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

摘要

A natural bacterial strain identified as Bacillus amyloliquefaciens MBAA3 using 16S rDNA partial genome sequencing has been studied for optimization of cellulase production. Statistical screening of media components for production of cellulase by B. amyloliquefaciens MBAA3 was carried out by Plackett–Burman design. Plackett–Burman design showed CMC, MgSO4 and pH as significant components influencing the cellulase production from the media components screened by Plackett-Burman fractional factorial design. The optimum concentrations of these significant parameters were determined employing the response surface central composite design, involving three factors and five levels was adopted to acquire the best medium for the production of cellulase enzyme revealed concentration of CMC (1.84?g), MgSO4 (0.275?g), and pH (8.5) in media for highest enzyme production. Response surface counter plots revealed that middle level of MgSO4 and middle level of CMC, higher level of CMC and lower level of pH and higher level of MgSO4 with lower level of pH increase the production of cellulase. After optimization cellulase activity increased by 6.81 fold. Presence of cellulase gene in MBAA3 was conformed by the amplification of genomic DNA of MBAA3. A PCR product of cellulase gene of 1500?bp was successfully amplified. The amplified gene was conformed by sequencing the amplified product and sequence was deposited in the gene bank under the accession number KF929416.Graphical AbstractResponse surface graph showing interaction effects between concentration of a CMC and MgSO4. b pH and CMC. c MgSO4 and pH