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. 2010 Jan;3(1):84-92.
doi: 10.1111/j.1751-7915.2009.00150.x. Epub 2009 Sep 18.

Hyperthermostable Acetyl Xylan Esterase

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Free PMC article

Hyperthermostable Acetyl Xylan Esterase

Katharina Drzewiecki et al. Microb Biotechnol. .
Free PMC article

Abstract

An esterase which is encoded within a Thermotoga maritima chromosomal gene cluster for xylan degradation and utilization was characterized after heterologous expression of the corresponding gene in Escherichia coli and purification of the enzyme. The enzyme, designated AxeA, shares amino acid sequence similarity and its broad substrate specificity with the acetyl xylan esterase from Bacillus pumilus, the cephalosporin C deacetylase from Bacillus subtilis, and other (putative) esterases, allowing its classification as a member of carbohydrate esterase family 7. The recombinant enzyme displayed activity with p-nitrophenyl-acetate as well as with various acetylated sugar substrates such as glucose penta-acetate, acetylated oat spelts xylan and DMSO (dimethyl sulfoxide)-extracted beechwood xylan, and with cephalosporin C. Thermotoga maritima AxeA represents the most thermostable acetyl xylan esterase known to date. In a 10 min assay at its optimum pH of 6.5 the enzyme's activity peaked at 90 °C. The inactivation half-life of AxeA at a protein concentration of 0.3 µg µl(-1) in the absence of substrate was about 13 h at 98 °C and about 67 h at 90°C. Differential scanning calorimetry analysis of the thermal stability of AxeA corroborated its extreme heat resistance. A multi-phasic unfolding behaviour was found, with two apparent exothermic peaks at approximately 100-104 °C and 107.5 °C. In accordance with the crystal structure, gel filtration analysis at ambient temperature revealed that the enzyme has as a homohexameric oligomerization state, but a dimeric form was also found.

Figures

Figure 1
Figure 1
SDS‐PAGE analysis of AxeA purification steps. Lane 1, molecular mass standard proteins; lane 2, crude extract of E. coli BL21(DE3)/pET24d (50 µg); lane 3, crude extract of E. coli BL21(DE3)/pET24d after heat treatment (3.5 µg); lane 4, crude extract of E. coli BL21(DE3)/pET24d‐axeA (40 µg); lane 5, crude extract of E. coli BL21(DE3)/pET24d‐axeA after heat treatment (9 µg); lane 6, pooled active fractions after Source 30 Q ion exchange chromatography (4 µg); lane 7, pooled active fractions after Phenyl Sepharose HP chromatography (2.4 µg); lane 8, pooled active fractions after Phenyl Sepharose HP chromatography (5.8 µg).
Figure 2
Figure 2
Size exclusion chromatography of purified recombinant AxeA, revealing active homodimeric and homohexameric forms of the enzyme. The elution volumes of the two symmetric peaks correspond to native molecular masses of 74.5 and 229.1 kDa respectively (for details see Experimental procedures). Insert: Oligomeric state of AxeA as derived from crystallographic data (PDB ID: 1vlq) which suggests 12 monomers arranged as two homohexamers in the asymmetric unit of the protein crystal.
Figure 3
Figure 3
pH dependence (at 70°C) and temperature dependence (at pH 5.5) of AxeA activity, using a 10 min assay and chemically acetylated xylan as the substrate.
Figure 4
Figure 4
Temperature inactivation kinetics of recombinant AxeA at 70°C, 90°C and 98°C. The purified enzyme (at a concentration of 0.3 µg µl−1) was incubated in the absence of substrate at the respective temperatures, samples were withdrawn and the residual activity was determined with pNP‐acetate as described in Experimental procedures.
Figure 5
Figure 5
Differential scanning calorimetry (DSC) unfolding trace of AxeA. The protein (0.6 mg ml−1) was heated in 50 mM sodium phosphate, pH 7.5 with a rate of 1°C min−1. The experimentally observed transition (solid line) was deconvoluted (smoothed solid line), yielding three species with Tm values of 100°C, 104°C and 107.5°C (dashed lines).
Figure 6
Figure 6
Working model showing the proposed metabolic functions encoded by the ORFs of the approximately 30 kb chromosomal gene cluster (TM0055–TM0077) for the breakdown and utilization of acetylated glucuronoxylan by T. maritima. The ORFs which are proposed to encode for the individual steps of the pathway are indicated next to the arrows. The proposed functional assignments of the ORFs are as follows (the relative orientation of the ORFs is indicated with ‘+’ and ‘−’ symbols): TM0055(+), α‐glucuronidase (aguA); TM0056(+), ABC‐transporter periplasmic binding protein; TM0057(+), ABC‐transporter ATP‐binding protein; TM0058(+), ABC‐transporter ATP‐binding protein; TM0059(+), ABC‐transporter permease protein; TM0060(+), ABC‐transporter permease protein; TM0061(−), endo‐1,4‐β‐xylanase [xynA, toga‐associated and partially released to the extracellular medium (Winterhalter and Liebl, 1995; Liebl et al. 2008)]; TM0062(−), hypothetical protein; TM0063(+), hypothetical protein; TM0064(+), glucuronate isomerase; TM0065(−), transcriptional regulator, IclR family; TM0066(−), 2‐dehydro‐3‐deoxyphosphogluconate aldolase/4‐hydroxy‐2‐oxoglutarate aldolase; TM0067(−), 2‐keto‐3‐deoxygluconate kinase; TM0068(−), d‐mannonate oxidoreductase; TM0069(−), mannonate dehydratase; TM0070(+), endo‐1,4‐β‐xylanase [xynB, XynB is thought to be present in the periplasm and also occurs extracellularly (Winterhalter and Liebl, 1995; Liebl et al., 2008)]; TM0071(−), ABC‐transporter periplasmic binding protein; TM0072(−), ABC‐transporter permease protein; TM0073(−), ABC‐transporter permease protein; TM0074(−), ABC‐transporter ATP‐binding protein; TM0075(−), ABC‐transporter ATP‐binding protein; TM0076(−), β‐xylosidase (bxlA); TM0077(−), acetyl xylan esterase (axeA). It is stressed that the proposed intracellular localization of AxeA has not been proven experimentally.

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