Microsoft Word - 55castellanos.docx CHEMICAL ENGINEERING TRANSACTIONS VOL. 64, 2018 A publication of The Italian Association of Chemical Engineering Online at www.aidic.it/cet Guest Editors: Enrico Bardone, Antonio Marzocchella, Tajalli Keshavarz Copyright © 2018, AIDIC Servizi S.r.l. ISBN 978-88-95608-56-3; ISSN 2283-9216 Molecular Detection and Characterization of Novel Lipase Genes of the Lipolytic Yeast Candida palmioleophila Zully Rodríguez-Mateusa, Katerine Vera Pachecoa, German Zafraa,b* a Universidad de Santander UDES, Grupo de Investigación en Ciencias Básicas y Aplicadas para la Sostenibilidad – CIBAS. Campus Lagos del Cacique, Calle 70 No. 55-210, Bucaramanga, Santander, 680003, Colombia. b Current address: Universidad Industrial de Santander, Escuela de Microbiología. Bucaramanga, Santander, 680002, Colombia. gzafra@udes.edu.co In this study we analyzed the genetic variability of lipase gene sequences from eight oil and grease-degrading strains of Candida palmioleophila and to relate it to their degrading ability. The genetic variability of lipase genes was analyzed by PCR-restriction fragment length polymorphism (PCR-RFLP) and low-stringency single specific primer- PCR (LSSP-PCR), in order to obtain specific DNA fingerprints from each strain, which were subsequently compared by bioinformatic programs. DNA fingerprints were contrasted to the ability of each strain to remove palm oil in liquid culture. The results showed that at least three genes encoding lipases are present in C. palmioleophila, two of them resembling the LIP2 and LIP6 genes of C. albicans. DNA fingerprints obtained by LSSP-PCR revealed differences in the sequences of C. palmioleophila lipase genes, which allowed to group the strains according to their degrading activity. C. palmioleophila strains SACL05, SACL08 and SACL11, which showed the highest removal of palm oil after 72 h (77 to 79 % removal), were grouped in a single clade in dendrograms. Similarly, strains SACL01, SACL03, SACL06 and SACL09, which showed intermediate removal activity of palm oil (54 to 76%) grouped in a different clade. This suggests the genetic variability in lipase genes is directly related to the differences found in the efficiency of degradation of oils. On the other hand, DNA fingerprints obtained by PCR-RFLP did not allow to differentiate the strains and did not generate changes in the bands patterns between the analyzed strains. In conclusion, this study reported for the first time the detection and characterization of lipase genes from the lipolytic yeast Candida palmioleophila, and their association to the degradation of oils. 1. Introduction Lipases are an important group of enzymes produced by a wide range of microorganisms, useful in different fields such as the pharmaceutical, industrial, chemical and environmental due to their regioselectivity, enantioselectivity and selectivity in the chain length of the substrate in which they act. For these reasons, there is a growing demand for identifying new lipolytic enzymes for the development and optimization of bioprocesses (Sharma et al. 2011). Lipases can be used in environmental applications, specifically in wastewater treatment processes from oil refineries, as toxic wastes are generated during all processing processes causing serious contamination in soils and water. These enzymes are produced by animals, plants, and microorganisms in general, which catalyze the ester-chain hydrolysis of triacylglycerols and release fatty acids and glycerol (Guncheva and Zhiryakova 2011). The most representative genera of microorganisms producing lipolytic enzymes are Candida, Pseudomonas, Burkholderia, Thermomyces, Rhizopus, Bacillus, Staphylococcus, Geobacillus, Acinetobacter, Rasltonia and Yarrowia (Bell et al. 2002). The lack of knowledge about new microorganisms exhibiting lipase activity has led to new investigations about the prospection of new lipolytic strains and to identify new lipases with biotechnological potential, especially for the mitigation of the environmental impact. Previous studies of our group have shown that different strains of the lipolytic yeast C. palmioleophila present different removal efficiencies of fats and oils under the same conditions, even though they show 100% similarity in their ribosomal sequences (Rodriguez-Mateus et al. 2016). These microorganisms could have a high potential for their use as bioremediation agents of effluents DOI: 10.3303/CET1864059 Please cite this article as: Rodriguez-Mateus Z., Vera Pacheco K., Zafra G., 2018, Molecular detection and characterization of novel lipase genes of the lipolytic yeast candida palmioleophila, Chemical Engineering Transactions, 64, 349-354 DOI: 10.3303/CET1864059 349 contaminated with fats and oils. The microbial degradation of fats and oils in these microorganisms is mainly modulated by extracellular lipases; however, to date, there are no studies about C. palmioleophila lipases, or their coding genes. The variability in these genes could significantly influence the activity and efficiency of these enzymes. Thus, the objective of this study was to detect and characterize genes encoding lipases in C. palmioleophila in order to correlate their genetic variations to their reported ability to metabolize grease and oils. 2. Materials and methods 2.1 Native degrading microorganisms Eight strains of Candida palmioleophila (SACL01, SACL03, SACL04, SACL05, SACL06, SACL08, SACL09, SACL11) and two strains of Bacillus sp. (SACB02 and SACB10), previously isolated from a grease trap of a palm oil refining process (Rodriguez-Mateus et al. 2016), were used in this study. All 10 strains showed lipolytic activity and the ability to remove grease, oils and organic matter present in wastewaters from palm oil extraction, either single or mixed (Agualimpia et al. 2016). In addition, genomic DNA from Candida albicans strain CA was used as a positive control during the amplification of LIP1, LIP2 and LIP6 genes. 2.2 Amplification and detection of lipase genes Yeast and bacterial genomic DNA extraction was performed by salting out (Miller et al. 1988) using a lysis buffer (10 mM Tris-Cl, 10 mM EDTA, 50 mM NaCl) containing 20 mg/ml proteinase K (Sigma-Aldrich, USA), 20 mg/ml lysozyme for bacteria (Merck, USA) or 67 mg/ml lyticase for yeast (Sigma-Aldrich, USA) to break down cell walls and membranes. Extracted DNA preparations were quantified and quality checked using a Nanodrop 2000 Spectrophotometer (Thermo Scientific, USA). PCR amplifications of four different lipase targets were carried out using the primers described in table 1. Due to the lack of available sequences for the lipase genes of C. palmioleophila in sequence repositories, primers targeting the open reading frame of the LIP1, LIP2 and LIP6 genes from the related yeast Candida albicans (Hube et al. 2000), as well as highly degenerate primers targeting the oxyanion hole and active-site regions of putative lipase genes (Bell et al. 2002), were used. LIP1, LIP2 and LIP6 sequences were amplified using a unique amplification protocol, consisting in a reaction mix composed of 1X PCR buffer, 2 mM MgCl2, 200 μM dNTPs, 1 U Taq DNA polymerase (Vivantis, Malaysia) and 1.5 μM of each primer. Amplification program consisted of 35 cycles of 94 °C (30 s), 52 °C (30 s) and 72 °C (1.5 min) and a final extension at 72 °C for 5 min. Oxyanion hole and active-site regions of putative lipase genes were amplified in a reaction mix composed of 1X PCR buffer, 2 mM MgCl2, 200 μM dNTPs, 1.5 U Taq DNA polymerase (Vivantis, Malaysia) and 4 μM of each primer (either OXF1/ACR1 or OXF1/ACR3 primer combination). Amplification program consisted of 45 cycles of 94 °C (30 s), 52 °C (30 s) and 72 °C (1.5 min) and a final extension at 72 °C for 7 min. PCR products were analyzed by 1 % agarose gel electrophoresis and visualized under UV light. Table 1. PCR primers used for the amplification of lipase sequences in C. palmioleophila Primer Sequence Target Expected Size of PCR product Reference OXF1 CCYGTKGTSYTNGTNCAYGG Oxyanion hole and active- site regions of putative lipase genes 250 bp Bell et al. (2002) ACR1 AGGCCNCCCAKNGARTGNSC OXF1 CCYGTKGTSYTNGTNCAYGG 250 bp Bell et al. (2002) ACR3 AGGCCRCCNTGNGARTGNSC LIP1a ACAAATTCACTGGGATCAAGAG Open reading frame of C. albicans LIP1 gene 545 bp Hube et al. (2000) LIP1b ATAAGTGACATGGACGTTACTG LIP2a TTTCCGACTTTGCTGTTCCAG Open reading frame of C. albicans LIP2 gene 589 bp Hube et al. (2000) LIP2b ATAATACTGCTTACAAGACCAAG LIP6a TTAAACCTGGTGCCAAAGCTG Open reading frame of C. albicans LIP6 gene 441 bp Hube et al. (2000) LIP6b TCGATGCCCTGGTGGTGAAC 350 2.3 LSSP-PCR and PCR-RFLP characterization of lipase genes Low-stringency single specific primer PCR (LSSP-PCR) reactions were carried out by purifying the 250 bp amplicons obtained with primer sets OXF1/ACR1 and OXF1/ACR3 and then performing a second round of amplification by using only one primer specific for one end of the fragment, under conditions of low stringency (Barreto et al. 1996). The reaction mix contained 1X PCR buffer, 1.5 U of Taq DNA polymerase (Vivantis, Malaysia), 200 µM dNTPs, 2 mM MgCl2 and 4 μM of primer OXF1 or ACR1. Amplification was achieved by using an initial denaturation at 94 °C for 5 min, followed by 35 cycles with denaturation at 94 °C for 30 s, annealing at 32 °C for 30 s and extension at 72 °C for 1 min. Ten microliters of each reaction were analyzed on 8 % nondenaturing polyacrylamide gels in TBE 0.5X buffer for 6 h at 110 V, being afterwards silver stained as described by Sanguinetti et al. (1994). Gels were photographed and analyzed using the Pyelph v4.0 program (Pavel and Vasile 2012) to create a binary data matrix of 0 and 1 representing the absence or presence of bands, respectively. Dendrograms were created using the UPGMA clustering method (Michener and Sokal 1957). PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) reactions were carried out by purifying amplicons obtained with the primer set LIP6a/LIP6b using the QIAquick PCR purification kit (QIAgen), and then performing a digestion with 2 units of AluI restriction enzyme (Vivantis, Malaysia) for 16 h at 37 °C. Fragment separation was performed on 2.5 % agarose gels stained with SYBR gold nucleic acid gel stain (Invitrogen, USA), and visualized under UV light. 3. Results and discussion 3.1 Detection of lipase genes in C. palmioleophila As shown in figure 1, the use of PCR primers flanking the oxyanion hole and active-site regions of putative lipase genes allowed to detect lipase gene sequences in all eight C. palmioleophila native strains. The OXF1/ACR1 primer combination produced the best results regarding band intensity and relative absence of unspecific bands, producing the expected amplicons of approximately 250 bp (Fig 1A). Using the same primer combination, Bell et al. (2002) obtained 250 bp amplicons which after being sequenced, showed high homology with lipases and phospholipase sequences; Terahara et al. (2010) reported also the detection of putative esterase genes from environmental DNA using primers OXF1-ACR1, whose sequences showed 32- 80% identity lipases and esterases of the α/β hydrolase family. In contrast, the use of OXF1/ACR3 primer combination did not produce unique bands, but rather a combination of PCR products ranging from 250 to 400 bp (Fig 1B). Such heterogeneity in PCR products was not unexpected, due to the degenerate nature of the primers and the stringency conditions used for PCR amplification. In spite of the above, it must be noted that this primer combination also allowed to detect lipase sequences in all C. palmioleophila strains, as well as in lipolytic Bacillus sp. strains. On the other hand, the use of specific primers designed to amplify lipase genes in C. albicans produced diverse results when tested on C. palmioleophila. The LIP1 gene was not detected in any of the C. palmioleophila strains, while LIP2 gene was detected only in strains SACL04, SACL09 and SACL11, but produced a shorter PCR amplicon of approximately 500 bp (Fig C, D). The LIP6 gene was instead detected in all eight C. palmioleophila strains, but amplifications also presented differences in PCR band number and size. Two different LIP6 bands of about 600 bp and 1,400 bp were detected, both of them being larger than the expected amplicon of 441 bp. In addition to suggesting differences in the LIP6 gene length between C. albicans and C. palmioleophila, this was also an indicative of clear differences among gene sequences. In C. albicans, five of the ten known lipase genes are located on the chromosome 1 (Hube et al. 2000; van het Hoog et al. 2007). LIP1, LIP2 and LIP6, the genes evaluated in this study, are also located on this chromosome. Together, the above results suggest that at least three genes encoding lipases are present in C. palmioleophila, two of them resembling the LIP2 and LIP6 genes of C. albicans. 3.2 Variability of lipase genes in C. palmioleophila and relation with palm oil removal LSSP-PCR and PCR-RFLP were used to detect variations within sequences from lipase genes of oil and grease degrading microorganisms. As shown in figure 2A, banding profiles obtained by LSSP-PCR allowed to detect small variations among the eight C. palmioleophila and two lipolytic Bacillus sp. strains when OXF1 primer was used. Overall, clustering analysis showed a correlation between LSSP-PCR profiles and the degradation activities reported previously for each of these strains (Rodriguez-Mateus et al. 2016). Specifically, dendrograms based on UPGMA cluster analysis separated banding profiles into two major clusters: one grouping most of the C. palmioleophila strains which showed the higher palm oil degradation efficiency (strains SACL11, SACL05 and SACL08) and the other grouping most of the C. palmioleophila strains with an average palm oil degradation efficiency (strains SACL01, SACL03, SACL06 and SACL09). This suggests that small differences in this gene could explain, at least in part, some of the differences 351 observed regarding oil degradation by these lipolytic strains as observed previously in hydrocarbon-degrading microorganisms (Zafra et al. 2016). Figure 1. PCR detection of lipase genes in strains of C. palmioleophila using different primer combinations. (A, B) Amplification of putative lipase genes with OXF1/ACR1 and OXF1/ACR3 primer combination, respectively. (C) Amplification of C. albicans LIP1 gene with primers LIP1a/LIP1b. (D) Amplification of C. albicans LIP2 gene with primers LIP2a/LIP2b. (E) Amplification of C. albicans LIP6 gene with primers LIP6a/LIP6b. M: 100pb opti-DNA weight marker (abm, Canada); CA: C. albicans strain CA. 352 Band profiles from lipolytic Bacillus sp. strains grouped into a separated cluster, even though they shared about more than 80 % of bands with those of C. palmioleophila. Only one strain (SACL04), which presented the lowest palm oil-removal activity (37 %), did not grouped into its corresponding cluster and instead showed similarity to the band profiles of strains with the higher removal percentages of palm oil (77 to 79 %). Since in this case the variability detected in the analyzed gene does not correlated with the palm oil removal efficiency, we hypothesize that oil removal efficiency in strain SACL04 could be more closely related to a different lipase gene, since up to ten lipase genes have been reported in Candida species and most of them are sensitive to different substrates (Guncheva and Zhiryakova 2011; Hube et al. 2000). On the contrary, LSSP signatures obtained with the ACR1 primer did not allowed to separate C. palmioleophila strains into different clusters, but as expected, these band profiles were notoriously different than those of Bacillus strains (Fig 2B). This could be an indicative of the marked differences between lipase genes from yeast and bacteria, as reported previously (Bell et al. 2002). Previously, the differences between the band profiles obtained with the OXF1 and ACR1 primers have also been reported to be prominent (Orozco et al. 2012). Band profiles obtained with primer ACR1 were 100% similar between the strains corresponding to each of the genus (Bacillus or Candida). On the other hand, due to the overall low amplicon length obtained for most of the PCR assays, PCR-RFLP typing was performed only on the purified 1,400 bp products from LIP6 gene amplifications in order to maximize the probability of finding restriction sites. The selection of AluI restriction endonuclease for RFLP-assays was based on the described LIP6 gene sequence of C. albicans deposited in GenBank (NCBI Reference Sequence NC_032089.1, region 2,114,091 to 2,115,482), in which we detected restriction sites for AluI restriction enzyme in silico but not for other common restriction enzymes (BamHI, EcoRI, HindIII, NotI). However, no restriction sites were detected in any of the C. palmioleophila strains by using this enzyme in PCR-RFLP assays (data not shown). Figure 2. LSSP-PCR signatures of putative lipase gene fragments from lipolytic strains of C. palmioleophila and Bacillus sp. Clustering was performed using the UPGMA method. (A) Banding patterns obtained with OXF1 primer. (B) Banding patterns obtained with ACR1 primer. Removal percentages of palm oil by each of the strains as described by Rodriguez-Mateus et al. (2016) are indicated. 353 4. Conclusions In conclusion, the results suggest that at least three genes encoding lipases are present in C. palmioleophila, two of them resembling the LIP2 and LIP6 genes of C. albicans. Moreover, we found that genetic variations in the oxyanion hole and active-site regions of a putative lipase gene were related to the ability to metabolize more or less efficiently crude palm oil by C. palmioleophila, which confirmed a key role of these enzymes on the differences in oil degradation by this lipolytic yeast. 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