Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 May 15;1748(2):222-30.
doi: 10.1016/j.bbapap.2005.01.003.

Heterologous Expression, Purification, Crystallization, X-ray Analysis and Phasing of the Acetyl Xylan Esterase From Bacillus Pumilus

Affiliations

Heterologous Expression, Purification, Crystallization, X-ray Analysis and Phasing of the Acetyl Xylan Esterase From Bacillus Pumilus

Ivet Krastanova et al. Biochim Biophys Acta. .

Abstract

Bacillus pumilus PS213 acetyl xylan esterase (AXE) acts as an accessory enzyme in the plant cell wall hemicellulose biodegradation pathway. It belongs to the carbohydrate esterase family 7 and hydrolyses the ester linkages of the acetyl groups in position 2 and/or 3 of the xylose moieties of the acetylated xylan fragments from hardwood. The enzyme displays activity towards a broad range of acetylated compounds including the antibiotic cephalosporin-C. In this study we report the heterologous expression, purification, physicochemical characterization and crystallization of the recombinant B. pumilus AXE. Remarkable improvement of the crystal quality was achieved by setting up crystallization conditions, at first established using the hanging drop vapor diffusion method, in a micro-batch experiment. Rod-like diffraction quality crystals were obtained using 10% PEG 6000, 0.1 M MES pH 6.0 and a wide range of LiCl concentrations (0.2-1.0 M) as precipitant agent. Two different crystal forms, both belonging to space group P2(1), were characterized, diffracting X-rays to 2.5 and 1.9 angstrom resolution. Successful molecular replacement showed 12 molecules in the asymmetric unit of either crystal forms that are arranged as two doughnut-like hexamers, each one encompassing a local 32 symmetry. A catalytic inactive mutant Ser181Ala of B. pumilus AXE was also engineered, expressed, purified and crystallized for functional and structural studies.

Similar articles

See all similar articles

Cited by 3 articles

Publication types

MeSH terms

LinkOut - more resources

Feedback