V. Ivanova

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.

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.

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.

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.

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.

HPLC study of starch hydrolysis products obtained with α-amylase from bacillus amyloliquefaciens and bacillus licheniformis

Two bacterial α-amylases from new industrial strains were studied: α-amylase fromBacillus amyloliquefaciens CCM 3502 (Czechoslovak) and thermostable α-amylase fromBacillus licheniformis 44MB82 (Bulgarian). The thermostable enzyme hydrolyzed starch mainly to dextrins, and after 1 h, 30% of the products were oligosaccharides. TheB. amyloliquefaciens enzyme produced more maltooligosaccharides than the first enzyme (B. licheniformis). Within 1 h, up to 80% of the substrate were hydrolyzed, giving different spectrum of oligosaccharides in comparison with the thermostable one.

Perspectives for the Production of Bioethanol from Lignocellulosic Materials

ABSTRACT The most common renewable fuel today and suitable alternative to replace fossil fuels is ethanol that can be blended with petrol or used as neat alcohol in engines. Ethanol is currently produced from sugar (Brazil) or grain (starch, USA). However, this raw material base will not be sufficient because the increasing demand for fuel ethanol and the lower than expected reduction of greenhouse gases. An alternative is the production of bioethanol from agroindustrial wastes containing abundant cellulosic fibers and carbohydrates such as grape pomace, sugar beet pomace, barley and rice straw, corncobs, sunflower stalks and heads, cotton waste, brewers spent grain, forest residues etc. Lignocellulosic raw materials and agroindustrial wastes minimize the potential conflict between land use for food (and feed) production and energy feedstock production. This review summarizes recent developments in the bioconversion processes, the new technologies required and the advances achieved in recent years to bring agricultural feedstock and lignocellulosic ethanol towards industrial production.

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.