Column Chromatography Free Purification of Recombinant α-Amylase from Bacillus licheniformis by Tagging with Hydrophobic Elastin Like Polypeptide

Abstract

Thermal sensitive and highly hydrophobic elastin like polypeptide (ELP) tends to undergo inverse transition cycling (ITC) which can be used for non chromatographic purification. The present study reports non-chromatographic purification of industrially important α-amylase tagged with ELP and compared with IMAC (Immobilized metal affinity chromatography) purified α-amylase. amyL-Gene encoding α-amylase from Bacillus licheniformis was cloned and expressed in E. coli BL21. The expressed protein with His-tag was purified through IMAC using Ni–NTA matrix. Three ELP genes encoding repeats of pentapeptide (Val-Pro-Gly-Val-Gly)n with variable length (V = 20, 21, 22) were synthesized through PCR using overlapping primers. To generate ELP tagged α-amylase, amyL was placed at N-terminal of ELPs and transformed to E. coli BL21 for expression. After ITC, ELP22 at 30 °C showed maximum yield. α-Amylase purification through ITC and IMAC showed 2.9 and 1.72-fold purification, respectively. Furthermore, physical parameters of ELP tagged α-amylase have shown improvement with working temperature and thermal stability in comparison to His-tag α-amylase. The k cat /K m for ELP tag and His-tag α-amylase was found to be 61.4 and 23.7 mg−1 min−1, respectively, which shows that ELP-tag increased the enzyme efficiency. In conclusion, the ELP-tag purification strategy can be applied to industrially relevant enzyme for purification by the non-chromatography method.