BRPI1004880A2 - Process for Obtaining 6-Aminopenicylic Acid (6-apa) - Google Patents

Abstract

PROCESS FOR OBTAINING 6-AMINOPENICILLANIC ACID (6-APA). A process for obtaining 6-aminopenicillanic acid (6-APA) by the simultaneous production and hydrolysis of penicillin G during the cultivation of Penicillium chrysogenum using penicillin G acylase immobilized on a biocatalyst agarose sphere containing magnetic particles is described. In the presence of the biocatalyst penicillin G is hydrolyzed, releasing the product of interest, 6-aminopenicitanic acid (6-APA) and phenyl acetic acid (AFA) which is recycled to the start of the process. The biocatalyst is recovered and reused several times.

Description

PROCESS FOR OBTAINING 6- AMINOPENICILLANIC ACID (6-APA) FIELD OF THE INVENTION

The present invention belongs to the field of processes for obtaining 6-aminopenicillanic acid (6-APA), more specifically to a process for obtaining 6-APA from the simultaneous production and hydrolysis of penicillin G during the cultivation of Penicillium chrysogenum using penicillin. G acylase immobilized on agarose sphere containing magnetic particles. BACKGROUND OF THE INVENTION

The production process of 6-aminopenicillanic acid (6-APA) has been long investigated and currently involves several steps. The first step for obtaining 6-APA is the production of natural penicillin by the fungus Penicillium chrysogenum. Penicillin is then extracted from the fermentation broth using butyl acetate or amyl acetate at low pH values (<2.5). To obtain the crystallized form as potassium salt, penicillin is extracted again to the aqueous phase at pH 7.0-8.0.

Potassic penicillin G (PG) is dissolved in aqueous medium and subjected to enzymatic hydrolysis releasing 6-APA and phenylacetic acid (AFA). AFA is extracted with organic solvent, while 6-APA is crystallized in aqueous medium by adjusting the pH at the isoelectric point (pH 3.6), where the solubility of this compound is minimal, according to the article cited by den Hollander, JL, Zomerdijk. , M., Straathof, AJJ, van der Wielen, LAM Continuous enzymatic penicillin G hydrolysis in countercurrent water-butyl acetate biphasic systems. Chemical Engineering Science, v. 57 (9), p. 1591 - 1598, 2002; Ferreira, J.S., Straathof, A.J.J., Li, X., Ottens, M., Franco, T.T., van der Wielen, L.A.M. Solution crystallization kinetics of 6-aminopenicillanic acid. Industrial & Engineering Chemistry Research, v. 45 (20), p. 6740-6744, 2006.

The method for obtaining 6-APA described above has been used successfully for many years due to its efficiency and low cost of the final product. However, the use of organic solvents makes the process environmentally unfavorable.

Many efforts have already been made to reduce, replace or eliminate the use of organic solvents from the 6-APA production process. However, most of the papers presented seek partial solutions to simplify the process. For example, modifications are suggested for penicillin extraction steps only or for 6-APA extraction only. In addition, developing a new method that does not interfere with the overall yield of 6-APA and the final cost of this product requires great effort.

Some examples of partial modifications to the 6-APA production process include: reactive penicillin extraction using high molecular weight amines, use of hydrolysis systems and simultaneous aqueous extraction, penicillin extraction in the presence of electric field, penicillin extraction in three liquid phases, 6-APA adsorption on Amberlite LA-2, trioctylamine, Aliquat-336, XAD16, XAD1180, XAD1600, anionic resin IRA 400 and secondary amines with molecular weight between 325 and 395. See the articles by Arnott, IA, Weatherley, LR The stability of penicillin G during recovery by electrically enhanced extraction. Process Biochemistry, v. 30 (5), p. 447-455, 1995; Hano, T .; Ohtake, T., Matsumoto, M., Ogawa, S., Hori, F. Extraction of penicillin with Iiquid surfactant membrane. Journal of Chemical Engineering of Japan, v. 23 (6), p. 772-775, 1990. van der Does, T., Kuipers, R.H, Kwant, G.J, Kerkhof, P.T, Kerkhof, J.H.P.M. Recovery of 6-amino-penicillanic acid from a mother liquor, useful as an intermediate in the production of beta-lactam antibiotics.

See also publications EP950660-A; WO9948895-A; WO9948895-A1; EP950660-A1; AU9930361-A and US2005020685-A.

Some modifications to the 6-APA production process are found in the patent literature, for example:

U.S. Patent 6,110,699 proposes to replace the organic solvent extraction steps, which isolate penicillin as a salt, by ultrafiltration steps of the fermentation broth. Enzymatic hydrolysis of penicillin is performed in the ultrafiltrate fermentative broth and the product (6-APA) is extracted by anion exchange column chromatography. The patent found differs from the present invention in the following point: Penicillin hydrolysis is performed only at the end of cultivation after filtration and ultrafiltration steps, while in the proposed invention hydrolysis is performed during P. chrysogenum cultivation.

In British publication GB892144 (A) penicillin amidase (penicillin hydrolyzing enzyme) is produced and the enzyme-containing fermentation broth is pretreated using different procedures (to separate biomass) and then used as the reaction medium for the hydrolysis of penicillin, ie the antibiotic in powder form is added to the fermentative broth containing the enzyme that will catalyze its hydrolysis, generating 6-APA and AFA. In this publication 6-APA is produced by the addition of penicillin to a complex medium, however this medium is used for the production of penicillin acylase (enzyme which hydrolyzes penicillin G). In the proposed process penicillin is hydrolyzed in a complex medium, however this medium is the fermentative broth of P. chrysogenum for antibiotic production.

In British publication GB1009028 (A) (1965) a solution of penicillin (potassium salt) or the penicillin contained in the fermentation broth is hydrolyzed by a preparation of penicillin amidase (fermentative broth where the enzyme produced after treatment eg filtration or bleaching). ) produced by Achromobacter sp. In this document 6-APA is produced by penicillin hydrolysis using preparation of penicillin amidase produced by Achromobacter sp. In the proposed process penicillin is hydrolyzed in a complex medium, however this medium is the fermentative broth of P. chrysogenum for antibiotic production.

U.S. Patent 3,260,653 describes the hydrolysis of penicillin by enzymatic extracts of E. coli fermentative broths. There are also comments on the use of penicillin acylase released by P. chrysogenum Q 176 for PG hydrolysis in the same medium in which it is produced. In this patent 6-APA is produced by penicillin hydrolysis using E.coli-produced penicillin amidase preparation. In the proposed process penicillin is hydrolyzed in a complex medium, however this medium is the fermentative broth of P. chrysogenum for antibiotic production.

Chinese publication CN101250247 describes a method of preparing magnetic microspheres for immobilizing penicillin acylase. This publication utilizes vinyl compounds as a functional monomer for polymer matrix preparation and produces microspheres, while the proposed invention utilizes agarose and produces 4 mm diameter spheres. Therefore, there is no similarity between this publication and the present invention.

Chinese publication CN1209454 describes the preparation of microbeads for immobilization of penicillin acylase. This publication uses agar gelatin as a polymeric matrix, while the proposed invention utilizes agarose and produces 4 mm diameter magnetic beads. Therefore, there is no similarity between this publication and the present invention.

Spanish Patent ES2005883 describes method of immobilizing penicillin acylase using small agarose particles. In this procedure the amino groups of the enzyme are covalently bonded to aldehyde groups on the agarose surface. The present invention makes use of this methodology, however additional steps have been included by dispersing the immobilized PGA-containing agarose particle and magnetic particles in an agarose solution (powder) at 60 ° C, then this solution is dripped into Vaseline to form spheres. 4 mm in diameter. In addition, the present invention includes the application of such immobilized PGA-containing magnetic beads for penicillin G hydrolysis simultaneous to their production by P. chrysogenum. Therefore, the present invention differs in many respects from the invention described herein.

Korean Patent KR860000698 describes a method of immobilizing "cells" which contain penicillin acylase using as a polymeric matrix carrageenan, cellulose acetate, agar and others. The proposed invention describes the immobilization of the "enzyme" using agarose. Penicillin hydrolysis in the proposed invention is performed concurrently with the production of the antibiotic, that is, within the fermentation oven (bioreactor), whereas in the Korean publication such hydrolysis is performed on a column containing the immobilized enzyme. Therefore, the Korean publication does not anticipate the concept of the present invention.

Polish patent PL274563 describes the production of penicillin acylase by a mutant Escherichia coli microorganism, while the proposed invention describes the method of immobilization of this enzyme and its application in penicillin hydrolysis in complex medium (Penicillium chrysogenum fermentation broth). GB2244711 (B0) describes the process of penicillin hydrolysis (in aqueous solution) using a fluidized reactor, while the proposed invention describes the process of penicillin hydrolysis in a stirred tank bioreactor during the cultivation of Penicillium chrysogenum. Other differences are the immobilization technique. In the document found the "cells" are immobilized by gel wrapping, while in the proposed invention the "enzyme" is immobilized by covalent bonding with the polymer matrix (400 μΐη agarose gel particles) and subsequently this is enveloped. derivative and magnetic particles in a 4 mm diameter sphere prepared from agarose powder. Therefore, this British patent does not anticipate the present invention.

U.S. Patent 5,679,539 describes chemical modification procedures for polyethylene and polypropylene for subsequent application as a support for various applications, including enzyme immobilization. The present invention uses the agarose polymer to immobilize the enzyme of interest: penicillin G acylase. Therefore, there is no similarity between the invention and this US patent.

Spanish Patent ES2005883 describes the method of immobilizing / stabilizing penicillin acylase using small agarose particles. In this method, covalent bonding occurs between enzyme amino groups and polymer aldehyde groups. Although the present invention makes use of this methodology, additional steps have been included to prepare 4 mm diameter spheres containing dispersed magnetic particles. Furthermore, the present application includes the application of such immobilized PGA-containing magnetic beads for the hydrolysis of penicillin G simultaneous to their production by P. chrysogenum. Therefore, the proposed invention differs in many respects from the Spanish publication. GB 1292097 describes the ability of penicillin acylase in optical resolution of racemic amino acid mixing. The present invention describes the method of immobilization of this enzyme and its application in the hydrolysis of penicillin in complex medium (Penicillium chrysogenum fermentation broth).

EP1088887 A1 discloses the method of immobilizing crosslinked proteins generating crosslinked enzyme aggregates (CLEAS). The present invention immobilizes the "enzyme" by covalently bonding with the polymer matrix (400 μηι agarose gel particles) and subsequently surrounds this derivative and magnetic particles in a 4 mm diameter sphere prepared from the agarose powder .

GB 1463513 deals with a method of crosslinking immobilization between the enzyme and a water immiscible solvent. The process of the present application immobilizes the "enzyme" by covalently bonding with the polymer matrix (400 μηι agarose gel particles) and subsequently surrounds this derivative and magnetic particles in a 4 mm diameter sphere prepared from the powder. of agarose.

U.S.6,280,983 B1 deals with immobilization of lipase by hybrid gel wrapping containing 30 to 50% gelatin and one of the following polymers: agarose, agar, pectin, sodium alginate or carrageenan. The present invention deals with a process where the "enzyme" is immobilized by covalent bonding with the polymeric matrix (400 μητι agarose gel particles) and subsequently surrounds this derivative and magnetic particles in a prepared 4 mm diameter sphere from the agarose powder.

The publications presented above describe the use of free penicillin acylase in P. chrysogenum fermentation broth, chemical methods, addition of penicillin in fermentation broths where penicillin acylase is produced, and enzyme immobilized in aqueous medium to produce 6-APA. These methods differ substantially from the concept of the present invention.

Thus, the technique still needs a 6-APA production process that perfects the process currently used in industry and still represents a technical advance over the different processes described in the literature.

Most of the 6-APA production processes described in the literature propose a partial modification of the process currently used by industry. For example, in some proposed processes only the penicillin extraction step is modified, in other processes only the penicillin hydrolysis step is modified (aqueous medium, penicillin acylase fermentation broth), still others only modify the preparation of the enzyme. be used (free enzyme, enzyme immobilized on different media). These partial modifications do not allow to reduce the number of steps of the production process by 6- APA.

The simultaneous production and hydrolysis of penicillin proposed in this process reduces the number of steps in the 6-APA production process currently used. In addition, the recirculation of AFA during P. chrysogenum cultivation reduces the cost of it. The development of a biocatalyst capable of catalyzing penicillin hydrolysis in the complex medium and conserving its activity, even after sterilization steps, associated with the apparatus developed to maintain the biocatalyst's physical integrity makes the present process interesting for the production of 6-APA without the use of organic solvents. SUMMARY OF THE INVENTION

Broadly, the process of producing 6-aminopenicillanic acid according to the invention comprises the steps of:

(a) providing biocatalyst with penicillin G acylase (PGA) immobilized on an agarose sphere containing magnetic particles;

(b) In a fermentation vessel containing Penicillium chrysogenum fungus cultivation and production medium, add between 0 and 12 hours phenyl acetic acid in the concentration range 0.1 to 1.0% (w / v) to initiate the process. penicillin G production;

(c) under fermentation conditions including stirring at 300-400 rpm, temperature at 28 to 23 ° C, oxygen saturation at 0 to 20% due to air flow at 1 to 4 L / min, reaction time at 12 to 240 hours and pH near neutrality, ferment the reaction broth to produce penicillin G;

d) add to the culture medium of c) the biocatalyst of step a) in a proportion of 2-3 g per liter of fermentation broth, between 12 and 36 hours, to hydrolyze the penicillin produced, being released the product of interest, 6- APA and the phenylacetic acid (AFA) co-product, the latter consumed by the fungus P. chrysogenum during the process;

e) separate the 6-APA;

f) separate the remaining AFA at the end of cultivation and recycle it to the fermentation vessel to start a new 6-APA production cycle;

g) recover and recycle the biocatalyst for step d).

The process may be conducted in batch mode or continuously.

The process can be conducted in agitated tank type reactors or alternatively in airlift type pneumatic reactors. Therefore, the present invention provides a process for obtaining 6-APA wherein AFA is recycled to be consumed by the P. chrysogenum fungus during the process-producing penicillin G1 production process.

The present invention also provides a process of obtaining 6-APA free from the use of organic solvent, with great environmental advantage.

The invention further provides a process for obtaining 6-APA which

uses an immobilized agarose ball biocatalyst containing

Magnetic particles that can be reused a large number of times.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying FIGURE 1 is a block diagram summarizing the process of the invention with recirculation of the AFA co-product during P. chrysogenum cultivation. DETAILED DESCRIPTION OF THE INVENTION

The present invention therefore comprises a process of producing 6-aminopenicillanic acid (6-APA) with simultaneous hydrolysis of penicillin G during cultivation of Penicillium chrysogenum using penicillin G acylase immobilized on magnetic agarose sphere containing magnetic particles.

To perform the present process, a biocatalyst was developed, it was applied to P. chrysogenum cultures in bioreactor and the biocatalyst performance on penicillin hydrolysis was analyzed.

The preparation of this biocatalyst is carried out in two steps described below. The first step consists of multipunctional covalent immobilization of penicillin G acylase (PGA) in agarose particles 400 μπ in diameter.

A standard procedure for immobilizing PGA on 400 μηι diameter agarose particles is used. One gram of glyoxyl agarose is added to 9 mL of an enzyme solution prepared in 100 mM carbonate buffer, pH 10, containing 100mM phenylacetic acid and 25% v / v glycerin. The suspension is kept under stirring until supernatant activity is zero. At the end of immobilization, sodium borohydride is added at a concentration of 1 mg / ml suspension to reduce the Schiff bases formed between enzyme and support and also to convert the remaining aldehydes to inert hydroxyls. The derivative is then washed first with 25 mM phosphate buffer, pH 5 and then with water and stored at 4 ° C.

The second stage consists of dispersion of 400 μπι diameter particles (containing immobilized PGA) into 4 mm diameter spheres.

To coat the small particles (400 μηπ), initially, an aqueous solution (10% w / v) of agarose powder is prepared, which is dissolved by heating the mixture to 95 ° C. Then 4 mL of this solution is cooled to 60 ° C and 1 g of particles containing immobilized PGA and 0.5 g of stainless steel wire are added. To form the 4 mm diameter spheres this suspension is dripped in petroleum jelly. The formed magnetic beads are washed with abundant distilled water and stirred under surfactant-containing solution (Tween, but not limited to) to completely remove the petroleum jelly, then washed again with abundant water and stored at 4 ° C. The immobilized enzyme is stained (blue) with Bradford reagent to confirm its dispersion throughout the volume of the bead. The application of biocatalyst in penicillin G hydrolysis during its production by P. chrysogenum in bioreactor is performed as follows: Biocatalyst sterilization

Before being added to the fermentation broth of P. chrysogenum the biocatalyst is subjected to a sterilization treatment. The established methodology for sterilizing the biocatalyst uses 50 μΜ iodine solution (to 1 g of catalyst 10 ml of iodine solution is added). The mixture is kept under mechanical agitation for minutes and then the biocatalyst is washed with sterile water in aseptic chamber. The sterile biocatalyst is added to an Erlenmeyer containing 100 mL of the culture medium described in Table 1 below and kept under stirring at 250 rpm for 24 h at 25 ° C. Then a plaque test for the medium containing the biocatalyst is performed to confirm its sterility. Once the absence of contaminants is verified, the biocatalyst is transferred to the bioreactor, which contains P. chrysogenum after a period between 12 and 36h of cultivation. Suitable cultivation times range from 12 hours to 240 hours. P. chrysogenum cultivation

The first step for the cultivation of Penicillium chrysogenum is the preparation of the medium for germination. The composition of this medium is shown in Table 1 below. TABLE 1

Components Concentration maceration water 2.0-5.5% v / v ammonium sulfate 0.1-1.0% w / v monobasic sodium phosphate 0.1-1.0% w / v calcium hydroxide 0.1-0.5% w / v calcium carbonate 0.0-0.4% w / v defoamer 0.1-1.0% w / v sucrose 1.0-5.0% w / vq water 100ml

The pH of the germination medium is adjusted to 7.0 using ammonium hydroxide and then transferred to the 500 mL Erlenmeyer and autoclaved for 15 minutes. After sterilization, the medium is cooled and 10 ml of Penicillium chrysogenum containing saline solution from spore scraping in a slanted tube containing growth and maintenance medium (agar, malt extract and peptone), which was stored at -50 ° C.

After 24 h in shaker at 250 rpm and 25 ° C, 100 mL of the inoculum is fully transferred to the bioreactor containing approximately 1.6 L of production medium (identical to that described in Table 1 above).

A scheme of the system used to perform P. chrysogenum cultivation is as described by Nucci, E.R., Souza, V.R., Silva, R.G., Reis, G..B., Giordano, R.L.C, Giordano, R.C., Cruz, A.J.G. Online Monitoring of Penicillin G Acylase (PGA) Production Using a Fuzzy Logic Algorithm. Chemical Product and Process Modeling, v. 4 (4), article 12. This system includes a bioreactor with impellers to facilitate the homogenization of the fermentation broth.

To avoid fragmentation of the biocatalyst spheres by the action of impellers contained within the P. chrysogenum culture apparatus. (bioreactor) a helicoid is used to prevent contact between these spheres and the impellers. The helicoid is attached to the cover of the cultivation bioreactor and constructed with stainless steel wire. In this system, the spheres containing the enzyme move only in the volume outside the helicoid, avoiding the loss of the biocatalyst by fragmentation.

The bioreactor used in the process of the invention for the manufacture of 6-APA is manufactured by Applikon Dependable Instruments BV (Netherlands) with a capacity of 2 L (not limited to this manufacturer or capacity) and coupled to a system of acquisition of data (National Instruments®) so that online variables are stored every second (pH, temperature, dissolved oxygen, stir rate, molar fraction of carbon dioxide in bioreactor exhaust gas).

In terms of reaction conditions of the fermentation broth in the bioreactor, the temperature is maintained in the range of 28-23 ° C with a typical value of 25 ° C and neutral or near neutral pH (7).

Air flow ranges from 1.0 to 4.0 L / min.

The culture medium is agitated at 300-400 rpm for this system. Dissolved oxygen (DO) is maintained between 0 and 20% saturation.

To the beginning of the reaction cycle, amount of AFA is added. Recirculation occurs during cultivation: phenyl acetic acid (AFA) is consumed by the fungus during the production of penicillin, which is excreted into the reaction medium. The biocatalyst hydrolyzes the penicillin produced by the fungus releasing 6-aminopenicillanic acid (6-APA) and AFA into the reaction medium.

The cycle proceeds with the consumption of AFA generated during the hydrolysis of penicillin by the fungus P. chrysogenum.

At the end of cultivation, the objective is to remove remaining AFA by hydrophobic adsorption and 6-APA by ionic interaction, according to state of the art technologies that are not the subject of the invention.

Figure 1 schematically illustrates the process of the invention. Biocatalyst performance analysis on penicillin hydrolysis in bioreactor

To evaluate the performance of the biocatalyst on penicillin hydrolysis during P. chrysogenum cultivation the substrate (penicillin) and product of interest (6-aminopenicillanic acid) concentration are monitored. 6-APA Quantification (PDAB Method)

6-APA is quantified by the p-dimethylaminobenzaldehyde (PDAB) method by adding 25μΙ_ aliquots of the fermentation broth to 2.5 mL of the PDAB solution. The absorbance obtained after 2.5 minutes of reaction is converted to 6-APA concentration using previously determined calibration curve. PG Quantification (Hydrolysis and PDAB)

During P. chrysogenum cultivation the PG is quantified by removing aliquots of 975 μΙ_ of fermentative broth and adding to these μΙ_ of free PGA solution (500 U / mL). This mixture is kept for minutes in a 37 ° C bath for total hydrolysis of PG. Considering the PG1 hydrolysis stoichiometry where each formed 6-APA molecule corresponds to a hydrolyzed PG molecule, the PDAB reagent is used to quantify 6-APA and the PG concentration is indirectly determined. During cultivation the PG is hrolled by the biocatalyst into the bioreactor, and to determine the concentration of PG remaining, samples at approximately 5 mL volumes are taken at specified times. Each sample is filtered to remove the fungus mass and a 25 μΙ_ aliquot of it is added to 2.5 mL of the PDAB solution to quantify 6-APA (from PG hydrolysis within the bioreactor). Then another aliquot of 975 μΙ_ of the same sample is subjected to complete free PGA hydrolysis and 25 μΐ_ of this is used to quantify 6-APA (from PG hydrolysis within the bioreactor + from free PG remaining hydrolysis) . The amount of total 6-APA (inside the bioreactor + produced by free PGA) minus what was inside the bioreactor (measured directly in PDAB) corresponds to the amount of PG remaining within the bioreactor.

The results obtained with the described process show that it is possible to obtain complete hydrolysis of penicillin during cultivation of P. chrysogenum, and furthermore that it is possible to recover the biocatalyst at the end of cultivation while maintaining enzymatic activity and sphere integrity.

From the foregoing, it is concluded that the present invention constitutes a technological development of interest to the pharmaceutical industry, as it has advantages such as: the reduction in the number of steps to obtain 6-APA, the elimination of the use of organic solvent in the process. and reduction in the overall process cost, mainly due to the recirculation of AFA in the production medium.

Claims (8)

A process for obtaining 6-aminopenicillanic acid (6-APA), characterized in that said process comprises the steps of: a) providing biocatalyst with penicillin G acylase (PGA) immobilized on an agarose sphere containing magnetic particles; (b) In a fermentation vessel containing Penicillium chrysogenum fungus cultivation and production medium, add between 0 and 12 hours phenyl acetic acid (AFA) in the concentration range from 0,1 to 1,0% (m / v) to start the penicillin G production process; (c) under fermentation conditions including stirring at 300-400 rpm, temperature at 28 to 23 ° C, oxygen saturation at 0 to 20% due to air flow at 1 to 4 L / min, reaction time at 12 to 240 hours and pH near neutrality, ferment in the oven of b) the reaction broth to produce penicillin G; d) add to the culture medium of c) the biocatalyst of step a) in a proportion of 2-3 g per liter of fermentation broth, between 12 and 36 hours, to hydrolyze the penicillin obtained, being released the product of interest, acid 6 -aminopenicillanic (6-APA) and the phenylacetic acid (AFA) co-product, the latter consumed by the fungus P. chrysogenum during the process; e) separating 6-aminopenicillanic acid (6-APA); f) separate the remaining AFA at the end of cultivation and recycle it to the fermentation vessel to start a new 6-APA production cycle; and g) recovering the biocatalyst and recycling it to step d. Process according to Claim 1, characterized in that said biocatalyst is prepared according to the following steps: a) covalently immobilizing the penicillin G acylase (PGA) on 400 μπι diameter agarose particles; b) Disperse 400 μηι diameter particles containing immobilized PGA into 4 mm diameter beads using aqueous (10% w / v) solution of dissolved agarose powder by heating to 95 ° C cool 4 mL of this solution to 60 ° C and add 1 g of particles containing immobilized PGA and 0.5 g of stainless steel wire obtaining a suspension and forming the 4 mm diameter beads by dripping said suspension into petroleum jelly obtaining magnetic beads washed with distilled water and stirred under solution containing surfactant to remove; the vaseline completely, then are washed again with water and stored at 4 ° C, and staining (blue) the enzyme immobilized with Bradford reagent to verify its dispersion throughout the volume of the sphere. Process according to Claims 1 and 2, characterized in that said biocatalyst is sterilized prior to use. Process according to Claim 1, characterized in that it is carried out continuously. Process according to Claim 1, characterized in that it is carried out in batch mode. Process according to Claim 1, characterized in that it is carried out in agitated and aerated tank reactors. Method according to Claim 1, characterized in that it is carried out in airlift-type pneumatic reactors. Process according to Claim 1, characterized in that the composition of the production medium contained in the fermentation vessel comprises, by mass, maceration water 2.0-5.5% v / v, ammonium sulphate 0.1-1.0%, monobasic sodium phosphate 0.1-1.0%, calcium hydroxide 0.1-0.5%, calcium carbonate 0.0-0.4%, defoamer 0 1-1.0%, sucrose 1.0-5.0% and water qsp 100ml.