Immobilization of Bacillus circulans MTCC 7906 for enhanced production of alkaline protease under batch and packed bed fermentation conditions
G Kocher, S Mishra
agar-agar entrapment, alkaline protease, bacillus circulans, packed bed, scanning electron microscopy
G Kocher, S Mishra. Immobilization of Bacillus circulans MTCC 7906 for enhanced production of alkaline protease under batch and packed bed fermentation conditions. The Internet Journal of Microbiology. 2008 Volume 7 Number 2.
Alkaline protease production by immobilized cells of
Alkaline proteases constitute an important group of industrial enzymes that are now used in a wide range of industrial processes viz; detergent, food, pharmaceutical, leather, and silk industries. Proteases account for 30% of the total worldwide enzyme production and 40% of the total worldwide enzyme sales (Sharma
Materials and Methods
Entrapment of cells in Agar-Agar and calcium alginate:
For entrapment of cells in Calcium alginate
Production of alkaline protease with immobilized cells
Batch production: The enzyme production was carried out under batch, repeated batch and packed bed fermentation conditions. The production media for immobilized cells of
The agar-agar and calcium alginate entrapped cells were used as inoculum for alkaline protease production medium (50ml in 250ml capacity erlenmeyer flasks). The flasks were incubated at 28°C for 120 h. Samples were withdrawn at regular intervals of 24 h and assayed for cell leakage and alkaline protease activity.
Production by repeated batch process: The reusability of
Packed bed production: Burettes (50ml capacity) were used as column reactors to which beads were filled upto different lengths (5-30 cm) to optimize the column length. The packed columns were filled with enzyme production medium upto 50 ml mark. The column were activated at room temperature for overnight (16 h), followed by flushing the spent production medium and fresh medium was then poured over the beads. The flow rate was regulated and spent medium was collected every hour and used as crude enzyme extracts. The spent medium was replaced with fresh medium continuously.
All the enzyme production experiments were planned statistically using Random Block Design where atleast three replications of each treatment were taken.
Extracellular Protease Activity: The protease activity of the periodic samples was determined in a reaction mixture consisting of 0.1 ml of crude enzyme, 2 ml of 0.5% azocasein (in carbonate-bicarbonate buffer, 0.1M, pH 9.5) and 0.9 ml of distilled water and incubated at 60°C for 15 minutes. The proteins were precipitated out thereafter by adding 3 ml of 5% ice-cold trichloroacetic acid (TCA) and free amino acids released by crude protease from casein hydrolysis were estimated by Lowry’s method. The protease activity was defined as Mol of tyrosine released per minute per ml of crude enzyme.
Study of cell leakage: The cell leakage from the production media were determined by viable cell plate count method. Periodic samples were serially diluted and plated on inoculum medium and incubated at 28±2°C for 48 h for determining the viable cell count.
Scanning Electron Microscopy: The
Results and Discussion
The batch production of alkaline protease by immobilized cells of
Batch production of alkaline protease
Effect of bead size: The effect of bead size was studied by approximately 150 beads each of agar agar and calcium alginate. And the data was fitted into TWO WAY ANOVA classification. The results presented in Table 1 reveal a better protease yield with low bead size of 6.25, 5.2 mm3 having statistically significant values of 16.6 and 18.4 IU having relative activity of 115.9 and 118.8% over free cells in case of agar agar and calcium alginate entrapped cells respectively. The better performance of entrapped cells over free cells has also been reported elsewhere (Beshay 2003, Adinaryana
Effect of entrapment material concentrations: The concentration of entrapment material is an important parameter and the concentration range of 2-4% studied in this study revealed 2% concentration of agar agar and 3% concentration of calcium alginate as the best for protease yield of 18.2 and 19.5 IU respectively (Table 1). Further increase in concentration of entrapment material decreased activity that may be attributed to diffusion limitation arising out of reduced pore size of beads at increased concentration of entrapment material (Elibol and Moreria ,2003; Rao
(1986) infact reported a better ethanol production with agar immobilized cells than alginate and -carrageenan.
Effect of inoculum size: The effect of inoculum size studied by varying the number of beads in production experiments showed increase in protease activities with increase in number of beads/flask at all the 5 levels (40-200 beads/flask) tested. While a significantly higher value of 19.9 IU was obtained with 200 beads/flask of agar agar, a maximum protease activity of 19.7 IU was achieved with similar number of calcium alginate beads. Thus, by batch production the protease activity was improved by 139.2 and 128.5 % over free cells in case of agar agar and calcium alginate beads respectively. Elsewhere, Beshay (2003) and Rao
Repeated batch production of alkaline protease
The reusability of beads was studied to determine their strength and performance. The results presented in Fig.1 reveal that agar agar beads behaved consistently over 9 consecutive cycles while calcium alginate beads were stable for 4 cycles only before the respective beads started disintegrating. The low stability of calcium alginate beads is attributed to the presence of potassium phosphate in the enzyme production medium that tends to dissolve the beads (Bajpai and Sharma, 2004). On the other hand, agar agar beads had no effect of this salt. Elsewher, Beshay (2003) and Elibol and Moreira (2003) reported maximum alkaline protease production in 3rd, and 4th cycle while Adinarayana
Packed bed production of alkaline protease
Earlier workers have suggested use of immobilized cells for continuous production (Beshay, 2003 and Adinaryana
A column/packed bed length of 30 cm was found to be optimum resulting in an overall increase in relative activity of 165.7 and 144.9% with agar gar and alginate entrapped cells respectively (Table 1). An optimum time of 36h and bed length of 30 cm used to standardize flow rate revealed a recovery of 10ml/h crude enzyme having 28 and 27 IU respectively with agar agar and alginate beads packed beds (Table 1). Thus, by using packed beds we were able to further enhance the protease activity by about 50% and reduce the production time from 100 to 36h. The study also revealed use of agar agar as an alternate equally effective entrapment material in place of calcium alginate the beads of which dissolve over the period of time in phosphate containing media.
Scanning Electron Microscopy (SEM) of immobilized cells
The surface structure of entrapped
The author are thankful to Dr. (Ms.) Rabinder Kaur of Nanoscience Laboratory, PAU, Ludhiana for help during Scanning Electron Microscopy (SEM) studies.