Fig 1.
(A) Cellulase enzyme activity (unit-ml-1) of culture extract of Bacillus subtilis TD11 incubated for different durations using 1% carboxymethyl cellulose (CMC) as a substrate at pH 7 and temperature 50°C (B) Gelatinase enzyme activity (unit-ml-1) of culture extract of B. subtilis TD11 incubated for different durations using 0.5% gelatine as a substrate at pH 8 and temperature 37°C (C) Pectinase enzyme activity (unit-ml-1) of culture extract of B. subtilis TD11 incubated for different durations using 1% pectin as a substrate at pH 5 and temperature 45°C (D) Protease enzyme activity (unit-ml-1) of culture extract of B. subtilis TD11 incubated for different durations using skimmed milk as substrate at room temperature (E) Chitinase enzyme activity (unit-ml-1) of culture extract of B. subtilis TD11 incubated for different durations using 0.5% colloidal chitin as a substrate at pH 5.0 and temperature 37°C. The experiments were conducted in triplicate and the data were analysed using one-way ANOVA and the means were compared with the help of Fisher least significant difference (LSD) test. The different small letters on each data point for each enzyme activity represent significant differences.
Table 1.
The stepwise purification and characteristics of cellulase enzyme produced by Bacillus subtilis TD11.
Fig 2.
(A) Elution profile of DEAE-cellulose ion exchange chromatography of cellulase produced by Bacillus subtilis strain TD11 using linear gradient concentrations of NaCl ranged between 0.1–0.5 mol at a flow rate was 20 ml.h-1 and (B) elution profile of gel filtration on Sephadex G-100 of cellulase produced by B. subtilis strain TD11 with 0.1 mol potassium phosphate buffer (pH 8.0) at a flow rate of 20 ml.h-1.
Fig 3.
SDS-PAGE analysis of cellulase enzyme purified from Bacillus subtilis TD11.
Lane M Shows Page Ruler Plus Pertained Protein Ladder (10–250 kDa). Lane 1 shows crude enzyme, Lane 2 shows fraction after DEAE-Cellulose ion exchange chromatography &Lane 3–14 shows purified fractions of cellulase 45 kDa after gel filtration (Sephadex G-100).
Fig 4.
(A) Effect of pH on the activity of cellulase purified from Bacillus subtilis TD11 using carboxymethyl cellulose as substrate at various pH values ranging from 3.0–7.0 at 50°C (B) The pH stability of cellulase purified from B. subtilisTD11, the enzyme was incubated at 50°C for 1 h with 1.0% (w/v) CMC at pH 3.0 to 11.0 (C) Thermal activity of cellulase purified from B. subtilis TD11 at 20 to 90°C at pH 7 after 20hrs of incubation while stability of cellulase at 30 to 80°C at pH 7 after 1 hr of incubation and (D) Relative thermal activity of cellulase purified from B. subtilis TD11 at 30 to 80°C at pH 7 after 20hrs of incubation. The assays were performed in triplicates and the results were expressed as mean ± SEM.
Table 2.
Substrate specificity of the cellulase purified from Bacillus subtilis TD11 using 1% (w/v) of the substrates.
Fig 5.
(A) Production of chitinase by Bacillus subtilis TD11 at different intervals of incubation (days after incubation) at pH 7 and 30°C, (B) Effect of temperature on chitinase production at pH 7 after 24 hr of incubation, (C) Effect of initial pH of the fermentation media on chitinase production after 24 hr of incubation, (D) Effect of different temperature on chitinase activity after 20 hr of incubation at pH 7 (E) SDS-PAGE analysis of Chitinase Enzyme. Lane M Show Bio basic wide range molecular weight Marker (Catalog# BSM 0661), Lane 1 shows crude enzymes, Lane 2 shows fraction after precipitation by 80% SAS, Lane 3 Shows enzyme column purified fraction and Lane 4 & 5 shows the purified fraction of Chitinase approximately 55 kDa after gel filtration chromatography (Sephadex G-100). The assays were performed in triplicates and the results were expressed as mean ± SEM.
Table 3.
The antifungal activity of purified chitinase produced by Bacillus subtilis TD11 using well diffusion assays against the supernatant containing metabolites of different plant pathogenic fungi including Fusarium oxysporum, Rhizoctonia solani, Colletotrichum Sp. and Aspergillus Sp.
Optical density was recorded at 405 nm using LEDTECH 96 ELISA Plate Reader.