FR2619824A1 - Process for immobilising beta -galactosidase - Google Patents

Abstract

Biotechnology. Process for adsorbing the beta -galactosidase produced by the strain Penicillium canescens F-178 on an inorganic support, the enzyme adsorbed on the support being bound by means of a 0.5-2.5 % glutaraldehyde solution. Application to the hydrolysis of lactose.

Description

 The present invention relates to biotechnology, and more particularly, to a method of immobilizing ss-galactosidase produced by a fungus mycelium.

 The invention finds its application in the production of glucose-galactose syrup, substitute of sugar value in different productions of the dairy industry, confectionery and bakery.

In the production of glucose-galactose syrup, ss-galactosidases of mycelial origin are used as biocatalyst during the hydrolysis of milk lactose and milk serum. However, the use of soluble ss-galactosidases has a number of drawbacks
1. it does not make it possible to obtain a high degree of hydrolysis of lactose (from 50 to 60);
2. it contaminates the finished product with protein additions, in particular ss-galactosidase itself
3. it allows trans-glycosilation and trans-galactosilation reactions which deteriorate the organoleptic qualities of the finished products
4. it is difficult to implement by a continuous automatic process of lactose hydrolysis.

 The use of immobilized B-galactosidases excludes the above-mentioned drawbacks.

A method of immobilizing ss-galactosidase from mycelium of Aspergillus niger is known on a mineral carrier, modified by adsorption of ortho or para-dianisidine which is then diazotized, by interaction of said carrier with the enzyme (Journal of Solid
Biochemistry phase, c. 1, NO 2, 1976, RA Messing, LF

Bialousz, EE Lindner, Aromatic amine coupling of
Aspergillus niger Lactase to controlled-pore silica with o-dianisidine). We then obtain immobilized ss-galactosidase products having an activity of 335 units of enzymatic activity of hydrolysis per gram of carrier (at a temperature of 500 ° C.) and a degree of fixation of 17 ".

 The unit of enzymatic hydrolysis activity is the activity of a quantity of enzyme hydrolyzing 1 micromole of lactose per minute at the temperature of 300C and at pH = 4.0. To calculate the activity at 500C, multiply the value of the activity at 300C by a coefficient equal to 2.5.

 The modification of said process for immobilizing ss galactosidase, consisting in the use at one of its stages of glutaraldehyde, results in the production of products having an activity of 443 to 620 units / g of carrier (at 500C ) and a degree of fixation of 25 to 31%. This product, used in the hydrolysis of a 5% lactose solution in a buffer solution at pH = 3.5 and at a temperature of 500C, has a half-inactivation period equal to 52 days.

 However, the use of an aromatic diamine, which is a blood poison, does not make it possible to have recourse to this process for the immobilization of enzymes intended for the food industry.

 We know a method of immobilizing ss-galactosidase produced by a mycelium of Aspergillus niger on hydrophobic carriers comprising a long aliphatic chain and phenyl radicals. The enzyme is attached to the phenyl radicals of the carrier by covalent attachment using glutaric aldehyde (French Patent Application 2,399,435).

 This process has not been widely applied because of the need to use a carrier modified by specific agents for the fixation of the enzyme, as well as to use surfactants for immobilization. hydrophilization of the surface of the carrier.

 A method of immobilizing the mycelial ss-galactosidase of Aspergillus niger is known on the porous aluminum oxide used as a carrier for 16-20 hours. In order to increase the quantity of adsorbed enzyme, either the enzyme or the surface of the carrier is treated with a triazine dye. The adsorbed enzyme is fixed with a glutaric aldehyde solution which is added to the enzyme solution before adsorption of the latter or 5 minutes after adsorption (USA, 3,873,425).

 The activity of ss-galactosidase obtained and immobilized in this way is equal to 7.6 units / g of product. The disadvantage of this process lies in the low activity of the immobilized product, the large expenditure of enzyme due to the fact that after the addition of the glutaraldehyde to the solution the enzyme forms conglomerates which cannot be used for their subsequent immobilization, as well as for the significant washing out of the adsorbed enzyme during its use for the hydrolysis of lactose in milk serum. Furthermore, improving adsorption requires the use of dyes, which is undesirable in the food industry.

 The problem posed in the present invention consists in achieving, by selection of the raw material and by modification of the parameters of the process, the immobilization of ss-galactosidase produced by a mycelium of fungus by obtaining a highly active and stable product during its use, and this in a shorter technological cycle and without the use of toxic substances.

 The method according to the invention for immobilizing ss-galactosidase produced by a fungus mycelium by adsorption of said ss-galactosidase from its solution on a mineral carrier with subsequent fixation of the ss-galactosidase adsorbed on the carrier by means of glutaric aldehyde immobilizes the ss-galactosidase produced by the Penicillium canescens F-178 strain and then fixes the ss-galactosidase adsorbed by a solution containing 0.5 to 2.5% glutaric aldehyde.

 Thanks to the present invention, it is possible to obtain an immobilized S-galactosidase product having an activity of 420-450 units / g of carrier (temperature 300C, pH = 4.0, lactose at 5.0%), of which the half-inactivation period is 136 days.

The claimed process makes it possible to simplify the immobilization process by eliminating the adsorption of a dye, and to reduce the duration of immobilization by 6 to 8 times compared to the process described in US patent no. 3,873 .426,
According to a variant of the invention, a granulated compound of formula Si02.n H20 where n is an integer is used as the mineral carrier, this compound having a specific surface of 70-100 m2 / g, with a grain size of 3155O0jim and pores of 40-6Onm.

 The use of the chosen siliceous carrier contributes to raising the activity of the product obtained and to strengthening its stability.

 To reduce by 20% the consumption of soluble ss-galactosidase and make possible the immobilization of the enzyme immediately after the stage of its production in culture, as well as to enhance the stability of the product (period of inactivation of the enzyme approximately 200 days), according to a variant of the invention, a solution containing 20-50 mg / ml of ss-galactosidase produced by a mycelium of fungus is used. Said ss-galactosidase is immobilized by adsorption several times on the same carrier until said ss-galactosidase is immobilized over the entire surface of the carrier, and the enzyme is fixed after each elemental adsorption with a solution of 1.0-2.5% glutaric aldehyde.

 The various aims and advantages of the present invention will be understood with the aid of the detailed description of the process for the immobilization of ss-galactosidase originating from a mycelium of fungus and of examples of implementation of this process.

 In the claimed process, it is proposed to immobilize the ss-galactosidase produced by the Penicillium canescens F-178 strain, the production technology of which is simple and accessible. The technology for producing said B-galactosidase comprises the stages of enzyme culture, ultra-concentration and drying with a spray dryer, unlike the technology for obtaining other ss-galactosidases in which implements, in addition to the stages mentioned, a purification on the carboxymethyl-cellulose, a separation by organic solvents, an affinity chromatography.

 The production of the ss-galactosidase of the Penicillium canescens F-178 strain is carried out by generally known methods, comprising the culture of the microscopic champion, the ultra-concentration of the culture liquid, the drying of the active enzyme product - the ss-galactosidase of the Penicillium canescens F-178 strain with a spray dryer, the subsequent dissolution of the product obtained in a buffer solution containing 0.2 M acetate of pH = 4.0 +0.1 until a sufficient concentration for immobilization of the enzyme, this concentration generally being greater than 130 mg / ml.

 The claimed process can be carried out on any known granulated mineral carrier, for example, on silica gels which have a specific surface of 28 m2 / g and a pore size of 75 nm, macroporous silicas (aerosils) of a specific surface of 26 to 120 m2 / g of carrier. The most advantageous is to use as a carrier a granulated porous body of formula SiO2.nH20, where n is an integer, which has a specific surface of 70 to 100 m2 / g, a grain size of 315 to 500 Zm and a pore size from 40 to 60 nm.

 The immobilization process is carried out according to the claimed invention, by adsorption of the B-galactosidase from the Penicillium canescens F-178 strain on a mineral carrier granulated at room temperature and over a sufficient time so that in the solution an equilibrium mass concentration is established of less than 4-6% of the initial concentration. The immobilization of said ss-galactosidase is then obtained over the entire surface of the carrier.

 The adsorbed ss-galactosidase is fixed on the carrier by means of a 0.5-2.5% solution of glutaric aldehyde, which stabilizes the enzyme on the carrier and allows the finished product to be used at temperatures up to 'at 40-600C.

 For concentrations of glutaraldehyde less than 0.5% in the fixing solution, it is possible to observe a washing of the enzyme of the carrier during its use for the hydrolysis of milk serum; for concentrations of glutaraldehyde greater than 2.5 μ, there is a reduction in the activity of the immobilized products of said ss-galactosidase due to the large quantity of chemical bonds which block the zones of activity of the enzyme.

 The claimed invention provides for the use of ss-galactosidase of the Penicillium canescens F-178 strain at a concentration of 20-50 mg / ml. Such a concentration is obtained after the stage of culture of the microscopic fungus during the separation of said 13-galactosidase. The exclusion of the ultraconcentration stages of the culture and drying liquid of the enzymatic product of said B-galactosidase makes it possible to simplify the immobilization process as a whole and to reduce the consumption of enzyme by 20%.

If we use the strain ss-galactosidase
Penicillium canescens F-178 with a concentration of 20-50 mg / ml to obtain highly active immobilized products, immobilization is carried out by several treatments of the carrier with alternating solutions of ss-galactosidase with a concentration of 20- 50 mg / ml and 1.0-2.5% glutaric aldehyde. Each of the solutions used successively, both of the enzyme and of the glutaraldehyde, must correspond to the concentration initially indicated. The number of treatments of the carrier with the enzyme and glutaraldehyde solutions is generally between 3 and 8, which ensures the immobilization of the enzyme over the entire surface of the carrier.

 If a concentration of glutaraldehyde less than 1% is used, the enzyme is washed out from the surface of the carrier during its use for the hydrolysis of lactose in milk serum; a concentration of glutaraldehyde greater than 2.5% leads to a reduction in the activity of the immobilized products of ss-galactosidase as a result of the formation of a large quantity of bonds which block the active centers of the enzyme.

 By bringing said ss-galactosidase immobilized several times at a temperature of 50-600C in an acetate buffer solution for 60 minutes the conservation of the activity of the immobilized product is 50% greater than that of a product obtained by immobilization in a single recovery of the ss-galactosidase contained in a high concentration solution.

 Example 1.

 Add to 0.8 g of ss-galactosidase enzyme product of the Penicillium canescens F-178 strain having an activity of 630 unit / g of product, dissolved in 5 ml of 0.2M acetate buffer solution and pH = 4 0.1 l / g of granulated siliceous carrier. Immobilize for 3 hours with stirring, at a temperature of 240C. Then add to the suspension of immobilized product 1 ml of 25% glutaric aldehyde solution. Carry out the treatment with glutaraldehyde for 1.5 hours. Wash the weakly adsorbed enzyme with a 2M NaCl solution, a 0.2M acetate buffer solution and with water.

The activity of immobilized ss-galactosidase is equal to 280 units / g of carrier.

 Example 2.

 0.8 g of ss-galactosidase enzyme product of the Penicillium canescens F-178 strain with an activity of 630 units / g of product, dissolved in 5 ml of 0.2M acetate buffer solution of pH L 4, 0, add 1 g of microporous granulated silica (specific surface of 25 to 120 m2 / g). Carry out the immobilization as in Example 1. Carry out the fixation of the immobilized product with 5 ml of 2.5% solution of glutaric aldehyde in a buffer solution 0.1 μM of phosphate of pH = 7.0.

The activity of the immobilized enzyme is 300 units / g of carrier.

 Example 3.

 With 1.2 g of ss-galactosidase enzyme product of the Penicillium canescens F-178 strain with an activity of 3000 units / g of the product, dissolved in 30 ml of 0.2M acetate buffer solution at pH = 4, 0, add 6 g of granulated siliceous carrier (SiO2.nH20, specific surface 70-100 m2 / g). Immobilize for 3 hours.

 Fix the immobilized product with glutaraldehyde for 1.5 hours, the concentration of glutaraldehyde in this case being 2%.

The activity of the immobilized product is 450 units / g of the carrier.

Example 4
To 60 g of ss-galactosidase enzyme product of the Penicillium canescens F-178 strain with an activity of 630 units / g of the product dissolved in 350 ml of 0.2M acetate buffer solution at pH = 4.0, add 100 g of granulated siliceous carrier (SiO2nH20 specific surface 70-100 2 / g), whose grain size is 315-500 and that of the pores at 40-60nm.

Immobilize for 2 hours at a temperature of 100C. Fix the immobilized product with 500 ml of 1% glutaraldehyde solution in a 0.1M phosphate buffer solution at pH = 7.0 for 1 hour. The activity of the product obtained is 220 units / g of the carrier.

 Place in a thermostated column 10 mm in diameter 1.7 g of wet ss-galactosidase product of the Penicillium canescens F-178 strain with an activity of 220 units / g of the carrier. Bring deproteinized and demineralized milk serum into the column with a 5% lactose content at a rate of 25 ml / h at a temperature of 50 μm.

Collect the lactose hydrolyzate at the outlet of the column and determine the glucose level, from which the degree of lactose hydrolysis is calculated. The degree of hydrolysis of lactose reaches 95-100%. Under conditions of uninterrupted operation, without cleaning or disinfection, for 36 hours the degree of hydrolysis of lactose does not change. The half-inactivation period of the immobilized ss-galactosidase of the strain
Penicillium canescens F-178, calculated using experimental data, is equal to 136 days.

 Example 5.

Load a column 10 mm in diameter with 0.3784 g of granulated siliceous carrier, identical to that of Example 4. Pass through the column in 3 hours 15 ml of ss-galactosidase solution of the strain
Penicillium canescens F-178 with a total activity of 1536 units. Treat the adsorbed enzyme for 1.5 hours with a solution of 1% glutaraldehyde in a phosphate buffer solution. The activity of the immobilized enzyme is equal to 320 units / g of carrier.

 Example 6.

Load a column 10 mm in diameter with 0.3626 g of granulated siliceous carrier (identical to that of Example 4). In 2 hours pass 15 ml of strain ss-galactosidase solution through the column
Penicillium canescens F-178 having a total activity of 1536 units. Then pass a 2% solution of glutaric aldehyde in a phosphate buffer solution through the column. The activity of the immobilized enzyme is 300 units / g.

 Example 7.

 To 5 ml of ss-galactosidase solution of the Penicillium canescens F-178 strain with a concentration of 20 mg / ml and a total activity of 90 units, add 1 g of mineral granular carrier, identical to that of example 4. Carry out the immobilization in 1.5 hours, mixing by oscillation. Then add the glutaraldehyde to a final concentration of 2.5% and mix again for 1 hour. Drain the solution and wash the immobilized enzyme with 80 ml of water, then pour the next portion (5 ml) of solution of said enzyme with a concentration of 20 mg / ml. Carry out adsorption and fixing as indicated above. Repeat the immobilization (adsorption and fixation) from 1 to 8 times.

The activity of ss-galactosidase immobilized after each immobilization is shown in Table 1.

 Example 8.

 To 5 ml of solution of B-galactosidase of the Penicillium canescens F-178 strain with a concentration of 30 mg / ml and a total activity of 135 units add 1 g of mineral granular carrier, identical to that of Example 4 Immobilize as described in Example 7. The activity of the immobilized ss-galactosidase products is shown in Table 1.

 Example 9.

 To 5 ml of B-galactosidase solution of the Penicillium canescens F-178 strain with a concentration of 40 mg / ml and total activity of 180 units, add 1 g of mineral granular carrier, identical to that of Example 7 The activity of the immobilized ss-galactosidase product is shown in Table 1.

 Example 10.

 To 5 ml of ss-galactosidase solution of the Penicillium canescens F-178 strain with a concentration of 50 mg / ml and total activity of 225 units, add 1 g of mineral granular carrier, identical to that of Example 4 Carry out the immobilization as in Example 7.

The activity of the ss-galactosidase products is shown in Table 1.

 Example 11. (Reference).

To 340 g of dry granulated mineral carrier add 1000 ml of ss-galactosidase solution of the strain
Penicillium canescens F-178 with a total activity of 270 units. Immobilize for 3 hours.

Then add 100 ml of 25% glutaric aldehyde solution to the reaction mixture. Fix for 1 hour. The activity of the immobilized product is 350 units / g, for a degree of fixation of the activity of 44%.

TABLE N 1
Activity of the products of the strain Agalactosidase
Penicillium canescens F-178, obtained by immobilization
multiple (examples 7,8,9,10) NO Number Units / g of Units / g Rate of ani- of product of fixation exem- bilisa- departure of duit obtained- according to ples nu activity
1 2 3 4 5
1 1. 90 - 90 100
2 2. 90 (180) 148 82
3 3. 90 (270) 187 73
4 4. 90 (360) 229 64
5 5. 90 (450) 256 57
6 6. 90 (540) 343 64
7 7. 90 (630) 350 56
8 8. 90 (720) 370 52
9 1. 135 - 132 98 10 2. 135 (270) 213 79 11 3. 135 (405) 257 63 12 4. 135 (540) 322 60 13 5. 135 (675) 360 53 14 1. 180 - 134 73 15 2. 180 (360) 212 59 16 3. 180 (540) 258 48 17 4. 180 (720) 350 49 18 1. 225 - 160 73 19 2. 225 (450) 266 59 20 3. 225 (675) 340 51
Example 12.

 To 5 ml of ss-galactosidase solution of the Penicillium canescens F-178 strain with a concentration of 130 mg / ml and a total activity of 600 units, add 1 g of granulated mineral carrier, identical to that of example 4. Carry out the adsorption for 3 hours.

Then add 0.5 ml of glutaraldehyde solution to the reaction mixture and fix for 1 hour. The activity of the immobilized product is 290 units / g of product for a degree of fixation, according to the activity, of 48%.

 Example 13.

 To 5 ml of ss-galactosidase solution of the Penicillium canescens F-178 strain with a concentration of 20 mg / ml and a total activity of 90 units, add 1 g of granulated mineral carrier, identical to that of example 4. Carry out immobilization for 1.5 hours. Then add 0.5 ml of 2556 glutaraldehyde solution to the mixture. Fix the immobilized product on the carrier as described in Example 7. The activity of the immobilized product is 350 units of product for a degree of fixation, according to the activity, of 56-60%.

 Example 14.

 Carry out the adsorption of the ss-galactosidase solution of the Penicillium canescens F-178 strain in the same way as in Example 13. Add 0.15 ml of 25% glutaraldehyde to the mixture. Fix the immobilized product on the carrier as in Example 7. The activity of the immobilized product is 360 units / g of product. After prolonged retention in a mixture of a 30% solution of isopropanol in a 2M NaCl solution, the betagalactosidase goes into solution and the solution shows ss-galactosidasic activity. In Examples 7 to 13, 13-galactosidasic activity is not observed.

 Example 15.

 Carry out the adsorption of ss-galactosidase on the granulated mineral carrier, identical to that of Example 4, in the same way as in Example 13. Then add to the mixture 1 ml of 25% glutaraldehyde solution. Fix the immobilized product on the carrier as in Example 7. The activity of the immobilized product is 280 units / g of product.

 Example 16.

 To 5 ml of ss-galactosidase solution of the Penicillium canescens F-178 strain of concentration 160 mg / ml and total activity 2400 units, add 1 g of granulated mineral carrier, identical to that of Example 4. Carry out the adsorption for 3 hours. Then add 0.5 ml of 25% glutaric aldehyde solution to the reaction mixture. Fix with glutaraldehyde on the carrier for 1 hour.

The activity of the immobilized product is 840 units / g of product, for a degree of conservation of the activity of 35%.

Example 17
To 5 ml of B-galactosidase solution of the Penicillium canescens F-178 strain with a concentration of 20 mg / ml and total activity of 300 units, add 1 g of granulated mineral carrier, identical to that of Example 4, Carry out the immobilization in the same way as in Example 7. After immobilization 6 times the activity of the product reaches 970 units / g of product, for a degree of conservation of the activity of 54%.

 Example 18.

 Load a 1.2 kg container of dry granulated mineral carrier, identical to that of Example 4 and add 6 liters of 13-galactosidase solution using a shower device. The concentration of the solution is 20 mg / ml and the total activity 10.8-104 units. Immobilize by circulating reagents. Carry out the adsorption for 30 minutes, then add 360 ml of glutaric aldehyde solution to the enzyme solution and fix for 45 minutes.

Repeat these operations 6 times. Wash in a closed circuit, as described in Example 7. The activity of the immobilized product is 360 units / g of product, for a degree of conservation of the activity of 47%.

 Example 19.

To 8 g of immobilized product of ss-galactosidase of the Penicillium canescens F-178 strain, obtained by single immobilization as in Example 11, add 80 ml of 0.2M acetate buffer solution, pH = 4.0, to the temperature of 600C. Every 10 minutes for one hour take samples and establish the activity according to a standard method. From the data as well
60 obtained calculate the inactivation constant K which is equal to 26 10 ~ 3 / min. in
Example 20.

 To 8 g of immobilized product of ss-galactosidase, obtained by immobilization several times, as in Example 7, add 80 ml of 0.2M acetate buffer solution, pH = 4.0, at the temperature of 60 ' vs. Every 10 minutes for one hour take samples and establish the activity according to a standard method. C-altered inactivation constant.

 K600 = 16.61O-3 / min.

Claims (3)

 1. Method for immobilizing ss-galactosidase produced by a mycelium of fungus by adsorption from its solution on a mineral carrier with subsequent fixation of the ss-galactosidase adsorbed on the carrier by means of glutaric aldehyde, characterized in that the immobilization of the B-galactosidase produced by the Penicillium canescens F-178 strain is carried out and in that the B-galactosidase adsorbed on the carrier is fixed with a 0.5-2 solution, 5% glutaraldehyde.  2. Method according to claim 1, characterized in that the mineral carrier is a porous granular compound of formula SiO2.nH20, where n is an integer, which has a specific surface of 70 to 100 m2 / g, a grain size from 315 to 500 µm and a pore size from 40 to 60 mm.  3. Method according to one of claims 1 and 2, characterized in that, when a solution of said ss-galactosidase is used at a concentration of 20 to 50 mg / ml, the immobilization of the B- is carried out galactosidase by its adsorption several times from solutions of the aforementioned concentration on the same carrier until the immobilization of said B-galactosidase over the entire surface of the carrier, by carrying out after each elementary adsorption the fixation of ss-galactosidase adsorbed with a 1.0-2.5% glutaric aldehyde solution.