WO2010103150A1 - Immobilised biocatalyst based on alginate for the biotransformation of carbohydrates - Google Patents

Immobilised biocatalyst based on alginate for the biotransformation of carbohydrates Download PDF

Info

Publication number
WO2010103150A1
WO2010103150A1 PCT/ES2010/070104 ES2010070104W WO2010103150A1 WO 2010103150 A1 WO2010103150 A1 WO 2010103150A1 ES 2010070104 W ES2010070104 W ES 2010070104W WO 2010103150 A1 WO2010103150 A1 WO 2010103150A1
Authority
WO
WIPO (PCT)
Prior art keywords
biocatalyst
reactor
immobilized
fructosyltransferase
enzyme
Prior art date
Application number
PCT/ES2010/070104
Other languages
Spanish (es)
French (fr)
Inventor
Lucía FERNÁNDEZ-ARROJO
Francisco José PLOU GASCA
Antonio Ballesteros Olmo
Miguel Alcalde Galeote
Patricia GUTIÉRREZ ALONSO
María FERNÁNDEZ LOBATO
Original Assignee
Consejo Superior De Investigaciones Científicas (Csic)
Universidad Autónoma De Madrid (Uam)
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Consejo Superior De Investigaciones Científicas (Csic), Universidad Autónoma De Madrid (Uam) filed Critical Consejo Superior De Investigaciones Científicas (Csic)
Publication of WO2010103150A1 publication Critical patent/WO2010103150A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2431—Beta-fructofuranosidase (3.2.1.26), i.e. invertase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01026—Beta-fructofuranosidase (3.2.1.26), i.e. invertase

Definitions

  • the present invention relates to biocatalysts comprising enzymes immobilized in alginate and their method of production.
  • the invention also refers to the use of said biocatalyst for biotransformation in which the substrate is a concentrated solution of a carbohydrate and can be carried out in a continuous reactor. Therefore, the present invention can be framed within the field of biotechnology industry.
  • Carbohydrates are the biological material very abundant in nature, they can be used as raw material for a wide variety of industrial processes, giving rise to other products with a lot of industrial and technological interest. For this, the enzymes involved in its transformation have an enormous interest.
  • prebiotic was introduced by Gibson and Roberfroid, who defined prebiotics as non-digestible food ingredients that beneficially affect the host by selective stimulation of the growth and / or activity of a limited group of bacteria in the colon.
  • prebiotics resist digestion in the upper part of the intestinal tract, and are metabolized by the endogenous bacteria of the colon.
  • the market-leading prebiotic molecules are fructooligosaccharides (FOS) (cf. PT. Sangeetha, MN Armes, SG Prapulla, Trends Food Sci. TechnoL 2005, vol. 16, pp. 442-457; AV Rao, J. Nutr. , 1998, vol. 80, pp. 1442S-1445S).
  • the FOS can exert a series of effects on the person who consumes them: control of constipation due to increased fecal mass, reduction of diarrhea episodes caused by rotavirus, improvement of symptoms of lactose intolerance, increase of the absorption of calcium, decrease in the mutagenic capacity of certain microbial enzymes such as nitro-reductase (associated with colon cancer), possible reduction of diseases related to dyslipidemia, etc.
  • the FOS currently marketed are made up of fructose molecules linked by ⁇ - (2-1) glycosidic bonds, with a terminal glucose molecule (cf. JW Yun, Enzvme Microb. TechnoL, 1996, vol. 19, pp. 107 -117), abbreviated as GF n , n being typically between 2 and 4 (1-kestose, nosy and 1 F-fructosylnistose).
  • GF n 1-kestose, nosy and 1 F-fructosylnistose
  • Recent studies show that the greatest prebiotic effect is exerted by the trisaccharide 1- kestose, in comparison with higher molecular weight compounds of the series (cf. O. Ba ⁇ uelos et al., Anaerobe. 2008, vol. 14, pp. 184- 189).
  • FOS are obtained by enzymatic synthesis from sucrose, using sucrose 1-fructosyltransferases (EC 2.4.1.9), beta-fructofuranosidases -invertases- (EC 3.2.1.26) or even levansacarasas (EC 2.4.1.10) (cf. LE Trujillo et al., Enzvme Microb. Technol. 2001, vol. 28, pp. 139-144). Using beta-fructofuranosidases the yield of synthesis products that is reached is very low, representing these less than 4% (w / w) of the total carbohydrates of the mixture.
  • FOS Fluorescence-Activated sugars
  • Aspergillus eg A. oryzae, A. ia pon i cus, A. niper, A. aculeatus, etc.
  • Aureobasidium eg A. pullulans
  • the large-scale production of prebiotic oligosaccharides synthesized by enzymatic route can be favored if the enzyme is immobilized on a solid support.
  • This process facilitates the separation of the biocatalyst from the medium with the consequent stop of the reaction, its reuse and, in some cases, an increase in its operational stability.
  • it allows to design continuous reactors of different configuration (fixed bed, fluidized bed, stirred tank, etc.).
  • Different immobilization methods have been studied for enzymes that act on carbohydrates: adsorption, covalent binding, crosslinking, granulation, entrapment, encapsulation, etc. As for the methods of immobilization by adsorption and granulation, these are usually inadequate because the reactions take place in an aqueous medium and the enzyme progressively detaches itself from the support (leaching).
  • alginate-based catalysts A very notable fact is that the specific activity (more specifically, the activity per unit volume) of the alginate-based catalysts is not very high, less than 10 U / ml gel (cf. KD Reh et al., Enzvme Microb. Tech., 1996, vol. 19, pp. 518-524). But without a doubt, the major drawback of alginate-based biocatalysts is the leaching of the enzyme during the course of the biotransformation, since the pores of the alginate network are excessively large for most of the enzymes, being, therefore, a very suitable method for the immobilization of whole cells (viable or not).
  • a first aspect of the present invention refers to a method of obtaining a biocatalyst (from now on the method of the invention), which comprises: a. immobilize a fungal enzyme by inclusion in a calcium alginate gel. b. Dry the immobilized biocatalyst obtained in step (a).
  • the drying is carried out at a temperature between 30 and 50 0 C, this temperature will depend on the thermostability of the enzyme immobilized.
  • fungal enzyme refers in the present invention to an enzyme obtained from a microorganism, more specifically by fungi, by techniques known to any person skilled in the art. These enzymes can have different activity depending on the type of transformation in which they act, for example, but without limitation they can have fructosyltransferase, ⁇ -fructofuranosidase, ⁇ -galactosidase, ⁇ -amylase or glucose isomerase activity.
  • the enzyme is fructosyltransferase or ⁇ -fructofuranosidase.
  • the enzyme When the enzyme is a fructosyltransferase, it preferably comes from a fungus of the genus Asperqillus or Aureobasidium. And more preferably of the species Aspergillus aculeatus.
  • the enzyme When the enzyme is a ⁇ -fructofuranosidase, it preferably comes from a fungus of the Rhodotorula, Schwanniomvces, Xanthophyllomvces or Saccharomvces genus. And more preferably of the Rhodotorula qracilis species.
  • a second aspect of the present invention refers to an immobilized biocatalyst obtainable by the process of the invention and comprising (from now on biocatalyst of the invention or of the DALGEE type ("Dr ⁇ ed alginate-entrapped enzyme")):
  • the biocatalyst of the invention can be used industrially to obtain oligosaccharides without requiring subsequent separation or purification steps.
  • Said biocatalyst, both in its gel state (“biocatalyst-gel”), prior to drying, and in its dried variant, can be packaged in a fixed bed reactor for the continuous production of fructooligosaccharides.
  • a concentrated carbohydrate solution for example sucrose, can be used as the input stream to the reactor.
  • the products resulting from the fructosyltransferase activity are fructooligosaccharides of the 1 F series (1-kestose, nistose, 1 F-fructosylnistose and 1 F-fructosyl-fructosylnistose), and their molar ratio can be controlled by varying the flow rate in the reactor.
  • the present invention involves a process for obtaining industrially viable fructooligosaccharides.
  • the composition of the reaction product in particular, its content in tri-, tetra-, penta- and hexasaccharides) can be regulated according to the residence time of the reactor.
  • the methodology presented is not only applicable to fructosyltransferases, but can also be extended to other enzymes that act on carbohydrates.
  • the beta-fructofuranosidase of Rhodotorula gracilis, ATCC1416 was immobilized following the steps described above (see examples).
  • this enzyme is capable of forming fructooligosaccharides under specific conditions, its main reaction is sucrose hydrolysis. This process is interesting for obtaining Fructose syrups as sweeteners from sucrose.
  • carbohydrates refers in the present invention to sugars capable of transforming to other compounds such as fructooligosaccharides, fructose syrup, among others. These sugars can be, but not limited to sucrose, lactose, maltose, glucose or starch.
  • the biocatalyst of the invention is used to obtain fructooligosaccharides or fructose syrup
  • Another aspect of the present invention relates to a carbohydrate hydrolysis process, comprising: a. pack the biocatalyst of the invention in a continuous fixed bed reactor; b. feed the continuous fixed bed reactor of step (a) with a carbohydrate solution in a concentration between 500 g / l and 700 g / l.
  • the reactor temperature is between 30 0 C and 40 0 C.
  • FIG. 2 represents the flow chart of our FOS production system, both with the biocatalyst-gel (2A) and with the DALGEE type (2B).
  • FIG. 3 shows the composition of sugars (in% w / w) of the outlet current of the reactor with the biocatalyst-gel of the Aspergillus aculeatus fructosyltransferase gel for 750 h.
  • the reaction conditions were: feed, 600 g / sucrose; reactor temperature, 35 0 C; flow of the input and output currents of the system, 0.84 ml / h; volume of biocatalyst-gel used, 25 ml.
  • FIG. 4 shows the effect of the work flow in the composition of the mixture of FOS at the outlet of the reactor (4A) and in the volumetric productivity, expressed in grams of FOS per liter of reactor and day (4B).
  • FIG. 5 shows the appearance of the biocatalyst-gel (upper image) and after undergoing the drying process (DALGEE, lower image).
  • FIG. 6 (6A and 6B) show scanning electron microscopy (SEM) micrographs of the DALGEE type biocatalyst.
  • FIG. 7 shows a chromatogram of a sample obtained from the reactor output current with the DALGEE biocatalyst of the Aspergillus aculeatus fructosyltransferase. Specifically, it corresponds to the sample taken at 622 hours of continuous operation of the reactor.
  • FIG. 9 shows the volumetric productivity, expressed in grams of FOS per liter of reactor and day, for the two forms of biocatalyst of Aspergillus aculeatus fructosyltransferase: biocatalyst-gel and DALGEE.
  • the operating conditions are the same as those described in Figure 6 for the biocatalyst-gel and in Figure 9 for the DALGEE type.
  • FIG. 10 shows the study of the leaching of both the biocatalyst-gel and the DALGEE type of the Aspergillus aculeatus fructosyltransferase.
  • the work protocol followed is represented and in the lower part the enzymatic activity in the supernatant after the successive washing cycles.
  • FIG. 11 shows a chromatogram of a sample obtained from the reactor output current with the DALGEE type biocatalyst of the Rhodotorula qracilis beta-fructofuranosidase ATCC1416. Specifically, it corresponds to the sample taken at 48 hours of continuous operation of the reactor.
  • EXAMPLE 1 Immobilization by entrapment of the Asperqillus aculeatus fructosyltransferase.
  • the fixed assets were subjected to two washing cycles (incubating the spheres for 40 min in 0.01 M sodium acetate buffer pH 5.6, at room temperature and with gentle magnetic stirring) in order to remove enzyme that had not been properly trapped in the gel of alginate.
  • the alginate spheres with the immobilized enzyme were stored at 4 0 C.
  • the loss of activity in the gelation process was measured from the determination of the activity of the supernatant and the washings, being estimated at 50%. However, this activity can be recovered and used for a subsequent immobilization process.
  • the volumetric activity of the biocatalyst obtained in this exemplary embodiment was 10 U / ml.
  • the release of reducing sugars was assessed by means of the dinitrosalicylic acid (DNS) method.
  • DNS dinitrosalicylic acid
  • the test was performed using a sucrose solution of 100 mg / ml, in 96-well plates with a flat bottom and a volume of 200 ⁇ l. In each well, 45 ⁇ l of 100 g / l sucrose solution (prepared in 0.02 M sodium acetate buffer, pH 5.6) and 5 ⁇ l of the enzyme solution were deposited. The plate was incubated for 20 minutes at 35 0 C and 200 rpm in an orbital shaking incubator (Vortemp 56, Labnet).
  • the biocatalyst of EXAMPLE 1 was used to pack 25 ml of a glass column (Amersham Pharmacia XK 16/20), thermostated at 35 0 C and connected to a Socratic double-piston pump (model 515, Waters).
  • the bioreactor was arranged as a fixed bed reactor, with an input and an output current.
  • the feed solution contained 600 g / l sucrose in 0.02 M sodium acetate buffer (pH 5.6), which was maintained with magnetic stirring and thermostatting at 35 ° C.
  • FIG. 2 represents the fixed bed reactors developed for these studies.
  • samples began to be collected at the exit of the column.
  • the samples were centrifuged for 5 min at 4300 xg using an eppendorf with filter Durapore of 0.45 ⁇ m (Millipore), and analyzed by high performance liquid chromatography.
  • a quaternary pump (Delta 600, Waters) and a Phenomenex column, Luna NH2 5 ⁇ m 100A (250 x 4.6 mm) were used.
  • the column was kept thermostatted at 25 0 C thanks to an oven (Timberline Instruments, Inc).
  • the mobile phase used was a mixture of acetonitrile / water, which was degassed with a continuous flow of Helium of 100 ml / min.
  • An evaporative light-scattering detector (ELSD, DDL Eurosep 31) operating at a temperature of 85 0 C and with N 2 nebulizer gas was used.
  • Data analysis was carried out with Waters Millennium 32 Software. The analyzes were performed operating in gradient mode according to the following program in Table 1:
  • FIG. 3 shows results obtained for the reactor packed with the biocatalyst-gel for 750 hours. Specifically, the percentage by weight referred to the total sugars in the mixture, over 750 hours of continuous operation of the reactor, is represented. This graph shows the high operational stability of the biocatalyst.
  • Table 2 shows the average composition, in grams per liter, of the reactor output current at a flow rate of 0.84 ml / h, once the steady state has been reached. Table 2. Average composition of the reactor output current packed with the gel biocatalyst, at a flow rate of 0.84 ml / h.
  • FIG. 4 shows how the composition of the reactor outlet can be regulated by varying the residence time, which is ultimately controlled through the flow rate.
  • a greater flow represents a shorter residence time and consequently a greater proportion of the products of low degree of polymerization (in particular, 1-kestose) in the mixture of the outlet.
  • a lower flow implies a longer residence time and therefore a greater proportion of the FOS with a higher degree of polymerization: tri-, tetra-, penta- and hexasaccharides.
  • a higher flow rate means a lower percentage of sucrose converted into products. Therefore, the volumetric productivity of the reactor (grams of total FOS per day and per liter of reactor) was altered by the work flow, as indicated graphically in the lower part of FIG. Four.
  • EXAMPLE 3 Obtaining a DALGEE type biocatalyst of the Asperqillus aculeatus fructosyltransferase.
  • the biocatalyst of Example 1 was allowed to air dry in a crystallizing glass, 35 0 C, for 3 days. After this time, the diameter of the Spheres obtained were 1 mm, which means a reduction in the volume of the biocatalyst of 96% (assuming spherical particles). The loss of activity in the drying process, per biocatalyst particle, was estimated at 50%. However, the volumetric activity of the dry biocatalyst was 140 U / ml, so there was an increase of approximately 13 or 14 times in the volumetric activity with respect to the biocatalyst-gel.
  • FIG. 5 shows the appearance of the biocatalyst spheres, before and after the drying process.
  • a HITACHI TM1000 scanning electron microscope (SEM) with a potential electron accelerator of 15 kV, and a high sensitivity BSE semiconductor detector was used.
  • FIG. 6 shows the morphology of the dry biocatalyst particles.
  • Table 1 shows the most outstanding aspects of the DALGEE type biocatalyst. Textural properties were studied by mercury porosimetry, using a Fisons Instruments Pascal 140/240 porosimeter. Samples were incubated at 60 0 C for 24 h before analysis.
  • the value of the contact angle of Hg (141 °) and the surface tension (484 mN / m) were selected to evaluate the Pressure / volume data by the Washburn equation, assuming a cylindrical pore model.
  • the particle size distribution was determined by analysis of the intrusion curve. From the porosity of the material and assuming spherical particles, the packing factor and the particle size distribution were calculated according to the Mayer-Stowe theory. Water content was determined by the Karl-Fischer method. It is observed that the DALGEEs have a very small pore volume (0.072 cm 3 / g), with a maximum in the pore size distribution of 8.3 nm. This data explains the low leaching of the DALGEE type biocatalysts, since most of the enzymes will not be able to escape through the pores. Table 3. Most outstanding characteristics of the DALGEE biocatalyst.
  • EXAMPLE 4 Obtaining fructooligosaccharides in a fixed bed reactor with DALGEE of the Aspergillus aculeatus fructosyltransferase.
  • the biocatalyst of EXAMPLE 3 was used to pack a 1 ml column (0.7 x 2.5 cm) thermostated at 35 0 C and connected to a pump socratic double piston.
  • the feed solution contained 600 g / l of sucrose in 0.02 M sodium acetate buffer (pH 5.6), which was maintained with magnetic stirring and thermostatting at 35 ° C.
  • the system arrangement did not include any recirculation current.
  • FIG. 2 represents the fixed bed reactors developed for these tests. After the system stabilization time, samples began to be collected at the exit of the column.
  • FIG. 7 shows a typical chromatogram of the reaction mixture at the outlet of the reactor containing the DALGEE biocatalyst.
  • FIG. 4 shows how the composition of the reactor outlet can be regulated by varying the residence time, which is ultimately controlled through the flow rate.
  • a greater flow represents a shorter residence time and consequently a greater proportion of the products of low degree of polymerization (in particular, 1-kestose) in the mixture of the outlet.
  • a lower flow implies a longer residence time and therefore a greater proportion of the FOS with a higher degree of polymerization: tri-, tetra-, penta- and hexasaccharides.
  • a higher flow rate means a lower percentage of sucrose converted into products. Therefore, the volumetric productivity of the reactor (grams of total FOS per day and per liter of reactor) was altered by the work flow, as indicated graphically in the lower part of FIG. Four.
  • FIG. 9 shows the volumetric productivity, expressed in grams of FOS per liter of reactor and day, for the two forms of biocatalyst, biocatalyst-gel and DALGEE, using a flow rate of 0.84 ml / h for the biocatalyst-gel and 0.60 ml / h for the DALGEE, during the 750 hours of operation of the bioreactor. It is appreciated that in the case of the biocatalyst-gel, the productivity is close to 100 g of FOS per liter of reactor and day, while with the DALGEE type, it reaches a value around 4000 g FOS per liter of reactor and day .
  • EXAMPLE 5 Study of the leaching of the biocatalyst-gel and DALGEE.
  • the immobilized biocatalysts of Aspergillus aculeatus fructosyltransferase were subjected to successive wash cycles with buffer solution (0.02 M sodium acetate pH 5.6).
  • buffer solution 0.2 M sodium acetate pH 5.6
  • biocatalyst particles were incubated for 1 h at 600 rpm and 35 0 C, with 500 .mu.l of buffer. After each wash, the 500 ⁇ l of solution was extracted, to evaluate the enzyme released, and replaced by fresh buffer solution for the next cycle.
  • the leaching percentage after the first cycle was 7.5% for the biocatalyst-gel and 5% for the DALGEE. From the third cycle, the leaching can be considered negligible, as shown in FIG. 10.
  • EXAMPLE 6 Obtaining a DALGEE type biocatalyst of the beta-fructofuranosidase from Rhodotorula qracilis ATCC1416.
  • the final activity of the mixture was approximately 12 U / ml.
  • the preparation of the biocatalyst-gel was performed as described in the
  • EXAMPLE 1 The loss of activity in the gelation process was measured from the determination of the activity of the supernatant and washes, estimated at 60%.
  • the volumetric activity of the biocatalyst obtained in this exemplary embodiment was 0.8 U / ml.
  • the biocatalyst obtained was subjected to a drying process as described in the
  • EXAMPLE 3 giving rise to a DALGEE type biocatalyst.
  • EXAMPLE 7 Hydrolysis of sucrose in a fixed bed reactor with DALGEE of the beta-fructofuranosidase of Rhodotorula gracilis ATCC1416.
  • the biocatalyst of EXAMPLE 6 was used to pack a 1.5 ml (0.6 x 5.3 cm) column, thermostated at 35 0 C and connected to a pump Socratic double piston.
  • the feed solution contained 600 g / l of sucrose in 0.02 M sodium acetate buffer (pH 5.6), which was maintained with magnetic stirring and thermostatting at 35 0 C. After the system stabilization time, samples were collected at Ia output of the column. The samples were analyzed as described in EXAMPLE 2.
  • FIG. 12 shows a typical chromatogram of the reaction mixture at the outlet of the reactor containing the DALGEE biocatalyst.
  • the reactor packaged with the biocatalyst DALGEE beta-fructofuranosidase from R. qracilis ATCC1416 maintained its initial activity for at least 48 hours.
  • the productivity obtained was estimated at 2600 g of reducing sugars per day and liter of reactor.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Dispersion Chemistry (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

Procedure of obtainment of a biocatalyst comprising: immobilisation of a fungal enzyme through inclusion in a calcium alginate gel and subsequent drying of the immobilised biocatalyst obtained in step (a). The invention furthermore relates to the biocatalyst obtained through the procedure of the invention and comprising fungal enzymes, preferably fructosyltransferase or β-fructofuranosidase, immobilised in alginate. Moreover the invention relates to the use of said biocatalyst for such biotransformation wherein the substrate is a concentrated solution of a carbohydrate and may be carried out in a continuous reactor.

Description

BIOCATALIZADOR INMOVILIZADO BASADO EN ALGINATO PARA LA BIOTRANSFORMACION DE CARBOHIDRATOS ALGINATE-BASED IMMOBILIZED BIOCATALIZER FOR CARBOHYDRATE BIOTRANSFORMATION
La presente invención se refiere a biocatalizadores que comprenden enzimas inmovilizadas en alginato y a su procedimiento de obtención.The present invention relates to biocatalysts comprising enzymes immobilized in alginate and their method of production.
Además, mediante el secado de dicho biocatalizador, Ia invención también se refiere al uso de dicho biocatalizador para Ia biotransformacion en las que el sustrato es una disolución concentrada de un carbohidrato y se puede llevar a cabo en un reactor continuo. Por tanto, Ia presente invención se puede encuadrar dentro del campo de Ia industria biotecnológica.Furthermore, by drying said biocatalyst, the invention also refers to the use of said biocatalyst for biotransformation in which the substrate is a concentrated solution of a carbohydrate and can be carried out in a continuous reactor. Therefore, the present invention can be framed within the field of biotechnology industry.
ESTADO DE LA TÉCNICA ANTERIORSTATE OF THE PREVIOUS TECHNIQUE
Los carbohidratos son el material biológico muy abundante en Ia naturaleza, se pueden utilizar como materia prima para una gran variedad de procesos industriales, dando lugar a otros productos con mucho interés industrial y tecnológico. Para ello, las enzimas implicadas en su transformación tienen un enorme interés.Carbohydrates are the biological material very abundant in nature, they can be used as raw material for a wide variety of industrial processes, giving rise to other products with a lot of industrial and technological interest. For this, the enzymes involved in its transformation have an enormous interest.
El campo de los oligosacáridos prebióticos, como ingredientes funcionales en alimentación y nutracéuticos, se ha desarrollado de manera espectacular en los últimos años, estos compuestos se obtienen mediante transformaciones enzimáticas de carbohidratos.The field of prebiotic oligosaccharides, as functional ingredients in food and nutraceuticals, has developed dramatically in recent years, these compounds are obtained by enzymatic carbohydrate transformations.
El término prebiótico fue introducido por Gibson y Roberfroid, quienes definieron a los prebióticos como ingredientes no digeribles de los alimentos que afectan beneficiosamente al huésped por una estimulación selectiva del crecimiento y/o actividad de un limitado grupo de bacterias en el colon. Los prebióticos resisten Ia digestión en Ia parte superior del tracto intestinal, y se metabolizan por las bacterias endógenas del colon. Las moléculas prebióticas líderes en el mercado son los fructooligosacáridos (FOS) (cfr. PT. Sangeetha, M. N. Armes, S. G. Prapulla, Trends Food Sci. TechnoL 2005, vol. 16, pp. 442-457; A.V. Rao, J. Nutr., 1998, vol. 80, pp.1442S-1445S). Los FOS pueden ejercer una serie de efectos sobre Ia persona que los consume: control del estreñimiento por aumento de Ia masa fecal, reducción de los episodios de diarreas causadas por rotavirus, mejora de los síntomas de intolerancia a Ia lactosa, aumento de Ia absorción de calcio, disminución de Ia capacidad mutagénica de ciertas enzimas microbianas como Ia nitro-reductasa (asociadas con el cáncer de colon), posible reducción de enfermedades relacionadas con dislipemias, etc.The term prebiotic was introduced by Gibson and Roberfroid, who defined prebiotics as non-digestible food ingredients that beneficially affect the host by selective stimulation of the growth and / or activity of a limited group of bacteria in the colon. Prebiotics resist digestion in the upper part of the intestinal tract, and are metabolized by the endogenous bacteria of the colon. The market-leading prebiotic molecules are fructooligosaccharides (FOS) (cf. PT. Sangeetha, MN Armes, SG Prapulla, Trends Food Sci. TechnoL 2005, vol. 16, pp. 442-457; AV Rao, J. Nutr. , 1998, vol. 80, pp. 1442S-1445S). The FOS can exert a series of effects on the person who consumes them: control of constipation due to increased fecal mass, reduction of diarrhea episodes caused by rotavirus, improvement of symptoms of lactose intolerance, increase of the absorption of calcium, decrease in the mutagenic capacity of certain microbial enzymes such as nitro-reductase (associated with colon cancer), possible reduction of diseases related to dyslipidemia, etc.
Los FOS que se comercializan actualmente están formados por moléculas de fructosa unidas por enlaces glicosídicos β-(2-1 ), con una molécula de glucosa terminal (cfr. J. W. Yun, Enzvme Microb. TechnoL, 1996, vol. 19, pp. 107-117), abreviándose como GFn, estando n típicamente comprendido entre 2 y 4 (1-kestosa, nistosa y 1F-fructosilnistosa). Estudios recientes muestran que el mayor efecto prebiótico es ejercido por el trisacárido 1- kestosa, en comparación con compuestos de mayor peso molecular de Ia serie (cfr. O. Bañuelos et al., Anaerobe. 2008, vol. 14, pp. 184-189).The FOS currently marketed are made up of fructose molecules linked by β- (2-1) glycosidic bonds, with a terminal glucose molecule (cf. JW Yun, Enzvme Microb. TechnoL, 1996, vol. 19, pp. 107 -117), abbreviated as GF n , n being typically between 2 and 4 (1-kestose, nosy and 1 F-fructosylnistose). Recent studies show that the greatest prebiotic effect is exerted by the trisaccharide 1- kestose, in comparison with higher molecular weight compounds of the series (cf. O. Bañuelos et al., Anaerobe. 2008, vol. 14, pp. 184- 189).
Los FOS se obtienen por síntesis enzimática a partir de sacarosa, utilizando sacarosa 1-fructosiltransferasas (EC 2.4.1.9), beta- fructofuranosidasas -invertasas- (EC 3.2.1.26) o incluso levansacarasas (EC 2.4.1.10) (cfr. L. E. Trujillo et al., Enzvme Microb. Technol. 2001 , vol. 28, pp. 139-144). Empleando beta-fructofuranosidasas el rendimiento de productos de síntesis que se alcanza es muy bajo, representando éstos menos del 4% (p/p) del total de carbohidratos de Ia mezcla. Sin embargo, con fructosiltransferasas el rendimiento que se puede alcanzar es mucho mayor, alcanzando valores próximos al 55-60% (p/p) de FOS referido al peso total de azúcares (cfr. M. Antosova, M. Polakovic, Chem. Pap. 2001 , vol. 55, pp. 350-358). En Ia actualidad, los FOS se producen industrialmente utilizando fructosiltransferasas del género Aspergillus (p. ej. A. oryzae, A. i a pon i cus, A. niper, A. aculeatus, etc.) o Aureobasidium (p. ej. A. pullulans) para generar oligosacáridos de cadena corta, de 3 a 5 unidades (cfr. C. Vannieeuwenburgh et al., Bioprocess Biosvst. Enq., 2002, vol. 25, pp. 13-20; CS. Chien et al., Enzvme Microb. TechnoL 2001 , vol. 29, pp. 252-257).FOS are obtained by enzymatic synthesis from sucrose, using sucrose 1-fructosyltransferases (EC 2.4.1.9), beta-fructofuranosidases -invertases- (EC 3.2.1.26) or even levansacarasas (EC 2.4.1.10) (cf. LE Trujillo et al., Enzvme Microb. Technol. 2001, vol. 28, pp. 139-144). Using beta-fructofuranosidases the yield of synthesis products that is reached is very low, representing these less than 4% (w / w) of the total carbohydrates of the mixture. However, with fructosyltransferases the yield that can be achieved is much higher, reaching values close to 55-60% (w / w) of FOS referred to Total weight of sugars (cf. M. Antosova, M. Polakovic, Chem. Pap. 2001, vol. 55, pp. 350-358). At present, FOS are produced industrially using fructosyltransferases of the genus Aspergillus (eg A. oryzae, A. ia pon i cus, A. niper, A. aculeatus, etc.) or Aureobasidium (eg A. pullulans) to generate short chain oligosaccharides, 3 to 5 units (cf. C. Vannieeuwenburgh et al., Bioprocess Biosvst. Enq., 2002, vol. 25, pp. 13-20; CS. Chien et al., Enzvme Microb. TechnoL 2001, vol. 29, pp. 252-257).
La producción a gran escala de oligosacáridos prebióticos sintetizados por vía enzimática puede verse favorecida si se inmoviliza Ia enzima en un soporte sólido. Este proceso facilita Ia separación del biocatalizador del medio con Ia consiguiente parada de Ia reacción, su reutilización y, en algunos casos, un aumento de su estabilidad operacional. Además, permite diseñar reactores continuos de diferente configuración (lecho fijo, lecho fluidizado, tanque agitado, etc.). Se han estudiado distintos métodos de inmovilización para enzimas que actúan sobre carbohidratos: adsorción, unión covalente, entrecruzamiento, granulación, atrapamiento, encapsulación, etc. En cuanto a los métodos de inmovilización por adsorción y granulación, éstos suelen ser inadecuados debido a que las reacciones tienen lugar en un medio acuoso y Ia enzima se desprende progresivamente del soporte (lixiviación).The large-scale production of prebiotic oligosaccharides synthesized by enzymatic route can be favored if the enzyme is immobilized on a solid support. This process facilitates the separation of the biocatalyst from the medium with the consequent stop of the reaction, its reuse and, in some cases, an increase in its operational stability. In addition, it allows to design continuous reactors of different configuration (fixed bed, fluidized bed, stirred tank, etc.). Different immobilization methods have been studied for enzymes that act on carbohydrates: adsorption, covalent binding, crosslinking, granulation, entrapment, encapsulation, etc. As for the methods of immobilization by adsorption and granulation, these are usually inadequate because the reactions take place in an aqueous medium and the enzyme progressively detaches itself from the support (leaching).
Uno de los métodos de inmovilización más estudiados para enzimas glicosídicas es el atrapamiento en geles de alginato calcico, debido a Ia facilidad para llevarlo a cabo, Ia ausencia de cambios conformacionales enOne of the most studied immobilization methods for glycosidic enzymes is the entrapment in calcium alginate gels, due to the ease of carrying it out, the absence of conformational changes in
Ia estructura de Ia enzima, el bajo precio de los materiales empleados y Ia elevada actividad recuperada. No obstante, esta metodología presenta todavía ciertos inconvenientes (cfr. U. Jahnz et al., Engineering and Manufacturinq for Biotechnoloqy, 2001 , vol. 4, pp. 293-307). Por un lado, Ia estabilidad mecánica del gel es limitada en reactores que presenten una alta tensión tangencial. Además, si se utilizan tampones fosfato o citrato, tiene lugar Ia pérdida de calcio, Io que origina un deterioro del biocatalizador. Por otro lado, el alginato puede ser biodegradado en el propio reactor o durante el almacenamiento cuando se trabaja en condiciones no estériles. Un hecho muy notorio es que Ia actividad específica (más concretamente, Ia actividad por unidad de volumen) de los catalizadores basados en alginato no es muy alta, inferior a 10 U/ml gel (cfr. K.D. Reh et al., Enzvme Microb. Tech., 1996, vol. 19, pp. 518-524). Pero sin lugar a dudas el mayor inconveniente de los biocatalizadores basados en alginato consiste en Ia lixiviación de Ia enzima durante el transcurso de Ia biotransformación, ya que los poros de Ia red de alginato son excesivamente grandes para Ia mayor parte de las enzimas, siendo, por tanto, un método muy adecuado para Ia inmovilización de células enteras (viables o no). En algunos casos se ha descrito Ia eliminación del agua contenida en las esferas de alginato calcico mediante un proceso de liofilización, sin embargo, en estos biocatalizadores tampoco se ha observado una reducción en el lixiviado de Ia enzima (cfr. S. S. Betigeri et al., Biomaterials. 2002, vol. 23, pp. 3627-3636).The structure of the enzyme, the low price of the materials used and the high activity recovered. However, this methodology still has certain drawbacks (cf. U. Jahnz et al., Engineering and Manufacturing for Biotechnoloqy, 2001, vol. 4, pp. 293-307). On the one hand, the mechanical stability of the gel is limited in reactors presenting a high tangential tension. In addition, if phosphate or citrate buffers are used, calcium loss occurs, which causes a deterioration of the biocatalyst. On the other hand, alginate can be biodegraded in the reactor itself or during storage when working under non-sterile conditions. A very notable fact is that the specific activity (more specifically, the activity per unit volume) of the alginate-based catalysts is not very high, less than 10 U / ml gel (cf. KD Reh et al., Enzvme Microb. Tech., 1996, vol. 19, pp. 518-524). But without a doubt, the major drawback of alginate-based biocatalysts is the leaching of the enzyme during the course of the biotransformation, since the pores of the alginate network are excessively large for most of the enzymes, being, therefore, a very suitable method for the immobilization of whole cells (viable or not). In some cases the elimination of the water contained in the calcium alginate spheres has been described by a lyophilization process, however, in these biocatalysts there has also been no reduction in the leaching of the enzyme (cf. SS Betigeri et al., Biomaterials, 2002, vol. 23, pp. 3627-3636).
Dada Ia importancia industrial de los oligosacáridos prebióticos, es deseable proporcionar enzimas y procedimientos para su obtención, que sean viables industrialmente.Given the industrial importance of prebiotic oligosaccharides, it is desirable to provide enzymes and processes for obtaining them, which are industrially viable.
DESCRIPCIÓN DE LA INVENCIÓNDESCRIPTION OF THE INVENTION
La presente invención proporciona biocatalizadores inmovilizados y un procedimiento para Ia obtención de dichos biocatalizadores, donde una de sus aplicaciones es Ia producción de oligosacáridos prebióticos, principalmente 1-kestosa (GF2), nistosa (GF3), 1 F-fructosilnistosa (GF4) y 1 F-fructosil-fructosilnistosa (GF5). Estos oligosacáridos prebióticos pueden ser utilizados como ingredientes funcionales en productos alimenticios, alimentos infantiles y/o alimentación animal.The present invention provides immobilized biocatalysts and a method for obtaining said biocatalysts, where one of its applications is the production of prebiotic oligosaccharides, mainly 1-kestose (GF2), nistose (GF3), 1 F-fructosylnistose (GF4) and 1 F-fructosyl-fructosylnistose (GF5). These prebiotic oligosaccharides can be used as functional ingredients in food products, baby food and / or animal feed.
También, pueden tener otras aplicaciones, como puede ser Ia obtención de jarabes de fructosa como edulcorantes a partir de sacarosa.Also, they may have other applications, such as obtaining fructose syrups as sweeteners from sucrose.
Por tanto, un primer aspecto de Ia presente invención se refiere a un procedimiento de obtención de un biocatalizador (a partir de ahora procedimiento de Ia invención), que comprende: a. inmovilizar una enzima fúngica por inclusión en un gel de alginato calcico. b. secar el biocatalizador inmovilizado obtenido en el paso (a).Therefore, a first aspect of the present invention refers to a method of obtaining a biocatalyst (from now on the method of the invention), which comprises: a. immobilize a fungal enzyme by inclusion in a calcium alginate gel. b. Dry the immobilized biocatalyst obtained in step (a).
En una realización preferida del procedimiento de invención, el secado se lleva a cabo a una temperatura de entre 30 y 50 0C, esta temperatura dependerá de Ia termoestabilidad de Ia enzima inmovilizada.In a preferred embodiment of the method of invention, the drying is carried out at a temperature between 30 and 50 0 C, this temperature will depend on the thermostability of the enzyme immobilized.
El término "enzima fúngica" se refiere en Ia presente invención a un enzima obtenida a partir de un microorganismo, más concretamente mediante hongos, mediante técnicas conocidas por cualquier experto en Ia materia. Estas enzimas pueden tener diferente actividad dependiendo del tipo de transformación en Ia que actúen, por ejemplo, pero sin limitarse pueden tener actividad fructosiltransferasa, β-fructofuranosidasa, β- galactosidasa, α-amilasa o glucosa isomerasa.The term "fungal enzyme" refers in the present invention to an enzyme obtained from a microorganism, more specifically by fungi, by techniques known to any person skilled in the art. These enzymes can have different activity depending on the type of transformation in which they act, for example, but without limitation they can have fructosyltransferase, β-fructofuranosidase, β-galactosidase, α-amylase or glucose isomerase activity.
En una realización preferida del procedimiento de Ia invención, Ia enzima es fructosiltransferasa o β-fructofuranosidasa.In a preferred embodiment of the process of the invention, the enzyme is fructosyltransferase or β-fructofuranosidase.
Cuando Ia enzima es una fructosiltransferasa, preferiblemente procede de un hongo de género Asperqillus o Aureobasidium. Y más preferiblemente de Ia especie Aspergillus aculeatus. Cuando la enzima es una β-fructofuranosidasa, preferiblemente procede de un hongo de género Rhodotorula, Schwanniomvces, Xanthophyllomvces o Saccharomvces. Y más preferiblemente de Ia especie Rhodotorula qracilis.When the enzyme is a fructosyltransferase, it preferably comes from a fungus of the genus Asperqillus or Aureobasidium. And more preferably of the species Aspergillus aculeatus. When the enzyme is a β-fructofuranosidase, it preferably comes from a fungus of the Rhodotorula, Schwanniomvces, Xanthophyllomvces or Saccharomvces genus. And more preferably of the Rhodotorula qracilis species.
Un aspecto positivo del biocatalizador de Ia invención es que presenta una alta actividad volumétrica, comparándolo con el biocatalizador-gel obtenido en el paso (a) del procedimiento descrito, convirtiéndolo en candidato idóneo para producir oligosacáridos prebióticos en reactores de un volumen reducido. Otro aspecto importante, a nivel industrial, es Ia alta estabilidad operacional del biocatalizador: mantiene su actividad catalítica en el reactor de lecho fijo durante largos tiempos de funcionamiento (> 700 h), trabajando a temperaturas próximas a 35 0C y empleando como corriente de alimentación una disolución de sacarosa de 600 g/l. Por otro lado, el biocatalizador de Ia invención no recupera su volumen inicial (previo al secado) cuando por él circula una solución concentrada de sacarosa (o de otro azúcar). Otra ventaja importante del biocatalizador seco frente al biocatalizador húmedo, es que las condiciones de almacenamiento son mucho menos exigentes, por su bajo contenido en agua, Io que Io hace más resistente a Ia biodegradación o contaminación microbiana. Además, el biocatalizador de Ia invención es también muy estable en presencia de disolventes orgánicos, incluyendo los de alta polaridad como el metanol. Este hecho podría conferir a los biocatalizadores de Ia invención excelente aplicabilidad en reacciones sintéticas en disolventes orgánicos. Por otro lado, Ia metodología desarrollada es aplicable a enzimas fuertemente glicosiladas, para las que los métodos de inmovilización covalente suelen dar lugar a valores muy bajos de actividad recuperada y actividad volumétrica.A positive aspect of the biocatalyst of the invention is that it has a high volumetric activity, comparing it with the biocatalyst-gel obtained in step (a) of the described procedure, making it an ideal candidate to produce prebiotic oligosaccharides in reactors of a reduced volume. Another important aspect, at the industrial level, is the high operational stability of the biocatalyst: it maintains its catalytic activity in the fixed bed reactor for long operating times (> 700 h), working at temperatures close to 35 0 C and using as a current of feed a sucrose solution of 600 g / l. On the other hand, the biocatalyst of the invention does not recover its initial volume (prior to drying) when a concentrated solution of sucrose (or other sugar) circulates through it. Another important advantage of the dry biocatalyst over the wet biocatalyst is that the storage conditions are much less demanding, due to its low water content, which makes it more resistant to biodegradation or microbial contamination. In addition, the biocatalyst of the invention is also very stable in the presence of organic solvents, including those of high polarity such as methanol. This fact could give the biocatalysts of the invention excellent applicability in synthetic reactions in organic solvents. On the other hand, the methodology developed is applicable to strongly glycosylated enzymes, for which covalent immobilization methods usually give rise to very low values of recovered activity and volumetric activity.
Por tanto, y debido a las características que confiere el procedimiento de obtención al biocatalizador, un segundo aspecto de Ia presente invención se refiere a un biocatalizador inmovilizado obtenible por el procedimiento de Ia invención y que comprende (a partir de ahora biocatalizador de Ia invención o de tipo DALGEE ("Dríed alginate-entrapped enzyme")):Therefore, and due to the characteristics conferred by the method of obtaining the biocatalyst, a second aspect of the present invention refers to an immobilized biocatalyst obtainable by the process of the invention and comprising (from now on biocatalyst of the invention or of the DALGEE type ("Dríed alginate-entrapped enzyme")):
- una enzima fúngica inmovilizada en un gel de alginato calcico.- a fungal enzyme immobilized in a calcium alginate gel.
El biocatalizador de Ia invención puede ser utilizado industrialmente para Ia obtención de oligosacáridos sin requerir etapas de separación o purificación posteriores. Dicho biocatalizador, tanto en su estado de gel ("biocatalizador-gel"), previo al secado, como en su variante desecada, puede ser empaquetado en un reactor de lecho fijo para Ia producción en continuo de fructooligosacáridos. Para ello se puede emplear como corriente de entrada al reactor, una disolución concentrada de carbohidrato, por ejemplo de sacarosa. En una realización particular, los productos resultantes de Ia actividad fructosiltransferasa son fructooligosacáridos de Ia serie 1F (1-kestosa, nistosa, 1F-fructosilnistosa y 1F-fructosil-fructosilnistosa), y su relación molar puede controlarse variando el caudal en el reactor.The biocatalyst of the invention can be used industrially to obtain oligosaccharides without requiring subsequent separation or purification steps. Said biocatalyst, both in its gel state ("biocatalyst-gel"), prior to drying, and in its dried variant, can be packaged in a fixed bed reactor for the continuous production of fructooligosaccharides. For this purpose, a concentrated carbohydrate solution, for example sucrose, can be used as the input stream to the reactor. In a particular embodiment, the products resulting from the fructosyltransferase activity are fructooligosaccharides of the 1 F series (1-kestose, nistose, 1 F-fructosylnistose and 1 F-fructosyl-fructosylnistose), and their molar ratio can be controlled by varying the flow rate in the reactor.
La presente invención conlleva un procedimiento de obtención de fructooligosacáridos viable industrialmente. Además, Ia composición del producto de reacción (en particular, su contenido en tri-, tetra-, penta- y hexasacáridos) puede ser regulada en función del tiempo de residencia del reactor.The present invention involves a process for obtaining industrially viable fructooligosaccharides. In addition, the composition of the reaction product (in particular, its content in tri-, tetra-, penta- and hexasaccharides) can be regulated according to the residence time of the reactor.
La metodología presentada no sólo es aplicable a fructosiltransferasas, sino que puede extenderse a otras enzimas que actúan sobre carbohidratos. En particular, Ia beta-fructofuranosidasa de Rhodotorula gracilis, ATCC1416, fue inmovilizada siguiendo los pasos anteriormente descritos (ver ejemplos). Aunque esta enzima es capaz de formar fructooligosacáridos bajo condiciones específicas, su reacción principal es Ia hidrólisis de sacarosa. Este proceso es interesante para Ia obtención de jarabes de fructosa como edulcorantes a partir de sacarosa.The methodology presented is not only applicable to fructosyltransferases, but can also be extended to other enzymes that act on carbohydrates. In particular, the beta-fructofuranosidase of Rhodotorula gracilis, ATCC1416, was immobilized following the steps described above (see examples). Although this enzyme is capable of forming fructooligosaccharides under specific conditions, its main reaction is sucrose hydrolysis. This process is interesting for obtaining Fructose syrups as sweeteners from sucrose.
Por tanto, otro aspecto de Ia presente invención se refiere al uso del biocatalizador de Ia invención, para Ia hidrólisis de carbohidratos.Therefore, another aspect of the present invention relates to the use of the biocatalyst of the invention, for carbohydrate hydrolysis.
El término "carbohidratos" se refiere en Ia presente invención a azúcares capaces de transformarse a otros compuestos como fructooligosacaridos, jarabe de fructosa, entre otros. Estos azúcares pueden ser, pero sin limitarse sacarosa, lactosa, maltosa, glucosa o almidón.The term "carbohydrates" refers in the present invention to sugars capable of transforming to other compounds such as fructooligosaccharides, fructose syrup, among others. These sugars can be, but not limited to sucrose, lactose, maltose, glucose or starch.
En una realización preferida, el biocatalizador de Ia invención se utiliza para Ia obtención de fructooligosacaridos o de jarabe de fructosaIn a preferred embodiment, the biocatalyst of the invention is used to obtain fructooligosaccharides or fructose syrup
Otro aspecto de Ia presente invención se refiere a un procedimiento de hidrólisis de carbohidratos, que comprende: a. empaquetar el biocatalizador de Ia invención en un reactor continuo de lecho fijo; b. alimentar el reactor continuo de lecho fijo del paso (a) con una disolución de carbohidrato en una concentración de entre 500 g/l y 700 g/l.Another aspect of the present invention relates to a carbohydrate hydrolysis process, comprising: a. pack the biocatalyst of the invention in a continuous fixed bed reactor; b. feed the continuous fixed bed reactor of step (a) with a carbohydrate solution in a concentration between 500 g / l and 700 g / l.
En una realización preferida del procedimiento de hidrólisis, Ia temperatura del reactor está entre 30 0C y 40 0C.In a preferred embodiment of the hydrolysis process, the reactor temperature is between 30 0 C and 40 0 C.
En otra realización preferida, cuando el carbohidrato es sacarosa y el biocatalizador es una fructosiltransferasa inmovilizada, en Ia reacción se obtienen fructooligosacaridos, que más preferiblemente se seleccionan de Ia lista que comprende trisacáridos, tetrasacáridos, pentasacáridos, hexasacáridos o cualquiera de sus combinaciones.In another preferred embodiment, when the carbohydrate is sucrose and the biocatalyst is an immobilized fructosyltransferase, in the reaction fructooligosaccharides are obtained, which are more preferably selected from the list comprising trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides or any combination thereof.
En otra realización preferida, cuando el carbohidrato es sacarosa y el biocatalizador es una β-fructofuranosidasa inmovilizada, en Ia hidrólisis se obtiene jarabe de fructosa.In another preferred embodiment, when the carbohydrate is sucrose and the Biocatalyst is an immobilized β-fructofuranosidase, in the hydrolysis fructose syrup is obtained.
A Io largo de Ia descripción y las reivindicaciones Ia palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en Ia materia, otros objetos, ventajas y características de Ia invención se desprenderán en parte de Ia descripción y en parte de Ia práctica de Ia invención. La siguiente descripción detallada, ejemplos y dibujos se proporcionan a modo de ilustración, y no se pretende que sean limitativos de Ia presente invención.Throughout the description and the claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following detailed description, examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.
DESCRIPCIÓN DE LAS FIGURASDESCRIPTION OF THE FIGURES
La FIG. 1 representa el proceso de inmovilización utilizado para obtener el biocatalizador-gel que se describe en esta invención.FIG. 1 represents the immobilization process used to obtain the biocatalyst-gel described in this invention.
La FIG. 2 representa el diagrama de flujo de nuestro sistema de producción de FOS, tanto con el biocatalizador-gel (2A) como con el tipo DALGEE (2B).FIG. 2 represents the flow chart of our FOS production system, both with the biocatalyst-gel (2A) and with the DALGEE type (2B).
La FIG. 3 muestra Ia composición de azúcares (en % p/p) de Ia corriente de salida del reactor con el biocatalizador-gel de Ia fructosiltransferasa de Aspergillus aculeatus durante 750 h. Las condiciones de reacción fueron: alimentación, 600 g/ de sacarosa; temperatura del reactor, 35 0C; caudal de las corrientes de entrada y salida del sistema, 0.84 ml/h; volumen de biocatalizador-gel empleado, 25 mi.FIG. 3 shows the composition of sugars (in% w / w) of the outlet current of the reactor with the biocatalyst-gel of the Aspergillus aculeatus fructosyltransferase gel for 750 h. The reaction conditions were: feed, 600 g / sucrose; reactor temperature, 35 0 C; flow of the input and output currents of the system, 0.84 ml / h; volume of biocatalyst-gel used, 25 ml.
La FIG. 4 muestra el efecto del caudal de trabajo en Ia composición de Ia mezcla de FOS a Ia salida del reactor (4A) y en Ia productividad volumétrica, expresada en gramos de FOS por litro de reactor y día (4B). La FIG. 5 muestra el aspecto del biocatalizador-gel (imagen superior) y después de someterse al proceso de secado (DALGEE, imagen inferior).FIG. 4 shows the effect of the work flow in the composition of the mixture of FOS at the outlet of the reactor (4A) and in the volumetric productivity, expressed in grams of FOS per liter of reactor and day (4B). FIG. 5 shows the appearance of the biocatalyst-gel (upper image) and after undergoing the drying process (DALGEE, lower image).
Las FIG. 6 (6A y 6B) muestran las micrografías de microscopía electrónica de barrido (SEM) del biocatalizador tipo DALGEE.FIG. 6 (6A and 6B) show scanning electron microscopy (SEM) micrographs of the DALGEE type biocatalyst.
La FIG. 7 muestra un cromatograma de una muestra obtenida de Ia corriente de salida del reactor con el biocatalizador DALGEE de Ia fructosiltransferasa de Aspergillus aculeatus. Concretamente, corresponde a Ia muestra tomada a las 622 horas de funcionamiento continuo del reactor.FIG. 7 shows a chromatogram of a sample obtained from the reactor output current with the DALGEE biocatalyst of the Aspergillus aculeatus fructosyltransferase. Specifically, it corresponds to the sample taken at 622 hours of continuous operation of the reactor.
La FIG. 8 muestra Ia composición de azúcares (en % p/p) de Ia corriente de salida del reactor con el biocatalizador DALGEE de Ia fructosiltransferasa de Aspergillus aculeatus durante 750 h. Las condiciones de reacción fueron: alimentación, 600 g/ de sacarosa; temperatura del reactor, 35 0C; caudal de las corrientes de entrada y salida del sistema, 0.6 ml/h; volumen de biocatalizador-gel empleado, 1 mi.FIG. 8 shows the composition of sugars (in% w / w) of the reactor output current with the DALGEE biocatalyst of the Aspergillus aculeatus fructosyltransferase during 750 h. The reaction conditions were: feed, 600 g / sucrose; reactor temperature, 35 0 C; flow of system input and output currents, 0.6 ml / h; volume of biocatalyst-gel used, 1 ml.
La FIG. 9 muestra las productividades volumétricas, expresadas en gramos de FOS por litro de reactor y día, para las dos formas de biocatalizador de Ia fructosiltransferasa de Aspergillus aculeatus: biocatalizador-gel y DALGEE. Las condiciones de operación son las mismas que las descritas en Ia figura 6 para el biocatalizador-gel y en Ia figura 9 para el tipo DALGEE.FIG. 9 shows the volumetric productivity, expressed in grams of FOS per liter of reactor and day, for the two forms of biocatalyst of Aspergillus aculeatus fructosyltransferase: biocatalyst-gel and DALGEE. The operating conditions are the same as those described in Figure 6 for the biocatalyst-gel and in Figure 9 for the DALGEE type.
La FIG. 10 muestra el estudio de Ia lixiviación tanto del biocatalizador-gel como del tipo DALGEE de Ia fructosiltransferasa de Aspergillus aculeatus. En Ia parte superior se representa el protocolo de trabajo seguido y en Ia parte inferior Ia actividad enzimática en el sobrenadante tras los sucesivos ciclos de lavado. La FIG. 11 muestra un cromatograma de una muestra obtenida de Ia corriente de salida del reactor con el biocatalizador tipo DALGEE de Ia beta-fructofuranosidasa de Rhodotorula qracilis ATCC1416. Concretamente, corresponde a Ia muestra tomada a las 48 horas de funcionamiento continuo del reactor.FIG. 10 shows the study of the leaching of both the biocatalyst-gel and the DALGEE type of the Aspergillus aculeatus fructosyltransferase. In the upper part the work protocol followed is represented and in the lower part the enzymatic activity in the supernatant after the successive washing cycles. FIG. 11 shows a chromatogram of a sample obtained from the reactor output current with the DALGEE type biocatalyst of the Rhodotorula qracilis beta-fructofuranosidase ATCC1416. Specifically, it corresponds to the sample taken at 48 hours of continuous operation of the reactor.
EJEMPLOSEXAMPLES
EJEMPLO 1 : Inmovilización por atrapamiento de Ia fructosiltransferasa de Asperqillus aculeatus.EXAMPLE 1: Immobilization by entrapment of the Asperqillus aculeatus fructosyltransferase.
Se prepararon 30 mi de una disolución de alginato sódico SG-300® al 4% (p/v) en agua. Para conseguir una solución homogénea, se sometió Ia mezcla a agitación vigorosa a temperatura ambiente. A continuación, se pesaron 25 g de Ia disolución de alginato y se Ie añadieron 25 mi de una disolución enzimática, previamente concentrada por ultrafiltración, que contiene una fructosiltransferasa de Aspergillus aculeatus (Ia disolución enzimática de partida es comercial, Pectinex Ultra-SPL®, procedente de Novozymes A/S, Dinamarca). La actividad final de Ia mezcla fue de aproximadamente 135 U/ml. Tras conseguir una mezcla homogénea (mediante agitación suave 100 rpm durante 40 mn.), Ia mezcla se dejó reposar 1 hora para eliminar las burbujas de aire y porsteriormente, se goteó con ayuda de una bomba peristáltica LKB-Pump P-1 (Pfizer- Pharmacia, Canadá) sobre 250 mi de una disolución 0.2 M de CaCl2 en tampón acetato sódico 50 mM (pH 5.6), manteniendo esta disolución bajo agitación magnética (200 rpm).30 ml of a solution of 4% (w / v) SG-300 ® sodium alginate in water was prepared. To achieve a homogeneous solution, the mixture was subjected to vigorous stirring at room temperature. Then, 25 g of the alginate solution were weighed and 25 ml of an enzyme solution, previously concentrated by ultrafiltration, containing a fructyltransferase of Aspergillus aculeatus (the starting enzyme solution is commercial, Pectinex Ultra-SPL®, were added). from Novozymes A / S, Denmark). The final activity of the mixture was approximately 135 U / ml. After obtaining a homogeneous mixture (by gentle stirring 100 rpm for 40 minutes), the mixture was allowed to stand for 1 hour to eliminate air bubbles and subsequently, dripped with the aid of a peristaltic pump LKB-Pump P-1 (Pfizer- Pharmacia, Canada) on 250 ml of a 0.2 M solution of CaCl2 in 50 mM sodium acetate buffer (pH 5.6), keeping this solution under magnetic stirring (200 rpm).
La FIG. 1 muestra un esquema del proceso de inmovilización utilizado. El diámetro de las esferas obtenidas fue de 3 mm (a este biocatalizador se Ie denomina "biocatalizador-gel"). Los factores que determinaron el tamaño del biocatalizador fueron Ia velocidad de goteo controlada por Ia bomba peristáltica y el diámetro del extremo final del tubo por el que goteaba Ia disolución. Una vez formadas las esferas de alginato calcico, se mantuvieron durante al menos 20 min en Ia disolución de CaCl2 para asegurar Ia resistencia mecánica del inmovilizado. Posteriormente el inmovilizado se sometió a dos ciclos de lavado (incubando las esferas durante 40 min en tampón acetato sódico 0.01 M pH 5.6, a temperatura ambiente y con agitación magnética suave) con el fin de eliminar enzima que no hubiera quedado convenientemente atrapada en el gel de alginato. Las esferas de alginato con Ia enzima inmovilizada se almacenaron a 4 0C.FIG. 1 shows a scheme of the immobilization process used. The diameter of the obtained spheres was 3 mm (this biocatalyst is called the "gel biocatalyst"). The factors that determined the size of the biocatalyst were the drip speed controlled by the pump peristaltic and the diameter of the end end of the tube through which the solution dripped. Once the calcium alginate spheres were formed, they were kept for at least 20 min in the solution of CaCl2 to ensure the mechanical strength of the fixed assets. Subsequently, the fixed assets were subjected to two washing cycles (incubating the spheres for 40 min in 0.01 M sodium acetate buffer pH 5.6, at room temperature and with gentle magnetic stirring) in order to remove enzyme that had not been properly trapped in the gel of alginate. The alginate spheres with the immobilized enzyme were stored at 4 0 C.
La pérdida de actividad en el proceso de gelificación se midió a partir de Ia determinación de Ia actividad del sobrenadante y los lavados, estimándose en un 50 %. No obstante, esta actividad puede recuperarse y utilizarse para un posterior proceso de inmovilización. La actividad volumétrica del biocatalizador obtenido en este ejemplo de realización fue de 10 U/ml.The loss of activity in the gelation process was measured from the determination of the activity of the supernatant and the washings, being estimated at 50%. However, this activity can be recovered and used for a subsequent immobilization process. The volumetric activity of the biocatalyst obtained in this exemplary embodiment was 10 U / ml.
Para Ia determinación de Ia actividad fructosiltransferasa de Ia enzima soluble se valoró Ia liberación de azúcares reductores mediante el método del ácido dinitrosalicílico (DNS). El ensayo se realizó empleando una disolución de sacarosa de 100 mg/ml, en placas de 96 pocilios de fondo plano y volumen de 200 μl. En cada pocilio se depositaron 45 μl de solución de sacarosa 100 g/l (preparada en tampón acetato sódico 0.02 M, pH 5.6) y 5 μl de Ia disolución con enzima. La placa se incubó durante 20 minutos, a 35 0C y 200 rpm en un incubador con agitación orbital (Vortemp 56, Labnet). Para Ia curva de calibrado se prepararon pocilios con distintas concentraciones de glucosa (de 0 a 2 g/l). Tras los 20 minutos, se añadieron 50 μl de DNS 10 g/l y se incubó Ia placa durante 30 minutos a 80 0C. Se llevaron a cabo dos controles: el primero en ausencia de enzima y el segundo en ausencia de sacarosa. Una vez que Ia microplaca se enfrió a temperatura ambiente, se añadieron 150 μl de agua IiIIi-Q y se midió Ia absorbancia a 540 nm en un lector de microplacas. Todas las muestras se prepararon por triplicado para minimizar errores. Una unidad de actividad enzimática (U) se definió como aquella capaz de formar un μmol de azúcar reductor por minuto bajo las condiciones de reacción anteriormente descritas. Para determinar Ia actividad de Ia enzima inmovilizada se incubaron a 35° C, en un tubo eppendorf, 5 esferas de biocatalizador con 0.5 mi de disolución de sacarosa 100 g/l. La mezcla de reacción se mantuvo a 200 rpm durante 20 min. A continuación, se pipetearon 50 μl del sobrenadante a un pocilio de Ia microplaca. Se añadieron 50 μl de Ia disolución de DNS y se incubó Ia placa durante 30 min a 80° C. El resto del procedimiento es el mismo que el descrito anteriormente.For the determination of the fructosyltransferase activity of the soluble enzyme, the release of reducing sugars was assessed by means of the dinitrosalicylic acid (DNS) method. The test was performed using a sucrose solution of 100 mg / ml, in 96-well plates with a flat bottom and a volume of 200 μl. In each well, 45 μl of 100 g / l sucrose solution (prepared in 0.02 M sodium acetate buffer, pH 5.6) and 5 μl of the enzyme solution were deposited. The plate was incubated for 20 minutes at 35 0 C and 200 rpm in an orbital shaking incubator (Vortemp 56, Labnet). For the calibration curve, wells with different glucose concentrations (from 0 to 2 g / l) were prepared. After 20 minutes, 50 μl of DNS 10 g / l was added and the plate was incubated for 30 minutes at 80 0 C. Two controls were carried out: the first in the absence of enzyme and the second in the absence of sucrose. Once the microplate was cooled to room temperature, 150 µl of IiIIi-Q water was added and the absorbance at 540 nm was measured in a microplate reader. All samples were prepared in triplicate to minimize errors. One unit of enzymatic activity (U) was defined as one capable of forming one μmol of reducing sugar per minute under the reaction conditions described above. To determine the activity of the immobilized enzyme, 5 biocatalyst spheres with 0.5 ml of 100 g / l sucrose solution were incubated at 35 ° C in an eppendorf tube. The reaction mixture was maintained at 200 rpm for 20 min. Then, 50 µl of the supernatant was pipetted into a well of the microplate. 50 µl of the DNS solution was added and the plate was incubated for 30 min at 80 ° C. The rest of the procedure is the same as described above.
EJEMPLO 2: Obtención de fructooligosacáridos en un reactor en lecho fijo con el biocatalizador-gel de Ia fructosiltransferasa de Asperqillus aculeatus.EXAMPLE 2: Obtaining fructooligosaccharides in a fixed bed reactor with the biocatalyst-gel of the Asperqillus aculeatus fructosyltransferase gel.
El biocatalizador del EJEMPLO 1 se utilizó para empaquetar 25 mi de una columna de vidrio (Amersham Pharmacia XK 16/20), termostatizada a 35 0C y conectada a una bomba ¡socrática de doble pistón (modelo 515, Waters). El biorreactor se dispuso como un reactor de lecho fijo, con una corriente de entrada y otra de salida. La solución de alimentación contenía 600 g/l de sacarosa en tampón 0.02 M acetato sódico (pH 5.6), que se mantuvo con agitación magnética y termostatización a 35 0C.The biocatalyst of EXAMPLE 1 was used to pack 25 ml of a glass column (Amersham Pharmacia XK 16/20), thermostated at 35 0 C and connected to a Socratic double-piston pump (model 515, Waters). The bioreactor was arranged as a fixed bed reactor, with an input and an output current. The feed solution contained 600 g / l sucrose in 0.02 M sodium acetate buffer (pH 5.6), which was maintained with magnetic stirring and thermostatting at 35 ° C.
La disposición del sistema no incluía ninguna corriente de recirculación. La FIG. 2 representa los reactores de lecho fijo desarrollados para estos estudios.The system layout did not include any recirculation current. FIG. 2 represents the fixed bed reactors developed for these studies.
Después del tiempo de estabilización del sistema, se comenzaron a recoger muestras a Ia salida de Ia columna. Las muestras fueron centrifugadas durante 5 min a 4300 x g usando un eppendorf con filtro Durapore de 0.45 μm (Millipore), y analizadas por cromatografía líquida de alta resolución. Se empleó una bomba cuaternaria (Delta 600, Waters) y una columna de Phenomenex, Luna NH2 5 μm 100A (250 x 4.6 mm).After the system stabilization time, samples began to be collected at the exit of the column. The samples were centrifuged for 5 min at 4300 xg using an eppendorf with filter Durapore of 0.45 μm (Millipore), and analyzed by high performance liquid chromatography. A quaternary pump (Delta 600, Waters) and a Phenomenex column, Luna NH2 5 μm 100A (250 x 4.6 mm) were used.
La columna se mantuvo termostatizada a 25 0C gracias a un horno (Timberline Instruments, Inc). La fase móvil empleada fue una mezcla de acetonitrilo/agua, que fue desgasificada con un flujo continuo de Helio de 100 ml/min. Se empleó un detector evaporativo de light-scatteríng (ELSD, DDL 31 Eurosep) que operó a una temperatura de 85 0C y con N2 como gas nebulizador. El análisis de datos se llevó a cabo con el Software Millenium 32 de Waters. Los análisis se realizaron operando en modo gradiente según el siguiente programa de Ia Tabla 1 :The column was kept thermostatted at 25 0 C thanks to an oven (Timberline Instruments, Inc). The mobile phase used was a mixture of acetonitrile / water, which was degassed with a continuous flow of Helium of 100 ml / min. An evaporative light-scattering detector (ELSD, DDL Eurosep 31) operating at a temperature of 85 0 C and with N 2 nebulizer gas was used. Data analysis was carried out with Waters Millennium 32 Software. The analyzes were performed operating in gradient mode according to the following program in Table 1:
Figure imgf000016_0001
Figure imgf000016_0001
La FIG. 3 muestra resultados obtenidos para el reactor empaquetado con el biocatalizador-gel durante 750 horas. Concretamente se representa el porcentaje en peso referido al total de azúcares en Ia mezcla, a Io largo de 750 horas de funcionamiento continuo del reactor. Esta gráfica muestra Ia alta estabilidad operacional del biocatalizador. La Tabla 2 muestra Ia composición media, en gramos por litro, de Ia corriente de salida del reactor a un caudal de 0,84 ml/h, una vez alcanzado el estado estacionario. Tabla 2. Composición media de Ia corriente de salida del reactor empaquetado con el biocatalizador-gel, a un caudal de 0.84 ml/h.FIG. 3 shows results obtained for the reactor packed with the biocatalyst-gel for 750 hours. Specifically, the percentage by weight referred to the total sugars in the mixture, over 750 hours of continuous operation of the reactor, is represented. This graph shows the high operational stability of the biocatalyst. Table 2 shows the average composition, in grams per liter, of the reactor output current at a flow rate of 0.84 ml / h, once the steady state has been reached. Table 2. Average composition of the reactor output current packed with the gel biocatalyst, at a flow rate of 0.84 ml / h.
Figure imgf000017_0001
Figure imgf000017_0001
La FIG. 4 muestra cómo Ia composición de Ia salida del reactor puede regularse variando el tiempo de residencia, que en definitiva se controla a través del caudal. Como se aprecia en Ia figura de Ia parte superior, un mayor caudal representa un menor tiempo de residencia y en consecuencia una mayor proporción de los productos de bajo grado de polimerización (en particular, 1-kestosa) en Ia mezcla de Ia salida. Por el contrario, un menor caudal implica un mayor tiempo de residencia y por ende una mayor proporción de los FOS con grado de polimerización más elevado: tri-, tetra-, penta- y hexasacáridos. Asimismo, un mayor caudal supone un menor porcentaje de sacarosa convertida en productos. Por tanto, Ia productividad volumétrica del reactor (gramos de FOS totales por día y por litro de reactor) se vio alterada por el caudal de trabajo, como se indica gráficamente en Ia parte inferior de Ia FIG. 4.FIG. 4 shows how the composition of the reactor outlet can be regulated by varying the residence time, which is ultimately controlled through the flow rate. As can be seen in the figure of the upper part, a greater flow represents a shorter residence time and consequently a greater proportion of the products of low degree of polymerization (in particular, 1-kestose) in the mixture of the outlet. On the contrary, a lower flow implies a longer residence time and therefore a greater proportion of the FOS with a higher degree of polymerization: tri-, tetra-, penta- and hexasaccharides. Also, a higher flow rate means a lower percentage of sucrose converted into products. Therefore, the volumetric productivity of the reactor (grams of total FOS per day and per liter of reactor) was altered by the work flow, as indicated graphically in the lower part of FIG. Four.
EJEMPLO 3: Obtención de biocatalizador tipo DALGEE de Ia fructosiltransferasa de Asperqillus aculeatus.EXAMPLE 3: Obtaining a DALGEE type biocatalyst of the Asperqillus aculeatus fructosyltransferase.
El biocatalizador del EJEMPLO 1 se dejó secar al aire en un cristalizador de vidrio, a 35 0C, durante 3 días. Tras este tiempo, el diámetro de las esferas obtenidas fue de 1 mm, Io cual supone una reducción del volumen del biocatalizador del 96% (suponiendo las partículas esféricas). La pérdida de actividad en el proceso de secado, por partícula de biocatalizador, se estimó en un 50 %. Sin embargo, Ia actividad volumétrica del biocatalizador seco fue de 140 U/ml, por Io que se produjo un aumento de aproximadamente 13 o 14 veces en Ia actividad volumétrica con respecto al biocatalizador-gel.The biocatalyst of Example 1 was allowed to air dry in a crystallizing glass, 35 0 C, for 3 days. After this time, the diameter of the Spheres obtained were 1 mm, which means a reduction in the volume of the biocatalyst of 96% (assuming spherical particles). The loss of activity in the drying process, per biocatalyst particle, was estimated at 50%. However, the volumetric activity of the dry biocatalyst was 140 U / ml, so there was an increase of approximately 13 or 14 times in the volumetric activity with respect to the biocatalyst-gel.
La FIG. 5 muestra el aspecto de las esferas de biocatalizador, antes y después del proceso de secado. Para realizar un estudio morfológico de los soportes, se empleó un microscopio electrónico de barrido (SEM) HITACHI TM1000 con un potencial acelerador de electrones de 15 kV, y un detector semiconductor BSE de alta sensibilidad. La FIG. 6 muestra Ia morfología de las partículas de biocatalizador seco. En Ia Tabla 1 se recogen los aspectos más sobresalientes del biocatalizador tipo DALGEE. Las propiedades texturales fueron estudiadas mediante porosimetría de mercurio, usando un porosímetro Fisons Instruments Pascal 140/240. Las muestras fueron incubadas a 60 0C durante 24 h antes del análisis. El valor del ángulo de contacto de Hg (141°) y Ia tensión superficial (484 mN/m) fueron seleccionados para evaluar los datos Presión/volumen por Ia ecuación Washburn, asumiendo un modelo de poro cilindrico. La distribución del tamaño de partícula fue determinada por análisis de Ia curva de intrusión. A partir de Ia porosidad del material y asumiendo partículas esféricas, el factor de empaquetamiento y Ia distribución del tamaño de partícula se calcularon de acuerdo con Ia teoría de Mayer- Stowe. El contenido en agua se determinó por el método Karl-Fischer. Se observa que los DALGEEs presentan un volumen de poro muy pequeño (0.072 cm3/g), con un máximo en Ia distribución de tamaños de poro de 8.3 nm. Este dato explica Ia baja lixiviación de los biocatalizadores tipo DALGEE, ya que Ia mayor parte de las enzimas no van a poder escapar a través de los poros. Tabla 3. Características más destacadas del biocatalizador DALGEE.FIG. 5 shows the appearance of the biocatalyst spheres, before and after the drying process. To perform a morphological study of the supports, a HITACHI TM1000 scanning electron microscope (SEM) with a potential electron accelerator of 15 kV, and a high sensitivity BSE semiconductor detector was used. FIG. 6 shows the morphology of the dry biocatalyst particles. Table 1 shows the most outstanding aspects of the DALGEE type biocatalyst. Textural properties were studied by mercury porosimetry, using a Fisons Instruments Pascal 140/240 porosimeter. Samples were incubated at 60 0 C for 24 h before analysis. The value of the contact angle of Hg (141 °) and the surface tension (484 mN / m) were selected to evaluate the Pressure / volume data by the Washburn equation, assuming a cylindrical pore model. The particle size distribution was determined by analysis of the intrusion curve. From the porosity of the material and assuming spherical particles, the packing factor and the particle size distribution were calculated according to the Mayer-Stowe theory. Water content was determined by the Karl-Fischer method. It is observed that the DALGEEs have a very small pore volume (0.072 cm 3 / g), with a maximum in the pore size distribution of 8.3 nm. This data explains the low leaching of the DALGEE type biocatalysts, since most of the enzymes will not be able to escape through the pores. Table 3. Most outstanding characteristics of the DALGEE biocatalyst.
Figure imgf000019_0001
Figure imgf000019_0001
EJEMPLO 4: Obtención de fructooligosacáridos en un reactor en lecho fijo con DALGEE de Ia fructosiltransferasa de Aspergillus aculeatus.EXAMPLE 4: Obtaining fructooligosaccharides in a fixed bed reactor with DALGEE of the Aspergillus aculeatus fructosyltransferase.
El biocatalizador del EJEMPLO 3 se utilizó para empaquetar una columna de 1 mi (0.7 x 2.5 cm), termostatizada a 35 0C y conectada a una bomba ¡socrática de doble pistón. La solución de alimentación contenía 600 g/l de sacarosa en tampón 0.02 M acetato sódico (pH 5.6), que se mantuvo con agitación magnética y termostatización a 35 0C. La disposición del sistema no incluía ninguna corriente de recirculación. La FIG. 2 representa los reactores de lecho fijo desarrollados para estos ensayos. Después del tiempo de estabilización del sistema, se comenzó a recoger muestras a Ia salida de Ia columna. Las muestras fueron centrifugadas durante 5 min a 4300 x g usando un tubo Eppendorf con filtro Durapore® de 0.45 μm (Millipore), y analizadas por cromatografía líquida de alta resolución como se describe en el EJEMPLO 2. La FIG. 7 muestra un cromatograma típico de Ia mezcla de reacción a Ia salida del reactor que contiene el biocatalizador DALGEE.The biocatalyst of EXAMPLE 3 was used to pack a 1 ml column (0.7 x 2.5 cm) thermostated at 35 0 C and connected to a pump socratic double piston. The feed solution contained 600 g / l of sucrose in 0.02 M sodium acetate buffer (pH 5.6), which was maintained with magnetic stirring and thermostatting at 35 ° C. The system arrangement did not include any recirculation current. FIG. 2 represents the fixed bed reactors developed for these tests. After the system stabilization time, samples began to be collected at the exit of the column. The samples were centrifuged for 5 min at 4300 xg using an Eppendorf tube with 0.45 µm Durapore ® filter (Millipore), and analyzed by high performance liquid chromatography as described in EXAMPLE 2. FIG. 7 shows a typical chromatogram of the reaction mixture at the outlet of the reactor containing the DALGEE biocatalyst.
La FIG. 4 muestra cómo Ia composición de Ia salida del reactor puede regularse variando el tiempo de residencia, que en definitiva se controla a través del caudal. Como se aprecia en Ia figura de Ia parte superior, un mayor caudal representa un menor tiempo de residencia y en consecuencia una mayor proporción de los productos de bajo grado de polimerización (en particular, 1-kestosa) en Ia mezcla de Ia salida. Por el contrario, un menor caudal implica un mayor tiempo de residencia y por ende una mayor proporción de los FOS con grado de polimerización más elevado: tri-, tetra-, penta- y hexasacáridos. Asimismo, un mayor caudal supone un menor porcentaje de sacarosa convertida en productos. Por tanto, Ia productividad volumétrica del reactor (gramos de FOS totales por día y por litro de reactor) se vio alterada por el caudal de trabajo, como se indica gráficamente en Ia parte inferior de Ia FIG. 4.FIG. 4 shows how the composition of the reactor outlet can be regulated by varying the residence time, which is ultimately controlled through the flow rate. As can be seen in the figure of the upper part, a greater flow represents a shorter residence time and consequently a greater proportion of the products of low degree of polymerization (in particular, 1-kestose) in the mixture of the outlet. On the contrary, a lower flow implies a longer residence time and therefore a greater proportion of the FOS with a higher degree of polymerization: tri-, tetra-, penta- and hexasaccharides. Also, a higher flow rate means a lower percentage of sucrose converted into products. Therefore, the volumetric productivity of the reactor (grams of total FOS per day and per liter of reactor) was altered by the work flow, as indicated graphically in the lower part of FIG. Four.
En Ia parte inferior de Ia FIG. 8, se muestra Ia composición de Ia corriente de salida en %(p/p), referido al peso total de azúcares en Ia mezcla, a Io largo de 700 horas de funcionamiento continuo del reactor empaquetado con el biocatalizador DALGEE. Esta gráfica muestra Ia alta estabilidad operacional del biocatalizador, ya se observa cómo Ia actividad catalítica del reactor permanece prácticamente invariable a Io largo de casi todo el ensayo. En Ia Tabla 4 se recogen las concentraciones medias de los distintos carbohidratos a Ia salida del reactor, una vez alcanzado el estado estacionario, utilizando un caudal de 0.6 ml/h. El reactor resultó ser estable durante al menos 750 h de trabajo. La productividad obtenida se estimó en 3680 g FOS por día y litro de reactor, tal como se puede apreciar en Ia FIG. 9.In the lower part of FIG. 8, the composition of the output current in% (w / w), based on the total weight of sugars in the mixture, is shown over 700 hours of continuous operation of the reactor packed with the DALGEE biocatalyst. This graph shows the high operational stability of the biocatalyst, it is already observed how the catalytic activity of the reactor remains virtually unchanged throughout most of the test. Table 4 shows the average concentrations of the different carbohydrates at the outlet of the reactor, once the steady state is reached, using a flow rate of 0.6 ml / h. The reactor turned out to be stable for at least 750 hours of work. The productivity obtained was estimated at 3680 g FOS per day and liter of reactor, as can be seen in FIG. 9.
Tabla 4. Composición media de Ia corriente de salida del reactor empaquetado con el biocatalizador DALGEE, a un caudal de 0.6 ml/h.Table 4. Average composition of the reactor output current packed with the DALGEE biocatalyst, at a flow rate of 0.6 ml / h.
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000020_0001
Figure imgf000021_0001
De esta forma, Ia FIG. 9 muestra las productividades volumétricas, expresada en gramos de FOS por litro de reactor y día, para las dos formas de biocatalizador, biocatalizador-gel y DALGEE, utilizando un caudal de 0.84 ml/h para el biocatalizador-gel y 0.60 ml/h para el DALGEE, durante las 750 horas de operación del biorreactor. Se aprecia que en el caso del biocatalizador-gel, Ia productividad es próxima a 100 g de FOS por litro de reactor y día, mientras que con el tipo DALGEE, alcanza un valor en torno a los 4000 g FOS por litro de reactor y día.In this way, FIG. 9 shows the volumetric productivity, expressed in grams of FOS per liter of reactor and day, for the two forms of biocatalyst, biocatalyst-gel and DALGEE, using a flow rate of 0.84 ml / h for the biocatalyst-gel and 0.60 ml / h for the DALGEE, during the 750 hours of operation of the bioreactor. It is appreciated that in the case of the biocatalyst-gel, the productivity is close to 100 g of FOS per liter of reactor and day, while with the DALGEE type, it reaches a value around 4000 g FOS per liter of reactor and day .
EJEMPLO 5: Estudio de Ia lixiviación del biocatalizador-gel y DALGEE.EXAMPLE 5: Study of the leaching of the biocatalyst-gel and DALGEE.
Se sometió a los biocatalizadores inmovilizados de Ia fructosiltransferasa de Aspergillus aculeatus a sucesivos ciclos de lavado con solución tampón (acetato sódico 0.02 M pH 5.6). En cada ciclo se incubaban 10 partículas de biocatalizador durante 1 h, a 600 rpm y 35 0C, con 500 μl de tampón. Tras cada lavado se extraían los 500 μl de solución, para evaluar Ia enzima liberada, y se sustituían por solución tampón fresca para el siguiente ciclo. El porcentaje de lixiviación tras el primer ciclo fue del 7.5% para el biocatalizador-gel y del 5% para el DALGEE. A partir del tercer ciclo, Ia lixiviación puede considerarse despreciable, como se representa en Ia FIG. 10.The immobilized biocatalysts of Aspergillus aculeatus fructosyltransferase were subjected to successive wash cycles with buffer solution (0.02 M sodium acetate pH 5.6). In each cycle 10 biocatalyst particles were incubated for 1 h at 600 rpm and 35 0 C, with 500 .mu.l of buffer. After each wash, the 500 μl of solution was extracted, to evaluate the enzyme released, and replaced by fresh buffer solution for the next cycle. The leaching percentage after the first cycle was 7.5% for the biocatalyst-gel and 5% for the DALGEE. From the third cycle, the leaching can be considered negligible, as shown in FIG. 10.
EJEMPLO 6: Obtención de biocatalizador tipo DALGEE de Ia beta- fructofuranosidasa de Rhodotorula qracilis ATCC1416.EXAMPLE 6: Obtaining a DALGEE type biocatalyst of the beta-fructofuranosidase from Rhodotorula qracilis ATCC1416.
Sobre 30 g de Ia disolución de alginato, preparada según se describe en el EJEMPLO 1 , se añadieron 30 mi de una disolución enzimática, de una beta-fructofuranosidasa extracelular de Rhodotorula qracilis ATCC1416. La producción de esta beta-fructofuranosidasa se llevó a cabo en cultivos de Rhodotorula gracilis ATCC1416 crecidos en medio mínimo para levaduras suplementado con maltosa. Los cultivos se realizaron en matraces de vidrio incubados a una temperatura comprendida entre 28-30 0C y con agitación orbital constante de 180-235 rpm. Las condiciones óptimas de crecimiento fueron 30 0C y 235 rpm. Se obtuvo Ia fracción libre de células por centrifugación, y se concentró utilizando un sistema de filtración tangencial (filtro de 30 kDa). Finalmente, el concentrado se sometió a diálisis frente a HCI-Tris 20 mM pH 7 durante 2 horas a una temperatura de 4 0C.About 30 g of the alginate solution, prepared as described in the EXAMPLE 1, 30 ml of an enzymatic solution, of an extracellular beta-fructofuranosidase of Rhodotorula qracilis ATCC1416 was added. The production of this beta-fructofuranosidase was carried out in cultures of Rhodotorula gracilis ATCC1416 grown in minimal medium for yeasts supplemented with maltose. The cultures were carried out in glass flasks incubated at a temperature between 28-30 0 C and with constant orbital agitation of 180-235 rpm. The optimal growth conditions were 30 0 C and 235 rpm. The cell-free fraction was obtained by centrifugation, and concentrated using a tangential filtration system (30 kDa filter). Finally, the concentrate was dialyzed against 20 mM Tris-HCl pH 7 for 2 hours at 4 0 C.
La actividad final de Ia mezcla fue de aproximadamente 12 U/ml. La preparación del biocatalizador-gel se realizó tal como se describe en elThe final activity of the mixture was approximately 12 U / ml. The preparation of the biocatalyst-gel was performed as described in the
EJEMPLO 1. La pérdida de actividad en el proceso de gelificación se midió a partir de Ia determinación de Ia actividad del sobrenadante y los lavados, estimándose en un 60 %. La actividad volumétrica del biocatalizador obtenido en este ejemplo de realización fue de 0.8 U/ml. El biocatalizador obtenido se sometió a un proceso de secado como se describe en elEXAMPLE 1. The loss of activity in the gelation process was measured from the determination of the activity of the supernatant and washes, estimated at 60%. The volumetric activity of the biocatalyst obtained in this exemplary embodiment was 0.8 U / ml. The biocatalyst obtained was subjected to a drying process as described in the
EJEMPLO 3 dando lugar a un biocatalizador tipo DALGEE. La pérdida de actividad en el proceso de secado, por partícula de biocatalizador, se estimó en un 45 %. La actividad volumétrica del biocatalizador seco fue deEXAMPLE 3 giving rise to a DALGEE type biocatalyst. The loss of activity in the drying process, per biocatalyst particle, was estimated at 45%. The volumetric activity of the dry biocatalyst was
15 U/ml, por Io que se produjo un aumento de aproximadamente 18 veces en Ia actividad volumétrica con respecto al biocatalizador-gel.15 U / ml, so there was an increase of approximately 18 times in the volumetric activity with respect to the biocatalyst-gel.
EJEMPLO 7: Hidrólisis de sacarosa en un reactor en lecho fijo con DALGEE de Ia beta-fructofuranosidasa de Rhodotorula gracilis ATCC1416.EXAMPLE 7: Hydrolysis of sucrose in a fixed bed reactor with DALGEE of the beta-fructofuranosidase of Rhodotorula gracilis ATCC1416.
El biocatalizador del EJEMPLO 6 se utilizó para empaquetar una columna de 1.5 mi (0.6 x 5.3 cm), termostatizada a 35 0C y conectada a una bomba ¡socrática de doble pistón. La solución de alimentación contenía 600 g/l de sacarosa en tampón 0.02 M acetato sódico (pH 5.6), que se mantuvo con agitación magnética y termostatización a 35 0C. Después del tiempo de estabilización del sistema, se comenzó a recoger muestras a Ia salida de Ia columna. Las muestras fueron analizadas como se describe en el EJEMPLO 2. Utilizando un caudal de 0.6 ml/h, Ia composición media a Ia salida del reactor fue de: 124 g/l fructosa, 147 g/l glucosa, 317 g/l sacarosa, 12 g/l 6-kestosa. La FIG. 12 muestra un cromatograma típico de Ia mezcla de reacción a Ia salida del reactor que contiene el biocatalizador DALGEE. El reactor empaquetado con el biocatalizador DALGEE beta- fructofuranosidasa de R. qracilis ATCC1416 mantuvo su actividad inicial durante al menos 48 horas. La productividad obtenida se estimó en 2600 g de azúcares reductores por día y litro de reactor. The biocatalyst of EXAMPLE 6 was used to pack a 1.5 ml (0.6 x 5.3 cm) column, thermostated at 35 0 C and connected to a pump Socratic double piston. The feed solution contained 600 g / l of sucrose in 0.02 M sodium acetate buffer (pH 5.6), which was maintained with magnetic stirring and thermostatting at 35 0 C. After the system stabilization time, samples were collected at Ia output of the column. The samples were analyzed as described in EXAMPLE 2. Using a flow rate of 0.6 ml / h, the average composition at the outlet of the reactor was: 124 g / l fructose, 147 g / l glucose, 317 g / l sucrose, 12 g / l 6-kestosa. FIG. 12 shows a typical chromatogram of the reaction mixture at the outlet of the reactor containing the DALGEE biocatalyst. The reactor packaged with the biocatalyst DALGEE beta-fructofuranosidase from R. qracilis ATCC1416 maintained its initial activity for at least 48 hours. The productivity obtained was estimated at 2600 g of reducing sugars per day and liter of reactor.

Claims

REIVINDICACIONES
1. Procedimiento de obtención de un biocatalizador, que comprende: a. inmovilizar una enzima fúngica por inclusión en un gel de alginato calcico. b. secar el biocatalizador inmovilizado obtenido en el paso (a).1. Procedure for obtaining a biocatalyst, comprising: a. immobilize a fungal enzyme by inclusion in a calcium alginate gel. b. Dry the immobilized biocatalyst obtained in step (a).
2. Procedimiento según Ia reivindicación 1 , donde el secado se lleva a cabo a una temperatura de entre 30 y 50 0C.2. Process according to claim 1, wherein the drying is carried out at a temperature of between 30 and 50 0 C.
3. Procedimiento según cualquiera de las reivindicaciones 1 o 2, donde Ia enzima es fructosiltransferasa o β-fructofuranosidasa.3. Method according to any of claims 1 or 2, wherein the enzyme is fructosyltransferase or β-fructofuranosidase.
4. Procedimiento según Ia reivindicación 3, donde Ia fructosiltransferasa se obtiene de un hongo de género Aspergillus.4. Method according to claim 3, wherein the fructosyltransferase is obtained from a fungus of the Aspergillus genus.
5. Procedimiento según Ia reivindicación 4, donde el hongo es de Ia especie Aspergillus aculeatus.5. Method according to claim 4, wherein the fungus is of the Aspergillus aculeatus species.
6. Procedimiento según Ia reivindicación 3, donde Ia β-fructofuranosidasa obtiene de un hongo de género Rhodotorula.6. Method according to claim 3, wherein the β-fructofuranosidase is obtained from a fungus of the Rhodotorula genus.
7. Procedimiento según Ia reivindicación 6, donde el hongo es de Ia especie Rhodotorula gracilis.7. Method according to claim 6, wherein the fungus is of the Rhodotorula gracilis species.
8. Biocatalizador inmovilizado obtenible por el procedimiento descrito en cualquiera de las reivindicaciones 1 a 7 y que comprende: una enzima fúngica inmovilizada en un gel de alginato calcico.8. Immobilized biocatalyst obtainable by the method described in any one of claims 1 to 7 and comprising: a fungal enzyme immobilized in a calcium alginate gel.
9. Uso del biocatalizador según Ia reivindicación 8, para Ia hidrólisis de carbohidratos. 9. Use of the biocatalyst according to claim 8, for carbohydrate hydrolysis.
10. Uso del biocatalizador según Ia reivindicación 9, donde los carbohidratos son sacarosa, lactosa, maltosa, glucosa o almidón.10. Use of the biocatalyst according to claim 9, wherein the carbohydrates are sucrose, lactose, maltose, glucose or starch.
11. Uso del biocatalizador según cualquiera de las reivindicaciones 9 o 10, para Ia obtención de fructooligosacaridos.11. Use of the biocatalyst according to any of claims 9 or 10, for obtaining fructooligosaccharides.
12. Uso del biocatalizador según cualquiera de las reivindicaciones 9 o 10, para Ia obtención de jarabe de fructosa.12. Use of the biocatalyst according to any of claims 9 or 10, for obtaining fructose syrup.
13. Procedimiento de hidrólisis de carbohidratos, que comprende: a. empaquetar un biocatalizador inmovilizado, descrito en Ia reivindicación 8, en un reactor de lecho fijo; b. alimentar el reactor continuo de lecho fijo del paso (a) con una disolución de carbohidrato en una concentración de entre 500 g/l y 700 g/l.13. Carbohydrate hydrolysis process, comprising: a. packaging an immobilized biocatalyst, described in claim 8, in a fixed bed reactor; b. feed the continuous fixed bed reactor of step (a) with a carbohydrate solution in a concentration between 500 g / l and 700 g / l.
14. Procedimiento según Ia reivindicación 13, donde Ia temperatura del reactor está entre 30 0C y 40 0C.14. Method according to claim 13, wherein the reactor temperature is between 30 0 C and 40 0 C.
15. Procedimiento según cualquiera de las reivindicaciones 13 o 14, donde el carbohidrato es sacarosa.15. Method according to any of claims 13 or 14, wherein the carbohydrate is sucrose.
16. Procedimiento según Ia reivindicación 15, donde el biocatalizador es una fructosiltransferasa inmovilizada.16. The method according to claim 15, wherein the biocatalyst is an immobilized fructosyltransferase.
17. Procedimiento según Ia reivindicación 16, donde en Ia hidrólisis se obtiene fructooligosacaridos que se seleccionan de Ia lista que comprende trisacáridos, tetrasacáridos, pentasacáridos, hexasacáridos o cualquiera de sus combinaciones.17. Method according to claim 16, wherein in the hydrolysis fructooligosaccharides are obtained which are selected from the list comprising trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides or any combination thereof.
18. Procedimiento según Ia reivindicación 15, donde el biocatalizador es una β-fructofuranosidasa inmovilizada.18. Method according to claim 15, wherein the biocatalyst is an immobilized β-fructofuranosidase.
19. Procedimiento según Ia reivindicación 18, donde en Ia hidrólisis se obtiene jarabe de fructosa. 19. Method according to claim 18, wherein in the hydrolysis fructose syrup is obtained.
PCT/ES2010/070104 2009-03-11 2010-02-24 Immobilised biocatalyst based on alginate for the biotransformation of carbohydrates WO2010103150A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ESP200930001 2009-03-11
ES200930001A ES2348990B1 (en) 2009-03-11 2009-03-11 IMMOBILIZED BIOCATALIZER BASED ON ALGINATE FOR THE BIOTRANSFORMATION OF CARBOHYDRATES.

Publications (1)

Publication Number Publication Date
WO2010103150A1 true WO2010103150A1 (en) 2010-09-16

Family

ID=42727827

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/ES2010/070104 WO2010103150A1 (en) 2009-03-11 2010-02-24 Immobilised biocatalyst based on alginate for the biotransformation of carbohydrates

Country Status (2)

Country Link
ES (1) ES2348990B1 (en)
WO (1) WO2010103150A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014044230A1 (en) 2012-09-18 2014-03-27 Centro De Ingenieria Genetica Y Biotecnologia Method for obtaining 1-kestose

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR900003710B1 (en) * 1988-06-09 1990-05-30 주식회사 미원 Method for immobilising of fracto transferase
US5215905A (en) * 1989-12-29 1993-06-01 Miwon Co., Ltd. Immobilization of fructosyltransferase on a basic, porous anion-exchange resin
ES2255847A1 (en) * 2004-12-16 2006-07-01 Consejo Superior Investig. Cientificas Novel enzyme for the production of prebiotic oligosaccharides
WO2007017537A1 (en) * 2005-07-29 2007-02-15 Universidad Autonoma De Madrid Novel enzyme with fructofuranosidase activity, which is used to obtain prebiotic oligosaccharides
WO2007074187A1 (en) * 2005-12-26 2007-07-05 Universidad Autónoma de Madrid Novel fructofuranosidase activity for obtaining the prebiotic oligosaccharide 6-kestose

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR900003710B1 (en) * 1988-06-09 1990-05-30 주식회사 미원 Method for immobilising of fracto transferase
US5215905A (en) * 1989-12-29 1993-06-01 Miwon Co., Ltd. Immobilization of fructosyltransferase on a basic, porous anion-exchange resin
ES2255847A1 (en) * 2004-12-16 2006-07-01 Consejo Superior Investig. Cientificas Novel enzyme for the production of prebiotic oligosaccharides
WO2007017537A1 (en) * 2005-07-29 2007-02-15 Universidad Autonoma De Madrid Novel enzyme with fructofuranosidase activity, which is used to obtain prebiotic oligosaccharides
WO2007074187A1 (en) * 2005-12-26 2007-07-05 Universidad Autónoma de Madrid Novel fructofuranosidase activity for obtaining the prebiotic oligosaccharide 6-kestose

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CRUZ, R. ET AL.: "Production of Fructooligosaccharides by the Mycelia of Aspergillus japonicus Immobilized in Calcium Alginate", BIORESOURCE TECHNOLOGY, vol. 65, no. 1-2, 1998, pages 139 - 143 *
DATABASE WPI Week 199123, Derwent World Patents Index; AN 1991-169323 *
GHAZI, I. ET AL.: "Immobilisation of Fructosyltransferase from Aspergillus aculeatus on Epoxy-Activated Sepabeads EC for the Synthesis of Fructo-Oligosaccharides", JOURNAL OF MOLECULAR CATALYSIS B ENZYMATIC, vol. 35, no. 1-3, 2005, pages 19 - 27 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014044230A1 (en) 2012-09-18 2014-03-27 Centro De Ingenieria Genetica Y Biotecnologia Method for obtaining 1-kestose
US10131927B2 (en) 2012-09-18 2018-11-20 Centro de Ingenieria Genética y Biotecnologia Method for obtaining 1-kestose
US10982246B2 (en) 2012-09-18 2021-04-20 Centro de Ingenierla Genética y Biotecnologia Method for obtaining 1-kestose

Also Published As

Publication number Publication date
ES2348990B1 (en) 2011-10-10
ES2348990A1 (en) 2010-12-21

Similar Documents

Publication Publication Date Title
Zhang et al. Bioinspired preparation of polydopamine microcapsule for multienzyme system construction
Górecka et al. Immobilization techniques and biopolymer carriers
Serra et al. A comparative study of periodic mesoporous organosilica and different hydrophobic mesoporous silicas for lipase immobilization
Castro et al. Screening and selection of potential carriers to immobilize Aureobasidium pullulans cells for fructo-oligosaccharides production
Ariaeenejad et al. An efficient nano-biocatalyst for lignocellulosic biomass hydrolysis: Xylanase immobilization on organically modified biogenic mesoporous silica nanoparticles
CN109554360B (en) Method for embedding thalli by using sodium alginate composite material
Güleç et al. Immobilization of Aspergillus oryzae β-galactosidase on low-pressure plasma-modified cellulose acetate membrane using polyethyleneimine for production of galactooligosaccharide
Singh et al. Carboxymethyl tamarind gum–silica nanohybrids for effective immobilization of amylase
Akay et al. An injectable alginate-based hydrogel for microfluidic applications
Shakeri et al. Efficient decolorization of an anthraquinone dye by recombinant dye-decolorizing peroxidase (rDyP) immobilized in silica-based mesocellular foam
Nawawi et al. A porous-cross linked enzyme aggregates of maltogenic amylase from Bacillus lehensis G1: Robust biocatalyst with improved stability and substrate diffusion
CN101397580A (en) Method for preparing low molecular weight chitosan under steady magnetic field condition
Xu et al. Immobilization of cellulase proteins on zeolitic imidazolate framework (ZIF-8)/polyvinylidene fluoride hybrid membranes
Zhang et al. Polymer–inorganic microcapsules fabricated by combining biomimetic adhesion and bioinspired mineralization and their use for catalase immobilization
CN102559648A (en) Immobilized enzyme using modified epoxy resin as carrier and preparation method and application thereof
Nikolić et al. Enzyme immobilization using two processing methods onto silica core-shell particles
Zhao et al. Immobilization of Papain on the Mesoporous Molecular Sieve MCM‐48
ES2348990B1 (en) IMMOBILIZED BIOCATALIZER BASED ON ALGINATE FOR THE BIOTRANSFORMATION OF CARBOHYDRATES.
Kamal et al. Immobilization of glucose isomerase onto radiation synthesized P (AA-co-AMPS) hydrogel and its application
Begum et al. Influence of silica architecture on the catalytic activity of immobilized glucose oxidase
CN107400693B (en) Method for producing astaxanthin by self-assembled siliceous body electrostatic spinning fibrous membrane immobilized cells
WO2007074187A1 (en) Novel fructofuranosidase activity for obtaining the prebiotic oligosaccharide 6-kestose
Kochanė et al. Starch hydrolysis using maltogenase immobilized via different techniques
Tümtürk et al. Covalent immobilization of a α‐amylase onto poly (methyl methacrylate‐2‐hydroxyethyl methacrylate) microspheres and the effect of ca2+ ions on the enzyme activity
Kovalenko et al. Catalytic properties of glucoamylase immobilized on synthetic carbon material Sibunit

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10750399

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10750399

Country of ref document: EP

Kind code of ref document: A1