Molecular cloning and optimization of selected culture conditions for production of Thermostable Alkaline Protease 50a (TAP50a) in Escherichia coli BL21 (DE3) and TOP10

• Main Author: Normazzaliana Ibrahim
• Format: UMK Etheses
• Language: English
• Published: 2022
• Online Access: http://discol.umk.edu.my/id/eprint/13394/1/Normazzaliana%20binti%20Ibrahim.pdf; http://discol.umk.edu.my/id/eprint/13394/

Protease is one of the important enzymes utilized in numerous industrial applications; thus, high production is required to meet the market demand. Thermostable alkaline protease 50a (TAP50a) gene was previously isolated from Bacillus subtilis and was successfully cloned and expressed in Escherichia coli BL21 (DE3) pLysS. The expression of the TAP50a enzyme in the recombinant host (53 U/mL) was higher than the wild type strain (18 U/mL). However, this host required two types of antibiotics which complicate the downstream processes and increase the overall production cost. Therefore, this research aimed to clone TAP50a in another potential host, expressing more enzymes at a lower production cost. TAP50a gene was cloned into two hosts, E. coli BL21 (DE3) and TOP10, and three culture conditions were optimized using response surface methodology (RSM) for the best enzyme production. First, the TAP50a gene was extracted from the previous host, amplified through polymerase chain reaction (PCR) and ligated into pGEX4T-1 plasmid. Then, the recombinant pGEX4T-1/50a was transformed into E. coli BL21 (DE3) and TOP10 competent cells. The positive clones were detected by forming clearing zones around the colonies on the Luria Bertani- skimmed milk agar (LB-SMA) plate after incubation at 60 °C for 5 h. The validation of clones was done through restriction digestion and PCR using specific primers incorporated with EcoR1 sites. The digested product showed two bands, 5200 bp and 1500 bp, referring to the TAP50a gene and pGEX4T-1 plasmid, while the PCR product exhibited one band with molecular size 1500 bp referring to the TAP50a gene. The expression of the TAP50a enzyme in both hosts was induced by 0.5 mM of isopropyl-β-D-thiogalactopyranoside (IPTG) at 30 °C. Box-Behnken Design (BBD) was applied to optimize the expression of the TAP50a enzyme by optimizing three parameters; agitation (rpm), inoculum size (%), and incubation time (h). The optimized condition for the optimum expression TAP50a enzyme in E. coli BL21 (DE3) was found to be at 184.06 rpm, 2.53 % inoculum size and 17.4 h incubation time yielding 70.33 U/mL of TAP50a. Meanwhile, the maximum expression of the TAP50a enzyme in E. coli TOP10 was 30.0 U/mL under optimized conditions at 199.90 rpm, 3.16% inoculum size, and 23.67 h of incubation time. The expression of TAP50a in both hosts presented 2.24 and 2.36-fold increment under optimized culture conditions compared to the non-optimized. Analysis of variance (ANOVA) revealed that the interaction of inoculum size and incubation time (X2X3) has a significant effect on the production of TAP50a enzyme in E. coli BL21 (DE3) since at p < 0.05. In E. coli TOP10, there was also a significant parameters interaction effect of parameters through the combination of agitation and inoculum size (X1X2) at p < 0.05. These findings suggested that E. coli BL21 (DE3) is a better host for the expression of xviii the TAP50a enzyme compared to E. coli TOP10. Therefore, further research should be conducted on this enzyme by optimizing nutritional factors to increase TAP50a enzyme production.