Produksi dan Pemurnian Endo-β-1,4-D-Xilanase Rekombinan Hasil Ekspresi Gen pET30a xynBTN63D di Escherichia coli BL21 (DE3) pada Media Minimal

Abstract

Endo-β-1,4-D-xylanase is an important enzyme produced from the pET30a xynBTN63D gene, which is expressed in E. coli BL21(DE3) using Luria Bertani (LB) medium. This enzyme helps break down xylan into smaller sugar units called xylooligosaccharides (XOS). XOS are well known as prebiotics that support the growth of good bacteria in the digestive system. However, using LB medium for large-scale enzyme production is expensive and not efficient for industrial use because of its complex and rich composition. Therefore, this study uses MTE as a more affordable alternative medium. MTE is a defined medium that contains essential nutrients such as carbon and nitrogen sources, inorganic salts, and trace elements. The enzyme produced in both LB and MTE is purified using affinity chromatography. Further analysis is done to measure protein content, enzyme activity, protein expression through SDS-PAGE, and the ability to break down XOS. The crude enzyme from MTE shows specific activity of 1.054 U/mg for beechwood xylan, 0.006 U/mg for pNP-A, and 0.006 U/mg for pNP-X. After purification, the specific activity increases to 3.993 U/mg, 0.010 U/mg, and 0.008 U/mg, respectively. These results show that MTE is a suitable and cost-effective medium for producing endo-β-1,4-D-xylanase.