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Optimization of cultivation media for heterologous gene expression of adamalysin-like melalloendopeptidase Bacillus pumilus.

Introduction

Zink-dependent metalloproteinases constitute a diverse group of enzymes within the metzincines clan, which includes both procariotic and eucariotic enzymes. Eucariotic metzinkins are mainly involved in regulatory processes, they execute highly specific proteolysis, activate and inactivate various bioactive molecules, are involved in processing of peptides, in embryonic development and differentiation of tissues [1,2]. Bacterial proteinases--analogs of eucariotic metzincines, which are an ideal model for research, are of particular interest. Searching for these enzymes among procariotes and their characteristics provides wide opportunities in science and in practice. Mainly, these proteins are found in Gram-negative bacteria. Genome sequencing has allowed to identify homologous genes in the genomes of bacilli. Cloning of the gene of metalloproteinase B. pumilus MprBp made it possible to identify and clean a corresponding protein. It was found that this enzyme belongs to the family adamalysine-like metalloproteinases of the metzincines clan and is currently the only protein of the bacilli isolated in a homogeneous state [3]. In the native strain of B. pumilus, metalloproteinase MprBp is a minor protein and is found in the culture fluid in trace quantities. For a substantial increase in the expression of protein, the gene of MprBp was cloned on plasmid pSA1 in protease deficient strain B. subtilis BG2036, which made it possible to obtain a sufficient amount of homogeneous enzyme for a detailed study of its physico-chemical, enzymatic, and structural features [3,4].

The purpose of the study was the optimization of the nutritional medium for production of metalloproteinase MprBp with the recombinant strain B. subtilis.

Materials and Methods

Erythromycin-resistant strain B. subtilis JB 2036 was used as the object of the research, bearing plasmid pSA1 with the gene of metalloproteinase B. pumilus 3-19 and deficient in its own extracellular proteolytic enzymes.

Plasmid-free strain B. subtilis JB 2036 provided by Professor E. Ferrarri (Genencor Int. Inc., USA) was used as the reference strain.

Initial nutrient medium for strain B. subtilis JB 2036 with plasmid pSA1 was the medium with the following composition (g/l): peptone--17, yeast extract--10, Ca[Cl.sub.2] X 2[H.sub.2]O, 0.1, MgS[O.sub.4] X 7[H.sub.2]O--0.1, NaCl--3, N[H.sub.4]Cl--0.1, MnS[O.sub.4]--0.1, pH of the medium 7.7 [5-7]. The medium was sterilized at 1 atm. Solutions of inorganic phosphate ([Na.sub.2]HP[O.sub.4]), casamino acids, amino acids, carbonhydrates, protein supplements (gelatin, albumin, and casein), and salts of divalent metals were sterilized separately and added into the medium before inoculation in indicated concentrations.

The recombinant strain was cultivated on a shaker (B. Braun, Germany) with swing intensity of 200 rpm at 37[degrees]C for 30 h. The medium volume to flask volume ratio was 1:7. Seed material was the 16 h inoculate (1% v/v). The culture fluid was separated from cells in a centrifuge for 20 min at 8,000 rpm.

The amount of biomass was determined using nephelometry on a photo-electrocalorimeter KFK-2 at 590 nm in a cuvette 1 cm thick and expressed in units of optical density.

Proteolytic activity of metalloproteinase was determined by splitting azocasein [8]. Measurement was made on a spectrophotometer (BioRad, USA) at 450 nm wavelength. The unit of activity was the amount of enzyme that hydrolyzed 1 pg of substrate for 1 min during the experiment, and was expressed in units/ml or in %. Productivity of the culture was defined as the ratio between the value of proteolytic activity to the unit of biomass, and expressed in arbitrary units (arb. units).

Microsoft Excel and the Statgraphics application were used for analysis of experimental data and for analysis of two-factor experiments data. For describing and comparing characteristics, 95% confidence intervals for the averages were built.

Results and Discussion

Study of the dynamics of growth and accumulation of proteolytic activity of metalloproteinases of the recombinant strain B. subtilis showed that the enzyme activity is manifested in the culture fluid at the 18th hour of growth, its level reaches its maximum at the 29-31 h of growth, which corresponds to the stationary phase of culture growth. Proteolytic activity of protease-free strain B. subtilis is found in trace quantities in comparison with the level of activity of the recombinant strain (Figure 1).

The synthesis of extracellular enzymes is determined by the composition of the cultivation medium. Induction of proteases occurs in environments with high content of organic compounds (peptone, casein hydrolysate, yeast extract). Also the composition of the medium often includes inorganic phosphorus-containing compounds that are necessary for the growth of the microorganism and for synthesis of essential compounds.

We found that in the original nutrient medium with peptone content of 17 g/l and inorganic phosphate of 0.3 g/l, the enzyme activity and productivity of the recombinant strain B. subtilis were insignificant. For the purpose of optimization of the nutrient medium composition, we performed a two-factor experiment where concentration of the main components of the nutrient medium varied in three levels: peptone--10, 20, and 30 g/l and inorganic phosphate--0.8, 1.2, and 1.6 g/l (Figure 2).

The results of the two experiments showed that the maximum activity of metalloproteinase was observed at concentration of peptone in the medium of 19 g/l and inorganic phosphate of 1.3 g/l, and the maximum production of the enzyme--at peptone concentration of 20 g/l and that of inorganic phosphate--1.45 g/l (Figure 2).

Effect of Protein Supplements on Metalloproteinase Production by Recombinant Strain B. subtilis

The presence of complex organic substrates in the medium has a stimulating effect on the enzyme biosynthesis. We introduced protein substrates, such as gelatin, albumin, and casein in concentrations from 0.1 to 2 g/l into the cultural medium with concentration of peptone of 20 g/l and that of inorganic phosphate--1.45 g/l. The presence of gelatin and albumin in the medium did not increase the enzyme activity and productivity of the recombinant strain B. subtilis. Introduction of casein at a concentration of 1 and 2 g/l into the environment increased the enzyme activity by 60%, and the culture production by 30-40% (Figure 3).

Since the introduction of casein cultivation in concentrations of 1-2 g/l into the medium had a positive influence on accumulation of activity of the extracellular enzyme and productivity of the culture, we performed a two-factor experiment in order to determine the optimal concentrations of casein and inorganic phosphate (Figure 4), Casein and inorganic phosphate was varied in three levels: casein--0.5, 1.0, and 1.5 g/l and inorganic phosphate--0.8, 1.2, and 1.6 g/l.

As a result, we found the optimal concentration of casein (1 g/l) in the medium that positively influences enzyme biosynthesis.

One of the ways of biosynthesis of extracellular enzymes is the regulation using the final product, which can be amino acids and casamino acids. In cultivation of microorganisms, casamino acids can serve as an additional source of both carbon and nitrogen. If casamino acids were introduced into the medium in concentration from 0.1 g/l to 0.5 g/l, the culture growth was at the level of the reference, and the presence of 0.1 g/l of casamino acids increased the activity of the enzyme 2 times, and the productivity 5 times. With increasing concentration of casamino acids to 1-2 g/l, the culture growth was increased 1.5-2 times, respectively; however, the enzyme activity and the culture productivity significantly decreased (Figure 5). The data obtained make it possible to conclude that the synthesis of metalloproteinase MprBp in the presence of large concentrations of casamino acids is sensitive to nitromethabolic repression.

Thus, in order to increase the production of metalloproteinase MprBp, it is appropriate to add 0.1 g/l of casamino acids into the nutrient medium.

Growth and development of microorganisms is stimulated by various supplements containing vitamins, amino acids, polypeptides, trace elements that promote intensive synthesis of proteolytic enzymes. For this purpose, the composition is often contains corn and yeast extracts, extract of flour soybeans and other substrates [9].

We studied the effect of protein additives (gelatin and albumin) on growth and productivity of the culture, and accumulation in the medium of metalloproteinase activity. It was shown that the addition of gelatin in a concentration of 0.1-2% to the cultivation medium significantly reduces enzyme activity and productivity of culture (Figure 6).

Albumin in the same range of concentrations increases the growth in culture of producer by 20% with 0.5% concentration in the environment. Enzyme activity and productivity of the culture with all studied concentrations decrease as compared to the reference. In this regard, it is impractical to introduce albumin and gelatin to the nutrient medium (Figure 6).

Effect of Amino Acids on Metalloproteinase Production by Recombinant Strain B. subtilis

Amino acids can serve as the source of both carbon and nitrogen and can have various influences on the growth of producer and on the activity of metalloproteinases. It has been noted that synthesis of metalloproteinases of bacilli is stimulated by amino acids such as leucine, isoleucine, tryptophan, glutamic acid, lysine, histidine, arginine added to the nutrient medium in the form of a mixture, or individually [10]. Thus, amino acids are involved in regulation of metalloproteinases synthesis, acting as the stimulant, and as the inducer of the enzyme synthesis.

We studied the influence of some individual amino acids on the growth of the producer and on the activity of metalloproteinase of the recombinant strain B. subtilis. Introduction of nonpolar hydrophobic amino acids (alanine, leucine), and aromatic amino acids of tryptophane causes a significant increase in both growth and activity of the enzyme and productivity of the culture. Alanine increases these figures only in the concentration of 0.1%: the activity increases 2 times and the productivity increases 4 times. With increasing concentration of amino acids, the indicators decrease sharply (Figures 7-11).

Leucine in the range of concentrations between 0.1% and 1% stimulates biosynthesis of metalloproteinase 1.4 times, and the productiveness of the culture increases with increasing concentration of amino acids, and reaches 200% with leucine concentration 1%. The effect of triptophane on metalloproteinase biosynthesis has been identified. When applied to the tryptophane medium at the concentration of 0.5-1%, the activity of the enzyme increases 1.5 times, and the productivity of the culture increases 1.6-2 times. However, strong inhibition of culture growth should be noted, which has a significant impact on productivity increase. Hydrophilic uncharged amino acids, amides of asparagine acid (asparagine) and glutamine acid (glutamine) in particular at low concentrations stimulate the synthesis of metalloproteinase. In the presence of asparagine at a concentration of 0.1% increases the activity of metalloproteinase and medium productivity 2.5 times, without compromising the growth of the producer biomass. However, at concentrations of 0.5% and 1%, these indicators reduce by 30% and 90%, respectively. Asparagine acid in concentrations of 0.1-0.5% has almost no effect on the activity and production of the enzyme, at a concentration of 1%, activity and productivity are reduced by 80% and 40%, respectively. The presence of glutamine in the concentration of 0.1% increases growth by 35%, activity and productivity--2 and 1.5 times, respectively. Addition of glutamine to the cultural medium in a concentration of 1% leads to a decline in the enzyme productivity 2 times. Glutamine acid in a concentration of 0.1% increases the growth of the producer by 40%, while the activity and productivity reduces by 10% and 30%, respectively. With increasing concentration of glutamine acid, the indicators decrease sharply (Figure 13).

Thus, the studied individual amino acids in low concentrations (0.1%) stimulate synthesis of metalloproteinases. With increasing concentration of these amino acids, the enzyme activity and the medium productivity significantly reduce.

The Influence of Carbohydrates on the Biosynthesis of Metalloproteinase by the Recombinant Strain B. subtilis

Often for growing producers of metalloproteinases, media that contain carbohydrates in a wide range of concentrations are used [11-14]. Carbohydrates stimulate growth and activity of metalloproteinases of some bacilli. However, this effect is not always the case. It was shown that under the influence of carbohydrates, particularly glucose, either inhibition of growth and biosynthesis of metalloproteinase or suppression only of enzyme synthesis occurs. In addition, it was noted that growth inhibition is sometimes stimulated formation of metalloproteinase [15]. We studied the influence of carbohydrates on the biosynthesis of metalloproteinase by the recombinant strain B. subtilis.

Monosaccharides (glucose and galactose) and disaccharides (maltose and lactose) in the range of concentrations between 0.1% and 1% have a positively influence upon the growth of the producer. The largest biomass growth is ensured by glucose in a concentration of 0.5% and 1% to 270% and 260%, respectively. Galactose in a concentration of 1% also increases the growth of the culture to 170% (Figure 12). These data are consistent with the literature data.

In case of glucose concentration of 0.1%, the enzyme activity remains at the reference level, while in the presence of other carbohydrates, activity slightly increased. Galactose in concentrations of 0.5% and 1% increases the activity of the enzyme by 40% and 20%, respectively, and the presence of other carbohydrates in the medium reduces enzyme activity (Figure 13). It should be noted that medium productivity in the presence of the studied carbohydrate in a concentration of 0.1% remains at the reference level, and in the presence of lactose, productivity falls by 40%. The increase in the concentration of carbohydrates in the medium to 1% leads to a drop in the metalloproteinase production by recombinant strain (Figure 14).

Thus, it was found that the studied carbohydrates can both stimulate synthesis of the enzyme (galactose) and call it repression, and with increasing concentration of carbohydrates, this effect increases.

The Influence of Divalent Metals Ions on Metalloproteinase Biosynthesis by Recombinant Strain B. subtilis

For cell growth and biosynthesis of enzymes, the presence of small amounts of bivalent metals ([Mg.sup.2+], [Ca.sup.2+]) ions, which are needed by microorganisms as electrolytes and for different catalytic reactions, is required in the medium [16]. In addition, some bacteria require ions of [Fe.sup.2+], [Zn.sup.2+], [Mn.sup.2+], [Co.sup.2+], [Cu.sup.2+], [Ni.sup.2+] in trace quantities, and higher concentrations (mM) have a toxic effect on growth and biosynthesis of the enzyme. In the literature, there is information about positive effect of [Ca.sup.2+], [Mg.sup.2+], and [Mn.sup.2+] ions on the production of proteolytic enzymes [17,18]. Thus, there is a reason to believe that these ions are involved in the mechanism of proteases secretion at the stage of release from the membrane into the medium [19]. In this regard, we studied catalytic activity of the enzyme depending on a concentration of bivalent metals ions added to the cultivation medium (Figure 15).

[Mg.sup.2+] ions have positive effect on enzyme activity. At a concentration of 7 mM, they increase enzyme activity by 25%. [Ca.sup.2+] ions at a concentration of 4-18 mm have no significant effect on metalloproteinase activity. Probably, calcium is involved in ensuring the stability of the enzyme molecule. [Mn.sup.2+] at a concentration of 2 mM considerably reduces enzyme activity. With concentration increasing, the inhibiting effect of [Mn.sup.2+] increases. [Fe.sup.2+] ions in a concentration of 0.5-2 mM considerably inhibits activity enzyme (Figure 15).

Conclusions

So, it was found during the experiment that, for the maximum production of metalloproteinases MprBp, it is practical to introduce into the production medium of recombinant strain of B. subtilispeptone at a concentration of 20 g/l and inorganic phosphate in a concentration of 1.45 g/l as main components, and casamino acid (0.1 g/l), leucine (1%), alanine (0.1%), asparagine or glutamine (0.1%), and [Ca.sup.2+] and [Mg.sup.2+] ions at a concentration of 7 mM and 14 mM, respectively, as additional components. Introduction of gelatin, albumin, tryptophan, aspartic and glutamic acids, carbohydrates, ions [Fe.sup.2+] and [Mn.sup.2+] into the cultural medium is impractical.

MprBp is a unique bacillary homologue of eucariotic adamalysines. A detailed study of its structure will help to learn more about evolution of these enzymes that are important for human health. The cultivation medium received in course of this work will make it possible to obtain the metalloproteinase MprBp in quantities that are sufficient for such studies.

Acknowledgments

This work was performed in accordance with the Russian Government Program of Competitive Growth of the Kazan Federal University. This work was supported by the subsidy allocated to the Kazan Federal University for the state assignment in the sphere of scientific activities.

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Natalia Leonidovna Rudakova *, Nelly Pavlovna Balaban, Ayslu Mirkasimovna Mardanova, Inna Borisovna Chastukhina, Margarita Rashidovna Sharipova

Kazan Federal University, Kremlevskaya Street 18, Kazan 420008, Russia

* Corresponding author: Rudakova NL, Kazan Federal University, Kremlevskaya Street 18, Kazan 420008, Russia; E-mail: natalialrudakova@mail.ru

Received: January 5, 2015; Accepted: February 17, 2015; Published: April 13, 2015
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Title Annotation:Research Article
Author:Rudakova, Natalia Leonidovna; Balaban, Nelly Pavlovna; Mardanova, Ayslu Mirkasimovna; Chastukhina, I
Publication:Biology and Medicine
Article Type:Report
Date:Apr 1, 2015
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