Isolation, characterization and identification of cellulase (Endo-β-1,4-glucanase) producing bacteria from diverse locations

Abstract

The objective of the study was to isolate cellulase producing bacteria from natural sources and identify the bacterial isolates that produce thermostable enzyme. Bacteria were isolated from samples collected from different locations like mango leaf litter, termite gut, composting yard (municipal solid waste, compost, and soil), soil from decaying paper waste, decaying wood, and saline coastal belt soil by enrichment in broth containing 1% cellulose and subsequent culturing on plates and the purified cultures stored in slants of the solidified agar medium of the same composition as enrichment medium. Primary screening of the isolates for cellulase (Endo β-1,4-glucanase) production was done by growing them on Carboxy Methyl Cellulose (CMC) agar medium and screening using indicator dye Congo red. Secondary screening of the positive isolates showing cellulolytic Index > 4 was done by crude cellulase enzyme extraction and assay using 1% CMC in 1N citrate buffer (pH 5.0) at different pH values (4.8- 7.0) and temperatures (35- 70℃). Cellulase activity (EA) in U/mL was calculated based on the absorbance change per min. which depends on the amount of reducing sugar produced by the enzyme catalysed conversion of CMC to reducing sugar. Isolates were characterized morphologically and biochemically and six isolates (Gram -ve: T1, MSW, C1, S2 and Gram +ve: W2, P) having significantly higher EA were selected for DNA extraction. DNA was quantified and the DNA of four isolates (MSW, S2, W2, P) were selected for sequence analysis. The 16S rDNA sequencing was carried out through PCR amplification and Blast using NCBI blast similarity search tool resulted in the identification of MSW, S2, W2 as E.coli, Sulfitobacter pontiacus, Bacillus subtilis respectively and P as Bacillus cereus and Rhodococcus erythropolis strain e1. Isolates showed significantly higher cellulase activity (EA) at pH 6.2 and at varying temperatures. Bacterial isolates P and C2 showed significantly higher activity at 40℃, MSW at 60℃ and others at 50℃ (EA ranging from 0.0054 – 0.0604 U/mL). Isolates W2, P, S2 had EA ranging from 0.006 - 0.03 U/mL at 70℃. This study showed that the cellulase producers could be grown at different temperatures (40 - 70℃) on cellulose based substrate. In this study the substrate specificity indicates that the crude enzyme is an endo- β-1,4-glucanase that plays a prominent role in initiating and sustaining the hydrolytic process and randomly cleaves cellulose into glucose and olygomeric polysaccharides. Hence by selecting samples with cellulosic substance from different locations with extreme environmental conditions like high temperature, high salinity it is possible to isolate cellulolytic bacteria producing cellulase enzymes that are stable at higher processing temperatures and other harsh conditions which is of importance in industrial applications.

Keywords: Endo-β-1,4-glucanase, Cellulolytic index, Cellulase activity, Thermostable