E. Dobreva

Purification and characterization of a thermostable alpha-amylase from Bacillus licheniformis

Abstract The extracellular alpha-amylase produced by the Bacillus licheniformis 44MB82-A strain was isolated. The enzyme was purified by two-phase separation in a PEG-Dextran system, followed by gel-filtration and ion exchange chromatography. Its molecular mass was determined as 58000 by SDS-PAGE and gel filtration. The enzyme was stable at pH values from 6.5 to 8.0 and its pH optimum was pH 6.0–6.5. The temperature optimum was determined as 90°C. The Michaelis constant for soluble starch was measured as 0.90 g l −1 . Thermostability was Ca 2+ -dependent. The half-life of the purified enzyme was 10 min at 85°C in buffer without Ca 2+ . The half-life at pH 6.5 with 1.0 mM CaCl 2 added was 30 min, and over 120 min with 5.0 mM CaCl 2 . The purified enzyme was strongly inhibited by N -bromosuccinimide (NBS) and by EDTA. Dithiothreitol and iodacetamide had no inhibitory effect on the purified enzyme.

Performance of a magnetically stabilized bed reactor with immobilized yeast cells

This paper is focused on the possibility to apply the magnetic stabilization technique in bioprocessing. The feasibility of a continuous ethanol fermentation process with immobilizedSaccharomyces cerevisiae cells in a magnetically stabilized bed (MSB) was demonstrated. The fermentation processes were carried out in an external magnetic field, transverse to the fluid flow. The flexibility to change the bed expansion owing to the independent change of the fluid flow and the field intensity (the “magnetization FIRST” mode) permitted the creation of fixed beds with different particle arrangements, which affected the bed porosity, the effective fluid-particle contact area, and the mass transfer processes on the particle-fluid interface. As a result, higher ethanol concentration, ethanol production, and glucose uptake rates than in conventional packed bed reactor were reached.

Immobilization of cells with nitrilase activity from a thermophilic bacterial strain.

Cells of the moderately thermophilic Bacillus sp. UG-5B strain, producing nitrilase (EC3.5.5.1), which converts nitriles directly to the corresponding acid and ammonia, were immobilized using different types of matrices and techniques. A variety of sol-gel silica hybrids were tested for entrapment and adsorption of bacterial cells as well as chemical binding on polysulphone membranes. Activation of the matrix surface with formaldehyde led to an increase in immobilization efficiency and operational stability of the biocatalysts. Among the supports screened, membranes gave the best results for enzyme activity and especially operational stability, with retention of 100% activity after eight reaction cycles.

Non-Porous Magnetic Supports for Cell Immobilization

Abstract A new method for covering magnetic particles with a stable non-porous layer of a material like zeolite or activated carbon was used for the preparation of support materials with good properties for the immobilization of yeast Saccharomyces cerevisiae cells. The immobilized cells can be used in batch and continuous alcoholic fermentation. A productivity of 35.6 g ethanol/ l · h was reached. The adsorption isotherms of the immobilized yeast cells were determined. Yeast cell immobilization on non-porous magnetic supports obeyed the Langmuir isotherm equation. Satisfactory results were obtained also from repeated batch fermentations with fixed cells on supports additionally treated with glutaraldehyde or by simple adsorption.

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.

Effect of temperature on some characteristics of the thermostable α-amylase from Bacillus licheniformis

The Vmax of an extracellular, thermostable α-amylase from Bacillus licheniformis 44MB82 were 5.70×10-3 and 9.70×10-3mM s-1 at 30 and 90°C, respectively, whereas the Km values were similar (0.9 mg ml-1) at both temperatures. Excluding dextrins, the dominant products from soluble starch and amylopectin hydrolysis contained less than six glucose residues. The enzyme hydrolysed amylopectin better than soluble starch. Increasing the temperature from 30 to 90°C was accompanied by an increase in the production of malto-oligosaccharides, especially maltotetrose, and this was related to the secondary hydrolysis of maltopentose and maltohexose.

Catalytic properties of immobilized purified thermostable α-amylase from Bacillus licheniformis 44MB82-A

Abstract Bacillus licheniformis 44MB82-A α-amylase was purified and immobilized on five silica supports. The relative activity of the immobilized amylase represented from 22 to 40% of the activity of the bound protein. The immobilized enzyme was used for repeated batch hydrolysis of soluble starch. Arylamine CPG and Spherosyl XOA 200 were found to be suitable as carriers and the enzyme immobilized on them retained 60% of its initial activity after reuse. Considerable changes in the hydrolysis products and the inability of the immobilized α-amylase to hydrolyse some malto-oligosaccharides were 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%.