2,716,084 United States Patent Office Patented Aug. 23, 1955 2 They conclude (supra, p. 1406) that and dex 2,716,084 tranase are formed only adaptively by their . We have shown in our pending applications above PRODUCTION OF ENDODEXTRANASEBY ASPERGLUS WENT identified, that the Aspergilli molds yield an or 5 endo- which, as pointed out above, attacks Wirginia Whiteside Carlson and Warner W. Carlson, Bir. glucosidic linkages of dextran to split it into segments or mingham, Ala., assignors to The Commonwealth En fractions made up of molecules of equal size and exhibits gineering Company of Ohio, Dayton, Ohio, a corporar a preference for the linkages interiorly of the dextran tion of Ohio molecule, i. e., it does not attack all linkages of the dex No Drawing. Application December 1, 1953, tran with equal ease. The Aspergii molds, therefore, yield an endo-dextranase which is different from dex Serial No. 395,582 tranases of the prior art as represented by that from the 13 Claims. (C. 195-66) Penicilium and Verticillium molds of Hultin and Nord strom discussed above. In our pending applications Serial Nos. 232,864, filed We find that the Aspergilli molds are also different in June 21, 1951, and 241,168, filed August 9, 1951, we have their behavior and environmental requirements. Whereas disclosed that when molds of the genus Aspergilli are cul the presence of dextran, of which at least 50%, and gen tivated on media containing inorganic salts and an ac erally more, of the molecular structural repeating linkages climating amount of dextran, the enzyme obtained is a between the anhydroglucopyranosidic units are of the "limit' endo-dextranase which is a hydrolytic agent for alpha-1,6 type, is required in the culture media for the dextran, attacking the glucosidic linkages of large dextran Peniciilia and Verticillia molds in order to obtain a dex molecules to split them into segments of substantially uni tranase, i. e., an enzyme capable of splitting the alpha-1,6 form and predeterminable size between the initial size linkages of dextran, we find that the Aspergilli molds do of the large molecule and a smaller size corresponding to not require dextran in the culture media for elaboration a molecular weight not substantially lower than 20,000, of our endo-dextranase. On the contrary, and surprisingly beyond which limit the endo-dextranase has little further in view cf the prior experience with other molds, the splitting effect on the dextran. This is an extremely im Aspergilli molds elaborate our endo-dextranase when cul portant discovery because, by correlating the conditions tivated in medium containing carbohydrates of varying under which the endodextranase is allowed to act on the molecular structural repeating alpha-1,6 to non-alpha-1,6 large dextrane molecules, such as those of so-called "na 30 linkages ratios including dextran in which the proportion tive' dextran obtained by enzyme synthesis of dextran of the alpha-1,6 type linkages is high and carbohydrates from in the presence or substantial absence of in which the proportion of alpha-1,6 linkages is not high. bacteria and cellular debris, it is possible to obtain dex Specifically, we find that the Aspergilli molds elaborate train segments or fractions of Substantially equal size and cur endodextranase in culture media which contain dex molecular weight above 20,000, which fractions are true 35 tran and also in media which contain no dextran but which homologs of the parent dextran polymer in that the de do contain, instead, either or glycogen. The endo pendent groups are retained intact. It is thus possible to dextranase elaborated by the Aspergilli molds in media obtain a "clinical' dextran for intravenous injection with containing appropriate inorganic Saits, an amino acids out resorting to acid of the native or high source, and either starch or glycogen, but no dextran, in molecular weight dextran and the disadvantages which 40 aqueous dilutions of from 1:10 to 1:100 attacks glucosidic attend it. As is known, acid attacks the large dextran linkages of large dextran molecules in the same manner molecules in an indiscriminate manner, to split it into as dees the endo-dextranase elaborated by those molds in non-uniform fractions or segments having a wide range dextran-containing culture media, to split the large mole of molecular weights and also cleaves off dependent cule into smaller segments or fractions having a Substan groups. The fractions or segments obtained by acid hy tially uniform molecular weight between the weight of drolysis of native or high molecular weight dextran are the dextran split and a lower limit of about 20,000, and not true homologs of the parent dextran polymer. the size of which, in the range stated, can be predetermined It is known, in enzymology, that, in cultivating molds by correlating the conditions under which the endo-dex to obtain an enzyme, the production of an enzyme having tranase acts on the dextra in and stopping the action in specific action on a given substrate can be favored by in any suitable way when the splitting has proceeded to the cluding an acclimating amount of the substrate to be sub desired extent. sequently acted on by the enzyme in the culture media The nutrient media for cuitivation of the Aspergilli for the . Not infrequently it happens that, if the may be an aqueous solution of metallic salts containing mold is cultivated in a medium containing a substrate or amino acids or a protein source of amino acids and the carbon source different from the substance to be acted starch or glycogen, and may also contain Supplements on subsequently by the enzyme, the enzyme will exhibit such as B-vitamins or yeast extract. The nutrient medium little if any action of the desired type on the latter sub may advantageously be an aqueous composition having a Stance. Thus, Hultin and Nordstrom report (Chemica total metailic salts content of between about 2.0 and 5.0 Scandinavic 3, 1405-1417, 1949) that when they culti gms./liter of water, about 2.0 to about 6.0 gms./liter of wated Penicillium and Verticillium molds in media con 60 peptone or an equivalent amount of amino acids or of an taining dextran as C source, they obtained an enzyme, a amino acid source other than peptone, e. g., protein hy dextranase, having the capacity to split dextran. They drolyzates such as casein hydrolyzate, and from 5.0 gms. show (supra, p. 1411 etc.) that their dextranase attacked all linkages of the dextran with equal ease. They ob to about 50.0 gms. of either dextran, soluble starch or tained, as the dextran cleavage products, reducing Sugars 65 glycogen, per liter. and oligo-saccharides, the latter being products having a The pH value of the media may vary between 4.0 and maximum molecular weight of 648, (C6H10O5)4O, i. e., 7.5, and for rapid elaboration of optimum amounts of low molecular weight products. However, these authors the endo-dextranase, is preferably controlled and main further report that when they cultivated their Penicillium tained in the range between neutrality and slightly on the and Verticillium molds in media containing starch as the alkaline side, i. e., between 7.0 and 7.5. The culture may sole C source (no dextran) they obtained an amylase effec be incubated at room temperature. tive on starch but having no splitting action on dextran. A typical medium in which the Aspergiili mold may be 2,716,084 3 4. cultivated to obtain rapid elaboration of the dextranase tion, Washington, D.C., and designated wentii has a composition as given below. ATTC 1023. Example I TYPICAL CULTURE MEDIUM The Aspergillus wenti was inoculated into a flask con Carbohydrate-salts solution: - . s taining a medium having the composition tabulated above Component: Grams/liter and containing 1.0% (about 5.0 gms.) of soluble starch. Peptone------5.0 The inoculated medium was incubated at room tempera MgSO4------10.0 ture for about seven days, after which it was filtered to NaCl------0.1 free it of the mold mat. The filtrate obtained contained FeSO4------. 0.1 O the endo-dextranase. MnSO4------O.O. KHPO4------0.01 Example II Sodium acetate------0.1 The Aspergillus wenti was inoculated into a flask con Potassium acetate------2.0 taining a medium having the composition tabulated above Dextran, starch or glycogen.------5.0 and containing 1.0% (about 5.0 gms.) of glycogen. The inoculated medium was incubated at room temperature B-vitamins: Milligrams/liter for about seven days, after which it was filtered to free it Nicotinic acid------1.0 of the mold mat. The filtrate obtained contained the Riboflavin------, 1.0 endo-dextranase. Thiamine------0.5 The endo-dextranase activity of the filtrate in splitting Ca pantothenate------0.5 high molecular weight dextran was proved by observa Pyridoxine------0.4. tion of the progressive decrease in flow time (and cal Folic acid------0.01 culation of the relative viscosity) of aqueous solutions Biotin------0.001 containing the dextran to be split and the filtrate over Species of Aspergilli which may be cultivated in these a period of time. media to obtain the endo-dextranase include A. ustus, Each filtrate was introduced into a separate No. 200 A. candidits, A. niger, A. tamaric, A. flavus-Oryzal, and Fenske-Ostwald viscometer containing an aqueous 4.5% A. wenti. At present, it is preferred to inoculate the cul solution of native dextran having a molecular weight ture media with a strain of A. wenti. calculated to be in the millions. The results are shown . When the endo-dextranase has been elaborated, the in the table, below: culture may be filtered to remove the mold mat and ob TABLE-DECREASE IN FLOWTIME tain a filtrate containing the endo-dextranase, which fil trate may be used as such, or after suitable dilution with Starch E38 SE,ae E,itrate water for splitting dextran or for any other purpose to 35 Time after Filtrate Addition Dilution. Dilution. Dilution which the endo-dextranase is adapted, or the latter may be of 100 of:0 of:0 isolated from other substances present in the filtrate by . selective solvent precipitation or by selective salting out. Seconds . . Seconds Seconds 58.0 58.0 58.0 The isolated endo-dextranase may be introduced into a 26.5 22.6 12.4 94.4 92.7 77.2 medium containing dextran to be split, or otherwise used 0.4 70,0 55-9 as such or in the form of an aqueous dilution. As noted 59.0 56.0 45.2 50,0 47.2 38.8 above, the endo-dextranase is effective in splitting dextran 40, 2 38.8 32.2 in aqueous medium in dilutions of 1:10 to 1:100 when the 35.8 34.5 30.5 culture media for the mold contains soluble starch or 33.5 32.3 29.4 3.4 30.4 28.4 glycogen and no dextran. The endo-dextranase elaborated 30. 29.5 28.0 in culture media containing an acclimating annount of the 24.2 24.5 23.9 dextran which may be native or high molecular weight NoTE:-The HO time of the viscometer was 14.0 seconds. The rela dextran (or a dextran of lower molecular weight) instead tive viscosity for each time interwa is obtained by dividing each of the of the carbohydrates mentioned usually is more active flow-time values by 14.0. and may be used in higher dilutions. Thus, the endo The progressive decrease in flow time (and in relative dextranase elaborated by Aspergilus wentii in a dextran viscosity) of the solution with time of action of the endo containing culture medium as given herein typically splits dextranase on the dextran being split is evidence that native dextran synthesized from sucrose by the enzyme the endo-dextranase attacked the alpha- 1,6 linkages of dextranslucrase into fractions of regular, Substantially uni- - the large dextran molecules of the native dextran, to form lower molecular weight very rapidly in dilutions of 5 5 split them into smaller segments or fractions. As will from 1:100 to 1:1000 in from one to five hours at room. be observed, the filtrates obtained from the culture con temperature, and may be used successfully in dilutions as taining soluble starch or glycogen compared very favor high as 1:10,000 to 1:100,000 with somewhat longer, re ably as to the dextran-splitting capacity thereof with the action periods. filtrate from the culture containing dextran. The compari Various modifications may be made in the composition GO son with the filtrate from the starch-containing medium of the specific medium given. Thus (1) the B-vitamins may be omitted; (2) the B-vitamins may be replaced by is particularly striking in view of the literature reports yeast extract in an amount of, for example, 0.4 gm./liter; that cultivation of other classes of molds in starch-con (3) the peptone may be replaced by casein hydrolyzate in taining media did not result in a dextranase or enzyme an amount of about 5.0 gm./liter with or without inclusion capable of splitting glucosidic linkages of dextran. Unlike of the B-vitamins; (4) a medium of the composition given the dextranase from the prior art molds referred to above, in which any one of the carbohydrates listed is present in our endo-dextranase is not formed only adaptively. an amount of from 5.0 to 50.0 gims./liter. It will be understood that the media for the cultivation The amounts of the various ingredients given are not of the Aspergilli molds and the conditions of the culti strictiy critical and may be varied somewhat, so long as vation may be varied, and that either the filtrate, the endo the nutrition requirements of the mold are satisfied so dextranase isolated therefrom, or an aqueous dilution of that the mold flourishes. the filtrate or of the precipitated endo-dextranase may The following examples wiil serve to illustrate the in be introduced into an aqueous medium containing any vention, it being understood that these examples are not appropriate concentration of dextran to be split, such as limitative. The mold used in the examples was similar to a 4% to 10% solution. Since these and other obvious the mold on file with the American Type Culture Collec variations and modifications may be made in the specific 2,716,084 5 6 details given, in practicing the invention, without depart ing the culture to remove the mold mat and leave a ing from the spirit and scope of the invention, it will be filtrate containing the endo-dextranase. understood that the invention is not intended to be limit 9. The method of obtaining a filtrate containing endo ed except as defined in the appended claims. dextranase having the capacity to attack glucosidic link What is claimed is: ages of large dextran molecules to thereby split the large 1. The method of obtaining a filtrate containing endo molecules into relatively low molecular weight segments, dextranase having the capacity to attack glucosidic link which comprises inoculating Aspergillus wentii into a ages of large dextran molecules to thereby split the large nutrient medium comprising an aqueous composition con molecules into relatively low molecular weight segments, taining the following constituents which comprises inoculating Aspergillus wenti into a nu 10 Carbohydrate-salts solution: trient medium comprising an aqueous composition con Component: Grams/liter taining dissolved metallic salts and, per liter of water, Peptone ------50 from about 2.0 gms. to about 6.0 gms. of a Substance MgSO4 ------10.0 selected from the group consisting of amino acids and NaCl ------0.1 protein sources of amino acids, and from about 5.0 gms. 5 FeSO4 ------0.1 to about 50.0 gms. of, as sole carbohydrate, a Substance MnSO4. ------0.01 selected from the group consisting of starch and glycogen, KH2PO4 ------0.01 incubating the culture until the endo-dextranase is elabo Sodium acetate------0.1 rated therein, and filtering the culture to remove the mold Potassium acetate------2.0 mat and leave a filtrate containing the endo-dextranase. 20 Starch ------5.0 2. The method according to claim 1, characterized in that the amino acids source in the nutrient medium is B-vitamins: Milligrams/liter peptone. Nicotinic acid------1.0 3. The method according to claim 1, characterized in Riboflavin ------1.0 that the carbohydrate in the nutrient medium is starch. 25 Thiamine ------0.5 4. The method according to claim 1, characterized in Ca pantothenate------0.5 that the carbohydrate in the nutrient medium is glycogen. Pyridoxine ------0.4 5. The method of obtaining a filtrate containing endo Folic acid------0.01 dextranase having the capacity to attack glucosidic link Biotin ------0.001 ages of large dextran molecules to thereby split the large 30 incubating the culture until the endo-dextranase is elab molecules into relatively low molecular weight segments, orated therein, and filtering the culture to remove the which comprises inoculating Aspergillus wentii into a mold mat and leave a filtrate containing the endo-dex nutrient medium comprising an aqueous solution of dis tranase. solved metallic salts containing B-vitamins, a protein 10. The method of obtaining a filtrate containing endo source of amino acids and, as sole carbohydrate, a Sub 35 dextranase having the capacity to attack glucosidic link stance selected from the group consisting of starch and ages of large dextran molecules to thereby split the large glycogen, incubating the culture until the endo-dextranase molecules into relatively low molecular weight segments, is elaborated therein, and filtering the culture to remove which comprises inoculating Aspergillus wentii into a the mold mat and leave a filtrate containing the endo nutrient medium comprising an aqueous composition con dextranase. 40 taining the following constituents 6. The method of obtaining a filtrate containing endo dextranase having the capacity to attack glucosidic link Carbohydrate-salts solution: ages of large dextran molecules to thereby split the large Component: Grams/liter molecules into relatively low molecular weight segments, Peptone ------5.0 which comprises inoculating Aspergillus wentii into a 45 MgSO4 ------10.0 nutrient medium comprising an aqueous solution of dis NaCl ------0.1 solved metallic salts containing yeast extract, peptone, FeSO4 ------0.1 and, as sole carbohydrate, a substance selected from the MnSO4 ------0.01 group consisting of starch and glycogen, incubating the KH2PO4------0.01 culture until the endo-dextranase is elaborated therein, 50 Sodium acetate------0.1 and filtering the culture to remove the mold mat and leave Potassium acetate------2.0 a filtrate containing the endo-dextranase. Glycogen ------5.0 7. The method of obtaining a filtrate containing endo B-vitamins: Milligrams/liter dextranase having the capacity to attack glucosidic link Nicotinic acid------1.0 ages of large dextran molecules to thereby split the large Riboflavin ------1.0 molecules into relatively low molecular weight segments, Thiamine ------0.5 which comprises inoculating Aspergillus wentii into a Ca pantothenate------0.5 nutrient medium comprising an aqueous solution of dis Pyridoxine ------0.4 solved metallic Salts containing B-vitamins, casein hy Folic acid------0.01 drolyzate and, as sole carbohydrate, a substance selected 60 Biotin ------0.00 from the group consisting of starch and glycogen, in incubating the culture until the endo-dextranase is elab cubating the culture until the endo-dextranase is elab orated therein, and filtering the culture to remove the orated therein, and filtering the culture to remove the mold mold mat and leave a filtrate containing the endo-dex mat and leave a filtrate containing the endo-dextranase. tranase. 65 11. The method of obtaining a filtrate containing endo 8. The method of obtaining a filtrate containing endo dextranase having the capacity to attack glucosidic link dextranase having the capacity to attack glucosidic link ages of large dextran molecules to thereby split the large ages of large dextran molecules to thereby split the large molecules into relatively low molecular weight segments, molecules into relatively low molecular weight segments, which comprises inoculating Aspergillus wentii into a which comprises inoculating an Aspergillus wentii into a 70 nutrient medium comprising an aqueous metallic salt solu nutrient medium comprising an aqueous solution of dis tion containing a substance selected from the group con Solved metallic salts containing B-vitamins, peptone and, sisting of amino acids and protein sources of amino acids as Sole carbohydrate, a substance selected from the group and as sole carbohydrate a substance selected from the consisting of Starch and glycogen, incubating the culture group consisting of starch and glycogen, incubating the until the endo-dextranase is elaborated therein, and filter 75 culture until the endo-dextranase is elaborated therein, 2,716,084 7 : 8 and filtering the culture to remove the mold mat and 2,597,019 Moyer ------May 20, 1952 leave a filtrate containing the endo-dextranase. 12. The method of claim 11 in which the sole carbo . . ; OTHER hydrate constituent of the medium is starch. Stokes et al., Jour. B acteriology, March 1944, pages 13. The method of claim 11 in which the sole carbo 293 through 299. . . . hydrate constituent of the medium is glycogen. References Cited in the file of this patent UNITED STATES PATENTS 2,482,055 Duggar ------Sept. 13, 1949 O