DE1237044B - Process to increase the glucogenic activity of raw, aqueous amyloglucosidase preparations from molds or bacteria - Google Patents

Description

Process for increasing the glucogenic activity of crude, aqueous Amyloglucosidase Preparations from Molds or Bacteria The invention relates to a method for increasing the glucogenic activity of crude, aqueous amyloglucosidase preparations from mold or bacteria. Here, carbohydrate-synthesizing enzymes, such as isomaltose synthetase and maltose transglucosidase, removed so that the purified Enzymes hydrolyze starch and its hydrolysis intermediates more effectively to glucose can.

The amyloglucosidase preparations treated according to the invention are with Help from fungi and bacteria, usually by submerged culture, or obtained by other methods. The enzyme preparation is usually in the form of a filtered or centrifuged fermentation broth, but can also be in other forms are present. These include (1) the entire fermentation mixture or culture inclusive the microorganisms, (2) the dried whole culture, (3) the dried fermentation broth, (4) the aqueous extract of the dried whole culture and (5) the concentrated one dried material, which is made up of by deposition of the starch hydrolyzing enzyme the filtered or centrifuged fermentation broth with a dehydrating agent, such as acetone or ethanol.

The main starch-hydrolyzing enzyme in preparations of the Invention is referred to differently. These names include y-amylase, Glucamylase, starch glucogenase, maltase and amyloglucosidase. This enzyme is different differs from other starch hydrolyzing enzymes by its ability to hydrolyze from starch to glucose without the simultaneous formation of substantial amounts of low molecular weight Hydrolysis intermediates such as maltose, maltotriose, higher sugars and soluble ones Dextrins. The effect of the enzyme appears to be that of the non-reducing one One glucose unit is removed at the end of a starch chain from one after the other. That Enzyme also hydrolyzes maltose, maltotriose, and other intermediate products of hydrolysis from starch to glucose.

Examples of microorganisms that can be grown to produce fermentation mixtures and fermentation broths containing amyloglucosidase in acceptable concentrations include the genus Aspergillus, Mucor, Clostridium and Rhizopus. The following subspecies provide amyloglucosidase in good yields: Aspergillus oryzae, Clostridium acetobutylicum, Rhizopus delemar, Aspergillus niger, Aspergillus phosnicis and Aspergillus fiavus. The production of amyloglucosidase in the form of whole cultures and fermentation broths described in the USA. Patents 2 557 078,2 881 115 and 2,893,921. The purification of a crude amyloglucosidase preparation, namely the water extract of Rhizopus delemar, is described in the Journal of the American Chemical Society, Vol. 73, pp. 3359-3365.

However, it has been found that following the procedures of the specified Amyloglucosidase preparations produced by patents for complete hydrolysis of starch or starch derivatives are not capable of glucose. With economical The graph shows the acceptable initial concentrations of starchy substrate Illustration of amyloglucosidase hydrolysis, in which the ratio of the weight of the generated glucose to the weight of the hydrolyzate dry matter on the ordinate has been plotted against the hydrolysis time on the abscissa, at about 0.86 either a constant level or "peaks" at around this value, the then fall off. The numerical value of D at the constant height or at the peak value is hereinafter referred to as glucogenic activity des - amyloglucosidase agent where D is the amount of glucose contained in the total solids in Indicates weight percent. The glucogenic effectiveness of an amyloglucosidase preparation but does not necessarily correspond to the uniformity of the means according to the teachings U.S. Patent 2,881,115.

In the production of crystalline glucose by hydrolysis of starch and its hydrolysis intermediates with the aid of amyloglucosidase according to the processes described in U.S. Patents 2,531,999, 2,567,000, 2,583,451 and 2,881,115 , it is generally desirable to have the greatest possible conversion or To achieve hydrolysis of the starchy substrate to glucose. A high degree of hydrolysis facilitates the crystallization of glucose from the concentrated hydrolyzate, because the hydrolysis products that do not consist of glucose (maltose, isomaitose, higher sugars and dextrins) inhibit the crystallization of glucose. A high degree of hydrolysis also increases the yield of glucose, while the amount of the often undesirable mother liquor solids is reduced. With the aid of the known amyloglucosidase preparations, useful enzymatic processes for the production of crystalline glucose from starch and from starch hydrolysis intermediates can be carried out. After hydrolysis of starch-like substrates with the conventional enzyme preparations, glucogenic activities of about 86 D on average were obtained. According to a further known process, amyloglucosidase preparations have been purified by treating the aqueous amyloglucosidase-containing solution with water-insoluble magnesium silicate and separating the product obtained after adsorption of the impurities, especially transglucosidase, and thereby increasing the glucogenic activity to DE values of a maximum of about 95010 obtain. In contrast, the process according to the invention is much simpler and cheaper, since smaller additives in dissolved form directly cause permanent inactivation of the impurities without the need for subsequent separation of the inactivated product and approximately the same level of glucogenic activities being obtained. Starch-like substrates show after hydrolysis with the. The proposed method treated conventional amyloglucosidase preparations an analytical value of 90 to 95 D. According to the method of the invention, an aqueous solution or dispersion of an amyloglucosidase preparation is mixed with a small proportion of a new cleaning agent, by means of which the undesired or contaminating carbohydrates synthesizing enzymes selectively deposited or inactivated will. The selective inactivation usually takes place by a deposition or agglomeration of the undesired enzymes or is accompanied by these phenomena, although the desired inactivation can also take place without a deposition. The inactivation, which takes place with or without separation, is not reversed later even if the temperature and the pH of the mixture of the amyloglucosidase preparation and the cleaning agent are increased. The mixture can accordingly be used for hydrolyzing starch and its hydrolysis intermediates to glucose without separating the liquor from the deposit. If necessary, however, a formed deposit can also be separated from the liquid which contains the amyloglucosidase in solution, whereupon the clarified liquid is used to hydrolyze starch or substrates obtained from starch to form glucose. In another method, either the clarified or unclarified aqueous amyloglucosidase preparation can be concentrated before use, evaporated to dryness, or dehydrated with a water-miscible organic liquid such as acetone or ethanol.

The amyloglucosidase preparation can be used before treatment with the detergent also contain water-insoluble substances, although the proportion of amyloglucosidase and the interfering carbohydrate synthesizing enzymes in the proposed one Process dissolve in the aqueous medium and therefore the reaction with the cleaning agent are accessible. However, the amyloglucosidase preparation is preferably in the form of a filtered or centrifuged fermentation broth, d. H. in the form of a clear if colored, aqueous solution is also used. With the proposed method you can however, amyloglucosidase preparations in the form of aqueous solutions of the enzymes are also used containing suspended, insoluble material, e.g. B. the entire fermentation culture.

In which. Process of the invention is preferred as the reaction medium Water is used, although other liquids such as acetone, ethanol, ethyl acetate and glycerin, may be present in the medium in small proportions.

As mentioned above, by the proposed treatment apparently one or more carbohydrate-synthesizing enzymes of the original Inactivated amyloglucosidase preparation, apparently one of these inactivated enzymes also includes maltose transglucosidase. Properties and mode of action of maltose transglucosidase are by P a n et a1 (Arch. Biochem. Biophys., 42, pp. 421 to 434) and by P a z u r and F r e n c h (J. Biol. Chem., 196, pp. 265 to 272).

The process according to the invention for increasing the glucogenic activity of crude, aqueous amyloglucosidase preparations from molds or bacteria is characterized in that they are treated at pH values above about 2 and below about 5 with an acid of the following structural formulas where R, an alkyl group with 10 to 15 carbon atoms, R2 an alkyl group with 3 to 5 carbon atoms, R3 an alkyl group of the formula CH3C (CH3) 2CH2C (CH3) 2 - or CH3C (CH3) 2CH2C (CH3} zCH2C (CH3) 2 -R4 is a 2-ethylhexyl group, R5 is an alkyl group with 14 to 18 carbon atoms, n = 0, 1 or 2 and m is an integer from 1 to 6, or a water-soluble salt of these acids is mixed.

The sodium salt of the acid is preferably used, but others are also used water-soluble salts are equally effective. These salts include the Salts of lithium, potassium, ammonia, lower alkyl amines and of quaternary ammonium hydroxides.

Process for the preparation of the sulphonic acids and sulfuric acid esters mentioned are in chapters 3 and 5 of "Surface Active Agents - Their Chemistry, and Technology" by A. M. S c h w a r t z and J. W. P e r r y, 1949, Interscience Publishers, Inc., described. The di-2-ethylhexyl ester of orthophosphoric acid can according to known Process are produced.

Examples of acids that correspond to the structural formulas given above, are monokerylbenzene monosulfonic acid, in which the keryl group has an alkyl group has an average of 15 carbon atoms and consists of a highly saturated kerosene has been obtained, monodecylbenzene monosulphonic acid, monopentadecylbenzene monosulphonic acid, monokeryltoluene monosulphonic acid, Monokerylxylene monosulfonic acid, monokerylcumol monosulfonic acid, monoisopropylnaphthalene monosulfonic acid, Monoisoamyl, naphthalene monosulfonic acid, the acidic ester of sulfuric acid of 7-ethyl-2-methyl-4-undecanol and the acid ester of sulfuric acid of 3,9-diethyl-6-tridecanol.

In the proposed method, the cleaning agent is used for the purpose Prepare a solution of moderate concentration, e.g. B. from 5 to 10 percent by weight of the material, preferably first dissolved in water, whereupon a suitable amount the solution is quickly mixed with the aqueous amyloglucosidase preparation to be cleaned will. The concentration of the solution is not essential, so that may also be Concentrations above and below 5 to 10 weight percent can be used can. According to another method, the detergent can be in dry or pure form are stirred into the aqueous amyloglucosidase preparation. Preferably however, a solution is used because of the likelihood of deposition or inactivation of part of the amyloglucosidase due to a local, high Concentration of detergent when adding the agent to the amyloglucosidase preparation can arise is reduced.

The detergents useful in the process have their largest Effective at pH values of around 4 down to pH values of 2 and below. Their effectiveness quickly decreases when the pH level is up in the treatment increases above 4, and is negligible at a pH value of 5. Weg the sensitivity of amyloglucosidase to low pH values and especially in the case of pH values of about 3 and below, the treatment is preferably carried out at a slightly higher pH, i.e. H. at a pH of 3 to 4. When the procedure is carried out at low pH values, the temperature is preferably low (e.g. at 20 ° C or below) to prevent the loss of amyloglucosidase from occurring Inactivation is kept as low as possible at low pH.

At the preferred pH of 3 to 4, a temperature is preferred used from 20 to 40 ° C. Lower temperatures down to the freezing point of the Means can optionally also be used, but this is due to the effort to generate the lower temperature, the advantage that achieved by the somewhat reduced loss of amyloglucosidase through inactivation will. Temperatures above 40 ° C can also occur during deposition or inactivation of the undesired enzymes are used, but values above 55 to 60 ° C are inappropriate because they lead to a temperature inactivation of amyloglucosidase to lead. The regulation of the temperature in the proposed method is on the one hand predominantly from the freezing point of the aqueous amyloglucosidase preparation and on the other hand determined by the inactivation of aryloglucosidase at 'elevated temperatures. The selective deposition or inactivation of the unwanted enzymes with the help of the cleaning agent is only slightly determined by the temperature between 0 and 60 ° C.

The weight ratio between detergent and the weight of the aqueous amyloglucosidase preparation is quite essential. It has been found that with aqueous amyloglucosidase preparations, the 10 to 150 units per cubic centimeter contain, the optimal proportion between 0.05 and 0.1 parts per 100 parts of the preparation lies. Below this range, the separation of impurities, d. H. the increase in glucogenic potency, while above this, decreased Area, some of the amyloglucosidase effectiveness is lost, which is likely on inactivation through the detergent without essential Increase in glucogenic activity is due. The lower and upper values for usable proportions of the detergent for amyloglucosidase preparations Up to 150 units of amyloglucosidase per cubic centimeter are around 0.01 or 0.2 Parts by weight, based on 100 parts by weight of aqueous amyloglucosidase preparation.

Wehn aqueous amyloglucosidase preparations containing more than 150 units Amyloglucosidase contained per cubic centimeter, according to the proposed method are to be cleaned, the above specified weight ratios of the cleaning agent be increased accordingly.

The amount of time it takes to substantially increase glucogenic effectiveness of the amyloglucosidase preparation is required by the protein precipitant pretty short. It is therefore only necessary to add the protein precipitant in the amyloglucosidase solution to distribute evenly. It has been found that a moderate agitation of 15 a duration of up to 30 minutes is appropriate, but a shorter duration is also sufficient is.

The method of the invention is for known amyloglucosidase preparations generally applicable. Although the commercially available amyloglucosidase preparations are common with the help of Aspergillus strains (Aspergillus niger, Aspergillus oryzae, Aspergillus phoenicis and Aspergillus flavus) is the method of the invention can also be used with amyloglucosidase preparations obtained by cultivating other microorganisms, which include representatives of Mucor, Clostridium and Rhizopus strains have been.

The following examples illustrate embodiments of the invention explained.

Example 1 This example illustrates the treatment of an amyloglucosidase preparation by the process of the invention obtained by filtering off an Aspergillus phoenicis culture broth prepared by the process of Example 1 of US Pat. No. 2,893,921. The amyloglucosidase activity of the filtered broth is 90 units per cubic centimeter as determined by the method described in column 2, lines 29 to 41 of the patent. 10 g of a 10% aqueous solution of the sodium salt of monokerylbenzene monosulfonic acid are stirred into 11 of the filtered broth, which has a temperature of 30 ° C. and a pH of 4. The mixture is brought to pH 3.5 with dilute hydrochloric acid, stirred for 30 minutes and then filtered through a coarse filter paper. Aliquots of the treated and original broth are then tested for their ability to hydrolyze acid-thinned corn starch paste or syrup by the following procedure: 100 cc aliquots of the diluted paste at 60 ° C (30 to 35 weight percent solids, 15 DE , pH 4, by carefully treating a corn starch slurry with a solids content of 35 °! o at a pH of 1.9, which has been adjusted with hydrochloric acid, in the autoclave, cooling and neutralizing to a pH of 4 with Soda made) are placed in a few 120 g bottles, which are then placed in an incubator kept at 60 ° C. 3.9 cc of the original broth are placed in one half of the bottles, while 4.2 cc of the treated broth are placed in the rest of the bottles, with the same volume of units as the 3.9 cc of the original broth is supplied. One of the two groups of bottles is then removed from the incubator after 48 hours, whereupon the glucose content is determined in this using the glucose oxidase method which is described in Analytical Chemistry, Vol . 31, p. 109 (1959). This procedure is repeated with separate pairs of bottles after incubation times of 72, 96 and 120 hours. The analysis results are given below Incubation period Glucose content Glucose content (Hours) (Original Enzyme) (Treated Enzyme) 48 86.0 89.3 72 85.7 91.1 96 86.2 94.2 120 86.7 94.9 If the above-described procedure is repeated under the same conditions but the pH of the treated broth is adjusted to 2, 4, 5 and 6 in separate experiments, the treatment is found to be practically ineffective at pH 6 that is, the treated broth and the original broth have practically the same starch hydrolyzing activity. It has also been found that at a pH of 2 a substantial part of the amyloglucosidase activity is lost.

If the procedure described in Example 1 is repeated, at separate treatment methods, however, 1, 5, 20 and 30g of the sulfonate solution are used are found that 1 / to part by weight of sulfonate per 1000 parts by weight of Amyloglucosidase broth represents about the lower limit of effectiveness and that the Amyloglucosidase effectiveness of the treated broth is substantially reduced if more than 2 parts by weight of the sulfonate used per 1000 parts by weight of the broth will.

Example 2 The process described in Example 1 is repeated at a pH of 3.5 with 0.1% precipitant, but the monokerylbenzene monosulphate is replaced by the sodium salt of monoisopropylnaphthalene monosulphonic acid. The following comparative values are obtained Incubation period Glucose content Glucose content (Hours) (Original Enzyme) (Treated Enzyme) 48 86.0 87.7 72 85.7 89.8 96 86.2 90.6 l20 i 86.7 91.9 Example 3 This example illustrates the use of a sulfonated higher alcohol to increase the glucogenic activity of an aqueous amyloglucosidase preparation obtained by growing Aspergillus niger. The fungus was grown according to the method described in U.S. Patent 2,557,078, column 4, lines 4-23. The filtered broth of the above culture contains 60 units of amyloglucosidase per cubic centimeter. The procedure described in Example 1 is repeated, except that the monokerylbenzene monosulfate is replaced by the sodium salt of the mono-3,9-diethyl-6-tridecanol ester of sulfuric acid and, in the comparative experiments, the volumes of the untreated and treated broths to 5.8 and 6, respectively , 0 cc have been changed. The analysis results are given below: I. Incubation period I Glucose content Glucose content (Hours) (Original Enzyme) (Treated Enzyme) 48 86.0 90.2 72 85.7 91.7 96 86.2 93.5 120 I 86.7 94.1 Example 4 In this example, the use of the di-2-ethylhexyl ester of orthophosphoric acid for purifying an aqueous amyloglucosidase preparation obtained by growing Aspergillus phoenicis by the method of Example 1 is described. The treatment process described in Example 1 is repeated at a pH of 3.5 and with 0.10% of the precipitant, but the monokerylbenzene monosulfonate is replaced by the sodium salt of the di-2-ethylhexyl ester of orthophosphoric acid. The analytical results obtained are shown below Incubation period Glucose content Glucose content (Hours) (Original Enzyme) l (Treated Enzyme) 48 86.0 88.5 72 86.0 89.0 96 86.2 92.1 120 86.7 - Example 5 In this example, the use of a naphthalene monosulfonic acid-formaldehyde condensate for purifying an aqueous amyloglucosidase preparation obtained by growing Aspergillus phoenicis by the method of Example 1 is illustrated. The treatment process described in Example 1 is repeated at a pH of 3.5 with 0.1% of the precipitant, but the monokerylbenzene monosulfonate is replaced by a sulfonated naphthalene-formaldehyde condensate. An example of such a commercially available condensate is a product sold by the National Aniline Division of Allied Chemical Corporation under the trade name "Naccotan A". The analytical results obtained are shown below Incubation period Glucose content Glucose content (Hours) (Original Enzyme) (Treated Enzyme) 48 86.0 I 88.4 72 86.4 91.6 96 86.2 92.5 120 87.2 93.2 Example 6 In this example, the use of an aryl alkyl ether sulfonate to purify an aqueous amyloglucosidase preparation obtained by growing Aspergillus phoenicis by the method of Example 1 is illustrated. The process described in Example 1 is repeated at a pH of 3.5 with 0.10% of the precipitating agent, but replacing the monokerylbenzene monosulfonate with the sodium salt of the sulfuric acid monoester of the formula is replaced. The analytical results obtained are given below: Incubation period Glucose content Glucose content (Hours) (Original Enzyme) (Treated Enzyme) 48 86.0 87.3 72 85.7 88.1 96 86.2 89.0 120 86.7 89.6 Example 7 The process described in Example 1 is repeated at a pH of 3.5 with 0.1% of the precipitant, but the solution of the monokerylbenzene monosulfonate is replaced by 40 g of a dilute sodium hydroxide solution containing 4 g of purified lignin. The following results are obtained Incubation period Glucose content Glucose content (Hours) (Original Enzyme) (Treated Enzyme) 48 83.3 90.4 72 84.5 89.2 96 85.7 91.0 120 86.1 92.7 As used herein, the term "starch" is intended to (1) any native starch that is either waxy or non-waxy and may be derived from the roots, stem or fruits of plants, (2) the separate fractions (amylose and amylopectin) of not denote waxy starches and (3) slightly modified starches which have been modified by oxidation, acid treatment, generation of derivatives or which have been modified by heat.

Claims (3)

Claims: 1. Process for increasing the glucogenic activity of crude, aqueous amyloglucosidase preparations from molds or bacteria, characterized in that these are treated at pH values above about 2 and below about 5 with an acid of the following structural formulas where R, an alkyl group with 10 to 15 carbon atoms, R2 an alkyl group with 3 to 5 carbon atoms, R3 an alkyl group of the formula CH3C (CH3) 2CH2C (CH3) z - or CH3C (CH3) 2CH2C (CH3) 2CH2C (CH3) 2 - R4 is a 2-ethylhexyl group, Rs is an alkyl group with 14 to 18 carbon atoms, n = 0, 1 or 2 and m is an integer from 1 to 6, or a water-soluble salt of these acids is mixed. 2. Procedure according to Claim 1, characterized in that an amyloglucosidase preparation up to at 150 units of amyloglucosidase per cubic centimeter the acids or their water-soluble ones Salts in an amount of 0.01-0.2 parts by weight per 100 parts by weight of amyloglucosidase preparation used. 3. The method according to claim 1, characterized in that temperatures of 20 to 40 ° C are maintained at pH values of 3 to 4. Considered publications German Auslegeschrift No. 1087 550.