BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Eryngase: a Pleurotus eryngii aminopeptidase exhibiting peptide bond formation activity Jiro Arima & Masanori Chiba & Tsuyoshi Ichiyanagi & Yukinori Yabuta & Nobuhiro Mori & Tadanori Aimi Received: 17 March 2010 / Revised: 28 April 2010 / Accepted: 29 April 2010 / Published online: 25 May 2010 # Springer-Verlag 2010 Abstract An aminopeptidase that has peptide bond formation activity was identified in the cell-free extract of carpophore of Pleurotus eryngii. The enzyme, redesignated as eryngase, was purified for homogeneity and characterized. Eryngase had a molecular mass of approximately 79 kDa. It showed somewhat high stability with respect to temperature and pH; it was inhibited by iodoacetate. Among hydrolytic activities toward aminoacyl-p-nitroanilides (aminoacyl-pNAs), eryng- ase mainly hydrolyzed hydrophobic L-aminoacyl-pNAs and exhibited little activity toward D-Ala-pNA and D-Leu-pNA. In terms of peptide bond formation activity, eryngase used various aminoacyl derivatives as acyl donors and acceptors. The products were all dipeptidyl derivatives. Investigation of time dependence on peptide synthesis revealed that some peptides that are not recognized as substrates for hydrolytic activity of eryngase could become good targets for synthesis. Furthermore, eryngase has produced opioid dipeptides–– L-kyotorphin (L-Tyr-L-Arg) and D-kyotorphin (L-Tyr-D- Arg)––using L-Tyr-NH 2 and D- and L-Arg-methyl ester respectively as an acyl donor and acceptor. Yield evaluation of kyotorphin synthesis indicated that the conversion ratio of substrate to kyotorphin was moderate: the value was estimated as greater than 20%. Keywords Aminopeptidase . Peptide bond formation . Pleurotus eryngii . Aminolysis Introduction Some peptidases exhibit peptide bond formation, i.e., reverse reaction or aminolysis of esters, thioesters, or amides) in accordance with their hydrolytic activity (Arima et al. 2006; Bratovanova and Petkov 1987; Oka and Morihara 1980; Yokozeki and Hara 2005). Using peptidases that possess peptide bond formation activity, several biologically active dipeptides have been synthesized. For instance, a precursor of aspartame, Z-L-Asp-L-Phe methyl ester (-OMe), was synthesized by reverse reaction of thermolysin (Nakanishi et al. 1987); also, L-Ala-L-Gln, an infusion material, can be synthesized by aminolysis reaction of serine aminopeptidase (Yokozeki and Hara 2005). Results of a recent study demonstrate that Streptomyces metalloaminopeptidase is applicable to produce L-Val-L- Tyr (inhibitor of angiotensin I converting enzyme), L-Ala- L-Tyr (infusion material), and aspartame in a one-pot synthesis (Arima et al. 2006). Moreover, we have synthesized biologically active dipeptides such as β-Ala- L-His (dipeptide with protective functions named carnosine; Arima et al. 2010a), cyclo (L-Pro-L-His) (anti-hyperglycemic activity; Arima et al. 2010b), and cyclo (D-Pro-L-Arg) (chitinase inhibitor) using serine peptidase from Streptomyces (Arima et al. submitted for publication). Although these peptidases show good per- formance in the synthesis of dipeptides, it is not possible for all biologically active dipeptides to be synthesized enzymatically. To give enzymatic dipeptide synthesis broader utility, obtaining additional information related to synthesis methods, including novel enzymes, is useful to develop potent biocatalysts for dipeptide synthesis. For this study, we identified an aminopeptidase that has peptide bond formation activity in the cell-free extract of carpophore of Pleurotus eryngii. We renamed the amino- Electronic supplementary material The online version of this article (doi:10.1007/s00253-010-2663-7) contains supplementary material, which is available to authorized users. J. Arima (*) : M. Chiba : T. Ichiyanagi : Y. Yabuta : N. Mori : T. Aimi Department of Agricultural, Biological, and Environmental Sciences, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan e-mail: arima@muses.tottori-u.ac.jp Appl Microbiol Biotechnol (2010) 87:1791–1801 DOI 10.1007/s00253-010-2663-7