Apparatus for preparing immobilized-enzyme beads and method for preparing immobilized-enzyme beads using same

Abstract

The present invention relates to an apparatus for preparing enzyme-immobilized beads used for preparation of tagatose, and a method for preparing enzyme-immobilized beads using the same. More specifically, the present invention relates to an apparatus for preparing enzyme-immobilized beads, comprising a nozzle with an inside diameter of 0.1-1 mm having a cylindrical lower end and comprising a cut-type liquid outlet (cut perpendicularly to the vertical axis of the nozzle) formed at the lower end, and a method for preparing enzyme-immobilized beads using the same.

Claims

1. An apparatus for preparing immobilized-enzyme beads, comprising: a) a first tank into which a mixed liquid containing an enzyme-containing material and an excipient is injected; b) a nozzle unit disposed at a lower end of the first tank and comprising a nozzle, the nozzle having an inner diameter of 0.1 mm to 1 mm and a cylindrical lower end and including a liquid outlet cut perpendicularly to a vertical axis of the nozzle at the lower end thereof; and c) a second tank disposed below the nozzle unit and containing a calcium chloride solution, wherein the distance between the surface of the calcium chloride solution in the second tank and the liquid outlet of the nozzle unit ranges from 0.1 m to 1.5 m.

2. The apparatus according to claim 1, wherein the nozzle unit comprises at least two nozzles.

3. The apparatus according to claim 1, wherein the first tank comprises an air inlet.

4. A method for preparing immobilized-enzyme beads using the apparatus for preparing immobilized-enzyme beads according to claim 1, wherein the method comprises: dropping the mixed liquid in the first tank through the nozzle unit onto the calcium chloride solution, thereby preparing immobilized-enzyme beads.

5. The method according to claim 4, wherein the mixed liquid injected into the first tank is adjusted to a viscosity of 3,000 cPs to 7,000 cPs.

6. The method according to claim 4, wherein the enzyme-immobilized beads have a spherical shape having a diameter of 0.5 mm to 3 mm.

7. A method for preparing immobilized-enzyme beads using the apparatus for preparing immobilized-enzyme beads according to claim 2, wherein the method comprises: dropping the mixed liquid in the first tank through the nozzle unit onto the calcium chloride solution, thereby preparing immobilized-enzyme beads.

8. The method according to claim 7, wherein the mixed liquid injected into the first tank is adjusted to a viscosity of 3,000 cPs to 7,000 cPs.

9. The method according to claim 7, wherein the enzyme-immobilized beads have a spherical shape having a diameter of 0.5 mm to 3 mm.

10. A method for preparing immobilized-enzyme beads using the apparatus for preparing immobilized-enzyme beads according to claim 3, wherein the method comprises: dropping the mixed liquid in the first tank through the nozzle unit onto the calcium chloride solution, thereby preparing immobilized-enzyme beads.

11. The method according to claim 10, wherein the mixed liquid injected into the first tank is adjusted to a viscosity of 3,000 cPs to 7,000 cPs, and a pressure of 0.1 kg/cm.sup.2 to 3 kg/cm.sup.2 is applied to the air inlet of the first tank.

12. The method according to claim 10, wherein the enzyme-immobilized beads have a spherical shape having a diameter of 0.5 mm to 3 mm.

13. The apparatus of claim 1, wherein the excipient is an alginate.

14. The method of claim 4, wherein the excipient is an alginate.

15. The method of claim 7, wherein the excipient is an alginate.

16. The method of claim 10, wherein the excipient is an alginate.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a schematic longitudinal sectional view of a lower end of a nozzle of an apparatus for preparing enzyme-immobilized beads according to one embodiment of the present invention.

(2) FIG. 2 is a schematic diagram of an apparatus for preparing enzyme-immobilized beads according to one embodiment of the present invention.

(3) FIG. 3 is a schematic diagram showing a process and apparatus for preparing enzyme-immobilized beads.

(4) FIG. 4 is a schematic longitudinal sectional view of a lower end of an injection needle of a typical apparatus for preparing enzyme-immobilized beads.

DETAILED DESCRIPTION OF THE INVENTION

(5) Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. A description of details apparent to those skilled in the art will be omitted for clarity.

(6) The present invention relates to an apparatus for preparing enzyme-immobilized beads used in preparation of tagatose and a method for preparing enzyme-immobilized beads using the same.

(7) More particularly, the present invention relates to an apparatus for preparing enzyme-immobilized beads, which includes a nozzle having an inner diameter of 0.1 mm to 1 mm and a cylindrical lower end and including a cut-type liquid outlet (cut perpendicularly to a vertical axis of the nozzle) formed at the lower end, and a method for preparing enzyme-immobilized beads using the same.

(8) According to one embodiment of the present invention, an apparatus for preparing enzyme-immobilized beads includes: a) a first tank into which a mixed liquid containing an enzyme-containing material and an excipient is injected; b) a nozzle unit disposed at a lower end of the first tank and including a nozzle, which has an inner diameter of 0.1 mm to 1 mm and a cylindrical lower end and includes a liquid outlet cut perpendicularly to a vertical axis of the nozzle at the lower end; and c) a second tank disposed below the nozzle unit and containing a calcium chloride solution.

(9) The first tank may include a tank which can receive the mixed liquid containing the enzyme-containing material and the excipient, and an inlet through which the mixed liquid and/or air can be injected into the tank.

(10) The mixed liquid injected into the first tank contains the enzyme-containing material and the excipient.

(11) The enzyme-containing material refers to an isomerase per se, or a material containing microbial cells or dead microbial cells capable of producing the isomerase, and the isomerase refers to an enzymatic isomerization of galactose into tagatose and may be an arabinose isomerase.

(12) In one embodiment, the microbial cells capable of producing the arabinose isomerase may include recombinant strains of the genus Corynebacterium.

(13) The recombinant strains of the genus Corynebacterium may be recombinant including a gene coding the arabinose isomerase derived from Thermotoga neapolitana which is a hyperthermophile.

(14) For details related to the recombinant strains of the genus Corynebacterium refer to Korean Patent No. 10-0872694.

(15) In some embodiments, the excipient is preferably an alginate, more preferably an alginate solution in which an alginate is dissolved in a silicon dioxide solution.

(16) According to the embodiment of the invention, the nozzle unit is disposed at the lower end of the first tank.

(17) The nozzle unit may include at least one nozzle.

(18) In some embodiment, the nozzle of the nozzle unit has a cylindrical lower end having an inner diameter from 0.1 mm to 1 mm, preferably from 0.2 mm to 0.5 mm, more preferably from 0.3 mm to 0.4 mm, and may include the cut-type liquid outlet formed at the lower end.

(19) The liquid outlet means an outlet through which the mixed liquid injected into the first tank is discharged, and the cut-type liquid outlet means that the liquid outlet is formed by cutting the lower end of the nozzle in the perpendicular direction to the vertical axis of the nozzle (see FIG. 1 which is a longitudinal sectional view of the lower end of the nozzle).

(20) A typical apparatus for preparing immobilized beads includes an injection needle (see FIG. 4) and is used to prepare pellet-type beads, thereby causing a problem of deterioration in isomerization efficiency. More specifically, when isomerization is performed using the pellet-type beads, a matrix liquid has a non-uniform moving load factor due to non-uniform pores when added, thereby causing deterioration in isomerization efficiency.

(21) In contrast, when the nozzle according to the present invention, which includes the cut-type liquid outlet at the lower end thereof and has an inner diameter of 0.1 mm to 1 mm, is used, uniform and smooth spherical beads are prepared and thus provide uniform pores, whereby the matrix liquid can also have a uniform moving load factor, thereby preventing disproportional use of an enzyme due to a difference in pores in a filling tower (a facility used in isomerization by filling the facility with the enzyme-immobilized beads, followed by injection of the matrix liquid).

(22) According to the present invention, the second tank includes a tank which can receive the calcium chloride solution, and an inlet into which the mixed liquid discharged from the nozzle unit is dropped and injected.

(23) In some embodiments, a distance between a surface of the calcium chloride solution in the second tank and the liquid outlet of the nozzle unit preferably ranges from 0.1 m to 1.5 m, more preferably from 0.5 m to 0.8 m.

(24) Within this range, since a dropping distance of the mixed liquid of the first tank from the nozzle unit can be appropriately adjusted, the mixed liquid can be formed into spherical beads while dropped and can be prevented from forming into elliptical beads due to collision with the surface of the calcium chloride solution in the event where the mixed liquid is dropped from an overly high place.

(25) The apparatus for preparing enzyme-immobilized beads according to the embodiment of the invention will be described in more detail with reference to FIG. 2.

(26) In FIG. 2, the apparatus for preparing enzyme-immobilized beads according to the embodiment of the present invention includes the first tank (a), the second tank (b), and the nozzle unit (c).

(27) The first tank (a) includes a mixed liquid and/or air inlet disposed at an upper end thereof. The first tank (a) may include one or more inlets, which may be used together or separately.

(28) The nozzle unit (c) is disposed at the lower end of the first tank (a) and may include at least one nozzle mounted therein.

(29) Since the nozzle is provided at an upper end (d) thereof with a mixed liquid inlet, the mixed liquid is injected (1) into the nozzle through the mixed liquid inlet while air is injected (2) into the first tank (a). Since the nozzle has the cylindrical lower end (e) and includes the cut-type liquid outlet formed at the lower end thereof, immobilized beads discharged through the liquid outlet can be formed in a smooth and uniform spherical shape while dropped (4).

(30) The immobilized beads discharged from the liquid outlet of the lower end of the nozzle are dropped (5) onto the calcium chloride solution contained in the second tank (b), and the distance between the lower end (e) of the nozzle and the surface of the calcium chloride solution in the second tank (b) may range from 0.5 m to 0.8 m.

(31) According to one embodiment of the invention, a method for preparing enzyme-immobilized beads using the apparatus for preparing enzyme-immobilized beads according to the present invention is provided.

(32) In preparation of enzyme-immobilized beads, a mixed liquid injected into the first tank may be prepared as follows: microbial cells capable of producing arabinose isomerase are killed, followed by centrifugation of a culture fluid of the microbial cells to recover dead microbial cells. Next, an alginate is dissolved to a concentration of 1.5% to 2.5% in a 0.4% to 1.0% silicon dioxide solution by stirring at 90±5° C. for 3 hours or more such that the alginate can be completely dissolved therein. After confirmation of complete dissolution of the alginate, the alginate solution is cooled to 35±5° C. by passing cooling water through a jacket. This process is performed to increase viscosity of the alginate while preventing damage to an enzyme upon mixing of microbial cells/enzyme.

(33) Next, the separated microbial cells are mixed with the alginate solution using a stirrer at 30 rpm to 60 rpm. Due to high viscosity of the alginate solution, when the rotational speed becomes excessively high upon stirring, there occurs a phenomenon in which bubbles are generated inside the mixed liquid and do not float up to an upper side of the liquid and air flows into the beads during preparation of the beads due to the bubbles, causing the beads to float to the upper side of the liquid. Thus, the stirring speed is advantageously adjusted to 30 rpm to 60 rpm.

(34) In preparation of the enzyme-immobilized beads, the mixed liquid injected into the first tank may have a viscosity of 3,000 cPs to 7,000 cPs. Within this range, uniform and smooth spherical beads can be prepared.

(35) In some embodiments, in preparation of the enzyme-immobilized beads, when the mixed liquid is discharged through the nozzle, a pressure from 0.1 kg/cm.sup.2 to 3 kg/cm.sup.2, preferably from 0.5 kg/cm.sup.2 to 2 kg/cm.sup.2, more preferably from 0.7 kg/cm.sup.2 to 1.7 kg/cm.sup.2 is applied thereto through the air inlet of the first tank.

(36) Within this range, the mixed liquid of the enzyme-containing material and the excipient can be passed in an appropriate amount through the nozzle and thus formed into the beads, and appropriate force can be applied to the mixed liquid while the mixed liquid is passed through the nozzle, whereby substantially spherical beads can be formed. As the shape of the beads is closer to a sphere, there are advantages in that the beads to which the enzyme is effectively immobilized can be obtained, and that subsequent isomerization can be efficiently performed due to an increased surface area of the beads.

(37) According to the present invention, in preparation of the enzyme-immobilized beads, after the mixed liquid of the enzyme-containing material and the excipient is dropped onto the calcium chloride solution, injection of air into the calcium chloride solution may be further performed such that the immobilized beads can maintain a spherical shape.

(38) Since there is a concern that the mixed liquid dropped onto the calcium chloride solution is pressed into a plate shape due to gravity and the beads pressed into the plate shape has low usability in isomerization due to a reduced surface area thereof, air may be injected into the calcium chloride solution to maintain the spherical shape of the beads.

(39) A method for preparing tagatose using the apparatus for preparing enzyme-immobilized beads according to the present invention will be briefly described hereinafter.

(40) 1) Starter microorganisms are inoculated as a seed.

(41) 2) Microbial cells obtained through seed culture are subjected to primary fermentation.

(42) 3) After completion of primary fermentation, secondary fermentation is performed.

(43) 4) After completion of secondary fermentation, tertiary fermentation is performed.

(44) 5) After completion of tertiary fermentation, quaternary fermentation is performed.

(45) 6) After completion of quaternary fermentation, a surfactant is introduced, followed by heating to induce the microbial cells death.

(46) 7) The microbial cells are isolated through centrifugation.

(47) 8) The recovered microbial cells are mixed with an alginate and silicon dioxide.

(48) 9) The mixed liquid is introduced into an immobilization apparatus to form beads.

(49) 10) The beads are stabilized by soaking in a galactose solution.

(50) 11) The beads are transferred to a fluidized bed reactor to fill the reactor column with the beads.

(51) 12) A galactose solution is transferred to the fluidized bed reactor to perform conversion into tagatose.

(52) In preparation of tagatose using the apparatus for preparing enzyme-immobilized beads according to the present invention, high-concentration and/or high-purity tagatose can be prepared. Advantageously, tagatose having a purity of 99% or more in terms of solid content can be prepared.

(53) Hereinafter, the present invention will be explained in more detail with reference to some examples. However, it should be understood that these examples are provided for illustration only and are not to be construed in any way as limiting the present invention.

EXAMPLE 1

(54) Preparation of Enzyme-Immobilized Beads

(55) (1) Preparation of Mixed Liquid of Enzyme-Containing Material and Excipient

(56) Microbial cells capable of producing an arabinose isomerase were killed, followed by centrifugation of a culture media of the microbial cells to recover the dead microbial cells. Next, an alginate was dissolved to a concentration of 2% in a 0.7% silicon dioxide solution by stirring at 90° C. for 3 hours or more such that the alginate could be completely dissolved in the solution. After confirmation of complete dissolution of the alginate, the alginate solution was cooled to 35° C. by passing cooling water through a jacket.

(57) Next, the alginate solution and the separated microbial cells were mixed using a stirrer at 50 rpm.

(58) (2) Discharge of Mixed Liquid

(59) The mixed liquid was confirmed to have a viscosity of 5,000 cPs, followed by injection into a first tank. Next, the alginate/microbial cells-mixed liquid was passed through a nozzle having an inner diameter of 0.3 mm and a cut shape at a distal end thereof by supplying air at a pressure of 1.2 kg/cm.sup.2, thereby dropping the mixed liquid in a spherical shape having a diameter of 1.7 mm onto a 1% calcium chloride solution contained in a second tank.

(60) (3) Formation of Immobilized Beads

(61) The beads discharged from the nozzle were dropped onto the calcium chloride solution separated from a liquid outlet of the nozzle by a distance of 0.6 m while supplying air onto the calcium chloride solution (air bubbling), and then transferred to a cooling tank, followed by curing at 2° C. for 24 hours.

(62) A process and apparatus for preparing the enzyme-immobilized beads are shown in FIG. 3.

LIST OF REFERENCE NUMERALS

(63) [Apparatus] a: First tank b: Second tank c: Nozzle unit d: Upper end of nozzle e: Lower end of nozzle

(64) [Process] 1: Inject mixed liquid 2: Inject air 3: Inject mixed liquid into mixed liquid inlet of upper end of nozzle 4: Discharge and drop immobilized beads from liquid outlet of lower end of nozzle 5: Drop immobilized beads onto calcium chloride solution in second tank