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2dcz
From Proteopedia
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(New page: 200px<br /><applet load="2dcz" size="450" color="white" frame="true" align="right" spinBox="true" caption="2dcz, resolution 1.90Å" /> '''Thermal Stabilizatio...)
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Revision as of 07:25, 21 November 2007
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Thermal Stabilization of Bacillus subtilis Family-11 Xylanase By Directed Evolution
Overview
We used directed evolution to enhance the thermostability of glycosyl, hydrolase family-11 xylanase from Bacillus subtilis. By combining random, point mutagenesis, saturation mutagenesis, and DNA shuffling, a, thermostable variant, Xyl(st), was identified which contained three amino, acid substitutions: Q7H, N8F, and S179C. The half-inactivation temperature, (the midpoint of the melting curves) for the Xyl(st) variant compared with, the wild-type enzyme after incubation for 10 min was elevated from 58 to, 68 degrees C. At 60 degrees C the wild-type enzyme was inactivated within, 5 min, but Xyl(st) retained full activity for at least 2 h. The, stabilization was accompanied by evidence of thermophilicity; that is, an, increase in the optimal reaction temperature from 55 to 65 degrees C and, lower activity at low temperatures and higher activity at higher, temperatures relative to wild type. To elucidate the mechanism of thermal, stabilization, three-dimensional structures were determined for the, wild-type and Xyl(st) enzymes. A cavity was identified around Gln-7/Asn-8, in wild type that was filled with bulky, hydrophobic residues in Xyl(st)., This site was not identified by previous approaches, but directed, evolution identified the region as a weak point. Formation of an, intermolecular disulfide bridge via Cys-179 was observed between monomers, in Xyl(st). However, the stability was essentially the same in the, presence and absence of a reducing agent, indicating that the increased, hydrophobicity around the Cys-179 accounted for the stability.
About this Structure
2DCZ is a Single protein structure of sequence from Bacillus subtilis with SO4 and DIO as ligands. Active as Endo-1,4-beta-xylanase, with EC number 3.2.1.8 Full crystallographic information is available from OCA.
Reference
Thermal stabilization of Bacillus subtilis family-11 xylanase by directed evolution., Miyazaki K, Takenouchi M, Kondo H, Noro N, Suzuki M, Tsuda S, J Biol Chem. 2006 Apr 14;281(15):10236-42. Epub 2006 Feb 8. PMID:16467302
Page seeded by OCA on Wed Nov 21 09:32:35 2007
Categories: Bacillus subtilis | Endo-1,4-beta-xylanase | Single protein | Kondo, H. | Miyazaki, K. | Noro, N. | Suzuki, M. | Takenouchi, M. | Tsuda, S. | DIO | SO4 | All beta
