DE1274549B - Process for purifying and increasing the glucogenic activity of aqueous amyloglucosidase preparations - Google Patents

Description

Process for purifying and increasing the glucogenic activity of Aqueous amyloglucosidase preparations The invention relates to a method for purification and increasing the glucogenic activity of aqueous amvolglucosidase preparations containing Removing carbohydrate-thickening enzymes as impurities from microorganisms.

It is known that arrryloglucosidase preparations which, for. According to U.S. Patents 2,557,07 @, 2,881,115 and 2,893,921 , starch or starch products such as e.g. B. with acid hydrolyzed starch, with α-amylase hydrolyzed starch Lz. The like., Do not hydrolyze completely to glucose. It is of course beneficial to achieve the greatest possible conversion or the greatest possible hydrolysis of the starch product to glucose within a short time. A high degree of hydrolysis facilitates the crystallization of the glucose from the concentrated hydrolyzate because the other hydrolysis products inhibit the crystallization of the glucose. A high degree of hydrolysis naturally increases the yield of glucose, while the yield of the mostly undesirable mother liquors decreases. Techniques for purifying and increasing the glucogenic activity of amvloglucosidase preparations are already known.

Atis is the documents of the French patent 1 299 760 the cleaning before. aqueous amyloglucosidase preparations at pH values of 2 to 5 and temperatures from 0 to 60 °: 'n with lignin. Tannic acid or water-soluble salts known of these substances. With relatively high losses of active material very active materials can be obtained there, which last for quite a long time Hydrolyses lead to high glucose levels, for example to glucose levels after 120 hours of 94.5. In contrast, the preparations obtainable according to the invention are essential more active, as they are already down to glucose levels of a comparable magnitude lead in a much shorter time.

To simplify the description, lignin, tannic acid are used below and water-soluble salts of these substances are referred to as Group A compounds.

In the earlier German patent 1237 044 a method for increasing the glucogenic activity of aqueous amyloglucosidase preparations is proposed, these being treated with acids of the following structural formulas at pH values above about 2 and below about 5 the definition of the above radicals being given below, or water-soluble salts of these acids mixed. A disadvantage of this process is that the preparation becomes cloudy. the removal of which leads to considerable material losses. Compared to this proposed preparation, too, the preparations obtainable according to the invention have the advantage of being more active if the starch is hydrolyzed for the same periods of time.

To simplify the description, the above are listed Acids or their salts designated as group B.

The method according to the invention for purifying and increasing the glucogenic activity of aqueous amyloglucosidase preparations containing carbohydrate-synthesizing enzymes as impurities from microorganisms by removing them at pH values of? to 5 and temperatures from 0 to 60 ° C with lignin, tannic acid, a v,:, -, is-soluble salt of these substances or an acid of the following structural formulas where R, an alkyl radical with 10 to 15 carbon atoms, R. = an alkyl radical with 3 to 5 carbon atoms, R; one of the radicals CH: 3C (CH:;) = CH = C (CH: #,). = -and CH:; C (CH: :) aCH; C (CH: s) = CH2C (CI-I :: ). = -. Ri der 2 _ Ätliylliexylrest, R; an alkyl radical with 14 to 13 carbon atoms, n = 0, 1 or 2 and an integer from 1 to 6, or a water-soluble salt of these acids mixed and then separated from the precipitate formed is characterized in that ligniri, tannic acid or a water-soluble salt of these substances and one of the acids given by structural formulas or a water-soluble salt of these acids is used.

So it was found that the common use of a detergent of group A together with a cleaning agent of group B the production of Amyloglucosidase preparations in an economical manner and without the formation of cloudiness made possible in the preparation. Surprisingly, it was found that the enzyme cleaning agents of group A in the presence of the enzyme detergents of class B the turbidity, the normally when using the Group B enzyme detergent alone arises, fails. When using compounds of groups A and B results in preparations with higher amyloglucosidase contents, d. i.e., the preparations have a higher effectiveness, which is evident from their saccharification ability from starch to larger amounts of glucose within a given period of time, than is the case with preparations made from identical starting preparations Treatment with either a group A agent alone or a group agent B were obtained. When using funds from groups A and B represents the removal of further transglucosidase from the amyloglucosidase priipate is another additional benefit. It was not to be expected that with the addition a group B agent that remains cloudy becomes a group agent A, with which there is no cloudiness, a surprisingly better cleaning of the preparation can basically be achieved without loss of amyloglucosidase and that per se very good results when using only the funds from group A or from group B can still be significantly improved.

According to the invention, a small proportion of an agent of the group A and an agent of group B with an aqueous amyloglucosidase preparation which contains colil hydrate synthesizing enzymes as impurities. That Amyloglucosidase preparation is then separated or physically separated from the precipitated or coagulated interfering enzymes separately. The liquid part that cleaned it up or treated amyloglucosidase preparation can be used as such for hydrolysis from starch and starch hydrolysis products to glucose used or concentrated, for Evaporated to dryness or with a water-miscible organic solvent. such as acetone or ethanol, must be dehydrated before it is used.

The most important starch-hydrolyzing enzyme in the preparations obtained according to the invention has various names. It is z. B. as;, - amylase, glucoamylase. Starch glucogenase, maltase and amyloglucosidase. The enzyme differs from other starch-hydrolyzing enzymes in its properties. To hydrolyze starch to glucose without significant amounts of low molecular weight hydrolytic intermediates being formed at the same time. like maltose, maltotriose, higher Sugar And Soluble L) e'Ctriiie .. That Enzyni Seems to \ viken in such a way that cs eii: z ° lne Glticosee; nli; fit, beginning ari your not @duziercaden end of one Starch cat, split off. 1311s Et: cynl '? #: Irfllysiert further Maltose. ILIaltotriosw Uli .. Between bClleilprodcihtc der- S :: ilk; -1 "ita ;; ae. The @ myloglricosicla = .wL.; T.cl- = l; tr, slic: i-findungsgc- mi1: @erwenclet tvertlerl., c. ~ 11 :: 11, we.rdelr from schim- I11elp11Lt'n undakt @ "ilera, ar, a @ 'ÖllnlICh by SUbniers- ferine'nt @? tion. but also in other processes ww) grooves. The, Lrlz`y'1npl '@ 4 @ Jar @ -t has pt "usually the For in a filtered or zenirifalgae fermentation solution, but can also be present in andccrl ° i-Uorin. For this belong 1. the entire fermentation mixture or fermentation culture including tles 1 @@ Iikroorganisnlus, ?. the dried whole culture, 3. the dried watering solution, 4. The aqueous extract of the -e dries completely aen culture Lind 1. Cinc ktlnlcitfr @ r'te;#ztrocl: _iiv-te Siibst @ tllZ, the by AustIhn cy @ s strength l: _vdi-civsiei-ends Enavnis from clcr atlier len oc @ t °: 7eritl il'ugierten GarlÖSCing with? A?. # Lll,: ItwaSS @@ rUI1 @ LTSml @ el, like Acetone or tthanei, is obtained. Wispicle for IVIikro @ rgariisn: er._. which in well-known Way under picture of complete fermentation gerrios or fermentation solutions that can be grown economically viable oil, bonzcritr-rltioiicn full Arny .. loghicosidase are .A.spel-gillcls, MLICOr. Clostridiuln undlüz. @ Lpu. The following types are Examples of good Erzouger -von Airlylogii.icosidase: Aspergillus oryzae, Clostridium acetobtit @, licuni, Rlii- ollus delemar, Aspcrgillus niger, Asper; gillus phoeni- cis, Aspergillus a @ varnori. AspergillUS I11wet1S Lind Aspergillus lavus. The production of Anivloglucosidae in the form of full: changing cultures -Iiad fermentation solutions is in the UNITED STATES. - Patents' 5.57 071 ;,? l # 8l 11 5 and @ 9; t) 21. The purification of vain crude AiiiS-logiticosidasepr; ipcirates. of the aqueous extract from Rhizopus deleniar, .is in Jour: i. .At the. Chem. Soc., 73, pp 3359-3365. The representationally indicated by the structural formulas given means of group B can either as free. Acid or its water-soluble salts be turned. The sodium salt of acids is the preferred form, but other water- soluble salts of these acids, such as the lithium, Potassium and ammonium salts and the salts with lower ones Alkyl amines and quaternary ammonium hydroxy which is also effective. Procedures according to which the above by the Structural formulas are given as sulfonic acids or Group B sulfuric acid esters are produced can, are in surface active agents, their chemi- stn, and Technology, by AM S c 1i wartz and J. W '. Pei- ry. 19: J9, Interscience Publishers, Inc., described in chapters 3 and 5. The di-2-ethyl hexyl ester of orthophosphoric acid can be ter way are produced. Examples of acids falling within the range of the above Structural formulas are: Monol; er @ Ibenzenesulfon- acid. where the keryl radical rides an alkyl flu an average of about 15 Kolilenstoffatornen, the is obtained from a highly @ -saturated luminous oil, h4orioclec@llic.nrolnlonosulfonsiicrre, Monopentade- Cylbenzolinoriostilfonsä ure, Monokeryltoluolmonosulfonsätrre, Monokervlxvlolmonosulfonsäuren c. MonokeryIccimolmonosulfonsäure, Monoisopropylnaphthalirrllionosulfonsätire, Monoisoainylnaphthalinmonosufonsäure. the acidic ester of sulfuric acid and 7-i #, thyl -? - methyl-4-undecanol and the acidic ester of sulfuric acid and 3,9-diethyl-6-tridecanol.

The detergents of group B are used in the proportions indicated in the earlier patent mentioned. An amyloglucosidase preparation with an enzyme activity of up to 150 units per cubic cell (-. R can be cleaned with about 0.01 to 0.2 parts by weight of an enzyme cleaning agent of group B. Per 100 parts by weight of amylogluccid) sidase preparation. When making stronger amyolglticosidase preparations, e.g. B. by precipitation of the enzyme from a fermentation solution, alcohol and.A, to dissolve the precipitate in a limited amount of water, the minimum amount by weight of cleaning agent given above must be increased in proportion to the increased effectiveness.

The means of group A consist of lignin, tannic acid or water-soluble Salt the same. An alkali lignin, which has been obtained from alkali: iolzpülpen, is the preferred form of lignin. It is insoluble in pure water, but soluble in aqueous solutions of strong alkalis such as sodium hydroxide. It's in trade in the »free (<or acid form available, which are dissolved in alkali tiluss, and in the sodium salt form, which dissolves in warm water. Any commercially available The form of raw or purified tannic acid is suitable. Tannic acid is soluble in water. These enzyme cleansers will all be in a concentration of about 0.01 to 1 (preferably 0.1 to 0.4) parts by weight per 100 parts by weight of aniyol glucosidase preparation applied with an enzyme activity of up to 150 units per cubic centimeter.

In particular, a cleaning agent from group A and a cleaning agent are used of group B 111 water to a single or two separate aqueous solutions Concentration dissolved, e.g. B. from 5 to 10 percent by weight. The concentration of However, solutions are not essential; they can also be used outside of this Area. The solution or solutions are then in the appropriate concentration rides the aqueous Aniyolglucosidase-. preparation mixed. According to a different procedure but can also use either a detergent or both in dry or pure State to be added to the aqueous amyloglucosidase preparation. The application in Solution is preferred because it reduces the likelihood of getting a Part of the amyloglucosidase is precipitated by local concentration of detergents or to inactivate.

Although the detergents in any order or at the same time can be added, cleaning agent B is preferred first and then that Detergent A added to the ainyloglcicosidase preparation to ensure the complete To ensure precipitation of all the turbidity caused by cleaning agent B. The time required for both Agent A and Agent B To increase the glucogenic effect of the amyloglucosidase preparation is quite good short. Also, only a short time is required to remove the cloudiness with detergent A. to remove that through the Cleaning agent B was created. It was found to have 15 to 30 minutes of moderate agitation with each detergent are appropriate, but that shorter times are sufficient.

The amyloglucosidase preparation is then coagulated or precipitated Enzyme and separated from the turbidity by treating with the enzyme detergents have arisen. The separation can be done by decanting off the clear protruding Liquid after a certain settling time or by filtering or centrifuging take place.

The cleaning agents used according to the invention have a pH range greatest effectiveness between about pH 4 and 2 or below. Your effectiveness decreases decreases rapidly when the pH rises above 4 and becomes vanishing at around pH 5 small. Because of the sensitivity of amyloglucosidase at low pH values, in particular at pH 3 and below, that is preferred for the treatment according to the invention pH in the upper part of the above range, i.e. H. at about 3 to 4. Furthermore it is when carrying out the method according to the invention at lower pH values expedient to keep the temperature low (e.g. at 20 ° C or below) in order to avoid loss of amyloglucosidase through inactivation.

The preferred temperature for the process according to the invention in the preferred pH value of 3 to 4 is between 20 and 40 ° C. If necessary, can lower temperatures down to the freezing point of the preparations are also used but the cost and difficulty of working at lower temperatures will decrease more significant than a small loss of amyloglucosidase due to pH inactivation. Temperatures above 40 "C can cause precipitation or inactivation of the undesired enzymes are used, but values are above 55 to 60-C inexpedient because the amyloglucosidase is then thermally inactivated. The temperature control in the process according to the invention is largely due to the freezing of the aqueous Amyloglucosidase preparations on the one hand and the inactivation of amyloglucosidase on the other hand, determined at elevated temperature. The selective precipitation or inactivation The unwanted enzymes with the detergent is caused by temperature changes only slightly affected in the range from 0 to 60 ° C.

The following examples explain the process according to the invention. Example 1 This example illustrates the application of the method according to the invention to an amyloglucosidase preparation obtained by filtering the culture solution from a fermentation batch from Aspergillus phoenicis, as in Example I of U.S. Patent 2,893,921. The amyloglucosidase activity of the filtered fermentation solution is 90 units per cubic centimeter, according to the information in column 2, lines 29 to 41, the method described in the patent specification. In 11 of the filtered fermentation solution were at 30 ° C and pH 4 5 g of a 10o; oigen aqueous solution (0.5 g dry solid substance) introduced by monokerylbenzene monosulfonic acid. The mixture was diluted with Hydrochloric acid brought to pH 3.5 and stirred for 30 minutes. Then 20 g of a diluted Sodium hydroxide solution containing 2 g of purified lignin, stirred into the solution. The mixture was brought to pH 3.5 with dilute hydrochloric acid and stirred for 30 minutes and then filtered through coarse filter paper. The clear filtrate did not contain any Cloudiness.

A second amyloglucosidase preparation was made in the same manner as made above, only the solution was not treated with lignin. The filtrate had a considerable content of opacifiers. The "turbidity" consisted of colloidal dispersion Substance that was formed when the detergent was added.

A third amyloglucosidase preparation was made in the same manner as made above, only the solution was made with lignin and not with monokerylbenzene monosulfonic acid treated. The clear filtrate did not contain any turbidity.

The original and the treated solutions were then tested for their ability to hydrolyze acid-diluted corn starch, paste or syrup as follows: Aliquots of the diluted paste of 100 cc (30 to 35 percent by weight solids, 15 DE, pH 4) were prepared by carefully treating a slurry of 35% solids made from corn starch at pH 1.9 with hydrochloric acid in the autoclave, cooling and neutralizing to pH 4 with soda), four 113 g bottles were measured at 60 ° C and placed in an incubator at 60 ° C posed. 4.2 cc of amyloglucosidase solution from each of the samples purified as described above were added to one bottle each and 3.9 cc of the unpurified amyloglucosidase solution was added to another bottle. After 72 hours, all bottles were removed from the incubator and examined for their glucose content by the glucose oxidase method described in Analytic Chemistry, 31 (1959), p. 109. The results are given below: Detergent glucose content Monokerylbenzene monosulfonic acid and lignin ......... ........... 93.9 Monokerylbenzenesulfonic acid ... 90.0 Lignin ..................: ........ 92.0 Without ........................... 87.5 The above example shows that diluted starch paste = an enzyme purified with both monokerylbenzene monosulfonic acid and lignin is converted to a much higher glucose content than an enzyme that has been purified with only one or none of these cleaning agents. The above example also shows that lignin is effective in removing the haze created by using monokerylbenzene monosulfonic acid as a detergent.

Example 2 Example 1 was repeated with practically the same results, except that the 2 g of lignin were replaced by 4 g of tannic acid. The solution, purified only with monokerylbenzene monosulfonic acid, contained turbidity after filtering. On the other hand, when tannic acid was used in conjunction with the monokerylbenzene monosulfonic acid, the filtrate contained no turbidity. The results of the 72 hour saccharification were as follows: Detergent glucose content Monokerylbenzene monosulfonic acid and tannic acid .................. 92.3 Monokerylbenzylmonosulfonic acid ... 90.0 Tannic acid ........................ 91.4 Without ............................ 87.5 Example 3 Example 1 was repeated with practically the same results, except that a sodium salt of monokerylbenzene monosulfonic acid in pearl form was used. The fermentation solution purified only with the sodium sulfonate was cloudy after filtering, while the solution purified with both cleaning agents gave a clear filtrate. The results of the 72 hour saccharification were as follows: Detergent glucose content Sodium salt of monokerylbenzene monosulfonic acid and lignin ...... 93.8 Sodium salt of monokerylbenzene monosulfonic acid ................ 89.2 Lignin ............................ 92.0 Without ............................ 87.5 Example 4.

Example 1 was repeated with practically the same results, where a monokerylbenzene monosulfonate and a lignin of different origin are used became. Purified only with the sodium salt of Monokerylbenzolmonosu) fonsäure Solution after filtration contained turbidity, while that with both detergents purified solution gave a cloud-free filtrate. Electrophoresis showed that the solution cleaned with both detergents did not contain any transglucosidase. Example Example 1 was repeated with practically the same results, only 0.5 g (dry weight) of a long-chain alcohol sulfate was used instead of monokerylbenzene monosulfonic acid used. The solution purified only with the sulfate contained after filtering a turbidity, while the solution cleaned with both cleaning agents is a turbidity-free one Filtrate resulted. Electrophoresis readings showed that those with both detergents jointly purified solution did not contain any transglucosidase.

Example 6 Example 1 was made with virtually the same results repeated, only 0.5 g (dry weight) of di - (') - ethylhexyl) sodium phosphate were used used in place of motiokerylbenzene monosulfonic acid. Those only with di- (2-ethyfhexyl) -sodium phosphate purified solution contained turbidity after filtering, while that with both Detergent-purified solution resulted in a cloud-free filtrate. Electrophoresis showed that the solution cleaned with both detergents was only a minor one Content of transglitcosidase contained.

Claims (3)

Claims: 1. A process for purifying and increasing the glucogenic activity of aqueous amyloglucosidase preparations containing carbohydrate-synthesizing enzymes as impurities from microorganisms by treating them at pH values of 2 to 5 and temperatures of 0 to 60 ° C with lignin, tannic acid, a water-soluble salt of these substances or an acid of the following structural formulas where R, an alkyl radical with 10 to 15 carbon atoms, R2 an alkyl radical with 3 to 5 carbon atoms, R; 3 one of the radicals CH, 3C (CHa @ 2CH2C (CH3) --- and CH3C (CHa> 2CH2C (CH: 3 ) 2CH2C (CHa @ 2 -, R1 the 2-ethylhexyl radical, R; an alkyl radical with 14 to 18 carbon atoms, fr = 0, 1 or 2 and an integer from 1 to 6, or a water-soluble salt of these acids mixed and are then separated from the precipitate formed, characterized in that lignin, tannic acid or a water-soluble salt of these substances and one of the acids indicated by structural formulas or a water-soluble salt of these acids are used. 2. The method according to claim 1, characterized characterized in that the lignin, the tannic acid or the water-soluble salt of these Substances according to one of the acids specified with structural formulas or a water-soluble one Salt of these acids- used. 3. The method according to claim 1 and 2, characterized in that the acids indicated with structural formulas or their water-soluble salts in an amount of about 0.01 to 0.2 parts by weight and the lignin, tannic acid or the water-soluble salt of these substances in one Amount of about 0.01 to 1.0 parts by weight per 100 parts by weight of the crude aqueous amyloglucosidase preparation used. References considered: Documentation of French patent No. 1299 760. Earlier patents considered: German patent No. 1237 044.