J. Microbiol. Biotechnol. (2011), 21(7), 667–674 doi: 10.4014/jmb.1103.03008 First published online 15 June 2011 A New Esterase, Belonging to Hormone-Sensitive Lipase Family, Cloned from Rheinheimera sp. Isolated from Industrial Effluent Virk, Antar Puneet 1 , Prince Sharma 2 * , and Neena Capalash 1 * Departments of Biotechnology and Microbiology , Panjab University, Chandigarh 160014, India Received: March 4, 2011 / Revised: April 16, 2011 / Accepted: April 25, 2011 The gene for esterase (rEst1) was isolated from a new species of genus Rheinheimera by functional screening of E. coli cells transformed with the pSMART/HaeIII genomic library. E. coli cells harboring the esterase gene insert could grow and produce clear halo zones on tributyrin agar. The rEst1 ORF consisted of 1,029 bp, corresponding to 342 amino acid residues with a molecular mass of 37 kDa. The signal P program 3.0 revealed the presence of a signal peptide of 25 amino acids. Esterase activity, however, was associated with a homotrimeric form of molecular mass 95 kDa and not with the monomeric form. The deduced amino acid sequence showed only 54% sequence identity with the closest lipase from Cellvibrio japonicus strain Ueda 107. Conserved domain search and multiple sequence alignment revealed the presence of an esterase/ lipase conserved domain consisting of a GXSXG motif, HGGG motif (oxyanion hole) and HGF motif, typical of the class IV hormone sensitive lipase family. On the basis of the sequence comparison with known esterases/ lipases, REst1 represents a new esterase belonging to the class IV family. The purified enzyme worked optimally at 50 o C and pH 8, utilized pNP esters of short chain lengths, and showed best catalytic activity with p-nitrophenyl butyrate (C 4 ), indicating that it was an esterase. The enzyme was completely inhibited by PMSF and DEPC and showed moderate organotolerance. Keywords: Rheinheimera, esterases, hormone-sensitive lipase Genus Rheinheimera [8] belongs to family Chromatiacae of Gammaproteobacteria, which also includes Alishewanella [12] and Alkalimonas [24]. Members of genus Rheinheimera are most abundant in marine and estuarine environments [1]. Rheinheimera is a very recently described genus and has been named after a German marine microbiologist, Gerhard Rheinheimer, in recognition of his work on marine and estuarine bacteria. The first species, R. baltica [8], was isolated from the Baltic Sea. Other species described include R. pacifica [31], R. perlucida [9], R. chironomi [16], R. texasensis [28], R. soli [33], and R. tangshanensis [38]. We have isolated a new species of Rheinheimera (GenBank Accession No. FJ645062) from industrial wastewaters and in this work are reporting a new esterase (REst1) from it. Bacteria produce different classes of lipolytic enzymes, including carboxylesterases (E.C. 3.1.1.1), which hydrolyze small ester-containing molecules at least partly soluble in water [4], true lipases (E.C. 3.1.1.3) that display maximal activity towards water-insoluble long-chain triglycerides, and various types of phospholipases. Being widely spread in several species, microbial esterases present high industrial potential and are often more useful than the enzymes from other sources owing to their substrate specificity, regioselectivity, and enantioselectivity, ability to remain active in organic solvents, high yields, and ease of genetic manipulation [17]. Esterases are applied in the synthesis of optically active compounds, pulp and paper industry, food processing, beverages, perfume industries, and degradation of synthetic materials. Bacterial class IV hormone-sensitive lipase (HSL) family displays striking amino acid sequence similarity to the mammalian HSL [19]. The bacterial proteins in the HSL family are only homologous to the catalytic domain of HSL. Their sizes are roughly comparable with the size of this catalytic domain. They are region 1, which contains a GXSXG motif that is conserved in most lipases/esterases and includes the active-site Ser residue, and region 2, which contains other two active-site residues (Asp and His). They show the characteristic α/β-hydrolase fold and the pentapeptide motif is usually located between a β- strand and the α-helix, forming an extremely sharp turn called ‘‘nucleophile elbow’’ [11]. *Corresponding author P. Sharma Phone: +91-172-2534091; Fax: +91-172-2541770; E-mail: princess@pu.ac.in N. Capalash Phone: +91-172-2534091; Fax: +91-172-2541770; E-mail: caplash@pu.ac.in