Cloning and expression of α-Amylase gene from Bacillus subtilis in Pichia pastoris and Escherichia coli.
Enzyme is a type of catalyst that present in living organisms used for many biotechnological functions in various industrial processing. It has a special characteristic that allows the chemical reaction to speed up without being altered, thus significantly improve the industrial productivity (Roy et al. 2012). Among various enzymes available in market, α-amylase has received a special attention in commercial production due to its widely used applications. α-Amylase contributed to 50% of the world enzyme production and has a great importance in many industries such as in ...view middle of the document...
Cloning Mechanism of α-Amylase Gene from Bacillus subtilis
There are several materials that were used in the cloning process of α-amylase gene from Bacillus subtilis PY22 namely plasmid Blunt II-Topo and E. coli as described by Karakas et al. (2010). The cloning process started off with the isolation of DNA fragment that encodes α-amylase of about 2.5 kbp using the MasterPure™ kits, which was then amplified by the PCR method. AmyFPmlI and AmyRKpn were both used as primers in order to introduced the PmlI and KpnI restriction sites to the PCR products.
After the PCR products were purified using the Qiagen, it was then ligated to the Blunt II-Topo plamid before transformed into E. coli. The E. coli was grown in Luria-Bertani (LB) medium that supplemented with antibiotic for easier selection. In another experiment carried out by Yin et al. (2003), the Bacillus subtilis RSKK strains were used to clone the α-amylase gene in E. coli XLl-Blue MRF. Approximately 1.7 kbp fragment DNA, which encodes the α-amylase was isolated and inserted into the pUC18 vector before transformed into E. coli. The E. coli was then grown in the medium that contains ampicillin for easier selection of transformed cells.
Expression of α-amylase gene in Escherichia coli and Pichia pastoris.
In order to meet the industrial demands for α-amylase enzyme, new alternative methods have been found, which can significantly improve the enzyme production with minimal cost. This involves the isolation of α-amylase gene from Bacillus subtilis and expressed it into another microorganism. In conventional method, Bacillus subtilis of specific strain was fermented in controlled medium and harvested for its enzyme (Nimkar et al. 2010). However, this method was not efficient as it requires longer time and the growth medium was very expensive. Besides, the purification of α-amylase produced from Bacillus subtilis was also difficult since the bacteria also produce other type of enzymes. Hence by isolating and expressing the specific gene that responsible for the production of α-amylase enzyme into another microorganism that easier to grow such as E. coli and Pichia pastoris will not only reduce the cost but also ease the enzyme purification (Nimkar et al. 2010).
Currently there are many trials have been carried out using this method involving several different microbes. However, at moment, the most commonly used host microbes are E. coli and Pichia pistoris. Pichia pistoris is a type of yeast that feed on methanol and does not normally produce α-amylase enzyme. It has been developed as a commercial expression system for recombinant protein due to its unique properties such as high growth rate and easier to grow (Bano et al. 2011). In the experiment carried out by Karakas et al. (2010), which used Pichia pastoris as host, they first digested the plasmid Blunt II-Topo that contain the fragment DNA from Bacillus subtilis as explained earlier with PmlI and KpnI before purified it. In order to...