Lipases (E.C 3.1.1.3) catalyze the hydrolysis of medium-and long-chain triglycerides, and a major characteristic is the sharp activation at the interface generated by a water-insoluble lipid substrate in aqueous solution [1]. Lipases can also catalyze ester synthesis and transesterification reactions with high regional and stereoselectivity in nonaqueous solvent systems [2-4]. These features allow lipases to be the most widely used enzymes in synthetic detergent additives, fine chemicals, and for precursors of chiral pharmaceutical and agrochemical production [5-7].

Organic solvents are usually utilized instead of water in some lipase-catalyzed reactions. There are many advantages that anhydrous organic solvents can offer, such as shifting the equilibrium to the synthetic direction, controlling or modifying enzyme selectivity by solvent engineering, increasing the solubility of substrates and the recovery of products in the organic phase, improving the thermal stability of enzymes, and suppressing undesirable water-dependent side reactions [8, 9]. However, organic solvents affect the activity and stability of enzymes to different degrees. Therefore, lipases that show high activity and stability in organic solvents are of interest [10-13].

At present, research on microbial lipases has mainly focused on strains with industrial application value such as Rhizopus, Aspergillus, Candida, Pseudomonas, and Bacillus [14-16]. There are few studies and reports on lipases from Paenibacillus. P. pasadenensis CS0611 was previously isolated from soils around chitin biological production factories in Shandong China and identified based on morphological characterization and 16s rDNA sequence analysis. The present paper describes the cloning, heterogeneous expression, and purification of a novel organic solvent-tolerant lipase from this strain. Consequently, detailed enzymatic properties of the recombinant enzyme were studied.

P. pasadenensis CS0611 was isolated by our group previously and stored at the China Center for Type Culture Collection (CCTCC M2014458). KOD FX (Toyobo, Japan) was used for PCR; pET-28a vector (Novagen, Germany) was used for the cloning, sequencing, and expression experiments; E. coli BL21 (DE3) was used as the expression host. Restriction enzymes Fast Digest Sal Ⅰ and BamH Ⅰ, T4 DNA ligase, and DNA and protein markers were purchased from Thermo Fisher Scientific (Wilmington, DE, USA). Synthesis of the primers and DNA sequencing were completed by Invitrogen (USA). The kits used in the construction of recombinant plasmids were purchased from Generay (Shanghai, China). Substrates (p-nitrophenyl fatty acid esters with varying acyl chain lengths), isopro-pyl-β-D-thiogalactopyranoside (IPTG), and kanamycin were purchased from Sigma-Aldrich (St. Louis, MO) or Aladdin (Shanghai, China). All other reagents and solvents were of analytical grade and used without further purification.

The cloning and transformation of the target gene were according to the methods described by Sambrook et al [17]. P. pasadenensis CS0611 was grown in Luria-Bertani (LB) medium (1% tryptone, 0.05% yeast extract, 1% NaCl) at 37 ℃ for about 15 h, and the genomic DNA was extracted and purified using a bacterial genomic DNA Kit according to the manufacturer's instructions. Two primers F1(5'-CGCGGATCCATGCGGAAGCAAAGCGAAAAGGA-3') and R1(5'-GCGTCGACAGAGTTTGCATAAATCCACATCTTGACCG-3') were designed based on the nucleotide sequences immediately upstream and downstream the known coding sequences of lp2252 and introduced BamH Ⅰ and Sal Ⅰ restriction sites in the 5' and 3' ends of the complete gene, respectively (underlined sequences correspond to the restriction sites). The amplification of the lp2252 gene was carried out by KOD FX DNA Polymerase using genomic DNA as a template. The PCR amplification was performed with the following cycles: one cycle of pre-denaturation at 94 ℃ for 2 min; 30 cycles of 98 ℃ for 10 s (denaturation), 45 ℃ for 30 s (annealing), and 68 ℃ for 1.5 min (extension); followed by one cycle of final extension at 68 ℃ for 7 min. The purified PCR product was digested with BamH Ⅰ and Sal Ⅰ, and inserted into the expression vector pET-28a previously digested with the same restriction enzymes to obtain the recombinant plasmid pET-28a-lp2252. The recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells using heat shock. The transformed cells were plated on LB agar (1% tryptone, 0.05% yeast extract, 1% NaCl, 2% agar) containing kanamycin (50 μg/mL) and incubated at 37℃ overnight. The positive transformed colonies were confirmed by sequencing after the plasmid extraction (using a high purity plasmid preparation kit according to manufacturer's instructions).

The recombinant cells were cultivated in 20 mL LB medium containing 50 μg/mL of kanamycin at 37 ℃, 180 r/min for 12 h. A 250-mL flask containing 100 mL LB broth medium supplemented with 50 μg/mL kanamycin was inoculated with 1% (v/v) of pre-cultured cells described above. Different concentrations of IPTG were added as an inducer at different OD600 values, and the cultivation continued at different temperatures to determine the optimum induction conditions. Recombinant cells were harvested by centrifugation (8000 r/min, 5 min) at 4 ℃. The cell pellet was suspended in 20 mmol/L phosphate buffer, pH 7.5 (10 mL). After ultrasonic disruption and centrifugation, the supernatant was filtered through a membrane filter with a pore size of 0.45μm and applied to a Bio-Scale^TM Mini Nuvia ^TM IMAC Ni-charged column (5 mL, Bio-Rad, USA). The column was washed with 10 volumes of distilled water. After equilibration with 10 volumes of binding buffer (20 mmol/L phosphate buffer, 500 mmol/L NaCl, and 10 mmol/L imidazole, pH 7.5), 5 mL of the crude enzyme preparation was loaded onto the column. The column was washed with 20 volumes of washing buffer (20 mmol/L phosphate buffer, 500 mmol/L NaCl, and 20 mmol/L imidazole, pH 7.5). The target protein was eluted with a 20–500 mmol/L imidazole gradient at a flow rate of 0.3 mL/min with elution buffer (20 mmol/L phosphate buffer, 500 mmol/L NaCl, and 500 mmol/L imidazole, pH 7.5). The active fraction was collected and concentrated with an ultra-filtration membrane (10 kDa, Millipore, Billerica, MA). The purity of lp2252 was analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described by Sambrook et al. [17].

Lipase activity was determined using p-nitrophenyl fatty acid esters as substrates [18]. The standard assay reaction mixture contained 20 mmol/L phosphate buffer (400 μL, pH 7), 10 mmol/L p-NPC₁₆ dissolved in isopropanol (50 μL), and purified enzyme (50 μL). 500 μL of 0.5 mol/L Na₂CO₃ was added to the reaction mixture after 5 min at 50 ℃ to terminate the reaction. One unit (U) of enzymatic activity is defined as the amount of enzyme required to produce 1 μmol of p-nitrophenyl per min (ε410 nm = 0.016 μL/mol). Unless specially mentioned, all measurements of lipase activity were conducted under standard conditions. Protein concentration was determined using the Bradford method with bovine serum albumin as the standard [19]. All assays were performed in triplicate and average values were taken.

The optimal temperature for activity was determined by measuring the hydrolytic activity at different temperatures (20–70 ℃). Thermostability was determined by measuring residual activity after incubation of the purified enzyme at temperatures ranging from 20 to 60 ℃ for different time intervals. The optimal pH was determined similarly at 50 ℃ in 50 mmol/L buffer solutions of pH values ranging from 2.0 to 10.0 (glycine-HCl: 2.0–3.0; HAc-NaAc: 3.0–5.0; Na₂HPO₄-citric acid: 5.0–8.0; Tris-HCl: 8.0–9.0; glycine-NaOH: 9.0–10.0). The effect of pH on enzyme stability was investigated by measuring the residual activity after pre-incubation of purified enzyme for different time intervals at 4 ℃ in Na₂HPO₄-citric acid buffer solutions (50 mmol/L, pH 3.0–8.0).

The effect of various metal ions, surfactants, and inhibitors on the lipase activity was determined by investigating the residual activity after incubating the purified lipase in phosphate buffer (20 mmol/L, pH 7.0) containing the following chemical agents at 30 ℃ for 30 min: Metal ions (2 mmol/L): Ca²⁺, Mg²⁺, Cu²⁺, Zn²⁺, Fe²⁺, Co²⁺, Mn²⁺, Fe³⁺; inhibitors (2 mmol/L): ethylene diamine tetra-acetic acid (EDTA) as a di-and tri-valent metal ion chelating agent, β-mercaptoethanol as a redox reagent, and phenyl methyl sulphonyl fluoride (PMSF) as a serine hydrolase inhibitor; and surfactants (0.1% (v/v)): Tween 80, Triton X-100, and SDS.

Substrate specificity was assayed using different p-nitrophenyl fatty acid esters with various acyl chain lengths such as p-NP butyrate (C₄), p-NP caproate (C₆), p-NP caprylate (C₈), p-NP decanoate (C₁₀), p-NP laurate (C₁₂), p-NP myristate (C₁₄), p-NP palmitate (C₁₆), and p-NP stearate (C₁₈) under standard conditions.

The Michaelis-Menten kinetic parameters K_m, V_max, K_cat, and K_cat/K_m of the lipase were calculated using p-nitrophenyl caprylate (p-NPC₈) and p-nitrophenyl palmitate (p-NPC₁₆) as substrates, respectively. The initial reaction velocity of different substrate concentration (50–400 mmol/L) was measured under standard assay conditions. Lineweaver-Burk plots were used to determine kinetic constants K_m and V_max.

To study the activity and stability of lp2252 in organic solvents, aliquots of the purified lipase were incubated in the presence of each organic solvent (30%, v/v) in phosphate buffer (20 mmol/L, pH 7.0). The residual activity was measured after incubating the mixture at 30 ℃ with constant shaking of 180 r/min for 1 h and 16 h.

The ester synthesis reaction was carried out in a 6-mL reaction system that contained 5 mL of various organic solvents respectively, and 0.28 mol/L of 1-octanol and octanoic acid. The added amounts of lipase and H₂O were 150 and 250 mg, respectively. The mixture was reacted at 40 ℃, 120 r/min for 12 h. Samples were withdrawn from the reaction medium and diluted with 5 mL of ethanol/acetone (1:1 v/v) and titrated with 0.05 mol/L NaOH in the presence of phenolphthalein (1%, w/v) for determination of the remaining acid. Ester conversion was calculated according to the equation below. The assays were performed in triplicate and average values were taken.

here, V, c, V₀, 0.915, 144.24 represent the volume of NaOH consumed during titration (mL), the concentration of NaOH (mol/L), the initial volume of octanoic acid (mL), the relative density of octanoic acid (g/mL), and the relative molecular mass of octanoic acid, respectively.

The full length of lp2252 was amplified from the genomic DNA of P. pasadenensis CS0611 using a pair of degenerate oligonucleotide primers. An open reading frame (ORF) of 1068 bp encoding a polypeptide of 356 aa residues was indicated after sequencing and gene analysis. The deduced recombinant lipase was composed of 405 amino acids including an additional 34 N-terminal and 15 C-terminal amino acid residues containing the 6 × His tags, which corresponded to a molecular weight of 44.3 kDa.

After amino acid sequence alignment and analysis with NCBI BLAST, lp2252 showed high similarity with alpha/beta hydrolase from Paenibacillaceae bacterium GAS479 (NCBI Reference Sequence: WP_090779060.1) and lysophospholipase from Thermobacillus sp. ZCTH02-B1 (GenBank: OUM94863.1), 60% and 53% respectively. A multisequence alignment of the deduced amino acid sequence of lp2252, Paenibacillaceae bacterium GAS479, and Thermobacillus sp. ZCTH02-B1 is shown in Fig. 1. We inferred that Ser, Asp, and His might be the catalytic triad (marked with red triangles). In addition, the conserved pentapeptide motif of Gly-X-Ser-X-Gly surrounding the active-site serine (marked by a rectangle) is typical of a/b hydrolases such as lipases and esterases.

Optimization of the expression conditions was obtained using properly controlled experiments as described above. After cultivation of recombinant cells at 37 ℃ in LB medium containing 50 μg/mL of kanamycin, IPTG was added to a final concentration of 0.1 mmol/L when the OD600 values reached 1.1. Then the culture was shaken for 16 h at 20 ℃, and cells were harvested by centrifugation. The His-tagged lipase was purified using an IMAC (immobilized metal affinity chromatography) Ni-charged column. As shown in Table 1, purification was achieved with an overall yield of 63.5% and a purification factor of 10.78. The molecular weight of the purified unfolded His-tagged recombinant lipase was estimated to be 45 kDa, which is similar to lipases from other species [20, 21]. This corresponded with the size of the purified protein by SDS-PAGE (Fig. 2).

Various temperatures (from 20 to 70 ℃) and pH values (from 3.0 to 10.0) were used to determine the effect of temperature and pH on the activity of lp2252. As shown in Fig. 3, the maximum activity was obtained at 50 ℃, and the purified enzyme kept more than 60% of its initial activity in a temperature range of 45–70 ℃. After incubation at temperatures from 20 to 50 ℃ for 20 h, the enzyme retained over 70% residual activity compared with the initial activity. However, when the incubated temperature reached 60 ℃, the residual activity decreased to less than 50%.

The purified enzyme showed an optimum hydrolytic activity at pH 7.0 (Fig. 4), which is similar to most lipases whose optimum pH are in a range of pH 7.0–8.0 [3]. There are some lipases that show the highest activity at alkaline pH, such as those from G. thermoleovorans YN (pH 9.5) [22] and Pseudomonas fluorescens JCM5963 (pH 9.0) [23]. As to stability at different pHs, the enzyme retained more than 85% of its initial activity after incubation at 4 ℃ in buffer (pH = 7.0 and 8.0) for 30 h. When the pH of the buffer decreased to 3.0–6.0, the residual activity was also more than 60% of the initial activity 30 h later. These results show that the recombinant enzyme is stable over a broad pH range of 3.0–8.0.

The effects of different metal ions, inhibitors, and surfactants on the activity of the purified enzyme are shown in Table 2. The presence of Ca²⁺ and Mg²⁺ strongly activated lipase activity up to 208% and 146%, respectively, compared to the control. This ion stimulation is similar to that of lipase from Acinetobacter sp. RAG-1 [24]. Cu²⁺ gave a moderate activation of 16%. In contrast, Zn²⁺, Fe²⁺, Co²⁺, Mn²⁺, and Fe³⁺ inhibited the activity of lp2252 lipase. The presence of Fe³⁺ made the relative activity of lp2252 decrease to 37% that of the control. In addition, the activity of lp2252 was strongly inhibited by the metal-chelating agent EDTA, which decreased to 39% of the control, suggesting that lp2252 is a metalloenzyme. This is consistent with many lipases reported previously [3]. PMSF almost inhibited the activity of lp2252 completely, which indicated the presence of a serine residue at the active site. β-mercaptoethanol as a redox reagent also had an inhibitory effect on lipase activity, suggesting the presence of disulfide bond(s) in the lipase structure. Non-ionic surfactants Triton X-100 and Tween-80 at 0.1% (v/v) increased the activity by 30% and 15%, respectively, which the anionic surfactant SDS inhibited the hydrolytic activity of lp2252 completely. Lipase from Bacillus thermoleovorans CCR11 [25] is enhanced in the presence of Triton X-100 and completely inhibited by SDS and Tween-80. Based on the results above, the appropriate concentration of metal ions like Ca²⁺ or Mg²⁺ and proper surfactants can be added to the reaction system to obtain higher hydrolytic activity.

As regards to substrate specificity (Table 3), the purified enzyme exhibited activity toward p-nitrophenyl fatty acid esters with a broad range of acyl chain lengths, and the highest activity was obtained with p-NPC₈. The hydrolytic activity against long-chain fatty acid esters of p-nitrophenyl was lower than those of short-and medium-chain lengths; activities were 51.8% (p-NPC₁₆) and 49.6% (p-NPC₁₈) of the maximum activity to p-NPC₈. A similar specificity has been reported for lipase from P. fluorescens JCM5963 [23], while some other lipases have shown preference for esters with shorter fatty acids (C₄ and C₆) [26, 27].

Kinetic parameters were determined by spectrophotometric assays using p-NPC₈ and p-NPC₁₆ as substrates. Lp2252 showed simple hyperbolic Michaelis-Menten kinetics for two substrates (Table 4). As shown, the recombinant lipase was more selective toward p-NPC₈ (K_m value of 0.12 mmol/L) than p-NPC₁₆ (0.34 mmol/L).

The stability and activity of lipases in organic solvents are important characteristics when they are used for chiral separation or organic synthesis. The relative hydrolytic activities of lp2252 in the presence of different organic solvents (30%, v/v) after incubation for 1 and 16 h are shown in Fig. 5. Among 13 different polar and non-polar organic solvents, whose empirical polarity parameters (E_T(30)) were from 0.006 to 0.762 [28], the activity of lp2252 increased in the presence of methanol (192.31%), ethanol (493.43%), butanol (154.78%), isopropanol (367.23%), tertiary butanol (134.14%), acetone (245.12%), and tetrahydrofuran (129.27%). After incubation for 16 h, the activation caused by ethanol (197.46%) and isopropanol (198.65%) was still distinct. However, lp2252 was inactivated to some extent in the presence of hexane (58.16%), cyclohexane (62.85%), and diethyl ether (69.42%). The polarity of these organic solvents was relatively high but less than water, so they can reduce the polarity of the environment allowing enzyme molecules to fold correctly and substrate and enzyme molecules to combine easily. However, the structure of lipase is typically hydrophilic in whole, with a hydrophobic core and hydrophilic surface. And its activity must be maintained by solvent with a certain polarity. When the solvent polarity is too low, the structure of the enzyme will be destroyed, leading to activity reduction or inactivation. Compared with a reported organic solvent-tolerant lipase, lp2252 activation by short carbon chain alcohols including isopropanol, methanol, and ethanol, as well as acetone, was remarkable. The relative activities of lipase from P. fluorescens JCM5963 [23] were about 110% after incubation for 1 h in the presence of each of these four solvents, respectively. For lipase from Galactomyces geotrichum Y05 [29], 50% (v/v) ethanol, acetone, isopropanol, and butanol decreased enzyme activity to 13%–27%. The activities and stabilities of different lipases are diverse in various organic solvents, and further mechanistic investigations are needed.

Fig. 6 shows the effects of various organic solvents on the lp2252-catalyzed synthesis of octyl octanoate. The ester conversion with decane (85.49%, logP 5.6) as the reaction medium was the highest, followed by isooctane (82.61%, logP 4.5), hexane (80.12%, logP 3.5), and cyclohexane (78.55%, logP 3.2). Other solvents with logP < 2.5 tended to give relatively low esterification. This was probably due to (1) high hydrophilicity solvents depriving the essential water around the enzyme present as a microaqueous layer, thereby affecting the active conformation of the enzyme, and (2) low polarity solvents enhancing dissociation of weak organic acid, leading to the reverse reaction (hydrolysis). Our observation is in agreement with previous results in which relatively low ester conversions happen in solvents with logP < 2.5 [30, 31].

In this study, a novel lipase gene from a newly isolated P. pasadenensis CS0611 strain was cloned, expressed, purified, and biochemically characterized, adding to our knowledge about the genes and enzymatic properties of lipases from Paenibacillus. The purified recombinant lipase exhibited high activity and stability in a range of temperatures and pH values. In addition, metal ions such as Ca²⁺ and Mg²⁺ had an activating effect on purified lipase to some extent. The most significant characteristic of lp2252 was its tolerance to a range of organic solvents. These features make it a promising candidate in the field of non-aqueous biocatalysis and for industrial lipase-catalyzed processes.