Immobilization of a Novel ESTBAS Esterase from Bacillus altitudinis onto an Epoxy Resin: Characterization and Regioselective Synthesis of Chloramphenicol Palmitate

Abstract

Novel gene estBAS from Bacillus altitudinis, encoding a 216-amino acid esterase (EstBAS) with a signal peptide (SP), was expressed in Escherichia coli. EstBASΔSP showed the highest activity toward p-nitrophenyl hexanoate at 50 °C and pH 8.0 and had a half-life (T1/2) of 6 h at 50 °C. EstBASΔSP was immobilized onto a novel epoxy resin (Lx-105s) with a high loading of 96 mg/g. Fourier transform infrared (FTIR) spectroscopy showed that EstBASΔSP was successfully immobilized onto Lx-105s. In addition, immobilization improved its enzymatic performance by widening the tolerable ranges of pH and temperature. The optimum temperature of immobilized EstBASΔSP (Lx-EstBASΔSP) was higher, 60 °C, and overall thermostability improved. T1/2 of Lx-EstBASΔSP and free EstBASΔSP at 60 °C was 105 and 28 min, respectively. Lx-EstBASΔSP was used as a biocatalyst to synthesize chloramphenicol palmitate by regioselective modification at the primary hydroxyl group. Conversion efficiency reached 94.7% at 0.15 M substrate concentration after 24 h. Lx-EstBASΔSP was stable and could be reused for seven cycles, after which it retained over 80% of the original activity.

Thermoalkalophilic recombinant esterase entrapment in chitosan/calcium/alginate‐blended beads and its characterization

“…53 Besides, immobilized esterase Lx-Est BAS ΔSP activity was reduced by 3% in the presence of Zn 2+ whereas Mg 2+ increased its activity by 8%. 45 The surfactant analysis result showed that SDS completely inhibited the immobilized esterase activity whereas Triton X-100 strongly decreased by 42% (Fig. 4).…”

“…50 An immobilized esterase Lx-Est BAS ΔSP also showed higher stability than free esterase Est BAS ΔSP and retained about 80% of residual activity at 60°C after 1 h of incubation. 45 Furthermore, two immobilized Mucor miehei esterase on core-shell magnetic beads via adsorption and covalent binding showed 80% and 90% of residual activity at 60°C, respectively. 27 There have been reported some studies on the thermal stability of free and immobilized esterase from various sources, showing much lower thermal stability, compared to the present study.…”

“…44 Also, an esterase from Bacillus altitudinis (Est BAS ΔSP) optimally worked at 50°C, whereas its immobilized form of the enzyme (Lx-Est BAS ΔSP) onto an epoxy resin exhibited maximum activity at 60°C. 45 A free and immobilized wheat esterase using glass fiber film optimally worked at 30 and 40°C, respectively. 46 Additionally, free esterase from Bacillus pumilus and its immobilized form on silane functionalized superparamagnetic nanoparticles (SNPs) had maximum activity at 37 and 45°C, respectively.…”

“…Similar to our findings, several studies have shown that different immobilization techniques did not change the optimal pH values of esterases from various sources. 43,45,49 Besides, immobilization of wheat esterase on glass fiber film decreased optimum pH by one unit, compared to the free form. 46 In contrast, immobilization of Mucor miehei esterase on core-shell magnetic beads increased the optimum pH by one unit, compared to its free form.…”

“…53 In another study, SDS and Triton X-100 dramatically reduced immobilized esterase Lx-Est BAS ΔSP activity by 80% and 62%, respectively. 45…”