Enzyme and Microbial Technology 32 (2003) 655–657 Can lipases hydrolyze a peptide bond? Tatsuo Maruyama a,b,d,∗ , Mitsutoshi Nakajima a , Hidemasa Kondo c , Kosei Kawasaki c , Minoru Seki b , Masahiro Goto d a National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642 Japan b Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyoku, Tokyo 113-8656 Japan c Structural Biology Group, Research Institute of Biological Science, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo, Hokkaido 062-8517 Japan d Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan Received 8 November 2002; received in revised form 12 December 2002; accepted 13 December 2002 Abstract Several research groups reported that lipase catalyzes peptide synthesis in organic solvents. Structural studies revealed that the catalytic triad of lipase consists of Ser, His, and Asp, the same as serine proteases. Lipase has a potential to have peptidase activity. In this report, we investigated the peptidase activity of lipases. Of 13 lipases of diverse origin tested, only commercially available porcine pancreatic lipase (PPL) exhibited peptidase activity. However, purification of PPL by gel permeation chromatography separated the peptidase and lipolytic activities of PPL. This study clearly demonstrated that all the lipases tested do not hydrolyze a peptide bond. © 2003 Elsevier Science Inc. All rights reserved. Keywords: Hydrolysis; Lipase; Peptidase activity; Serine protease 1. Introduction Lipases catalyze the hydrolysis of triacylglycerols to free fatty acids and glycerol. At the 1990s, great progress with the three-dimensional structure of lipase revealed that the catalytic triad of lipase consists of three amino acids, Ser, His, and Asp (or Glu), the same as serine proteases [1]. There are several reports that porcine pancreas lipase (PPL), espe- cially in crude type, catalyzes peptide synthesis in organic solvents [2–6], and that protease can catalyze the hydrolysis and the transesterification of esters [7]. Thus indicating the functions and characteristics of lipase and protease over- lap. Further, several reports describe the amidase activity of lipase [8–10]. It thus is reasonable to conjecture that lipase has the potential to hydrolyze peptide bonds. If so, this will provide information of significance to the field of enzymology, and will also extend the application of lipase. However, there is almost no report that lipase catalyzed the hydrolysis of a peptide bond. In this study, we discussed the structural similarity between lipase and serine protease, and investigated whether lipase can hydrolyze a peptide bond. ∗ Corresponding author. Tel.: +81-92-642-3578; fax: +81-92-642-3575. E-mail address: tmarutcm@mbox.nc.kyushu-u.ac.jp (T. Maruyama). 2. Materials and methods PPLs (crude and highly pure, types II and VI-S), Rhizomucor miehei and Candida rugosa (type VII) were purchased from Sigma, St. Louis, MO; another PPL and human pancreatic lipase from the Elastin Products Com- pany, Inc., Owensville, MO; Rhizopus delemar lipase from Seikagaku Kogyo Co., Ltd.; and lipase Saiken 100 (from Rhizopus japonicus) from Nagase Biochemicals Ltd., Osaka, Japan. Chromobacterium viscosum lipase with high purity was a gift from Asahi Chemical Industry Ltd., Tokyo, Japan; cutinase (from Fusarium solani) with over 90% purity was kindly supplied by Unilever Research Vlaaridin- gen, The Netherlands. Lipase PLG (from Alcaligens spp.) was kindly supplied by Meito Sangyo, Nagoya, Japan. Lipase from Bacillus subtilis 168 (BsL) was expressed in Bacillus megaterium WH320, where the DNA fragment con- taining lipA gene was introduced plasmid vector pWH1520 (BoBiTec, Germany). The extracellular lipase was purified from the culture supernatant using ion exchange chromatog- raphy (MacroPrep High-S and Uno-Q, Bio-Rad Laborato- ries). The lipase activity was assayed with p-nitrophenyl butylate as a substrate. The active fraction was lyophilized prior to the evaluation of peptidase activity. The N-terminal amino acid sequence was determined as AEHNPVVMV, 0141-0229/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0141-0229(03)00053-X