Enzyme and Microbial Technology 98 (2017) 86–95 Contents lists available at ScienceDirect Enzyme and Microbial Technology jo urnal homepage: www.elsevier.com/locate/emt A novel thermophilic and halophilic esterase from Janibacter sp. R02, the first member of a new lipase family (Family XVII) a a a a b Agustín Castilla , Paola Panizza , Diego Rodríguez , Luis Bonino , Pilar Díaz , a a,∗ Gabriela Irazoqui , Sonia Rodríguez Giordano a Bioscience Department, Facultad de Química, Universidad de la República, Gral. Flores 2124, 11800 Montevideo, Uruguay b Dept. Genetics, Microbiology & Statistics, University of Barcelona, Av. Diagonal 643, 08028 Barcelona, Spain a r t i c l e i n f o a b s t r a c t Article history: Janibacter sp. strain R02 (BNM 560) was isolated in our laboratory from an Antarctic soil sample. A remark- Received 15 July 2016 able trait of the strain was its high lipolytic activity, detected in Rhodamine-olive oil supplemented plates. Received in revised form Supernatants of Janibacter sp. R02 displayed superb activity on transesterification of acyl glycerols, thus 24 November 2016 being a good candidate for lipase prospection. Considering the lack of information concerning lipases Accepted 31 December 2016 of the genus Janibacter, we focused on the identification, cloning, expression and characterization of Available online 31 December 2016 the extracellular lipases of this strain. By means of sequence alignment and clustering of consensus nucleotide sequences, a DNA fragment of 1272 bp was amplified, cloned and expressed in E. coli. The Keywords: resulting recombinant enzyme, named LipJ2, showed preference for short to medium chain-length sub- Esterase ◦ Janibacter strates, and displayed maximum activity at 80 C and pH 8–9, being strongly activated by a mixture of + + Thermophilic Na and K . The enzyme presented an outstanding stability regarding both pH and temperature. Bioin- Halophilic formatics analysis of the amino acid sequence of LipJ2 revealed the presence of a consensus catalytic triad and a canonical pentapeptide. However, two additional rare motifs were found in LipJ2: an SXXL ␤ -lactamase motif and two putative Y-type oxyanion holes (YAP). Although some of the previous features could allow assigning LipJ2 to the bacterial lipase families VIII or X, the phylogenetic analysis showed that LipJ2 clusters apart from other members of known lipase families, indicating that the newly isolated Janibacter esterase LipJ2 would be the first characterized member of a new family of bacterial lipases. Published by Elsevier Inc. 1. Introduction enzymes, since they display characteristics that are ideal to indus- trial processes such as thermal stability and tolerance to harsh Lipases (EC 3.1.1.3) and carboxylesterases (E.C. 3.1.1.1) are reaction conditions [8,9]. Several thermophilic lipases have been enzymes that hydrolyze the carboxyl ester bonds in acyl-glycerides. characterized to date, from both, Bacteria [10] and Archaea [11], They differentiate on the length of their substrate; while lipases with the later comprising the most thermostable enzymes. Only prefer long chain acylglycerols, esterases hydrolyze short chain a few lipases from Archaea have been characterized and they glycerol esters. Despite this difference, they are collectively referred show extremely high optimal temperatures, ranging from 90 to ◦ to as lipases or “lipolytic enzymes” [1,2]. Microbial lipases are the 100 C [11,12]. Most thermostable lipases characterized in Bac- third largest group of industrial biocatalysts after bacterial amy- teria have been isolated from Bacillus strains, showing optimal ◦ lolytic enzymes and proteases [3]. They are used in biotechnological temperatures between 50 and 70 C [10] like other characterized applications in different sectors such as pharmaceutical, cosmetic, bacterial enzymes. Some exceptions are the lipases isolated from food, detergent, leather, textile, paper and biodiesel industries Caldanaerobacter subterraneus, Thermoanaerobacter thermohydro- [4–7]. The increasing demand for environmentally friendly indus- sulfuricus DSM7021 [13], and Marinobacter lipolyticus SM19 [2], ◦ trial processes has encouraged the use of biocatalysts in industry, with optimal temperatures ranging from 75 to 80 C. Highly ther- and has triggered new research in this field. Much work has mostable lipases are mostly produced by thermophilic bacteria, been done on the discovery and characterization of thermophilic although some mesophilic bacteria like Burkholderia cepacia [14], Pseudomonas [15,16], and Amycolatopsis mediterranei [17], have also been reported to produce thermostable enzymes, including ∗ a hyperthermophilic enzyme from Pseudomonas [18]. Corresponding author. E-mail address: [email protected] (S. Rodríguez Giordano). http://dx.doi.org/10.1016/j.enzmictec.2016.12.010 0141-0229/Published by Elsevier Inc. A. Castilla et al. / Enzyme and Microbial Technology 98 (2017) 86–95 87 Table 1 Despite the large number of microbial lipases identified, cloned, Primers used for lipJ2 gene identification and cloning. and characterized to date, they are still narrowed to a small por- tion of the microbial diversity. Scarce information concerning the Primer Sequence lipases of the genus Janibacter is available. A few strains have JNB 18b 5 -CGACAGGTGCGCCCC GTC-3 been isolated from different environments and have shown lipoly- JNB18RVG72 5 -CCGCCGCCCTGCCAGTAGCC-3 tic activity [19,20]. Recently, a mono and di-acyl glycerol lipase SP2FwLipJ pos112 5 -GGCACCCACACCTACATGGTCC-3 SP1RvLipJ pos112 5 -GGACCATGTAGGTGTGGGTGCC-3 MAJ1 from Janibacter HTCC2649, belonging to Family I.7, has been SP2RvLipJ pos34 5 -CGTACTCGACGAGGCTCTCCATCTG −3’ isolated and characterized. The optimum activity of this enzyme ◦ SP1FwLipJ pos34 5 -CAGATGGAGAGCCTCGTCGAGTACG-3 occurred at pH 7.0 and 30 C, and it retained 50% of the optimum Arb1-1 5 -GGCCACGGGTCGTCTAGTCA NNNNNNNNNN CGACG-3 ◦ activity at 5 C, indicating that MAJ1 is a cold-active lipase [21]. Arb1-2 5 -GGCCACGGGTCGTCTAGTCA NNNNNNNNNN CGTCG-3 Arb1-3 5 -GGCCACGGGTCGTCTAGTCA NNNNNNNNNN GTCGG-3 Janibacter strains are halotolerant microorganisms [22,23], and Arb1-4 5 -GGCCACGGGTCGTCTAGTCA NNNNNNNNNN TCGAC-3 thus, they can constitute a source of halophilic enzymes. In addition Arb1-5 5 -GGCCACGGGTCGTCTAGTCA NNNNNNNNNN CGGCG-3 to thermostable lipases, halophilic enzymes are also interesting Arb1-6 5 -GGCCACGGGTCGTCTAGTCA NNNNNNNNNN TCGGC-3 for biotechnological applications [24]. Their ability to work in the Arb2 5 -GGCCACGGGTCGTCTAGTCA-3 presence of low water activity could be useful in harsh industrial LipJ2 Fw 5 -ATGACCTCCATGACCAAGCCGCCGTC-3 LipJ2 Rv 5 -TCACAGACGCCAGCAGGAGTTGACC-3 environments where enzymatic reactions need to be carried out in organic solvents or in the presence of other chemicals. Janibacter sp. strain R02 (BNM 560) was previously isolated in AP-PCR was performed according to the procedure reported by our laboratory from an Antartic soil sample. A remarkable trait of Das et al. [29], although primers were modified to fit Janibacter this strain was its high extracellular lipolytic activity, thus being genome. For lipJ2 identification, degenerated primers JNB 18b and a good candidate for lipase prospection. Herein we describe the JNB18RVG72 (Table 1) were used to amplify a central consensus identification, cloning, expression and characterization of a novel region, using Janibacter R02 genomic DNA as a template. AP-PCR esterase, LipJ2, from Janibacter sp. R02. This enzyme displays an ◦ and sequencing allowed identification of the complete lipJ2 gene. optimal temperature of 80 C, being extremely thermostable. In Specific primers SP1RvLipJ pos112 and SP1FwLipJ pos34 were addition, it is alkalophilic and halophilic, constituting the first used along with degenerate primers Arb1-1 to Arb1-6 to amplify report of an extremophilic enzyme described from the genus Jani- the external sequences. The 3 five bases long random sequences bacter. The discovery of LipJ2 and the knowledge of its particular for primers Arb1-1 to 1–6 were designed based on the most com- characteristics, along with a phylogenetic and structural analy- mon five bases motifs found in Janibacter genome. Nested PCR sis, provided evidence to propose the existence of a new family was then performed with Arb2 and either SP2FwLipJ pos112 or of bacterial lipases. Following the criteria and the nomenclature SP2RvLipJ pos34 in order to specifically amplify the upstream and established by Arpigny and Jaeger [1], LipJ2 would be the first mem- downstream DNA fragments. Sequencing of all amplicons was per- ber assigned to a newly proposed bacterial lipase family designated formed at Macrogen, Korea. ContigExpress was used to assemble as family XVII [25,26]. the amplified DNA sequences (Vector NTI, version 8; Invitrogen, Carlsbad, CA). The sequence of lipJ2 gene was deposited in GenBank 2. Materials and methods under accession number KX096709. Specific primers LipJ Fw NdeI and LipJ Rv HindIII were designed 2.1. Strains and growth conditions for PCR amplification of the complete lipJ2 gene. Amplified DNA was purified from agarose gels and ligated to the Nde I/HindIII digested Janibacter sp. R02 was isolated from an Antarctic soil sam- pET22 b+ vector. The resulting recombinant plasmid, pET22b-LipJ2 ple. Lipolyitic activity was assayed in Rhodamine-Olive Oil plates was transformed into E. coli BL21(DE3) and E. coli Rossetta for and by hydrolysis of triolein with the extracellular enzyme frac- expression. tion. Identification of the strain