Molecular detection and cloning of bacterial thermophilic cellulase gene isolated from hot spring of Gilgit Baltistan
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Abstract
Microbial cellulases have been found to be used in diverse industrial processes such as juice clarification. This research aimed to isolate cellulase producing thermophilic bacteria from Tatta Pani hot spring located at Gilgit Baltistan, Pakistan and to clone the cellulase gene in a better host. The collected samples were serially diluted and spread on nutrient agar medium for the isolation of bacteria. The bacterial colonies were then purified by streaking and their growth and enzyme production potential were checked. The potential cellulase gene responsible for the cellulase production were then cloned in Escherichia coli DH5α. A total of 20 cellulase producing bacteria were isolated, in which four isolates were selected for further processing based on their higher efficiency. The bacteria such as Rhodobacter thermarum, Bacillus safensis, Bacillus paramycoides and Bacillus albus were identified using 16S RNA gene sequencing. The optimum temperature and pH for Rhodobacter thermarum was 60ºC and 9, respectively. Gene responsible for cellulase production was amplified using specific primers and transformed to Escherichia coli (DH5α strain) using thermo scientific clone JET PCR cloning kit (00715987, k1232, Lithuania). Then three different types of juices (apple, pear and orange) were clarified using both the wild and recombinant cellulases and their clarification efficiency was determined. The cloned strain showed high efficiency of juice clarification in comparison to the wild strain. The isolation and cloning of these thermostable cellulase producers present promising potential for enhancing industrial processes, including biofuel production and food processing. This study contributes to the development of sustainable biotechnological solutions and benefits the scientific community by providing new insights into enzyme engineering for industrial applications.
Keywords: Applications; Cloning; Juice clarification; Microbial Cellulase; Thermophilic bacteria