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Characterization and engineering of carbohydrate-active enzymes for biotechnological applications
KTH, School of Biotechnology (BIO), Industrial Biotechnology. (Christina Divne)
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.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:8
Keyword [en]
se conversion, galacto-oligosaccharides, thermostability, propeptide
National Category
Structural Biology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-165613ISBN: 978-91-7595-511-7 (print)OAI: oai:DiVA.org:kth-165613DiVA: diva2:808679
Public defence
2015-05-26, FB55, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

QC 20150429

Available from: 2015-04-29 Created: 2015-04-29 Last updated: 2015-04-29Bibliographically approved
List of papers
1. Biochemical and structural characterization of a thermostable beta-glucosidase from Halothermothrix orenii for galacto-oligosaccharide synthesis
Open this publication in new window or tab >>Biochemical and structural characterization of a thermostable beta-glucosidase from Halothermothrix orenii for galacto-oligosaccharide synthesis
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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.

Keyword
beta-glucosidase, beta-galactosidase, Halothermophile, Halothermothrix, Lactose conversion, Galacto-oligosaccharides, Biochemical characterization, Structural analysis
National Category
Microbiology
Identifiers
urn:nbn:se:kth:diva-162963 (URN)10.1007/s00253-014-6015-x (DOI)000350028600017 ()25173693 (PubMedID)2-s2.0-84922434915 (Scopus ID)
Funder
Swedish Research Council
Note

QC 20150331

Available from: 2015-03-31 Created: 2015-03-26 Last updated: 2017-12-04Bibliographically approved
2. Engineering a polyextremophilic Halothermothrix orenii β-glucosidase for improved galacto-oligosaccharide synthesis
Open this publication in new window or tab >>Engineering a polyextremophilic Halothermothrix orenii β-glucosidase for improved galacto-oligosaccharide synthesis
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(English)Manuscript (preprint) (Other academic)
National Category
Natural Sciences
Identifiers
urn:nbn:se:kth:diva-165617 (URN)
Note

QS 2015

Available from: 2015-04-29 Created: 2015-04-29 Last updated: 2015-04-29Bibliographically approved
3. High-resolution crystal structure of a polyextreme GH43 glycosidase from Halothermothrix orenii with alpha-L-arabinofuranosidase activity
Open this publication in new window or tab >>High-resolution crystal structure of a polyextreme GH43 glycosidase from Halothermothrix orenii with alpha-L-arabinofuranosidase activity
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2015 (English)In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 71, no Pt 3, 338-45 p.Article in journal (Refereed) Published
Abstract [en]

A gene from the heterotrophic, halothermophilic marine bacterium Halothermothrix orenii has been cloned and overexpressed in Escherichia coli. This gene encodes the only glycoside hydrolase of family 43 (GH43) produced by H. orenii. The crystal structure of the H. orenii glycosidase was determined by molecular replacement and refined at 1.10Å resolution. As for other GH43 members, the enzyme folds as a five-bladed β-propeller. The structure features a metal-binding site on the propeller axis, near the active site. Based on thermal denaturation data, the H. orenii glycosidase depends on divalent cations in combination with high salt for optimal thermal stability against unfolding. A maximum melting temperature of 76°C was observed in the presence of 4M NaCl and Mn2+ at pH 6.5. The gene encoding the H. orenii GH43 enzyme has previously been annotated as a putative α-l-arabinofuranosidase. Activity was detected with p-nitrophenyl-α-l-arabinofuranoside as a substrate, and therefore the name HoAraf43 was suggested for the enzyme. In agreement with the conditions for optimal thermal stability against unfolding, the highest arabinofuranosidase activity was obtained in the presence of 4M NaCl and Mn2+ at pH 6.5, giving a specific activity of 20-36μmolmin-1mg-1. The active site is structurally distinct from those of other GH43 members, including arabinanases, arabinofuranosidases and xylanases. This probably reflects the special requirements for degrading the unique biomass available in highly saline aqueous ecosystems, such as halophilic algae and halophytes. The amino-acid distribution of HoAraf43 has similarities to those of mesophiles, thermophiles and halophiles, but also has unique features, for example more hydrophobic amino acids on the surface and fewer buried charged residues.

Place, publisher, year, edition, pages
International Union of Crystallography, 2015
Keyword
glycoside hydrolase, five-bladed beta-propeller, arabinofuranosidase, Halothermothrix orenii, halothermophile
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-164102 (URN)10.1107/S2053230X15003337 (DOI)000351157900016 ()25760712 (PubMedID)2-s2.0-84924655292 (Scopus ID)
Funder
Swedish Research Council Formas, 2013-1741Swedish Research Council, 2013-5717
Note

QC 20150419

Available from: 2015-04-13 Created: 2015-04-13 Last updated: 2017-12-04Bibliographically approved
4. Crystal structures of Phanerochaete chrysosporium pyranose 2-oxidase suggest that the N-terminus acts as a propeptide that assists in homotetramer assembly
Open this publication in new window or tab >>Crystal structures of Phanerochaete chrysosporium pyranose 2-oxidase suggest that the N-terminus acts as a propeptide that assists in homotetramer assembly
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2013 (English)In: FEBS Open Bio, E-ISSN 2211-5463, Vol. 3, 496-504 p.Article in journal (Refereed) Published
Abstract [en]

The flavin-dependent homotetrameric enzyme pyranose 2-oxidase (P2O) is found mostly, but not exclusively, in lignocellulose-degrading fungi where it catalyzes the oxidation of β-. d-glucose to the corresponding 2-keto sugar concomitantly with hydrogen peroxide formation during lignin solubilization. Here, we present crystal structures of P2O from the efficient lignocellulolytic basidiomycete Phanerochaete chrysosporium. Structures were determined of wild-type PcP2O from the natural fungal source, and two variants of recombinant full-length PcP2O, both in complex with the slow substrate 3-deoxy-3-fluoro-. β-. d-glucose. The active sites in PcP2O and P2O from Trametes multicolor (TmP2O) are highly conserved with identical substrate binding. Our structural analysis suggests that the 17°C higher melting temperature of PcP2O compared to TmP2O is due to an increased number of intersubunit salt bridges. The structure of recombinant PcP2O expressed with its natural N-terminal sequence, including a proposed propeptide segment, reveals that the first five residues of the propeptide intercalate at the interface between A and B subunits to form stabilizing, mainly hydrophobic, interactions. In the structure of mature PcP2O purified from the natural source, the propeptide segment in subunit A has been replaced by a nearby loop in the B subunit. We propose that the propeptide in subunit A stabilizes the A/B interface of essential dimers in the homotetramer and that, upon maturation, it is replaced by the loop in the B subunit to form the mature subunit interface. This would imply that the propeptide segment of PcP2O acts as an intramolecular chaperone for oligomerization at the A/B interface of the essential dimer.

Keyword
Crystal structure, Lignin degradation, Oligomerization, Propeptide, Pyranose 2-oxidase, Thermostability
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-139900 (URN)10.1016/j.fob.2013.10.010 (DOI)000339569800078 ()2-s2.0-84888118395 (Scopus ID)
Funder
Swedish Research Council, 2008-4045 2011-5768
Note

QC 20140115

Available from: 2014-01-15 Created: 2014-01-15 Last updated: 2017-12-06Bibliographically approved

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