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.

Extracellular xylanase production by two thermophilic alkali-tolerant Bacillus strains in batch and continuous cultures.

Xylanase production of newly isolated thermophilic alkali-tolerant Bacillus sp. strain SP and strain BC was investigated in batch and continuous cultures. Enzyme synthesis was inducible with both strains and was observed only in xylan-containing media. Xylan from oat spelt is a better inducer than xylan from birch for strain Bacillus sp. BC while such difference was not observed for strain SP. Compared with batch cultures xylanase production of both strains increased about two times and its rate became more than four times faster in continuous cultures at a dilution rate of 0.2 h(-1).

Influence of culture conditions on thermostable lipase production by a thermophilic alkalitolerant strain of Bacillus sp.

The effect of different culture conditions on thermostable lipase production by Bacillus sp. was studied in shake flasks. A maximum enzyme activity of 67-75 nkat/mL was observed in a medium consisting of 0.5% soybean flour and 0.1% stearyl glycerol esters or natural fats. A lipase activity of about 117 nkat/mL was established when the cultivation was carried out in a laboratory fermentor at 20% minimal dissolved oxygen level, the enzyme production being increased 1.5 fold compared to that in a flask culture.

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

The V max of an extracellular, thermostable α-amylase from Bacillus licheniformis 44MB82 were 5.70×10(-3) and 9.70×10(-3) MM s(-1) at 30 and 90°C, respectively, whereas the K m 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.

Characterisation of a thermostable alpha-amylase from Bacillus brevis.

Biochemical characterization of a novel heat-stable alpha-amylase, produced by a thermophilic strain of Bacillus brevis, has been made. The pattern of the enzyme action on different substrates was studied. It was found that reducing groups were rapidly liberated from amylopectin, soluble and insoluble starch compared to amylose and glycogen. B. brevis alpha-amylase acted via endo-attack producing mainly maltopentaose during the first hour of hydrolysis. The enzyme showed high activity towards maltohexaose and maltoheptaose. The alpha-amylase from B. brevis had a neutral pI and was found to be a glycoprotein, containing 9.2% (by mass) neutral sugars. The enzyme protein possessed a unique high glycine content. Calcium or sodium ions in appropriate concentrations were required for enzyme thermostability.

Purification and general biochemical properties of thermostable pullulanase from Bacillus stearothermophilus G-82.

Thermostable extracellular pullulanase, produced by Bacillus stearothermophilus G-82 was purified to homogeneity from supernatants of continuous culture by ultrafiltration, ammonium sulphate precipitation, chromatography on Sephadex G-100, and DEAE cellulose. A mol wt of 53,000 was determined by gel filtration and 56,000 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The isoelectric point (pI) was 4.2. The pullulanase contained predominantly acidic amino acids. The enzyme was optimally active at a temperature of 60 degrees C and pH 7.0. It preserved 100% of its activity after 10 min treatment at 60 degrees C. The thermostability was considerably increased in the presence of pullulan. Ca2+ did not increase activity or thermostability. Enzyme activity was fully inhibited by N-bromosuccinimide and partially by phenylmethylsulfonyl fluoride. Bacillus stearothermophilus G-82 pullulanase was able to hydrolyze alpha 1-6 as well as alpha 1-4 glucosidic bonds in pullulan, amylopectin, amylose, glycogen, and dextrin. The enzyme showed highest affinity to pullulan (Km = 0.14).

Effect of carbon source and dissolved oxygen level on cell growth and pullulanase production byBacillus stearothermophilus G-82.

Cell growth and extracellular pullulanase production ofBacillus stearothermophilus G-82 were investigated in batch culture using a defined medium with glucose, maltose, pullulan or amylopectin as carbon source. Maximum enzyme activity was with pullulan or amylopectin. Cell growth in batch culture was better under oxygen unlimited conditions, while higher total and specific enzyme activities, using pullulan or amylopectin, were obtained in oxygen-limited conditions. Enzyme accumulation took place in the late growth phase. The highest enzyme production of 300 U/I was reached when pullulan was used as carbon source in conditions of oxygen limitation.

Functional half-life of the alpha-amylase mRNA of Bacillus licheniformis.

Some aspects of the regulation of alpha-amylase synthesis in Bacillus licheniformis CCM 2205 were investigated. The effect of actinomycin D and chloramphenicol was studied at the level of RNA transcription and translation. alpha-amylase synthesis in Bacillus licheniformis CCM 2205 was practically not altered during the first 20 min after the addition of actinomycin D, although RNA synthesis was almost completely blocked. In contrast to RNA polymerase inhibitor, chloramphenicol stopped immediately the synthesis of alpha-amylase. By using the least squares method the mean half-life of alpha-amylase mRNA was calculated to range from 7.5 to 8.4 min. the mean half-life of cell protein mRNA was determined to range from 2.6 to 3.8 min. Having in mind the immediate effect of chloramphenicol on the alpha-amylase synthesis, it can be concluded that de novo protein synthesis is required in the case of actinomycin D resistant residual synthesis.

Kinetics of alpha-amylase production in a continuous culture of Bacillus licheniformis.

As found during continuous cultivation of Bacillus licheniformis on a semisynthetic medium (glucose or maltose as C source), the specific rate of alpha-amylase production is proportional to growth rate but is repressed by higher substrate concentrations. Besides glucose or maltose, peptone was also used as an alternative carbon source during cultivation. The specific rate of production of the enzyme on maltose is half that found with glucose.