DEVELOPMENT OF ELISA AND ENZYME-LINKED IMMUNOFILTRATION ASSAY (ELIFA)METHODS FOR MONITORING CYCLODEXTRIN GLYCOSYLTRANSFERASE (CGTASE) PRODUCTION AND BACTERIAL-GROWTH IN BACILLUS-MACERANS BATCH CULTURES

Immunochemical methods were developed for monitoring cyclodextrin (CB) glycosyltransferase (CGTase)! production and growth of an industrial CD-producing Bacillus macerans strain. Extracellular concentrations of CGTase released into a non-transparent culture medium during a 44 h l ong fermentation were detected by an indirect antigen inhibition enzym e-linked immunosorbent assay (ELISA). The ELISA was sensitive (minimal detection level 6 ng ml(-1)) and highly reproducible (coefficients of variation less than or equal to 1.2 and 5.9%, within-runs and between -runs, respectively) compared to assays of CGTase activity (coefficien ts of variation less than or equal to 4.2 and 7.0%, respectively). The ELISA, in combination with enzyme activity measurements, was useful t o detect the decrease in the specific CGTase activities after 36 h of incubation, which was clearly indicative of the proteolytic degradatio n of CGTase. B. macerans cell numbers were estimated using an enzyme-l inked immunofilter assay (ELIFA). The assay took less than 1 h and the coefficients of variation within and between-runs (2.9-6.4%) were con siderably less than for viable counting (10.6-15.4%). In the exponenti al phase of growth, ELIFA results correlated more closely with the cel l counting based on total protein than with viable counts. Nevertheles s, in the phase of cell lysis, the bacterial cell number was systemati cally underestimated by ELIFA in comparison to both viable cell number and total protein determinations. Thus cell antigens detected with im munological procedures might be lost during the transition from vegeta tive cells to spores. On the other hand, the ELIFA procedure was speci fic for B.,macerans cells and was a better indicator of the onset of t he different growth phases than the cell numbers calculated from the p rotein assay. (C) 1998 Elsevier Science B.V. All rights reserved.