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Biochemical and structural characterization of a thermostable beta-glucosidase from Halothermothrix orenii for galacto-oligosaccharide synthesis
KTH, School of Biotechnology (BIO).
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2015 (English)In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 99, no 4, 1731-1744 p.Article in journal (Refereed) Published
Abstract [en]

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 beta-glucosidases (BGL) with significant beta-galactosidase (BGAL) activity can be used to convert lactose to glucose (Glc) and galactose (Gal), and most retaining BGLs also synthesizemore 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 beta-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 beta-galactosidase activity at pH 6.0. The temperature optimum is in the range of 65-70 degrees C, and HoBGLA also shows excellent thermostability at this temperature range. The main GOS products from HoBGLA transgalactosylation are beta-D-Galp-(1 -> 6)-D-Lac (6GALA) and beta-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.

Place, publisher, year, edition, pages
2015. Vol. 99, no 4, 1731-1744 p.
Keyword [en]
beta-glucosidase, beta-galactosidase, Halothermophile, Halothermothrix, Lactose conversion, Galacto-oligosaccharides, Biochemical characterization, Structural analysis
National Category
URN: urn:nbn:se:kth:diva-162963DOI: 10.1007/s00253-014-6015-xISI: 000350028600017PubMedID: 25173693ScopusID: 2-s2.0-84922434915OAI: diva2:799790
Swedish Research Council

QC 20150331

Available from: 2015-03-31 Created: 2015-03-26 Last updated: 2015-04-29Bibliographically approved
In thesis
1. Characterization and engineering of carbohydrate-active enzymes for biotechnological applications
Open this publication in new window or tab >>Characterization and engineering of carbohydrate-active enzymes for biotechnological applications
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Extremozymes are enzymes produced by microorganisms that live in extreme habitats. Due to their higher stability, extremozymes is attracting interest as biocatalysts in various industrial processes. In this context, carbohydrate-active extremozymes can be used in various processes relevant to the paper, food and feed industry.

In this thesis, the crystal structure, biochemical characterization and the capacity to synthesize prebiotic galacto-oligosaccharides (GOS) were investigated for a β-glucosidase (HoBGLA) from the halothermophilic bacterium Halothermothrix orenii. The wild-type enzyme displays favorable characteristics for lactose hydrolysis and produces a range of prebiotic GOS, of which β-D-Galp-(1→6)-D-Lac and β-D-Galp-(1→3)-D-Lac are the major products (Paper I).

To further improve GOS synthesis by HoBGLA, rational enzyme engineering was performed (Paper II). Six enzyme variants were generated by replacing strategically positioned active-site residues. Two HoBGLA variants were identified as potentially interesting, F417S and F417Y. The former appears to synthesize one particular GOS product in higher yield, whereas the latter produces a higher yield of total GOS.

In Paper III, the high-resolution crystal structure and biochemical characterization of a hemicellulase (HoAraf43) from  H. orenii is presented. HoAraf43 folds as a five-bladed β-propeller and displays α-Larabinofuranosidase activity. The melting temperature of  HoAraf43 increases significantly in the presence of high salt and divalent cations, which is consistent with H. orenii being a halophile.

Furthermore, the crystal structures of a thermostable tetrameric pyranose 2-oxidase from Phanerochaete chrysosporium (PcP2O) were determined to investigate the structural determinants of thermostability (Paper IV). PcP2O has an increased number of salt links between subunits, which may provide a mechanism for increased stability. The structures also imply that the N-terminal region acts as an intramolecular chaperone during homotetramer assembly.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2015. 57 p.
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:8
se conversion, galacto-oligosaccharides, thermostability, propeptide
National Category
Structural Biology
Research subject
urn:nbn:se:kth:diva-165613 (URN)978-91-7595-511-7 (ISBN)
Public defence
2015-05-26, FB55, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 13:00 (English)

QC 20150429

Available from: 2015-04-29 Created: 2015-04-29 Last updated: 2015-04-29Bibliographically approved

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