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Biotechnology and Applied Biochemistry (2003) 37, (165–171) (Printed in Great Britain)

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Regulatory effects of alanine-group amino acids on serine alkaline protease production by recombinant Bacillus licheniformis

Pnar Çalk*¹,Arzu Bayram† andTunçer H. Özdamar†

*Industrial Biotechnology Laboratory, Department of Chemical Engineering, Middle East Technical University, 06531 Ankara, Turkey, and †Ankara University Biotechnology Research Center, Industrial Biotechnology Department, Tandoan, 06100 Ankara, Turkey

Key words: alanine, amino acid, fermentation, leucine, serine alkaline protease, valine.

Abbreviations used: SAP, serine alkaline protease; t, bioreactor cultivation time; C , initial alanine concentration.

¹To whom correspondence should be addressed (e-mail pcalik@metu.edu.tr).

Influences of the concentration and addition time of alanine-group amino acids, i.e. alanine, leucine and valine, on serine alkaline protease (SAP) synthesis were investigated by Bacillus licheniformis (DSM 1969) carrying pHV1431::subC in a defined medium to identfy the amino acids creating intracellular reaction-rate limitation in SAP production. While the precursors of alanine-group amino acids, pyruvate and alanine, did not affect SAP production considerably within the range 0–15 mM, the addition of leucine decreased both SAP production and cell formation, because of the inhibition of valine synthesis. Although valine inhibits reactions starting with pyruvate towards 2-oxo-isovalerate, due to conversion of valine into 2-oxo-isovalerate and from 2-oxo-isovalerate to leucine, valine did not inhibit leucine synthesis. Val (7.5 mM) supply at t = 0 h increased SAP activity to an activity of 1070 units · cm^-3 which was 1.3-fold higher than that of the reference production medium. The highest cell growth yield on substrate (Y_X/S) was obtained as 0.24 g · g^-1 with the supply of alanine; and the highest product formation yield on substrate was obtained as 0.134 units · g^-1 with the supply of valine. By using the results obtained, strategies for increasing SAP production and complex medium design were also discussed.

Received 9 September 2002/18 November 2002; accepted 29 November 2002

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