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Keywords:

  • cold-active esterase;
  • Psychrobacter cryohalolentis ;
  • cryopeg;
  • permafrost

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References

A psychrotrophic gram-negative bacterium Psychrobacter cryohalolentis K5T was previously isolated from a cryopeg within Siberian permafrost and its genome has been completely sequenced. To clone and characterize potential cold-active lipases/esterases produced by Pcryohalolentis K5T, we have identified their potential genes by alignment with amino acid sequences of lipases/esterases from related bacteria. One of the targets, EstPc, was cloned and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant protein was produced with a 6x histidine tag at its C-terminus and purified by nickel affinity chromatography. Purified recombinant protein displayed maximum esterolytic activity with p-nitrophenyl butyrate (C4) as a substrate at 35 °C and pH 8.5. Activity assay conducted at different temperatures revealed that EstPc is a cold-adapted esterase which displayed more than 90% of its maximum activity at 0–5 °C. In contrast to many known cold-active enzymes, it possesses relatively high thermostability, preserving more than 60% of activity after incubation for 1 h at 80 °C. It was activated by Ca2+, Mn2+, and EDTA whereas Zn+2, Cu+2, Co+2, Ni+2, and Mg+2 inhibited it. Various organic solvents (ethanol, methanol and others) inhibited the enzyme. Most non-ionic detergents, such as Triton X-100 and Tween 20 increased the lipase activity while SDS completely inhibited it.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References

Cryopegs are the lenses of overcooled sodium chloride water brines derived from ancient marine sediments and sandwiched within permafrost (Tolstikhin & Tolstikhin, 1974). They remain liquid at the in situ temperature of −10 °C as a result of their high salt content (170–300 g L−1) and represent the specific ecological niche inside permafrost grounds (Gilichinsky et al., 2005). Microbial community of cryopegs from the northeastern Arctic region was previously studied (Gilichinsky et al., 2003; Shcherbakova et al., 2004; Kochkina et al., 2007). One of the isolates, Gram-negative bacterium Psychrobacter cryohalolentis K5T, was characterized on the morphological and biochemical levels (Bakermans et al., 2006) and its genome has been completely sequenced (GenBank accession number CP000323). Proteomic studies (Bakermans et al., 2007) as well as transcriptomic data obtained from the work on related Psychrobacter arcticus (Bergholz et al., 2009) revealed different mechanisms of their adaptation to permafrost environment, including modification of transport systems, changes in translation machinery, energy metabolism, and others.

Synthesis of cold-adapted enzymes is one of the key adaptive strategies of psychrophilic and psychrotrophic bacteria (Gerday et al., 2000). Such enzymes have recently received increased attention, owing to their relevance both for basic and applied research (Alquati et al., 2002; Feller & Gerday, 2003). Because of their high catalytic activity at low temperatures, cold-active enzymes are expected to be useful as additives to detergents, biocatalysts for biotransformation of labile compounds at cold temperatures, and bioremediation of polluted soils and wastewaters at low and moderate temperatures (Gerday et al., 2000; Joseph et al., 2007, 2008).

Lipases/esterases (triacylglycerol acyl hydrolases, E.C. 3.1.1.3) catalyze the hydrolysis of acylglycerols and widely exist in animal tissues, plants and microorganisms, including fungi, bacteria, and archaea (Jaeger et al., 1999; Reetz, 2002). Cold-active lipases/esterases have been isolated from different psychrophilic and psychrotrophic bacteria, including bacteria of genus Psychrobacter (Arpigny et al., 1993; Kulakova et al., 2004; Zhang et al., 2007; Chen et al., 2011). In our study, we have cloned novel esterase gene from P. cryohalolentis K5T and characterized the recombinant protein overproduced in Escherichia coli to study its thermostability and optimum catalytic conditions.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References

Bacterial strains

Psychrobacter cryohalolentis strain K5T was provided by All-Russian Collection of Microorganisms (B-2378 Type strain). Escherichia coli strains XL-1 Blue (Stratagene) and BL21(DE3); (Novagen) were used for the cloning and expression steps, respectively.

Gene cloning and sequence analysis

All DNA manipulations were performed by standard methods (Sambrook & Russell, 2001). The genomic DNA-containing cell lysate from P. cryohalolentis K5T was prepared for PCR by boiling of 20 μL of overnight culture grown on agar plates with TSB medium (Difco) and 2% NaCl for 5 min in 50 μL of PCR buffer for Taq DNA polymerase (Fermentas). The esterase gene (without the first 28 codons and terminator codon) was amplified using primers Pc0023F 5′-ATAATACATATGATAAATACCACCCAAAAGATTATTC (with NdeI restriction site underlined) and Pc0023R 5′-ACATGTCGACGTTCTTTAACCCTTCACGAAAC (with SalI restriction site underlined) designed on the basis of the available genomic sequence (http://www.genome.jp/dbget-bin/www_bget?gn:T00350 ). Reaction mixture contained Pfu DNA polymerase buffer with 2 mM MgCl2 (Fermentas, Lithuania), 0.2 mM dNTP mix, 50 pmol each of Pc0023F and Pc0023R, and 5 μL of cell lysate in a total volume of 50 μL. PCR was performed with an initial denaturation at 95 °C for 5 min; followed by 30 cycles of 95 °C for 45 s, 55 °C for 45 s, 72 °C for 90 s; and a final extension at 72 °C for 10 min, using 1 : 4 mix of the high fidelity Pfu DNA polymerase (0.5 u) + Taq DNA polymerase (2 u; Fermentas, Lithuania). PCR product was digested for 2 h at 37 °C with NdeI and SalI restriction enzymes (Fermentas), purified with MinElute Gel Extraction Kit (Qiagen), and cloned into pET-32a(+) vector (Novagen) cut by NdeI and XhoI and purified the same way.

Insertion in the resulting plasmid pET32a(+)/EstPc was verified by sequencing in both directions using standard primers T7prom and T7term with ABI PRISM® BigDye™ Terminator v. 3.1 kit on ABI PRISM 3100-Avant (Genome Centre, Russia).

Homology search was performed by blast (Altschul et al., 1990). Multiple protein alignments were produced with BioEdit software (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). For the analysis of signal sequence cleavage sites SignalP (http://www.cbs.dtu.dk/services/SignalP/) and LipoP (http://www.cbs.dtu.dk/services/LipoP/) were used. DNA and protein statistics were analyzed with Sequence Manipulation Suite (http://www.bioinformatics.org/sms2).

Optimization of recombinant EstPc expression

For the optimization of IPTG concentration and induction time, E. coli BL21 (DE3) cells containing pET32a(+)/EstPc were grown in 5 mL of LB medium at 37 °C, 250 r.p.m. for 10 h. The inoculum (1%, v/v) was transferred into 5 mL of LB medium, and after the cell culture reached optical density of 0.8 at 560 nm, the cells were induced with different concentrations (0.05, 0.1, 0.2 mM) of IPTG at 27 °C for 2 or 4 h. For the optimization of induction temperature, cells were induced with 0.1 mM IPTG at optical density of 0.8 at 560 nm and incubated at different temperatures (27 and 37 °C) for 4 h. The esterase expression level was verified by 13% SDS-PAGE.

Purification of recombinant esterase

Escherichia coli BL21 (DE3) cells harboring the recombinant pET32a (+)/EstPc plasmid were grown at 37 °C and 250 r.p.m. After the cell culture's optical density at 560 nm reached 0.8, the expression of the recombinant EstPc esterase with the C-terminal 6x His-tag was induced with 0.1 mM IPTG. After incubation at 27 °C and 200 r.p.m. for 4 h, induced cells were harvested by centrifugation (7000 g, 15 min at 4 °C) and resuspended in buffer A (50 mM Tris–HCl, pH 8.0, 200 mM NaCl). The cell suspension was sonicated at 50% output (from 200W) on an ice bath for 5 min (10 s on, 1 min off). Cell debris was removed by centrifugation at 34 000 g for 20 min at 4 °C. Purification of the recombinant esterase was carried out by a one-step Ni-affinity chromatography on a Ni-Sepharose FastFlow resin (GE Healthcare).

After sonication, the supernatant solution was loaded on a Ni-affinity column, which had already been equilibrated with buffer A containing 10 mM imidazole. The column was extensively washed with buffer B (20 mM Tris–HCl, pH 8.0, 400 mM NaCl and 20 mM imidazole) to remove any nonspecifically bound proteins. The target protein was eluted with buffer C (20 mM Tris–HCl, pH 8, 200 mM NaCl and 300 mM imidazole). Fractions were analyzed by SDS-PAGE on 13% separating gel. Fractions containing the target protein were combined and extensively dialyzed against buffer D (20 mM Tris–HCl, pH 8.0, 50 mM NaCl).

Esterase activity and protein concentration assay

Esterase activity assay was performed using p-nitrophenyl butyrate (p-NPB) as a substrate (Kulakova et al., 2004). The reaction mixture contained 50 μL of substrate (5 mM p-NPB ester in 2-propanol), 945 μL of buffer (20 mM Tris–HCl buffer, pH 8.0, with 0.5% (w/v) Triton X-100 and 0.1 M NaCl), and 5 μL (1 μg) of purified EstPc. After incubation at 25 °C for 15 min, reaction was stopped by adding 200 μL 10% SDS solution. Absorbance at 415 nm was determined with Model 680 Microplate Reader (Bio-Rad). One unit of esterase activity was defined as a release of 1 μmol of p-nitrophenol per min. Protein concentration was measured with Protein Assay Kit (Bio-Rad) with BSA used as a standard.

Characterization of the esterase

Analytical and preparative gel filtration was performed on a Superdex 75-10/300GL (GE Healthcare) column with flow rate 0.4 mL min−1 in 100 mM Tris–HCl, pH 8.0, 150 mM NaCl. Protein gel electrophoresis was carried out according to Laemmli with Coomassie R-250 staining (Laemmli, 1970).

The optimum reaction temperature was determined by measuring the esterase activity at different temperatures (0–45 °C) under standard assay conditions. The esterase thermostability was evaluated by measuring the residual esterase activity after incubating the esterase at different temperatures (−20 to +90 °C) for 1 h. The optimum pH conditions were determined after incubating the esterase in 50 mM potassium phosphate (pH 5.6–8.0) or 50 mM Tris–HCl (8.0–9.5). Salinity effect was evaluated by activity measurements of the enzyme in 50 mM Tris–HCl buffer (pH 8.0) containing a specific NaCl concentration (0–1.75 M). The effect of various metal ions (Zn+2, Ca+2, Ni+2, Cu+2, Co+2, Mg+2, and Mn+2) and other additives (PMSF, EDTA, and mercaptoethanol) on the esterase activity was assessed by incubating EstPc in 50 mM Tris–HCl, pH 8.0, supplemented with a final concentration of 1 mM of the individual ion solution. Detergent resistance of EstPc was estimated by determining residual activity after 30-min incubation at 5 °C in 50 mM Tris–HCl (pH 8.0) containing 0.5% and 0.05% (w/v) of various detergents (SDS, Triton X-100, Tween 20, and CHAPS). The effect of various organic solvents (methanol, ethanol, acetonitrile, DMSO, and DMFA) on EstPc was estimated by determining residual activity after 30-min incubation at 25 °C of esterase in 50 mM Tris–HCl (pH 8.0) containing 5% and 10% (w/v) of each solvent. Substrate specificity of the esterase was studied at 25 °C and pH 8.0 on p-nitrophenyl ester substrates with variable chain length (C2–C16; Lee et al., 1993).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References

Esterase gene sequence analysis

EstPc gene was identified in P. cryohalolentis K5T genome as a homologue of Psychrobacter immobilis lipase (Arpigny et al., 1993). The level of similarity between these two proteins comprises 89%, with 78% of identical residues. Later lipase sequences with higher similarity to EstPc were published including those from Psychrobacter sp. G (97% and 98%) and Psychrobacter sp. C 18 (79% and 90%; Xuezheng et al., 2010; Chen et al., 2011; Fig. 1).

image

Figure 1. The alignment of deduced amino acid sequence of EstPc from Psychrobacter cryohalolentis K5T with sequences of other lipases/esterases: D2XSH3, Psychrobacter sp. G lipase; D4P8C9, lipase from Psychrobacter sp. C 18; P24640, Moraxella sp. strain TA144 lipase3; Q02104, lipase from Psychrobacter immobilis; Q1M315, lipase from uncultured soil bacterium; Q2KTB5, Psychrobacter sp. 7195 lipase. Amino acid residues belonging to the catalytic triad are indicated by ● and to the oxyanion hole are indicated by ▲.

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EstPc gene consists of 948 bp and encodes a polypeptide of 315 amino acid residues. Total GC content of the gene is 43.99%. The predicted protein has a molecular weight of 34 562.7 Da and an isoelectric point of 6.55. Analysis with SignalP and LipoP tools revealed the presence of a potential signal sequence with cleavage site between 18 and 19 amino acid residues (VVG-CT). The mature protein in this case begins with a single cysteine residue in the molecule. Another possible cleavage site was predicted between 27 and 28 amino acid residues (TLA-INT).

EstPc gene was amplified from the genomic DNA of P. cryohalolentis K5T with gene-specific oligonucleotide primers. Sequencing of the cloned gene revealed its 100% identity with published genomic sequence.

Expression of EstPc in E. coli

To avoid potential issues associated with incorrect targeting of secretory protein in heterologous expression system and its proteolytic degradation, we have amplified the predicted mature coding sequence of EstPc without first 27 codons from the genomic DNA of P. cryohalolentis K5T. The cloned EstPc gene was placed under the control of a strong T7lac promoter by cloning into pET32a(+). The optimal expression conditions for EstPc carrying a His6 tag at its C-terminus were determined in the pilot experiments. Induction was performed with 0.1 mM IPTG for 4 h at 27 °C to promote correct folding and solubility of the recombinant protein. Under these conditions, the recombinant enzyme was produced at high level mostly in soluble active form based on the results of SDS-PAGE (Fig. 2a) and esterase activity assay.

image

Figure 2. EstPc expression and purification: (a) SDS-PAGE analysis of uninduced culture (lane 2), culture after 4 h induction (lane 3) and the purified protein after Ni-affinity column (lane 4). Lane 1 – protein MW markers (Fermentas). (b) Analytical gel-filtration elution profile of EstPc on a Superdex 75-10/300GL column.

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Purification and characterization of the recombinant EstPc

Purification of the recombinant EstPc using the Ni-affinity chromatography resulted in a 3.7-fold increase in target protein purity. The purified EstPc appeared on SDS-PAGE as a single band with an apparent molecular mass of about 33 kDa, consistent with the calculated mature EstPc (28–315) molecular weight (32 985.6 Da; Fig. 2a). Gel-filtration chromatography of purified EstPc on calibrated column (Fig. 2b) confirmed the monomeric nature of this protein. As a result, an active recombinant esterase was obtained with 56% yield and the high specific activity of 8767 U mg−1.

Effects of substrates, pH, and temperature on esterase activity

The substrate specificity assay indicated that short- and medium-chain fatty acids of p-nitrophenyl esters were better substrates for EstPc. It displayed the highest esterolytic activity with the p-NP butyrate substrate. Activity toward the p-NP palmitate constituted only 2% of the maximum activity (Fig. 3).

image

Figure 3. Substrate specificity of the purified EstPc. Lipase assay was performed using p-nitrophenyl esters of fatty acid with varying carbon chain length (C2, C4, C8, C10, C12, and C16). The activity toward p-nitrophenyl butyrate was taken as 100%. Data are given as means ± SD, n = 3.

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The purified EstPc was active in Tris–HCl buffer, pH 8–9.5, and potassium phosphate buffer, pH 6–8. However, the maximum activity was observed in Tris–HCl buffer at pH 8.5 (Fig. 4).

image

Figure 4. Effect of pH on the activity of EstPc. After its incubation with 0.25 mM p-nitrophenyl butyrate as a substrate in various pH buffers at 25 °C for 15 min, the enzyme activity was measured. The enzyme activity in Tris–HCl (pH 8.0) was taken as 100%. Buffers used (final concentration 50 mM) were potassium phosphate (image pH 5.6–8.0) and Tris–HCl (image pH 8.0–9.5). Data are given as means ± SD, n = 3.

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Purified esterase EstPc showed its maximum activity toward p-NPB (C4) at 35 °C, when assayed at pH 8.0. At 0–30 °C, esterase activity was constantly high and amounted 82–97% of maximum. At the temperatures above the optimum, the activity decreased sharply. It was 45% at 40 °C and only 8% at 45 °C (Fig. 5).

image

Figure 5. Temperature dependence of EstPc esterase activity. The enzyme activity was determined with p-nitrophenyl butyrate as a substrate after 15-min incubation at various temperatures. Activity value obtained at 35 °C was taken as 100%. Data are given as means ± SD, n = 3.

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Thermostability of the recombinant EstPc was determined by preincubating the enzyme at different temperatures (−20, 4, 20, 30, 40, 60, 80, 90 °C) for 1 h. The remaining esterase activity was measured at 25 °C. It revealed that the recombinant esterase EstPc was very stable at temperature from −20 to 30 °C and relatively thermostable at higher temperatures (Fig. 6). After incubation at 40 °C, the esterase activity decreased to 87%. When the EstPc was incubated at 60 and 80 °C for 1 h, the esterase activity reduced to 72% and 63% of the initial activity, respectively. And only when the protein was incubated at 90 °C for 1 h, its activity dropped to 27% of the initial level. Taken together, EstPc possesses a typical temperature optimum of cold-adapted lipases/esterases and relatively high thermostability.

image

Figure 6. Effect of temperature on the enzyme stability. The enzyme was incubated in 50 mM Tris–HCl (pH 8.0) at −20, 5, 20, 30, 40, 60, 80, and 90 °C for 15, 30, 45, and 60 min, respectively and the remaining esterase activity was measured with p-nitrophenyl butyrate as a substrate. Please notice that the esterase activities are basically the same at −20, 4, 20, and 30 °C. Data are given as means ± SD, n = 3.

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Effects of salinity, metal ions, detergents, and organic solvents on esterase activity

The esterase activity of purified EstPc increased sharply in the presence of 0.25 M NaCl (180% of the activity in the salt-free buffer). Further increase in NaCl concentration from 0.25 to 1.75 M had a very little effect on its activity (Table 1).

Table 1. Effect of different concentration of NaCl on EstPc esterase activity
NaCl concentration (M)Relative activitya (%)
  1. Data are given as means ± SD, n = 3.

  2. a

    EstPc (1 μg) was incubated in 50 mM Tris–HCl (pH 8.0) containing a specific NaCl concentration (0–1.75 M). Relative activity was determined with 0.25 mM p-nitrophenyl butyrate at 25 °C.

None100.0
0.25179.8 ± 6.1
0.5174.6 ± 2.5
0.75178.5 ± 1.1
1.0179.0 ± 2.6
1.25178.6 ± 1.4
1.5183.4 ± 5.7
1.75179.4 ± 3.1

To avoid possible interference of Ni2+ ions leakage from Ni-Sepharose the following experiments were performed on EstPc preparation additionally purified with gel filtration on a Superdex 75-10/300GL column. The activity of purified EstPc esterase was partially inhibited by Mg2+ (remaining activity, 94%), Co2+ (93%), Ni2+ (74%), Cu2+ (49%) ions, 2-mercaptoethanol (69%) and strongly inhibited by Zn2+ ion and phenylmethylsulfonyl fluoride (to 0.8% and 6.7% of the activity without additives, respectively). Mn2+ ion, Na2N3, and EDTA had enhancement effect on the EstPc esterase activity (121% and 134% of the initial activity respectively) and Ca2+ ion had no effect on it.

The presence of non-ionic detergents Triton X-100, Tween 20, and CHAPS increased EstPc esterase activity by 16–20% while SDS completely inhibited it (Table 2).

Table 2. Effect of various detergents on EstPc esterase activity
DetergentRelative activitya (%)
Concentration 0.05% (w/v) Concentration 0.5% (w/v)
  1. Data are given as means ± SD, n = 3.

  2. a

    EstPc (1 μg) was incubated in 50 mM Tris–HCl (pH 8.0) buffer containing each detergent at 5 °C for 30 min. Remaining activity was determined with 0.25 mM p-nitrophenyl butyrate at 25 °C.

None100.0100.0
Triton X-100119.1 ± 0.2117.1 ± 0.8
Tween 20121.2 ± 0.8116.7 ± 0.1
CHAPS120.5 ± 1.2116.5 ± 0.1
SDS00

All tested organic solvents present at 5% and 10% concentrations reduced the activity of purified EstPc esterase activity by factor of 1.5–3 with the only exception of DMSO that was found to have no effect on EstPc activity (Table 3).

Table 3. Effect of organic solvents on EstPc esterase activity
Organic solventRelative activitya (%)
Concentration 5% (w/v) Concentration 10% (w/v)
  1. Data are given as means ± SD, n = 3.

  2. a

    EstPc (1 μg) was incubated in 50 mM Tris–HCl (pH 8.0) buffer containing each organic solvent for 30 min. Remaining activity was determined with 0.25 mM p-nitrophenyl butyrate in at 25 °C.

None100.0100.0
Acetonitrile28.0 ± 0.51.6 ± 0.3
Ethanol88.3 ± 1.260.0 ± 0.6
Methanol92.0 ± 1.569.0 ± 1.3
DMSO105.4 ± 2.195.0 ± 2.1
DMF45.7 ± 0.79.0 ± 0.8

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References

In this study, we performed cloning and characterization of a new esterase EstPc from a psychrotrophic bacteria P. cryohalolentis K5T. This strain was isolated from a 110 000-year-old Siberian cryopeg (Bakermans et al., 2006). It was shown that the optimal growth temperature of P. cryohalolentis was 22 °C; the minimal and maximal growth temperatures were −10 °C and 30 °C, respectively. It was capable of growth at the pH range from 6.0 to 9.5 and at salinity from 0 to 1.7 M NaCl. Biochemical tests revealed the production of the lipase (C14) and esterase (C4) by this microorganism (Bakermans et al., 2006). Later, the total genomic sequence of this bacterium was obtained (http://www.genome.jp/dbget-bin/www_bget?gn:T00350), which prompted us to begin the cloning and biochemical characterization of its enzymes.

It was shown that numerous Psychrobacter strains harbor lipase/esterase genes, particularly encoding cold-adapted lipases/esterases (Arpigny et al., 1993, 1995; Yumoto et al., 2003; Kulakova et al., 2004; Zhang et al., 2007; Chen et al., 2011). Phylogenetic analysis indicates that some of these cold-adapted lipases/esterases have closely related amino acid sequences (Fig. 1) and, together with EstPc, belong to Family V (or Moraxella esterase 3 like family) of lipolytic enzymes (Arpigny & Jaeger, 1999). Amino acid sequences of lipases/esterases comprising this family are characterized by several features, including dipeptide HG located in the N-terminal part of the protein and GXSXG motive containing the active serine residue (Fig. 1). In EstPc, this motive is surrounded by additional glycine residues, possibly reflecting the enhanced conformational mobility of the active site characteristic for the cold-adapted phenotype. Lys to Lys + Arg ratio in EstPc is 0.78, which is also typical for cold-adapted proteins (Borders et al., 1994). Additional functional determinants that can be predicted on the basis of structural homology are amino acid residues forming the catalytic triad (S142, D264, H292) and oxyanion hole (F76, M143).

Examination of EstPc amino acid sequence with LipoP tool revealed potential presence of lipoprotein signal on its N-terminus. This post-translational modification is of wide occurrence in bacteria and has many potential functions. Lipoprotein signal sequences are characterized by several structural features, including positively charged N-terminus, hydrophobic core, and conserved ‘lipobox’ before Cys residue (Braun & Wu, 1993; Babu et al., 2006). All these features are present in EstPc signal sequence indicating high probability of its lipoprotein nature. After its modification with N-acyl-S-diacylglyceryl moiety and cleavage with SPaseII between Gly and Cys, N-terminal Cys can be anchored to the membrane. The exact localization of lipoprotein in the inner or outer membrane is supposed to be determined with amino acid residues just after N-terminal Cys (+2, +3, +4). Asp in +2 position governs the protein to the inner membrane, while Ser and other residues cause their accumulation in the outer membrane (Yamaguchi et al., 1988; Seydel et al., 1999). Amino acid sequence of EstPc containing threonines in +2 and +3 positions presumably promotes its outer membrane localization in P. cryohalolentis K5T.

Overexpression of the truncated EstPc (28–315) in E. coli BL21 (DE3) led to the production of a recombinant protein with apparent molecular weight of approximately 33 kDa. Most of the known Psychrobacter lipases/esterases have been reported to have a similar molecular weight. (Zhang et al., 2007; Xuezheng et al., 2010; Chen et al., 2011). Expression level was relatively high allowing us to obtain about 30 mg of active enzyme from 1 L of culture.

Our recombinant cold-active esterase showed high relative activity with short- to medium-chain fatty acids (C4 – 100% and C8 – 73%). Esters of longer-chain fatty acids were poor substrates (C12 – 8.3% and C16 – 2%) of EstPc. It differed from those obtained for Psychrobacter sp. C18 LipX lipase (Chen et al., 2011), which showed maximum relative activity with myristate and low activity with butyrate. The substrate specificity of EstPc was similar to one of Pseudomonas sp. Strain B11-1 LipP (Choo et al., 1998) lipases, which also showed maximum activity toward C4 ester.

Maximum EstPc esterase activity was observed at pH 8.5, similar to other alkaline lipases/esterases isolated from psychrotrophs Psychrobacter sp. C 18 (Chen et al., 2011), Pseudomonas sp. strain B11-1 (Choo et al., 1998), Acinetobacter baumannii BD5 (Park et al., 2009) and Psychrobacter sp. 7195 (Zhang et al., 2007). The purified EstPc activity was increased in the presence of NaCl, but was not substantially affected by changing salt concentration from 0.25 to 1.75 M. LipX activity also increased in the presence of NaCl (Chen et al., 2011), but the effect was not as pronounced as in the case of EstPc. Probably this result is related to the host strain P. cryohalolentis K5T isolated from an ultrasaline (2 M NaCl) cryopeg environment (Gilichinsky et al., 2005).

The optimal temperature for the EstPc activity was 35 °C, which is similar to that of lipases from Acinetobacter baumannii BD5 (Park et al., 2009), Pseudomonas sp. KB700A (Rashid et al., 2001) and lower than that of LipP from Pseudomonas sp. B11-1 (Choo et al., 1998) and Lip3 from Moraxella sp. TA144 (Feller et al.,1991). The EstPc exhibited 91.5% and 92.5% of its highest activity at 5 and 10 °C, respectively in contrast to LipX (Chen et al., 2011) whose relative activity at these temperatures was < 30%.

EstPc displayed enhanced thermal stability compared to previously reported for cold-adapted lipases/esterases. After incubation at 60 and 80 °C for 1 h, the esterase activity of EstPc reduced respectively to 72% and 63% of its initial value. Only after incubation at 90 °C for 1 h, a substantial reduction in enzyme activity to 27% was observed. It should be noted that the lipase activity of LipX reduced more rapidly to 30% and 20% after incubation at 65 and 70 °C for 1 h (Chen et al., 2011). Also KB-Lip activity reduced more than 70% after incubation for only 5 min at 60 °C (Rashid et al., 2001) and LipP after incubation at 70 °C for 30 min was totally inactivated (Choo et al., 1998).

We also examined effects of various compounds on the enzyme activity. It has been reported that metal ions and metal ion chelators affect the lipase activity (Chakraborty & Paulraj, 2009). Our data showed that the EstPc esterase activity was partially inhibited by Mg2+, Co2+, Ni2+, Cu2+ ions and strongly inhibited by Zn2+ ion. Mn2+ ion and EDTA had stimulating effect on the EstPc activity and Ca2+ ion had no effect on it. It is known that Zn2+ ion also strongly inhibited the LipX, LipP and LipA1 (Chen et al., 2011; Choo et al.,1998, Zhang et al., 2007). Interestingly, the presence of EDTA inactivated the majority of related enzymes (LipX, LipA1, and PsyEst) and had no effect on LipP (Chen et al., 2011; Choo et al., 1998, Kulakova et al., 2004; Zhang et al., 2007).

It should be noted that up to now, no three-dimensional structure has been yet solved for any of the members of Family V lypolytic enzymes. Construction of an efficient expression system for new cold-active esterase from P. cryohalolentis K5T is a prerequisite for all structural studies of this enzyme and other related proteins and creates the basis for their optimization. Our current work on the cloning and purification of the new cold-active esterase from P. cryohalolentis K5T is one of the first attempts to characterize the enzyme representing Siberian permafrost bacterial community. The obtained results indicate that recombinant esterase EstPc exhibits high activity at low temperature and relative stability at high temperatures. These properties make EstPc an attractive candidate for various biotechnological applications; for example, it can be included in detergent preparations and used for bioremediation of polluted soils and waters in cold regions. The obtained results also prove that the unique microbial community of cryopegs may be an important resource for prospecting novel cold-active lipases/esterases and other enzymes.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References

The authors thank V. Shcherbakova for providing the strain P. cryohalolentis K5T. This work was supported by Russian Academy of Sciences program ‘The origin of the biosphere and the evolution of geo-biological systems. The microbial biosphere'.

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  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References
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