Over-expression and properties of a purified recombinant Bacillus licheniformis lipase: a comparative report on Bacillus lipases Mulalo B. Nthangeni a, *, Hugh-George Patterton b , Andre ´ van Tonder a , Wilma P. Vergeer c , Derek Litthauer a a Department of Microbiology and Biochemistry, University of the Free State, P.O. Box 339, Bloemfontein, 9300, South Africa. b Present address: Department of Biochemistry, University of Cape Town, University Private Bag, Rondebosch, 7701, South Africa c National Control Laboratory, University of the Free State, P.O. Box 339, Bloemfontein, 9300, South Africa Received 21 September 2000; received in revised form 15 January 2001; accepted 30 January 2001 Abstract The gene coding for an extracellular lipase of Bacillus licheniformis was cloned using PCR techniques. The sequence corresponding to the mature lipase was subcloned into the pET 20b(+) expression vector to construct a recombinant lipase protein containing 6 histidine residues at the C-terminal. High-level expression of the lipase by Escherichia coli cells harbouring the lipase gene-containing expression vector was observed upon induction with IPTG at 30°C. A one step purification of the recombinant lipase was achieved with Ni-NTA resin. The specific activity of the purified enzyme was 130 units/mg with p-nitrophenyl-palmitate as substrate. The enzyme showed maximum activity at pH 10 –11.5 and was remarkably stable at alkaline pH values up to 12. The enzyme was active toward p-nitrophenyl esters of short to long chains fatty acids but with a marked preference for esters with C 6 and C 8 acyl groups. The amino acid sequence of the lipase shows striking similarities to lipases from Bacillus subtilis and Bacillus pumilus. Based on the amino acid identity and biochemical characteristics, we propose that Bacillus lipases be classified into two distinct subfamilies of their own. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Bacillus; Lipase; Sequence; Over-expression; Purification 1. Introduction Lipases (E.C. 3.1.1.3) constitute a group of enzymes having the ability to hydrolyse triacylglycerols at a lipid- water interface [1]. They remain active in a variety of organic solvents, where they can catalyse various transfor- mations other than the hydrolytic reaction by which they are defined [2]. Microbial lipases have attracted considerable attention owing to their biotechnological potential, ranging from their use as additives in laundry detergents to ste- reospecific biocatalysis [3]. Lipases are secreted into culture medium by many fungi and bacteria and have been purified and extensively characterized in pursuit of lipases with novel properties for industrial applications. Alkaliphilic and thermophilic microorganisms have been the focus of a number of investigations into sources of lipases that are stable and function optimally at extreme alkaline pH values and high temperatures. Isolates of Ba- cillus species have been found to produce lipolytic enzymes under alkaline conditions [4]. Lipases from B. subtilis [5] and B. pumilus [6] have been of particular interest as they exhibit optimal activity and stability at extreme alkaline pH values (pH values above 9.5). These enzymes, however, are thermolabile. This is in contrast to lipases from B. stearo- thermophilus [7], B. thermocatenulatus [8] and B. thermo- leovorans [9], which are thermotolerant, and display max- imal activity and stability at moderate alkaline pH values (pH 7–9.5). The genes encoding these Bacillus lipases have been sequenced and translated into amino acid sequences, and were found to lack the characteristic Gly-X-Ser-X-Gly motif centered on the lipase active serine residue. Purification of Bacillus lipases for biochemical charac- terization and crystallographic studies is difficult, mainly due to stringent regulatory mechanisms of their synthesis, which often result in low levels of production. For example, purification of about 100 mg of B. subtilis lipase requires the * Corresponding author. Tel.: +27-51-401-2274; fax: +27-51-444- 3219. E-mail address: nthangen@micro.nw.uovs.ac.za (M.B. Nthangeni). www.elsevier.com/locate/enzmictec Enzyme and Microbial Technology 28 (2001) 705–712 0141-0229/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S0141-0229(01)00316-7