Ilma Hiroko Higuti

Colorimetric determination of alpha and beta-cyclodextrins and studies on optimization of CGTase production from B. firmus using factorial designs

Cyclodextrin glycosyltransferase (EC 2.4.1.19, CGTase) production from B. firmus, isolated from soil of Curitiba, PR, was optimized in shake flask using an experimental design approach. The CGTase was produced when the carbon source was starch and b-CD, but when simple sugars such as glucose, galactose, lactose, sucrose, and maltose were used, there was no enzyme production. CGTase production was the same with either organic nitrogen or inorganic nitrogen source. CGTase activity decreased 2-fold when incubation temperature was increased from 28 to 37 ° C, and decreased 2.1- fold when the initial pH was lowered from 10.3 to 7.4. The colorimetric determinations of a - and b -CD were analyzed as a non-linear relationship and the equilibrium constant for a -CD/methyl orange and b -CD/phenolphthalein complexes were 7.69 x 103 L / mol and 2.33 x 103 L/ mol, respectively.

Occurrence of extended-spectrum beta-lactamases in Enterobacteriaceae isolated from hospitalized patients in Curitiba, southern Brazil

Production of extended-spectrum beta-lactamases (ESBL) by enterobacteria is an important resistance mechanism against antimicrobial beta-lactamics. We tested 498 bacterial strains isolated from two tertiary-care teaching hospitals for ESBL production, using screening breakpoints for aztreonam and third generation cephalosporins, according to CLSI recommendations. Among these isolates, 155 were positive for the ESBL screening test, and 121 (78%) were confirmed by the clavulanic acid combination disk method. We found a high frequency of ESBL (24%) among Enterobacteriaceae, with a frequency of 57.4% for Klebsiella pneumoniae, 21.4% for Klebsiella oxytoca, and 7.2% for E. coli. In other members of Enterobacteriaceae, non-Klebsiella and non-E. coli, the prevalence was 21.6%. Ceftriaxone and cefotaxime showed a higher sensitivity in the screening test (99.2%) when compared to ceftazidime, aztreonam and cefpodoxime. However, cefotaxime/cefotaxime plus clavulanic acid showed a higher sensitivity in the confirmatory test (96.7%).

Studies on alkalophilic CGTase-producing bacteria and effect of starch on cyclodextrin-glycosyltransferase activity

The production of b- and g -CD by CGTase from Bacillus firmus was studied, in regard to the effect of the source and concentration of starch on the yield of CD and CGTase activity. A cyclic activity of accumulation and consumption of b -CD and g -CD occurred during the bacterial growth. CGTase was more active when citrate buffer, pH 5.5 was used. No differences were found for production of g -CD with the use of commercial starch flour when compared with corn starch (p>0.05). The use of commercial starch flour resulted in decreased conversion of starch to b-CD (all the 4 starch sources were statistically different, p<0.05). The best results for b -CD production were obtained with the use of corn starch. The specific activity of CGTase was not affected by starch concentration, different source of starch nor by the presence of glutamic acid, a CD-complexing agent.

Occurrence of coliforms in water samples of the Perequê and Penedo Rivers in Paraná, Brazil

The Pereque and Penedo rivers flow through the plains of Praia de Leste and fall into the sea between the municipalities of Pontal do Sul and Ponta do Poco. Both of these rivers belong to the sub-basin of Paranagua Bay, and are constituent parts of the hydrographic basin of the Atlantic Ocean (State of Parana). Four sites along the Pereque River (Stations 1-4) and one along the Penedo River (Station 5) were monitorated for water pollution over a 12-month period. During the period of December 1995 to November 1996, three water samples were collected from each site, placed in sterilized bottles and transported to the laboratory in ice-boxes. They were analyzed for the occurrence of total and fecal coliforms, temperature and pH. Fecal pollution was detected throughout the period of study. Coliform contamination was smaller in Station 1 and 5 than in any other sites, probably due to lesser influence from urbanized areas. Water at Station 4 was strongly contaminated due to its close proximity to a sewage canal. This study demonstrated that the Pequere and Penedo rivers, freshwater shrimps habitats, were heavily contaminated with fecal indicator bacteria, and this occurrence was not related with temperature or pH.