Analysis of an industrial production suspension of Bacillus lentus subtilisin crystals by powder diffraction: a powerful quality-control tool.
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 = 75.74 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 × 20 µm was subsequently used to study the largest crystals present in the suspension, with diffraction data being collected from two single crystals (∼20 × 20 × 60 µm) to resolutions of 1.40 and 1.57 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 = 71.37 Å and a = 52.72, b = 57.13, c = 65.86 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 1.17 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.
Department of Chemistry, Technical University of Denmark, Kemitorvet 207, DK-2800 Kgs. Lyngby, Denmark.
Christian G
CG
Frankaer
Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5DD, England.
Olga V
OV
Moroz
Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5DD, England.
Johan P
JP
Turkenburg
Novozymes A/S, Krogshøjvej 36, DK-2880 Bagsvaerd, Denmark.
Stein I
SI
Aspmo
Novozymes A/S, Krogshøjvej 36, DK-2880 Bagsvaerd, Denmark.
Majbritt
M
Thymark
Novozymes A/S, Krogshøjvej 36, DK-2880 Bagsvaerd, Denmark.
Esben P
EP
Friis
Department of Chemistry, Technical University of Denmark, Kemitorvet 207, DK-2800 Kgs. Lyngby, Denmark.
Kenny
K
Stahl
Novozymes A/S, Krogshøjvej 36, DK-2880 Bagsvaerd, Denmark.
Jens E
JE
Nielsen
Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5DD, England.
Keith S
KS
Wilson
Department of Chemistry, Technical University of Denmark, Kemitorvet 207, DK-2800 Kgs. Lyngby, Denmark.
Pernille
P
Harris
D001407:Bacillus / Q000201:enzymology*; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D033362:Powder Diffraction / Q000379:methods*; D017434:Protein Structure, Tertiary; D020860:Subtilisin / Q000032:analysis / Q000737:chemistry*
D020860:Subtilisin
Savinase; crystal contacts; crystal packing; microcrystalline suspension; protein polymorphs; protein powder diffraction; quality control; single-crystal analysis