Heterologous expression, purification, immobilization and characterization of recombinant α-amylase AmyLa from Laceyella sp. DS3

• Published in: International journal of biological macromolecules Vol. 132; pp. 1274 - 1281
• Main Authors: El-Sayed, Ahmed K.A., Abou-Dobara, Mohamed I., El-Fallal, Amira A., Omar, Noha F.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.2019
• Subjects: Heterologous expression; Immobilization; α-Amylase; SDS-PAGE; Km; orf; AmyLa; ANOVA; Vmax; IPTG; Immobilization; ITS; α-Amylase; LSD; Heterologous expression
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2019.04.010

AmyLa α-amylase gene from Laceyella sp. DS3 was heterologously expressed in E. coli BL21. E. coli BL21 maximally expressed AmyLa after 4 h of adding 0.02 mM IPTG at 37 °C. The recombinant AmyLa α-amylase was purified 2.19-fold through gel filtration and ion exchange chromatography. We immobilized the purified recombinant AmyLa α-amylase on four carriers; chitosan had the best efficiency. The recombinant free and the immobilized AmyLa α-amylase showed optimum activity in the pH ranges of 6.0–7.0 and 4.0–7.0, respectively and possessed an optimum temperature of 55 °C. The free enzyme had activation energy, Km, and Vmax of 291.5 kJ, 1.5 mg/ml, and 6.06 mg/min, respectively. The immobilized enzyme had activation energy, Km, and Vmax of 309.74 kJ, 6.67 mg/ml, and 50 mg/min, respectively. The immobilized enzyme was calcium-independent and insensitive (relative to the free enzyme) to metals. It could also be reused for seven cycles.