Gene cloning, heterologous expression, and partial characterization of a novel cold-adapted subfamily I.3 lipase from Pseudomonas fluorescence KE38

A novel cold-active true lipase from Pseudomonas sp. KE38 was cloned, sequencing and expressed in E. coli by degenerate PCR and genome walking technique. The open reading frame of the cloned gene encoded a polypeptide chain of 617 amino acids with a confirmed molecular weight of 64 kD. Phylogenetic analysis of the deduced amino acid sequence of the lipase indicated that it had high similarity with lipases of subfamily Ι.3 of bacterial lipases. Recombinant lipase was purified in denatured form as inclusion bodies, which were then renatured by urea followed by dialysis. Lipase activity was determined titrimetrically using olive oil as substrate. The enzyme showed optimal activity at 25 °C, pH 8.5 and was highly stable in the presence of various metal ions and organic solvents. Low optimal temperature and high activity in the presence of methanol and ethanol make this lipase a potential candidate for transesterification reactions and biodiesel production.


Results
Partial amplification of the LipI.3_KE38 gene. DNA sequences of already sequenced lipase genes of Pseudomonas species that are phylogenetically closely related to P. fluorescens were aligned, and two degenerate primers (KE38Lip_F and KE38Lip_R) were designed to regions that showed a high level of sequence identities (Fig. 1). Although 5′ ends of the aligned lipase sequences contained a high level of identity, 3′ ends were poor in identity, forcing us to design the 3′ primer to a region around the middle of the sequence. Sequence homology analysis of a PCR amplified fragment of the expected size (1105 bp) cloned into pTZ57RT/A confirmed successful amplification of the 5′ region of a lipase gene.
In order to amplify the 3′ end of the LipI.3_KE38 gene, genome walking technique 41 was employed by using isolated genomic DNA. This technique is based on a nested PCR-based strategy for extending a known sequence region to its uncharacterized flanking region. A walker primer which is partially degenerate (Semi2) and a set of nested gene-specific primers (LIP7, LIP5, LIP4) were used in three successive rounds of nested PCR reaction (Fig. 2a). The walker primer was expected to bind randomly to several sites on the bacterial chromosome, potentially generating many nonspecific as well as specific PCR products. Specific products were then further amplified in the second and third round of PCR using second and third nested gene specific primers, respectively eliminating nonspecific artifacts.
During our genome walking attempt, no bands over 1000 bp were observed after the first and second-round PCR while the third round PCR produced a band of approximately 1600 bp (Fig. 2b), which was cut from agarose gel and sequenced from both ends using the primers LIP4 and Semi2. Sequence analysis revealed a complete overlap between the 5′ end of the cloned fragment and the 3′ end of the previously sequenced 1105 bp partial lipase gene, providing the evidence of correct amplification. Furthermore, sequence homology analysis using BLAST also revealed that 3′ end of the cloned fragment had high homology to the 3′ end of extracellular lipase genes from various Pseudomonas species confirming the specific amplification of the 3′ end region of the LipI.3_KE38 full lipase gene (Fig. 2c). This sequence was then used for the completion of the LipI.3_KE38 lipase gene sequence. New primers, LIP_start and LIP_stop, were designed for the amplification of the whole LipI.3_KE38 open reading frame to be cloned into an expression vector.

Partial purification of the recombinant lipase by inclusion body isolation. Culture supernatants
of IPTG induced E. coli BL21(DE3) pET28-LipI.3_KE38 had low lipase activity compared to culture supernatants of P. fluorescens KE38 which displayed high levels of lipase activity on plate assays, indicative of the production of lipase as unsecreted inclusion bodies in E.coli BL21(DE3) pET28-LipI.3_KE38 (data not shown). Therefore, the expressed lipase was subjected to partial purification by inclusion body isolation from both the soluble and insoluble fractions of the lysed cells and analyzed by SDS PAGE. As expected, inclusion bodies were only present in the insoluble fraction and had an estimated molecular weight of around 64 kDa (Fig. 6a). This was consistent with the predicted molecular weight of the LipI.3_KE38 encoded by the cloned lipase gene.

Determination of the optimum lipase production time.
For the determination of optimum recombinant lipase production time, E. coli BL21(DE3) pET28a-LipI.3_KE38 cells were grown at 37 °C for 10 and then induced by IPTG. Samples were collected just before, and every 2 h after induction, and purified lipase from each sample were analyzed by SDS-PAGE (Fig. 6b). Maximum lipase production started from the second hour and continued until the sixth hour post-induction, followed by a drop in production from the 8-h postinduction. Therefore, the maximum production time was determined to be 6 h post-induction.
Partial characterization of the recombinant lipase. Determination of the optimum temperature and pH. The activity of the recombinant lipase was determined at various pH and temperature values. Consistent with most other subfamily I.3 lipases (Table 1), LipI.3_KE38 displayed its maximum activity towards olive oil at pH 8.5 and retained around 80% of its activity at pH 9.0, showing its alkaline nature (Fig. 7a). However, outside this range, activity quickly dropped down to around 20% at pH 8.0 and 10, and to less than 5% at pH 7.0 and 11. On the other hand, maximum activity of the enzyme was recorded at 25 °C and while it retained 80% of its maximum activity at 30 °C, the activity dropped to 50% below and above these values at 20 and 35 °C. However, the enzyme still retained 35-40% of its maximum activity at 10 and 15 °C (Fig. 7b).
Determination of the effects of organic solvents and metal ions on the activity of lipase. The activity of the recombinant lipase on olive oil was measured in the presence of various organic solvents (Fig. 8a). While methanol and acetone did not affect the activity of the lipase, the activity was increased to 125 and 135% in the presence of ethanol and acetonitrile respectively. Although the enzyme retained more than half of its activity in the presence of n-hexane, iso-propanol and ethyl acetate considerably diminished the activity of the enzyme to 10 and 30% of the maximum activity respectively. Among the metal ions tested (Fig. 8b), NaCl did not affect the activity of the enzyme. However, CuSO 4 , KNO 3 and CaCl 2 caused an increase of 160%, 118%, and 120% respectively in enzyme activity, whereas Pb(NO 3 ) 2 , MgCl 2 , and ZnCl 2 led to 25, 15 and 20% decrease respectively in the activity of the enzyme. EDTA (a metal chelator) on the other hand, led to a sharp decrease in enzyme activity as was expected since it is well known that subfamily I.3 lipases require the presence of calcium metal ions for proper folding and thus the activity of the enzyme 42 .

Discussion
In this study, we cloned and expressed a novel cold-active lipase enzyme in E. coli from Pseudomonas sp. KE38. We used degenerate PCR and genome walking technique to achieve the sequencing of the whole lipase gene. The deduced amino acid sequence of LipI.3_KE38 indicated that it had high similarity with lipases of subfamily Ι.3 of bacterial lipases according to the classification and properties of bacterial lipases 4,42 . The N-terminal domain contained the GXSXG motif which included Ser207 Asp255, and His313 of the catalytic triad. The C-terminal domain contained the putative secretion signals and several repeats of the GGXGXDXUX sequence that forms a β-roll motif in the presence of Ca 2 , which was implicated to facilitate the correct folding of the enzyme 47,48 .
Expression of the lipase involved the use of E. coli BL21(DE3) and pET28a system which adds histidine tags to the N-terminal of the expressed protein in order to facilitate its single-step purification through the affinity of the histidines to metal ions. However, our attempts using metal affinity failed probably due to the predominantly hydrophobic nature of the lipase protein and the formation of inclusion bodies due to the over-expression of the recombinant protein. Therefore, the expressed lipase was subjected to purification by inclusion body isolation There are some advantages of formation of inclusion bodies. They have different size and density as compared with cellular contaminants so that they can be easily isolated from cells. Also, the expressed protein in the form of inclusion bodies is usually highly resistant to proteolytic cleavage by cellular proteases 49 .
As we mentioned previously, LIPI.3_KE38 was clustered with cold-active lipase members of the subfamily I.3 lipases. Indeed, LipI.3_KE38 showed optimal activity at 25 °C which was similar to the cold-active members of the subfamily I.3 lipases, which had an optimal temperature range of 20-30 °C (Table 1). Although its activity sharply dropped below 10 °C, the enzyme also showed substantial activity (33-50%) at around 10-20 °C, an important property for reactions involving heat-labile substrates and/or products. On the other hand, the enzyme displayed about 80% and 50% of its maximum activity at 30 °C and 35 °C respectively making it a candidate enzyme even for warm temperature applications.
Previously reported subfamily I.3 lipases exhibited their highest activity at alkaline pH around 8-9 (Table 1) and LipI.3-KE38 was not an exception showing optimum activity at 8.5 and 80% of maximum at pH9. Below and above these pH values, the activity of the enzyme declined sharply. High activity at alkaline pH is a desired property for candidate enzymes to be used in detergent industries 50 . Table 1. Comparison of the properties of LipI.3_KE38 with other lipases belonging to the same family, I.3. nd not determined, ne no effect on lipase activity. a Accession numbers from NCBI database. −: negative effect on lipase activity. +: positive effect on lipase activity. www.nature.com/scientificreports/ Although the activity LipI.3_KE38 was increased in the presence of Ca 2+ (which is a common feature for all subfamily I.3 lipases), it was the only subfamily I.3 lipase with increased activity (over 150%) in the presence of Cu 2+ . The activity of the lipase was not affected significantly in the presence of various other metal ions tested except Pb 2+ . However, EDTA inhibited the activity of the enzyme which is reminiscent of all subfamily I.3 lipases. Moreover, LipI.3_KE38 was able to retain a high level of activity in the presence of various organic solvents. Indeed, its activity was remarkably increased by ethanol and acetonitrile, while methanol and acetone did not have any effect on the activity of the lipase.
In recent years, there has been a considerable number of cold active subfamily I.3 lipase discoveries (Table 1). Although the main properties of LipI.3_KE38 is common with other cold active lipases, the nucleotide and amino acid sequence differences can provide valuable information for studies focused on lipase protein function and structure. We also believe that LipI.3_KE38 may have the potential to be used in biodiesel production using methanol and ethanol or in other transesterification or lipolysis reactions requiring the use of low temperature and alkaline pH.
Conserved motifs are shown in underlined bold capital letters, and amino acids unique to LipI.3_KE38 are shown in grey boxes. Cold active enzymes are indicated with "*" sign. The sequences were obtained from NCBI (http://www.ncbi.nlm.nih.gov/) and aligned by the Clustall Omega software (www.clust all.org).
Scientific Reports | (2020) 10:22063 | https://doi.org/10.1038/s41598-020-79199-w www.nature.com/scientificreports/  To obtain the complete sequence of the lipase gene, genome walking technique described by Guo and Xiong was employed in three rounds of PCR reactions 41 using lipase specific forward nested primers LIP7 (5′-ACG TGA TCA ACG ACC TGC TGG-3′), LIP5 (5′-CCA ATG GCT TGT CGG GAA AAG-3′) and LIP4 (5′-CCA AAG AGT CGG CCA CCG -3′), and a common degenerate reverse walker primer Semi-2 (5′-GCC TTA AGG CCT ANGARM-SNCCNAG-3′). First-round PCR was performed as follows: 50 ng of plasmid pTZ57R-5′Lip (template DNA), 10 μl each of LIP7-Semi2 primers (2 μM), 10 μl of dNTP mix (2 μM), 10 μl of 10X Taq DNA Polymerase buffer, 12 μl of MgCl 2 (25 μM), 0.5 μl of Taq DNA Polymerase (5U/μl) and finally 46.5 μl dH 2 O were mixed in a total volume of 100 μl. The conditions for PCR amplification were as follows: an initial denaturation step at 94 °C for 2 min; followed by 30 cycles including denaturation at 94 °C for 30 s, gradient primer annealing at 55 °C for 1 min and elongation at 72 °C for 2 min and also final elongation at 72 °C for 10 min. Then, 1 μl of the first-round PCR mixture was used as a template for the second round PCR amplification. The reaction mixture www.nature.com/scientificreports/ and PCR conditions were the same as first-round PCR, except the LIP5-Semi2 primer pair was used instead of LIP7-Semi2. Two μl of the second round PCR mixture was used as a template for the third round PCR in which the LIP4-Semi2 primer pair was used. A DNA band over 1 kb obtained after the 3rd round of PCR was cut from agarose gel, purified, and directly sequenced at both directions using primers LIP4, and Semi-2. The complete lipase sequence was deposited in GenBank with accession number MT344965. Complete lipase gene was amplified by PCR using primers LIP_Start (5′-CAT ATG GGT GTG TAT GAC TAC AAG AAC-3′), and LIP_Stop (5-'TCA GGC AAT CAC AAT CCC TGT ACC -3′) containing the recognition sequences for the restriction enzymes Bgl II and BamHI respectively as follows: 100 ng of genomic DNA, 5 μl each of each primer (2 μM), 5 μl of dNTP mix (2 μM), 5 μl of 10× Pfu DNA Polymerase Buffer, 1 μl of Pfu DNA Polymerase (5U/ μl) and finally 26 μl of dH 2 O were mixed in a total volume of 50 μl. The conditions for PCR amplification were as follows: an initial denaturation step at 94 °C for 2 min; followed by 30 cycles including denaturation at 94 °C for 30 s, primer annealing at 55 °C for 1 min and elongation at 72 °C for 5 min with final elongation at 72 °C for 15 min. The band at 1800 bp corresponding to the size of the lipase gene was purified, cloned into pJET1.2/ blunt cloning vector according to the manufacturer's instructions, and sequenced.
Then, the lipase gene was removed from this construct by Nde I/Bgl II double digestion and ligated into BamHI/Nde I double digested and gel purified pET28a expression vector. Ligated products were used for the transformation of E. coli DH5α. Plasmid pET28a bearing KE38 Lipase gene (pET28a-LipI.3_KE38) was then purified and used for the transformation of E. coli BL21(DE3) cells for the expression of the lipase gene by induction with IPTG.

Partial purification of recombinant lipase by inclusion body isolation. Inclusion body isolation
was performed by the slight modification of a previously described method 46 . Two milliliters of an overnight culture of the E. coli BL21(DE3) pET28a-LipI.3_KE38 supplemented with kanamycin was used to seed 1 L culture and incubated overnight at 37 °C. Recombinant lipase expression was induced by the addition of IPTG at the final concentration of 1 mM after the optical density (OD) of the culture medium at 600 nm was reached to Lipase activity assay. Lipase activity was determined titrimetrically using olive oil as substrate. 100 µl purified enzyme solution was added to the 20 ml assay substrate containing 5% (w/v) olive oil and 50 mM Tris-HCl, pH 8.5, and incubated at 25 °C for 20 min while liberated fatty acids were continuously titrated with 0.01 mol/l NaOH using automatic titration instrument TitraLab 854 (Radiometer Analytical). The reaction mixture without the enzyme was titrated in the same way and used as blank. One 'lipase unit' was defined as the amount of the enzyme that released one μmol fatty acid per min under standard assay conditions of triplicate experiments All the assays were done in triplicate, and data were expressed as mean ± SD. www.nature.com/scientificreports/ Determination of temperature and pH optima of the lipase. To determine the optimum temperature of the purified extracellular lipase, which was performed prior to optimum pH determination, enzyme activity was assayed at temperatures ranging from 5 to 40 °C for 30 min, at otherwise standard conditions. To determine the optimum pH, activity was assayed using standard conditions for 30 min at various pH from 6 to 10 using different buffers (potassium phosphate for pH 5-7, Tris-HCL for pH 7-9, and Glycine-NaOH for pH 9-10).
Effect of various metal ions and organic solvents on lipase activity. The effects of metal ions (Na +1 , K +1 , Mg +2 , Ca +2 , Zn +2 , and metal chelator EDTA, each at 1 mM), and organic solvents (methanol, ethanol, acetone, acetonitrile, n-hexane, and 2-propanol at a concentration of 30% (v/v)) on lipase activity were investigated at standard assay conditions. Scientific Reports | (2020) 10:22063 | https://doi.org/10.1038/s41598-020-79199-w www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.