JPH0569509B2 - - Google Patents

Description

[Detailed description of the invention]

The use of thermostable α-amylase as a thinning agent during starch liquefaction is of great importance in the production of sugar sweeteners. Enzymatic liquefaction of starch materials reduces the formation of undesirable by-products and
It also allows the use of low levels of salt, which is desirable since high levels of salt in the final glucose syrup involve additional manufacturing costs for its removal by ion exchange resins. The usefulness of enzymes such as α-amylase for liquefying starches at elevated temperatures depends primarily on the thermostability of the enzyme, since exposure to heat causes its irreversible denaturation. This modification results in safe loss of biocatalytic activity. Thermostable α-amylases obtained from thermophilic microorganisms have been used to hydrolyze, liquefy and/or convert starch-containing materials to starch hydrolysates. For example, US Pat. No. 3,654,081 and US Pat.
No. 3912590 describes the liquefaction of starch at high temperatures using a thermostable α-amylase produced by a bacterium of the species Bacillus licheniformis. Many commercially available alpha-amylase products are based on bacterial sources such as Bacillus subtilis, B. licheniformis, B. stearothermophilus, Bacillus
Obtained from B. coagulans.
Fungal α-amylases have very limited applicability in starch liquefaction at high temperatures because they are generally not considered thermostable enzymes. Enzymatic liquefaction of starch is usually carried out at elevated temperatures to increase the amenity of the starch to enzymatic hydrolysis. The loss of biocatalytic activity described above can occur even with thermostable alpha-amylases at elevated temperatures preferred for the liquefaction process. Therefore, to increase its thermal stability,
Add stabilizers to enzymes. Thus, Wallerstein's U.S. patent no.
No. 905029 discloses that high-temperature saccharification of starch by enzymatic methods is enhanced by the addition of calcium sulfate to the starch-containing medium. The use of calcium ions to render α-amylase thermostable is generally accepted in the art, but has certain drawbacks, such as the problems discussed above associated with adding salt to starch. Most recently, in U.S. Pat. No. 3,654,081, it was shown that starch can be easily and completely liquefied by adding calcium and/or sodium salts to an aqueous starch slurry, preferably containing an alpha-amylase. Disclosed. In U.S. Pat. No. 3,912,590, an aqueous starch suspension was prepared from α
- A method for the combined liquefaction and thinning of starch is disclosed, consisting of treatment at elevated temperatures with amylase. US Pat. No. 4,284,722 claims a heat and acid stable alpha-amylase derived from a bacterium of the species Bacillus stearothermophilus. Pace and others are authors of the Journal of Biological Chemistry.
(logical chemistry), the thermal and guanidine hydrochloride denaturation of lysozyme was studied at various concentrations of tri-N-acetylglucoseamine, which binds specifically to the active site of native lysozyme, and the presence of thermal and guanidine hydrochloride reported that it results in readily appreciable stability of the protein against salt denaturation. Adding chloride ions to an aqueous solution of α-amylase,
Although this has been found to provide some thermal stability, the main purpose of such addition is the regulation of the PH value. In U.S. Pat. No. 4,497,867, preservation of protease with Subtilisin Carlsberg consists of adding calcium ions and an aqueous carboxylate selected from the group consisting of formate, acetate, propionate and mixtures thereof to a solution of the enzyme. A method is disclosed for increasing the shelf life of. U.S. Pat. No. 4,318,818 contains (a) 0 to about 75% detersive surfactant; (b) pure enzyme, preferably about a proteolytic enzyme.
0.025 to about 10%; (c) 0% to low molecular weight primary or secondary alcohol
about 60%; (d) a short chain length carboxylate, preferably about 60%;
Discloses a stabilized aqueous enzyme composition consisting of 0.1% to about 10%; (e) a soluble calcium salt in an amount sufficient to provide 0.1 to 10mM calcium ions; and (f) the remainder water. . In a typical commercial operation, starch is liquefied using a thermostable α-amylase at a temperature range of 80°C to about 110°C and a pH value of 6 or above. Generally, calcium salts (50-150 ppm Ca ⁺⁺ ) are added to further stabilize the enzyme at elevated temperatures. These conditions for enzymatic liquefaction of starch have three drawbacks. Firstly, the hydrolysis of starch at pH values above 6.0 and temperatures of 80°C or above promotes the isomerization of the reducing groups of the starch resulting in the production of maltose during the subsequent conversion step, resulting in the final glucose causing a decrease in yield. Second, the optimal PH value used for saccharification of liquefied starch to produce glucose for subsequent conversion to fructose is generally about 4.5 for Aspesgillus type glucoamylase; For Rhizopus type enzymes it is approximately 5.0. Thus, if starch is to be liquefied at a pH value of 6.0 or higher, the pH value must be lowered before saccharification using glucoamylase. this
PH value adjustment, due to the addition of acid, increases the salt content of the resulting glucose syrup and thus increases the expenditure on ion exchange resins required to remove excess salts. For liquefaction carried out at a pH value of 6.0 or lower, additional calcium ions (approx.
(up to 500ppm) must be added. This makes it difficult to completely remove added calcium ions from the glucose syrup produced by starch saccharification when glucose isomerase is used to produce high fructose corn syrup. It would be desirable, and it is an object of the present invention, to provide a new method of increasing the thermal stability of alpha-amylase in aqueous solution. A further object of the invention is to provide stabilized α-amylase compositions produced by this method. A further object of the invention is that there is no need to add calcium ions and that low levels of calcium (25
The object of the present invention is to provide such a composition that can be used to liquefy starch (~50 ppm). Another object is to provide such a composition which can be used for high temperature liquefaction of starch with pH values as low as 5.0. The present invention is a method of increasing the thermostability of bacterial alpha-amylase. This method involves stabilizing amounts of amphiphiles in an aqueous solution of α-amylase.
−le). The invention also includes alpha-amylase solutions stabilized by this method and their use in starch liquefaction. Alpha-amylase useful in the present invention is produced by culturing suitable microorganisms in a nutrient growth medium to generate extracellular enzymes. Amphiphile stabilizers can be added before or after removing the biomass or concentrating the enzyme solution. However, in a typical experiment, the microbial cells are removed from the alpha-amylase-containing medium by conventional methods, such as centrifugation, decantation and/or filtration.
The liquid is then concentrated using ultrafiltration and vacuum evaporation to obtain an aqueous solution of enzyme with the desired activity level. This solution is preferably a solution stabilized by the method of the invention. Increasing the thermal stability of the α-amylase solution is achieved by adding a stabilizing amount of an amphiphile to this solution. The term amphiphile refers to a molecule or ion having a polar (water-soluble) head and a non-polar (water-insoluble) tail. In addition, the amphiphile must itself be large enough for each end to undergo a dissolution reaction. The general structural formula of amphiphiles is shown below:

embedded image where X is a polar or ionic group, e.g.
COO ^- , NH ₄ ⁺ , OH, SO ₄ =, SO ₃ = or
represents CONH ₂ and R is a nonpolar moiety. Thus, different groups of amphiphiles are considered useful in contributing to the thermostability of alpha-amylase. Examples of useful amphiphiles are alcohols such as ethyl alcohol, propyl alcohol, butyl alcohol, benzyl alcohol; amines such as benzylamine, butylamine, ethylamine, hexylamine; and amides such as butylamide, acetamide,
Phenylacetamide and benzamide. While not intending to find a particular theory of how the invention operates, it is believed that amphiphiles contribute to the thermal stability of α-amylase due to electrostatic and hydrophobic interactions. In a preferred embodiment, the ionic group of the amphiphile is such that the ionic moiety has the formula R-COO ^-- wherein R is H or an aliphatic or aromatic group containing from 2 to 14 carbon atoms. It is a carboxylate represented by: More particularly, R can be methyl, ethyl, n- or isopropyl, n- or isobutyl, phenyl or substituted phenyl. Amphiphiles containing carboxylates can be introduced into the aqueous solution of α-amylase in the form of the corresponding carboxylic acid, but in order to increase the solubility with the acid salts, e.g. Preference is given to using sodium or calcium salts. Carboxylate ions, typically in their sodium salt form, are added to an aqueous solution of α-amylase in a stabilizing amount, ie, an amount that significantly increases the resistance of the enzyme to thermal inactivation. Analysis of a commercially available α-amylase preparation showed that the preparation contained 0.02 acetate ions per million units of enzyme activity, expressed in MWU, measured as shown in Example 1.
It has been shown that it can contain up to g. Presumably, the presence of acetate ions is a result of the addition of acetic acid for pH adjustment, and its presence at this low concentration is insufficient to confer meaningful thermostability to α-amylase. Typically, the concentration of acetate is at least about 2.89 x acetate per mole of alpha-amylase, using a molecular weight of 58,000 Daltons for alpha-amylase for calculation purposes.
10 should be ³ mol, this value is the enzyme activity 100
Corresponds to 0.24g of acetate per 10,000 units. The minimum levels of other amphiphiles required for effective stabilization of the enzyme will vary depending on the particular material used, but can be readily determined without experimentation. When acetate is used as a stabilizing agent, the preparation of the stabilized α-amylase preparation involves adding the sodium salt of the acetate ion to an aqueous concentrate of α-amylase and adding 170,000-180,000 MWU/ml to 100 ml of an enzyme concentrate with activity. against sodium acetate 10
This is typically done by adding to a final concentration of g. In other embodiments, the amphiphile is added to the starch before or after the addition of the alpha-amylase in an amount sufficient to provide an equivalent ratio of amphiphile to alpha-amylase. The method of carrying out the invention is further illustrated by the following examples. Example 1 A sample of α-amylase was prepared by culturing a mutant strain of Bacillus licheniformis (described as ATCC 53376 under the Budapest Treaty) in a suitable nutrient growth medium. After fermentation, the microbial cells were removed by conventional methods, leaving the extracellular enzymes in solution. The enzyme containing solution was then purified using ultrafiltration and vacuum evaporation to 340,000−
360000MWU/g (Modified Wohlgemuth Unit/g)
concentrated to the desired activity level. α-Amylase activity was obtained using a blue equivalent reduction of starch-iodine complexes and measured by determining the hydrolysis of soluble starch. In a typical experiment, 2% soluble starch buffered to a pH of 5.4
ml and 14 ml of enzyme appropriately diluted with 4 ml of water.
Culture was carried out in a water bath maintained at 40°C. At timed intervals (5-30 minutes after enzyme addition), 1 ml of the test material was removed and injected into a tube containing 5 ml of diluted iodine solution and mixed by inversion. Next, the developed color is made by Hellige No. 600-
A DA daylight colorimetric illuminator was used to monitor the approach of reaction endpoint and enzyme activity, which was monitored as modified Wohlgemuth units. 1 Modified Wohlgemuth unit (MWU) is calculated using the following formula:
It is the activity of sizing 1 ml of soluble starch to a defined blue value within 30 minutes under analytical conditions: MWU/g = 100 x 30/T x W where 100 = starch in each culture mix mg number of 30 = defined glue purification time, min T = time required to reach the end point, min W = weight of enzyme added to the culture mixture of 1 ml of diluted enzyme specimen, g Sodium acetate (CH ₃ COONa), the pH of the solution
Varying amounts were added to the appropriately diluted (340000 MWU/ml...1700 MWU/ml or 0.2 mg protein/ml) enzyme solution, keeping the value at 5.8.
The enzyme activity of each solution was determined as previously described. The enzyme solution was then heated to 90°C for 10 minutes. After heating, the activity was measured again to determine what effect the addition of sodium acetate had on the thermostability of α-amylase. The results of this experiment are shown in Table 1 for various levels of sodium acetate.

Table 1 From Table 1 it can be determined that the addition of acetate ions in the form of sodium acetate increases the thermostability of α-amylase. Furthermore, the stabilization observed was up to Na acetate per million units of enzyme activity.
It increased with increasing acetate concentration up to 2.35 g, and at this concentration level the thermal stability decreased again. Example 2 Sodium salts of monocarboxylic acids, such as sodium formate (HCOONa), sodium acetate (CH ₃ COONa), sodium propionate (CH ₃ CH ₂ COONa) and sodium butyrate (CH ₃ CH ₂ CH ₂ COONa) were added to 40 mg of protein. Enzyme solution containing /ml (α-amylase activity 340000MWU/ml)
ml) to a final concentration of 1.22M. After appropriate dilution (1 x 100), enzyme solution, pH value 5.8,
was heated to 95° C. and the thermostability of the enzyme was determined at various intervals after heating by determining the residual activity of the diluted enzyme. The time required to cause 50% inactivation (half-life t1/2) was calculated from a logarithmic plot of percentage residual activity versus culture time.
The results of this calculation are shown in Figure 1. From Figure 1, α
- The effect of increasing aliphatic chain length on thermostabilizing amylase can be seen. Increasing the aliphatic chain length resulted in a proportional increase in increasing the thermostability of the enzyme in aqueous solution at 95°C and PH value 5.8. Thermal stabilization of the enzyme followed the following order: formate < acetate < propionate < butyrate. The half-life of the formate-treated enzyme at 95°C and pH value 5.8 was 1.8 minutes compared to 1.8 minutes for the control under the same conditions.
It took 6.3 minutes. However, the introduction of a nonpolar methyl group relative to the hydrogen in the formate caused a significant increase in the thermostability of the enzyme (half-life = 10.5 min). Furthermore, increasing the length of the alkyl chain resulted in only a minimal increase in the half-life of the enzyme under the conditions tested (1.4 min/CH ₂ group). It has thus been shown that compounds containing non-polar aliphatic chains (tails) and polar carboxylate groups (heads) protect enzymes from denaturation at high temperatures. Example 3 In this experiment, organic acids containing non-polar aromatic groups were also tested. Sodium benzoate (C ₆ H ₅ COONa), Sodium phenyl acetate (C ₆ H ₅ CH ₂ COONa), Sodium phenyl propionate (C ₆ H ₅ CH ₂ CH ₂ COONa) and Sodium diphenyl acetate (C ₁₂ H ₁₀ CHOONa) ) to an enzyme solution (40 mg/ml protein, α-amylase activity,
340,000 MWU/ml) to a final concentration of 1.22M. In addition, these aromatic acids have a pH value of 5.8 at 95℃.
- resulted in a significant increase in the thermostability of amylase. The results of this experiment are summarized in Table 2; from this table it was found that benzoic acid had the lowest value for stabilization among the aromatic carboxylic acids tested, while diphenylacetate anion had the highest value. .

[Table] Example 4 Sodium acetate was added to α-amylase concentrate containing 40 mg/ml of protein (α-amylase activity,
340000MWU/ml) with a final concentration of 1.22M (10% w/
v) was added. Next, use sodium hydroxide to adjust the pH value of the enzyme solution to 5.0, 5.6, 6.0, 7.0, 8.0.
and adjusted to 9.0. After 30 min, the enzyme was further diluted (1 x 100 times) and heated to 95 °C for various time intervals.
Residual enzyme activity was measured. Thermal stability was measured at 85°C with pH values of 5.0 and 5.5. For comparison, the influence of PH value on the thermostabilization of the enzyme in the absence of sodium acetate was determined. The experimental results are shown in the third
Shown in the table.

[Table] The outstanding features of the above results indicate that: (1) The treatment of α-amylase with an amphiphile containing a carboxylic acid group and an aliphatic or aromatic non-polar residual group is (2) Increase in chain length of nonpolar groups increases thermal stability; (3) Aromatic residues have a greater effect than aliphatic residues. Although the claimability of the present invention is not based on a specific structure, the essential structure required for the observed stabilizing effect is a polar head that is inactivated by the polar functional group of the enzyme. and consisting of non-organic polar hydrocarbon tails (aliphatic or aromatic). Hydrocarbon tails may also protect certain areas of the enzyme molecule from surrounding water molecules. This gives the enzyme molecule the necessary rigidity for structural resistance to thermal denaturation. The formate ion (HCOO ^- ) was found to be considerably less effective than the anions provided by acetate, propionate or butyrate. The interaction of hydrogen in the formate anion with water in the bulk tends to weaken the interaction between the enzyme and the anion, while the methyl group in the acetate shows a significant increase in thermal stabilization energy, May interact with adjacent hydrophobic side chains of proteins. Benzoic acid is the least effective of the aromatic anions tested. It is possible that the double bond nature of the bond between the carboxyl group and the benzene ring may limit the suitability of the carboxylate ion for interaction with polar residues of the enzyme molecule. The additional stabilization free energies for increasing the thermostability of α-amylase due to added carboxylate salts were calculated and shown in Table 4.

[Table] a = Relative stabilization due to added ligand b = Stabilization free energy is denoted by -R T In F, where T = 368K F = Relative stability factor R = Gas constant, 1.987 calories/mol/ Degree t 1/2° = Half-life in the absence of ligand From Table 4 it can be determined that the addition of carboxylate to α-amylase increases the half-life of this enzyme. t1/2 in the presence of the added artifact vs. (t1/2°) in its absence
The relative stabilization factor, expressed as a ratio, increases with increasing carboxylic acid chain length. This relative stabilization factor can be converted into additional stabilizing free energy using the relationship △G° = − RT In F: where T is the temperature at which the enzyme is heated (°K);
R is the gas constant and F is the relative stabilization factor. Formate gives a free energy of stabilization of 0.9 KCal/mol. This is thought to occur primarily through electrostatic interactions with positively charged groups of the enzyme, and the effect is primarily entropic, resulting from the release of interactions of water molecules by the charged groups. . The additional stabilization by about 0.6 KCal/mol due to the butyl group (in butyrate) results from enhanced hydrophobic interactions, which also have a primarily entropic contribution to the free energy. The influence of chain length increases and aromatic rings are very quiet reactive partners in interactions in the sense that they can direct interacting molecules towards optimal interaction sites. In both cases, the interaction appears to be specific (albeit weak) with electrostatic and hydrophobic components. It is cooperative interactions that can provide greater stabilization than can be provided by individual interactions. In order to remain inactive for long periods of time at high temperatures or to withstand high temperature exposure, active molecules interact with the enzyme in the interval regions, not necessarily in the active site, but in all similar regions that help enforce the enzyme conformation. It was shown that they interact. Although surface interactions are not determined, the reinforcing effect of pendant nonpolar groups indicates internalization of interactions. Example 5 The α-amylase stabilized by the method of the invention is particularly suitable for the liquefaction and conversion of starch in the production of maltodextrin and the subsequent production of glucose using glucoamylase. . In this example, U.S. Patent No. 3654081
A high temperature jet cooking process was used to liquefy the starch using the thermostable alpha-amylase described in the patent. In a typical experiment, 60 pounds of corn starch was suspended in water to a starch concentration of 35-37% DSB, and the PH value was adjusted to 5.0 using acetic acid.
170000MWU/ml [Taka-
Therm) L-170] Sodium acetate with activity
Takatherm prepared at 10%
Therm) thermostable α-amylase was added at 0.05% dry solids basis (DSB). Next, the starch slurry is transferred to the pilot plant, steam, jet,
Gelatinization was carried out in a pilot plant steam jet cooker at 140° C. for 15 seconds. The gelatinized starch was blown directly into a temperature controlled vessel maintained at 95°C. 0.10% DSB Takartherm L- to give a total concentration of 20400 MWU per 100 g of starch.
170000 (prepared with 10% sodium acetate)
After addition, liquefaction was continued for 20 minutes and then cooled to 85°C. The starch was then held at 85°C for an average of 120 minutes. During the liquefaction period, dextrose equivalents (DE) were monitored using a standard titration method. The above experiment was carried out using Takatherm L-170.
PH value in the presence of 150 ppm of added calcium
6.5 and repeated in the presence of 500 ppm calcium at a pH value of 5.0. In any case, no reverse change in starch was observed. The results are shown in Table 5 below; in the table the term "spin residue" refers to the unhydrolyzed starch recovered after centrifugation.

[Table] Residue%
Residue% Residue% Residue%

Claims (1)

Claims: 1. A method for increasing the thermostability of bacterial α-amylase comprising adding an amphiphile to an aqueous solution of α-amylase, wherein the amphiphile has the formula R-COO ^- , R is an aliphatic or aromatic group containing 1 to 14 carbon atoms, and the calculated molecular weight of α-amylase is
The carboxylate is present at a concentration of at least 2.89 moles per mole of α-amylase as 58,000 Dantons, and the aqueous solution contains no more than about 50 ppm calcium ions and contains 1 to 6 carbon atoms.
A method for increasing the thermostability of bacterial α-amylase, characterized in that it does not contain alkanol. 2 α-amylase was extracted from Bacillus subtilis.
subtilis), Bacillus licheniformis (B.
licheniformis), B. stearothermophilus or B. stearothermophilus
A method according to claim 1, which is derived from a microorganism of the species B. coagulans. 3. The method according to claim 2, wherein the microorganism is Bacillus licheniformis species. 4. Claim 1, wherein R is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, phenyl or substituted phenyl.
The method described in section. 5. The method of claim 1, wherein the carboxylate-containing amphiphile is added to the aqueous solution of α-amylase in the form of its alkali metal salt. 6. The method according to claim 5, wherein the alkali metal is sodium. 7. The amphiphile is acetate, and the acetate is added to the α-amylase aqueous solution until the concentration of acetate ions is at least 0.24 g per million units of enzyme activity expressed in modified Wohlgemuth units. The method described in section.