JPH03195489A - Production of spherical formed product of immobilized biocatalyst - Google Patents

Abstract

PURPOSE:To obtain the subject formed product having high strength, resistant to water and useful for bio-reactor, etc., by successively containing a liquid droplet of a specific mixed aqueous solution with a cation-containing compound and a compound having syneretic action to polyvinyl alcohol. CONSTITUTION:The objective formed product can be produced by containing (A) a droplet of a mixed solution containing (i) a biocatalyst, (ii) a polyvinyl alcohol having a polymerization degree of >=2,800 and (iii) a water-soluble polymeric polysaccharide capable of forming a gel in contact with at least one kind of cation with (B) an aqueous solution containing a cation-containing compound to form the component A in spherical form and containing the spherical component A with (C) a liquid containing a compound exhibiting syneretic action to polyvinyl alcohol, thereby gelatinizing the polyvinyl alcohol. Preferably, the component (iii) is sodium alginate, the component B is CaCl2 and the component C is a sulfate.

Description

DETAILED DESCRIPTION OF THE INVENTION A. Part 1 The present invention relates to a method for producing a molded article on which biocatalysts such as enzymes and microorganisms are immobilized for use in bioreactors and the like.

ing.

One method for immobilizing biocatalysts is entrapping immobilization, in which the biocatalyst is wrapped in a polymeric material. Examples of polymeric materials commonly used in this method include agar, alginate, carrageenan, and polyacrylamide. , polyvinyl alcohol, photocurable resin, etc. Among these, it is known that polyvinyl alcohol (hereinafter sometimes abbreviated as PVA) hydrogel can be used as an excellent immobilization carrier by entrapping a biocatalyst.

On the other hand, reaction vessels in which biocatalysts are immobilized by comprehensive immobilization to perform biological reactions include fixed beds (packed beds), fluidized beds, stirred vessels, etc., and the immobilization carriers used in these reaction vessels are The shape allows for a wide active surface;
A spherical shape is desirable because of its good fluidity, good filling effect, and ease of handling.

In particular, from an industrial practical standpoint, it is important that a spherical immobilization carrier with high strength can be produced easily and at low cost.

Conventionally, alginate, which has been widely used as an immobilized support for biocatalysts, easily forms a spherical gel when a sodium alginate aqueous solution is dropped into an aqueous solution containing cations such as Ca'''' and Al'' at room temperature. It is an inexpensive and easy-to-use gel.However, alginate gels can be destroyed by phosphates, and the gel itself can be eroded and dissolved when used for wastewater treatment.Alginate gels also have poor mechanical strength. It is not always sufficient and is often damaged during long-term use in a reaction tank.

Gels formed from photocurable resins and acrylamide can also be used after being made into spheres at room temperature;
Since the raw material price is very expensive compared to gel, it is difficult to use (Y, H, JO (ilIi
Value (7) High biological reaction zOIJ11:IiA.

Conventionally, as a method for molding PVA gel into a sphere, PV
A method of freezing and thawing an aqueous solution, a method of dropping a PVA aqueous solution into a solvent or a saturated boric acid aqueous solution to form a gel, and a method of dropping a PVA aqueous solution into a sulfate aqueous solution and solidifying it are known.

Problems to be Solved by C1 In order to produce spherical PVA gel, the freeze-thaw method requires freezing equipment such as a freezer, and requires a large amount of energy and time for freezing. Large amount of spherical PV
In order to continuously produce A-gel, this method cannot be said to be preferable in terms of equipment and energy.

Furthermore, the method of molding a PVA aqueous solution into spheres in an organic solvent such as methanol or an acidic/basic solvent such as boric acid is also expected to have an adverse effect on the biocatalyst due to the solvent. is an undesirable method and limits its scope of application.

Furthermore, a method in which a PVA aqueous solution is directly dropped into a liquid containing a compound having a synergic effect on PVA, such as a sulfate aqueous solution, has the disadvantage that a perfectly spherical gel cannot be obtained due to the slow coagulation rate.

Furthermore, conventional PVA gels using PVA with a degree of polymerization of about 1000 to 2000 have a problem in that some PVA dissolves into water, and it has been desired to improve the durability of the -layer.

The present invention solves the above-mentioned problems, and allows PVA gel to be produced without using a freezing operation that is disadvantageous in terms of equipment or energy, and without using a solvent that has an adverse effect on the biocatalyst. Taking advantage of its inherent characteristics of high water content and porousness, it has become possible to produce a spherical biocatalyst-immobilized molded product with high strength and high water resistance.

D. The spherical PVA gel obtained according to the present invention has a high affinity with biocatalysts, has strength and durability that can be applied to various reaction formats, and is also water resistant and chemical resistant. Excellent in sex,
It has desirable characteristics as an entrapping immobilization support for biocatalysts.

(A) Biocatalyst, (B) Degree of polymerization 2800 or more + 7) PV
A, (C) By bringing a mixed aqueous solution containing a water-soluble polymeric polysaccharide capable of gelling upon contact with at least one type of cation with an aqueous cation-containing solution, a sphere close to a true sphere can be easily formed. PV that is formed, has a solid surface immediately, does not allow the spherical molded products to fuse together, and has sufficient strength for subsequent handling of the molded product.
A spherical molded product of the Ali aqueous solution is obtained.

The obtained spherical molded product of the PVA mixed aqueous solution is separated from the cation-containing aqueous solution, washed with water, and then immersed in an aqueous solution of a PVA compound having a synergic action. As a result, PVA gels and a spherical PVA gel is obtained.

It was thus discovered that a truly spherical PVA gel could be obtained by performing the two-step process of molding and gelation.

The PVA gel obtained as described above has the characteristics inherent to PVA gel, is spherical and has high strength, and the method for producing it is easier and cheaper than the conventional method for producing spherical PVA gel.

Hereinafter, the method for manufacturing the spherical biocatalyst-immobilized molded article of the present invention will be explained in more detail.

There are no particular restrictions on the biocatalyst (A) used in the present invention, and any microorganisms and enzymes can be immobilized according to the present invention. Typical examples of microorganisms include molds such as the genus Aspergillus and the genus Rhizopus; the genus Pseudomonas, the genus Acetobacter, and the genus Streptomyces. , Nijierisia (
Bacteria such as the genus Esch-erichia; yeasts such as the genus Saccharomyces and the genus Candida. Typical examples of enzymes include lactate hydrogenase (1, 1, 2, 3) and lactate oxidase (1, 1!).

2), glucose oxidase (1,1J,4), formate dehydrogenase (1,2,1,2), aldehyde dehydrogenase (1,2,1J), aldehyde oxidase (1,2,3,1), xanthine oxidase (1,2,3,2), pyruvate oxidase (1,24°3), pyruvate reductase (L,2
．． 4.1), cortisone-α-reductase (1,3
, 1, 4), acyl Co^-dehydrogenase (13
,99,3), 3-ketosteroid Δ1-dehydrogenase (lJ, 99.4), 3-ketosteroid Δ4-
Dehydrogenase (1,3°99.5), L-alanine dehydrogenase (1,4,1°1), L-glutamate dehydrogenase (1,4,1°3), L-amino acid oxidase (1,4,3, 2), D-amino acid oxidase (1,4,3,3), pyridoxal phosphate oxidase (1,4,3,5), catalase (1,
11,1,6), catechol methyltransferase (2,1,1,6), carnitine acetyltransferase (2J, 1.7), acetyl CO^acetyltransferase (2,3,1,9), asbertate aminotransferase Elase (2,6,1,1)
, alanine aminotransferase (2,6°1.2
), pyridoxamine pyruvate transferase (
2゜6.1), hexokinase (2,7,11),
glucokinase (2,7,1,2), fructokinase (2,7,1,4), phosphoglucokinase (2,7
, 1, 1 (1), phosphofructokinase (2, 7,
1,11), pyruvate kinase (2,7,1,40
), carboxylesterase (3,1,1゜1), arylesterase (3,1,1,2), lipase (
3,1,1,3), phospholipase A (3,1,1
, 4), acetyl esterase (3, 1, 1, 6),
Cholesterol esterase (3,1,1,13),
glucoamylase (3,2,1,3), cellulase (
3,2,1,4), inulase (3,2,1,7)
, α-glucosidase (3,2,1°20), β-glucosidase (3,2,1,21), α-galactosidase (3,2,1,22), β-galactosidase (
3,2,1,23), invertase (3,2,1,
26), pepsin (3,4,4,1), trypsin (3
, 4, 4, 4), Chymotrypsin A (3, 4, 4, 5)
), calapsin A (3°4), papain (3,4,4
, 40), thrombin (3,4,4゜13), amidase (3,5,1,4), urease (3,5゜1.5)
), penicillin acidase (3,5,1,11),
Aminoacylase (3,5,1,14), adenine deaminase (3,5,4,2), A, T, P,
pyruvate decarboxylase (4,1,1,1), oxalate decarboxylase C4,1,1,2), tryptophan decarboxylase (4,1,1,27) ), aldolase (4,1
, 2, 13), maletotosudase (4, 1, 3,
2), tryptophan synthase (4,2,1°20)
, Aspergillus (4,3,1,l), lysine racemase (5,1,1,5), glucose-6-phosphate isomerase (5,3,1,9), steroid Δ-isomerase (5,3 , :(,1), v-cucinyl-CoA synthetase (,6,2,1,5), [(Note) The number in parentheses represents the enzyme number], and the like.

(B) PVA used in the present invention has an average degree of polymerization of 28
00 or more, preferably 30H or more, and the degree of saponification is 98.
Completely saponified PVA with a saponification degree of 5 mol % or more, preferably 99.85 mol % or more is desirable from the viewpoint of gel formation. Furthermore, as the PVA of the present invention, various known modified PVAs can be used within the range that does not impede the purpose of the present invention.

The higher the concentration of the PVA aqueous solution, the stronger the gel produced; however, as long as the required gel strength is obtained, a lower PVA concentration is advantageous in terms of raw material cost. Although it is necessary to select an appropriate concentration depending on the type and concentration of additive components other than PVA, the temperature of the PVA mixed aqueous solution, and the droplet formation method, when dropping the PVA mixed aqueous solution at room temperature, the PVA concentration is 0.3. When the content is 10 vt%, spheroidization is easy and a practically sufficient gel strength can be obtained.

In the present invention, (C) water-soluble polymeric polysaccharides capable of gelling upon contact with at least one cation include, specifically, water-soluble alginate, carrageenan, mannan, chitosan, etc. However, sodium alginate is particularly preferred.

The concentration of (C) a water-soluble polymer polysaccharide capable of gelling upon contact with at least one cation, preferably sodium alginate, added to the PVA aqueous solution is 0.2 to 4 vt% based on water; Preferably it is 0.5 to 2 wt%. If it is less than 0.2vt%, the spheroidization ability of the PVA mixed aqueous solution is poor, and if it is more than 4vt%, a hard spherical molded product can be obtained, but the solution viscosity increases and the cost of raw materials increases. This is undesirable.

In the present invention, cation-containing compounds include, specifically, metal cations such as calcium ions, magnesium ions, strontium ions, barium ions, aluminum ions, potassium ions, cerium ions, and nickel ions; cations such as ammonium ions; Examples include compounds containing at least one of them, and among them, compounds containing divalent or higher cations are preferred, and CaC1* is particularly desirable.

In the present invention, examples of the aqueous solution of a polyvinyl alcohol compound having a synergic effect include sodium sulfate, ammonium sulfate, potassium sulfate, magnesium sulfate, aluminum sulfate, sodium citrate, ammonium citrate, potassium citrate, magnesium citrate, and citric acid. Examples include liquids containing at least one of compounds such as aluminum, sodium tartrate, ammonium tartrate, potassium tartrate, magnesium tartrate, and aluminum tartrate, but sulfate aqueous solutions are particularly preferred from the viewpoint of cost.

Next, a method for preparing a spherical molded article using each of the above-mentioned components will be explained.

(B) PVA with a degree of polymerization of 2800 or higher and (C) A desired biocatalyst is mixed into an aqueous solution of a water-soluble polymeric polysaccharide capable of gelling upon contact with at least one type of cation, and the mixture is mixed with PVA by stirring. Prepare an aqueous solution.

In addition, a microorganism culture medium, a reinforcing agent to increase the strength of the immobilization carrier, a filler to adjust the specific gravity of the produced gel, etc. may be added to this PVA mixed aqueous solution within a range that does not inhibit the gelation of PVA. good.

The above PVA mixed aqueous solution is, for example, dropped from a tubular cap or sprayed from a spray cap to form droplets, and then the droplets are mixed with a cation-containing compound,
Contact is preferably made with an aqueous solution containing CaCIt. P
When the droplets of the VA mixed aqueous solution come into contact with the C1CL aqueous solution, they become spherical due to surface tension, and the outermost surface of the sphere solidifies into a thin film, forming a spherical molded product of the PVA mixed aqueous solution.

The diameter of the spherical molded product can be adjusted from 1 mm to 1 mm by adjusting the diameter of the cap, the spray pressure, and the viscosity of the PVA mixed aqueous solution.
Can be arbitrarily changed to 0■■. The CaCl aqueous solution may be left in standing water, but by forcibly stirring it with a stirrer or the like, the reaction rate between the molded PVA mixed aqueous solution and the CaCl1g aqueous solution can be accelerated, and fusion of the spherical molded products can be almost completely prevented. In practice, when producing a large amount of PVA gel,
In order to make the diameter of the spherical molded product uniform, a pump or the like can be used for extrusion to drip the PVA mixed aqueous solution. For example, if a roller pump is used that compresses a flexible tube and delivers the liquid, a constant amount of liquid is discharged from the mouthpiece, making it easy to obtain a uniform spherical molded product.

The PVA aqueous solution molded product obtained by spheroidizing CaCl in an aqueous solution is separated from the CaCl5 aqueous solution, washed with water, and then immersed in a NatSO aqueous solution. The concentration of hatsOt aqueous solution is LOOg
/Q or higher is sufficient, but a saturated solution is desirable to obtain a stronger PVA gel. The immersion time is preferably 10 minutes or more, preferably 30 minutes or more. By immersing it in NatSO, aqueous solution, PVA gels and a spherical PVA gel is obtained.

The PVA gel thus obtained has a strength that will not deform or break over a long period of time in various types of reaction vessels, and can be operated continuously without being attacked by water or various chemical solutions. It has practical utility as a biocatalyst immobilized molded product.

E, L91 Hereinafter, the present invention will be specifically explained with reference to Examples, but the present invention is not limited to these Examples.

Example! Polyvinyl alcohol (PVA) manufactured by Kuraray Co., Ltd. (average degree of polymerization 3300, degree of saponification 99.85 mol%)
After washing with warm water at ℃ for about 1 hour, add water to PVA so that the PVA concentration is 10vt%, make the total amount 400g, and adjust the pH to 6.
Adjusted to. This was treated in an autoclave at 120°C for 30 minutes to dissolve PVA, and then allowed to cool to room temperature. Add 200% of 4% sodium alginate aqueous solution to this PVA aqueous solution.
Activated sludge bacteria (concentration M
200g of LSS 80000a+g/12) was added and thoroughly stirred.

These mixed solutions were pumped at a rate of lsQ/min using a roller pump using a vinyl tube with an inner diameter of 2 m@φ and a syringe needle with an inner diameter of 0.8 mm attached to the tip, and stirred with a stirrer.
, 5so(!/12) was dropped into an aqueous solution of calcium chloride (CaCL) from a height of 5c- on the ice surface.
It immediately became spheroidized and precipitated in the aClt aqueous solution. These spheroidized PVA mixture moldings were separated from the CaClt aqueous solution, washed lightly with distilled water, and then immersed in a saturated Na, So, aqueous solution stirred with a stirrer for 60 minutes, giving them an opaque brown color. A rich spherical gel was obtained. This gel is shaped into spheres and is not sticky. The particle size was 3 to 3.5 .phi.

Regarding the PVA gel obtained in this way, P in water
The elution amount of VA was measured. 30g of gel to 300g of water
g was added thereto, and the mixture was stirred at 30°C for 7 days. PV at this time
The elution amount of A was 1.2 g per Ikg of gel. Furthermore, this gel was taken out, washed lightly with water, drained, added 300 g of water, and stirred at 30°C for 7 days.
The elution amount of PVA at this time is 0.0g per 1kg of gel.
The amount of PVA eluted was very small.

Comparative Example 1 A gel was prepared in the same manner as in Example 1 except for using PVA manufactured by Kuraray Co., Ltd. (average degree of polymerization 1740, degree of saponification 99.85 mol%).

The resulting gel was a non-tacky, opaque brown, flexible spherical gel with a diameter of 3-3.5 mg+.

Regarding the PVA gel thus obtained, Example 1
The amount of PVA eluted into water was measured in the same manner as described above. The elution amounts for the first 7 days and the next 7 days were 9.0 g and 1.1 g per kg of gel, respectively, which were large amounts.

Comparative Example 2 Polyvinyl alcohol (PVA) manufactured by Kuraray Co., Ltd. (average degree of polymerization 1740, degree of saponification 99J5 mol%) was heated at 40°C.
After washing with warm water for about 1 hour, water was added to the PVA so that the PVA concentration was 10 wt%, the total amount was 400 g, and the pH was adjusted to 6. This was treated in an autoclave at 120°C for 30 minutes to dissolve PVA, and then allowed to cool to room temperature. In addition, Kuraray Okayama Factory (1-2 Kaigandori, Okayama City, Okayama Prefecture)
Activated sludge bacteria (concentration M L S S 80,000
Add 200g of eg/Q) and add water to bring the total amount to 800g.
g and stirred thoroughly. These mixed solutions were pumped at a rate of 1 m12/min using a roller pump using a vinyl pipe with an inner diameter of 2 mm and a syringe needle with an inner diameter of 0.8 mm attached to the tip, and the mixture was stirred with a stirrer to give a solution of 40 g/Q in concentration. By dropping the mixture into an acid aqueous solution from a height of 5 cm above the ice surface and immersing it for 24 hours, a favorable reaction of PVA with boric acid occurred, and a spherical gel was obtained.

Regarding the PVA gel thus obtained, Example 1
The amount of PVA eluted into water was measured in the same manner as described above. The elution amounts for the first 7 days and the next 7 days were 19.5 g and 9.8 g per kg of gel, respectively, which were extremely large amounts.

F, [1OxL As is clear from the above examples, the biocatalyst-immobilized molded product made of PVA with a high degree of polymerization according to the present invention has high strength and sufficient water resistance, and can be used in bioreactors, etc. It has extremely high industrial value.

Claims (4)

[Claims] (1) A mixture containing (A) a biocatalyst, (B) polyvinyl alcohol with a degree of polymerization of 2800 or more, and (C) a water-soluble polymeric polysaccharide capable of gelling upon contact with at least one cation. By bringing droplets of the aqueous solution into contact with an aqueous solution containing a cation-containing compound, the mixed aqueous solution is formed into a spherical shape, and then by contacting this with a liquid containing a compound having a synergic effect of polyvinyl alcohol, A method for producing a spherical biocatalyst-immobilized molded article, which is obtained by gelling polyvinyl alcohol. (2) The method for producing a spherical biocatalyst-immobilized molded article according to claim 1, wherein (C) the water-soluble polymeric polysaccharide capable of gelling upon contact with at least one cation is sodium alginate. (3) The method for producing a spherical biocatalyst-immobilized molded article according to claim 1 or 2, wherein the cation-containing compound is calcium chloride. (4) The method for producing a spherical biocatalyst-immobilized molded article according to any one of claims 1 to 3, wherein the compound having a synergic effect on polyvinyl alcohol is a sulfate.