Analysis of an industrial production suspension of Bacillus lentus subtilisin crystals by powder diffraction: a powerful quality-control tool.
Acta Crystallogr D Biol Crystallogr
; 70(Pt 4): 1115-23, 2014 Apr.
Article
em En
| MEDLINE
| ID: mdl-24699655
ABSTRACT
A microcrystalline suspension of Bacillus lentus subtilisin (Savinase) produced during industrial large-scale production was analysed by X-ray powder diffraction (XRPD) and X-ray single-crystal diffraction (MX). XRPD established that the bulk microcrystal sample representative of the entire production suspension corresponded to space group P212121, with unit-cell parameters a = 47.65, b = 62.43, c = 75.74â
Å, equivalent to those for a known orthorhombic crystal form (PDB entry 1ndq). MX using synchrotron beamlines at the Diamond Light Source with beam dimensions of 20 × 20â
µm was subsequently used to study the largest crystals present in the suspension, with diffraction data being collected from two single crystals (â¼20 × 20 × 60â
µm) to resolutions of 1.40 and 1.57â
Å, respectively. Both structures also belonged to space group P2(1)2(1)2(1), but were quite distinct from the dominant form identified by XRPD, with unit-cell parameters a = 53.04, b = 57.55, c = 71.37â
Å and a = 52.72, b = 57.13, c = 65.86â
Å, respectively, and refined to R = 10.8% and Rfree = 15.5% and to R = 14.1% and Rfree = 18.0%, respectively. They are also different from any of the forms previously reported in the PDB. A controlled crystallization experiment with a highly purified Savinase sample allowed the growth of single crystals of the form identified by XRPD; their structure was solved and refined to a resolution of 1.17â
Å with an R of 9.2% and an Rfree of 11.8%. Thus, there are at least three polymorphs present in the production suspension, albeit with the 1ndq-like microcrystals predominating. It is shown how the two techniques can provide invaluable and complementary information for such a production suspension and it is proposed that XRPD provides an excellent quality-control tool for such suspensions.
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01-internacional
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MEDLINE
Assunto principal:
Bacillus
/
Subtilisina
/
Difração de Pó
Idioma:
En
Revista:
Acta Crystallogr D Biol Crystallogr
Ano de publicação:
2014
Tipo de documento:
Article
País de afiliação:
Dinamarca