title: Engineering a thermostable Halothermothrix orenii β-glucosidase for improved galacto-oligosaccharide synthesis.
abstract: Lactose is produced in large amounts as a by-product from the dairy industry. This inexpensive disaccharide can be converted to more useful value-added products such as galacto-oligosaccharides (GOSs) by transgalactosylation reactions with retaining β-galactosidases (BGALs) being normally used for this purpose. Hydrolysis is always competing with the transglycosylation reaction, and hence, the yields of GOSs can be too low for industrial use. We have reported that a β-glucosidase from Halothermothrix orenii (HoBGLA) shows promising characteristics for lactose conversion and GOS synthesis. Here, we engineered HoBGLA to investigate the possibility to further improve lactose conversion and GOS production. Five variants that targeted the glycone (-1) and aglycone (+1) subsites (N222F, N294T, F417S, F417Y, and Y296F) were designed and expressed. All variants show significantly impaired catalytic activity with cellobiose and lactose as substrates. Particularly, F417S is hydrolytically crippled with cellobiose as substrate with a 1000-fold decrease in apparent k cat, but to a lesser extent affected when catalyzing hydrolysis of lactose (47-fold lower k cat). This large selective effect on cellobiose hydrolysis is manifested as a change in substrate selectivity from cellobiose to lactose. The least affected variant is F417Y, which retains the capacity to hydrolyze both cellobiose and lactose with the same relative substrate selectivity as the wild type, but with ~10-fold lower turnover numbers. Thin-layer chromatography results show that this effect is accompanied by synthesis of a particular GOS product in higher yields by Y296F and F417S compared with the other variants, whereas the variant F417Y produces a higher yield of total GOSs.
authors.affiliation: AlbaNova University Center, School of Biotechnology, KTH Royal Institute of Technology, Roslagstullsbacken 21, S-10691, Stockholm, Sweden.
authors.forename: Noor
authors.initials: N
authors.lastname: Hassan
authors.affiliation: Food Biotechnology Laboratory, BOKU-University of Natural Resources and Life Sciences Vienna, 1190, Vienna, Austria.
authors.forename: Barbara
authors.initials: B
authors.lastname: Geiger
authors.affiliation: AlbaNova University Center, School of Biotechnology, KTH Royal Institute of Technology, Roslagstullsbacken 21, S-10691, Stockholm, Sweden.|Department of Medical Biochemistry and Biophysics, Karolinska Institute, Scheelelaboratoriet, Scheeles väg 2, S-17177, Stockholm, Sweden.
authors.forename: Rosaria
authors.initials: R
authors.lastname: Gandini
authors.affiliation: Microbial Gene Research and Resources Facility, School of Biomolecular and Physical Sciences, Griffith University, Brisbane, QLD 4111, Australia.
authors.forename: Bharat K C
authors.initials: BK
authors.lastname: Patel
authors.affiliation: Food Biotechnology Laboratory, BOKU-University of Natural Resources and Life Sciences Vienna, 1190, Vienna, Austria.
authors.forename: Roman
authors.initials: R
authors.lastname: Kittl
authors.affiliation: Food Biotechnology Laboratory, BOKU-University of Natural Resources and Life Sciences Vienna, 1190, Vienna, Austria.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Haltrich
authors.affiliation: Food Biotechnology Laboratory, BOKU-University of Natural Resources and Life Sciences Vienna, 1190, Vienna, Austria.
authors.forename: Thu-Ha
authors.initials: TH
authors.lastname: Nguyen
authors.affiliation: AlbaNova University Center, School of Biotechnology, KTH Royal Institute of Technology, Roslagstullsbacken 21, S-10691, Stockholm, Sweden.|Department of Medical Biochemistry and Biophysics, Karolinska Institute, Scheelelaboratoriet, Scheeles väg 2, S-17177, Stockholm, Sweden.
authors.forename: Christina
authors.initials: C
authors.lastname: Divne
authors.affiliation: AlbaNova University Center, School of Biotechnology, KTH Royal Institute of Technology, Roslagstullsbacken 21, S-10691, Stockholm, Sweden. tantc@kth.se.|Department of Medical Biochemistry and Biophysics, Karolinska Institute, Scheelelaboratoriet, Scheeles väg 2, S-17177, Stockholm, Sweden. tantc@kth.se.
authors.forename: Tien Chye
authors.initials: TC
authors.lastname: Tan
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism*; D044222:Halothiobacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D009844:Oligosaccharides / Q000096:biosynthesis*; D015202:Protein Engineering; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D009844:Oligosaccharides; D001617:beta-Glucosidase; D007785:Lactose; D005690:Galactose
keywords: Galacto-oligosaccharides; Halothermophile; Halothermothrix; Lactose conversion; Transglycosylation mutants; β-Galactosidase; β-Glucosidase