Production of alkaline protease with immobilized cells of bacillus subtilis PE-11 in various matrices by entrapment technique

The purpose of this investigation was to study the effect ofBacillus subtilis PE-11 cells immobilized in various matrices, such as calcium alginate, k-Carrageenan, ployacrylamide, agar-agar, and gelatin, for the production of alkaline protease. Calcium alginate was found to be an effective and suitable matrix for higher alkaline protease productivity compared to the other matrices studied. All the matrices were selected for repeated batch fermentation. The average specific volumetric productivity with calcium alginate was 15.11 U/mL/hour, which was 79.03% higher production over the conventional free-cell fermentation. Similarly, the specific volumetric productivity by repeated batch fermentation was 13.68 U/mL/hour with k-Carrageenan, 12.44 U/mL/hour with agar-agar, 11.71 U/mL/hour with polyacrylamide, and 10.32 U/mL/hour with gelatin. In the repeated batch fermentations of the shake flasks, an optimum level of enzyme was maintained for 9 days using calcium alginate immobilized cells. From the results, it is concluded that the immobilized cells ofB subtilis PE-11 in calcium alginate are more efficient for the production of alkaline protease with repeated batch fermentation. The alginate immobilized cells ofB subtilis PE-11 can be proposed as an effective biocatalyst for repeated usage for maximum production of alkaline protease. Published: October 21, 2005     

Investigations on alkaline protease production with B. subtilis PE-11 immobilized in calcium alginate gel beads

Bacillus subtilis PE-11 cells were immobilized in calcium alginate and used for the production of alkaline protease. The influence of alginate concentration, different cations, concentration of cation, curing time, bead diameter and nutrient strength on alkaline protease production and stability of biocatalyst were investigated. Repeated batch fermentations of immobilized cells in shake flasks were carried out with the optimized parameters such as; 3% alginate, 0.25 M calcium chloride with 1 h curing time, 3.24 mm bead diameter and 0.75% glucose and 0.75% peptone as nutrients. The results indicated that, a good level of enzyme was maintained for a period of about 9 days. The immobilized cells of B. subtilis PE-11 in calcium alginate are more efficient for the production of alkaline protease with repeated batch fermentation.     

Production of xylanase by immobilized Trichoderma reesei SAF3 in Ca-alginate beads

In the present study, the optimum conditions for the production of xylanase by immobilized spores of Trichoderma reesei SAF3 in calcium alginate beads were determined. The operational stability of the beads during xylanase production under semi-continuous fermentation was also studied. The influence of alginate concentration (1, 2, 3, and 4%) and initial cell loading (100, 200, 300, 400, and 500 beads per flask) on xylanase production was considered. The production of xylanase was found to increase significantly with increasing concentration of alginate and reached a maximum yield of 3.12 ± 0.18 U ml⁻¹ at 2% (w/v). The immobilized cells produced xylanase consistently up to 10 cycles and reached a maximum level at the forth cycle (3.36 ± 0.2 U ml⁻¹).     

Isolation and screening of Streptomyces sp. Al-Dhabi-49 from the environment of Saudi Arabia with concomitant production of lipase and protease in submerged fermentation

In this study, Streptomyces sp. Al-Dhabi-49 was isolated from the soil sample of Saudi Arabian environment for the simultaneous production of lipase and protease in submerged fermentation. The process parameters were optimized to enhance enzymes production. The production of protease and lipase was found to be maximum after 5 days of incubation (139.2 ± 2.1 U/ml, 253 ± 4.4 U/ml). Proteolytic enzyme increases with the increase in pH up to 9.0 (147.2 ± 3.6 U/ml) and enzyme production depleted significantly at higher pH values. In the case of lipase, production was maximum in the culture medium containing pH 8.0 (166 ± 1.3 U/ml). The maximum production of protease was observed at 40 °C (174 ± 12.1 U/ml) by Streptomyces sp. Lipase activity was found to be optimum at the range of temperatures (30–50 °C) and maximum production was achieved at 35 °C (168 ± 7.8 U/ml). Among the evaluated carbon sources, maltose significantly influenced on protease production (218 ± 12.8 U/ml). Lipase production was maximum when Streptomyces sp. was cultured in the presence of glucose (162 ± 10.8U/ml). Among various concentrations of peptone, 1.0% (w/v) significantly enhanced protease production. The lipase production was very high in the culture medium containing malt extract as nitrogen source (86 ± 10.2 U/ml). Protease production was maximum in the presence of Ca²⁺ as ionic source (212 ± 3.8 U/ml) and lipase production was enhanced by the addition of Mg²⁺ with the fermentation medium (163.7 ± 6.2 U/ml).     

Production of thermostable and neutral glucoamylase using immobilized <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis>

Thermomucor indicae-seudaticae was immobilized in alginate, κ-carrageenan, agarose, agar, polyacrylamide and loofah (Luffa cylindrica) sponge (as such or coated with alginate/starch/Emerson YpSs agar), and used for the production of glucoamylase in submerged fermentation. The mycelium developed from alginate-immobilized sporangiospores secreted higher glucoamylase titres (22.7 U ml⁻¹) than those immobilized in other gel matrices and the freely growing mycelial pellets (18.5 U ml⁻¹). Loofah network provided a good support for mycelial growth, but the enzyme production was lower than that attained with alginate beads. Glucoamylase production increased with inoculum density and the optimum levels were achieved when 40 calcium alginate beads (∼5 × 10⁶ immobilized spores) were used to inoculate 50 ml production medium. The alginate bead inoculum displayed high storage stability at 4°C and produced comparable enzyme titres up to 120 days. The glucoamylase production by hyphae emerged from the immobilized sporangiospores was almost stable over eight batches of repeated fermentation. Scanning electron micrographs of alginate beads, after batch fermentation, revealed extensive mycelial growth inside and around the beads.     

Protease production by thermo-alkaliphilic novel gut isolate Kitasatospora cheerisanensis GAP 12.4 from Gryllotalpa africana

In course of searching for proteolytic microbes from the gut of Gryllotalpa africana, a potent isolate GAP 12.4 was screened and identified as Kitasatospora cheerisanensis having protease activity 46.8?±?1.52?U/ml. Optimum conditions for the protease production (605.3?±?9.7?U/ml) were 7-d cultivation, 5% inoculum, pH 9.5, 55?°C, 150?rpm, and supplementation with 0.8% glucose and 0.6% ammonium sulfate. Surfactants such as SDS, EDTA, Tween 80 and Triton X-100 showed positive effect on enzyme production. Addition of biotin (50?μg/ml) promotes enzyme production maximally (674.15?±?4.13?U/ml). Further enhancement on addition of casein hydrolysate and molasses to the production medium was 709.20?±?7.53?U/ml and 744.26?±?9.71?U/ml, respectively. The isolate was also able to utilize agro-industries waste, green gram husk in solid-state fermentation for enzyme production (1675.02?±?21.58?U/ml). This thermo-alkaliphilic isolate may be a potent candidate for low cost protease production through management of agro-residues. It is the first report of protease production by a member of actinobacteria under the Kitasatospora genus.     

Thermus 2S from Thai hot springs: isolation and immobilization

Three strains of thermophilic bacteria producing extracellular protease have been isolated from hot springs in Chiang Mai, Thailand. The bacterium producing the highest amount of protease has been selected and identified as belonging to the genusThermus, and is tentatively calledThermus 2S. The isolate is a Gram-negative, rod shaped bacterium. It exhibited maximum growth around 60°C at pH 7. Entrapment of the microbial cells in calcium alginate maintained the cell viability. Protease production from immobilized cells using 2 g wet cells per 10 ml 3% (w/v) sodium alginate was higher than that from a free-cell system using 2% inoculum.     

Physical Factors and Reuse Affect the Production of Cholesterol Oxidase in Reactors Containing Calcium Alginate-Immobilized Rhodococcus equi No. 23

Summary Some physical factors including initial pH of medium, cultivation temperature and shaking speed as well as reuse affecting the production of cholesterol oxidase (CholOx) in reactors containing calcium alginate-immobilized cells of Rhodococcus equi No. 23 were investigated. Results revealed that the free cells showed the maximum CholOx in the culture with an initial pH of 5.0, while culture inoculated with immobilized cells exhibited a broad pH range, 6.0–9.0, for maximum CholOx production. The immobilized and free cells produced the maximum CholOx in the culture incubated at 30 and 25°C, respectively. The CholOx production decreased upon increasing the cultivation temperature. Increasing CholOx activity was also noted for both immobilized and free cells of R. equi No. 23 in the culture with increasing shaking speed. Under the optimal culture conditions, that were established, a higher maximum CholOx production of 0.94 unit/ml was found for immobilized R. equi No. 23 compared to that of 0.84 unit/ml for free cells after 48 h of cultivation. Furthermore, no gel leakage was noted after re-use of the calcium alginate-immobilized R. equi No. 23 for seven consecutive 48 h batch culture. The CholOx production in the seventh cycle was about 60.4% of that obtained in the first cycle.     

Protease production by free and immobilized cells of the cold-adapted yeast Cryptococcus victoriae CA-8

The present study was performed to produce the protease using free and immobilized cells of locally isolated cold-adapted psychrotolerant yeast Cryptococcus victoriae CA-8. Cell immobilization was performed using sodium alginate as entrapping agent. The best conditions for enzyme production by both free and immobilized cells of the yeast were temperature of 15°C and initial pH of 8.0. The optimal incubation times were 72 and 96 h for immobilized and free cells, respectively. Immobilized cells were reused in 3 successive reaction cycles without any loss in the maximum protease activity. Little decreases in the protease activity were observed in 4 and 5 cycles. Under the optimized conditions, the maximum enzyme activities were determined as 12.1 and 13.5 U/mL for free and immobilized cells, respectively. This is a first attempt on cold-active alkaline protease production by free and/or immobilized cells of yeasts. Besides, the protease activity of the yeast C. victoriae CA-8 was investigated for the first time in the present study.     

Cell immobilization technique for the enhanced production of alpha-galactosidase by Streptomyces griseoloalbus

Streptomyces griseoloalbus was immobilized in calcium alginate gel and the optimal immobilization parameters (concentrations of sodium alginate and calcium chloride, initial biomass and curing time) for the enhanced production of alpha-galactosidase were determined. The immobilization was most effective with 3% sodium alginate and 0.1M calcium chloride. The optimal initial biomass for immobilization was approximately 2.2g (wet wt.). The alginate-entrapped cells were advantageous because there was a twofold increase in the enzyme yield (55 U/ml) compared to the highest yield obtained with free cells (23.6 U/ml). Moreover, with immobilized cells the maximum yield was reached after 72 h of incubation in batch fermentation under optimal conditions, whereas in the case of free cells the maximum enzyme yield was obtained only after 96 h of incubation. The alginate beads had good stability and also retained 75% ability of enzyme production even after eight cycles of repeated batch fermentation. It is significant that this is the first report on whole-cell immobilization for alpha-galactosidase production.     

Ethanol fermentation of mahula (<Emphasis Type="Italic">Madhuca latifolia</Emphasis>) flowers using free and immobilized bacteria <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> MTCC 92

Mahula (Madhuca latifolia L.) is a deciduous tree commonly found in the tropical rain forests of Asian and Australian continent. Corolla, the edible part of its flowers, is rich in fermentable sugar (37 ± 0.23%; on dry weight basis). Batch fermentation of mahula flowers was carried out using Zymomonas mobilis MTCC 92 free cells and cells immobilized in calcium alginate matrix. The ethanol productions were 122.9 ± 0.972 and 134.6 ± 0.104 g/kg flowers on dry weight basis using free and immobilized cells, respectively, after 96 h of fermentation, which showed that cells entrapped in calcium alginate matrix yielded 8.7% more ethanol than free cells. Further, the immobilized cells were physiologically active up to three more cycles of fermentation producing 132.7 ± 0.095, 130.5 ± 0.09 and 128.7 ± 0.056 g ethanol per kg flower in first, second and third cycle, respectively.     

Characterization of a multifunctional feather-degrading <Emphasis Type="Italic">Bacillus subtilis</Emphasis> isolated from forest soil

In this study, we isolated and characterized a novel feather-degrading bacterium that shows keratinolytic, antifungal and plant growth-promoting activities. A bacterium S8 was isolated from forest soil and confirmed to belong to Bacillus subtilis by BIOLOG system and 16S rRNA gene analysis. The improved culture conditions for the production of keratinolytic protease were 0.1% (w/v) sorbitol, 0.3% (w/v) KNO₃, 0.1% (w/v) K₂HPO₄, 0.06% (w/v) KH₂PO₄ and 0.04% (w/v) MgCl₂·6H₂O (pH 8.0 and 30°C), respectively. In the improved medium containing 0.1% (w/v) feather, keratinolytic protease production was around 53.3 ± 0.3 U/ml at 4 day; this value was 10-fold higher than the yield in the basal feather medium (5.3 ± 0.1 U/ml). After cultivation for 5 days in the improved medium, intact feather was completely degraded. Feather degradation resulted in free –SH group, soluble protein and amino acids production. The concentration of free –SH group in the culture medium was 15.5 ± 0.2 μM at 4 days. Nineteen amino acids including all essential amino acids were produced in the culture medium; the concentration of total amino acid produced was 3360.4 μM. Proline (2809.9 μM), histidine (371.3 μM) and phenylalanine (172.0 μM) were the major amino acids released in the culture medium. B. subtilis S8 showed the properties related to plant growth promotion: hydrolytic enzymes, ammonification, indoleacetic acid (IAA), phosphate solubilization, and broad-spectrum antimicrobial activity. Interestingly, the strain S8 grown in the improved medium produced IAA and antifungal activity, indicating simultaneous production of keratinolytic and antifungal activities and IAA by B. subtilis S8. These results suggest that B. subtilis S8 could be not only used to improve the nutritional value of feather wastes but also is useful in situ biodegradation of feather wastes. Furthermore, it could also be a potential biofertilizer or biocontrol agent applicable to crop plant soil.     

Production of pullulanase by free and immobilized cells of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> NRC22 in batch and continuous cultures

The production of extracellular pullulanase by Bacillus licheniformis NRC22 was investigated using different fermentation modes. In batch culture maximal enzyme activity of 18 U/ml was obtained after 24 h of growth. In continuous fermentation by the free cells, maximal reactor productivity (4.15 KU/l/h) with enzyme concentration of 14.8 U/ml and specific productivity of 334.9 U/g wet cells/h was attained at a dilution rate of 0.28/h, over a period of 25 days. B. licheniformis NRC22 cells were immobilized on Ca-alginate. The immobilization conditions with respect to matrix concentration and cell load was optimized for maximal enzyme production. In repeated batch operation, the activity of the immobilized cells was stable during the 10 cycles and the activity remained between 9.8 and 7.7 U/ml. Continuous production of pullulanase by the immobilized cells was investigated in a packed–bed reactor. Maximal reactor productivity (7.0 KU/h) with enzyme concentration of 16.8 U/ml and specific productivity of 131.64 U/g wet cells/h was attained at dilution rate of 0.42/h. The enzyme activity in the effluent started to decline gradually to the level of 8.7 U/ml after 25 days of the operation.     

Evaluation of various parameters of calcium-alginate immobilization method for enhanced alkaline protease production by Bacillus licheniformis NCIM-2042 using statistical methods

Calcium-alginate immobilization method for the production of alkaline protease by Bacillus licheniformis NCIM-2042 was optimized statistically. Four variables, such as sodium-alginate concentration, calcium chloride concentration, inoculum size and agitation speed were optimized by 2(4) full factorial central composite design and subsequent analysis and model validation by a second-order regression equation. Eleven carbon, 11 organic nitrogen and seven inorganic nitrogen sources were screened by two-level Plackett-Burman design for maximum alkaline protease production by using optimized immobilized conditions. The levels of four variables, such as Na-alginate 2.78%; CaCl(2), 2.15%; inoculum size, 8.10% and agitation, 139 rpm were found to be optimum for maximal production of protease. Glucose, soybean meal and ammonium sulfate were resulted in maximum protease production at 644 U/ml, 720 U/ml, and 806 U/ml when screened for carbon, organic nitrogen and inorganic nitrogen sources, respectively, using optimized immobilization conditions. Repeated fed batch mode of operation, using optimized immobilized conditions, resulted in continuous operation for 12 cycles without disintegration of beads. Cross-sectional scanning electron microscope images have shown the growth pattern of B. licheniformis in Ca-alginate immobilized beads.     

Potential application of immobilized streptokinase extracted from Streptococcus equinus VIT_VB2

Streptokinase purified from Streptococcus equinus VIT_VB2 isolated from bovine milk sample was immobilized in various solid supports namely entrapment in agarose gel, calcium alginate beads and gelatin gel by cross-linking with formaldehyde. Immobilization of streptokinase in calcium alginate beads showed maximum efficiency (81.8?±?1.06%) when compared with entrapment with agarose gel (55.6?±?2.17%) and cross-linked gelatin formaldehyde gel (71.0?±?1.54%). The purified SK activity was expressed maximum in calcium alginate (1%) and gelatin gel (0.25%) with 1292.68?±?1.33 and 1121.9?±?1.2?U?mL^?1, respectively. Similarly, SK entrapped in gelatin gel and calcium alginate showed maximum in vitro blood clot lysis activity with 77.67?±?2.64% and 76.16?±?2.72%, respectively. The immobilized SK in gelatin gel showed complete clot lysis within 15?min; hence, this application of the study could be used in the treatment of superficial thrombophlebitis, phlebitis, and venous thrombosis. These beads were used for three repeated cycles to check the conversion of substrates into their products, and we concluded that SK can be immobilized in the suitable matrices. Therefore, this helps in the drug-delivery strategies in highly efficient way, moreover, economically competent process in the pharmaceutics.     

Thermostable amylase production by immobilized thermophilic Bacillus sp.

Agar, agarose and alginate immobilized cells of thermophilic Bacillus sp. WN11 produced 7.0, 6.2, and 10.5 U/ml of thermostable amylase in shake flasks, respectively. Alginate entrapped cells released high level of amylase (10.5 U/ml) than freely suspended cells (8.0 U/ml). Amylase production was stable in five successive batches with productivity of 9-12 U/ml for alginate, 5.5-8.8 U/ml for agar, and 4.8-8.0 for agarose immobilized cells.     

固定化芽孢菌产碱性蛋白酶的研究

本文报导了用海藻酸钙固定地衣芽孢杆菌(Bacilluslicheniformis),发酵生产碱性蛋白酶的研究。固定化细胞颗粒在低浓度的玉米粉、黄豆饼粉为原料的培养基中发酵24h,酶活高达1724u/ml,充分利用并节省了原材料,缩短了发酵周期。发酵液菌浓度的测定结果表明,固定化细胞凝胶粒细菌的渗漏程度较低,有利于提取工艺的简化。     

Immobilization of Bacillus amyloliquefaciens MBL27 cells for enhanced antimicrobial protein production using calcium alginate beads

Cell immobilization is one of the common techniques for increasing the overall cell concentration and productivity. Bacillus amyloliquefaciens MBL27 cells were immobilized in calcium alginate beads and it is a promising method for repeated AMP (antimicrobial protein) production. The present study aimed at determining the optimal conditions for immobilization of B. amyloliquefaciens MBL27 cells in calcium alginate beads and the operational stability for enhanced production of the AMP. AMP production with free and immobilized cells was also done. In batch fermentation, maximum AMP production (7300 AU (arbitrary units)/ml against Staphylococcus aureus) was obtained with immobilized cells in shake flasks under optimized parameters such as 3% (w/v) sodium alginate, 136?mM CaCl2 with 350 alginate beads/flask of 2.7-3.0?mm diameter. In repeated cultivation, the highest activity was obtained after the second cycle of use and approx. 94% production was noted up to the fifth cycle. The immobilized cells of B. amyloliquefaciens MBL27 in alginate beads are more efficient for the production of AMP and had good stability. The potential application of AMP as a wound healant and the need for development of economical methods for improved production make whole cell immobilization an excellent alternative method for enhanced AMP production.     

Cellulase production by free and immobilized <Emphasis Type="Italic">Aspergillus terreus</Emphasis>

Aspergillus terreus, isolated from rotting bagasse, showed comparable cellulolytic activities when grown either in the free or immobilized states with cellulose as the sole carbon source. The cultural and nutritional requirements for maximum cellulase production by the organism either in the free or immobilized states were similar, except an increase in the temperature optimum from 30 to 40°C, occurred upon immobilization. In the free state, the maximum filter paper hydrolase, carboxymethylcellulase and β-glucosidase activities produced were 2.1, 13.6, and 3.2 U/ml, respectively, while in the immobilized state, the levels were 1.8, 12.0, and 2.4 U/ml. Production of cellulolytic enzymes by immobilized cells was influenced by the surface area of the support material. In addition, cells in the immobilized state sustained enzyme production for a much longer period with a 4.5-fold increase in productivity during repeated batch when compared to free cells.     

Semicontinuous cultivation of immobilized Claviceps purpurea

Summary Mycelia of Claviceps purpurea CBS 164.59 were immobilized in 2%, 4%, and 8% calcium alginate. Alkaloid production by free cells declined after 60 days, while immobilized cells retained their activity for 200 days. The cumulative alkaloid production for all fermentation cycles using 8% calcium alginate immobilized mycelia was 25 times higher than that from free cells. The best yields of the ergopeptide ergometrine were reached with 4% gel immobilized mycelia, while higher gel concentrations caused a shift in the alkaloid biosynthesis towards high clavine alkaloid production.Beginning with the third cycle of reincubation the immobilized mycelia showed a marked tendency to fragmentize into vacuolated arthrosporoid-like structures and produced violet-black pigments so that the beads recalled sclerotial structures of parasitically living Claviceps.Dedicated to Prof. Dr. K. Esser to his 60th birthday