Abstract
A moderately thermotolerant bacterial strain was isolated from the hot spring of Tatta Pani (AJ and K) Pakistan and was designated as Bacillus subtilis strain GQ 301542 after biochemical, morphological and 16S rDNA sequence analysis. This strain and its catabolite repression resistant mutant CRM197 were utilized for the study of different production kinetic parameters of both endoglucanase and cellobiohydrolase. Time course study on one monomeric (glucose), one dimeric (maltose) and two polymeric substrates (α-cellulose and wheat straw) was carried out at different time intervals (4 - 28 h, after each 4 h) for determining the maximum enzyme productivity on a particular substrate. Maximum rate of endoglucanase production by the mutant (53.1 IU/L/h) was significantly (P = 0.0007) higher than that (23.7 IU/L/h of the parental organism following their growth on glucose in Dubos salts medium while the optimum product yields (Yp/s) was calculated as 69.0 IU/g S (parent) and 82.3 IU/g S (mutant) for cellobiohydrolase production. Deoxy-D-glucose resistant mutant was significantly (p = 0.03 to 0.0007) improved over its parental strain with respect to some substrate consumption and all product formation parameters and can easily degrade cellulosic biomass for production of fermentable carbohydrates. Key words: Cellobiohydrolase, endoglucanase, thermotolerant, Bacillus subtilis.
Highlights
Cellulases are defined by their ability to cut the -1,4 glycosidic bonds
A thermotolerant cellulose degrading strain was isolated from leaves and mud samples from hot spring of Tatta Pani, Azad Jammu and Kashmir (AJ and K), Pakistan by enrichment technique using Dubos salts medium supplemented with -cellulose and after incubation on a vibratory shaker at 50°C for 72 h
The strain was further confirmed by 16S rRNA analysis by using universal primers P1 and P6 (Chun and Bae, 2000) which amplified the desired gene when DNA sample of the TP- strain was used as a template in a polymerase chain reaction (PCR) reaction, for which following parameters were adopted: an initial denaturation at 95°C (5 min), followed by 35 cycles of 95°C for 1 min, 50°C (1 min), 72°C (1 min), with a final extension at 72 °C for 10 min
Summary
Cellulases are defined by their ability to cut the -1,4 glycosidic bonds. The complete enzyme hydrolysis of cellulosic materials needs different types of cellulases: endoglucanase (1,4- -D-glucan-glucanohydrolase; EC 3.2.1.4), exocellobiohydrolase Majority of studies on cellulase production have focused on fungi, with relatively lesser emphasis on bacterial sources (Bhat, 2000; Camassola et al, 2004; Kotchoni et al, 2003). Bacteria, due to their high diversity, faster growth and capability to produce highly thermostable enzymes, are ideal for use in industries as highly potent and robust sources of industrially important enzymes (Bhat, 2000; Camassola et al, 2004; Haki and Rakshit, 2003; Kotchonios et al, 2003; Vielle and Zeikus, 2001). Responses of variation of process parameters were measured by observing their influence on different growth kinetic parameters with relation to substrate utilization and product formation by employing a moderately thermophilic Bacillus strain on available and low cost substrate compared with and slowly metabolizable substrates, respectively
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