Development of ELISA and enzyme-linked immunofiltration assay (ELIFA) methods for monitoring cyclodextrin glycosyltransferase (CGTase) production and bacterial growth in Bacillus macerans batch cultures

Abstract

Immunochemical methods were developed for monitoring cyclodextrin (CD) 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 long fermentation were detected by an indirect antigen inhibition enzyme-linked immunosorbent assay (ELISA). The ELISA was sensitive (minimal detection level 6 ng ml−1) and highly reproducible (coefficients of variation ≤1.2 and 5.9%, within-runs and between-runs, respectively) compared to assays of CGTase activity (coefficients of variation ≤4.2 and 7.0%, respectively). The ELISA, in combination with enzyme activity measurements, was useful to detect the decrease in the specific CGTase activities after 36 h of incubation, which was clearly indicative of the proteolytic degradation of CGTase. B. macerans cell numbers were estimated using an enzyme-linked immunofilter assay (ELIFA). The assay took less than 1 h and the coefficients of variation within and between-runs (2.9–6.4%) were considerably less than for viable counting (10.6–15.4%). In the exponential phase of growth, ELIFA results correlated more closely with the cell counting based on total protein than with viable counts. Nevertheless, in the phase of cell lysis, the bacterial cell number was systematically underestimated by ELIFA in comparison to both viable cell number and total protein determinations. Thus cell antigens detected with immunological procedures might be lost during the transition from vegetative cells to spores. On the other hand, the ELIFA procedure was specific for B. macerans cells and was a better indicator of the onset of the different growth phases than the cell numbers calculated from the protein assay.