Abstract
Pullulanase (EC 3.2.1.41) is an important debranching enzyme that plays a critical role in maximizing the abundant energy present in branched polysaccharides. Its unique ability to efficiently degrade branched polysaccharides makes it crucial in industries like biofuels, food, and pharmaceuticals. Therefore, discovering microbes that produce pullulanase and thrive in harsh industrial conditions holds significant potential for optimizing large-scale bioprocessing. This unique property has made pullulanase an important enzyme in the industry. Thus, the search for microbes that have the pullulanase production properties and capacity to withstand harsh industrial conditions will be of high industrial relevance. Therefore, this study aimed to amplify, sequence, and molecularly characterize the pullulanase gene encoding extracellular pullulanase in Bacillus licheniformis strain FAO.CP7 (Accession No: MN150530.1.) which was obtained from cocoa pods using several bioinformatics tools. The amplified PulA gene had a nucleotide sequence of 2247 base pairs encoding a full-length open reading frame (ORF) pullulanase protein of 748 amino-acids residues with molecular weight 82.39 kDa and theoretical isoelectric point of 6.47, respectively. The deduced pullulanase protein had an aliphatic index of 77.66. Using BLASTp, the deduced amino acid sequence of the pullulanase gene showed 85% homologies with those from B. licheniformis strains. Multiple sequence alignment of PulA protein sequence showed that it contains YNWGYNP motif which is also found in all type I pullulanase protein sequences analysed. The restriction mapping of the gene showed that it can be digested with several restriction enzymes. Further analysis revealed that the deduced protein had a hydrophobicity score of - 0.37 without a transmembrane helix. Overall, this study revealed the PulA gene of B. licheniformis strain FAO.CP7 obtained from cocoa pods and its deduced protein show significant potential for enhancing starch bioprocessing. With further optimization, it could offer substantial benefits to starch-based biotechnological industries.