Granules comprising isomaltulose synthase

Abstract

The invention provides granules comprising A) at least one enzyme selected from at least one of the groups selected from transferases of EC 2, hydrolases of EC 3, lyases of EC 4 and isomerases of EC 5, B) at least one polymer selected from C.sub.1-C.sub.10-alkyl acrylate polymer, C.sub.1-C.sub.10-alkyl methacrylate polymer and C.sub.1-C.sub.10-alkyl acrylate-C.sub.1-C.sub.10-alkyl methacrylate copolymer, preferably C.sub.1-C.sub.10-alkyl acrylate-C.sub.1-C.sub.10-alkyl methacrylate copolymer and C) at least one inorganic carrier material.

Claims

1. Granules comprising: A) at least one isomaltulose synthase of EC 5.4.99.11, B) at least one polymer selected from the group consisting of a C.sub.1-C.sub.10-alkyl acrylate polymer, a C.sub.1-C.sub.10-alkyl methacrylate polymer, and a C.sub.1-C.sub.10-alkyl acrylate-C.sub.1-C.sub.10-alkyl methacrylate copolymer, and C) at least one inorganic carrier material.

2. The granules according to claim 1, wherein the isomaltulose synthase is present in the form of whole-cell catalysts or in the form of disrupted cells.

3. The granules according to claim 1, wherein the polymer has a mass-average molecular weight of 100000 to 1500000 g/mol.

4. The granules according to claim 1, wherein the polymer is an ethyl acrylate/methyl methacrylate copolymer.

5. The granules according to claim 1, wherein the inorganic carrier material is at least one selected from the group consisting of silicas and aluminum silicates.

6. The granules according to claim 1, wherein, based on the total weight of the granules, the at least one polymer B) is present in an amount of from 0.1% by weight to 80% by weight and the at least one inorganic carrier material C) is present in an amount of from 20% by weight to 95% by weight.

7. The granules according to claim 1, wherein the granules have a specific activity of 1 to 1000 U, based on the total weight of the granules.

8. The granules according to claim 1, wherein the at least one polymer is a C.sub.1-C.sub.10-alkyl acrylate-C.sub.1-C.sub.10-alkyl methacrylate copolymer.

9. The granules according to claim 1, wherein the polymer has a mass-average molecular weight of 500000 to 1000000 g/mol.

10. The granules according to claim 4, wherein the ethyl acrylate/methyl methacrylate copolymer is poly(ethyl acrylate-co-methyl methacrylate) 2:1.

11. The granules according to claim 5, wherein the silica is a precipitated silica.

12. The granules according to claim 5, wherein the aluminum silicate is a zeolite.

13. The granules according to claim 1, which have a half-value particle size (d50) of 100 to 2000 m.

14. The granules according to claim 1, which have a d10 value of greater than or equal to 50% of a half-value particle size (d50), a d90 value of less than or equal to 150% of the half-value particle size (d50) or both.

15. The granules according to claim 1, wherein at least one selected from the group consisting of a d10 value, a half-value particle size (d50), and a d90 value is reduced by less than 10% after 60 minutes shaking at 400 rpm in a medium, relative to a d10 value, a half-value particle size (d50), and a d90 value of the granules prior to the shaking.

Description

(1) The following figures are a component of the examples:

(2) FIG. 1: Specific activity (U/g) based on the main product (isomaltulose in mol) which is formed per minute under defined conditions (40% sucrose (starting material), RT, pH 6, 100 rpm), by 1 g of granules or by a fermentation liquor comprising P. rubrum with a biomass equivalent to the granules.

(3) FIG. 2: Graph of the particle size distribution of the preparation according to the invention (untreated or after incubation on a shaker at 400 rpm) as a function of distribution density.

(4) FIG. 3: Graph of the particle size distribution of the non-inventive preparation (untreated or after incubation on a shaker at 400 rpm) as a function of distribution density.

(5) FIG. 4: Graph of the conversion of sucrose into fructose and glucose.

EXAMPLES

Example 1

Production of a Preparation According to the Invention

(6) 100 ml of a 30% strength by weight dispersion of an ethyl acrylate/methyl methacrylate copolymer (Eudragit NM, Evonik Industries AG) are homogenized with 130 ml of dry mass 6% by weight strength fermentation liquor of P. rubrum and 62 g of carrier material consisting of Sipernat 320 (Evonik Industries AG) in a kneader. The homogeneous mixture is then extruded using an extruder (Extruder 20, Caleva) and granulated using a spheronizer (Spheronizer 250, Cavela).

(7) The resulting granules are dried overnight at room temperature and a residual moisture of 30% (w/w) is adjusted, ascertained by dry weighing.

(8) Based on total dry weight, the particles comprise 30% by weight of ethyl acrylate/methyl methacrylate copolymer, 8% by weight of fermentation dry mass and 62% by weight of sipernat.

Example 2

Apparent Activity

(9) Determination of the apparent activity of the preparation according to Example 1.

(10) The determination of the activity takes place in three separate batch mixtures. For this, in each case 1 g of the dried granules is placed into a 15 ml reaction tube and supplied with 10 ml of 40% (w/w) sucrose solution. Incubation takes place on a shaker with 100 rpm at RT and a pH of 6. The specific activity U/g was ascertained after an incubation time of 120 min by determining the product concentration. This was ascertained here in each case with the help of HPLC analysis. The specific activity of fermentation liquor containing P. rubrum was determined analogously, with a biomass equivalent to the granules present.

(11) The results are shown in FIG. 1.

Example 3

Mechanical Stability

(12) The mechanical stability of the preparation according to Example 1 is assessed by means of the determination of the d10 [m], d50 [m] and the d90 [m] value of the particle size distribution from the distribution density function. This was determined visually under standard conditions with the help of a Retsch Camsizer.

(13) The determination of the particle size distribution takes place untreated and also after incubation for 30 and 60 minutes in a 50 ml reaction vessel with in each case 25 g of granules and 25 g of medium on a shaker (400 rpm) in the medium and subsequent drying. It is clear, particularly as a result of the virtually unchanged dl 0 [m] value, which is to be deduced from the distribution density function, that the preparation is stable and no particle fragments are formed. Accordingly, 10% of the particles of the particle collective prior to incubation have a diameter of less than 881 m and, after incubation for 60 minutes, a diameter of less than 866 m.

(14) Particle Size DistributionPreparation According to the Invention

(15) TABLE-US-00001 d(10) d(50) d(90) Preparation according to the invention [m] [m] [m] Sample 1 In medium - 0 min 881 1017 1267 Sample 2 In medium - 30 min 818 980 1160 shaker, 400 rpm Sample 3 In medium - 60 min 866 983 1183 shaker, 400 rpm

Example 4

Production of a Preparation According to the Invention Comprising Saccharomyces cerevisiae with the Enzyme Invertase (EC 3.2.1.26)

(16) The production of the preparation takes place as in Example 1, except that the biocatalytic active component used is 130 ml of a dry mass 6% by weight strength suspension comprising standard commercial Saccharomyces cerevisiae. The Saccharomyces cerevisiae whole cells contain inter alia the enzyme invertase (EC 3.2.1.26) from the group of hydrolases of EC 3, which hydrolytically cleaves sucrose into fructose and glucose. To investigate the apparent activity, the preparation was placed into a fixed-bed reactor which has side septums for sampling. The starting material used was a 40% (w/w) sucrose solution which was adjusted to a pH of 6. The starting material was fed in at an LHSV [h1] of 0.2, which is formed by the quotient of volume stream [m3/h] and the catalyst volume used [m3]. In order to achieve a steady state in the fixed-bed reactor, sampling was carried out after a run-in phase of 72 h. Since sampling was carried out along the length of the reactor, the conversion of the starting material can be monitored over the dimensionless reactor length x/X []. The sample composition was ascertained in each case with the help of HPLC analysis. The results are shown in FIG. 4.

Comparative Example 1 (not According to the Invention)

Production of a Preparation not According to the Invention

(17) Production of a preparation according to WO95/22606 (Novo Nordisk NS): Method for production of an immobilized enzyme preparation and use of the immobilized enzyme preparation.

(18) 65 g of Celkate T-21 are added in powder form to a high-speed mixer. To this, 25 g of liquid fermentation liquor comprising sucrose isomerase are added to the powder continuously and with running impellor. Then, a further 50 g of liquid fermentation liquor comprising sucrose isomerase, combined with 3% (w/w) Kollidon K25 polyvinylpyrrolidone (BASF) are added. The resulting granules are dried overnight at room temperature and sieved. The residual moisture content is adjusted to 10%.

Comparative Example 2 (not According to the Invention)

Mechanical Stability

(19) Determination of the mechanical stability of the preparation according to Comparative example 1. The measurement was carried out analogously to Example 1. Results are shown in FIG. 3. The considerable shift in the d10 value [m] shows that before the incubation 10% of the particle collective is less than 784 m and after incubation less than 389 m. This means that the preparation not according to the invention disintegrates into smaller particle fragments as a result of the mechanical stress on the shaker.

(20) TABLE-US-00002 d(10) d(50) d(90) Preparation not according to the invention [m] [m] [m] Sample 1 In medium - 0 min 784 1024 1355 Sample 2 In medium - 30 min 695 1004 1290 shaker, 400 rpm Sample 3 In medium - 60 min 389 874 1092 shaker, 400 rpm