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Crystals 2018, 8(1), 13; https://doi.org/10.3390/cryst8010013

In Situ Random Microseeding and Streak Seeding Used for Growth of Crystals of Cold-Adapted β-d-Galactosidases: Crystal Structure of βDG from Arthrobacter sp. 32cB

1
Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, Stefanowskiego 4/10, 90-924 Lodz, Poland
2
Department of Molecular Biotechnology and Microbiology, Faculty of Chemistry, Gdansk University of Technology, Narutowicza 11/12, 80-233 Gdansk, Poland
*
Author to whom correspondence should be addressed.
Received: 29 November 2017 / Revised: 28 December 2017 / Accepted: 29 December 2017 / Published: 1 January 2018
(This article belongs to the Special Issue Biological and Biogenic Crystallization)
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Abstract

There is an increasing demand for cold-adapted enzymes in a wide range of industrial branches. Nevertheless, structural information about them is still scarce. The knowledge of crystal structures is important to understand their mode of action and to design genetically engineered enzymes with enhanced activity. The most difficult task and the limiting step in structural studies of cold-adapted enzymes is their crystallization, which should provide well-diffracting monocrystals. Herein, we present a combination of well-established crystallization methods with new protocols based on crystal seeding that allowed us to obtain well-diffracting crystals of two cold-adapted β-d-galactosidases (βDGs) from Paracoccus sp. 32d (ParβDG) and from Arthrobacter sp. 32cB (ArthβDG). Structural studies of both βDGs are important for designing efficient and inexpensive enzymatic tools for lactose removal and synthesis of galacto-oligosaccharides (GOS) and hetero-oligosaccharides (HOS), food additives proved to have a beneficial effect on the human immune system and intestinal flora. We also present the first crystal structure of ArthβDG (PDB ID: 6ETZ) determined at 1.9 Å resolution, and compare it to the ParβDG structure (PDB ID: 5EUV). In contrast to tetrameric lacZ βDG and hexameric βDG from Arthrobacter C2-2, both of these βDGs are dimers, unusual for the GH2 family. Additionally, we discuss the various crystallization seeding protocols, which allowed us to obtain ParβDG and ArthβDG monocrystals suitable for diffraction experiments. View Full-Text
Keywords: β-d-galactosidase; cold-adapted; lactose removal; microseeding; protein crystallization; crystal structure β-d-galactosidase; cold-adapted; lactose removal; microseeding; protein crystallization; crystal structure
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Rutkiewicz-Krotewicz, M.; Pietrzyk-Brzezinska, A.J.; Wanarska, M.; Cieslinski, H.; Bujacz, A. In Situ Random Microseeding and Streak Seeding Used for Growth of Crystals of Cold-Adapted β-d-Galactosidases: Crystal Structure of βDG from Arthrobacter sp. 32cB. Crystals 2018, 8, 13.

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