Date Published: February 15, 2012
Author(s): Bala Muntari, Azura Amid, Maizirwan Mel, Mohammed S Jami, Hamzah M Salleh.
Bromelain, a cysteine protease with various therapeutic and industrial applications, was expressed in Escherichia coli, BL21-AI clone, under different cultivation conditions (post-induction temperature, L-arabinose concentration and post-induction period). The optimized conditions by response surface methodology using face centered central composite design were 0.2% (w/v) L-arabinose, 8 hr and 25°C. The analysis of variance coupled with larger value of R2 (0.989) showed that the quadratic model used for the prediction was highly significant (p < 0.05). Under the optimized conditions, the model produced bromelain activity of 9.2 U/mg while validation experiments gave bromelain activity of 9.6 ± 0.02 U/mg at 0.15% (w/v) L-arabinose, 8 hr and 27°C. This study had innovatively developed cultivation conditions for better production of recombinant bromelain in shake flask culture.
The use of highly purified proteins for therapeutic purposes has been in existence for many decades (Paul, 2004). Enzymes, mostly proteases, constitute the largest portion of these purified proteins for industrial and therapeutic applications. Proteases are enzymes that catalyze the hydrolysis of peptide linkages in proteins. They have wide applications in food, pharmaceutical and detergent industries. In fact, these enzymes constitute about 60% of all commercial enzymes in the world (Lucia and Tomas, 2010). Recently, microbial enzymes have been substituting those from other sources and might now account for almost 90% of the total market (Illanes, 2008). This is due to the fact that microbial cells are excellent systems for enzyme production. Thus, there is a great stimulation for extensive research works on recombinant proteins (Illanes, 2008).
There are several strategies that are being employed to improve culture cultivation conditions for expressing soluble recombinant proteins in E. coli. The screening of experimental conditions for the improvement of enzyme expression coupled with the RSM experimental design serve as vital tool for analyzing the influence of cultivation conditions on the expression of recombinant bromelain. The design had proved to be effective in determining the important induction conditions that have significant effect on recombinant bromelain production in E. coli BL21-AI. Using face centered central composite design (FCCCD), the optimum induction conditions for high bromelain activity were induction temperature (27°C), L-arabinose concentration (0.15% w/v) and post-induction period of 8 hr. The results presented in this research for the analysis of cultivation conditions for the expression of recombinant bromelain corroborate the applicability of experimental design to the field of molecular biology.
The authors declare that they have no competing interests.
1. All the tests were carried out at 37°C, OD600 nm of 0.4, 0.2% (w/v) L-arabinose and 4 hr post-induction period (besides the specific tested conditions).