caa a22 4500 605503737 CHVBK 20210128100612.0 cr unu---uuuuu 210128e20150201xx s 000 0 eng 10.1007/s00253-014-6015-x doi (NATIONALLICENCE)springer-10.1007/s00253-014-6015-x Biochemical and structural characterization of a thermostable β -glucosidase from Halothermothrix orenii for galacto-oligosaccharide synthesis [Elektronische Daten] [Noor Hassan, Thu-Ha Nguyen, Montira Intanon, Lokesh Kori, Bharat Patel, Dietmar Haltrich, Christina Divne, Tien Tan] 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-Galp-(1→6)-d-Lac (6GALA) and β-d-Galp-(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. The Author(s), 2014 β -glucosidase nationallicence β -galactosidase nationallicence Halothermophile nationallicence Halothermothrix nationallicence Lactose conversion nationallicence Galacto-oligosaccharides nationallicence Biochemical characterization nationallicence Structural analysis nationallicence Hassan Noor KTH Royal Institute of Technology, School of Biotechnology, Albanova University Center, Roslagstullsbacken 21, S-10691, Stockholm, Sweden aut Nguyen Thu-Ha Food Biotechnology Laboratory, BOKU University of Natural Resources and Life Sciences, A-1190, Vienna, Austria aut Intanon Montira Food Biotechnology Laboratory, BOKU University of Natural Resources and Life Sciences, A-1190, Vienna, Austria aut Kori Lokesh Microbial Gene Research and Resources Facility, Griffith University, School of Biomolecular and Physical Sciences, 4111, Brisbane, QLD, Australia aut Patel Bharat Microbial Gene Research and Resources Facility, Griffith University, School of Biomolecular and Physical Sciences, 4111, Brisbane, QLD, Australia aut Haltrich Dietmar Food Biotechnology Laboratory, BOKU University of Natural Resources and Life Sciences, A-1190, Vienna, Austria aut Divne Christina KTH Royal Institute of Technology, School of Biotechnology, Albanova University Center, Roslagstullsbacken 21, S-10691, Stockholm, Sweden aut Tan Tien KTH Royal Institute of Technology, School of Biotechnology, Albanova University Center, Roslagstullsbacken 21, S-10691, Stockholm, Sweden aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/4(2015-02-01), 1731-1744 0175-7598 99:4<1731 2015 99 253 https://doi.org/10.1007/s00253-014-6015-x text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-014-6015-x text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Hassan Noor KTH Royal Institute of Technology, School of Biotechnology, Albanova University Center, Roslagstullsbacken 21, S-10691, Stockholm, Sweden aut NATIONALLICENCE 700 1- Nguyen Thu-Ha Food Biotechnology Laboratory, BOKU University of Natural Resources and Life Sciences, A-1190, Vienna, Austria aut NATIONALLICENCE 700 1- Intanon Montira Food Biotechnology Laboratory, BOKU University of Natural Resources and Life Sciences, A-1190, Vienna, Austria aut NATIONALLICENCE 700 1- Kori Lokesh Microbial Gene Research and Resources Facility, Griffith University, School of Biomolecular and Physical Sciences, 4111, Brisbane, QLD, Australia aut NATIONALLICENCE 700 1- Patel Bharat Microbial Gene Research and Resources Facility, Griffith University, School of Biomolecular and Physical Sciences, 4111, Brisbane, QLD, Australia aut NATIONALLICENCE 700 1- Haltrich Dietmar Food Biotechnology Laboratory, BOKU University of Natural Resources and Life Sciences, A-1190, Vienna, Austria aut NATIONALLICENCE 700 1- Divne Christina KTH Royal Institute of Technology, School of Biotechnology, Albanova University Center, Roslagstullsbacken 21, S-10691, Stockholm, Sweden aut NATIONALLICENCE 700 1- Tan Tien KTH Royal Institute of Technology, School of Biotechnology, Albanova University Center, Roslagstullsbacken 21, S-10691, Stockholm, Sweden aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/4(2015-02-01), 1731-1744 0175-7598 99:4<1731 2015 99 253