ES2214933B1 - HYDROLYSIS OF LACTOSE WITH IMMOBILIZED THERMORESISTENT LACTASE AND ITS METHOD OF OBTAINING. - Google Patents

Abstract

Hydrolysis of lactose with immobilized heat-resistant lactase and its method of production. A lactose hydrolysis process catalyzed by immobilized-stabilized recombinant derivatives of {be} -galactosidase from Thermus sp. (strain t2) fused with poly-histidine tails (imac / {be} -galactosidase) cloned in E. coli CECT 5970. The procedure is of interest to the dairy and biotechnology industry by applying in different dairy derivatives, be it milk, whey or any other compound with lactose to avoid problems of intolerance and organoleptic improvement. In relation to the Environment, eliminate the highly polluting spill of lactose (a very reducing sugar), transforming this waste into a product of a certain added value (a glucose and fructose syrup). The enzyme is heat resistant has no impurities and its preparation procedure allows its purification in one step and is done with a mesophilic microorganism.

Description

Lactose hydrolysis with lactase thermo-resistant immobilized and its method of obtaining.

Technical sector

The claimed invention is framed mainly within the agri-food area, although it is also It relates to the biotechnological and environmental areas. In the area agrifood is intended to reduce or eliminate lactose from the milk, dairy products and dairy by-products, to avoid intolerance problems and improve organoleptic properties, and improve fermentation processes, using very tools various related to biotechnology and technology enzymatic Regarding the environmental impact, it is intended eliminate the highly polluting spill of lactose (a sugar very reductive), transforming this waste into a product of a certain added value (a glucose and fructose syrup).

State of the art

The β-D-galactosidase (EC 3.2.1.23), also called lactase, has as its primary function the lactose hydrolysis in D-galactose and D-glucose Therefore, the fundamental application of β-D-galactosidases are circumscribes to the field of dairy products and by-products [Pivarnik, L.F .; Senecal, A.G. and Rand, A.G. 1995. "Hydrolytic and transgalactosylic activities of commercial β-galactosidase (lactase) in food processing ". Adv. Food Nutrit. Res. 38, 1-102]. In addition, this enzyme has transgalactosylase activity and is used for the synthesis of galactosyl oligosaccharides, compounds with a growing interest in the agri-food area and Pharmaceutical

As a non-enzymatic alternative to hydrolysis with β-D-galactosidases it You can use chemical hydrolysis, but it produces no effects desirable (e.g., loss of nutrients, organoleptic defects, etc.) [Gekas, V. and López-Leiva, M. 1985. "Hydrolisis of lactose: A literature review". Process Biochem 20, 1-12].

The β-galactosidases are used in the agri-food industry to reduce the content in lactose from dairy products and derivatives, which has a varied interest.

First, the most complex problem would be make these foods accessible to those affected by hypolactasia, which constitute 70% of the world's population, for which we will need to hydrolyze 100% milk lactose [Savaiano, D.A. and Kotz, C. 1988. "Recent advances in the management of lactose intolerance ". Contemp. Nutr. 13, 10; Wolin, M.J. 1981. "Fermentation in the numen and human large intestine. "Science 213, 1463-1468; Phillips, M.C. and Bríggs, G.M. 1975. "Milk and its role in the American diet ". J. Dairy Sci. 58, 1751-1763; Birge, S.J .; Keutmann, H.T .; Cuatrecases, P. and Whedon, G.D. 1963 "Osteoporosis, intestinal lactase deficiency and low dietary calcium intake ". N. Engl. J. Med. 276, 445-448].

In other uses, the total hydrolysis requirement may be diminished. For example, the use of β-galactosidases to revalue dairy sera by transforming them into glucosegalactose syrups [Mahoney, RR 1985. "Modification of lactose and lactosecontaining dairy products with β-galactosidase". In " Developments in Dairy Chemistry: Lactose and minor constituents ". Fox, F. (Ed), pp. 69-108. Elsevier, New York; Short, JL 1975. "Prospects for the utilization of deproteinated whey in New Zealand-A review." NZJ Dairy Sci. Technol. 13,181-194], avoiding what - on the other hand - would be a serious environmental problem, due to the high biological oxygen demand of the lactose of the mentioned sera, and since only half of the one produced is processed by pouring the rest into river channels [Pivarnik, LF; Senecal, AG and Rand, AG 1995. "Hydrolytic and transgalactosylic activities of commercial? -Galactosidase (lactase) in food processing". Adv. Food Nutrit Res. 38, 1-102] (to get an idea of the problem, 1 kg of cheese requires, on average, 10 L of milk).

On the other hand, milk with hydrolyzed lactose they are better substrates for fermentation, so that this process has interest to accelerate the production of derivatives fermented milk (cheeses, yogurts ..). it is also interesting consider that lactose has a reduced solubility when compares it with that of galactose and glucose, so that its hydrolysis prevents problems of appearance of lactose crystals in condensed milk, ice cream, etc. (in addition to adding a sweet taste to food).

Another implication, not hydrolytic if not transglycosilic, would be food and pharmaceutical: synthesis of transglycosylation galactosi1-oligosaccharides [Zárate, S. and López-Leiva, M.H. 1990. "Oligosaccharide formation during enzymatic lactose hidrolysis: A literature review ". J. Food Prot. 53, 262-268], may be used for food interest purposes [Pivamik, L.F .; Senecal, A.G. and Rand, A.G. 1995. "Hydrolytic and transgalactosylic activities of commercial β-galactosidase (lactase) in food processing ". Adv. Food Nutrit. Res. 38, 1-102] and pharmacist [Nilsson K.G.I., 1988 "Enzymatic synthesis of oligosaccarides "Trends Biotechnol, 6, 256-264].

Preparations of (? -Galactosidase that are applied to food processing, mainly come from molds and mesophilic yeasts [Kuntz, LA, 1993. "Dairy enzymes: The biochemical benefit". Food Prod. Des. 3, 59-72; Gekas, V. and López-Leiva, M. 1985. "Hydrolysis of lactose: A literature review". Process. Biochem. 20, 1-12; Rao, DR 1997. "Oral supplements to improve lactose digestion and tolerance." Food Sci. Technol. Int. 3, 87-92] All of them, and others not collected, perform lactose hydrolysis at most at 55 ° C, and have other proteins in their composition as impurities that deteriorate the sensory quality of food on which they are processed [Mahoney, RR 1985. "Modification of lactose and lactose-containing dairy products with? -galactosidase". In " Developments in Dairy Chemistry: Lactose and minor constítuents ". F. Fox (Ed), pp. 69-108 Elsevier, New York.] Having very thermostable and pure enzymes would allow for pasteurization to be combined On or other heat treatments with the elimination of lactose and preserve the product from undesirable modifications. This would reduce production costs and avoid risks of bacterial contamination in the process (as we said, milk with hydrolyzed lactose is a better substrate for the growth of microorganisms). An interesting source of this type of enzymes would be thermophilic organisms. Being able to produce these enzymes in mesophilic microorganisms would allow them to substantially reduce their production costs, since incubation at high temperatures or in very complex media would not be necessary [Tennisi, E. 1997. "In industry, extrephiles begin to make their mark" . Science 276, 705-7061].

Also, in addition to having an enzyme with good properties, it is necessary to have systems of enzyme immobilization as pure as possible, well purifying it previously or associating the immobilization and purification, and so that we have a high volumetric load (i.e. immobilize a lot of active protein) and, if possible, improve its properties (for example, thermostability, kinetics, etc). Most commercial lactases present very serious inhibition problems when facing concentrations of order of 5% lactose (such as milk) and even more pronounced if used in hydrolysis of lactose concentrates (similar to whey concentrates), which reduces its usefulness.

The only known Thermus β-galactosidase patents are the following:

United States Patent No. 5,283,189 (February 1, 1994) : "β-Galactosidase from Thermus sp .". Inventors: Mitsunori Takase, Oomiya; Kouki Horikosi, Tokyo (Japan). The potential use of the enzyme is primarily food. The invention consists in the production of the enzyme from three strains of Thermus sp ., Other than strain T2, by a conventional method of fermentation of these thermophilic microorganisms.

Spanish Patent No. 9701759 (August 7, 1997) : "A process for producing β-galactosidase from Thermus sp . (Strain T2) in host cells, and purifying it in a single chromatographic step." Inventors : A. Vián; AV Carrascosa; JL García and E. Cortés. It claims a different sequence from the present invention, and a different purification system, so it is a different invention.

In the present invention the production of β-galactosidase is made by the fermentation of a recombinant mesophilic microorganism. Saying microorganism contains the gene that codes for the β-galactosidase from Thermus sp . T2, together with a module that codes for 6 histidines that allow purification and subsequent immobilization by chromatography of affinity with metal ions ("Ion metal affinity chromatography ", IMAC), differently from the one claimed in the aforementioned patent ofThermus sp. T2 Therefore the originality of the present invention against the Spanish Patent No. 9701759 (August 7, 1997) mentioned above remains safeguarded.

Likewise, systems of patent protected transglycosylation, not always designed with commercial β-galactosidases. The applications of the synthesized oligosaccharides claimed they are food, medical and pharmaceutical, and in their obtaining considers heat resistance to be able to perform the process under aseptic conditions [Pivamik, L.F .; Senecal, A.G. Y Rand, A.G. 1995. "Hydrolytic and transgalactosylic activities of commercial? -galactosidase (lactase) in food processing ". Adv. Food Nutrit. Res. 38, 1-102]. The patents are as follows:

one.

European Patent No. 88312,328.3 (Kobayashi, Y .; Kan, T. and Terashima, T. 1988. "Method for producing processed milk containing galactooligosaccharides").

2.

Japanese Patent No. 6394,987 (Okonogi, S .; Tomita, M .; Tomimura, T .; Tamura, Y. and Mizota, T. 1988. "Enzymic manufacture of β-D-galactopyranosyl- (1-6) - β-L-galactopyranosyl- (1-4) -D-fructose from lactulose ").

3.

International Patent WO 8909,275 (Nilsson, KGI 1989. "Enzymic sinthesis of complex carbohydrates").

Four.

US Patent No. 4,944,952 (Kobayashi, Y .; Kan, T. and Terashima, T. 1988. "Method for producing processed milk containing galactooligosaccharides").

5.

Japanese Patent No. 04218,362 (Oki, J .; Kobayashi, K .; Hashimoto, K. and Kubota, A. 1990. "Vinegar containing galactooligosaccharides").

The β-galactosidase gene of Thermus sp . T2, whose sequence and production system are claimed in the present invention, had been cloned and expressed previously in Escherichia coli by other authors, for scientific and non-commercial purposes [Koyama, Y .; Okamoto, S. and Furukawa, K. 1990 "Cloning of α and β-Galactosidase from an extreme thermophile, Thermus strain T2, and their expression in Thermus thermophilus HB27". Appl. Environ. Microbiol 56, 2251-2254]. Subsequently, its production was carried out in Escherichia coli JM109, together with a choline binding domain that allowed it to be purified in a single chromatographic step [Vian A, Carrascosa AV, Garcia JL, Cortes E. 1997. "A procedure to produce \ Thermus sp . beta-galactosidase (strain T2) in host cells, and purify it in a single chromatographic step. " Spanish patent 9701759. 7-8-97; Vian A, Carrascosa AV, Garcia JL, Cortes E. 1997 Structure of the beta-galactosidase gene from Thermus sp . strain T2: expression in Escherichia coli and purification in a single step of an active fusion protein. Appl. Environm. Microbiol 64,2187-21911 The present invention also allows, although differently from the aforementioned and with very interesting improvements, the production of β-galactosidase in E. coli , whereby the incubation is easier because it is not made 70 at 80 ° C, which is the optimum growth temperature of Thermus sp . T2, as well as not requiring special microbiological culture media or a long fermentation time. It also makes its immobilization possible, so the enzyme does not have to be removed from the substrate after acting, and several cycles can be used, with the consequent economic savings. On the other hand, the choline binding domain could present certain feeding problems, coming from a pathogenic organism.

Description of the invention

Total hydrolysis (more than 99%) of lactose in milk and whey was achieved using different immobilized derivatives of the enzyme β-galactosidase of Thermus sp . T2 To reach this final objective it was necessary to solve a series of problems.

The strain Thermus sp . T2 was isolated in hot springs of Yellowstone National Park, showed an optimal growth temperature of 70 ° C, with stirring and pH 7.7 and showed β-galactosidetic activity, with an optimum at 80 ° C and pH 5 [Ulrich, JT; MacFeters, GA and Temple, KL 1972. "Induction and characterization of β-galactosidase in an extreme thermophile". J. Bacteriol. 110, 691-698]. The strain, due to its morphological and culture characteristics, was in principle ascribed to the Thermus aquaticus species, but a recent study suggests that the strain belongs to the Thermus oshimai species [Ishiguro, M .; Kaneko, S .; Kuno TO.; Koyama, S .; Park, GG; Sakakibara, Y .; Kusakabe, 1 .; Kobayashi, H. 2001. Purification and characterization of the recombinant Thermus sp . Strain T2 alpha-galactosidase expressed in Escherichia )]. This strain is deposited in the American Type Culture Collection (ATCC 27737). The claimed invention uses DNA from this strain: that of the β-galactosidase enzyme gene.

The strain deposited in the Spanish Type Culture Collection (CECT) and protected by the Budapest Treaty, with deposit number 5970 corresponds to E. coli MC1116, host of the recombinant plasmid pBGT3. This strain produces a mutein of the enzyme β-galactosidase from Thermus sp . T2, with a module for IMAC, so in the future we will call it IMAC / β-galactosidase.

Purification was performed using supports IMAC produced to measure to avoid two serious problems that affected the selective adsorption of the enzyme and its desorption final when working with this multimeric enzyme: adsorption from native proteins to supports (which can contaminate the final preparation and forces to use larger columns to purify the same amount of enzymes) and enzyme binding multi-subunits (which caused such adsorption strong enzyme that greatly hindered its desorption final). In addition, it was necessary to consider purification effects inactivating certain cations (Cu).

Covalent immobilization was performed on epoxyacrylic resins with a high degree of crosslinking prepared in the presence of porogenic agents. Subsequently, some epoxide groups were modified with different compounds capable of adsorbing proteins (aminophenylboronic, IDA, IDA-metal ion, ethylenediamine) to alter the area of the protein involved in immobilization [C. Mateo, G. Fernández-Lorente, 0. Abian, R. Fernández-Lafuente, & JM Guisán. 2000. "Multifunctional epoxy-supports: a new tool to improve the covalent immobilization of proteins. The promotion of physical adsorptions of proteins on the supports before their covalent linkage". Biomacromolecules 1 , 739-745.]. Reversible immobilizations of the enzyme were also carried out on supports activated with polyethyleneimine, achieving a very strong, slightly distorting immobilization of the enzyme [Mateo, C., Abian, O, Fernández-Lafuente, R., & Guisán, JM 2000 "Reversible enzyme immobilization via a very strong and non-distorting adsorption on supports-polyethylene imine composites " Biotechnol. Bioeng 68 , 98-105]

Brief Description of the Invention

The application of commercial β-galactosidases to milk, dairy products and discharges has two drawbacks fundamental: the thermosensitivity of enzymes so far in use, and the impurities that these enzymes usually have in the Commercial preparations This prevents the use of enzymes in processes that include moderate heat treatments (p. e. pasteurization), and causes undesirable changes in the product respectively. In addition, as discussed above, product inhibitions may produce partial hydrolysis of the lactose, insufficient for some uses.

The present invention consists in the design and implementation of a process for the hydrolysis of lactose using β-galactosidase derivatives of Thermus sp . T2 in E. coli MC1116, and purified by immobilized metal ion affinity chromatography, and immobilize it on suitable supports (in a single step or in two) and its application to the hydrolysis of different dairy derivatives (from milk to dairy sera). The fact of producing the enzyme in this mesophilic microorganism is cheaper than in Thermus strains, among other reasons, because it is not necessary to incubate at 70 ° C to obtain the active enzyme.

Detailed description of the invention Enzyme Production

Plasmid pBGT3 was constructed to express the β-galactosidase activity of Thermus sp . T2 in a heterologous host (Figure 1). This plasmid pBGT3 overproduces the β-galactosidase of Thermus sp . T2 modified so that it can be purified by immobilized metal ion affinity chromatography. The modification consists of introducing six histidine residues at the N-terminal end of the protein of interest. The resulting protein chimera monomer, IMAC / β-galactosidase, has a predicted relative molecular mass of 73 kDa (Figure 2).

The heterologous host used was strain MC1116 of the Escherichia coli species, which lacks? -Galactosidase activity by carrying a complete deletion of the lactose operon. Said host was transformed with the plasmid pBGT3, obtaining as a final result the E. coli strain MC1116 (pBGT3). This strain is deposited in the CECT (Spanish Type Culture Collection) with registration number 5970 and protected in accordance with the provisions of the Budapest Treaty.

Next, the production and obtaining of extracts enriched in β-galactosidase from Thermus sp . (strain T2), in the form of IMAC / β-galactosidase protein chimera. E. coli MC1116 cells (pBGT3) were grown in a previously selected medium containing carbon and nitrogen sources and mineral salts, at a temperature of 30 ° C, for 14-18 hours. The cells were collected by centrifugation and broken by a French press. The treated extract was centrifuged, obtaining an expensive supernatant, enriched in thermostable β-galactosidase activity.

The activity test was then carried out thermostable enzyme β-galactosidase IMAC / R-galactosidase recombinant, obtained in the last step. Such activity was measured at 25 and 70 ° C for 10 minutes. using 2-nitrophenyl-? -D-galactopyranoside (ONPG) as a substrate at an approximate concentration of 13 mM.

Enzyme Purification

In the purification process they were used IMAC columns, to take advantage of having an enzyme to which poly-histidine tails had been added. Conventional protocols are based on protein adsorption  native and the target protein on the IMAC column and performing the purification by selective desorption (the chimeric enzyme adsorbs more strongly to the column).

This presents some problems, first the possible co-elution with native protein rich in Histidines, the increase in the volume of the spine needed to purify similar amounts of enzyme (since in addition to adsorbing our enzyme adsorb many others).

Therefore, we have noticed how to achieve purification by selective adsorption of the protein Diana. For them we rely on the hypothesis that while natural proteins can only be adsorbed on IMAC supports to through interactions between VARIOUS His and VARIOUS chelate groups, polyHis tailed proteins could be adsorbed through interactions of several His queues with ONE ONLY group chelate

On the other hand, multimeric enzymes (as it is the case in question) can be adsorbed to an IMAC support to through several subunits (two, three or more), originating a binding so strong that subsequently the desorption of the enzyme is very complex

The solution to both problems is to prepare IMAC brackets with very few metal chelates, sufficient for coat the protein support but not for a joint multi-point or multi-subunits.

In the case of this enzyme, the adsorption more rapid was achieved using as metal the Cu, being the union very selective if performed in the presence of 25 mM of imidazole and a support with only about 12 micromoles of chelate groups per gram of support. However, using this metal two arose problems: the first, and most serious, was that the traces of Cu desorbed from the support inactivated the enzyme, the second, that at despite the low level of activation adsorption occurred multi-subunits in adsorption processes of more than 10 minutes, which which made desorption very difficult. This problem was fixed. using Ni or Co. as metal

Covalent enzyme immobilization

Since this is a multimeric enzyme, it could have interest immobilize it through the maximum subunits to prevent these from being desorbed during the reaction (contaminating milk and decreasing the activity of the derivative). Therefore, supports were used whose internal morphology were large areas surrounding empty spaces (resins epoxyacrylics with a high degree of polymerization produced in presence of porogenic agents), in this way a Good enzyme-support geometric congruence.

On the other hand, it was important to choose suitable groups to perform multi-subunit covalent immobilizations. The glyoxyl and epoxide groups may be the most suitable, since they react with primary amino groups (and thiols and hydroxyls in the case of the epoxide), are attached through short arms to the support. A description of the advantages of these groups can be found in [Mateo, C., Abian, O, Fernández-Lafuente, R., & Guisán, JM 2000 "Increase in conformational stability of enzymes immobilized on epoxy-activated supports by favoring additional multipoint covalent attachment " Enzyme Microb. Technol 26 , 509-515].

However, epoxy groups barely react with soluble enzymes, immobilize enzymes through a mechanism in two stages, a first rapid physical adsorption and a subsequent covalent immobilization.

On the other hand, although the first immobilization can be carried out under mild conditions (neutral pH), if we intend to force the co-valent union between the enzyme (already immobilized covalently through a few bonds) and the support it will be necessary to use conditions of pH, T , etc., where the reactive groups of the protein (especially the most abundant on the surface, the Lys are reactive). In addition, we can think that the reaction between two rigid and non-complementary structures will require the correct alignment of the groups to be reacted, so that both time and temperature will play a key role in determining the degree of multi-point covalent bonding. [Mateo, C., Abian, O, Fernández-Lafuente, R., & Guisán, JM 2000 "Increase in conformational stability of enzymes immobilized on epoxy-activated supports by favoring additional multipoint covalent attachment" Enzyme Microb. Technol 26 , 509-515].

Using new epoxy-multifunctional supports, we can vary the cause of adsorption and in that way we can vary the area of the enzyme involved in immobilization, [C. Mateo, G. Fernández-Lorente, O. Abian, R. Fernández-Lafuente, & JM Guisán. 2000. "Multifunctional epoxy-supports: a new tool to improve the covalent immobilization of proteins. The promotion of physical adsorptions of proteins on the supports before their covalent linkage". Biomocromolecules . 1 , 739-745] thus being able to vary its stability, activity, inhibition, specificity, ...

Covalent immobilization and purification of the enzyme in a single He passed

Since the target enzyme is fused with tails of polyHis and that have heterofunctional supports epoxide-chelate, an elegant alternative to preparation of pure enzyme derivatives would be to prepare supports mixed in which the specificity of IMAC adsorption is combined (point 4.2.2.) and the possibilities of co-valent multipunual junction of the epoxy groups (point 4.2.3.).

This strategy, in addition to saving time (by do both processes in one step) can have other advantages. In some cases, pure enzymes may present a reduced stability, or they may tend to add hindering their driving. However, depending on the technological capabilities of companies, it may be preferable to use the enzyme directly purified supplied by a second company. Therefore, in this different solutions are given for companies with different needs

Stabilization of the quaternary structure of enzymes and protein

A very serious problem in food technology, which has a lower incidence in other enzyme applications, is the possible desorption of multimeric protein subunits not covalently bound to the support. In addition to causing the inactivation of the enzyme of interest, these subunits pass into the food, being able to alter their properties, produce allergic reactions, etc. It is interesting to stabilize not only the target enzyme, but any other possible contaminant that can contaminate, even at a very low level, the final derivative. Therefore, the different covalent derivatives have been treated with polyfunctional polymers (dextran aldehyde), following a strategy described in [Fernández-Lafuente, R., Rodríguez. V., Mateo, C., Penzol, G Hernández-Justiz. O., Irazoqui, G., Villarino, A., Ovsejevi, K., Batista, F. & Guisan, JM 1999. "Strategies for the stabilization of multimeric enzymes via immobilization and post-immobilization techniques" J. Mol. Cat. B: Enzymatic , 7 , 181-189]. The use of polymers of large size and with numerous functional groups reduce some problems of cross-linking with bifunctional reagents: competition between unipuncture modification and cross-linking, covering a wide variety of distances well and, by involving large areas of the protein, can easily involve several subunits .

Reversible enzyme immobilization

Given the great evolution of molecular genetics, microbiology and fermentation systems, it is possible that when working with very stable enzymes (such as thermophilic ones) to which it is not necessary to apply additional stabilization techniques , the price of the support exceeds the cost of the enzyme. Therefore, it may be convenient to develop methods of reversible immobilization, in which the enzyme is very strongly bound to the support, without decreasing its stability, and the enzyme can be desorbed once it has been inactivated (using very drastic conditions, such as acids or diluted bases, guanidine, extreme ionic strength, ...). To achieve this type of very strong and non-distorting joints, we propose the use of supports coated with polyfunctional polymers. In this way, for each support group we can introduce many functional groups, and being a flexible polymer, we do not force the enzyme to adapt to the support, but to the polymer to adapt to the enzyme [Matthew, C., Abian, O, Fernández-Lafuente, R., & Guisán, JM 2000 "Reversible enzyme immobilization via a very strong and non-distorting adsorption on supports-polyethylene imine composites" Biotechnol. Bioeng 68 , 98-105].

Detailed description of the drawings Figure 1 Scheme of the method followed to construct a plasmid that expresses, in a mesophilic heterologous host, the structural gene that codes for the thermophilic microorganism beta-galactosidase Thermus sp . T2

Plasmids, which are represented linearized, are not drawn to scale, but their size is expressed in kb (kilobases). For details, see the text (Example 1). Only the most important restriction sites and structures are indicated to better understand the figure. The origins of replication ( ori ), the ampicillin resistance gene (Ap R), the repressor gene of the lac operon ( lac l or lac l q alleles) are indicated. Large arrows indicate the promoters: trc P in pTrcHisB and the two series promoters Ipp P and lac P / O in pBGT1. The module that allows purification by IMAC has been designated as ATG (His) 6.

Examples of embodiment of the invention

Explanatory, but in no case limiting to this patent, in the examples set forth in Below is the experimentation needed to Develop this patent.

Example 1 A process of hydrolysis of lactose catalyzed by immobilized-stabilized recombinant derivatives of β-galactosidase from Thermus SP . (strain T2) fused with poly-histidine tails (Imac / β-galactosidase) cloned in E. coli cect 5970 1.1 Construction of a plasmid to express the β-galactosidase activity of Thermus sp . T2 in the mesophilic host Escherichia coli MC1116

The constructed plasmid is called pBGT3 and for its realization recombinant DNA techniques were used Conventional [Sambrook, J., Frítsch, E.F. and Maniatis, T. 1989. Molecular Cloning. CSHL Press, Cold Spring Harbor, New York], which know any person moderately versed in this matter.

To construct pBGT3, the expression vector pTrcHisB and the plasmid pBGT1 were used. The pTrcHisB expression vector is commercial, and can be purchased from the company InVitroGen (Leek, The Netherlands). Plasmid pBGT1 [Vian A, Carrascosa AV, Garcia JL, Cortes E. 1997. "A procedure to produce β-galactosidase from Thermus sp . (Strain T2) in host cells, and purify it in a single chromatographic step." Spanish patent 9701759. 7-8-97; Vian A, Carrascosa AV, Garcia JL, Cortes E. 1997 Structure of the beta-galactosidase gene from Thermus sp . strain T2: expression in Escherichia coli and purification in a single step of an active fusion protein. Appl. Environm. Microbiol 64,2187-2191] overproduces in E. coli the β-galactosidase that can be considered native to Thermus sp . T2

The pTrcHisB vector was digested with restriction enzymes Xho l and Hin dlll, the large fragment being purified. Plasmid pBGT1 was digested with the enzymes Sall and Hindlll, the ca fragment being purified. 2.3 kb containing the bgaA gene of Thermus sp . T2

The purified fragments were covalently bound with T4 DNA ligase. The union between the compatible ends Sal l and Xho l means that neither of the two initial recognition sequences is recovered. With the above-mentioned ligation mixture competent cells of E. coli MC1116 were transformed [Casadaban MJ, Martinez-Arias A, Shapira SK, Chou J. Beta-galactosidase gene fusions for analyzing gene expression in Escherichia coli and yeast Methods Enzymol., 100: 293-308]. Plating was done on LB agar with ampicillin, X-Gal (5-Bromo-4-chloro-3-indolyl-? -D-galactopyranoside) and IPTG (isopropyl-? -D-1-thiogalactopyranoside), incubating at 37 ° C A transforming colony was chosen.

Such a colony was reisolated and a minipreparation of its plasmid DNA. After checking that the pattern restriction with various enzymes was as expected, a Final verification by sequencing.

The sequence check of pBGT3 was done using the oligonucleotide OBGT-6. Saying oligonucleotide, whose sequence is

5'-GACAGTAGCAGCGCACG-3 '

it is located about 250 bp of the ATG towards the 3 'zone of the wild bga A gene, extending from positions 1561 to 1545 of the published sequence in which the bgaA gene of Thermus sp ., strain T2 [Vian A, Carrascosa] AV, García JL, Cortes E. 1997 Structure of the beta-galactosidase gene from Thermus sp . strain T2: expression in Escherichia coli and purification in a single step of an active fusion protein. Appl. Environm. Microbiol 64,2187-21911 In this way it was possible to check the sequence of the first 200 nucleotides of the gene and the presence of the coding area for the six histidine residues in translational fusion with the previous one (SEQ ID 1).

1.2 Production of the chimeric protein consisting of Thermus sp T2? -Galactosidase linked to a module with poly-histidines (IMAC /? -Galactosidase)

E. coli CECT 5970 cells were grown for 18 hours at 37 ° C in one liter of LB with ampicillin (125 mg / ml) with shaking at 250 rpm. The culture was centrifuged at 6000 rpm (GSA rotor) in a Beckman centrifuge (model J21B) for 15 minutes at 20 degrees Celsius. The pelleted cells were resuspended in 45 ml of 50 mM sodium phosphate buffer at pH 7 and lysed through the French press three times, applying a pressure of 1100 PSI. Next, the extract was centrifuged at 15,000 rpm (SS34 rotor) for 20 minutes, at 20 degrees Celsius, the sediment being discarded.

To check if the new protein produced (the chimera), had been produced correctly, studies were conducted comparative biochemicals with the corresponding native protein, finding quite coincident results.

1. Substrates : Hydrolyzes ONPG with a specific activity of 158,500 units at pH 6.5 and at 70 ° C. Hydrolyzes lactose with a specific activity of 25,400 units.

2. Reaction pH: The optimal pH of the hydrolysis is between 5.5 and 6.5. Activity is clearly detected in a pH range of 5 to 7.

3. Optimum temperature : the maximum was reached at 80ºC at pH 7.

4. Thermostability : The soluble enzyme retains 50% of the activity after heating for 120 minutes at 80 ° C and pH 6.5, and retains 50% of the activity after heating 7 hours at 70 ° C and pH 6.5. The enzyme immobilized / purified in a single step by sepabeads-IDA-Co, retains 70% of the activity after 56 hours at 70 ° C and pH 6.5.

5. Inhibition by reaction products : Up to 75% of the enzyme activity is conserved in the presence of 100 mM glucose. Galactose, at a concentration of 50 mm, decreases the activity by 80%. At 70 ° C and pH 6.5 (when tested with the soluble enzyme). The immobilized enzyme is more protected than the respective soluble enzyme. At 100 mM glucose the immobilized derivative retained more than 90% of its activity (non-competitive inhibition) and 100 mM galactose decreases by 30%.

6. Conservation The enzyme can be stored at 4 ° C immobilized / purified on sepabeads-IDA-epoxide supports for more than 12 months, retaining more than 95% of its initial activity.

1.3 Purification of the chimeric protein consisting of Thermus sp T2? -Galactosidase attached to a module with poly-histidines (IMAC /? -Galactosidase) using immobilized copper chelates supports

Once the crude extract was obtained, its purification was followed, in previously activated agarose supports with a low concentration of iminodiacetic acid (IDA) (12 µmoles of reactive groups / ml of gel) [Armisen, P., Mateo, C., Cortes, E., Barredo, JL, Salto, F., Ten, B., Rodes, L., Garcia, JI, Fernández-Lafuente, R., Guisán, JM 1999 "Selective adsorption of poly-His tagged glutarayl acylase on tailor- made metal chelate supports " J. Chromat . A, 848, 61-70] and chelated with Cu 2+, Ni 2+ and Co 2+. Adsorption of the protein to the support was performed at pH 7 and 25 ° C. Adsorption kinetics for each metal were analyzed individually. Table 1 shows that the maximum adsorption rate was achieved with Cu, so this support was selected at first.

TABLE 1 Adsorption kinetics of the β-galactosidase of Thermus sp . T2 on chelated supports activated with copper, nickel cobalt, reaction at room temperature (25º)

Adsorption of the protein to the support (%) Weather (min) Ag-IDA-Cu Ag-IDA-Ni Ag-IDA-Co 0 0 0 0 5 85 65 73 10 100 88 92 30 - 100 100 60 - - - 150 - - - 1440 - - -

To check the effectiveness of the process adsorption, electrophoresis were performed on polyacrylamide gels in the presence of SDS (12%) and beta galactosidase activity assays and protein determination by the Bradford method, found that 100% of our enzyme of interest (IMAC / β-galactosidase), stuck to the support but that, more than 70% of other proteins present in the extract, also adhered to it. To solve this situation, we try to improve the selectivity of the process through the addition of small concentrations of imidazole (25mM), followed of a small wash of the support also with imidazole (50mM), when we use the copper chelate support, as a support for purify. In the end we check that 100% of the β-galactosidase adsorbed to the support and no other protein (proteases, etc ...) adhered to the same.

The desorption of the IMAC / β-galactosidase from the support was then carried out, using increasing concentrations of imidazole (from 10 to 100 mM), finding that the first traces of pure enzyme eluted at 60 mM of imidazole (8% of the enzyme) and that at 100 mM eluted 100% of the enzyme IMAC / β-galactosidase from Thermus sp . T2, and the conditions are described in table 2.

TABLE 2 Purification of the chimeric protein IMAC / β-galactosidase from Thermus sp T2, in batch and at 25 ° C, starting from 1 L of culture. Desorption was performed immediately after adsorption. One unit has been defined as, the amount of enzyme that hydrolyzes 1 µm of substrate per minute under standardized reaction conditions

Stage Activity Prot. Performance. Specific Factor (U / ml) (mg / ml) Recovered U. (U / mg) purification Abstract raw 17.4 7.5 100 2.32 one Ag-IDA-Cu 15.84 0.26 91 62.1 26

Once purified, the enzyme was stored at 4 ° C for later experiments. After 24 hours stored, we saw that it had lost more than 60% of its activity. I identify as a cause of this fall in activity to the presence of traces of Soluble cu.

On the other hand, it was found that if the adsorption is performed in longer periods of time (that is, if you don't know desorbed the enzyme immediately after adsorption), this He was able to join the support very strongly, hindering his subsequent desorption

For these two reasons, the supports with copper chelates for purification of this enzyme.

TABLE 3 Study of the bond strength during adsorption of IMAC / β-galactosidase from Thermus sp . T2, in IDA-Cu supports, activated with 12 µmoles of reactive groups / ml of support, in the process of purification

Weather from [Imidazole for desorber IMAC / β-galactosidase adsorption (minutes) the enzyme (mM)] desorbed (%) 5 100 100 30 100 fifteen '' 200 23.5 '' 300 28.8

1.4 Purification of the chimeric protein consisting of Thermus sp T2? -Galactosidase linked to a module with poly-histidines (IMAC /? -Galactosidase) with cobalt and nickel chelates

In view of the inactivating effect of certain cations on the enzyme, the effect of the other cations was evaluated on the stability of the enzyme, finding that only Cu It had a negative effect.

Therefore, copper was replaced by cobalt or nickel.

The agarose supports modified with chelates of cobalt or nickel, had the same amount of reactive groups that the case of copper chelate, that is, they were substituted with 12 µmoles of reactive groups per milliliter of gel.

TABLE 4 Effect of desorption of small traces of supports containing metal cations, on the β-galactosidase activity of the IMAC / β-galactosidase Thermus sp . T2, during the purification processes

System Activity β-galactosidase residual by cation effect (%) Sodium Phosphate Buffer 100 (+ -2) Support Ag-IDA-Zn 100 (+ -2) Support Ag-IDA-Ni 100 (+ -2) Support Ag-IDA-Co 100 (+ -2) Support Ag-IDA-Cu 25 (25 + -2)

The purification processes with these supports They were performed at room temperature and in batch. The results of This experiment is summarized in Table 5.

TABLE 5 Purification of the IMAC / β-galactosidase chimeric protein from Thermus sp. T2, in batch and at 25ºC, on agarose supports-IDA-Ni and Co

Stage Activity Prot. Performance. Specific Factor (U / ml) (mg / ml) Recovered U. (U / mg) Purification Abstract raw 17.4 7.5 100 2.32 one Ag-IDA-Ni 15.8 0.33 92 48.3 21.2 Ag-IDA-Co 16.2 0.36 93.2 44.5 twenty

Making use of these two chelate supports for carry out the purification, we get very similar results to those obtained with copper chelate (yields, activity specific and purification factor), but with the difference of that, we eliminate the problems of lost activity, found when it was purified with copper chelate. Being the more selective cobalt and nickel (the adsorption kinetics of the  support protein, they are slower than in copper), adsorption unspecific of other proteins to the support, it looks pretty hindered, so, the final purification protocol with These chelates remained: During the adsorption of the protein, you we add 25mM of imidazole to the reaction medium and followed by direct desorption with 100 mM imidazole (the intermediate step of washed with 50 mM imidazole, as in copper, it is done unnecessary).

Finally, it was found that by prolonging the adsorption time was not significantly increased the absorption force, that is, it seemed that it was only adsorbed by a tail of polyHis.

1.5 Immobilization of IMAC / β-galactosidase from Thermus sp ., T2 purified on epoxyacrylic-epoxy supports modified with boronate, ethylenediamine and with metal chelates

Fresh enzyme samples were immobilized purified on conventional epoxy supports (at 1 M phosphate sodium), epoxy-amino, epoxy-aminophenylboronic and epoxy-IDA-Co (in these 3 cases at 25 mM sodium phosphate), at pH 7 and 22 ° C for 24 h. They came to immobilize up to 60 mg of enzyme per ml of packed support (in all cases with the exception of standard support (at 1 M of sodium phosphate) keeping the activity of the enzyme.

To end the reaction and hydrophilize the support, the epoxide support was reacted with 3M glycine at pH 8.5 for 24 h. (4ml of glycine / ml of support). In the case of the supports with boronic groups obtained the maximum stability, far superior to that of the soluble enzyme, being those of EDA and support standard, the least stable, while the IDA-Co allowed to obtain more stable derivatives than the soluble enzyme, only slightly less stable than those obtained with boronic.

Incubation of the best derivatives at pH alkaline before blocking the support with glycine allowed to improve even more its stability without significant loss of activity., keeping the boronate-epoxide support as the That offered the best results.

1.6 One-step purification and immobilization of the chimeric protein consisting of? -Galactosidase from Thermus sp . T2 attached to a module with poly-histidines (IMAC / β-galactosidase), in sepabeads-IDA-Co-epoxide supports

One way to solve the enzyme trend pure to add would be to associate the purification processes and immobilization For this, supports were used. epoxi-IDA-Co to which the impure enzyme. The support design used the information obtained in the previous examples: very low activation with groups Support IDA, even less than before, since now it was not possible to include imidazole as it would covalently bind to epoxy groups, and very low affinity cations. Using supports with only 5 micromoles of groups IDA-Co, was able to immobilize and stabilize a amount of beta galactosidase equivalent to what could be immobilize using freshly purified enzyme (about 60 mg).

1.7 Stabilization of the quaternary structure of the IMAC / β-galactosidase of Thermus sp . T2, and possible contaminating enzymes

It was found that even the most stable derivatives released protein to the medium when boiled in the presence of SDS, which suggested that some enzyme subunits were not covalently attached to the support. The treatment of derivatives with dextrans it allowed to avoid this problem, but it caused a Very important fall in activity (up to 80%). That's why it made the modification in the presence of protective agents. If the modification was made in the presence of lactose, the fall of Activity was very small. After optimizing the time, tºC and size of dextran, the following was established as the optimal protocol: incubation of the derivative with molecular weight dextrans 71.4 KD and protection with 1M lactose, reaction at 4 ° C for approximately 6 hours with continuous agitation.

The derivative thus treated no longer released subunits after boiling in SDS, and its thermal stability was similar to that of the untreated derivative.

1.8 Reversible immobilization of the enzyme IMAC / β-galactosidase Thermus sp ., T2 pure on rigid supports coated with PEI

100 mg of freshly purified protein was added to 10 ml of PEI support prepared as described in [Mateo, C., Abian, O, Fernández-Lafuente, R., & Guisán, JM 2000 "Reversible enzyme immobilization via a very strong and non-distorting adsorption on supports-polyethylene imine composites " Biotechnol. Bioeng 68, 98-105], at pH 7 and 25 ° C, The enzyme was previously dialyzed against 100 volumes of 50 mM phosphate pH 7. Immobilization was performed very quickly and without affecting the activity of the enzyme. Stability studies showed that the enzyme was much more stable than the soluble enzyme.

On the other hand, desorption experiments they showed that the union was very strong (much more, in fact, than conventional supports DEAE agarose type, although it could be removed the whole enzyme using 1M NaCl and an intense wash with water distilled

In this way, the support could be reused as many times as desired.

1.9 Hydrolysis of concentrated solutions of pure lactose with enzyme IMAC / β-galactosidase from Thermus sp . Pure soluble T2 and various immobilized derivatives

A solution of 5% lactose in TAMPÓN NOVO pH 6.5 was prepared at different temperatures. Then, 8 U of soluble or immobilized-stabilized enzyme was incubated in 10 ml of this solution on boronate-epoxide or IDA-Co-epoxide supports. It can be seen that the enzyme was capable of performing hydrolysis even at 70 ° C
(Figure 6).

However, while the soluble enzyme performed hydrolysis of the last 10% lactose very slowly, the derivatives were able to do it in a much more linear way, more in the case of boronate-epoxide than in the case of the IDA-Co epoxide. Kinetic studies demonstrated that while the soluble enzyme had values of 3.1 km and of noncompetitive glucose inhibition constants of 49.9 and competitive by 3.1 galactose the derivative made on IDA-Co-epoxide showed some Km values of 7.7, Ki for glucose of 61.1 and Ki for galactose 11.7 while immobilized on boronate-epoxide they showed constants of Km 6.6; Ki glucose 99.1 and Ki galactose 12.5 That is, immobilization not only served to improve the enzyme stability, but allowed, by gentle distortions of the active center, decrease the inhibition produced for the different products, improving in this way and in a way very significant the possibilities of using this enzyme to scale industrial.

1.10 Hydrolysis of lactose in milk and in cheese whey catalyzed by immobilized derivatives of IMAC / β-galactosidase enzyme from Thermus sp . Pure T2

The derivative behavior was studied sepabeads-boronate during the reaction of hydrolysis of a semi-skimmed commercial milk (since a milk With a high fat content it would make the derivative action difficult during the reaction), with 4.8% lactose and 8 U of the derivative at 70 ° C The amount of milk in the reactor was 10 ml. After About 10 minutes 100% hydrolysis was reached. A sample of cheese whey was also hydrolyzed therein conditions that the reaction with milk. The serum had a content in lactose of approximately 5%. As seen in the graph, it reached 100% hydrolysis at 25 minutes of reaction.

<110> SUPERIOR INVESTIGATION COUNCIL SCIENTISTS

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CARRASCOSE SANTIAGO, ALFONSO V.

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GUISAN SEIJAS, JOSE MARIA

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Claims (15)

1. Recombinant chimeric β-galactosidase enzyme derivative of Thermus sp. T2, characterized in that:

(to)

presents the amino acid sequence described in SEQ ID NO: 2, with a module (IMAC) with a queue of histidines (IMAC / β-galactosidase),

(b)

is purified by affinity chromatography on metal chelates in supports containing transition metals,

(C)

is stabilized and immobilized by covalent bonds or adsorption physics on suitable supports (in a single step or in two) Y

(d)

it has the capacity to hydrolyze and transglycosilar lactose

2. Recombinant chimeric β-galactosidase enzyme derivative of Thermus sp . T2 according to claim 1, characterized in that the method of obtaining comprises the following steps:

(to)

isolation of the DNA fragment that contains the nucleotide sequence that codes for the β-galactosidase of Thermus sp . T2 by digestion of plasmid pBGT1 with restriction enzymes,

(b)

digestion of plasmid pTrcHisB which contains the nucleotide sequence that encodes a module with a tail of 6 histidines, which allows purification by affinity chromatography with metal chelates,

(C)

DNA fragment fusion from (a) to plasmid residue pTrcHisB of (b) obtaining a new plasmid, pBGT3, which contains a nucleotide sequence SEQ ID NO: 1 new, which codes for a protein chimeric β-galactosidase with tail of histidines: IMAC / β-galactosidase,

(d)

cell transformation host with said recombinant plasmid;

(and)

host cell culture transformed into a suitable culture medium and under conditions that allow the production of β-galactosidase IMAC / β-galactosidase chimeric,

(F)

selective adsorption purification of the chimeric β-galactosidase enzyme with the target polyhistidine tail by affinity chromatography at metal chelates (e.g. Co or Ni)

(g)

immobilization-stabilization on rigid supports (highly crosslinked epoxy acrylic resins in presence of porogenic agents, porous glass, other silicates, random, cellulose, etc.), containing as groups for the union covalent aldehyde or epoxide groups, and which at the same time may have groups to physically adsorb enzymes (e.g., derivatives of boronate, groups with positive charge, groups with negative charge, chelates, ...) and

(h)

additional stabilization if it comes from the quaternary structure by subsequent crosslinking with polyfunctional polymers, covalent with dextran-aldehyde or physical with type polymers polyethyleneimine

3. Recombinant chimeric β-galactosidase enzyme derivative of Thermus sp . T2, according to claims 1 and 2, characterized in that the immobilization-stabilization is carried out at alkaline pH to force covalent bonding. 4. Recombinant chimeric β-galactosidase enzyme derived from Thermus sp . T2, according to claims 1 and 2, characterized in that the immobilization-stabilization uses supports with epoxy groups and that after the reaction are blocked with hydrophilic groups (amino acids, amino alcohols, sugars, thioamines, etc.). 5. Recombinant chimeric β-galactosidase enzyme derivative of Thermus sp . T2, according to claims 1 and 2, characterized in that the immobilization, stabilization is performed using epoxy or aldehyde supports -IDA-Co, Ni or Zn. 6. Recombinant chimeric β-galactosidase enzyme derivative of Thermus sp . T2, according to claims 1 and 2, characterized in that the immobilization is carried out because it is physically adsorbed on supports coated with polycationic polymers of the polyethyleneimine, polyallylamine, aminated dextran or other amino polysaccharides type. 7. Recombinant chimeric β-galactosidase enzyme derivative of Thermus sp . T2 according to claim 2 characterized in that the stabilization of the quaternary structure uses dextran aldehyde to achieve the chemical cross-linking of the subunits, performed in the presence of protective agents (eg, substrates) to avoid distortion of the active center of the enzyme. 8. Recombinant chimeric β-galactosidase enzyme IMAC / β-galactosidase from Thermus sp . T2 characterized SEQ ID NO: 2. 9. Nucleotide sequence characterized by the sequence SEQ ID NO: 1 encoding a chimeric β-galactosidase protein according to 8. 10. Transformed host cells characterized by containing the plasmid pBGT3 according to claim 2, which contains SEQ ID NO: 1, preferably the pure Escherichia coli strain CECT 5970, its mutants or its transformed derivatives. 11. Method of enzymatic treatment of lactose, sugars or structural analogues, characterized in that

(to)

the biocatalyst (enzyme) is used, the derivative of the recombinant chimeric β-galactosidase enzyme of Thermus sp . T2 purified, immobilized-stabilized according to claims 1-7,

(b)

with preferential use between 25 and 75 ° C.

12. Method of enzymatic treatment of lactose, sugars or structural analogs according to claim 10, characterized in that the hydrolysis of lactose concentrates (from 1 to 40%) is carried out up to 75 ° C from pH 4 to pH 8 by means of the purified biocatalyst (enzyme) , immobilized-stabilized according to claims 1-7. 13. Method of enzymatic treatment of lactose, sugars or structural analogues according to claim 10, characterized in that the hydrolysis thereof is carried out in whole, skimmed or semi-skimmed milk up to 75 ° C by means of the purified, immobilized-stabilized biocatalyst (enzyme) according to the claims 1-7. 14. Method of enzymatic treatment of lactose, sugars or structural analogs according to claim 11, characterized in that the hydrolysis thereof is carried out in dairy whey, concentrated or not, or permeate of whey of different origins (pH 4-8) and up to 75 ° C by means of the purified, immobilized-stabilized biocatalyst (enzyme) according to claims 1-7. 15. Method of enzymatic treatment of lactose, sugars or structural analogs according to claim 11, characterized in that it is carried out in any liquid containing them, at pH 4 to 8 to 75 ° C, characterized in that the immobilized, purified biocatalyst (enzyme) is used -stabilized according to claims 1-7.