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. 2019 Jul 8;18(1):122.
doi: 10.1186/s12934-019-1169-y.

Characterization of an Acetyl Xylan Esterase From the Marine Bacterium Ochrovirga Pacifica and Its Synergism With Xylanase on Beechwood Xylan

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Free PMC article

Characterization of an Acetyl Xylan Esterase From the Marine Bacterium Ochrovirga Pacifica and Its Synergism With Xylanase on Beechwood Xylan

Sachithra Amarin Hettiarachchi et al. Microb Cell Fact. .
Free PMC article

Abstract

Background: Acetyl xylan esterase plays an important role in the complete enzymatic hydrolysis of lignocellulosic materials. It hydrolyzes the ester linkages of acetic acid in xylan and supports and enhances the activity of xylanase. This study was conducted to identify and overexpress the acetyl xylan esterase (AXE) gene revealed by the genomic sequencing of the marine bacterium Ochrovirga pacifica.

Results: The AXE gene has an 864-bp open reading frame that encodes 287 aa and consists of an AXE domain from aa 60 to 274. Gene was cloned to pET-16b vector and expressed the recombinant AXE (rAXE) in Escherichia coli BL21 (DE3). The predicted molecular mass was 31.75 kDa. The maximum specific activity (40.08 U/mg) was recorded at the optimal temperature and pH which were 50 °C and pH 8.0, respectively. The thermal stability assay showed that AXE maintains its residual activity almost constantly throughout and after incubation at 45 °C for 120 min. The synergism of AXE with xylanase on beechwood xylan, increased the relative activity 1.41-fold.

Conclusion: Resulted higher relative activity of rAXE with commercially available xylanase on beechwood xylan showed its potential for the use of rAXE in industrial purposes as a de-esterification enzyme to hydrolyze xylan and hemicellulose-like complex substrates.

Keywords: Acetyl xylan esterase; Beech wood xylan; Marine bacteria; Ochrovirga pacifica; Synergism.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Nucleotide and deduced amino acid sequence of AXE. The N-terminal signal sequence is underlined; the acetyl esterase domain (aa 60 to 274) is highlighted; asterisk indicates a stop codon
Fig. 2
Fig. 2
SDS-PAGE analysis of rAXE. M molecular weight marker, BI whole cell lysate before induction, AI whole cell lysate after induction (incubated at 20 °C for 16 h with 180 rpm agitation), P purified rAXE using His·Bind® Resin Chromatography Kit
Fig. 3
Fig. 3
Effects of pH and temperature on rAXE activity. a Effect of temperature on relative enzyme activity (relative activity was calculated using activity at 50 °C as 100%). b Thermal stability assay (relative activity was calculated using activity of untreated enzyme as 100%). c Effect of pH on activity at 50 °C (relative activity was calculated using activity at pH 8.0 as 100%). d pH stability assay (relative activity was calculated using activity of enzyme treated with pH 8.0 buffer as 100%). Data are shown as mean ± standard deviation (sd), n = 3
Fig. 4
Fig. 4
a Effect of various metal ions on the relative activity of rAXE. The enzyme reaction was performed with final concentrations of 1 mM and 5 mM for each metal ion. The activity in the absence of a metal ion was taken as the control (100%). b Effect of NaCl on the relative activity and stability of rAXE. The activity at 0 M NaCl in the reaction mixture was taken as the control (100%). Data are presented as mean ± standard deviation (sd), n = 3
Fig. 5
Fig. 5
Synergism of AXE with a commercially available xylanase on beechwood xylan as the substrate. The reaction mixtures were prepared in Eppendorf tubes containing 1% beechwood xylan in phosphate buffer (pH 8.0) and incubated over the time at 50 °C. Combination of rAXE and xylanase was included 1.7 U of rAXE and 5 U of commercially available endo-1,4-β-xylanase derived from A. niger. Only rAXE and xylanase contained reaction mixtures were included 1.7 U and 5 U respectively. Relative activity was determined with the activity of rAXE + xylanase at 120 min as 100%. Data are given as means ± SD, n = 3

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