Castilla Agustín, Panizza Paola, Rodríguez Diego, Bonino Luis, Díaz Pilar, Irazoqui Gabriela, Rodríguez Giordano Sonia
Bioscience Department, Facultad de Química, Universidad de la República, Gral. Flores 2124, 11800 Montevideo, Uruguay.
Dept. Genetics, Microbiology & Statistics, University of Barcelona, Av. Diagonal 643, 08028 Barcelona, Spain.
Enzyme Microb Technol. 2017 Mar;98:86-95. doi: 10.1016/j.enzmictec.2016.12.010. Epub 2016 Dec 31.
Janibacter sp. strain R02 (BNM 560) was isolated in our laboratory from an Antarctic soil sample. A remarkable trait of the strain was its high lipolytic activity, detected in Rhodamine-olive oil supplemented plates. Supernatants of Janibacter sp. R02 displayed superb activity on transesterification of acyl glycerols, thus being a good candidate for lipase prospection. Considering the lack of information concerning lipases of the genus Janibacter, we focused on the identification, cloning, expression and characterization of the extracellular lipases of this strain. By means of sequence alignment and clustering of consensus nucleotide sequences, a DNA fragment of 1272bp was amplified, cloned and expressed in E. coli. The resulting recombinant enzyme, named LipJ2, showed preference for short to medium chain-length substrates, and displayed maximum activity at 80°C and pH 8-9, being strongly activated by a mixture of Na and K. The enzyme presented an outstanding stability regarding both pH and temperature. Bioinformatics 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 β-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.
詹氏菌属菌株R02（BNM 560）是我们实验室从一份南极土壤样本中分离得到的。该菌株的一个显著特征是其在添加了罗丹明 - 橄榄油的平板上检测到的高脂肪酶活性。詹氏菌属R02的上清液对酰基甘油的酯交换反应表现出卓越的活性，因此是脂肪酶勘探的良好候选对象。鉴于关于詹氏菌属脂肪酶的信息匮乏，我们专注于该菌株胞外脂肪酶的鉴定、克隆、表达及特性研究。通过对共有核苷酸序列进行序列比对和聚类分析，扩增出一个1272bp的DNA片段，将其克隆并在大肠杆菌中表达。所得的重组酶命名为LipJ2，它对短至中链长度的底物表现出偏好，在80°C和pH 8 - 9时活性最高，受到Na和K的混合物强烈激活。该酶在pH和温度方面都具有出色的稳定性。对LipJ2氨基酸序列的生物信息学分析揭示了一个共有催化三联体和一个典型的五肽。然而，在LipJ2中还发现了另外两个罕见的基序：一个SXXLβ - 内酰胺酶基序和两个假定的Y型氧阴离子洞（YAP）。尽管上述一些特征可能使LipJ2归属于细菌脂肪酶家族VIII或X，但系统发育分析表明LipJ2与已知脂肪酶家族的其他成员聚类不同，这表明新分离的詹氏菌酯酶LipJ2将是一个新的细菌脂肪酶家族中首个被表征的成员。
