BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Co-expression of the lipase and foldase of Pseudomonas aeruginosa to a functional lipase in Escherichia coli Bhawna Madan & Prashant Mishra Received: 29 February 2008 / Revised: 6 July 2009 / Accepted: 6 July 2009 / Published online: 21 July 2009 # Springer-Verlag 2009 Abstract The lipA gene, a structural gene encoding for protein of molecular mass 48 kDa, and lipB gene, encoding for a lipase-specific chaperone with molecular mass of 35 kDa, of Pseudomonas aeruginosa B2264 were co- expressed in heterologous host Escherichia coli BL21 (DE3) to obtain in vivo expression of functional lipase. The recombinant lipase was expressed with histidine tag at its N terminus and was purified to homogeneity using nickel affinity chromatography. The amino acid sequence of LipA and LipB of P. aeruginosa B2264 was 99–100% identical with the corresponding sequence of LipA and LipB of P. aeruginosa LST-03 and P. aeruginosa PA01, but it has less identity with Pseudomonas cepacia (Burkholderia cepacia) as it showed only 37.6% and 23.3% identity with the B. cepacia LipA and LipB sequence, respectively. The molec- ular mass of the recombinant lipase was found to be 48 kDa. The recombinant lipase exhibited optimal activity at pH8.0 and 37°C, though it was active between pH5.0 and pH9.0 and up to 45°C. K m and V max values for recombinant P. aeruginosa lipase were found to be 151.5±29μM and 217± 22.5μmol min -1 mg -1 protein, respectively. Keyword Lipase . Co-expression . Foldase . P. aeruginosa . E. coli Introduction Lipases (triacylglycerol ester hydrolases, E.C. 3.1.1.3) cata- lyse the hydrolysis of long-chain triacylglycerols in aqueous media, whereas in non-aqueous media, they catalyse the esterification and transesterification reactions (Zaks and Klibanov 1988; Klibanov 2001). Owing to the properties like wide substrate specificity, enantio- and regioselectivity, they have applications in organic synthesis, in detergent formulations and in the food and pharmaceutical industries (Reetz 2002; Jaeger and Eggert 2002; Chand and Mishra 2003; Madan and Mishra 2009). Whilst lactonising lipase of Pseudomonas sp. has been used in the synthesis of macrolide antibiotics (Ihara et al. 1991), Pseudomonas aeruginosa lipase has exhibited amide hydrolysing activity (Fujii et al. 2005) and high enantioselectivity towards hydrolysis of optically active trifluoroethylated (3′-indolyl) thiacarboxylic esters, novel plant growth regulators (Kato et al. 1999) and chiral model substrate 2-methyldecanoic acid p-nitrophenyl ester (Liebeton et al. 2000). In spite of various potential applications of Pseudomonas lipases, overexpression of functional lipase has been a lim- iting factor, as lipases from Pseudomonas species require an assistant protein, a lipase-specific chaperone to fold into an active conformation (Arpigny and Jaeger 1999; Rosenau et al. 2004). The foldase for lipase (chaperone) is encoded in the same operon along with the structural gene of lipase (lipA). Lipase-specific chaperone is either required during or after translation. In Pseudomonas sp. Strain KWI-56 activator (chaperone) is required at the time of lipase synthesis (Yang et al. 2000). The functional expression of Pseudomonas lipase has been achieved by in vitro refolding of lipase using various approaches (Jorgensen et al. 1991; Oshima-Hirayama et al. 1993; Hobson et al. 1993; Ihara et al. 1995; Yang et al. 2000; Traub et al. 2001). So far, in vivo expression of functional P. aeruginosa lipase in heterologous host Escherichia coli has not been achieved, and hence, engineering of lipases for improved properties has been limited to homologous host. In homologous host, the enantioselectivity of P. aeruginosa lipase towards the B. Madan : P. Mishra (*) Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India e-mail: pmishra@dbeb.iitd.ac.in Appl Microbiol Biotechnol (2010) 85:597–604 DOI 10.1007/s00253-009-2131-4