Biosynthesis of cyclodextrin glucosyltransferase by the free and immobilized cells of Bacillus cereus NRC7 in batch and continuous cultures.

AIMS: The objective of this study was to enhance the production of cyclodextrin glucanotransferase (CGTase) produced by a local isolate Bacillus cereus NRC7. METHODS AND RESULTS: In batch culture, maximal CGTase activity (69·0 U ml(-1)) was reached after 24-h incubation period. In continuous production of CGTase by the free cells of B. cereus NRC7, maximal reactor productivity (11·76 KU l(-1) h(-1)), with enzyme concentration of 49·0 U ml(-1) and specific productivity of 904·6 U per g wet cells per h, was attained at dilution rate of 0·24 h(-1), over a period of 640 h. Bacillus cereus NRC7 cells were immobilized on chitosan. The immobilization conditions with respect to matrix concentration and maximal cell loading were optimized for maximal CGTase production. In repeated batch operation, the activity of the immobilized cells was stable during ten cycles and the activity remained between 51 and 55 U ml(-1). In packed-bed reactor, the immobilized cells showed maximal productivity (27·18 KU l(-1) h(-1)) with enzyme concentration of 54·63 U ml(-1) and specific productivity of 151·89 U per g wet cells per h at dilution rate of 0·5 h(-1). The half-life of the immobilized cells was higher than 20 days. CONCLUSIONS: Continuous fermentation by the immobilized cells in packed-bed reactor is an appropriate potential technique for B. cereus NRC7 CGTase production that gave maximum productivity (27·18 KU l(-1) h(-1)), which was 9·47-, 2·31-, 12·24- and 12·94-fold higher than the free cells in batch, free cells in continuous, immobilized cells in batch and repeated batch cultures, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that evaluates CGTase productivity, in different fermentation modes, in terms of specific productivity (U per gram cells per h). In continuous fermentation by immobilized cells, maximal levels of CGTase productivity are higher than the previously reported values.

Repair of experimental plaque-induced periodontal disease in dogs.

Forty mongrel dogs were used in this study for induction of periodontal disease by placing subgingival silk ligatures affecting maxillary and mandibular premolar teeth during a 12-month period. Experimental premolar teeth received monthly clinical, radiographic, and histometric/pathologic assessments. The results demonstrated significant increases in scores and values of periodontal disease parameters associated with variable degrees of alveolar bone loss. The experimental maxillary premolar teeth exhibited more severe and rapid rates of periodontal disease compared with mandibular premolar teeth. Histometric analysis showed significant reduction in free and attached gingiva of the experimental teeth. Histopathological examination of buccolingual sections from experimental premolar teeth showed the presence of rete pegs within the sulcular epithelium with acanthosis and erosive changes, widening of the periodontal ligament, and alveolar bone resorption. Various methods for periodontal repair were studied in 194 experimental premolar teeth exhibiting different degrees of periodontal disease. The treatment plan comprised non-surgical (teeth scaling, root planing, and oral hygiene) and surgical methods (closed gingival curettage, modified Widman flap, and reconstructive surgery using autogenous bone marrow graft and canine amniotic membrane). The initial non-surgical treatment resulted in a periodontal recovery rate of 37.6% and was found effective for treatment of early periodontal disease based on resolution of gingivitis and reduction of periodontal probing depths. Surgical treatment by closed gingival curettage to eliminate the diseased pocket lining resulted in a recovery rate of 48.8% and proved effective in substantially reducing deep periodontal pockets. Open root planing following flap elevation resulted in a recovery rate of 85.4% and was effective for deep and refractory periodontal pockets. Autogenous bone graft implantation combined with canine amniotic membrane as a biodegradable membrane was used in 18 premolar teeth and failed to improve advanced furcation defects in most teeth.

Stabilization of cellobiase by covalent coupling to soluble polysaccharide.

Cellobiase from Aspergillus niger was glycosylated by covalent coupling to cyanogen bromide activated dextran. The conjugated enzyme retained 62% of the original specific activity exhibited by the native cellobiase. The optimum pH as well as the pH stability of the conjugated form remain almost the same as for the native enzyme. Compared to the native enzyme, the conjugated form exhibited a higher optimal reaction temperature and energy of activation, a higher K(m) (Michaelis constant) and lower Vmax (maximal reaction rate), and improved thermal stability. The thermal deactivation of the native and conjugated cellobiase obeyed the first-order kinetics. The calculated half-life values of heat inactivation at 60, 70 and 80 degrees C was 10.7, 6.25, and 4.05 h, respectively, whereas at these temperatures the native enzyme was less stable (half-life of 3.5, 1.69, and 0.83 h, respectively). The deactivation rate constant at 80 degrees C for the conjugated cellobiase is about 7.9 x 10(-2) h-1, which is lower than that of the native enzyme (36.0 x 10(-2) h-1). The activation energy for denaturation of the native enzyme is about 10.58 kcal/mol, which is 7.25 kcal/mol lower than that of the conjugated enzyme. The effect of different surfactants and some metal ions on the activity of the conjugated cellobiase has been investigated.

Purification and properties of three cellobiases from Aspergillus niger A20.

Three cellobiases, here called cellobiase A, B, and C, from the culture filtrate of Aspergillus niger A20, were purified by precipitation with ammonium sulphate, gel filtration through Sephadex G-75, and column chromatography of DEAE-cellulose. The purified enzymes were homogeneous on polyacrylamide disk electrophoresis. The mol wt of the purified enzymes were estimated by SDS-gel electrophoresis to be 88,000, 80,000, and 71,000 for cellobiases A, B, and C, respectively. The enzymes were active at pH 4.5 and 55-60 degrees C. The pattern of their amino acid compositions showed high contents of aspartic acid, glutamic acid, threonine, serine, and glycine. The apparent K(m) values for cellobiose were 0.9, 1.63, and 1.0 mM for cellobiases A, B, and C, respectively. Calcium ions stimulated cellobiases B and C, and Co2+ and Mg2+ ions stimulated cellobiase A. The purified enzymes hydrolyzed cellobiose and aryl-beta-D-glucosides, but they had no action on sucrose, maltose, and cellulose. The three cellobiases catalyzed transglycosylase reaction, and the major product formed from cellobiose was tetramer of glucose.

Utilization of water hyacinth cellulose for production of cellobiase-rich preparation by Aspergillus niger 1.

Production of a cellobiase-rich preparation by Aspergillus niger 1 was achieved using water hyacinth cellulose as the sole carbon source in the culture medium. Production of cellobiase, carboxymethylcellulase (CMC-ase) and filter paper (FP)-cellulase was favoured by controlling the pH of the culture medium during fermentation at 5.0. Sodium citrate (0.5%), sodium phytate (0.1%), Tween-80 (0.2%, v/v) and asparagine (0.07%) had stimulating effects on the productivity of cellobiase, CMC-ase and FP-cellulase. Potassium dihydrogen phosphate doubled the yield of CMC-ase but had a slight effect on FP-cellulase and cellobiase. Wheat bran had a pronounced stimulating effect on the production of cellobiase and CMC-ase. The combined effects of these stimulators resulted in an enzyme preparation rich in cellobiase and contained 18.5, 0.29 and 2.21 U/ml of cellobiase, FP-cellulase and CMC-ase, respectively. A high cellobiase/FP-cellulase ratio of 63.8:1 was thus obtained with the fungal enzyme preparation. The cellobiase activity was maximal at pH 5.0 and showed good thermostability.

Immobilization of Aspergillus niger NRC 107 Xylanase and beta-Xylosidase, and Properties of the Immobilzed Enzymes.

Aspergillus niger NRC 107 xylanase and beta-xylosidase were immobilized on various carriers by different methods of immobilization, including physical absorption, covalent binding, ionic binding, and entrapment. The immobilized enzymes were prepared by physical adsorption on tannin-chitosan, ionic binding onto Dowex-50W, covalent binding on chitosan beads through glutaraldehyde, and entrapment in polyacrylamide had the highest activities. In most cases, the optimum pH of the immobilized enzymes were shifted to lower than those of free enzymes. The optimum reaction temperature of immobilized xylanase was shifted from 50C to 52.5-65C, whereas that of immobilized beta-xylosidase was shifted from 45C to 50-60C. The Km values of immobilized enzymes were higher than those of native enzymes. The operational stability of the immobilized enzymes was evaluated in continuous operation in packed-bead column-type reactors. The enzymes covalently bounded to chitosan showed the highest operational stability. However, the enzymes immobilized by physical absorption or by ionic binding showed a low operational stability. The enzymes entrapped in polyacrylamide exhibited lower activity, but better operational stability.

Production and properties of fibrinolytic enzyme fromStreptomyces sp. NRC 411.

Of 16Streptomyces spp. investigated for the production of extracellular fibrinolytic enzyme, one species was chosen as the most promising producer. Using shaken cultures grown for 7 days, optimal conditions for enzyme production were pH 6.0, 5% (w/v) starch as carbon source, (NH4)2SO4 and soybean flour as nitrogen sources and KH2PO4 at 1.2 g/l. Maximal activity of the crude enzyme was at pH 6.0 and 45°C. Holding the enzyme at 37°C for 2 h decreased the activity by only 10%.