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Three thermotolerant Bacillus isolates from the Philippines - identified as Bacillus subtilis (1280), Bacillus velezensis (1573), and Bacillus amyloliquefaciens (10105) - exhibited cellulase activity in carboxymethylcellulose (CMC) agar plate. Moreover, the expected size (approximately 1,500 bp) putative endoglucanase gene was amplified from all three local isolates. The endoglucanase gene amplified from a Bacillus licheniformis isolate (1320) was also used in the gene sequence analysis. A colorimetric dinitrosalicylic (DNS) acid assay revealed that the total endoglucanase activity of B. velezensis 1573 at 0.168 µM reducing sugar/min is significantly higher than the endoglucanase activity of the B. subtilis 1280 at 0.0673 µM rs/min and B. amyloliquefaciens 10105 at 0.0579 µM rs/min. Endoglucanase gene sequence analysis showed the highest pairwise identity between the endoglucanase gene of B. velezensis 1573 and B. amyloliquefaciens 10105 at 97.4%. The endoglucanase gene of B. licheniformis 1320 has the lowest pairwise identity at 74.6%, 76.2%, and 75.4% with B. amyloliquefaciens 10105, B. subtilis 1280, and B. velezensis 1573, respectively. Predicted amino acid sequences of the endoglucanases revealed variations at the signal peptide, catalytic core (CC) domain, linker, and carbohydrate-binding module (CBM). The endoglucanase of B. subtilis 1280 has a 506D insertion at the CBM domain. None of these variations, however, are located in regions implicated in catalytic reaction and substrate binding. Although the predicted protein structures using MODELLER revealed endoglucanases with good fit and high homology, variations in amino acid sequences may have resulted in the differences in enzyme activity and stability. The study generated gene and protein sequences as well as amplified genes that can be used as a starting template for enzyme improvement by rational design and site-specific mutagenesis or directed evolution using DNA shuffling. [ABSTRACT FROM AUTHOR]

Culture-independent molecular-based approaches can be used to identify genes of interest from environmental sources that have desirable properties such as thermo activity. For this study, a putative thermo stable endoglucanase gene was identified from a mixed culture resulting from the inoculation of Brock-CMcellulose (1%) broth with mudspring water from Mt. Makiling, Laguna, Philippines that had been incubated at 90 °C. Genomic DNA was extracted from the cellulose-enriched mixed culture and endo1949 forward and reverse primers were used to amplify the endoglucanase gene, which was cloned into pCR-script plasmid vector. Blastn alignment of the sequenced insert revealed 99.69% similarity to the glycosyl hydrolase, sso1354 (CelA1; Q97YG7) from Saccharolobus solfataricus. The endoglucanase gene (GenBank accession number MK984682) was determined to be 1,021 nucleotide bases in length, corresponding to 333 amino acids with a molecular mass of ~ 37 kDa. The endoglucanase gene was inserted into a pET21 vector and transformed in E. coli BL21 for expression. Partially purified recombinant Mt. Makiling endoglucanase (MM-Engl) showed a specific activity of 187.61 U/mg and demonstrated heat stability up to 80 °C. The thermo-acid stable endoglucanase can be used in a supplementary hydrolysis step to further hydrolyze the lignocellulosic materials that were previously treated under high temperature-dilute acid conditions, thereby enhancing the release of more glucose sugars for bioethanol production.