THERMOSTABLE RECOMBINANT ESTERASE PRODUCTION IN 3-L STIRRED TANK BIOREACTOR, PURIFICATION AND CHARACTERIZATION

Abstract

Thermophilic lipases/esterases are currently attracting enormous attention because of their biotechnological potential. It was thus aimed in the present study to optimize enzyme on large scale production, to purify and characterize the produced enzyme. The est fragment of Geobacillus sp. AZ1 (ac: KM823656) was cloned directly by PCR into the pCYTEXP1 expression vector under the control of lambda promoter. Good intracellular expression of the studied gene was obtained in Escherichia coli DH5α. Optimization of the recombinant protein production was carried out using a 3L bench-top bioreactor with a stirred tank. The results showed that the fermenter condition and induction by shifting temperature from 37ºC to 42ºC at OD. 0.7 resulted in a 2-fold increase. The expressed protein was fused C-terminally with 6x-his tag to allow one step purification using IMAC (Immobilized Metal Affinity Chromatography). A 28kD protein was achieved. The kinetic characterization of the purified enzyme exhibited maximum activity at 50ºC and pH 7.4. The enzyme had a considerable thermal stability. The percentage of remained activity after 1h exposure to 50, 55 and 60ºC reached to 50, 42 and 27%, and retained more than 60% of its original activity at 65ºC for 15min. However, exposure of crude enzyme to these conditions showed a complete stability and more than 90% of activity. The enzyme was also highly stable in a pH range of 3-10 for 24h. The enzyme activity was promoted in the presence of Co+2, K+1, Ca+2 and Fe+2 and was inhibited by Mn+2, Mg+2, Cu+2, Hg+2, Zn+2. Diethyl-ether, and acetone but hexane enhanced the activity. On the contrary nbutanol, DMSO, ethanol, isopropanol, glycerol, methanol and chloroform, reduce the enzyme activity. EGTA, DTT, EDTA, PMSF and SDS decreased the enzyme activity, whereas the presence of urea, oxidizing and reducing agents, some non-ionic surfactants increased the enzyme activity. The values of Km and Vmax as calculated from the Lineweaver- Burk plot were 12.66 mM and 333.33 U/mg protein respectively.