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Biotechnology and Applied Biochemistry (2002) 36, (71–76) (Printed in Great Britain)

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Boilysin and thermolysin in dipeptide synthesis: a comparative study

Diana Kühn, Peter Dürrschmidt, Johanna Mansfeld and Renate Ulbrich-Hofmann¹

Department of Biochemistry and Biotechnology, Institute of Biotechnology, Martin-Luther University, Kurt-Mothes-Str. 3, D-06120 Halle, Germany

Key words: aspartame, Bacillus stearothermophilus, neutral protease, organic solvent, specificity.

Abbreviations used: BLN, boilysin; OMe, methyl ester; TFA, trifluoroacetic acid; TLN, thermolysin; WT, wild-type; Z, N-(benzyloxycarbonyl).

¹To whom correspondence should be addressed (e-mail Ulbrich-Hofmann@biochemtech.uni-halle.de).

Boilysin (BLN) is an engineered, highly thermostable neutral protease from Bacillus stearothermophilus. Its high resistance is based on the stabilization of a surface loop (amino acid residues 55–69) by eight amino acid exchanges, including the introduction of a disulphide bond. In the present study, BLN was compared with the well-known and structurally related thermolysin (from B. thermoproteolyticus) with respect to their dipeptide- synthetic properties. The synthesis of the aspartame precursor N-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-Asp-Phe-OMe) was used as the model reaction to study its enantioselectivity as well as the influence of neutral salt, temperature and calcium- ion concentration on peptide synthesis. The reactions were performed in homogeneous reaction systems containing DMSO or aliphatic alcohols. Furthermore, the substrate specificity in the synthesis of Z-Asp-X-OMe (X = Phe, D-Phe, Ala, Ile, Leu, Met, Tyr or Val) was examined. The two enzymes showed no difference in time course of reaction by the use of salts as activators or different alcohols as co-solvents. Both enzymes showed a high enantioselectivity towards the amino component in the reaction. They were strongly activated by NaCl, whereas the final product yields were strongly decreased at high NaCl concentrations. Aliphatic alcohols act as an inhibitor, but allow higher product yields compared with purely aqueous medium. Differences between the two enzymes are found at higher temperatures ( 60 °C) in the absence or at low concentrations of Ca²⁺ ions; BLN proved superior under these conditions, because its stability is less dependent on Ca²⁺ ions. The substrate specificities of BLN and thermolysin at the position follow the same tendencies, with differences in the initial rates for the conversion of Z-Asp with Ile-OMe, Leu-OMe and Val-OMe.

Received 12 February 2002/13 May 2002; accepted 27 May 2002

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