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

Maria Rutkiewicz-Krotewicz,Agnieszka Pietrzyk-Brzezinska,Marta Wanarska,Anna Bujacz,Hubert Cieslinski

doi:10.3390/cryst8010013

Maria Rutkiewicz-Krotewicz, Agnieszka Pietrzyk-Brzezinska + Show 3 more

Open Access

https://doi.org/10.3390/cryst8010013

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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.

Highlights

Introduction βD-Galactosidases (EC 3.2.1.23) are widely used in the food industry as they catalyze the hydrolysis of terminal non-reducing β-D-galactose residue in β-D-galactosides
In contrast to commercially available mesophilic β-D-galactosidase from Kluyveromyces lactis, the cold-active enzyme could make it possible to reduce the risk of mesophilic contamination and save energy during the industrial process connected with lactose hydrolysis [10,11,12]
The crystal structure of ParβDG has been already reported in our previous article [36] that focused on its structural analysis (PDB ID: 5EUV)

Summary

Introduction

D-Galactosidases (EC 3.2.1.23) are widely used in the food industry as they catalyze the hydrolysis of terminal non-reducing β-D-galactose residue in β-D-galactosides. They are especially relevant in the dairy industry due to their ability to catalyze the hydrolysis of lactose, a natural substrate. Cold-active β-D-galactosidases (βDGs) have become a focus of attention because of their ability to eliminate lactose from refrigerated milk, convert lactose to glucose and galactose (decreasing its hygroscopicity), and eliminate lactose from dairy industry pollutants associated with environmental problems. In contrast to commercially available mesophilic β-D-galactosidase from Kluyveromyces lactis, the cold-active enzyme could make it possible to reduce the risk of mesophilic contamination and save energy during the industrial process connected with lactose hydrolysis [10,11,12]

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