JPH0328194B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field] The present invention relates to a method for hydrolyzing high-molecular organic substances such as starch and inulin using an enzyme, and in particular, the present invention relates to a method for hydrolyzing high-molecular organic substances such as starch and inulin, and in particular, using a solution of a hydrolase with low purity or dilution. This invention relates to a method for hydrolyzing molecular organic substances.

[Conventional technology]

Currently, glucose and high fructose sugar are mass-produced by hydrolyzing starch using α-amylase and glucoamylase sugar starch hydrolase (amylase). There is also active research into hydrolyzing cellulose-containing substances into cellulose and glucose using cellulases. Enzymes, including these hydrolytic enzymes, are generally used by culturing microorganisms in liquid and separating and purifying the culture filtrate.
The culture filtrate contains various inorganic salts, low-molecular organic substances, and high-molecular organic substances as impurities, and often contains proteolytic enzymes (proteases) that destroy target enzymes. For this reason, separation and purification of enzymes is essential when producing useful substances using enzymatic reactions. Enzyme purification is generally performed through a complex process that combines various physical and chemical methods such as molecular sieves, ion exchange chromatography, salting out, and PH precipitation, which requires an extremely large amount of labor, time, and
Requires expenses. On the other hand, it has been previously known that amylase selectively adsorbs to the substrate starch, and a method in which amylase is adsorbed to starch particles and then desorbed has also been reported. However, in desorption, a desorption solution such as a salt solution or an acid-alkali solution is used, and the recovery rate of amylase is low, and removal and concentration of the salt and acid-alkali are required as post-treatment.

Therefore, in view of the above-mentioned drawbacks, the present inventors conducted intensive research on a highly practical method for hydrolyzing polymeric substances using a relatively dilute solution with low purity such as a culture filtrate containing a hydrolase.

[Purpose of the invention]

The purpose of the present invention is to use a low-purity or relatively dilute solution containing a hydrolyzing enzyme of a polymeric substance, to adsorb the enzyme contained in the solution onto a solid polymeric substrate, and to obtain an enzyme-substrate complex. The object of the present invention is to provide a method for efficiently hydrolyzing a polymeric substrate without introducing impurities into the reaction system and without diluting the reaction solution by using the enzyme as an enzyme agent.

[Structure of the invention]

The present invention provides a method for hydrolyzing a polymeric organic substance with a hydrolase, in which an aqueous solution of a hydrolase as an enzyme agent and an insoluble or slightly soluble solid polymer substrate are heated above the freezing point, and the two do not substantially react with each other. This is a method for hydrolyzing high-molecular organic substances, which is characterized by using an enzyme-substrate complex obtained by contacting the substance at a temperature below that temperature.

The inventors changed their thinking from conventional methods by bringing the bacterial culture filtrate containing α-amylase into contact with starch granules at low temperatures where virtually no reaction occurs, and then separating and washing the resulting α-amylase/starch granule complex. Thereafter, this complex was directly added to a starch solution as α-amylase and reacted. As a result, they found that it is possible to efficiently hydrolyze not only the starch substrate but also the starch granules used as an adsorbent without introducing impurities contained in the culture filtrate into the reaction system.

The present invention is applicable not only to starch hydrolysis but also to various hydrolysis reactions. Examples include dextrin and oligoglucose hydrolysis, cellulose hydrolysis, inulin hydrolysis, and protein hydrolysis.

As the adsorbent for the enzyme used, a solid polymer substrate that is insoluble or sparingly soluble at temperatures above the freezing point and below at which substantially no reaction occurs (specifically 0 to 10°C) is used. Specific examples include starch grains in general such as potatoes, sweet potatoes, and corn;
For cellulose, cellulose and lignocellulose-containing powder or fine fibers are used, for inulin, plant inulin grains and inulin crystals are used, and for protein, casein, collagen, etc. are used. The allowable temperature for the solubility of the adsorbent solid polymer substrate varies depending on the type of enzyme, target recovery rate of the enzyme, temperature, coexisting solutes, pH, etc., but it is 0.1% or less, preferably 0.01%.
It is as follows. This is because if the solubility is high, the amount of enzyme molecules bound to dissolved substrate molecules cannot be recovered as an enzyme/solid substrate complex. The amount of substrate used as an adsorbent cannot be specified because it varies depending on the type and concentration of the enzyme, the concentration of impurities other than the target enzyme, especially the concentration of inorganic salts or polymer electrolytes, and pH. However, in practice, enzyme-containing liquid
It is used in a range of 20wt% or less.

As the enzyme, hydrolytic enzymes shown in the following examples can be used. amylases that use starch or dextrin as substrates, i.e. α-amylase;
Glucoamylase, β-amylase, etc. can be used. Other examples include cellulase that uses cellulose or lignocellulose as a substrate, inulase that uses inulin as a substrate, and various proteases that use protein as a substrate.

Enzyme-containing fluids are mainly biological secretions,
That is, culture filtrates, body fluids, digestive juices, etc., and extracts from living organisms are used. In addition, enzyme-containing liquids produced in the use of enzymes include, for example, reaction-completed liquids containing reaction products and residual enzymes, and solutions containing enzymes in enzyme separation and purification processes.

The method of bringing the enzyme-containing liquid into contact with the solid substrate for adsorbent is not particularly limited, and various known methods are fully applicable. For example, a solid substrate may be added to the enzyme-containing liquid as an adsorbent, or the enzyme-containing liquid may be passed through a packed bed of solid substrate for the adsorbent. The temperature and time during contact will depend on the characteristics of the enzyme, the characteristics of the solid substrate for the adsorbent,
It is appropriately selected depending on the concentration of the enzyme and the solid substrate for the adsorbent, the composition of impurities other than the target enzyme, etc. Generally, the temperature is above freezing and below 10 degrees Celsius, and the time is less than 10 minutes.

The method for separating the enzyme/solid substrate complex is not particularly limited, and known solid separation methods are fully applicable. When an enzyme-containing solution and a solid substrate are brought into contact with each other,
Sedimentation and filtration methods using centrifugal force or gravity are used. Mother liquor is attached to the separated enzyme/solid substrate complex, but if it is desired to further remove impurities from the engraving liquid, the enzyme/solid substrate complex may be washed. The cleaning method is not particularly limited, and various known methods can be applied. Water is usually used as the detergent, but it can be selected as appropriate depending on the type of enzyme and solid substrate and the properties of the complex. For example, salts, acids,
Solutions containing alkalis may also be used.

Enzyme and solid substrate complexes are used to hydrolyze polymeric organic substances as substrates, but even if the substrates are chemically the same as the solid substrate for adsorbents, they may have different degrees of polymerization or branching. It's okay. Like a solid substrate for an adsorbent, it does not need to be solid at the reaction temperature, but may be a solution. For example, an α-amylase-containing liquid is brought into contact with corn starch granules at a low temperature, and the mixture is solid-liquid separated to obtain an α-amylase/corn starch granule complex. The complex thus obtained is α−
When used in the reaction as amylase, the substrate may be the same corn starch granules, potato starch granules, or solubilized starches thereof. The type of starch may be amylose or amylopectin.
Alternatively, a glucoamylase/corn starch granule complex may be used, and corn starch granules or a dextrin solution may be used as a substrate. Note that reaction conditions are appropriately selected depending on the type of enzyme used, substrate, purpose, etc.

Next, an example of an embodiment of the present invention will be explained using the flow shown in FIG.

A medium solution is placed in a culture tank 2 for hydrolytic enzyme-producing bacteria, and after culturing for a predetermined period of time at a predetermined temperature, pH, and atmosphere, the culture solution 1 is stored in a storage tank 4 via a transfer pipe 3. The culture solution 1 in the storage tank is led to a centrifuge 6 through a pipe 5, the culture filtrate 11 passes through a pipe 7 to a culture filtrate storage tank 12, and the bacterial slurry 8 passes through a pipe 10 and is stored in a bacterial slurry storage tank 9. be done. If the enzyme is secreted outside the bacterial cells, the main culture filtrate 11 is used as the enzyme raw material solution. Next, the culture filtrate 11 is led to the enzyme and solid substrate complex adjustment tank 14 via the pipe 13, and brought into contact with the solid substrate slurry 20 supplied from the solid substrate slurry storage tank 21 via the pipe 16,
Forms an enzyme/solid substrate complex. The generated enzyme/solid substrate complex suspension 15 is led to a centrifugal filter 19 via a transfer pipe 17 and separated into individual liquids. The filtrate passes through piping 29 and is stored in storage tank 28 . Next, the water 21 is led from the water storage tank 22 to the centrifugal filter 19 through the piping 18, the enzyme/solid substrate complex in the centrifugal filter 19 is washed, and the washing waste liquid is led to the storage tank 28 through the piping 29, where it is combined with the filtrate. It is stored in the storage tank 28.
The recovered enzyme/solid substrate complex 25 is stored in a storage tank 26 via piping 24. The enzyme/solid substrate complex slurry 25 is led to a reaction tank 32 via a pipe 30, mixed with the raw material substrate slurry 20 supplied from a raw material substrate slurry storage tank 21 via a pipe 23, and retained under a predetermined condition for a predetermined time under stirring. to hydrolyze the substrate. For heating at this time, for example, water vapor 33 may be supplied to the reaction tank 32 through the pipe 34. After the reaction is completed, the reaction solution 35 is taken out of the system.

〔Example〕

Hereinafter, the effects of the present invention will be explained in more detail using Examples and Comparative Examples.

Example 1 Soluble starch 1.5%, polypeptone 0.5%, yeast extract 0.5%, monopotassium phosphate 0.7%, dibasic sodium phosphate 0.35%, magnesium sulfate heptahydrate
Dispense 1.52 kg of liquid medium (PH6.5) containing 0.01% and tap water into a culture tank with an internal volume of 5, and sterilize it at 120°C for 20 minutes.

In addition to this, heat-resistant α-
Amylase-producing bacteria (Clostridium bacteria,
80 g of a bacterial cell suspension of Micro-Ken Bacteria No. 7918) was added. Next, the gas phase was replaced with argon gas, and then cultured under anaerobic conditions. The cells were cultured for 46 hours while automatically adjusting the pH in the culture tank to 6.0 and the temperature to 60°C. This culture solution was centrifuged at 6000 rpm for 10 minutes to remove bacterial cells, yielding an α-amylase activity of 5.2 units/ml culture filtrate 1.5. Main culture filtrate contains 1.1% inorganic salt,
Contains 2.4% organic matter other than α-amylase. In particular, the organic matter contains about 0.3% volatile fatty acids, mainly acetic acid and propionic acid, so it has an unpleasant acid odor and sulfide odor.

Next, the main culture filtrate (containing 0.5α-amylase 2600 units, pH 6.0) was cooled to 5° C., 10 g of pharmacopoeia corn starch was added, and the mixture was kept under stirring for 1 minute. The above liquid was centrifuged (3000 rpm, 5 minutes) to obtain 30 g of a slurry of α-amylase/starch complex and 0.51 g of a clear liquid. 30g of the above slurry 100g of distilled water
ml (5°C, PH6.2), mixed, and centrifuged (3000 rpm, 5 minutes) to obtain 30 g of washed composite slurry and 100 ml of washing waste liquid. The above clarified liquid and washing waste liquid were combined and the α-amylase activity was measured. As a result, the α-amylase activity that was not adsorbed to starch was less than 52 units, so 98% of the α-amylase in the test culture filtrate was measured. It is estimated that it was recovered as an α-amylase/starch complex.

Next, add the above α-amylase to a mixture of 100 g of corn starch and 230 ml of 60°C warm water.
29g of starch complex was added. Then, at 80℃ for 30
After heating for 1 minute, 359 g of a dextrin solution with a DE (Dextrose Equivalent Value: DE=direct reducing sugar (labeled as glucose)/solid content x 100) of 10.2 was obtained.This reaction solution had no odor or color derived from the culture filtrate. It is a translucent solution with a starch odor. In this example, the reaction solution contained 19 ml of α-amylase/starch complex.
(Dilution ratio = 359/330≒1.09The definition of dilution ratio is: Substrate + volume of enzyme or enzyme-substrate complex/volume of substrate x 100. [Similarly below]) Very slight dilution. It is possible to react at a concentration almost close to that of the substrate in the test substrate solution.

Example 2 357 g (α-amylase activity 2540 units) of the DE10.2 reaction solution obtained in Example 1 was cooled to 5°C. To this was added 10 g of pharmacopoeia corn starch and held for 1 minute. Centrifuge the above solution (3000 rpm, 5 minutes)
Slurry of α-amylase/starch complex 30g
and 337 g of clear liquid was obtained. α- of 30g of the above slurry
Measurement of amylase activity resulted in 2390 units (recovery rate
94%). Next, topical corn starch
Add the above α-
30g of amylase/starch complex was added to obtain 330g of reaction solution. This was reacted at 80℃ for 30 minutes,
A reaction solution with a DE of 10.0 was obtained. This reaction solution has no odor or color derived from the culture filtrate, and is a translucent solution with a starch odor. In this example, the reaction solution was only slightly diluted by the 20 ml of water contained in the α-amylase/starch complex (dilution ratio = 330/300≒1.10), and was different from Example 1. Similarly, the reaction can be performed at a concentration close to the substrate concentration of the test substrate solution.

Comparative Example 1 Washed α prepared in the same batch as Example 1
- 30 g of slurry of amylase/starch complex was packed into a column with a filter. Elution was performed from the top of the column with 2M saline (PH9) 0.5 to obtain an eluate containing α-amylase at 0.5. Next, this solution was filtered and concentrated to 0.1 with a pore size molecular weight 2+ ¹⁰⁴ molecular sheep membrane, and washing with water was repeated twice to finally obtain a desalted α-amylase-containing eluate with a pore size of 0.2. The α-amylase activity in the above solution was measured, and it was confirmed that 90% of the used enzyme activity was recovered. Then,
The above α-amylase-containing eluate was added to a mixture of 100 g of locally produced corn starch and 230 ml of 60°C warm water to prepare a reaction mixture of 530 ml. This was reacted at 80°C for 30 minutes to obtain a reaction solution with DE10.1. This reaction solution has no odor or color derived from the culture filtrate, and is a translucent solution with a starch odor. In this comparative example, as in Examples 1 and 2, no coloration or odor is observed in the reaction solution, but the enzyme solution is diluted by the amount of water added to the reaction system (dilution ratio = 530/300≒1.61). has.

Comparative Example 2 Culture filtrate prepared in the same batch as Example 1 0.5
was used as a test enzyme and added to a mixture of 100 g of locally processed corn starch and 230 ml of 60°C warm water to prepare 830 g of a reaction mixture. This was reacted at 80°C for 30 minutes to obtain a reaction solution with DE9.2. This reaction solution has an acid odor derived from the culture filtrate and is colored yellow. Furthermore, since the culture filtrate is directly added to the reaction system as the enzyme solution, the reaction solution is diluted to a large extent by the amount of water in the culture filtrate (dilution ratio = 830/330≈2.52).

Example 3 Soluble starch 1.5%, polypeptone 0.5%, yeast extract 0.5%, potassium phosphate 0.7%, sodium phosphate 0.35%, magnesium sulfate heptahydrate
Dispense a liquid medium (PH7.0) containing 0.01% and tap water into 15 100ml flasks each with an internal volume of 500ml.
Sterilize at 120°C for 15 minutes. To this, 10 ml of an aerobic culture of an α-amylase producing bacterium belonging to the genus Bacillus subtilis that had been aerobically cultured in the same medium was dispensed and cultured with shaking at 37°C for 15 hours (120 strokes/min). . This culture solution was centrifuged at 6000 rpm for 10 minutes to remove bacterial cells and α-amylase activity was determined.
1.5 of the culture filtrate with 8.0 units/ml was obtained. The main culture filtrate contains 1.1% of inorganic salts and 1.2% of organic substances other than α-amylase. In particular, organic matter contains about 0.5% of volatile fatty acids, mainly acetic acid and butyric acid, and has an unpleasant sour odor. Next, the main culture filtrate was cooled to 5° C., 10 g of potato starch was added, and the mixture was kept under stirring for 1 minute. The above solution was centrifuged (3000 rpm, 5 minutes) to obtain 25 g of washed α-amylase/starch complex slurry and 100 ml of washing waste liquid. The above clarified liquid and washing waste liquid were combined and the α-amylase activity was measured. As a result, the α-amylase activity that was not adsorbed to starch was less than 250 units, so 99% of the α-amylase in the test culture filtrate was measured. It is estimated that it was recovered as an α-amylase/starch complex.

Next, add 100g of topical potato starch and 230g of warm water at 50℃.
ml of the above α-amylase/starch complex was added to obtain 355 g of a reaction solution. this
As a result of heating at 50℃ for 1 hour, DE after reaction was 12.3
showed that. This reaction solution has no odor or color derived from the culture filtrate, and is an almost transparent solution with a starch odor. In this example, the reaction solution was only diluted by the amount of water of 15 ml contained in the complex, and the reaction could be carried out at a concentration almost close to the test substrate concentration (dilution ratio = 355/330≈1.08).

Comparative Example 3 Culture filtrate prepared in the same batch as Example 1 0.5
Use local method potato starch 100 as the test enzyme as it is.
g and 230 ml of 50°C warm water, add the reaction mixture
Adjusted to 830g. This was reacted at 50℃ for 1 hour,
A reaction solution of DE9.2 was obtained. In this comparative example, the reaction solution had an acid odor derived from the culture filtrate and was colored yellowish brown. Furthermore, since the culture filtrate is directly added to the reaction system as the enzyme solution, the reaction solution is diluted to a large extent by the amount of water in the culture filtrate (dilution ratio = 830/330≈2.51).

Example 4 Aspergillus orizae
Dilute solution of glucoamylase (3.5 units/ml, PH6.8, inorganic salt concentration 0.01) originating from ATCC 1003)
%) 0.41 was cooled to 10° C., 10 g of potato starch was added and kept under stirring for 2 minutes. The above solution was centrifuged (3000 rpm, 5 minutes), and 25 g of washed glucoamylase/starch complex slurry and washing waste liquid were added.
Obtained 100ml. Combine the above clarified liquid and washing waste liquid,
As a result of measuring glucoamylase activity, the glucoamylase activity that was not adsorbed to starch was less than 120 units, so it was estimated that 91% of the glucoamylase in the test culture filtrate was recovered as a glucoamylase/starch complex. .

Next, add the above glucoamylase/starch complex to a mixture of 100 g of soluble starch and 230 ml of 50°C warm water.
29g was added to obtain 355g of reaction solution. This is 55
As a result of reacting at ℃ for 40 hours, a sugar solution with DE90.2 was obtained.

This reaction solution is colorless and translucent, with no odor other than starch odor. In this example, the reaction solution was only slightly diluted by the amount of water contained in the complex, 19 ml (dilution ratio = 355/330≈1.07).

Comparative Example 4 Glucoamylase solution of the same batch as Example 4
0.4 as the test enzyme, soluble starch 100
g and 230 ml of 50°C hot water and the pH was adjusted to 4.0 to prepare 730 g of a reaction mixture. This at 55℃
The reaction was carried out for 44 hours to obtain a sugar solution with DE90.5. In this comparative example, since the glucoamylase solution is directly added to the reaction system, the reaction solution is diluted to a large extent by the amount of water in the added glucoamylase solution (dilution ratio = 730/330≈2.21).

Example 5 Liquid medium 2 containing 1.5% inulin, 0.1% yeast extract, 0.05% potassium phosphate, 0.01% dipotassium phosphate, 0.05% magnesium sulfate heptahydrate, 0.2% ammonium sulfate, and tap water, PH
5.7) into a culture tank with an internal volume of 51 cm and sterilize at 120℃ for 20 minutes. This was then cultured anaerobically in the same medium as the inulase producing bacterium (Saccharomyces fragilis).
100 g of a cell suspension of Saccharomyces fragilis IF0-0288) was added and cultured at 30°C for 48 hours under anaerobic conditions while automatically adjusting the pH to 5.7. Next, this culture solution was centrifuged at 6000 rpm for 5 minutes to remove bacterial cells, and a culture filtrate with an inulase activity of 1.1 units/ml was obtained. Main culture filtrate contains 0.25 inorganic salts.
%, Contains 0.95% organic matter other than inulase. In particular, the organic matter contains 0.3% ethyl alcohol and acetic acid, and has an alcohol odor and a weak acid odor.

Next, 0.5 l of the main culture filtrate (containing 550 units of inulase, pH 5.8) was cooled to 2°C. 5 g of inulin powder was added to this and kept under stirring for 1 minute. The above solution was centrifuged (3000 rpm, 5 minutes) to create a slurry of 19 g of inulase/inulin complex and a clear solution.
Obtained 0.5ml. 19g of the above slurry and 20g of cold water at 1℃
ml, mixed, centrifuged (3000 rpm, 5 minutes) and washed 21 g of complex slurry and washing waste liquid
Obtained 22ml. Combine the above clarified liquid and washing waste liquid,
As a result of measuring inulase activity, the amount of inulase activity that was not adsorbed to starch was less than 62 units, so it was estimated that 89% of the inulase in the test culture filtrate was recovered as an inulase-inulin complex.

Next, 190 ml of water was added to 10 g of inulin and dissolved by heating to 80°C. This inulin solution was cooled to 50°C, and the above-mentioned inulase-inulin complex was added thereto and reacted at 50°C for 3 hours. The DE after the reaction was measured and found to be 82.3.

This reaction solution is a colorless and transparent solution without any alcohol odor or yeast odor derived from the culture filtrate. In this example, the reaction solution was only diluted by the amount of water of 11 ml contained in the complex, and the reaction could be performed at a concentration almost close to that of the test substrate solution (dilution ratio = 221/200≒ 1.11). ).

〔Effect of the invention〕

According to the present invention, even if the raw enzyme solution contains impurities or the enzyme concentration is dilute, the substrate can be increased without introducing impurities into the reaction system and without diluting the reaction system. Can hydrolyze molecular substances.

[Brief explanation of drawings]

FIG. 1 is a flow diagram showing an example of the present invention. 1... Enzyme-producing bacteria culture solution, 2... Culture tank, 3,
5...Culture solution transfer piping, 4...Culture solution storage tank, 6...
...Centrifugal separator, 7,13...Culture filtrate transfer piping,
8... bacterial cell slurry, 9... bacterial cell slurry storage tank,
10... Microbial cell slurry transfer piping, 11... Culture filtrate, 12... Culture filtrate storage tank, 14... Enzyme/solid substrate complex preparation tank, 15... Enzyme/solid substrate complex suspension, 16... solid substrate slurry transfer piping,
17... Enzyme/solid substrate complex suspension transfer tube, 1
8...Water transfer piping, 19...Centrifugal filter, 20...
... Starch slurry, 21 ... Starch slurry storage tank, 2
2... Water storage tank, 23... Raw material substrate transfer piping, 24
... Enzyme/solid substrate complex slurry transfer piping, 2
5... Enzyme/solid substrate complex slurry, 26...
Enzyme/solid substrate complex slurry storage tank, 27...filtrate and washing wastewater, 28...waste liquid storage tank, 29...filtrate and washing wastewater transfer piping, 30...enzyme/solid substrate complex slurry transfer piping, 31...substrate Hydrolysis reaction liquid, 32...reaction tank, 33...steam,
34...Steam supply piping, 35...Reaction liquid.

Claims (1)

[Scope of Claims] 1. In a method of hydrolyzing a polymeric organic substance with a hydrolase, an aqueous solution of a hydrolase and an insoluble or poorly soluble solid polymer substrate are used as enzyme agents at a temperature exceeding the freezing point and both substantially A method for enzymatic hydrolysis of high-molecular organic substances, characterized by using an enzyme-substrate complex obtained by contacting the substance at a temperature below which no reaction occurs. 2. The method for hydrolyzing a high-molecular organic substance using an enzyme according to claim 1, characterized in that starch, cellulose, inulin, or protein is used as the high-molecular organic substance. 3. The method for hydrolyzing a polymeric organic substance according to claim 1, characterized in that α-amylase, glucoamylase, cellulase, inulase, or protease is used as the hydrolyzing enzyme. 4 Starch, cellulose,
2. A method for hydrolyzing a high-molecular organic substance according to claim 1, characterized in that inulin and casein are used.