Cloning of the Endoglucanase Gene from a <i>Bacillus amyloliquefaciens</i> PSM 3.1 in <i>Escherichia coli</i> Revealed Catalytic Triad Residues Thr-His-Glu

Abstract

Problem statement: An Indonesian marine bacterial isolate, Bacillus amyloliquefaciens PSM 3.1 was isolated for hydrolyzing cellulose. A 1500-bp nucleotide fragment was amplified from the chromosomal DNA by the use of primers directed against the conserved sequence of Bacilli endoglucanase genes obtained from GenBank. Approach: The fragment was cloned and expressed in Escherichia coli. Results: The endoglucanase gene (eglII gene) had an open reading frame of 1500 nucleotides encoding a protein of 499 amino acids. The EglII protein belonged to Glycosyl Hydrolase family 5 (GH5) with a Cellulose Binding Module 3 (CBM 3). The structure model of the EglII protein revealed that the catalytic residues seemed to be Glu169 (as proton donor) and Glu257 (as nucleophile) and the catalytic triad residues were Thr256, His229 and Glu169. The EglII endoglucanase exhibited an optimum pH of 6.0 and temperature of 50°C and the enzyme tolerated to high salt concentration. Conclusion/Recommendations: This EglII endoglucanase is a promising candidate for many applications in biomass degradation.

Application of Statistically Based Experimental Designs to Optimize Cellulase Production and Identification of Gene

“…Amplification of cellulase gene among several sets of primers tried during the study, the best amplification was observed by the primer set CelF This CelF: 5′-ATGAAACGGTCAATCTC-3′ and CelR 5′-CTAATTTGGTTCTGTTCCC-3′ set of primer was tried following the sequence of cellulase gene from B. amyloliquefaciens PSM 3.1 [32]. Among rest of sets of primers, sufficient amplification of cellulase gene could not be achieved as some set of primers showed non specific amplification of gene while a few others amplified DNA fragments of very minute base pair size (~ 300).…”

“…The PCR products obtained using B. amyloliquefaciens PSM 3.1 chromosomal DNA as template were about 1500 bp and other non-specific bands were obtained [32]. Amplification of cellulase gene was reported in which sequence specific primers for cellulase genes were used and significant amplification was achieved from the cDNA and the corresponding bands were found in the gel [33].…”

“…Purity of DNA was judged on the basis of optical density ratio at 260:280 nm [32]. The DNA having ratio between 1.8 and 2.0 was considered to be of good purity.…”

“…For the confirmation of cellulase gene the gene fragment was amplified by PCR from genomic DNA using gene specific set of primers: celF (5′-ATGAAACGGTCAATCTC-3′) and celR (5′ - CTAATTTGGTTCTGTTCCC-3′) [32]. PCR was carried out in a final reaction volume of 25 µl in 200 µL capacity thin wall PCR tube in Eppendorf Thermal Cycler.…”

Effect of CMC and MCC as Sole Carbon Sources on Cellulase Activity and eglS Gene Expression in Three Bacillus subtilis Strains Isolated from Corn Stover

“…In particular, several species of the genera Bacillus produce cellulases, including strains of B. cereus, B. subtilis, B. licheniformis, B. amyloliquefaciens, and alkaliphilic Bacillus (Horikoshi 1997;Bischoff et al 2006;Nurachman et al 2010;Yan et al 2011;Asha and Sakthivel 2014). These enzymes have shown properties of industrial interest such as halotolerance and stability in the presence of surfactants and metal ions (Zhu et al 2011).…”