Recombinant acetylxylan esterase of Halalkalibacterium halodurans NAH-Egypt: molecular and biochemical study

Abstract

Acetylxylan esterase plays a crucial role in xylan hydrolysis as the acetyl side-groups restrict endoxylanase action by stearic hindrance. In this study, an acetylxylan esterase (AXE-HAS10: 960 bp & 319 a.a) putative ORF from Halalkalibacterium halodurans NAH-Egypt was extensively studied through heterologous overexpression in Escherichia coli, biochemical characterization, and structural modeling. The AXE-HAS10 tertiary structure was predicted by the Local Meta Threading Server. AXE-HAS10 belongs to the carbohydrate esterase Family 7. Purified to homogeneity AXE-HAS10 showed specific activity (36.99 U/mg), fold purification (11.42), and molecular mass (41.39 kDa). AXE-HAS10 showed optimal pH (8.5) and temperature (40 oC). After 15 h of incubation at pH 7.0–9.0, AXE-HAS10 maintained 100% activity. After 120 min at 35 and 40 oC, the retained activity was 80 and 50%, respectively. At 10 mM Mn2+, Fe3+, K+, and Ca2+ after 30 min, retained activity was 329 ± 15, 212 ± 5.2, 123 ± 1.4, and 120 ± 3.0%, respectively. After 30 min of preincubation with triton x-100, SDS, and CTAB at 0.1% (v/v), the retained activity was 150 ± 19, 88 ± 4, and 82 ± 7%, respectively. At 6.0 M NaCl after 30 min, retained activity was 58%. A 1.44-fold enhancement of beechwood xylan hydrolysis was achieved by AXE-HAS10 and Penicillium chrysogenum DSM105774 β-xylanase concurrently. Present data underpins AXE-HAS10 as a promising AXE for industrial exploitation.

A molecular study on recombinant pullulanase type I from Metabacillus indicus

“…The purification of the recombinantly expressed Pull_Met was carried out using a previously reported procedure with minor modifications (Embaby and Mahmoud 2022 ; Mahmoud et al 2021 ). In brief, the resultant soluble portion of cell lysate containing 100 mg of crude protein was loaded onto a 2 mL Ni 2+ -NTA affinity matrix.…”

Single‐Step Purification of Catalase Enzyme From Human Blood Erythrocytes Using Affinity Chromatography Technique

“…For the optimization of equilibration, washing, and elution buffers of the affinity column, each condition was prepared as described in this section, and 10 mL of hemolysate was used for each. The equilibration buffer was prepared with 25 mM Tris-Base/0.1 M NaCl in different pH values (9.5-9-8.5-8-7.5-7-7-6.5-6-6-5.5-5) [ 16 ]. The column was equilibrated with each buffer individually, and the optimum pH was determined to be 8.5.…”