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. 2016 Sep;5(3):143-155.

Purification and Biochemical Properties of a Thermostable, Haloalkaline Cellulase From Bacillus licheniformis AMF-07 and Its Application for Hydrolysis of Different Cellulosic Substrates to Bioethanol Production

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Free PMC article

Purification and Biochemical Properties of a Thermostable, Haloalkaline Cellulase From Bacillus licheniformis AMF-07 and Its Application for Hydrolysis of Different Cellulosic Substrates to Bioethanol Production

Fatemeh Azadian et al. Mol Biol Res Commun. .
Free PMC article

Abstract

A thermophilic strain AMF-07, hydrolyzing carboxymethylcellulose (CMC) was isolated from Kerman hot spring and was identified as Bacillus licheniformis based on 16S rRNA sequence homology. The carboxymethylcellulase (CMCase) enzyme produced by the B. licheniformis was purified by (NH4)2SO4 precipitation, ion exchange and gel filtration chromatography. The purified enzyme gave a single band on SDS- PAGE with a molecular weight of 37 kDa. The CMCase enzyme was highly active and stable over broad ranges of temperature (40-80ºC), pH (6.0-10.0) and NaCl concentration (10-25%) with an optimum at 70ºC, pH 9.0 and 20% NaCl, which showed excellent thermostable, alkali-stable and halostable properties. Moreover, it displayed high activity in the presence of cyclohexane (134%) and chloroform (120%). Saccharification of rice bran and wheat bran by the CMCase enzyme resulted in respective yields of 24 and 32 g L-1 reducing sugars. The enzymatic hydrolysates of rice bran were then used as the substrate for ethanol production by Saccharomyces cerevisiae. Fermentation of cellulosic hydrolysate using S. cerevisiae, reached maximum ethanol production about 0.125 g g-1 dry substrate (pretreated wheat bran). Thus, the purified cellulase from B. licheniformis AMF-07 utilizing lignocellulosic biomass could be greatly useful to develop industrial processes.

Keywords: Bioethanol; Cellulosic hydrolysate; Characterization; Purification.

Figures

Figure 1
Figure 1
The phylogenetic tree for Bacillus licheniformis AMF-07 and related strains based on the 16S rRNA sequence. The tree was drawn using MEGA 4.0 with computing linearized tree
Figure 2
Figure 2
Effect of (a) temperature, (b) pH and (c) NaCl on the activity and stability of the purified CMCase AMF-07. The activity of purified CMCase was measured at different temperatures, pH and NaCl concentrations using a phosphate buffer (pH 7.5). Relative activity was defined as the percentage of activity detected with respect to the maximum enzyme activity. For determining the stability, the activity of the enzyme without any treatment was taken as 100%.
Figure 3
Figure 3
Time course profile of enzyme production by Bacillus licheniformis AMF-07 on wheat bran and rice bran. Results are the mean of three replicates
Figure 4
Figure 4
Calibration curve of ethanol standards (a), Changes in sugar concentration and ethanol contents of wheat bran pretreatment over the fermentation period by Saccharomyces cerevisiae (b

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