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      Biochemical and structural characterization of a thermostable β-glucosidase from Halothermothrix orenii for galacto-oligosaccharide synthesis

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          Abstract

          Lactose is a major disaccharide by-product from the dairy industries, and production of whey alone amounts to about 200 million tons globally each year. Thus, it is of particular interest to identify improved enzymatic processes for lactose utilization. Microbial β-glucosidases (BGL) with significant β-galactosidase (BGAL) activity can be used to convert lactose to glucose (Glc) and galactose (Gal), and most retaining BGLs also synthesize more complex sugars from the monosaccharides by transglycosylation, such as galacto-oligosaccharides (GOS), which are prebiotic compounds that stimulate growth of beneficial gut bacteria. In this work, a BGL from the thermophilic and halophilic bacterium Halothermothrix orenii, HoBGLA, was characterized biochemically and structurally. It is an unspecific β-glucosidase with mixed activities for different substrates and prominent activity with various galactosidases such as lactose. We show that HoBGLA is an attractive candidate for industrial lactose conversion based on its high activity and stability within a broad pH range (4.5–7.5), with maximal β-galactosidase activity at pH 6.0. The temperature optimum is in the range of 65–70 °C, and HoBGLA also shows excellent thermostability at this temperature range. The main GOS products from HoBGLA transgalactosylation are β- d-Gal p-(1→6)- d-Lac (6GALA) and β- d-Gal p-(1→3)- d-Lac (3GALA), indicating that d-lactose is a better galactosyl acceptor than either of the monosaccharides. To evaluate ligand binding and guide GOS modeling, crystal structures of HoBGLA were determined in complex with thiocellobiose, 2-deoxy-2-fluoro- d-glucose and glucose. The two major GOS products, 3GALA and 6GALA, were modeled in the substrate-binding cleft of wild-type HoBGLA and shown to be favorably accommodated.

          Electronic supplementary material

          The online version of this article (doi:10.1007/s00253-014-6015-x) contains supplementary material, which is available to authorized users.

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          Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants

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            High-throughput production of human proteins for crystallization: The SGC experience

            Producing purified human proteins with high yield and purity remains a considerable challenge. We describe the methods utilized in the Structural Genomics Consortium (SGC) in Oxford, resulting in successful purification of 48% of human proteins attempted; of those, the structures of ∼40% were solved by X-ray crystallography. The main driver has been the parallel processing of multiple (typically 9–20) truncated constructs of each target; modest diversity in vectors and host systems; and standardized purification procedures. We provide method details as well as data on the properties of the constructs leading to crystallized proteins and the impact of methodological variants. These can be used to formulate guidelines for initial approaches to expression of new eukaryotic proteins.
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              Microbial beta-glucosidases: cloning, properties, and applications.

              Beta-glucosidases constitute a major group among glycosylhydrolase enzymes. Out of the 82 families classified under glycosylhydrolase category, these belong to family 1 and family 3 and catalyze the selective cleavage of glucosidic bonds. This function is pivotal in many crucial biological pathways, such as degradation of structural and storage polysaccharides, cellular signaling, oncogenesis, host-pathogen interactions, as well as in a number of biotechnological applications. In recent years, interest in these enzymes has gained momentum owing to their biosynthetic abilities. The enzymes exhibit utility in syntheses of diverse oligosaccharides, glycoconjugates, alkyl- and aminoglucosides. Attempts are being made to understand the structure-function relationship of these versatile biocatalysts. Earlier reviews described the sources and properties of microbial beta-glucosidases, yeast beta-glucosidases, thermostable fungal beta-glucosidase, and the physiological functions, characteristics, and catalytic action of native beta-glucosidases from various plant, animal, and microbial sources. Recent efforts have been directed towards molecular cloning, sequencing, mutagenesis, and crystallography of the enzymes. The aim of the present article is to describe the sources and properties of recombinant beta-glucosidases, their classification schemes based on similarity at the structural and molecular levels, elucidation of structure-function relationships, directed evolution of existing enzymes toward enhanced thermostability, substrate range, biosynthetic properties, and applications.
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                Author and article information

                Contributors
                +46-73-8425695 , tantc@kth.se
                Journal
                Appl Microbiol Biotechnol
                Appl. Microbiol. Biotechnol
                Applied Microbiology and Biotechnology
                Springer Berlin Heidelberg (Berlin/Heidelberg )
                0175-7598
                1432-0614
                31 August 2014
                31 August 2014
                2015
                : 99
                : 1731-1744
                Affiliations
                [ ]KTH Royal Institute of Technology, School of Biotechnology, Albanova University Center, Roslagstullsbacken 21, S-10691 Stockholm, Sweden
                [ ]Food Biotechnology Laboratory, BOKU University of Natural Resources and Life Sciences, A-1190 Vienna, Austria
                [ ]Microbial Gene Research and Resources Facility, Griffith University, School of Biomolecular and Physical Sciences, Brisbane, QLD 4111 Australia
                [ ]Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030 USA
                [ ]Department of Medical Biochemistry and Biophysics, Scheelelaboratoriet, Karolinska Institute, Scheeles väg 2, S-17177 Stockholm, Sweden
                Article
                6015
                10.1007/s00253-014-6015-x
                4322223
                25173693
                ae00f6d0-925f-4223-8133-2f7aa55644df
                © The Author(s) 2014

                Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

                History
                : 21 May 2014
                : 4 August 2014
                : 5 August 2014
                Categories
                Biotechnologically Relevant Enzymes and Proteins
                Custom metadata
                © Springer-Verlag Berlin Heidelberg 2015

                Biotechnology
                β-glucosidase,β-galactosidase,halothermophile,halothermothrix,lactose conversion,galacto-oligosaccharides,biochemical characterization,structural analysis

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