Heterologous expression, purification, crystallization, X-ray analysis and phasing of the acetyl xylan esterase from Bacillus pumilus

Krastanova, I.; Guarnaccia, Corrado; Zahariev, Sotir; Degrassi, Giuliano; Lamba, Doriano

doi:10.1016/j.bbapap.2005.01.003

Cited by 17 publications

(12 citation statements)

References 34 publications

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“…Especially for GAE, 60% of triacetin were deacetylated in 2 min. For AXE's deacetylation of different model compounds, representing acetylated polysaccharides and non-polysaccharide compounds, was reported before for chitosan, chitin [19], and cephalosporin-C [20].…”

Section: Deacetylationmentioning

confidence: 55%

Enzymatic Systems for Cellulose Acetate Degradation

et al. 2017

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Cellulose acetate (CA)-based materials, like cigarette filters, contribute to landscape pollution challenging municipal authorities and manufacturers. This study investigates the potential of enzymes to degrade CA and to be potentially incorporated into the respective materials, enhancing biodegradation. Deacetylation studies based on Liquid Chromatography-Mass Spectrometry-Time of Flight (LC-MS-TOF), High Performance Liquid Chromatography (HPLC), and spectrophotometric analysis showed that the tested esterases were able to deacetylate the plasticizer triacetin (glycerol triacetate) and glucose pentaacetate (cellulose acetate model compound). The most effective esterases for deacetylation belong to the enzyme family 2 (AXE55, AXE 53, GAE), they deacetylated CA with a degree of acetylation of up to 1.8. A combination of esterases and cellulases showed synergistic effects, the absolute glucose recovery for CA 1.8 was increased from 15% to 28% when an enzymatic deacetylation was performed. Lytic polysaccharide monooxygenase (LPMO), and cellobiohydrolase were able to cleave cellulose acetates with a degree of acetylation of up to 1.4, whereas chitinase showed no activity. In general, the degree of substitution, chain length, and acetyl group distribution were found to affect CA degradation. This study shows that, for a successful enzyme-based deacetylation system, a cocktail of enzymes, which will randomly cleave and generate shorter CA fragments, is the most suitable.

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Section: Deacetylationmentioning

confidence: 55%

Enzymatic Systems for Cellulose Acetate Degradation

et al. 2017

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“…These enzymes are not active on acetylgalactomannan residues (Biely et al 1997a, 1997b; Hakulinen et al 2000; Altaner et al 2003; Tenkanen et al 2003). The CE class-6 AcXEs are considered as real AcXEs, as they precipitate the acetylxylan from the solution, but reports also suggest that CE class-6 AcXEs exhibit a broad substrate specificity (Blum et al 1999; Bitto et al 2005; Krastanova et al 2005; Lopez-Cortés et al 2007). The CE class-7 AcXEs exhibit a strong ability to DE acetylate wide range of acetylated carbohydrate substrates; however, studies report that intracellular localisation of the CE class-7 enzymes does not support their classification as AcXE as they do not get to interact with the polymeric substrates.…”

Section: Microbial Enzymes Depolymerisation Of Hemicellulosementioning

confidence: 99%

“…This might propose that these enzymes take part in the degradation of the acetylxylan in its final stages. The CE class-7 AXE’s exhibit a very low positional specificity, these enzymes also exhibit a cephalosporin C deacetylase activity, respectively (Vincent et al 2003; Kremnický et al 2004; Krastanova et al 2005; Biely et al 2007; Biely 2012; Levisson et al 2012). The activity of AcXe is affected by the substrate and positional specificity, respectively.…”

Section: Microbial Enzymes Depolymerisation Of Hemicellulosementioning

confidence: 99%

Understanding the structural and functional properties of carbohydrate esterases with a special focus on hemicellulose deacetylating acetyl xylan esterases

Kameshwar

Qin

2018

Mycology

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Acetyl and methyl esterifications are two major naturally found substitutions in the plant cell-wall polysaccharides. The non-cellulosic plant cell-wall polysaccharides such as pectin and hemicellulose are differentially esterified by the O-acetyl and methyl groups to cease the action of various hydrolytic enzymes secreted by different fungi and bacterial species. Thus, microorganisms have emerged with a special class of enzymes known as carbohydrate esterases (CE). The CE catalyse O-de, N-deacetylation of acetylated saccharide residues (esters or amides, where sugars play the role of alcohol/amine/acid). Carbohydrate active enzyme (CAZy) database has classified CE into 16 classes, of which hemicellulose deacetylating CE were grouped into eight classes (CE-1 to CE-7 and CE-16). Various plant biomass degrading fungi and bacteria secretes acetyl xylan esterases (AcXE); however, these enzymes exhibit varied substrate specificities. AcXE and xylanases-coupled pretreatment methods exhibit significant applications, such as enhancing animal feedstock, baking industry, production of food additives, paper and pulp, xylitol production and biorefinery-based industries, respectively. Thus, understanding the structural and functional properties of acetyl xylan esterase will significantly aid in developing the efficient AcXE with wide range of industrial applications.

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“…Acetylxylan and xylooligosaccharides are deesterified by acetylxylan esterases (EC 3.1.1.72). These enzymes have been found and isolated from many genera, including Trichoderma [4], Aspergillus [5], Penicillium [6], Schizophyllum [7], Rhodotorula [8], Streptomyces [9], Fibrobacter [10], Bacillus [11], Hypocrea [12], Volvariella [13], Neocallimastix [14] and Thermobifida [15]. …”

Section: Introductionmentioning

confidence: 99%

Constitutive Expression of Thermobifida fusca Thermostable Acetylxylan Esterase Gene in Pichia pastoris

Yang

Lin

Chen

et al. 2010

IJMS

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A gene encoding the thermostable acetylxylan esterase (AXE) in Thermobifida fusca NTU22 was amplified by PCR, sequenced and cloned into the Pichia pastoris X-33 host strain using the vector pGAPZαA, allowing constitutive expression and secretion of the protein. Recombinant expression resulted in high levels of extracellular AXE production, as high as 526 U/mL in the Hinton flask culture broth. The purified enzyme showed a single band at about 28 kDa by SDS-polyacrylamide gel electrophoresis after being treated with endo-β-N-acetylglycosaminidase H; this agrees with the predicted size based on the nucleotide sequence. About 70% of the original activity remained after heat treatment at 60 °C for three hours. The optimal pH and temperature of the purified enzyme were 8.0 and 60 °C, respectively. The properties of the purified AXE from the P. pastoris transformant are similar to those of the AXE from an E. coli transformant.

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Heterologous expression, purification, crystallization, X-ray analysis and phasing of the acetyl xylan esterase from Bacillus pumilus

Cited by 17 publications

References 34 publications

Enzymatic Systems for Cellulose Acetate Degradation

Enzymatic Systems for Cellulose Acetate Degradation

Understanding the structural and functional properties of carbohydrate esterases with a special focus on hemicellulose deacetylating acetyl xylan esterases

Constitutive Expression of Thermobifida fusca Thermostable Acetylxylan Esterase Gene in Pichia pastoris

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