ES2288076B1 - PROCEDURE FOR OBTAINING THERMOPHILIC ENZYMES WITH LIPOLITIC ACTIVITY OF MICROORGANISMS OF THE GENERO THERMUS. - Google Patents

Abstract

Procedure for obtaining thermophilic enzymes with lipolytic activity of microorganisms of the thermus genus. The procedure consists in growing microorganisms of the Thermus genus in a growth medium prepared with a mineral-medicinal thermal water and subjecting the post-incubated to sequential stages of freezing, thawing and centrifugation. The result of this procedure is to obtain thermophilic enzymes with lipolytic activity in the liquid, extracellular. The procedure and the product of its application are of interest in the industries of manufacturing enzymes, pharmaceuticals, fine chemicals or research and development activities.

Description

Procedure for obtaining thermophilic enzymes with lipolytic activity of microorganisms of the Thermus genus.

Technical sector

The procedure described is immediate application in the enzyme manufacturing industry, the product obtained (lipolytic enzymes) thermophilic) to the pharmaceutical, fine chemical or research and development activities.

State of the art

Lipolytic enzymes (lipases, esterases) they present a great variety of industrial applications in very diverse sectors, such as the manufacture of detergents, aromas and homoquiral pharmaceutical principles, industry food, paper and pulp, leather, and even in the Sewage Treatment.

One of the biggest problems encountered in the Enzyme utilization as industrial biocatalysts is its deactivation at high temperatures, which means an increase in the associated operating costs. Strategies have been developed to obtain thermostable enzymes, such as the variation of its molecular structure by directed mutagenesis or by modification surface chemistry, or the application of techniques immobilization that also allows to recover and reuse the biocatalyst

Thermophilic microorganisms grow to temperatures above 45 ° C, and in some cases (hyperthermophiles) even above 90 ° C. Enzymes produced by these microorganisms have a high thermal stability and resistance to chemical denaturants (chaotropic salts, organic solvents, detergents). These properties make it possible to use in temperature processes high with minimal losses of activity, with the consequent increase in reaction rate and solubility of reagents Various thermophilic enzymes of potential application in biotechnological processes, highlighting the amylolytic enzymes, cellulases, xylanases, chitinase, proteases, DNA polymerases, reverse transcriptases, ligases, glucose isomerases and alcohol dehydrogenases.

The ability of various strains of the Thermus genus to produce enzymes with lipolytic activity, which can be released into the culture medium, has recently been indicated. Two enzymes, of molecular weights 32 and 64 kDa, have been identified that have high activity at alkaline pH and high temperature, as well as exceptional thermostability and stability in the presence of high concentrations of organic solvents. Although aspects related to small-scale cultivation, purification and characterization conditions have been described, there are no registered procedures for obtaining them with a view to their industrial application.

Some articles are specified below related:

BECKER RJ, STARZYK MJ ( 1984 ) Morphology and rotund body formation in Thermus aquaticus . Microbes 41, 115-129.

BERGER JL, LEE BH, LACROIX C. ( 1995 ) Identification of new enzyme activities of several strains of Thermus species. Appl. Microbool Biotechnol 44, 81-87.

DOMÍNGUEZ A., SANROMÁN A., FUCIÑOS P., RÚA ML, PASTRANA L., LONGO MA ( 2004 ) Quantification of intra- and extracellular thermophilic lipase / esterase production by Thermus sp. Biotechnol Lett . 26, 705-708.

FUCIÑOS P., ABADÍN CM, SANROMÁN A., LONGO MA, PASTRANA L., RÚA ML ( 2005 ) Identification of extracellular lipases / esterases produced by Thermus thermophilus HB27: partial purification and preliminary biochemical characterization. J. Biotechnol . 117, 233-241.

JAEGER KE, REETZ MT ( 1998 ) Microbial Jipases form versatile tools for biotechnology. TIBTECH 16, 396-403.

LAGARDE D., NGUYEN HK, RAVOT G., WAHLER D., REYMOND JL, HILLS G., VEIT T., LEFEVRE F. ( 2002 ) High-throughput screening of thermostable esterases for industrial bioconversions. Organic Process Res. Develop. 6 , 441-445.

NIEHAUS F., BERTOLDO C., KAHLER M., ANTRANIKIAN G. ( 1999 ) Extremophiles as a source of novel enzymes for industrial application. Appl. Microbe/. Biotechnol 51, 711-729.

SCHIRALDI C., DE ROSA M. ( 2002 ) The production of biocatalysts and biomolecules from extrephiles. TIBTECH 20, 515-521.

Description of the invention Brief Description of the Invention

A procedure for obtaining enzymes with lipolytic activity by culture of microorganisms of the Thermus genus is proposed, consisting in the design of a culture medium and the appropriate operating conditions for the production of a high level of extracellular lipolytic activity, as well as in the definition of a system for the recovery in the extracellular fluid of the enzymes produced.

Detailed description of the invention

The obtaining of thermophilic enzymes with lipolytic activity is based on growing a microorganism of the Thermus genus in a suitable medium and conditions, and subsequently recovering the enzymes in the extracellular liquid by freezing / thawing and centrifugation stages.

The details of the procedure are the following:

Composition of the culture medium

The culture medium used consists of a dissolution in thermal mineral-medicinal water of high mineralization and with a high dry residue content of a source of carbon such as glucose or other simple sugar, a source of nitrogen as a yeast extract or other protein hydrolyzate and a mixture of salts, all at the appropriate concentrations to promote the growth of the microorganism and the production of enzymes with lipolytic activity, whose pH is initially adjusted to A value close to neutrality. When sources of complex nitrogen the addition of the source of carbon to the culture medium.

Cultivation to obtain inoculum

The inoculum for the production process is obtained by axenic culture of a microorganism of the Thermus genus in the medium described above by the addition of preserved microorganism cells frozen to Erlenmeyer flasks with a loading ratio of less than 50% and without the need for forced aeration After homogenizing the mixture, it is incubated with constant stirring and controlled temperature between 50 and 90 ° C for a time that allows a sufficient level of cell growth to be achieved.

Then the biomass is recovered by centrifugation, removing excess moisture, and retaining frozen until later use as inoculum.

Culture for obtaining enzyme

This stage of the process is carried out in sterile conditions, and all the material and solutions used must be previously sterilized in autoclave.

When it comes to obtaining small volumes of enzyme the culture of the microorganism can be carried out following the same procedure described in the preparation of the inoculum. On the contrary, for volumes of culture medium greater than one liter, it is introduced into a fermenter equipped with aeration system. In all cases, it is necessary to leave a sufficient free volume in the containers to facilitate the transfer of oxygen. The medium is inoculated with a certain amount of Thermus active biomass, obtained as indicated in the previous section. After homogenizing the mixture, it is incubated with constant stirring and controlled temperature between 50 and 90 ° C for a time that allows a sufficient level of cell growth to be achieved.

Enzyme Recovery

The cell suspension obtained in the culture of Enzymatic production is frozen in order to disintegrate cellular structures where the enzyme could be retained and facilitate recovery. Once thawed at temperature ambient biomass is separated by centrifugation, and the supernatant, in which the enzymatic activity is found.

Detailed description of the figures

Figure one

Evolution of biomass production and extracellular lipolytic activity in Thermus thermophilus HB27 cultures on medium prepared with Milli-Q quality ultrapure water (black symbols) and thermal mineral-medicinal water (white symbols). Cultures were performed in Erlenmeyer flasks of 2 L capacity, with 400 mL of culture medium, incubated at 70 ° C under the conditions described in Example 1.

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Figure 2

Evolution of the production of biomass (black symbols) and extracellular lipolytic activity (white symbols) in Thermus thermophilus HB27 cultures carried out in a stirred tank reactor of 5 L capacity, with 3 L of culture medium prepared with water thermal medicinal miner, incubated at 70 ° C under the conditions described in Example 2. The two dashed lines indicate the start of operation in continuous mode with a flow rate of 1 L / day (residence time \ = 72 h) , and the change to feed culture medium prepared with Milli-Q quality ultrapure water.

Figure 3

Comparison between the extracellular lipolytic activity detected in various cultures of Thermus thermophilus HB27 when post-incubations are subjected only to a centrifugation stage to separate the biomass (black bars) or to subsequent stages of freezing, thawing and centrifugation (gray bars), as indicated at
Example 3

Examples of embodiment of the invention Example 1 Production of enzymes with lipolytic activity by Thermus thermophilus HB27, in small volume and with culture media prepared in different types of water

In this first example, the influence of the type of water used in the preparation of the culture medium on the evolution of biomass production and extracellular lipolytic activity in Thermus thermophilus HB27 cultures, carried out according to the described procedure is compared.

The culture media used consisted of solutions of the following compounds, in the amounts indicated: tripticase (8 g / L), yeast extract (4 g / L) and NaCl (3 g / L). The pH was initially adjusted to a value of 7.5 ± 0.2 by adding 1 M NaOH. Two media were used, the first prepared in Milli-Q quality ultrapure water and the second in a high thermal medicinal mineral water mineralization and high dry residue content, whose physicochemical properties and composition are indicated in Table 1.

The inoculum for the production process was obtained by culturing the microorganism in 250 mL Erlenmeyer flasks, in which 50 mL of the same culture medium was introduced. They were covered with cellulose caps, to allow passive aeration of the cultures. 1.5 mg (dry weight) of Thermus thermophilus HB27 active biomass, previously prepared, was added to each flask, which corresponds to an inoculum ratio of 30 mg of cells (dry weight) / L. After homogenizing the mixture, it was incubated for 24 hours or until an adequate level of growth of the microorganism (cellular concentration of approximately 1 g / L) in an orbital incubator at a constant temperature of 70 ° C and a stirring speed of 100 rpm. To check the level of cell growth samples were taken periodically and centrifuged for 10 min at 5000 xg and 4 ° C to separate the biomass, which was determined, once washed to remove the remains of culture medium, gravimetrically by
heavy

The produced cell mass recovered, always under sterile conditions, by centrifugation for 10 minutes at 5000 x g and 4 ° C. It was then distributed in sterile vials, removing moisture with the help of a vacuum pump, and kept at -20 ° C until later use as inoculum.

Enzyme production cultures were carried out in Erlenmeyer flasks of 2 L capacity, in which 400 mL of the culture media described were introduced. The flasks were covered with cellulose caps, which allow passive aeration of the cultures. To each flask was added a inoculum ratio of Thermus thermophilus HB27, previously prepared as indicated, of 30 mg of cells (dry weight) / L. After mixing properly, it was incubated for 32 hours in an orbital incubator at a constant temperature of 70 ° C and a stirring speed of 100 rpm. Periodically, samples were taken that were used to determine cell growth and enzymatic activity in the extracellular fluid. The biomass was separated from the samples and its concentration was determined identically to that used in the inoculum preparation cultures. The supernatant obtained in the centrifugation was used to determine the enzymatic activity, by measuring the hydrolysis of p- nitrophenyl laurate at 65 ° C and pH 8.0 (Fuciños et al ., 2005).

The results of this procedure are shown in the Figure 1, expressed as a function of the evolution along the time of biomass concentration and lipolytic activity extracellular, in the two culture media tested. Of them deduces the aptitude of the process for the production of enzymes with lipolytic activity in the described medium, preparing the same in a thermal mineral medicinal water with high dry residue content. In this case, they are reached at 24 h from incubation the maximum levels of enzymatic activity, with values close to 130 U / L. This implies an interesting level of production, given the low levels of expression in the production of extracellular enzymes commonly found in microorganisms Thermophiles

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It should be noted that when the culture medium is prepared in Milli-Q quality ultrapure water, the maximum values of enzymatic activity are obtained later (at 28-32 h of culture), and are significantly lower than those reached in the other medium (85 U / L at 32 h, 33% lower than those detected in medicinal mineral water at the same time of cultivation). With regard to biomass, the profiles are similar in both cases, with final levels around 1.4-1.6 g / L, being slightly higher in crops made in medicinal mineral water. The adequacy of the culture medium proposed for the production of extracellular lipolytic enzymes by microorganisms of the genus Thermus on a small scale is therefore illustrated, highlighting the convenience of using a thermal mineral-medicinal water of high dry residue content for the preparation of the medium. .

Example 2 Production of enzymes with lipolytic activity by Thermus thermophilus HB27 in stirred tank reactor, in discontinuous and continuous mode, with culture media prepared in different types of water

This example shows the evolution of cell growth and the production of extracellular lipolytic activity in Thermus thermophilus HB27 cultures, carried out according to the procedure described in a 5 L capacity fermenter, equipped with a mechanical shovel and systems agitator Temperature control and sterile air inlet. Throughout the culture time, means prepared in a manner analogous to that indicated in Example 1 have been used sequentially, with two types of water (thermal medicinal miner described in Table 1 and Milli-Q ultrapure quality).

Initially, 3 L of culture medium prepared in thermal medicinal mineral water were introduced into the reactor, which was inoculated with 90 mg (dry weight) of Thermus thermophilus HB27 biomass previously prepared as in example 1. The culture was incubated at a constant temperature of 70 ° C, with a stirring speed of 650 rpm, and an air flow of 1 L / min.

After 72 hours of cultivation, the process in continuous mode, with introduction of a current 1 L / day of culture medium prepared in water thermal medicinal miner and extraction thereof reactor content flow. This corresponds to a time of residence in the reactor (ta) of 72 hours.

The system was maintained in these conditions until 312 hours of cultivation, at which time the feeding medium was replaced by another prepared in Milli-Q quality ultrapure water, keeping the rest of the conditions the same (inlet and outlet flow, agitation , aeration, temperature). The process was monitored until 504 hours of
culture.

Samples were taken throughout the process periodic inside the reactor (period in discontinuous mode, between 0 and 72 hours) or from the reactor output current (period in continuous mode, from 72 hours), for determination of biomass concentration and lipolytic activity extracellular

In all cases, the samples were centrifuged for 10 minutes at 5000 xg and 4 ° C, to separate the biomass. The supernatant was used to determine the enzymatic activity, by measuring the hydrolysis of p- nitrophenyl laurate at 65 ° C and pH 8.0 (Fuciños et al ., 2005). The solid residue, once washed to remove the remains of culture medium, was used to determine the biomass gravimetrically by weighing.

The results obtained are shown in the Figure 2, expressed as a function of the evolution along the time of biomass concentration and lipolytic activity extracellular The moments of dashes are indicated by dashed lines. start of continuous operation mode, and change of means of feed culture (medium prepared in water quality thermal or ultrapure medicinal miner Milli-Q). From them the fitness of the Enzyme production scaling procedure thermophilic lipolytics, reaching discontinuously afterwards 72 h incubation levels of enzyme activity around 130 U / L and biomass around 1.2 g / L. They are obtained, therefore, values similar to those detected in small-scale crops as described in example 1, although more times are required crop lengths As indicated above, this implies a acceptable level of production, given the low levels of expression in the production of enzymes commonly found in thermophilic microorganisms. In addition, the viability is demonstrated of the continuous mode of operation for enzyme production lipolytic according to the described procedure, maintaining levels means of enzymatic activity and biomass in the output stream of the reactor around 230 U / L and 1.4 g / L, respectively.

Finally, the effect derived from the replacement of the feed of culture medium prepared in thermal medicinal mineral water with another formulated in Milli-Q quality ultrapure water stands out. From the moment the change is carried out, a progressive decrease in biomass is observed, and especially in the extracellular enzymatic activity detected in the reactor's output current. These results confirm what is indicated in Example 1, regarding the convenience of using thermal mineral-medicinal water for the preparation of the
culture.

Example 3 Recovery of enzymes in extracellular fluid

This example shows the effect of introduce freeze / thaw stages prior to separation of biomass from crops, in order to increase the recovery percentage of the enzymes produced.

The microorganisms of the Thermus genus often form known clusters with the name of multicellular bodies, consisting of a variable number of cells surrounded by a membrane formed by the fusion of the outer membranes of each cell (Becker and Starzyk, 1984). A certain amount of enzyme can accumulate inside these structures, which is not possible to recover in the extracellular liquid if the separation of the biomass is carried out by simple centrifugation. The incorporation of previous stages of freezing / thawing induces the rupture of the membranes that surround these aggregates, and therefore the release of the retained enzymes, virtually maintaining the integrity of the individual cells. Cell suspensions from cultures performed analogously to that described in Example 1 underwent two types of treatment. On the one hand, the suspensions were centrifuged for 10 minutes at 5000 xg and 4 ° C directly after the end of the culture to separate the biomass. The supernatant was used to determine the extracellular enzymatic activity, by measuring hydrolysis of p- nitrophenyl laurate at 65 ° C and pH 8.0 (Fuciños et al .,
2005).

On the other hand, the suspensions were frozen to -40 ° C for 24 h, after which they were thawed at temperature ambient. Then, they were centrifuged for 10 minutes at 5000 x g and 4 ° C, to separate the biomass. The supernatant was used to determine the enzymatic activity, as in the previous case.

The results obtained with samples from 24 h crops grown in prepared media with thermal medicinal mineral water and ultrapure water from Milli-Q quality are shown in Figure 3. It can be observe how the freeze / thaw process leads to a significant increase in the rate of enzyme recovery in the extracellular fluid, which increases between 60 and 150% in samples from crops made in water quality thermal and ultrapure medicinal miner Milli-Q, respectively.

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TABLE 1 Water features thermal medicinal miner used in the preparation of the culture medium used in Examples 1, 2 and 3

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Claims (3)

1. A procedure for obtaining thermophilic enzymes with lipolytic activity from microorganisms of the Thermus genus consisting of the cultivation of the microorganism in a growth medium prepared with a mineral-medicinal thermal water and the recovery of the activity by means of a freezing procedure of The crops 2. A method according to claim 1 characterized by the cultivation in mineral-medicinal water of microorganisms of the Thermus genus and the recovery in the extracellular liquid of the lipolytic enzymes produced. 3. A process according to claims 1 and 2 characterized by the recovery of lipolytic enzymes produced in liquid culture by sequential stages of freezing, thawing and centrifugation.