed to the action of the protease at 37° C. for 20 minutes and the increase in the light absorbence at the wave length of 275 mil lirnicrons during the reaction was measured; and that the absorbence corresponding to 1 milligram equivalent of tyrosine at »the same w-ave length was «taken as the unit of enzymilc' activity. The abbreviation “/mg. P.” means per milligram protein. 6 was gradually added, while cooling,y ca. 20 percent by volume of `cooled acetone, and the mixture was allowed to stand in an ice chest. After 24 hours, crystals began to appear and the crystallization was completed after 4 to 5l days. The protease activity of the crystalline enzyme thus obtained was: m2563222' 275 m: ses/mg. P. It was found that about 10 percent of the protease activi-ty in the culture íiltrate was preserved in .the crystalline prod uct. It was also observed that the speciiic activity of the product was ca. 20' times as high as that of the culture Tram-etes cínnabarína (Jacq.) Fr. (IPO-6139) was ñltrate. employed instead of Trametes sanguínea (L. ex Fr.) In .the event that the dialyzed solution «is colored, the Lloyd in the procedure precedingly set forth in this ex color-imparting impurities can be eliminated by passing 15 ample, acid protease powder was again obtained. The the colored solution through a tower packed with a Weak strains of Trametes sanguínea (L. ex Fr.) Lloyd and base anion exchange resin (eg. Duoli-te A-7, manufac Tmm-etes cinnaibarina (Jacq.) Fr. used in the present tured and sol-d by Chemical Process Co., U.S.A.) which example are on deposit at ATCC under accession numbers can be produced, for example, by »the method described ATCC-14622 and ATCC-14623, respectively. 20 by Kunin, supra, pages 87-88 and 97. The thus decolored Example 2 solution is then further processed as precedingly described. The crystalline acid protease obtained in this example Poria vaporaria (Fr. non Pers.) Cooke (IPO-7044) shows ythe following properties: was inoculated in 5 liters of the culture medium of pH (1) Its optimum pH is about 2.3 to 2.5 as shown in 5.2 which consisted of 5 percent of glucose, 0.3 percent of polypepton-e, 0.15 percent of potassium dihydrogen phos 25 FIG. l of the accompanying drawings and its optimum temperature is about 55 to 60° C. as shown in FIG. phate, 0.05 percent of magnesium sulfate (7 hydrate), 5-when the activity is tested against casein as substrate. 0.2 percent of yeast extract, 2 milligrams per liter of It also shows the optimum »pH 2.8 against hemoglobin thiamine hydrochloride and water and then incubated as shown in FIG. 2 and the optimum pH 3.4 against egg for 6 days under shaking at a temperature from 28 to 30° C. After the incubation, the culture broth was ñlte-red 30 albumin as shown in FIG. 3; (2) `It is stable in the pH range from 2.0 to 6.5 as to obtain filtered broth. The protease activity of the shown in FIG. 4, and loses the activity by heating at iiltered broth was determined through a modiñcation of 70° C. lfor 10 minutes without substrate as shown in Kunin’s method by the use of lactate buffer solution of FIG. 6; pH 2.8 to be: 35 (3) Its ultraviolet absorption spectrum shows the curve of .a typical protein which has the maximum absorption at the wave length of 277-280 millimicrons, as shown in Ethanol W-as added to the filtered broth to obtain 20 FIG. 7; grams of enzymic powder preserving the acid protease activity. The strain of Porìa vaporaría (Fr. non Pers.) Cooke used in the present example is on deposit at ATCC under number ATCC-14624. Example 3 (4) I-ts uniformity is coníirmed through electrophoresis 40 by Tiselius’ apparatus, where 15 percent of the enzymic protein in phosphate buifer (,u=0.1) of pH 6.08 is sub jected to an electric field of 72 volts at the current of l0 mil-liamperes for 3 hours; (5) The uniformity of the protease was ¿further estab Trametes sanguínea (L. ex Fr.) Lloyd (ATCC-14622; 45 lished through the analysis by ultracentrifuge in acetate buffer (n=0.l) of pH 5.0 at a speed of 9.667><10'2 rounds .the same strain as used in Example l) Was inoculated in 30 liters of the culture medium of a pI-I of 3.0‘ to 3.2 in per second for 75 minutes. a tank, «the medium consisting of 5 percent of dextrin, 2 9.75><10“13, Vfrom which the molecular weight of the sedimentation constant was percent of cornsteep liquor, 0.15 percent of potassium acid protease was calculated to be about 3.0)(104; (6) The elementary analysis of the protease: dihydrogen phosphate, 0.05‘ percent of magnesium sul 50 fate (7 hydrate), 2 milligrams per liter of thiamine hy dnochloride, and water, and then incubated for 6 days under agitation and aeration at a temperature of 25 to C, percent H, percent N, percent 30° C. After the incubation, \ he culture broth was filtered` to obtain filtered broth. Acetone was added to the ñltered 55 broth to give 150 grams of white enzyme powder. The (1) ____________________________________ ._ (2) ____________________________________ __ (3)__-_ ___ 44 18 44 16 __... 7. 12 7. 12 _\ __ 14. 2 12. 78 13.37 enzyme powder was dissolved in a small amount of hydrochloric acid-sodium acetate bulfer solution of pI-I 3.5 in the molar concentration `of 0.02 mole, and the solu (7) The infrared absorption spectrum of the protease tion was allowed to pass through a column of weak acid 60 is shown in FIG. 8 and the significant absorption bands carboxylic yacid-type cation exchanger (“Duolite CS 101”), which had been pretreated with the buffer solu t-ion of the same constitution as above-used, and then the protease absorbed in the resin was eluted with the hy drochlori‘c acid-Sodium acetate buffer solution of pH 65 5.5 in the molar concentration of 0.5 mole. From the eluate, fractions showing protease activity were collected and submitted to fractional precipitation by ammonium in microns are as follows: 3.0 (strong) 3.38 (middle) 6.105 (broad, strong) 6.55 (broad, strong) 6.88 (weak) 7.15 (broad, middle) 8.12 (middle), and 9.3 (broad, weak) sulfate. Precipitates obtained at between 40 and 80 per ment saturation of ammonium sulfate were collected and 70 (8) It is easily soluble in water but insoluble in or dissolved in a small amount of said buffer solution of pH ganic solvents such as methanol, ethanol, acetone, ether, 3.5 in the molar concentration of 0.02 mole, and the solu benzene, petroleum ether, etc.; and tion was subjected `to dialysis for 24 hours against the (9) Crystal form of the protease is usually needle or buffer solution of the same constitution as the solvent. Into the solution remaining Within the diaphragm, there 75 -platelet shape. 3,097,145 Having thus disclosed the invention, what is claimed is: 1. A method for producing acid protease, which com (4) sta-bility in the pH range Ifrom 2.0 to 6.5, as indi cated in FIG. 4 of the drawings; prises incubating a member selected from the group con (5) maximum absorption, in its ultraviolet absorption sisting of Trametes sanguínea (L. eX Fr.) Lloyd, Trametes cinnabarz'na (Jacq.) Fr. and Porìa vapomria (Fr. non 5 Pers.) Cooke in an aqueous medium containing assimi lable carbon sources, digestible nitrogen sources and other nutrients necessary for the growth of the microorganism at a temperature of from 20 to 35° C. under aerobic conditions until the acid protease is substantially accumu 10 lated in the culture broth, ‘and recovering the accumulated acid protease ‘from the liquid part of the culture broth. ATCC-14623. 4. The method as claimed in claim 1, wherein the microorganisrn is Poria vapora‘rìa (Fr. non Pers.) Cooke, 20 ATCC~14624~ 5. A crystalline acid protease, metabolite of an acid protease-producing microorganism belonging to the family Polyporaceae, which has the following properties: (l) optimum activity, when tested against casein as 25 substrate, at a pH of about 2.3 to 2.5, as indicated in =as substrate, at a pH of about 2.8, as indicated in as substrate, at a pH ‘of about 3.4, as indicated in FIG. 3 of the drawings; (8) infrared absorption spectrum as shown in FIG. 8 3.0 (strong), 3.38 (middle), 6.05 (broad, strong), 6.55 (broad, strong), 6.88 (weak), 7.15 (broad, middle), 8.12` (middle), and 9.3 (broad, weak); (9) soluble in water but insoluble in organic solvents methanol, ethanol, acetone, ether, benzene and petro leum ether; (10) elementary analysis being between 44.16 and 44.18% by weight carbon, about 7.12% by weight hydrogen and between 12.78 and 14.2% by weight nitrogen, the latter as determined by the Dumas method. FIG. 1 of the drawings; (2) `optimum activity, when tested against hemoglobin » of the drawings; (7) molecular weight .of about 3.0)(104; are as follows: microorganism is Trametes sanguínea (L. ex Fr.) Lloyd, ATCC-44622. 15 3. The method as claimed in claim 1, wherein the microorganism is Trametes cìnnabarina (Jacq.) Fr., FIG. 2 of the drawings; microns, as indicated in FIG. 7 of the drawings; (6) 'loss of activity upon being heated at 70° C. for 10 minutes without substrate, as indicated in FIG. 6 wherein the significant ‘absorption bands in microns 2. The method as claimed in claim 1, wherein the (3) optimum activity, when tested against egg albumin l spectrum, at the wave length of 277-280 milli 30 References Cited in the ñle of this patent Gonshvili: Chem. Abst., 46, 4591d (1952). Levine et al.: Journal of Bacteriology, 1953, pages 10-15, page 11 particularly relied on.