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Cloning, expression, purification, and characterization of lipase 3646 from thermophilic indigenous Cohnella sp. A01
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Bahram pooreydy Golaki, Saeed Aminzadeh ⇑, Ali Asghar Karkhane ⇑, Parisa Farrokh, Seyed Hossein Khaleghinejad, Asal Akhavian Tehrani, Sina Mehrpooyan
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Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14965/161, Tehran, Iran
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Article history: Received 27 July 2014 and in revised form 27 September 2014 Available online xxxx Keywords: Cloning Expression Purification Thermostable lipase Cohnella sp. A01
a b s t r a c t Lipases form one of the most important groups of biocatalysts used in biotechnology. We studied the lipase from the bacterium Cohnella sp. A01 due to the versatility of thermophilic lipases in industry. In this study lipase 3646 gene from the thermophilic bacterium Cohnella sp. A01 was expressed in Escherichia coli and the enzyme was purified by a two-steps anion exchange chromatography. The purified lipase appeared to have a molecular weight of approximately 29.5 kDa on SDS–PAGE. The values of Km and Vmax, calculated by the Michaelis–Menten equation, were 1077 lM and 61.94 U/mg, respectively. The kinetic characterization of the purified enzyme exhibited maximum activity at 70 °C and pH 8.5. Activities at 50, 55 and 60 °C for 120 min were measured 58%, 47% and 41%, respectively. The enzyme was also highly stable at the pH range of 8.5–10.0 for 180 min. The effect of EDTA indicated that the enzyme is not a metalloenzyme. The stability of lipase 3646 in the presence of organic solvents, detergents, metal ions and inhibitors suggested that this lipase could be exploited in certain industries such as detergent and leather. Lipase 3646 was determined structurally to be 37.5% a-helix, 12.8% b-sheet, 22.7% b-turn and 27% random coil. Ó 2014 Published by Elsevier Inc.
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Introduction
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Lipases (triacylglycerol acyl hydrolases, E.C. 3.1.1.3) have a wide range of applications in a variety of industries such as food (dairy, biosensors for food, bakery, fats and oil) [1], biodiesel [2], cosmetic, pharmaceutical, leather, textile, detergent, and paper [3,4]. Since these enzymes catalyze both the hydrolysis and the synthesis of a great variety of esters [5], and inter and trans-esterification [6] such as the resolution of racemic mixtures [7,8] they are considered as multipurpose enzymes. Lipases belong to a/b hydrolase superfamily and most of them are serine enzymes in which the active site has a hydrogen bond network consisting of a catalytic triad of serine–histidine–aspartic/glutamic acid [9]. The serine residue usually is located in the conserved pentapeptide GlyXaa-Ser-Xaa-Gly motif. However, it has been shown that in various bacilli the first glycine in the conserved pentapeptide has been replaced by an alanine residue [10,11]. The active sites of the majority of bacterial lipases are covered by an a-helical flexible ‘‘flap’’ that can change from closed to open conformation when
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⇑ Corresponding authors. E-mail addresses: [email protected] (S. Aminzadeh), [email protected] (A.A. Karkhane).
the enzyme is absorbed by the lipid–water interface [12]. The phenomenon of increased lipase activity at the lipid–water interface is known as the interfacial activation [13]. Lipases are widely present in plants, animals and microorganisms [11], but those from thermophiles, due to their high stability at elevated temperatures and under severe operational and/or storage conditions, have become the subject of special interest for biotechnological applications [14,15]. Since the thermophilic bacteria are expected to produce more heat-stable enzymes than mesophilic microorganisms, they are usually used as a source of thermostable enzymes [16]. Implementation of biotechnological processes at high temperatures has many advantages: high solubility of substrates (in particular for poorly soluble or polymeric molecules) which can shift the equilibrium to a higher product yield, higher reaction rates, increased availability of substrates, decreased risk of microbial contamination and lower viscosity of reaction mixtures which in turn reduces the costs related to pumping, filtration and centrifugation [17,18]. In the present study, we report cloning and overexpression of a novel lipase gene from thermophilic indigenous Cohnella sp. A01 (isolated by Aminzadeh et al. from shrimp farming ponds (unpublished data). We also report purification and characterization of the overexpressed enzyme in Escherichia coli BL21.
http://dx.doi.org/10.1016/j.pep.2014.10.002 1046-5928/Ó 2014 Published by Elsevier Inc.
Please cite this article in press as: B.p. Golaki et al., Cloning, expression, purification, and characterization of lipase 3646 from thermophilic indigenous Cohnella sp. A01, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.10.002
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Materials and methods
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Materials
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High pure DNA purification kit was purchased from BIONEER (Seoul, Korea). NdeI, EcoRI and pTZ57R/T plasmid were provided by Fermentas (Glen Burnie, MD, USA). High pure plasmid purification kit was purchased from Roche. E. coli DH5a and E. coli BL21 (DE3) strains, and vector pET-26b (+) were obtained from Invitrogen (Carlsbad, CA, USA). 1-Anilino naphthalene-8-sulfonate (ANS), p-nitrophenyl esters and p-nitrophenol (pNP) were purchased from Sigma (St. Louis, USA). DE52 resin was provided from Whatman. All other chemicals were purchased from Merck (Darmstadt, Germany). Software VMD 1.9 (University of Illinois, Urbana-Champaign, USA), Gene Runner 4.0 (Lynnon Biosoft, Canada), GraphPad Prism 5 (San Diego, CA, USA), were used for displaying and analysis of the protein structure [19], DNA sequence analysis, and creation of the Michaelis–Menten curve and calculation of the Km and Vmax, respectively.
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Methods
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Cloning of lipase gene from Cohnella sp. A01 Cohnella sp. A01 was used as the lipase gene source. The bacterial cells were cultured in nutrient broth medium at 60 °C for three days and used for DNA extraction. Lipase gene was obtained by PCR using the forward primer with NdeI recognition site (50 -TA CATATGCGCAAGGGCGGCTAC-30 ) and the reverse primer with EcoRI recognition site (50 -ATGAATTCTCATTACCCGCGGTTCTTGCC CG-30 ). The amplified fragment with a size of 807 bp was ligated into pTZ57R/T vector and sequenced using M13/pUC forward and M13/pUC reverse sequencing primers. Recombinant plasmid (pAKP-AT) was digested with endonucleases NdeI and EcoRI and the fragment containing the lipase gene was ligated into the expression vector pET-26b (+) which had been digested with the same enzymes. Finally, the ligated plasmid pAKP-3646 was transformed into E. coli BL21.
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Expression and purification After transformation a single colony was grown overnight at 37 °C in LB medium containing 30 lg/ml of kanamycin then transferred into a fresh medium. Protein expression was induced by final concentration of 0.5 mM IPTG after 3 h of incubation at 37 °C when the optical density at 600 nm (OD600) reached 0.6. Subsequently cells were collected by centrifugation (9000g for 10 min at 4 °C) and resuspended in 25 ml of 100 mM Tris-base buffer (pH 8.5). After that the cells were sonicated on ice for six sets of twominute pulses (100 mHz) with 10-s intervals. Crude cell extracts were centrifuged (9000g for 20 min at 4 °C) and the supernatant was used for protein purification. The purification method consisted of two steps: ion-exchange chromatography using DE52 resin in pH 5.5, and the same column in pH 4.3. Step 1: 40 ml of supernatant was dialyzed overnight in 50 mM Tris–HCl buffer (pH 5.5) at 4 °C. The dialyzed solution was centrifuged (10,000g for 15 min at 4 °C) and was applied on the DE52 column pre-equilibrated with the same buffer. The column was washed with three 50 mM Tris–HCl buffers (pH 5.5) containing 270, 330 and 500 mM of NaCl, respectively. The fractions were collected and assayed for lipase activity and analyzed for purity by SDS–PAGE. The fraction containing 330 mM NaCl eluent was retained for further purification. Step 2: 60 ml of the fraction containing 330 mM NaCl eluent from the previous step was dialyzed overnight in 50 mM Tris– HCl buffer (pH 4.3) at 4 °C. The dialyzed solution was centrifuged
(10,000g for 15 min at 4 °C) and the supernatant was applied on a DE52 column pre-equilibrated with the same buffer (pH 4.3). In this step other proteins were bound to column but lipase 3646 was eluted without binding to column and the flow-through was collected. All fractions were assayed for lipase activity and visualized using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) [20]. Protein bands were stained by Coomassie brilliant blue R250.
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Protein assay
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The protein concentration was estimated according to Bradford’s method using bovine serum albumin as standard solution [21].
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Circular dichroism
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Purified lipase 3646 was analyzed with spectropolarimeter J-715 (Jasco, Tokyo, Japan) equipped with a thermostatically controlled cell holder. Samples were dialyzed against 10 mM Tris-base buffer at pH 8.5. Circular dichroism (CD) spectrum was measured at a protein concentration of 0.2 mg/ml, using a 1-mm path length quartz cell. Secondary structure of the enzyme was monitored in the region between 190–250 nm.
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Lipase activity and substrate specificity
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Enzyme activity was estimated by a spectrophotometric assay with p-nitrophenyl palmitate (pNPP) as substrate. A variety of substrates, including pNPP, were examined in a mixture containing 3 mM p-nitrophenyl esters, 50 mM Tris-base buffer (pH 8.5), 1% acetonitrile (v/v) and 4% 2-propanol (v/v). Enzyme solution was added to the pNPP solution and reaction progress was monitored at 410 nm per minute at 70 °C using a spectrophotometer (Beckman, DU 530 from USA) with pNP as the standard solution. One unit of enzyme activity was defined as the amount of the enzyme that liberated 1 lmol of pNP per minute under the standard assay conditions.
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Effect of pH on the lipase activity and stability
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pH profile was studied with p-nitrophenyl palmitate assay at a pH range of 3.0–12.0 by using 50 mM acetate (pH 3.6–5.6), 50 mM phosphate (pH 5.8–8.0), and 50 mM glycine (pH 8.6–10.6) buffers. Enzyme stability in different pHs was measured at pH 5, 8.5 and 10 by using the same buffers. Enzyme activity was measured at 70 °C and pH 8.5 every 30 min for six times.
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Effect of temperature on the lipase activity and stability
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The effect of temperature on the lipase activity was studied by preincubation of the enzyme at a temperature range of 30–100 °C in 50 mM Tris-base buffer (pH 8.5). For the measurement of thermostability, the enzyme was incubated at 55, 60, 65 and 70 °C temperatures for 120 min in 50 mM Tris-base buffer (pH 8.5) and the activity was measured every 30 min.
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Effect of metal ions, inhibitors, detergents, organic solvent on the lipase activity
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The effect of metal ions (1 mM) on lipase activity was investigated using Zn2+, Cu2+, Fe2+, Mg2+, Mn2+, Na+, K+ and Ca2+. 5 mM of phenyl methyl sulfonyl fluoride (PMSF), diethyl pyrocarbonate (DEPC), iodoacetamide and EDTA were used as inhibitors. The influence of 1% (v/v) surfactants (SDS, tween-20, 40, 65 and 80,
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Please cite this article in press as: B.p. Golaki et al., Cloning, expression, purification, and characterization of lipase 3646 from thermophilic indigenous Cohnella sp. A01, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.10.002
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triton X-100 and deoxycholate) and 30% (v/v) of organic solvents (methanol, ethanol, 2-propanol, glycerol, b-mercaptoethanol, aceton, chloroform, n-hexan, n-heptan and diethylether) on the enzyme activity were examined. The enzyme was treated with these compounds at room temperature for 120 min. The relative activity was subsequently measured at 70 °C and pH 8.5 by pNPP substrate. The activity of the untreated enzyme without additives was considered 100%.
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Determination of substrate specificities
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Substrate specificity was analyzed under optimum conditions (pH 8.5 and 70 °C) using p-nitrophenyl butyrate (pNPB; C4), p-nitrophenyl hexanoate (pNPH; C6), p-nitrophenyl octanoate (pNPO; C8), p-nitrophenyl decanoate (pNPD; C10), p-nitrophenyl myristate (pNPM; C14), p-nitrophenyl palmitate (pNPP; C16) and p-nitrophenyl stearate (pNPS; C18). The activity was determined at 410 nm for 1 min incubation at 70 °C.
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Michaelis–Menten constants
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Lipase assays were performed in 50 mM Tris-base buffer (pH 8.5) at 70 °C with a range of 0.01–1.5 mg/ml of concentration of the pNPP. Michaelis–Menten plot was used to determine Km and Vmax in GraphPad Prism 5 software. The effect of substrate concentration (pNPP) on the reaction rate was examined for 1 min at pH 8.5 using a spectrophotometric method.
CD spectroscopy was used to elucidate the secondary structure content of the purified enzyme. The CD spectrum of secondary structure analysis showed that the lipase 3646 contained 37.5% a-helix, 12.8% b-sheet, 22.7% b-turn and 27% random coil. While prediction of secondary structure showed 30.6 a-helix, 20.9% b-sheet, 51.5% b-turn and random coil. There is 7–8% different between results of prediction and Circular dichroism that this different is normal.
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Results and discussion
Effect of temperature on lipase activity and stability
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Cloning of lipase 3646
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Cloning of the lipase gene into cloning vector (pAKP-TA) was confirmed by sequencing, and subsequently the gene was successfully subcloned into the expression vector pET-26b (+) (pAKP3646). Using Gene Runner software, the sequence analysis indicated that the fragment encoded a putative protein of 268 amino acids with a molecular mass of 29.5 kDa. The gene of lipase 3646 with the accession number JX833623 was recorded in GenBank database. BLAST of lipase 3646 gene in NCBI showed 53% identity to the sequences of the triacylglycerol lipase from Paenibacillus sp. HGF5 (GenBank Accession No. EGG38175). The protein structure prediction indicated that this enzyme belongs to the a/b hydrolase superfamily. Lipase 3646 contains the consensus sequence Gly-His-Ser-leu-Gly with the catalytic triad of Ser 133, His 212 and Asp189. In addition, there is an active site flap
The purified recombinant lipase exhibited maximum activity at 70 °C and was highly active in the temperature range of 50–80 °C (Fig. 3A). The optimum temperature for lipase 3646 was similar to the lipases from Bacillus sp. L2 [26], Thermoanaerobacter thermohydrosulfuricus and Caldanaerobacter subterraneus subsp. tengcongensis [27] which is higher than that of the thermostable lipases from Thermomyces lanuginosus [28] and Geobacillus thermodenitrificans IBRL-nra [25]. As shown in Fig. 3B, more than 45% of lipase 3646 activity maintained after incubation at 50, 55 and 60 °C for 90 min. However, after incubation at 65 °C for 60 min remaining activity was about 45%. Incubation at 70 °C for 30 min dampened the activity to 33%. The stability of lipase 3646 at 65 °C for 30 min was higher than that of BTL2 lipase from Bacillus thermocatenulatus (30 min at 60 °C) [29] and the lipase from Bacillus stearothermophilus L1 (30 min at 65 °C) [30]. In addition, the enzyme was more stable in 60 and 65 °C for 60 min than
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spanning from amino acid 73 to 84. Three dimensional structure of lipase 3646 was predicted using Phyre 2 server and visualized by VMD 1.9 software (Fig. 1).
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Purification of the recombinant lipase
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The recombinant lipase was purified in two steps. Chromatography for the first step purification is shown in Fig. 2A. A single band with an apparent molecular mass of about 29.5 kDa was detected by SDS–PAGE electrophoresis (Fig. 2B). Although three to four-steps purification was followed by most of the researchers [22,23], we were able to purify the enzyme using one resin and just by changing the pH. Purification of lipase 3646 using DE52 resin gave a yield of 38% with a purification factor of 13.4 (Table 1), while other studies achieved the final yields of 2.5 [24], 8.9 [25] and 19.7% [22].
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Circular dichroism spectral analysis of lipase 3646
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Fig. 1. 3D structure of lipase 3646. The a/b hydrolase structure of lipase 3646 is shown as new cartoon representation. 3D structure of the nucleophilic elbow, active site flap and active site amino acids.
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Fig. 2. (A) Purification profile of lipase 3646 using DE52 ion exchange chromatography by 270, 330 and 500 mM NaCl, respectively. (B) SDS–PAGE of expressed and purified protein encoded by lipase 3646 gene. lane M, protein molecular mass marker; lane 1, total protein before induction with IPTG; lane 2, total protein after induction with IPTG; lanes 3, 4 and 5, column content eluted with 270, 330 and 500 mM NaCl, respectively in the first step of purification; lane 6, column content eluted after the second step of purification (for details please see the M&M section).
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the lipase of Bacillus sp. L2 (60 min at 60 and 65 °C) [26]. Hydrophobic amino acids increase proteins thermostability [31]. 45.5% of amino acids in Lipase 3646 are hydrophobic which could be the reason of the thermostability of this enzyme. Since most of the industrial processes operate at temperatures above 50 °C, the importance of thermostable enzymes such as lipases is significantly enhanced [22] and many heat-stable lipases have been isolated from various sources such as Pseudomonas fluorescens [32] and Bacillus species [22,23].
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Effect of pH on lipase activity and stability
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The stability of lipase 3646 in alkaline pH justified it as a potential alkaline lipase in the degreasing process in leather industry [33]. The effect of pH on the enzyme activity and stability is shown
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in Fig. 3C and D. Lipase 3646 was the most active in the pH range of 7–9, with more than 70% of its lipolytic activity. Maximal activity was observed at pH 8.5. The pH profile of lipase 3646 was similar to the lipases from Bacillus subtilis [11] and Antrodia cinnamomea BCRC 35396 [34]. Optimum pH for lipase 3646 activity also was higher than those of the thermostable lipase from G. thermodenitrificans IBRL-nra [25], Pf2001lipase from Pyrococcus furiosus [35], and Rhizopus oryzae lipase (ROL) [4]. The enzyme showed high stability in alkaline pH. Remaining activity after 180 min of treatment at pH 10 was more than 60%, while after treatment at pH 8.5 and 5, it was below 40%. Furthermore, stability of lipase 3646 after treatment at pH 10 for 120 min was more than those of the L2 lipase from Bacillus sp (30 min at pH 8.5 and 10) [26], and the lipase from Geobacillus sp. TW1 (15 min at pH 10) [5].
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Effect of organic solvents, metal ions, inhibitors and detergents on lipase activity
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The enzyme activity in the presence of various organic solvents, metal ions, inhibitors and detergents is shown in Table 2. The enzyme activity was not significantly changed in the presence of the methanol, ethanol, 2-propanol, n-hexane and n-heptane but was decreased in the presence of the acetone, chloroform and diethylether. Klibanov in 1997 reported that in most cases activity was more declined by organic solvents rather than water because of the changes in destabilization of the enzyme, protein flexibility, or diffusional limitations [36]. Decrease in the activity in the presence of acetone and chloroform has also been reported for LipSBRN2 [9] and the lipase from Rhizopus oryzae [37]. Lipases from Candida rugosa, Candida antarctica and Pseudomonas cepacia were inactivated by 10% methanol after 30 min of treatment [38]. The enzyme activity was decreased to 38% for lipase from Pseudomonas sp. B11-1 by 30% ethanol [39]. It was also reduced to 66% for LipSBRN2 by 30% 2-propanol [9]. In the case of 30% b-mercaptoethanol the activity dropped dramatically, however in the presence of 10% 2-mercaptoethanol it fell slightly down to 89%. Reduction in lipase activity to 64% in the presence of 1 mM 2-mercaptoethanol has been reported previously [22]. The remaining activity was increased by glycerol by the amount of 10%. The effects of metal ions on lipase activity are shown in Table 2. Metal ions play important roles on the structure and function of enzymes. For example Ca2+ increases the amount of alpha-helix and beta-sheet content in Pseudomonas fluorescence SIK W1 lipase [40]. In the present study, the remaining activity was more than 75% in the presence of Zn2+, Ca2+ and Na+, in addition it was more than 60% after treatment with the Cu2+, Mg2+ and Mn2+. Treatment with Fe2+ and K+ lowered the enzyme activity to below 60% of the control value. Arnaldo et al. also reported similar results about the effects of 1 mM metal ions such as of Zn2+, Cu2+, Fe2+, Mg2+, Mn2+, Na+, and Ca2+ on lipase activity, especially the decrease in activity down to 33 and 74% by Fe2+ and Ca2+, respectively [41]. 1 mM of Mg2+ and Zn2+ inactivated Lipase from Bacillus coagulans BTS-3 [24]. The addition of 1 mM of metal ions such as Mg2+, Fe2+ and Cu2+ to L2 lipase, dampened the activity to about 30% and Zn2+, K+ and Na+ also reduced the activity more than 50% of the control value [26]. An increase in the lipase activity in a short time (15 min) and a reduction of the activity in a long time by Ca2+
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Table 1 The efficiency of purification of recombinant lipase. Steps
Volume (ml)
Total protein (mg)
Total activity (units)
Specific activity (units/mg)
Purification (fold)
Yield (%)
Crude DE52 (pH = 5.5) DE52 (pH = 4.3)
37 63 63
47.51 5.48 1.36
83.62 58.10 31.96
1.76 10.63 23.51
1.00 6.02 13.35
100 69.52 38.00
Please cite this article in press as: B.p. Golaki et al., Cloning, expression, purification, and characterization of lipase 3646 from thermophilic indigenous Cohnella sp. A01, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.10.002
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Fig. 3. The effect of pH and temperature on the recombinant lipase. (A) The effect of temperature on the activity of the recombinant lipase. (B) Thermostability of the recombinant lipase. (C) The effect of pH on the activity of the recombinant lipase. (D) pH stability of the recombinant lipase (For more details please see M&M section).
Table 2 Effect of some organic solvents, metal ions, inhibitors, detergents and substrate chain length on the activity of lipase 3646. Effector molecule Organic solvent (30%) None Acetone Methanol Ethanol 2-Propanol n-hexane n-heptane 2-Mercaptoethanol (30%) 2-Mercaptoethanol (10%) Chloroform Diethylether Glycerol Metal ions (1 mM) None Zn2+ Cu2+ Fe2+ Mg2+ Mn2+ K+ Ca2+ Na+
345 346 347 348 349 350 351 352 353 354
Relative activity (%) 100 ± 1.7 58.0 ± 2.39 94.3 ± 4.2 92.1 ± 3.4 88.4 ± 4.47 94.0 ± 3.0 93.5 ± 3.1 0 ± 0.0 89.2 ± 3.5 74.8 ± 1.0 75.6 ± 2.9 110.3 ± 4.6 100 ± 1.9 85.3 ± 2.3 61.3 ± 3.5 35.7 ± 1.1 69.3 ± 3.0 65.2 ± 1.5 59.8 ± 3.3 81.6 ± 4.5 79.8 ± 2.6
has been previously reported [42]. Therefore, the decrease in lipase 3646 activity by Ca2+ probably is due to the treatment of the enzyme with Ca2+ for a long time (120 min). Iodoacetamide is an irreversible inhibitor of enzymes with a cysteine in their catalytic sites. In this assay, iodoacetamide had no effect on the enzyme activity which suggests that lipase 3646 does not have a cysteine in its active site. The activity was inhibited in the presence of 5 mM PMSF, showing that the catalytic triad is inactivated through covalent modification of serine by PMSF. Furthermore, the enzyme activity was decreased to 38% by 5 mM
Effector molecule Effectors (5 Mm) None EDTA Iodoacetamide PMSF DEPC Detergents (1%) None SDS Tween 20 Tween 40 Tween 65 Tween 80 Triton x100 Deoxycholate Substrate specificity pNPB (C4) pNPH (C6) pNPO (C8) pNPD (C10) pNPM (C14) pNPP (C16) pNPS (C18)
Relative activity (%) 100 ± 2.1 88.6 ± 2.5 100 ± 1.9 8.7 ± 2.3 37.7 ± 3.5 100 ± 2.4 0.0 ± 0.0 92.6 ± 1.5 87.8 ± 3.3 87.1 ± 4.6 42.45 ± 1.9 20.4 ± 3.4 93.6 ± 3.9 100 ± 3.8 58.8 ± 4.3 24.9 ± 2.1 31.4 ± 3.3 19.0 ± 2.4 74.7 ± 3.6 83.1 ± 2.5
DEPC. These effects suggest that lipase 3646 indeed have a catalytic triad containing serine and histidine residues. The sharp decline in activity by PMSF and DEPC have also been reported by other researchers [26,41]. The enzyme activity was decreased only 11% in the presence of 5 mM EDTA. This indicated that the enzyme is not a metalloenzyme [22]. The effect of detergents was determined after 120 min of treatment at room temperature. Remaining activities were 93%, 92%, 88%, and 87% in the presence of deoxycholate, tween 20, tween 40, and tween 65, respectively. The decrease in the activity to
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Fig. 4. Michaelis–Menten plot for purified lipase 3646.
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42% by tween 80 may be due to the long acyl ester chains of this detergent, making a substrate for the enzyme to form a competitive inhibitor. These results also suggested that the enzyme has a preference for C18 esters (Tween 80) over C16 (Tween 40) and C12 (Tween 20). It has been reported previously that lipases can use tween 80 as a substrate [41,43]. Remaining activity was reduced to 20% in the presence of Triton 100 and was dramatically decreased by SDS.
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Lipase specificity
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Table 2 demonstrates the substrate specificity of the lipase 3646 with p-nitrophenyl esters of varying acyl chain lengths from C4 to C18. The highest activity was observed by pNPB (100%) and high activities were obtained by pNPS (C18), pNPP (C16) and pNPH (C6), respectively. These results suggested that lipase 3646 has a strong catalytic ability toward the substrates with short and long acyl chains and low activity for those substrates with mediumchain fatty acyl esters. The high lipase activity toward long-chain fatty acid esters indicated that lipase 3646 is a true lipase in contrast to esterases that hydrolyze exclusively short chain fatty acyl esters [10].
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Michaelis–Menten plot
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The kinetic parameters were determined using Michaelis– Menten plot (Fig. 4). The values of Km and Vmax with pNPP substrate were calculated 1077 lM and 61.94 U/mg, respectively.
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Conclusion
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Thermophilic lipases which facilitate the performance of industrial reactions in higher temperatures are widely used in many industries such as leather and detergent. In this study, we cloned the lipase 3646 gene from thermophilic bacterium Cohnella sp. A01 and the corresponding protein was expressed, purified and characterized. Lipase 3646 possesses the conserved region Gly-His-Ser-Leu-Gly and amino acids Ser and His in its active site. Lipase 3646 had a strong catalytic ability toward substrates with long acyl chains and showed appropriate activity in high temperatures and alkaline pH. Not only this lipase was not a metalloenzyme, but also its stability in the presence of several organic solvents, metals ions, inhibitors and detergents indicated that this enzyme could be exploited in certain industries as explained before. Structure–function relationship of this enzyme is being investigated further.
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Acknowledgements
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We would like to thank Dr. R.H. Sajedi and Mr.R. Mohseni for technical supports.
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References
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Contents lists available at ScienceDirect
Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep 5 6
Cloning, expression, purification, and characterization of lipase 3646 from thermophilic indigenous Cohnella sp. A01
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Bahram pooreydy Golaki, Saeed Aminzadeh ⇑, Ali Asghar Karkhane ⇑, Parisa Farrokh, Seyed Hossein Khaleghinejad, Asal Akhavian Tehrani, Sina Mehrpooyan
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Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14965/161, Tehran, Iran
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Article history: Received 27 July 2014 and in revised form 27 September 2014 Available online xxxx Keywords: Cloning Expression Purification Thermostable lipase Cohnella sp. A01
a b s t r a c t Lipases form one of the most important groups of biocatalysts used in biotechnology. We studied the lipase from the bacterium Cohnella sp. A01 due to the versatility of thermophilic lipases in industry. In this study lipase 3646 gene from the thermophilic bacterium Cohnella sp. A01 was expressed in Escherichia coli and the enzyme was purified by a two-steps anion exchange chromatography. The purified lipase appeared to have a molecular weight of approximately 29.5 kDa on SDS–PAGE. The values of Km and Vmax, calculated by the Michaelis–Menten equation, were 1077 lM and 61.94 U/mg, respectively. The kinetic characterization of the purified enzyme exhibited maximum activity at 70 °C and pH 8.5. Activities at 50, 55 and 60 °C for 120 min were measured 58%, 47% and 41%, respectively. The enzyme was also highly stable at the pH range of 8.5–10.0 for 180 min. The effect of EDTA indicated that the enzyme is not a metalloenzyme. The stability of lipase 3646 in the presence of organic solvents, detergents, metal ions and inhibitors suggested that this lipase could be exploited in certain industries such as detergent and leather. Lipase 3646 was determined structurally to be 37.5% a-helix, 12.8% b-sheet, 22.7% b-turn and 27% random coil. Ó 2014 Published by Elsevier Inc.
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Introduction
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Lipases (triacylglycerol acyl hydrolases, E.C. 3.1.1.3) have a wide range of applications in a variety of industries such as food (dairy, biosensors for food, bakery, fats and oil) [1], biodiesel [2], cosmetic, pharmaceutical, leather, textile, detergent, and paper [3,4]. Since these enzymes catalyze both the hydrolysis and the synthesis of a great variety of esters [5], and inter and trans-esterification [6] such as the resolution of racemic mixtures [7,8] they are considered as multipurpose enzymes. Lipases belong to a/b hydrolase superfamily and most of them are serine enzymes in which the active site has a hydrogen bond network consisting of a catalytic triad of serine–histidine–aspartic/glutamic acid [9]. The serine residue usually is located in the conserved pentapeptide GlyXaa-Ser-Xaa-Gly motif. However, it has been shown that in various bacilli the first glycine in the conserved pentapeptide has been replaced by an alanine residue [10,11]. The active sites of the majority of bacterial lipases are covered by an a-helical flexible ‘‘flap’’ that can change from closed to open conformation when
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⇑ Corresponding authors. E-mail addresses: [email protected] (S. Aminzadeh), [email protected] (A.A. Karkhane).
the enzyme is absorbed by the lipid–water interface [12]. The phenomenon of increased lipase activity at the lipid–water interface is known as the interfacial activation [13]. Lipases are widely present in plants, animals and microorganisms [11], but those from thermophiles, due to their high stability at elevated temperatures and under severe operational and/or storage conditions, have become the subject of special interest for biotechnological applications [14,15]. Since the thermophilic bacteria are expected to produce more heat-stable enzymes than mesophilic microorganisms, they are usually used as a source of thermostable enzymes [16]. Implementation of biotechnological processes at high temperatures has many advantages: high solubility of substrates (in particular for poorly soluble or polymeric molecules) which can shift the equilibrium to a higher product yield, higher reaction rates, increased availability of substrates, decreased risk of microbial contamination and lower viscosity of reaction mixtures which in turn reduces the costs related to pumping, filtration and centrifugation [17,18]. In the present study, we report cloning and overexpression of a novel lipase gene from thermophilic indigenous Cohnella sp. A01 (isolated by Aminzadeh et al. from shrimp farming ponds (unpublished data). We also report purification and characterization of the overexpressed enzyme in Escherichia coli BL21.
http://dx.doi.org/10.1016/j.pep.2014.10.002 1046-5928/Ó 2014 Published by Elsevier Inc.
Please cite this article in press as: B.p. Golaki et al., Cloning, expression, purification, and characterization of lipase 3646 from thermophilic indigenous Cohnella sp. A01, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.10.002
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Materials and methods
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Materials
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High pure DNA purification kit was purchased from BIONEER (Seoul, Korea). NdeI, EcoRI and pTZ57R/T plasmid were provided by Fermentas (Glen Burnie, MD, USA). High pure plasmid purification kit was purchased from Roche. E. coli DH5a and E. coli BL21 (DE3) strains, and vector pET-26b (+) were obtained from Invitrogen (Carlsbad, CA, USA). 1-Anilino naphthalene-8-sulfonate (ANS), p-nitrophenyl esters and p-nitrophenol (pNP) were purchased from Sigma (St. Louis, USA). DE52 resin was provided from Whatman. All other chemicals were purchased from Merck (Darmstadt, Germany). Software VMD 1.9 (University of Illinois, Urbana-Champaign, USA), Gene Runner 4.0 (Lynnon Biosoft, Canada), GraphPad Prism 5 (San Diego, CA, USA), were used for displaying and analysis of the protein structure [19], DNA sequence analysis, and creation of the Michaelis–Menten curve and calculation of the Km and Vmax, respectively.
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Methods
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Cloning of lipase gene from Cohnella sp. A01 Cohnella sp. A01 was used as the lipase gene source. The bacterial cells were cultured in nutrient broth medium at 60 °C for three days and used for DNA extraction. Lipase gene was obtained by PCR using the forward primer with NdeI recognition site (50 -TA CATATGCGCAAGGGCGGCTAC-30 ) and the reverse primer with EcoRI recognition site (50 -ATGAATTCTCATTACCCGCGGTTCTTGCC CG-30 ). The amplified fragment with a size of 807 bp was ligated into pTZ57R/T vector and sequenced using M13/pUC forward and M13/pUC reverse sequencing primers. Recombinant plasmid (pAKP-AT) was digested with endonucleases NdeI and EcoRI and the fragment containing the lipase gene was ligated into the expression vector pET-26b (+) which had been digested with the same enzymes. Finally, the ligated plasmid pAKP-3646 was transformed into E. coli BL21.
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Expression and purification After transformation a single colony was grown overnight at 37 °C in LB medium containing 30 lg/ml of kanamycin then transferred into a fresh medium. Protein expression was induced by final concentration of 0.5 mM IPTG after 3 h of incubation at 37 °C when the optical density at 600 nm (OD600) reached 0.6. Subsequently cells were collected by centrifugation (9000g for 10 min at 4 °C) and resuspended in 25 ml of 100 mM Tris-base buffer (pH 8.5). After that the cells were sonicated on ice for six sets of twominute pulses (100 mHz) with 10-s intervals. Crude cell extracts were centrifuged (9000g for 20 min at 4 °C) and the supernatant was used for protein purification. The purification method consisted of two steps: ion-exchange chromatography using DE52 resin in pH 5.5, and the same column in pH 4.3. Step 1: 40 ml of supernatant was dialyzed overnight in 50 mM Tris–HCl buffer (pH 5.5) at 4 °C. The dialyzed solution was centrifuged (10,000g for 15 min at 4 °C) and was applied on the DE52 column pre-equilibrated with the same buffer. The column was washed with three 50 mM Tris–HCl buffers (pH 5.5) containing 270, 330 and 500 mM of NaCl, respectively. The fractions were collected and assayed for lipase activity and analyzed for purity by SDS–PAGE. The fraction containing 330 mM NaCl eluent was retained for further purification. Step 2: 60 ml of the fraction containing 330 mM NaCl eluent from the previous step was dialyzed overnight in 50 mM Tris– HCl buffer (pH 4.3) at 4 °C. The dialyzed solution was centrifuged
(10,000g for 15 min at 4 °C) and the supernatant was applied on a DE52 column pre-equilibrated with the same buffer (pH 4.3). In this step other proteins were bound to column but lipase 3646 was eluted without binding to column and the flow-through was collected. All fractions were assayed for lipase activity and visualized using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) [20]. Protein bands were stained by Coomassie brilliant blue R250.
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Protein assay
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The protein concentration was estimated according to Bradford’s method using bovine serum albumin as standard solution [21].
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Circular dichroism
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Purified lipase 3646 was analyzed with spectropolarimeter J-715 (Jasco, Tokyo, Japan) equipped with a thermostatically controlled cell holder. Samples were dialyzed against 10 mM Tris-base buffer at pH 8.5. Circular dichroism (CD) spectrum was measured at a protein concentration of 0.2 mg/ml, using a 1-mm path length quartz cell. Secondary structure of the enzyme was monitored in the region between 190–250 nm.
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Lipase activity and substrate specificity
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Enzyme activity was estimated by a spectrophotometric assay with p-nitrophenyl palmitate (pNPP) as substrate. A variety of substrates, including pNPP, were examined in a mixture containing 3 mM p-nitrophenyl esters, 50 mM Tris-base buffer (pH 8.5), 1% acetonitrile (v/v) and 4% 2-propanol (v/v). Enzyme solution was added to the pNPP solution and reaction progress was monitored at 410 nm per minute at 70 °C using a spectrophotometer (Beckman, DU 530 from USA) with pNP as the standard solution. One unit of enzyme activity was defined as the amount of the enzyme that liberated 1 lmol of pNP per minute under the standard assay conditions.
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Effect of pH on the lipase activity and stability
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pH profile was studied with p-nitrophenyl palmitate assay at a pH range of 3.0–12.0 by using 50 mM acetate (pH 3.6–5.6), 50 mM phosphate (pH 5.8–8.0), and 50 mM glycine (pH 8.6–10.6) buffers. Enzyme stability in different pHs was measured at pH 5, 8.5 and 10 by using the same buffers. Enzyme activity was measured at 70 °C and pH 8.5 every 30 min for six times.
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Effect of temperature on the lipase activity and stability
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The effect of temperature on the lipase activity was studied by preincubation of the enzyme at a temperature range of 30–100 °C in 50 mM Tris-base buffer (pH 8.5). For the measurement of thermostability, the enzyme was incubated at 55, 60, 65 and 70 °C temperatures for 120 min in 50 mM Tris-base buffer (pH 8.5) and the activity was measured every 30 min.
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Effect of metal ions, inhibitors, detergents, organic solvent on the lipase activity
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The effect of metal ions (1 mM) on lipase activity was investigated using Zn2+, Cu2+, Fe2+, Mg2+, Mn2+, Na+, K+ and Ca2+. 5 mM of phenyl methyl sulfonyl fluoride (PMSF), diethyl pyrocarbonate (DEPC), iodoacetamide and EDTA were used as inhibitors. The influence of 1% (v/v) surfactants (SDS, tween-20, 40, 65 and 80,
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triton X-100 and deoxycholate) and 30% (v/v) of organic solvents (methanol, ethanol, 2-propanol, glycerol, b-mercaptoethanol, aceton, chloroform, n-hexan, n-heptan and diethylether) on the enzyme activity were examined. The enzyme was treated with these compounds at room temperature for 120 min. The relative activity was subsequently measured at 70 °C and pH 8.5 by pNPP substrate. The activity of the untreated enzyme without additives was considered 100%.
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Determination of substrate specificities
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Substrate specificity was analyzed under optimum conditions (pH 8.5 and 70 °C) using p-nitrophenyl butyrate (pNPB; C4), p-nitrophenyl hexanoate (pNPH; C6), p-nitrophenyl octanoate (pNPO; C8), p-nitrophenyl decanoate (pNPD; C10), p-nitrophenyl myristate (pNPM; C14), p-nitrophenyl palmitate (pNPP; C16) and p-nitrophenyl stearate (pNPS; C18). The activity was determined at 410 nm for 1 min incubation at 70 °C.
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Michaelis–Menten constants
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Lipase assays were performed in 50 mM Tris-base buffer (pH 8.5) at 70 °C with a range of 0.01–1.5 mg/ml of concentration of the pNPP. Michaelis–Menten plot was used to determine Km and Vmax in GraphPad Prism 5 software. The effect of substrate concentration (pNPP) on the reaction rate was examined for 1 min at pH 8.5 using a spectrophotometric method.
CD spectroscopy was used to elucidate the secondary structure content of the purified enzyme. The CD spectrum of secondary structure analysis showed that the lipase 3646 contained 37.5% a-helix, 12.8% b-sheet, 22.7% b-turn and 27% random coil. While prediction of secondary structure showed 30.6 a-helix, 20.9% b-sheet, 51.5% b-turn and random coil. There is 7–8% different between results of prediction and Circular dichroism that this different is normal.
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Results and discussion
Effect of temperature on lipase activity and stability
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Cloning of lipase 3646
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Cloning of the lipase gene into cloning vector (pAKP-TA) was confirmed by sequencing, and subsequently the gene was successfully subcloned into the expression vector pET-26b (+) (pAKP3646). Using Gene Runner software, the sequence analysis indicated that the fragment encoded a putative protein of 268 amino acids with a molecular mass of 29.5 kDa. The gene of lipase 3646 with the accession number JX833623 was recorded in GenBank database. BLAST of lipase 3646 gene in NCBI showed 53% identity to the sequences of the triacylglycerol lipase from Paenibacillus sp. HGF5 (GenBank Accession No. EGG38175). The protein structure prediction indicated that this enzyme belongs to the a/b hydrolase superfamily. Lipase 3646 contains the consensus sequence Gly-His-Ser-leu-Gly with the catalytic triad of Ser 133, His 212 and Asp189. In addition, there is an active site flap
The purified recombinant lipase exhibited maximum activity at 70 °C and was highly active in the temperature range of 50–80 °C (Fig. 3A). The optimum temperature for lipase 3646 was similar to the lipases from Bacillus sp. L2 [26], Thermoanaerobacter thermohydrosulfuricus and Caldanaerobacter subterraneus subsp. tengcongensis [27] which is higher than that of the thermostable lipases from Thermomyces lanuginosus [28] and Geobacillus thermodenitrificans IBRL-nra [25]. As shown in Fig. 3B, more than 45% of lipase 3646 activity maintained after incubation at 50, 55 and 60 °C for 90 min. However, after incubation at 65 °C for 60 min remaining activity was about 45%. Incubation at 70 °C for 30 min dampened the activity to 33%. The stability of lipase 3646 at 65 °C for 30 min was higher than that of BTL2 lipase from Bacillus thermocatenulatus (30 min at 60 °C) [29] and the lipase from Bacillus stearothermophilus L1 (30 min at 65 °C) [30]. In addition, the enzyme was more stable in 60 and 65 °C for 60 min than
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spanning from amino acid 73 to 84. Three dimensional structure of lipase 3646 was predicted using Phyre 2 server and visualized by VMD 1.9 software (Fig. 1).
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Purification of the recombinant lipase
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The recombinant lipase was purified in two steps. Chromatography for the first step purification is shown in Fig. 2A. A single band with an apparent molecular mass of about 29.5 kDa was detected by SDS–PAGE electrophoresis (Fig. 2B). Although three to four-steps purification was followed by most of the researchers [22,23], we were able to purify the enzyme using one resin and just by changing the pH. Purification of lipase 3646 using DE52 resin gave a yield of 38% with a purification factor of 13.4 (Table 1), while other studies achieved the final yields of 2.5 [24], 8.9 [25] and 19.7% [22].
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Circular dichroism spectral analysis of lipase 3646
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Fig. 1. 3D structure of lipase 3646. The a/b hydrolase structure of lipase 3646 is shown as new cartoon representation. 3D structure of the nucleophilic elbow, active site flap and active site amino acids.
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Fig. 2. (A) Purification profile of lipase 3646 using DE52 ion exchange chromatography by 270, 330 and 500 mM NaCl, respectively. (B) SDS–PAGE of expressed and purified protein encoded by lipase 3646 gene. lane M, protein molecular mass marker; lane 1, total protein before induction with IPTG; lane 2, total protein after induction with IPTG; lanes 3, 4 and 5, column content eluted with 270, 330 and 500 mM NaCl, respectively in the first step of purification; lane 6, column content eluted after the second step of purification (for details please see the M&M section).
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the lipase of Bacillus sp. L2 (60 min at 60 and 65 °C) [26]. Hydrophobic amino acids increase proteins thermostability [31]. 45.5% of amino acids in Lipase 3646 are hydrophobic which could be the reason of the thermostability of this enzyme. Since most of the industrial processes operate at temperatures above 50 °C, the importance of thermostable enzymes such as lipases is significantly enhanced [22] and many heat-stable lipases have been isolated from various sources such as Pseudomonas fluorescens [32] and Bacillus species [22,23].
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Effect of pH on lipase activity and stability
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The stability of lipase 3646 in alkaline pH justified it as a potential alkaline lipase in the degreasing process in leather industry [33]. The effect of pH on the enzyme activity and stability is shown
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in Fig. 3C and D. Lipase 3646 was the most active in the pH range of 7–9, with more than 70% of its lipolytic activity. Maximal activity was observed at pH 8.5. The pH profile of lipase 3646 was similar to the lipases from Bacillus subtilis [11] and Antrodia cinnamomea BCRC 35396 [34]. Optimum pH for lipase 3646 activity also was higher than those of the thermostable lipase from G. thermodenitrificans IBRL-nra [25], Pf2001lipase from Pyrococcus furiosus [35], and Rhizopus oryzae lipase (ROL) [4]. The enzyme showed high stability in alkaline pH. Remaining activity after 180 min of treatment at pH 10 was more than 60%, while after treatment at pH 8.5 and 5, it was below 40%. Furthermore, stability of lipase 3646 after treatment at pH 10 for 120 min was more than those of the L2 lipase from Bacillus sp (30 min at pH 8.5 and 10) [26], and the lipase from Geobacillus sp. TW1 (15 min at pH 10) [5].
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Effect of organic solvents, metal ions, inhibitors and detergents on lipase activity
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The enzyme activity in the presence of various organic solvents, metal ions, inhibitors and detergents is shown in Table 2. The enzyme activity was not significantly changed in the presence of the methanol, ethanol, 2-propanol, n-hexane and n-heptane but was decreased in the presence of the acetone, chloroform and diethylether. Klibanov in 1997 reported that in most cases activity was more declined by organic solvents rather than water because of the changes in destabilization of the enzyme, protein flexibility, or diffusional limitations [36]. Decrease in the activity in the presence of acetone and chloroform has also been reported for LipSBRN2 [9] and the lipase from Rhizopus oryzae [37]. Lipases from Candida rugosa, Candida antarctica and Pseudomonas cepacia were inactivated by 10% methanol after 30 min of treatment [38]. The enzyme activity was decreased to 38% for lipase from Pseudomonas sp. B11-1 by 30% ethanol [39]. It was also reduced to 66% for LipSBRN2 by 30% 2-propanol [9]. In the case of 30% b-mercaptoethanol the activity dropped dramatically, however in the presence of 10% 2-mercaptoethanol it fell slightly down to 89%. Reduction in lipase activity to 64% in the presence of 1 mM 2-mercaptoethanol has been reported previously [22]. The remaining activity was increased by glycerol by the amount of 10%. The effects of metal ions on lipase activity are shown in Table 2. Metal ions play important roles on the structure and function of enzymes. For example Ca2+ increases the amount of alpha-helix and beta-sheet content in Pseudomonas fluorescence SIK W1 lipase [40]. In the present study, the remaining activity was more than 75% in the presence of Zn2+, Ca2+ and Na+, in addition it was more than 60% after treatment with the Cu2+, Mg2+ and Mn2+. Treatment with Fe2+ and K+ lowered the enzyme activity to below 60% of the control value. Arnaldo et al. also reported similar results about the effects of 1 mM metal ions such as of Zn2+, Cu2+, Fe2+, Mg2+, Mn2+, Na+, and Ca2+ on lipase activity, especially the decrease in activity down to 33 and 74% by Fe2+ and Ca2+, respectively [41]. 1 mM of Mg2+ and Zn2+ inactivated Lipase from Bacillus coagulans BTS-3 [24]. The addition of 1 mM of metal ions such as Mg2+, Fe2+ and Cu2+ to L2 lipase, dampened the activity to about 30% and Zn2+, K+ and Na+ also reduced the activity more than 50% of the control value [26]. An increase in the lipase activity in a short time (15 min) and a reduction of the activity in a long time by Ca2+
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Table 1 The efficiency of purification of recombinant lipase. Steps
Volume (ml)
Total protein (mg)
Total activity (units)
Specific activity (units/mg)
Purification (fold)
Yield (%)
Crude DE52 (pH = 5.5) DE52 (pH = 4.3)
37 63 63
47.51 5.48 1.36
83.62 58.10 31.96
1.76 10.63 23.51
1.00 6.02 13.35
100 69.52 38.00
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Fig. 3. The effect of pH and temperature on the recombinant lipase. (A) The effect of temperature on the activity of the recombinant lipase. (B) Thermostability of the recombinant lipase. (C) The effect of pH on the activity of the recombinant lipase. (D) pH stability of the recombinant lipase (For more details please see M&M section).
Table 2 Effect of some organic solvents, metal ions, inhibitors, detergents and substrate chain length on the activity of lipase 3646. Effector molecule Organic solvent (30%) None Acetone Methanol Ethanol 2-Propanol n-hexane n-heptane 2-Mercaptoethanol (30%) 2-Mercaptoethanol (10%) Chloroform Diethylether Glycerol Metal ions (1 mM) None Zn2+ Cu2+ Fe2+ Mg2+ Mn2+ K+ Ca2+ Na+
345 346 347 348 349 350 351 352 353 354
Relative activity (%) 100 ± 1.7 58.0 ± 2.39 94.3 ± 4.2 92.1 ± 3.4 88.4 ± 4.47 94.0 ± 3.0 93.5 ± 3.1 0 ± 0.0 89.2 ± 3.5 74.8 ± 1.0 75.6 ± 2.9 110.3 ± 4.6 100 ± 1.9 85.3 ± 2.3 61.3 ± 3.5 35.7 ± 1.1 69.3 ± 3.0 65.2 ± 1.5 59.8 ± 3.3 81.6 ± 4.5 79.8 ± 2.6
has been previously reported [42]. Therefore, the decrease in lipase 3646 activity by Ca2+ probably is due to the treatment of the enzyme with Ca2+ for a long time (120 min). Iodoacetamide is an irreversible inhibitor of enzymes with a cysteine in their catalytic sites. In this assay, iodoacetamide had no effect on the enzyme activity which suggests that lipase 3646 does not have a cysteine in its active site. The activity was inhibited in the presence of 5 mM PMSF, showing that the catalytic triad is inactivated through covalent modification of serine by PMSF. Furthermore, the enzyme activity was decreased to 38% by 5 mM
Effector molecule Effectors (5 Mm) None EDTA Iodoacetamide PMSF DEPC Detergents (1%) None SDS Tween 20 Tween 40 Tween 65 Tween 80 Triton x100 Deoxycholate Substrate specificity pNPB (C4) pNPH (C6) pNPO (C8) pNPD (C10) pNPM (C14) pNPP (C16) pNPS (C18)
Relative activity (%) 100 ± 2.1 88.6 ± 2.5 100 ± 1.9 8.7 ± 2.3 37.7 ± 3.5 100 ± 2.4 0.0 ± 0.0 92.6 ± 1.5 87.8 ± 3.3 87.1 ± 4.6 42.45 ± 1.9 20.4 ± 3.4 93.6 ± 3.9 100 ± 3.8 58.8 ± 4.3 24.9 ± 2.1 31.4 ± 3.3 19.0 ± 2.4 74.7 ± 3.6 83.1 ± 2.5
DEPC. These effects suggest that lipase 3646 indeed have a catalytic triad containing serine and histidine residues. The sharp decline in activity by PMSF and DEPC have also been reported by other researchers [26,41]. The enzyme activity was decreased only 11% in the presence of 5 mM EDTA. This indicated that the enzyme is not a metalloenzyme [22]. The effect of detergents was determined after 120 min of treatment at room temperature. Remaining activities were 93%, 92%, 88%, and 87% in the presence of deoxycholate, tween 20, tween 40, and tween 65, respectively. The decrease in the activity to
Please cite this article in press as: B.p. Golaki et al., Cloning, expression, purification, and characterization of lipase 3646 from thermophilic indigenous Cohnella sp. A01, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.10.002
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Fig. 4. Michaelis–Menten plot for purified lipase 3646.
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42% by tween 80 may be due to the long acyl ester chains of this detergent, making a substrate for the enzyme to form a competitive inhibitor. These results also suggested that the enzyme has a preference for C18 esters (Tween 80) over C16 (Tween 40) and C12 (Tween 20). It has been reported previously that lipases can use tween 80 as a substrate [41,43]. Remaining activity was reduced to 20% in the presence of Triton 100 and was dramatically decreased by SDS.
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Lipase specificity
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Table 2 demonstrates the substrate specificity of the lipase 3646 with p-nitrophenyl esters of varying acyl chain lengths from C4 to C18. The highest activity was observed by pNPB (100%) and high activities were obtained by pNPS (C18), pNPP (C16) and pNPH (C6), respectively. These results suggested that lipase 3646 has a strong catalytic ability toward the substrates with short and long acyl chains and low activity for those substrates with mediumchain fatty acyl esters. The high lipase activity toward long-chain fatty acid esters indicated that lipase 3646 is a true lipase in contrast to esterases that hydrolyze exclusively short chain fatty acyl esters [10].
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Michaelis–Menten plot
386 388
The kinetic parameters were determined using Michaelis– Menten plot (Fig. 4). The values of Km and Vmax with pNPP substrate were calculated 1077 lM and 61.94 U/mg, respectively.
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Conclusion
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Thermophilic lipases which facilitate the performance of industrial reactions in higher temperatures are widely used in many industries such as leather and detergent. In this study, we cloned the lipase 3646 gene from thermophilic bacterium Cohnella sp. A01 and the corresponding protein was expressed, purified and characterized. Lipase 3646 possesses the conserved region Gly-His-Ser-Leu-Gly and amino acids Ser and His in its active site. Lipase 3646 had a strong catalytic ability toward substrates with long acyl chains and showed appropriate activity in high temperatures and alkaline pH. Not only this lipase was not a metalloenzyme, but also its stability in the presence of several organic solvents, metals ions, inhibitors and detergents indicated that this enzyme could be exploited in certain industries as explained before. Structure–function relationship of this enzyme is being investigated further.
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Acknowledgements
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We would like to thank Dr. R.H. Sajedi and Mr.R. Mohseni for technical supports.
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References
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Please cite this article in press as: B.p. Golaki et al., Cloning, expression, purification, and characterization of lipase 3646 from thermophilic indigenous Cohnella sp. A01, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.10.002
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