Can lipases hydrolyze a peptide bond?

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 Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyoku, Tokyo 113-8656 Japan 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 b

c

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

2. Materials and methods

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), especially 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 overlap. 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.

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 Company, 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 Vlaaridingen, 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 containing lipA gene was introduced plasmid vector pWH1520 (BoBiTec, Germany). The extracellular lipase was purified from the culture supernatant using ion exchange chromatography (MacroPrep High-S and Uno-Q, Bio-Rad Laboratories). 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,

∗

Corresponding author. Tel.: +81-92-642-3578; fax: +81-92-642-3575. E-mail address: [email protected] (T. Maruyama).

0141-0229/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0141-0229(03)00053-X

656

T. Maruyama et al. / Enzyme and Microbial Technology 32 (2003) 655–657

which was identical with that of BsL produced by B. subtilis 168 [11]. This lipase, of greater than 90% purity, was used for the tests. Peptidase activity was assayed using N-benzoyl-l-tyrosine p-nitroanilide (Bz-Tyr-pNA) as a substrate. A solution containing Bz-Tyr-pNA was prepared by firstly adding 0.3 ml of acetone solution of Bz-Tyr-pNA (2 mmol/l) to 2.7 ml of Tris–HCl buffer containing CaCl2 2 mmol/l and NaN2 2 mmol/l. To this Bz-Tyr-pNA solution, 0.52 ml of enzyme solution (1.5 mg/ml) was added. The hydrolysis of Bz-Tyr-pNA at 25 ◦ C was followed by continuous measurement of the increase in absorbance at 405 nm (due to the release of p-nitroaniline from Bz-Tyr-pNA). The release of p-nitroaniline (␮mol/min) was defined as peptidase activity (U). Lipolytic activity was assayed as follows. Lipase catalyzed hydrolysis reactions of emulsified tributyrin were carried out at 25 ◦ C in a 20 ml aqueous solution (adjusted to pH 7.0 by NaOH) containing glycerol (8.0 vol.%), gum arabic (1.0 g/l), NaCl (0.050 mol/l), KH2 PO4 (0.50 mmol/l) and 0.1 ml tributyrin as a substrate. The reaction medium was initially homogenized at 10,000 rpm for 5 min using a Polytron PT 3000 (Kinematica AG, Switzerland). The hydrolysis reaction was started by adding a lipase solution. Release of free butyric acid was monitored by continuous titration with NaOH solution (0.05 mol/l) with a pH-stat (632 Digital pH-Meter, 614 Impulsomat and 665 Dosimat, Metrohm Ltd., Switzerland). The release of butyric acid (␮mol/min) from tributyrin was defined as lipolytic activity (U). Purification by gel permeation chromatography (GPC) [12] was performed using a chromatographic system Akta-Prime (Amersham-Pharmacia Biotech, NJ). Two hundred units of PPL (Elastin Products Company, Inc., Owensville, MO) was dissolved in 2.5 ml of buffer containing 0.5 mol/l NaCl, 0.025 mol/l Tris–HCl, pH 8.1, 2 mmol/l CaCl2 , and 2 mmol/l NaN2 . The PPL solution (2 ml) was filtrated with a 0.45 ␮m filter (DISMIC-25CS; Toyo Roshi Kaisha, Ltd., Tokyo) and then applied to a column of Sephacryl S-200 (2.6 cm × 60 cm; Amersham Pharmacia Biotech), equilibrated, and developed with Tris–HCl buffer. The flow rate was 1.0 ml/min, and fractions of 10 ml each were collected.

3. Results and discussion As noted in Section 1, the functions and characteristics of lipase and serine protease overlap. The unique property of lipase, which differs from serine protease, is based on the physical state of the substrate. Many lipases act specifically in a heterogeneous medium (interfacial activation), the lipid substrate being dispersed as an emulsion in the aqueous medium. They act at the lipid–water interface, and tend not to act on water-soluble substrates [13]. Brady et al. (1990) reported, for the first time, the three-dimensional structure of lipase [1]. The structure of R. miehei lipase has an ␣-helical

Fig. 1. Superposition of catalytic triad from cutinase (dark color) with ␣-chymotrypsin (light color). Residues have been fitted using least-squares.

fragment (termed the “lid”) which covers the active site. They discussed that the unique property of lipase (interfacial activation) could be explained by opening the lid. The lid is supposed to prevent a water-soluble substrate from accessing the active site of lipase and perhaps to distinguish lipase from serine protease. However, Martinez et al. (1992) reported that there is lipase (cutinase from F. solani) not having a lid [14]. The catalytic triad of the cutinase also consists of Ser, His and Asp and is accessible by solvent. The cutinase may be said to be structurally close to serine protease because of the solvent-accessible catalytic triad. We then compared the catalytic triads of the cutinase and serine protease. ␣-Chymotrypsin [15] was adopted as a model of serine protease. Fig. 1 shows the superposition of the catalytic triad from cutinase with ␣-chymotrypsin. This superimposing indicated that the spatial arrangements of cutinase catalytic triad were similar to those of ␣-chymotrypsin, which agreed with the previous reports [1]. The structural similarities and the reports on lipase-catalyzed peptide synthesis [2–6] allowed us to suspect that lipase, especially cutinase, could hydrolyze a peptide bond. Many lipases are inactive for water-soluble substrates in an aqueous buffer, but a few (cutinase, etc.) hydrolyze water-soluble substrates [16]. Peptidase activity is usually assayed using water-soluble substrates. Therefore, to assess peptidase activity, we selected two bacterial lipases that hydrolyze water-soluble substrates, in addition to several commercially available mammalian, fungal, and bacterial lipases. Thirteen lipases from different suppliers and of diverse origins were tested for peptidase activity using Bz-Tyr-pNA. Their activity is summarized in Table 1. Of the 13 lipases, only PPLs (crude and highly pure types) obviously displayed the peptidase activity. Unlike our expectations, lipases without a lid (cutinase and lipase from B. subtilis 168) did not display peptidase activity. The peptidase activity of PPLs depended on the lot number (data not shown), but all tested lots showed peptidase activity to some degree.

T. Maruyama et al. / Enzyme and Microbial Technology 32 (2003) 655–657 Table 1 Peptidase activity of various lipases Lipase origin

Peptidase activity (U/mg)

Porcine pancreas from Sigma (crude, type II) Porcine pancreas from Sigma (type VI-S, lot no. 68H7440) Porcine pancreas from Elastin Products Company (lot no. 12744) Human pancreas R. miehei R. japonicus R. delemar Thermomyces lanuginosa C. viscosum Alcaligenes spp. Cutinase from F. solani B. subtilis 168 C. rugosa

0.0069 0.016 0.097 None None None None None None None None None None

657

shows the elution profile of the GPC on Sephacryl S-200. Fig. 2A shows UV absorbance at 280 nm for each fraction. Small peaks in Fig. 2A indicate that this PPL preparation contains various compounds. Fig. 2B shows the lipolytic and peptidase activities of each fraction. Lipolytic activity was found in fractions 17 and 18, but peptidase activity was mainly in fractions 20–22. These results demonstrate that PPL itself does not hydrolyze Bz-Tyr-pNA, and that commercial PPL preparations, even those of high purity, contain peptidase as an impurity. Our present study demonstrates that lipases do not hydrolyze the peptide bond, although lipase and serine protease have many points in common. Commercial PPL preparations, even those of high purity, were found to contain peptidase as an impurity. Our finding suggests that the peptide synthesis catalyzed by lipases should be studied using highly purified lipase preparations. We are currently investigating the machinery difference between lipases and serine proteases to reveal why the lipases do not have peptidase activity.

Acknowledgments This work was supported by the Bio-oriented Technology Research Advancement Institution, Japan. We thank Dr. M. Burg-Koorevaal (Unilever B.V.), Dr. K. Mogi (Nippon Lever B.V.), Asahi Chemical Industry Ltd., and Meito Sangyo Ltd. for providing lipase samples. References

Fig. 2. Elution profile of porcine pancreatic lipase on Sephacryl S-200. (A) UV absorbance at 280 nm and (B) lipolytic and peptidase activity.

These results agree with previous reports that commercially available PPL catalyzed the synthesis of peptide [2]. However, there remains a possibility that these PPLs contain other enzymes as impurities, although two of the PPLs used in this study had commercially high purity and the Elastin Products Company states that their PPL does not contain any protease (as set out in their catalogue). PPL from the Elastin Products Company was selected for further purification because they state that their PPL does not hydrolyze casein. The PPL was purified by GPC, following the procedure reported by Garner and Smith [12]. Fig. 2

[1] Brady L, Brzozowski AM, Derewenda ZS, Dodson E, Dodson D, Tolley S, et al. Nature 1990;343:767–70. [2] Margorin AL, Klibanov AM. J Am Chem Soc 1987;109:3802–4. [3] West JB, Wong CH. Tetrahedron 1987;28:1629–32. [4] Kawashiro K, Kaiso K, Minato D, Sugiyama S, Hayashi H. Tetrahedron 1993;49:4541–8. [5] Zhang LQ, Zhang YD, Xu L, Li XL, Yang XC, Xu GL, et al. Enzyme Microb Technol 2001;29:129–35. [6] So JE, Kang SH, Kim BG. Enzyme Microb Technol 1998;23:211–5. [7] Okazaki S, Kamiya N, Goto M. Biotechnol Prog 1997;13:551–6. [8] Smidt H, Fischer A, Fischer P, Schmid RD. Biotechnol Tech 1996;10:335–8. [9] Castro MS, Gago JVS. Tetrahedron 1998;54:2877–92. [10] Mahmoudian M, Lowdon A, Jones M, Dawson M, Wallis C. Tetrahedron Asymmetry 1999;10:1201–6. [11] Lesuisse E, Colson C, Schanck K. Eur J Biochem 1993;216:155–60. [12] Garner CW, Smith LC. J Biol Chem 1972;247:561–5. [13] Martinelle M, Hult K. In: Woolley P, Petersen SB, editors. Lipases. Cambridge: The University Press; 1994. p. 159–80. [14] Martinez C, De Geus P, Lauwereys M, Matthyssens G, Cambillau C. Nature 1992;356:615–8. [15] Tsukada H, Blow DM. J Mol Biol 1985;184:703–11. [16] Carvalho CM, Aires-Barros M, Cabral MS. Biotechnol Bioeng 1999;66:17–34.