DE1417575A1 - Process for making acid-resistant protease - Google Patents

Description

Method for producing acid-resistant protease The invention relates to a method for producing acid-resistant protease with a. optimal pH of about 2.7 by growing Kuro-k mold strains in an assimilable carbon and nitrogen containing = @ eermedum: In US Pat. No. 2,848,3'j1 is: a method for: creating a new proteolytic Enzyme with the help of Aspergillus usamii, Aspergillus saitoi and the like 'oehrleen ,, 1., s enzyme has an optimal pH of 2.7 and is grown in a liquid culture medium containing 3% wheat bran and 2% defatted soybeans ..

The invention is concerned with the composition of the for manufacture this enzyme most suitable (liquid or solid) nutrient medium .. There were Already numerous studies on the formation of protease using it made by mold Aspergillus on proteases, their activity in the neutral to alkaline range. At - the formation of protease, its activity and optimal pH value is approximately 3.0 and which is resistant to acids is, however, the yield is low and, moreover, the number of usable types of mold for this purpose are limited. It is known that some species of mold of the black Aspergillus type produce proteases that are approximately acid-resistant Contain protease with an optimal pH of about 2.7, and that the acid-resistant Protease can be isolated by a specific purification process; see USA patent 2,848,371; F. Yoshida, Bul. Agr. Chem. Soc., Japan 20, 233 (1956).

To date, there has been little systematic research into education the protease with the optimal pH of approximately 3.0 and an acid resistance with the exception that Gorbach, among other things, discussed the formation of a proteae by cultivating Aspergillus niger with an optimal pH of about 4.9 have reported in liquid. (See G. Gorbach and 0.G. Koch, Arch, for microbiology 23, 265 and 284 (1955).

Systematic studies on the cultivation conditions for the generation of an acid-resistant protease using black mold Aspergillus from the mold group Kuro-Köji (cf.- Sakaguchi, Iizuka and Yamaguchi, J. Applied Mycology (Unix. Of Hokkaido), 3, 54 ( 1949); ibid. 3_, (1: 950); and 4; 1 (1950); Journal of the Agricultural Chemical Society of Japan 24, 138 (1.951) showed that obtaining the most acid-resistant protease using black mold Aspergillus can be increased by 40 to almost 90 liters compared to controls. The special feature of this method is that according to the invention, the C / N ratio of the nutrient medium for the Aspergillus Usamii ATCC 14331 or Aspergillus saitoi ATCC 14332 is below 3.2 for the strain Aspergillus aureus ATCG 14334 or Aspergillus awamori ATCC 14335 to about 5, and for the strain Aspergillus inuii ATGC 14335 or Aspergillus nakasawai AM 14336 to about 8 and further 0.25 to stick The position of the mold species in the classification of Aspergillus is shown in the following table.

(K. Sakaguchi, H. Iizukä and S. Yamazaki: J. Agr. Chem. Soc., Japan, Vol, 24, 138, 1951; and H. Iizuka: J. General and Applied Microbiology, Vol. 1, No. 1, 10, 1955) (1) Conidia wall in the state of maturity with colored stripes ---- A. niger group; (1) Conidia wall. Smooth, rough or prickly ---- Kuro-Koji- Mold group; (2) Black colonies -______-_____ ö ____________ (2) Colonies with brown or olive shading- ___e_________________________________ (3). Nitrite Assimilates + -_____________ - __ (4) Is aterigmata over 3011 ---------- A.batatae, Saito; (4) 1 st sterigmata 13-25ju ------------ --------- --------- (5); (5) Yellow pigment is produced ---- A. usamii nov. sP .; (5) Yellow pigment is not produced ------- -------- --------------- -------.-- A, usamii var. R-17 Nov.var :; (3) 'No nitrite assimilates ---------------- --------- (6); (6) 1 st sterigmata 15-23 / u, conidiophores 2-3 mm or more ---------------------- säitoi-riov. sP. ; (6) 1 st '*; sterigmata over 16 ju, colonies about outer-brown ---- _-..-___________ saitöi var. R @ = fi6nov. var. (6) 1 st sterignate 10-13, u, conidiophores below 1.5 mm, Honidien head huddled together ...... A. saitoi var Kagoshima nov. var .; (7) Colonies with olive shadows, nitrite assimilates ----- --------_ ------- _ ---- _ --------------------------- _ -@.(G) (8) Sterigmata mostly in double series, but often mixed with simple series in the same tip, 1 st sterigmata 5 ------------------- A. inuii nov. sP .; (8) Sterigmata, usually in double series ---------------- -------------- _ ---------------- A. inuli var.K-19 'nov. var .; (e). Conical heads small -------- A. inuii värP-7 Nov. var .; (7) Colonies with olive shadows, nitrite assimilates ----- -------------------------------------------------- - (9) (g) @ Nitrite Assimilates ---------------- A. aureus Näkazawa, A. aureus var, minor Nakazawa and Shimo, A. awamori var. fumeus Hakazawa and Shimo, A. aureus F. sP. .R-2; (9) without nitrite assimilates ------------------------------ (10); (10) yellow-orange pigment in the 1 "lycel, conidia heads in the ordinary colonies not visible - A.nakazawa..nov "sP ,; (10) __ no yellow-orange pigment ---------------------------- _-_ --- .- A. awamori Näkazawa and Shimo, lA._awamori var. Minimus Nakazawa and Shimo, A._awamori var..piceu -Nakazawa and: -Shimo, A. awamori var. Fuscus Nakazawa and Shimo, A.aureus var. Acidus Nakazawa and Shimo , A. awamori var. Mirinus Nakazawa and Shimo, A. Awamori.F. sP..F-5, R-9, H-2 and R-1. + Nitrite assimilation was tested using the following medium: 30 g sucrose, NaN02 1.5 g, 1 g "K2HP04;0.5'g KCZ, 0.059 MgS04 # 7. H2O, 0.05 g FeS04 in 1 1 distilled water .

The nitrite-assimilating species grow in a day or two at 30 up to 35 ° C, while the non-assimilating species do not even after 5 to 10 days or show little growth in the upper part of the agar level.

The strains used in the following examples belong to the Kuro Koji mold group, which is obtained at the Institute of Applied Microbiology, University of Tokyo as well the group Aspergillus flavas-oryrae, listed in the Arima Research Zaboratory, Universty of Tokyo, and- the group Aspergllus sojae, which is in the Noda Institute for Scientific Research.

The enzyme solution from the solid cultivation is prepared as follows: 5 g of wheat bran are mixed with 35 ml of water and sterilized in the usual way under pressure. The bran is then inoculated with a mold and hatched for about 64 hours at 30 ° C. After extraction with 50 ml of aqueous hydrochloric acid (pH 2.7) and filtration, the filtrate is treated with n / 10 acetate buffer (pH 2.7) for the preparation of the diluted to 10 times the volume. The amount of each compound added to the starting wheat bran culture medium is expressed in percent by weight based on the wheat bran.

The enzyme solution from the (immersion) culture in liquid is prepared as follows: 3 g of a mixture of wheat bran and defatted soybeans are mixed with 10 ml of water in a shake flask with a capacity of 500 ml and sterilized under pressure in the usual way. The mixture is inoculated into a mold strain and hatched at 300C in a shaker that operates at 140 vibrations per minute. Some time after the inoculation, the mixture is filtered and the filtrate. diluted to 10 times the volume with n / 10 acetate pepper (pH 2, 'T), wöbe. the enzyme solution is obtained. _-The protease sample is carried out as follows for the enzyme solution used according to the invention. The enzyme solution containing the protease acts on a substrate clay 2% milk casein (Haaieretein) at 30 ° C for 10 minutes, namely according to the Teränder-auson's method. After filtering off the precipitate of the precipitate formed by the addition of triehloroacetic acid, the. One ml of Folin's reagent is added to the filtrate and the color that occurs is measured colorimetrically at 660e using a photometer (Hitachi, Ztd., EPO-B type), in QO.D. Measured: Preliminary tests have shown that 50 to 709 of the amount of water irrigation and cultivation conditions of 62 to 64 hours at 3000 are sufficient for solid cultivation. Furthermore, the relationships between the amount of acid protease formed and the ratio of wheat bran to defatted soybeans in the (exchange) cultivation in liquid were determined for the determination of the base medium for different strains of the Kuro-Ko ji-Sc @ immel group. The results are shown in FIG. These experiments show that Aspergillus Ussaii, Lapergillus Sait and other strains prefer nitrogen sources in concentrated form, that Aspergillus inuii, Aspergillus Nakazawai prefer nitrogen sources in equally low concentrations, and Aspergillus aureus, Aapergillus axamoxi and other strains prefer stiocarbon sources: The following explains the effect of the addition of various types of nitrogen-containing compounds on the formation of the acid protease of black mold aspergillua according to the invention. . Fig. 2 is a graph showing the effect of Lie adding various kinds of nitrogen-containing compounds to form the acid protease using Aspergillus Saitoi in solid cultivation. The experiment shows that the formation of the acid protease is noticeably increased by adding various types of nitrogen-containing compounds, and that the preferred C / N ratio of the medium (ratio of carbon% to nitrogen% in the medium) depends on the type of odorous substances Compound somewhat: fluctuates, but is generally around 85 and that mainly inorganic nitrogen sources, such as z: $. Nitrates and ammonine salts, * are extremely effective for the formation of proteases, even in comparatively small amounts. Therefore, the use of inorganic nitrogen sources - especially from an economic point of view, is very advantageous over the use of organic nitrogenous sources :; In addition to the nitrogenous compounds, some potassium salts, calcium chloride and others seem to be weakly effective. Different carbon sources and different metal salts are, however, a hindrance to the formation of the protease.

Figure 3 shows a graphical representation of those formed over time Acid Protease, with 0.59% sodium nitrate and 1% ammonium chloride added.

There is some evidence of the effectiveness of the addition of inorganic nitrogen sources to solid cultivation in producing the acid protease. Only Sakamot et al have found that the addition of ammonium sulfate increases the formation of the enzyme in the case of Penicillium, namely by 122% compared to the control (see J. Fermentation Technology 35, 1957)).

The following table lists typical strains of black Schimtel aapergillus of the Kuro-Koji-Schimmel group, which show a remarkably increased formation of acid protease through the addition of inorganic nitrogen sources. Table 1 typical strains showing an increased production of Acid protease when an -inorganic am- show monium salt or nitrate. Aspergillus Usami (ATCC No. 14331) Aspergillus Saitoi (ATCC No. 14332) Aspergillus .lnuii (ATCC No.`14333) Aspergillus Aureus - (-ATCC No: 14334) Aspergllus Awamori (ATCC No. 14333) Aspergillus Nekazawai (ATCC No. 14336) The effects of inorganic nitrogen compounds related to the formation of different types of proteases, using the black Schimmelaspergillus species Asper% -gillus saitoi, Aspergillus oryzae flavus and "other loper-gillus- types are apparent from Table 2. As it takes the acid protease Formation only in black mold aspergillus through the addition of inorganic nitrogen sources, otherwise only the alkaline proteae of PR 7.5 increases. For the formation of the protein from black mold aspergillus by (tauces) cultivation in a liquid according to the method according to the invention, various amounts of inorganic nitrogen sources in the form of ammonium chloride or sodium nitrate were added to the above-mentioned medium, which contains organic nitrogen sources in increased concentration. The: * results for the black Aspergillus mold saitoi are shown in Table 3 below. Table 3 Effect of the concentration of inorganic 'nitrogen compounds in the medium on the formation of protease using animals from Aspergillus saitoi (30 ° C, $ 7 140 rpm) Addition of Nitrogen compound dungen (9 @) Ammonium chloride sodium nitrate C / N pH protease yield, C / N PH protease yield; Control -% 0 3.15 4.7 0.268 100 - 0.25 2.18 497 0.324 128 2.41 4.3 0.252 94 0 9 5 1t60 4.5 0, .372 139 1.95 4.5 0.322 120 1.0 1.07 4.5 0.416- 155 1.42 4.0 0.380 142 1.5 0.80 4.6 0.424 158 1.17 4.6 0.396 148 2.0 0.64 4.8 0.412 154 0.91 4.3 0.378 141 Defatted soybeans 0.5 2.70 3.8 0.235 87.7 1-, 0 2.39 3.6 0.229 85.4 The C / N ratio in the medium used in the experiment is considerably lower in known methods (especially in the medium with a higher nitrogen content). The experiment shows that an extremely favorable and maximum yield is achieved if 1% ammonium chloride or sodium nitrate is added to the base medium containing concentrated organic nitrogen compared to the control. .

Have been defatted soybeans, one of the organic nitrogen-containing Sources added to the control, so the yield is lower.

The formation of the acid protease in the above base medium (C / B ratio 3.2), which is mixed with 1% different ammonium salts and nitrates, is carried out by shaking culture for 63 hours. The results are shown in Table 4. Table 4 Effect of different types of inorganic nitrogen compounds (1%) as an additive to the medium in the Generation of the acid rotease using Asper-. giilus.eaitoi Z30 C, 140 rpm) 63 hours 87 hours . Nitrogen Protesse: --- '' PröäB`` links . GIN pH 0.D. Yield pH 0.D. Yield., Control 3915 3.5 0.150 100 3.9- 09181 100 NH401 _ 1.07 4i3 09261 174 497. - 09-334 174 (NH4) 2S04 1.22 3.9 0.229 153 4.4 0.275 138 NH4 @ 03 0987 4.1 0.199 132 498 ` 09328 165 (N1.4) H2M4 1.65 3.6 0.181 124 4.1 0.330 166 (NH4) 2HP04 1.22 4.0 0.058 39 3.6 0.282. 142 . NaN03 1.42 4.1 0.269 149 KN0 1955 .4490 0 09 239 132 NH4 citrate. 1.64. 3.8 0.067 44 3.8 0.244 122 NH4-Ta * at 1948 492 09018 73 492 09246 124 From the experiment 4 results, DAB, the addition of A 'onium salt formation of the Söureprotease at the short Kultivierungsdeuer ste% siege and that the addition of inorganic Stickstottquelle after a 87-hour cultivation, a 40 increased to 809 yield results in Fg. Is a graphical representation of the change in the amount of protease formed as a function of time with the addition of 1% ammonium salt and 1% nitrate.

Different types of mold - the Kuro-Koji mold group - act in a liquid medium under the above optimal culture conditions with a reduced C / N ratio and the addition of inorganic nitrogen sources: The results of the formation of the acid protease are shown in Table 5 below. Many studies have already been carried out to regulate the pH value in the cultivation of bacterial species. Examples of this are the fermentation of amylase from Aspergillus niger NRRL-337 (EH Le Mense et al, J. Bact: '54 ° -149 47), acid amylase from Aspergillus-awamori var, fumeus (Minoda, J. of 17 Chem. Soc. of Japan, 35, 479 and 481 (1961 ", Protease aua Aepergillus oryzae (ME Magwell, Aust. J .: Sci. Res. B5, 42 (-1952)) and protease from Aepergillus reger (G. Gorbach and 0.G. 'Koch, Arch. Für Mikribiol. 23., 265 (1955) j.. R Regarding the effect of the pH value, .Rauf @ the formation of the acid protease with an optimal PR of 2, 7 from black mold aspergillus of the Kuro = Koji-mold group with liquid cultivation, the following was found: The protease produced according to the invention with a resistance to acids is stable within a broad range of 2.5 to 6.o if fine -Substrate. Is available,. (VBl.

F. Yoshida and M. Nagasawa, Bull: Agr. Chem. Soc. Japan 20, 257 (1956j). The changes in pH as a function. Ton de, time in cultivation of Aspergillus Ksitoi in a medium with highly concentrated nitrogen and with the addition of further inorganic nitrogen sources are shown in FIG. In general, the pH decreases to 3.0 to 4.0 after about 40 hours and increases then gradually increased to 5.0. If nitrate is added, this is delayed Decrease in pH up to 40 hours and the curve shows a similar course like control. As stated above, when a medium is added, it tends to be physiological acidic salt to the acid and with the addition of a physiologically alkaline salt to the Alkali out.

However, there is no remarkable change in the formation of the acid protease in a medium with highly concentrated nitrogen with the addition of various kinds of salts and the use of black mold Aspergillus such as Aspergillus usaaii, Aspergillus saitoi or the like. In the case of Aspergillus aureus, Aspergillus awamori, Aspergillus inuii or the like, a rapid increase of the PG value is noted after 60 hours and the acid protease obtained increases with the Anati.eg the pH to 6.0 or above rapidly. Accordingly, the Pb value is set in the formation of the acid protease using black Schimelaspergillus so that the initial pH value of about 6.0 cheating and thus is suitable for the development of black Aspergillns. The lowest pH value is reached after about 40 hours and controlled in such a way that it does not drop below 2.5 to -5 and cultivation is carried out after about 40 hours within the pff range from 2.5 to 6.0, in which the acid protease from black Aspergillus is stable.

The invention will now be further explained with the aid of the following technical exemplary embodiment: Example The hunting was carried out as follows #.

To 100 il. 1.8 kg of wheat germ and 1.2 kg of defatted soybeans were added to the water. The mixture was used as a control medium. A corresponding mixture was enriched with 1 kg of NH4Cl and used as the test medium. A conventional sterilization media were exposed to the control and test brock, and then cooled: The cooled OremisoW were inoculated with ATCC 14332 and Asp saitoi Belgftutrg.. and stirring for 80 times at 30 ° C in the incubator. After the cultivation , the obtained culture was filtered and the filtrate is appropriately acidified Water with a pH of 2.7 diluted. 1 * n3 -der dilute solution was mixed with 1 0s3 2% casein solution with a p8 value of 2.7 mixed and the mixture left for 10 minutes rest for a long time at 300 ° C. The mixture was added 2 c33 a 0; 4-M- Trichloroacetic acid is added to the reactions in mixture The mixture was then filtered. To 1 am3 of the filtrate, 5 cm3 of 0.4 M.Batriuakarbonat- solution and then 1 cm 3 Folin reagent (fivefold dag) zpetzt. The mixture was left for 20 minutes 300C stand. The protease activity of the mixture was determined as optical adsorption value of 600 au using a SPOO-B photoseters of la. Hitaehi Ltd. measured. In the following data resulted:. Control test culture growth in item no Protease activity 5 5 of the Cosasteddum 7.680 z 105 13.368 = 10. 74

Claims (1)

A process for the production of acid-resistant protease with an optimal pH of about 2.7 by cultivating Kuro-koji mold strains in a nutrient medium containing assimilable carbon and nitrogen, characterized in that the C / N ratio of the nutrient medium for the Aspergillus usamii ATCC 14331 strain or Aspergillus saitoi ATCC 14332 to below 3.2 for the Aspergillus aureus ATCC 14334 or Aspergillus awamori ATCC 14335 strain to about 5, and for the Aspergillus inuii ATCC 14333 or Aspergillus nakazawai ATCC 14336 strain to about 8 and others Adding 0.25 to 3.096 inorganic nitrogen compound to the nutrient medium.