Alexandra Tonkova

Bacterial cyclodextrin glucanotransferase

Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) is an enzyme which catalyzes intramolecular (cyclizing) and intermolecular (coupling, disproportionation) transglycosylation as well as having a hydrolytic action on starch and cyclodextrins. By a cyclizing reaction, the enzyme converts starch and related α-1, 4-glucans to cyclodextrins which are widely utilized in food, pharmaceutical, and chemical industries. The present review attempts to summarize the reported data concerning the bacterial producers of CGTase, growth cultural conditions providing optimal enzyme biosynthesis in batches, repeated batch and continuous cultivation of free and immobilized cells, as well as some physicochemical and biochemical characteristics of the enzyme, CGTase immobilization, and enzyme structure.

Thermostable exo-inulinase production by semicontinuous cultivation of membrane-immobilized Bacillus sp. 11 cells

Abstract Growing cells of thermophilic Bacillus sp. 11, producer of a thermostable exo-inulinase, were immobilized on formaldehyde-activated polysulphone membranes. The biocatalysts obtained showed 1.5–2 fold higher enzyme yields (inulinase and invertase activities) than those of free cells. Active cell growth and no alterations of the membrane structure were observed by scanning electron microscopy (SEM) after 10 days (five cycles) repeated batch cultivation. The residual enzyme activities of the biocatalysts were 60–90% of their initial activities at the end of fifth run of semicontinuous cultivation.

Influence of the immobilization conditions on the efficiency of α-amylase production by Bacillus licheniformis

Abstract Bacillus licheniformis 44MB82-A cells were immobilized in alginate and agar gels. The optimal immobilization parameters (gel concentration, initial cell quantity, biomass age, bead size and solidification prolongation) were determined. The immobilization procedure was most effective at a gel concentration of 4% using cells from a 12 h culture. The optimal initial cell quantity was found to be 0·6–3·0% in agar gel and 0·4% in Ca-alginate gel with bead sizes of 3·0 and 5·0 mm, respectively. An enzyme yield of 1100 U ml −1 culture medium was reached in batch fermentation with agar-entrapped cells under optimal conditions. This activity represented 135% of the corresponding yield obtained with free cells. Significant increases (2·2-fold) in the enzyme yields in the fourth cycle of repeated-batch runs with cells entrapped in agar gel pellets with bead size 5·0 mm was observed.

Characterization and cultural conditions of a novel cyclodextrin glucanotransferase‐producing Bacillus stearothermophilus strain

A cyclodextrin glucanotransferase (CGTase)‐producing thermophilic bacterium (strain 2/2) was iso‐lated from Bulgarian hyperthermal springs with a temperature of 102 °C and a pH value of 8.0—8.8. The strain was aerobic, Gram‐positive, and endospore forming with a temperature optimum of about 60 °C and a pH optimum of about 9.0. According to morphological, cultural and biochemical characteristics, and molecular properties, the isolate was identified as an alkalotolerant member of the species Bacillus stearothermophilus.

Immobilization of Bacillus licheniformis cells, producers of thermostable α-amylase, on polymer membranes

Cells of Bacillus licheniformis 44MB82-G immobilized on different polymer membranes were used for production of thermostable α-amylase. The α-amylase yields of the membrane-immobilized cells were affected by the reactive chemical groups of the carriers and the spacer size. Formaldehyde-activated polysulphone membranes (PS-FA) were the most suitable for effective immobilization. The highest amylase yield (62% increase of the control) and operational stability (97% residual activity after 480 h repeated batch cultivation) were obtained with this system. This was confirmed by scanning electron micrographs. An additional increase of α-amylase production by PS-FA-membrane immobilized cells was achieved in a fluidized-bed reactor.

Thermostable α-amylase from derepressed Bacillus licheniformis produced in high yields from glucose

Abstract High yields of thermostable α-amylase was produced by Bacillus licheniformis 44MB82-G, resistant to glucose catabolite repression, on the basis of inexpensive raw materials and glucose as a main carbon source. The optimal parameters for the α-amylase production were an agitation rate of 500 rpm, constant air-flow rate (1 vvm) and cultivation temperature 40°C. An enzyme activity of 4800–5000 U/ml culture medium was reached in 96–120 h. The α-amylase preparation had the following characteristics: α-amylase activity 55 000 U/ml, high thermostability (98% residual α-amylase activity after 10 min treatment at 90°C), protein content 88 mg/ml and dry substances 30%.

Screening of a growing cell immobilization procedure for the biosynthesis of thermostable α-amylases

Studies were carried out on α-amylase production with immobilized cells of twoBacillus strains. High yields of thermostable αamylases were obtained byBacillus licheniformis 44MB82-G, resistant to glucose catabolite repression and a thermophileBacillus brevis 174, after repeated batch cultivation (270–600 h) of the immobilized biocatalysts. Various cell immobilization techniques were compared, including entrapment in gel matrices (Ca-alginate,x-carrageenan, agar, and their combinations with polyethylene oxide), adsorption on cut disks of polymerized polyethylene oxide, and fixation on formaldehyde activated acrylonitrile-acrylamide membranes. The optimal immobilization parameters (gel and biocatalyst concentration, initial cell quantity) were determined. Among the gels and supports tested, agar,x-carrageenan, agar/polyethylene oxide gels, and the membranes were found to be suitable for immobilization and biocatalysts with high operational stabilities were obtained. An enzyme yield of 2750 U/mL culture medium was reached in the fifth repeated batch run with membrane-immobilizedBacillus licheniformis cells. This activity represented 176% of the corresponding yield obtained in batch fermentation with free cells. Higher amylase yields than the activity of the control were reached in all experiments and repeated batch runs with immobilizedBacillus brevis cells.

Agar gel immobilization ofBacillus brevis cells for production of thermostable α-amylase

Growing cells of a thermophilic strain ofBacillus brevis, producer of thermostable α-amylase, were immobilized by entrapment in agar gel. Optimum immobilization conditions for effective α-amylase production in batch fermentations were established (gel concentration 3%, initial biomass concentration in the gel 0.8% (W/V), and preculture age—late exponential phase). The dynamics of α-amylase synthesis by the biocatalysts obtained under the optimal conditions was compared with that of free cells and the operational stability of the biocatalysts was studied in semicontinuous cultivation experiments. Maximum α-amylase yields (252% of the control) were achieved after the second cycle of cultivation. Scanning electron microscopy was used to characterize the bacteria entrapped in agar gel.

Production and Properties of a Bacterial Thermostable Exo-inulinase

Abstract Enzyme production of newly isolated thermophilic inulin-degrading Bacillus sp. 11 strain was studied by batch cultivation in a fermentor. The achieved inulinase and invertase activi ties after a short growth time (4.25 h) were similar or higher compared to those reported for other mesophilic aerobic or anaerobic thermophilic bacterial producers and yeasts. The investigated enzyme belonged to the exo-type inulinases and splitted-off inulin, sucrose and raffinose. It could be used at temperatures above 65 °C and pH range 5.5-7.5. The obtained crude enzyme preparation possessed high thermostability. The residual inulinase and inver tase activities were 92-98% after pretreatment at 65 °C for 60 min in the presence of substrate inulin.

Characterisation of cyclodextrin glucanotransferase fromBacillus circulans ATCC 21783 in terms of cyclodextrin production

Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) fromBacillus circulans ATCC 21783 was purified by ultrafiltration and a consecutive starch adsorption. Total enzyme yield of 75.5% and purification factor of 13.7 were achieved. CGTase was most active at 65°C, possessed two clearly revealed pH-optima at 6.0 and 8.6 and retained from 75 to 100% of its initial activity in a wide range of pH, between 5.0 and 11.0. The cyclising activity was enhanced by 1 mM CaCl2 or 4 mM CoCl2. The enzyme was thermostable up to 70°C, and 64% of the original activity remained at 70°C after 30 min heat treatment. Up to 41% conversion into cyclodextrins was obtained from 40 g l−1 starch without using any additives. This CGTase produced two types of cyclodextrins, beta and gamma, in a ratio 73:27 after 4 h reaction time at 65°C. This feature of the enzyme could be of interest for industrial cyclodextrin production.