PURIFICATION AND CHARACTERIZATION OF AN ALKALINE CELLULASE PRODUCED BYBacillus subtilis(AS3)

Abstract

An extracellular alkaline carboxymethycellulase (CMCase) from Bacillus subtilis was purified by salt precipitation followed by anion-exchange chromatography using DEAE-Sepharose. The cell-free supernatant containing crude enzyme had a CMCase activity of 0.34 U/mg. The purified enzyme gave a specific activity of 3.33 U/mg, with 10-fold purification and an overall activity yield of 5.6%. The purified enzyme displayed a protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular size of 30 kDa, which was also confirmed by zymogram analysis. The enzyme displayed multisubstrate specificity, showing significantly higher activity with lichenan and β-glucan as compared to carboxymethylcellulose (CMC), laminarin, hydroxyethylcellulose, and steam-exploded bagasse, and negligible activity with crystalline substrate such as Avicel and filter paper. It was optimally active at pH 9.2 and temperature 45°C. The enzyme was stable in the pH range 6-10 and retained 70% activity at pH 12. Thermal stability analysis revealed that the enzyme was stable in temperature range of 20°C to 45°C and retained more than 50% activity at 60°C for 30 min. The enzyme had a Km of 0.13 mg/ml and Vmax of 3.38 U/mg using CMC as substrate.

Modification of Different Pulps by Homologous Overexpression Alkali-Tolerant Endoglucanase in Bacillus subtilis Y106

“…Some studies have also focused on promoting the alkaline or alkali-tolerant enzyme activity, for example, Deka et al . (2013 a, b) enhanced extracellular alkaline cellulase production from Bacillus subtilis by optimizing physical parameters and the CMCase activity was promoted from 0.43 IU/ml to 0.56 IU/ml at pH 6.0 30, 31 . Ariffin et al .…”

Novel perspectives for evolving enzyme cocktails for lignocellulose hydrolysis in biorefineries

“…A more cost-effective process alternative is simultaneous saccharification and fermentation (SSF) in which the two steps of enzymatic hydrolysis and fermentation are carried out together in a vessel. As suggested by Das et al, [14] SSF processes utilizing recombinant enzymes have the potential to minimize the production costs and maximizing the volumetric productivity in the bio-ethanol industry. Utility cost of enzymatic hydrolysis is low compared to acid or alkaline hydrolysis because enzyme hydrolysis is usually conducted at mild conditions (pH 4.8 and temperature 45-50°C) and does not have a corrosion problem [15].…”

Scale up and efficient bioethanol production involving recombinant cellulase (Glycoside hydrolase family 5) from Clostridium thermocellum

“…B. subtilis inoculum was prepared by transferring a loop full of culture from nutrient agar slant in 5 mL of nutrient broth and incubated for 18 h at 37°C and 180 rpm. 2% (v v -1 ) of the fresh inoculum was transferred to 50 mL of the optimised medium containing (g L -1 ): CMC, 18; peptone, 8; yeast extract, 5; K 2 HPO 4 , 1; MgSO 4 .7H 2 O, 0.25; and NaCl, 5 [8] in 250 mL Erlenmeyer flask and incubated at 37°C for 48 h followed by centrifugation at 10,000g for 15 min at 4°C. The cell free supernatant was used as the crude enzyme for saccharification.…”

“…Bacterial cellulases are also potential candidates as they can withstand harsh conditions such as high temperature, sugar, salt and ethanol concentrations during lignocellulose degradation and can metabolize wide range of sugars improving the process of ethanol production [7]. Bacillus subtilis (AS3) produce cellulases which are alkaline, thermostable, show wide range of pH stability and substrate specificity for both soluble and crystalline substrates such as avicel [8]. The optimum temperature for cellulase activity of B. subtilis (AS3) is close to that of recombinant and fungal cellulase [8].…”

“…Bacillus subtilis (AS3) produce cellulases which are alkaline, thermostable, show wide range of pH stability and substrate specificity for both soluble and crystalline substrates such as avicel [8]. The optimum temperature for cellulase activity of B. subtilis (AS3) is close to that of recombinant and fungal cellulase [8]. The cellulosome of Clostridium thermocellum is known to have one of the highest rates of cellulose utilization till date reported, that displays 50-fold higher specific activity than the corresponding Trichoderma reesei system against crystalline cellulose [9].…”