Experimento

Biotechnology Letters (2005) 27: 661–664 DOI 10.1007/s10529-005-4476-9

Ó Springer 2005

Increased erythromycin production by alginate as a medium ingredient or immobilization support in cultures of Saccharopolyspora erythraea
J. Hamedib*, F. Khodagholib & A. Hassani-Nasabb
Microbial Biotechnology Laboratory, Department of Biology, Faculty of Science, University of Tehran, Tehran, Iran*Author for correspondence (Fax: +98-21-6405141; E-mail: jhamedi@khayam.ut.ac.ir)
Received 11 January 2005; Revisions requested 27 January 2005; Revisions received 7 March 2005; Accepted 18 March 2005

Key words: alginate, erythromycin, immobilization, Saccharopolyspora erythraea, shear

Abstract Erythromycin production by Saccharopolyspora erythraea immobilized in 2% (w/v) calcium alginate orgrown in medium containing 20 g sodium alginate/l inoculated with free cells was almost twice more than that of the control. S. erythraea did not consume alginate, agar, dextran, silicon antifoaming agent or cyclodextrin as a carbon source, although, all of these increased the production of erythromycin. Highest titer of erythromycin (2.3 times more than that of the control) was achieved in mediumcontaining 1 g agar/l.

Introduction Alginate is a polymer containing 1, 4-linked bD-mannuronic acid and a-L-guluronic acid residues of widely varying composition and sequential arrangement. It used to immobilize cells and enzymes. It also acts as an elicitor which enhances growth of fungal (Radman et al. 2004), plant (Akimoto et al. 1999) and animal cell cultures (Kawada et al. 1999). Althoughalginate may improve growth of Bifidobacterium, an obligatory anaerobic bacterium, (Akiyama et al. 1992), there is no report of its effects on other bacteria including actinomycetes. Actinomycetes are the source of more than 67% of all discovered antibiotics (Berdy 1995). Some of these antibiotics can also be produced using immobilized cells (Veelken & Pape 1982; Adinarayana et al. 2004;Bandyopadhyay et al. 1993; Devi & Sridhar 1999). However, to our knowledge, the effect of alginate on these processes has been neglected. In this research for the first time, we have compared the effect

of alginate and some other polymers on the erythromycin production by Saccharopolyspora erythraea.

Materials and methods Bacterial strain and media Saccharopolyspora erythraea UT110 (a mutant obtained by UVradiation of S. erythraea DSMZ240517) was used. The sporulation medium was oat-meal agar. The composition of the defined fermentation medium used for erythromycin production was as follows (g/l): glucose 50, NaNO3 18.5, KH2PO4 3, K2HPO4 7. In addition, 2 ml trace metals solution with the following composition was added (g/l): MgSO4Æ7H2O 0.25, FeSO4Æ 7H2O 0.025, CuCl2 00053, CoCl2 0.00055, CaCl2Æ2H2O 0.0138, ZnCl2 0.0104, MnCl2 0.0002 and Na2MoO4 0.0003. The pH was adjusted to 7 ± 0.1 by using 5 M KOH (Clark et al. 1995). Dextran, sodium alginate, agar, silicon antifoaming agent

662 (Merck) and cyclodextrin were added to the fermentation medium as indicated in the text. Immobilization of S. erythraea UT110 spores in calcium alginate beads A spore suspension of S. erythraea UT110 wasmixed with a 2% (w/v) sodium alginate solution, dropped into 0.2 M CaCl2 at 4 °C, stirred for 2 h, then washed twice by sterile saline. All these steps were carried out under aseptic conditions. Cultural method Spore suspension of S. erythraea UT110 or cellscontaining beads were inoculated into 1000 ml Erlenmeyer flask containing 150 ml fermentation medium and incubated at 30 °C for 5 days at 220rpm. Erythromycin assay Erythromycin Total erythromycin was extracted from fermentation broth with chloroform and was mixed with Bromophenol Blue. The absorbance of organic phase was measured at 415 nm. Concentration of erythromycin A was determined by HPLC at 205 nm; column, C18; mobile phase, acetonitrile methanol 0.2 M ammonium acetate water (45:10:10:35, by vol.) at 1 ml/min; column at 40 °C;...