Mi crobiol. Res. (1998) 153, 157-162 © Gustav Fischer Ve rlag Thermostable a-amylase production by Bacillus licheniformis cells immobilized on polyacrylates with cyclic carbonate groups in the side chain E. Dobreva, V. Ivanova, M. Stefanova, A. Tonkova, L. Kabaivanova, D. Spassova Inst itute of Microbiology, BulgarianAcademy of Sciences, 26 Acad. G. B onchev str., 1113 Sofi a, Bulgaria Telephone:+ 359-2713 -31-63, fax:+359-2-700 109 Accepted: January 22, 1998 Abstract A considerable increase (up to 35%) of thermostable a-amy- lase yield was achieved by Bacillus licheniformis cells, immo- bilized on polymers containing cyclic carbonate groups in comparison to the synthesis by free cells. The increase of enzyme production was dependent on the manner of cell immobili zation and on the kind of polymers used. The a-amy- lase yield by immobilized "growing" cells was higher than that reached by immob ilized "washed" cells. The homopolymer poly [(2-oxo-1 ,3-dioxolan-4-yl) methyl 2-propenoate] and especiallyits methyl derivative were found to be more suitable for bacterial cell immobilization than the copolymer N-vinyl- pyrrolydone poly [(2-oxo-1,3-dioxolan-4-yl) methyl 2-prope- noate]. After 576h (4 cycles) of repeated batch fermentations , all biocat alysts used showed approxi mate ly equal a-amylase activity (2000 U/ml), correspondi ng to 60-80% residual activity d epending on the kindof the biocatalyst. Key words: Immobilization - Bacillus licheniformis - ther- mostable a -amylase-polymers with cyclic carbonate group s Introduction The simplest immobilization procedure for biological substances consists of their direct contact with preform- ed reactive polymers, such as maleic or methacrylic an- hydride based copolymers (Levin et al. 1964; Zingaro and Uziel 1970; Zaborsky 1973). However these sup- ports were synthesized in nonaqueous solvents and de- compose in water. In their turn, polymers containing cy- Corresponding author: E. Dobreva clic carbonate groups would have been of interest as car- riers because of the high reactivity and selectivity of di- oxolan ring towards aminogroups. These assumptions were based on the successful applications of cyclic car- bonate derivatives of cellulose (Barker et al. 1971) or dextran (Doane et al. 1968) for immobilization of differ- ent enzymes such as trypsin, dextranase (Kennedy and Zamir 1973; Cheetham and Richards 1973). However these supports had a rather short spacer and the toxic ethyl-chloroformiate was used in their syn- thesis. Polymers with a cyclic carbonate in the side chain had been obtained after a radical polymerization of acrylic monomers (Couvert and Bross 1990) but to our knowledge there are no studies on their use for immobi- lization of biological materials. The purpose of this study was to investigate the possibilities for thermostable a -amylase production by Bacillus lichenif ormis cells immobilized on polyacry- lates with 1,3-dioxolan-2-on (cyclic carbonate) side group. Materials and methods Microorganism and media. Bacillus licheniformis 44 MB 82 G strain, resistant to catabolite repression, pro- ducer of thermostable a -amylase was used (Tonkova et al. 1989; Tonkova 1991). Nutrient broth supplement- ed with 1% (w/v) soluble starch and 2% (w/v) glucose was used as seed medium. The fermentation medium contained [in % (w/v)]: glucose-6 .0; beef extract "Lab- Lemco powder"-1.5; peptone-1.5; K 2HPO c 1.04; corn steep liquor-0.66; CaCI 2 - O.II ; pH 7.0-7.2. Microbiol. Res. 153 (1998) 2 157