DE3733055A1 - METHOD FOR IMMOBILIZING THE (BETA) MUSHROOM GALACTOSIDASE - Google Patents

Abstract

Fungal beta -galactosidase produced by Penicillium canescens strain F-178 is adsorbed on an inorganic carrier, such as granulated silicon dioxide, and is fixed by means of a 0.5-2.5% solution of glutaldehyde.

Description

The present invention relates to biotechnology and relates in particular to a method for immobilizing the β- fungal galactosidase.

The registered invention is used in the Production of glucose-galactose syrup - a wholesome one Sugar substitute in a number of productions the milk, confectionery and bakery industry.

In the production of glucose-galactose syrup, β- mushroom galactosidases are used as biocatalysts in the hydrolysis of milk and whey lactose. However, the use of soluble β- fungal galactosidases has a number of disadvantages:

• 1) a high degree of hydrolysis does not succeed To achieve lactose (50 to 60%);
• 2) the end product is contaminated with protein admixtures with β- galactosidase;
• 3) when using the soluble β- galactosidases, the reaction of trans-glycosylation and trans-galactosilation takes place, which deteriorate the taste properties of the end products;
• 4) the exploitation of the soluble β- galactosidases complicates the development of a continuous automated hydrolysis process of lactose.

The use of the immobilized β- galactosidases eliminates these disadvantages.

A method for immobilizing the β- fungal galactosidase Aspergillus niger onto an inorganic support, which has been modified by adsorption of ortho- or para-dianisidine with subsequent diazotization, by reacting this carrier with the ferment is known (Journal of Solid-Phase Biochemistry, vol . l, No. 2, 1976, RA Messing, LF Bialousz, RE Lindner "Aromatic amine coupling of Aspergillus niger lactase to controlled-pore selica with o-dianisidine").

Preparations of immobilized β- galactosidase with an activity of 335 units / g carrier (at a temperature of 50 ° C.) and a degree of binding of 17% are obtained.

The modification of this procedure, which in the exploitation of glutaric acid aldehyde in one of the stages, led to the preparation of the preparations with an activity from 443 to 620 units / g carrier (at a temperature of 50 ° C) and a degree of binding of 25 to 31%. (One unit of activity is thought to be so much of the ferment, the 1 µmol lactose per 1 min at one temperature of 30 ° C and a pH of 4.0 hydrolyzed. To convert the activity at a temperature of 50 ° C it is necessary to measure the value of the activity at a temperature of 30 ° C with a coefficient of 2.5 to multiply.) This preparation had hydrolysis of the 5% lactose solution in the buffer with a pH of 3.5 and one Temperature of 50 ° C the half inactivation time of 52 days.

The use of aromatic diamine, the one Blood toxin represents, but does not allow, the said Use methods for immobilizing the ferment, intended for the food industry.

A method is known for immobilizing the Aspergillus niger β- fungal galactosidase on hydrophobic supports with a long aliphatic chain and phenyl radicals. The ferment is fixed on the phenyl radical of the support by the covalent bond method with the aid of glutaric acid aldehyde (FR, A, 78 22 681).

This method was not widely used because it is associated with the need for one with specific ones Modified carrier for fixation of the reagents Exploit ferments and surfactants Hydrophilization of the surface of the support during immobilization to use.

It is a method for immobilizing the b -Pilzgalaktosidase Aspergillus niger on the porous aluminum oxide is known which is used as a carrier within 16 to 20 hours. To increase the amount of the adsorbed ferment, it or the surface of the carrier is treated with a triazine dye. The adsorbed ferment is fixed with the glutaric acid aldehyde solution which is added to the ferment solution before the adsorption of the ferment or 5 minutes after the adsorption (US, A, 38 73 426).

The activity of the immobilized β- galactosidase obtained in this way is 7.6 β- galactosidase units / g of preparation. The disadvantage of the method consists in a low activity of the immobilized preparation, in a large consumption of the ferment, because after the addition of the glutaric acid aldehyde solution the ferment forms conglomerates which are unsuitable for repeated immobilization, as well as in a significant washing away of the adsorbed ferment in the process of Utilization of whey lactose during hydrolysis. It is also necessary to use dyes to improve adsorption, which is also undesirable for the food industry.

The present invention is based on the task of realizing the immobilization of the β- fungal galactosidase by producing a highly active and stable preparation for a shorter technological cycle and without using toxic substances by the choice of the raw material and the change in the process conditions.

This object is achieved in that in the process for immobilizing the β- mushroom galactosidase, which is achieved by adsorbing the said β- mushroom galactosidase from its solution onto an inorganic support and subsequently fixing the adsorbed β- galactosidase on the support with the aid of the glutaric acid aldehyde the immobilization of the β- fungal galactosidase of the Penicillium canescens F-178 strain is carried out and the fixation of the adsorbed β- galactosidase is carried out with a 0.5 to 2.5% glutaric acid aldehyde solution.

The β- fungal galactosidase of the Penicillium canescens F-178 strain used according to the invention was deposited on November 23, 1981 with the Tsentralny Muzei Promyshlennykh Mikroorganizmov Instituta "Vniigenetika" (Accession No. 20171).

The present invention makes it possible to prepare the immobilized β- galactosidase preparation with an activity of 420 to 450 units / g of carrier (at a temperature of 30 ° C., a pH of 4.0, the 5.0% lactose ) that has a semi-inactivation time of 136 days.

The method according to the invention enables the immobilization process by eliminating the adsorption  of the dye and simplify the immobilization time 6 to 8 times that of the US patent No. 38 73 426 to shorten the registered procedure.

According to the invention, it is expedient as an organic carrier a granulated compound of the formula SiO₂ · n H₂O, where n is an integer which has the specific surface area of 70 to 100 m² / g, with a granule size of 0.315 to 0.500 mm and a pore size of Use 400 to 600 Å.

The use of the registered silicon oxide carrier contributes to increasing the activity and stability of the preserving preparation.

In order to reduce the consumption of the soluble β- galactosidase by 20% and to be able to use the ferment for immobilization immediately after the cultivation step, and to increase the stability - the inactivation time of the ferment is approximately 200 days - it is expedient to use it according to the invention the solution with a concentration of the said β- mushroom galactosidase from 20 to 50 mg / ml the immobilization of the β- mushroom galactosidase by its multiple adsorption from the solutions with the concentration mentioned on one and the same carrier until the immobilization of the said β- mushroom galactosidase on the whole Realize the surface of the support by performing the fixation with a 1.0 to 2.5% glutaric acid aldehyde solution after each adsorption.

Further objects and advantages of the present invention will become apparent from the detailed description of the method for immobilizing the β- fungal galactosidase and the examples for realizing this method which follows.

In the registered procedure, we propose to immobilize the β- fungal galactosidase from the Penicillium canescens F-178 strain, the production technology of which is simple and accessible. The production technology of the β- mushroom galactosidase mentioned includes the stages of cultivation of the ferment, the ultraconcentration and the drying in the atomizing dryer, in contrast to the production technology of other β- mushroom galactosidases, where, in addition to the steps listed above, cleaning of carboxymethyl cellulose, precipitation with organic solvents, affine chromatography can be exploited.

The production of the β- fungal galactosidase from the Penicillium canescens F-178 strain is carried out using the customary technology which involves the cultivation of the microscopic fungus, the ultraconcentration of the culture fluid and the drying of the fermentation preparation of the β- fungal galactosidase from the Penicillium canescens F-178 strain in an atomizing dryer then dissolving the preparation obtained in 0.2 mol of acetate buffer with a pH of 4.0 ± 0.1 until reaching a concentration required for the immobilization of the ferment, which is generally at least 130 mg / ml.

It is possible to carry out the process according to the invention on any known inorganic granulated carrier, for example silica gels which have a specific surface area of 28 m² / g carrier and a pore size of 750 Å, or macroporous aerosils with a specific surface area of 26 to 120 m² / g carrier to realize. However, it is more appropriate to use a porous granular compound of the formula SiO₂ · n H₂O, where n is an integer having a specific surface area of 70 to 100 m² / g, with a granule size of 0.315 to 0.500 mm and a pore size of Use 400 to 600 Å.

The immobilization process is carried out according to the invention by adsorption of the β- galactosidase from the strain Penicillium canescens F-178 onto an inorganic granulated carrier at room temperature and over a period of time which is sufficient for the formation in the solution of the equilibrium concentration of at most 4 to 6% of the initial concentration . The immobilization of the β- fungal galactosidase mentioned is achieved on the entire surface of the support.

The β- fungal galactosidase adsorbed on the carrier is fixed with the aid of a 0.5 to 2.5% glutaric acid aldehyde solution, which leads to stabilization of the ferment on the carrier, which allows this preparation to be used at elevated temperatures of 40 to 60 ° C to use.

At a concentration of the glutaric acid aldehyde of less than 0.5%, the ferment can be rinsed away from the carrier in the process and used in whey hydrolysis; at a concentration of the glutaric acid aldehyde of over 2.5%, the activity of the immobilized preparations of the β- fungal galactosidase mentioned is reduced as a result of a large amount of the chemical bonds which cover the active center of the ferment.

The proposed invention provides the possibility of using the β- fungal galactosidase from the Penicillium canescens F-178 strain at a concentration of 20 to 50 mg / ml. Such a concentration is reached after the stage of cultivation of the microscopic fungus in the production of the β- fungal galactosidase mentioned. The elimination of the stages of ultraconcentration of the culture liquid and the drying of the ferment preparation of the above-mentioned β- fungal galactosidase make it possible to simplify the immobilization process on the whole and to reduce the consumption of the ferment by 20%.

When using the β- fungal galactosidase from the Penicillium canescens F-178 strain in a concentration of 20 to 50 mg / ml for the production of the highly active immobilized preparations, the immobilization by treating the inorganic carrier is repeated several times in a certain order with solutions of the β- fungal galactosidase a concentration of 20 to 50 mg / ml of the glutaric acid aldehyde (1.0 to 2.5% solution) realized. Each subsequent solution of both the ferment and the glutaric acid aldehyde must correspond to an initially mentioned concentration. The multiplicity of treatment of the carrier with the solutions ferment-glutaric acid aldehyde is generally 3 to 8, as a result of which the ferment is immobilized on the entire surface of the carrier.

When glutaric acid aldehyde is used in this case in a concentration below 1%, the ferment is washed away from the carrier in the process of its use in the hydrolysis of whey lactose; the concentration of the glutaric acid aldehyde above 2.5% leads to a reduction in the activity of the immobilized preparations of the β- fungal galactosidase due to a large amount of the bonds forming which cover the active center of the ferment.

When the multiply immobilized β- fungal galactosidase is incubated at a temperature of 50 to 60 ° C in the acetate buffer within 60 minutes, the activity of the immobilized preparation is maintained 50% higher than with the once-immobilized β- fungal galactosidase from the highly concentrated Solution was obtained.

Example 1

To 0.8 g of fermentation preparation of the β- galactosidase from the strain Penicillium canescens F-178 with an activity of 630 units / g of preparation, which was dissolved in 5 ml of 0.2 molar acetate buffer with a pH of 4.0, 1 g of a granulated siliceous carrier was added. The immobilization is carried out with stirring at a temperature of 24 ° C. within 3 hours. Afterwards, 1 ml of 25% glutaric acid aldehyde solution is added to the suspension of the immobilized preparation. The treatment with glutaric acid aldehyde is carried out within 1.5 hours. The weakly adsorbed ferment is rinsed off with 2 molar NaCl solution, 30% isopropanol solution in 1 molar NaCl and with 0.2 molar acetate buffer solution and water. The activity of the immobilized β- galactosidase is 280 units / g of carrier.

Example 2

To 0.8 g of fermentation preparation of the β- galactosidases of the strain Penicillium canescens F-178 with an activity of 630 units / g of preparation, which was dissolved in 5 ml of 0.2 molar acetate buffer with a pH of 4.0, 1 g of granulated macroscopic silica (the specific surface is 26 to 120 m² / g) was added. The immobilization is carried out similarly to Example 1. The immobilized preparation is crosslinked with 5 ml of 2.5% glutaric acid aldehyde solution in 0.1 molar phosphate buffer with a pH of 7. The activity of the immobilized ferment is 300 units / g of carrier.

Example 3

To 1.2 g of fermentation preparation of β- galactosidase from the strain Penicillium canescens F-178 with an activity of 3000 units / g of preparation, which was dissolved in 30 ml of 0.2 molar acetate buffer with a pH of 4.0, 6 g of a granulated siliceous support (SiO₂ · n H₂O; the specific surface is 70 to 100 m² / g) is added. The immobilization is carried out within 3 hours.

The immobilized preparation is linked with Performed glutaric acid aldehyde within 1.5 hours, the concentration of the glutaric acid aldehyde in it is Trap 2%. The activity of the immobilized preparation is 450 units / g carrier.

Example 4

To 60 g of fermentation preparation of the β- galactosidase from the strain Penicillium canescens F-178 with an activity of 630 units / g of preparation which was dissolved in 350 ml of 0.2 molar acetate buffer with a pH of 0.4, 100 g of a granulated siliceous carrier (SiO₂ · n H₂O; the specific surface is 70 to 100 m² / g) with a granule size of 0.315 to 0.5 mm, a pore size of 400 to 600 Å. The immobilization is carried out at a temperature of 10 ° C. within 2 hours. The immobilized preparation is crosslinked with 500 ml of 1% glutaric acid aldehyde solution in 0.1 molar phosphate buffer with a pH of 7.0 within 1 hour. The activity of the preparation obtained is 220 units / g of carrier.

1.7 g of moist preparation of the immobilized β- fungal galactosidase from the strain Penicillium canescens F-178 with an activity of 220 units / g of carrier are introduced into a thermostated column of 10 mm in diameter. The whey containing no proteins and mineral salts with a lactose content of 5% is fed into the column at a rate of 25 ml / h. given at a temperature of 50 ° C. The lactose hydrolyzate is collected at the column outlet and the glucose content is determined, according to which the degree of hydrolysis of lactose is calculated. The degree of hydrolysis of lactose achieved is 95 to 100%. With continuous work within 36 days without cleaning and disinfection, the degree of hydrolysis of the lactose does not change. The calculated from the experimental data Halbinaktivisierungszeit the immobilized β -Glaktosidase of the strain Penicillium canescens F-178 is 136 days ago.

Example 5

0.3784 g of a granulated silica support similar to that of Example 4 is introduced into a 10 mm diameter column. 15 ml solution of the β- fungal galactosidase from the strain Penicillium canescens F-178 with an activity of 1536 units / 15 ml preparation solution are passed through the column within 3 hours; the adsorbed ferment is treated with 1% glutaric acid aldehyde solution in the phosphate buffer within 1.5 hours. The activity of the immobilized ferment is 320 units / g of carrier.

Example 6

0.3626 g of a granulated silica support similar to that of Example 4 is introduced into a 10 mm diameter column. 15 ml of solution of the β- fungal galactosidase from the strain Penicillium canescens F-178 with an activity of 1536 units / 15 ml of preparation solution are passed through the column within 2 hours. A 2% glutaric acid aldehyde solution in the phosphate buffer is then passed through the column. The activity of the immobilized ferment is 300 units / g.

Example 7

To 5 ml of β- galactosidase solution from the strain Penicillium canescens F-178 with a concentration of 20 mg / ml and an activity of 90 units / 5 ml of preparation solution is added 1 g of a granulated inorganic carrier similar to Example 4. The immobilization is carried out with stirring and shaking within 1.5 hours. The glutaric acid aldehyde is then added until a final concentration of 2.5% is reached and stirring is continued for an hour. The solution is then poured off and the immobilized ferment is washed through with 80 ml of water and the next portion (5 ml) of the said ferment is added at a concentration of 20 mg / ml. The adsorption and fixation with glutaric acid aldehyde are carried out as described above. The immobilization (adsorption and fixation) is repeated 1 to 8 times. The activity of the immobilized β- galactosidase after immobilization is shown in Table 1.

Example 8

To 5 ml of β- galactosidase solution of the strain Penicillium canescens F-178 with a concentration of 30 mg / ml and an activity of 135 units / 5 ml of preparation solution is added 1 g of a granulated inorganic carrier similar to Example 4. The immobilization is carried out similarly to Example 7.

The activity of the immobilized β- galactosidase preparations is shown in Table 1.

Example 9

To 5 ml of β- galactosidase solution of the strain Penicillium canescens F-178 with a concentration of 40 mg / ml and an activity of 180 units / 5 ml of preparation solution is added 1 g of a granulated inorganic carrier similar to Example 4. The immobilization is carried out similarly to Example 7.

The activity of the immobilized β- galactosidase preparation is shown in Table 1.

Example 10

To 5 ml of β- galactosidase solution of the strain Penicillium canescens F-178 with a concentration of 50 mg / ml and an activity of 250 units / 5 ml of preparation solution is added 1 g of a granulated inorganic carrier similar to Example 4. The immobilization is carried out similarly to Example 7.

The activity of the immobilized β- galactosidase preparations is shown in Table 1.

Example 11 (control)

1000 ml of β- galactosidase solution from the strain Penicillium canescens F-178 with an activity of 270 units / 1000 ml of preparation solution are added to 340 g of a dry granulated inorganic carrier. The immobilization is carried out within 3 hours. 100 ml of 25% glutaric acid aldehyde solution are then added to the reaction mixture. The fixation is carried out within 1 hour. The activity is 350 units / g of immobilized preparation with a degree of binding after the activity of 44%.

Table 1

Activity of the β- galactosidase preparations of the strain Penicillium canescens F-178, which were obtained by the multiple immobilization method (Examples 7, 8, 9, 10)

Example 12

To 5 ml of β- galactosidase solution of the strain Penicillium canescens F-178 with a concentration of 130 mg / ml and an activity of 600 units / 5 ml of preparation solution is added 1 g of a granulated inorganic carrier similar to Example 4. The adsorption is carried out within 3 hours. Then 0.5 ml of 25% glutaric acid aldehyde solution is added to the reaction mixture. The fixation is carried out within 1 hour. The activity of the immobilized preparation activity of 40%.

Example 13

To 5 ml of β- galactosidase solution of the strain Penicillium canescens F-178 with a concentration of 20 mg / ml and an activity of 90 units / 5 ml of preparation solution is added 1 g of a granulated inorganic carrier similar to Example 1. The immobilization is carried out within 1.5 hours. Then 0.5 ml of 25% glutaric acid aldehyde solution is added to the reaction mixture. The immobilized preparation is fixed on the carrier analogously to Example 7. The activity of the immobilized preparation is 350 units / g preparation with a degree of binding after the activity of 56 to 60%.

Example 14

The adsorption of the β- galactosidase solution of the strain Penicillium canescens F-178 is carried out similarly to Example 13. Afterwards, 0.15 ml of 25% glutaric acid aldehyde solution is added to the reaction mixture. Fixation of the immobilized preparation on the carrier is carried out similarly to Example 7. The activity of the immobilized preparation is 360 units / g preparation. If the mixture is left to stand for a long time in a mixture of the 30% isopropanol solution in 2 molar NaCl solution, β- galactosidase dissolves and the solution has a β- galactosidase activity. In Examples 7 to 13 no β- galactosidase activity was detected.

Example 15

The adsorption of the β- galactosidase from the solution onto a granulated inorganic carrier which is similar to the carrier described in Example 4 is carried out similarly to Example 13. Then 1 ml of 25% glutaric acid aldehyde solution is added to the mixture. Fixation of the immobilized preparation on the carrier is carried out similarly to Example 7. The activity of the immobilized preparation is 280 units / g preparation.

Example 16

To 5 ml of β- galactosidase solution from the strain Penicillium canescens F-178 with a concentration of 160 mg / ml and an activity of 2400 units / 5 ml of preparation solution is added 1 g of a granulated inorganic carrier which is similar to the carrier described in Example 4 , admitted. The adsorption is carried out within 3 hours. Then 0.5 ml of 25% glutaric acid aldehyde solution is added to the reaction mixture. The fixation is carried out using the glutaric acid aldehyde on the support within 1 hour. The activity of the immobilized preparation is 840 units / g preparation with a degree of maintained activity of 35%.

Example 17

1 g of a granulated inorganic carrier is added to 5 ml of β- galactosidase solution of the strain Penicillium canescens F-178 with a concentration of 20 mg / ml and an activity of 300 units / 5 ml of preparation solution. The immobilization is carried out similarly to Example 7. The activity of the preparation after 6-fold immobillization is 970 units / g preparation with a degree of maintained activity of 54%.

Example 18

1.2 kg of dry granulated inorganic carrier, which is similar to the carrier described in Example 4, are placed in a container. 6 liters of β- galactosidase solution are added through the shower device. The concentration of the solution is 20 mg / ml and the activity is 10.8 · 10⁴ units / 6 liters of preparation solution. The immobilization is carried out by circulating the reactants. The adsorption is carried out within 30 minutes, after which 360 ml of 25% glutaric acid aldehyde solution are added to the fermentation solution and the fixation is carried out within 45 minutes. The operations are repeated 6 times. The washing through is carried out similarly to Example 7 by circulating the reactants. The activity of the immobilized preparation is 360 units / g preparation with a degree of maintained activity of 47%.

Example 19

To 8 g of immobilized β- galactosidase from the strain Penicillium canescens F-178, which was obtained by single immobilization similarly to Example 11, 80 ml of 0.2 molar acetate buffer solution (pH 4.0) are added at a temperature of 60 ° C too. Samples are taken every 10 minutes within 1 hour and the activity of the preparation obtained is determined according to the standard methodology. The inactivation constant is calculated based on the data found:
K = ^{26.10 -3} min ^-1 .

Example 20

80 ml of 0.2 molar acetate buffer solution (pH 4.0) at a temperature of 60 ° C. are added to 8 g of the immobilized β- galactosidase preparation, which was obtained by repeated immobilization similarly to Example 7. Samples are taken every 10 minutes within 1 hour and the activity of the preparation obtained is determined according to the standard methodology. The inactivation constant is calculated based on the data found:
K = 16.6 · 10 ^-3 min ^-1 .

Claims (4)

1. Method for immobilizing the β- fungal galactosidase, realized by

• - Their adsorption from the solution on an inorganic support;
• - The subsequent fixation of the adsorbed β- galactosidase on the support with the help of glutaric acid aldehyde;

characterized in that the immobilization of the β- fungal galactosidase of the Penicillium canescens F-178 strain is carried out and the fixation of the β- galactosidase adsorbed on the carrier is carried out with 0.5 to 2.5% glutaric acid aldehyde solution. 2. The method according to claim 1, characterized in that a porous granulated compound of the formula SiO₂ · n H₂O, in which n is an integer having a specific surface area of 70 to 100 m² / g, with a granule size of as the inorganic carrier 0.315 to 0.500 mm and a pore size of 400 to 600 Å. 3. The method according to claim 1, 2, characterized in that when using the solution with a concentration of said β- fungal galactosidase from 20 to 50 mg / ml the immobilization of the β- fungal galactosidase

• - By their repeated adsorption from the solutions with the concentration mentioned on one and the same carrier
• - is implemented on the entire surface of this support until the immobilization of the β- fungal galactosidase mentioned,
• - By carrying out the fixation with 1.0 to 2.5% glutaric acid aldehyde solution after each adsorption.