Exploring thermostable glycoside hydrolases: Amylases and beta-glucosidases

Pernilla Turner

Exploring thermostable glycoside hydrolases: Amylases and beta-glucosidases

Pernilla Turner

Biotechnology

Research output: Thesis › Doctoral Thesis (compilation)

Abstract

Two putative cyclodextrinases (CDases) were amplified from two moderate thermophiles isolated in Iceland: Anoxybacillus flavithermus and Laceyella sacchari. They were cloned, expressed and characterized. The expression of the former was optimized in E. coli by using a solubility-enhancing tag, NusA, and tuning of inducer concentration in the expression strain Tuner(DE3). The CDases were optimally active at around 57 ºC and had a poor thermal stability. AfCda13 had significantly higher activity for cyclodextrins and formed a dimer in solution, whereas LsCda13 existed as a monomer, which likely lowered the activity for cyclodextrins.

Two beta-glucosidases, members of glycoside hydrolase families 1 and 3 and originating from the extreme thermophile Thermotoga neapolitana were cloned and expressed in E. coli. The apparent thermal unfolding temperatures were around 90 ºC and 102 ºC for TnBgl3B and TnBgl1A, respectively. TnBgl3B hydrolyzed para-nitrophenyl-glucoside with a Vmax at 90 ºC of 93 ± 13 U/mg and a Km of 0.11 ± 0.03 mM.

TnBgl3B was tested for its transglycosylating ability by forming hexyl- and octyl-beta-D-glucoside. The selectivity of alcoholysis compared to hydrolysis was higher than for other reported enzymes. Both beta-glucosidases were also tested in reactions cleaving off glucose from quercetin-glycosides extracted from onion by subcritical water. They both performed better than the commercially available almond beta-glucosidase.

Predicted catalytic amino acids of TnBgl3B were replaced with non-catalytic glycines. The aspartate nucleophile is highly conserved in GH3 and the D242G mutant lost all activity. The acid/base residue is less conserved and two residues were mutated, Glu458 and Asp461. Mutant E458G had low but significant activity, while D461G lost almost all activity, which points to the latter residue as the catalytic acid/base of TnBgl3B. However, further analyses including structural information should confirm this.

TnBgl3B was crystallized and the structure was solved by multiple wavelength anomalous diffraction using selenomethionyl crystals in addition to native crystals. X-ray diffraction data was collected to 2.4 Å resolution and the structure has been solved to 2.7 Å. Manual model building and refinement is in progress.

Original language	English
Qualification	Doctor
Awarding Institution	Biotechnology
Supervisors/Advisors	Holst, Olle, Supervisor
Award date	2007 Feb 1
Publisher	Department of Biotechnology, Lund University
ISBN (Print)	978-91-89627-51-2
Publication status	Published - 2007

Bibliographical note

Defence details

Date: 2007-02-01
Time: 13:15
Place: Hörsal A, Kemicentrum, Getingevägen 60, Lunds Tekniska Högskola

External reviewer(s)

Name: Svensson, Birte
Title: Professor
Affiliation: Technical University of Denmark

---

<div class="article_info">Pernilla Turner, Antje Labes, Olafur H. Fridjonsson, Gudmundur O. Hreggvidson, Peter Schönheit, Jakob K. Kristjansson, Olle Holst and Eva Nordberg Karlsson. 2005. Two novel cyclodextrin-degrading enzymes isolated from thermophilic bacteria have similar domain structures but differ in oligomeric state and activity profile Journal of Bioscience and Bioengineering, vol 100 pp 380-390. Elsevier</div>
<div class="article_info">Pernilla Turner, Olle Holst and Eva Nordberg Karlsson. 2005. Optimized expression of soluble cyclomaltodextrinase of thermophilic origin in Escherichia coli by using a soluble fusion-tag and by tuning of inducer concentration Protein Expression and Purification, vol 39 pp 54-60. Elsevier</div>
<div class="article_info">Pernilla Turner, Carina Nilsson, David Svensson, Olle Holst, Lo Gorton and Eva Nordberg Karlsson. 2005. Monomeric and dimeric cyclomaltodextrinases reveal different modes of substrate degradation Biologia, Bratislava, vol 60 pp 79-87. Slovak Academic Press</div>
<div class="article_info">Pernilla Turner, David Svensson, Patrick Adlercreutz and Eva Nordberg Karlsson. 2007. A novel variant of Thermotoga neapolitana beta-glucosidase B is an efficient catalyst for the synthesis of alkyl glucosides by transglycosylation Department of Biotechnology, Lund University, Sweden (submitted)</div>
<div class="article_info">Charlotta Turner, Pernilla Turner, Gunilla Jacobson, Knut Almgren, Monica Waldebäck, Per Sjöberg, Eva Nordberg Karlsson and Karin E. Markides. 2006. Subcritical water extraction and beta-glucosidase-catalyzed hydrolysis of quercetin glycosides in onion waste Green Chemistry, vol 8 pp 949-959. Royal Society of Chemistry</div>
<div class="article_info">Pernilla Turner, Anna Lundell, Erik Kanders, Christina Wennerberg, Derek Logan and Eva Nordberg Karlsson. 2007. Crystallization, preliminary X-ray diffraction analysis and mutation of active site residues of Thermotoga neapolitana beta-glucosidase B Department of Biotechnology, Lund University, Sweden (manuscript)</div>

Subject classification (UKÄ)

Industrial Biotechnology

Free keywords

Bioteknik
Biotechnology
Proteiner
beta-glucosidase
thermostability
activity
cyclodextrinase
enzymology
crystallization
enzymologi
expression
glycoside hydrolase
Proteins
transglycosylation

Cite this

@phdthesis{df445a2a3f1e4b6d95e017e9e5fe8ae6,

title = "Exploring thermostable glycoside hydrolases: Amylases and beta-glucosidases",

abstract = "Two putative cyclodextrinases (CDases) were amplified from two moderate thermophiles isolated in Iceland: Anoxybacillus flavithermus and Laceyella sacchari. They were cloned, expressed and characterized. The expression of the former was optimized in E. coli by using a solubility-enhancing tag, NusA, and tuning of inducer concentration in the expression strain Tuner(DE3). The CDases were optimally active at around 57 ºC and had a poor thermal stability. AfCda13 had significantly higher activity for cyclodextrins and formed a dimer in solution, whereas LsCda13 existed as a monomer, which likely lowered the activity for cyclodextrins. Two beta-glucosidases, members of glycoside hydrolase families 1 and 3 and originating from the extreme thermophile Thermotoga neapolitana were cloned and expressed in E. coli. The apparent thermal unfolding temperatures were around 90 ºC and 102 ºC for TnBgl3B and TnBgl1A, respectively. TnBgl3B hydrolyzed para-nitrophenyl-glucoside with a Vmax at 90 ºC of 93 ± 13 U/mg and a Km of 0.11 ± 0.03 mM. TnBgl3B was tested for its transglycosylating ability by forming hexyl- and octyl-beta-D-glucoside. The selectivity of alcoholysis compared to hydrolysis was higher than for other reported enzymes. Both beta-glucosidases were also tested in reactions cleaving off glucose from quercetin-glycosides extracted from onion by subcritical water. They both performed better than the commercially available almond beta-glucosidase. Predicted catalytic amino acids of TnBgl3B were replaced with non-catalytic glycines. The aspartate nucleophile is highly conserved in GH3 and the D242G mutant lost all activity. The acid/base residue is less conserved and two residues were mutated, Glu458 and Asp461. Mutant E458G had low but significant activity, while D461G lost almost all activity, which points to the latter residue as the catalytic acid/base of TnBgl3B. However, further analyses including structural information should confirm this. TnBgl3B was crystallized and the structure was solved by multiple wavelength anomalous diffraction using selenomethionyl crystals in addition to native crystals. X-ray diffraction data was collected to 2.4 {\AA} resolution and the structure has been solved to 2.7 {\AA}. Manual model building and refinement is in progress.",

keywords = "Bioteknik, Biotechnology, Proteiner, beta-glucosidase, thermostability, activity, cyclodextrinase, enzymology, crystallization, enzymologi, expression, glycoside hydrolase, Proteins, transglycosylation",

author = "Pernilla Turner",

note = "Defence details Date: 2007-02-01 Time: 13:15 Place: H{\"o}rsal A, Kemicentrum, Getingev{\"a}gen 60, Lunds Tekniska H{\"o}gskola External reviewer(s) Name: Svensson, Birte Title: Professor Affiliation: Technical University of Denmark --- <div class={"}article_info{"}>Pernilla Turner, Antje Labes, Olafur H. Fridjonsson, Gudmundur O. Hreggvidson, Peter Sch{\"o}nheit, Jakob K. Kristjansson, Olle Holst and Eva Nordberg Karlsson. 2005. Two novel cyclodextrin-degrading enzymes isolated from thermophilic bacteria have similar domain structures but differ in oligomeric state and activity profile Journal of Bioscience and Bioengineering, vol 100 pp 380-390. Elsevier</div> <div class={"}article_info{"}>Pernilla Turner, Olle Holst and Eva Nordberg Karlsson. 2005. Optimized expression of soluble cyclomaltodextrinase of thermophilic origin in Escherichia coli by using a soluble fusion-tag and by tuning of inducer concentration Protein Expression and Purification, vol 39 pp 54-60. Elsevier</div> <div class={"}article_info{"}>Pernilla Turner, Carina Nilsson, David Svensson, Olle Holst, Lo Gorton and Eva Nordberg Karlsson. 2005. Monomeric and dimeric cyclomaltodextrinases reveal different modes of substrate degradation Biologia, Bratislava, vol 60 pp 79-87. Slovak Academic Press</div> <div class={"}article_info{"}>Pernilla Turner, David Svensson, Patrick Adlercreutz and Eva Nordberg Karlsson. 2007. A novel variant of Thermotoga neapolitana beta-glucosidase B is an efficient catalyst for the synthesis of alkyl glucosides by transglycosylation Department of Biotechnology, Lund University, Sweden (submitted)</div> <div class={"}article_info{"}>Charlotta Turner, Pernilla Turner, Gunilla Jacobson, Knut Almgren, Monica Waldeb{\"a}ck, Per Sj{\"o}berg, Eva Nordberg Karlsson and Karin E. Markides. 2006. Subcritical water extraction and beta-glucosidase-catalyzed hydrolysis of quercetin glycosides in onion waste Green Chemistry, vol 8 pp 949-959. Royal Society of Chemistry</div> <div class={"}article_info{"}>Pernilla Turner, Anna Lundell, Erik Kanders, Christina Wennerberg, Derek Logan and Eva Nordberg Karlsson. 2007. Crystallization, preliminary X-ray diffraction analysis and mutation of active site residues of Thermotoga neapolitana beta-glucosidase B Department of Biotechnology, Lund University, Sweden (manuscript)</div>",

year = "2007",

language = "English",

isbn = "978-91-89627-51-2",

publisher = "Department of Biotechnology, Lund University",

type = "Doctoral Thesis (compilation)",

school = "Biotechnology",

}

TY - THES

T1 - Exploring thermostable glycoside hydrolases: Amylases and beta-glucosidases

AU - Turner, Pernilla

N1 - Defence details Date: 2007-02-01 Time: 13:15 Place: Hörsal A, Kemicentrum, Getingevägen 60, Lunds Tekniska Högskola External reviewer(s) Name: Svensson, Birte Title: Professor Affiliation: Technical University of Denmark --- <div class="article_info">Pernilla Turner, Antje Labes, Olafur H. Fridjonsson, Gudmundur O. Hreggvidson, Peter Schönheit, Jakob K. Kristjansson, Olle Holst and Eva Nordberg Karlsson. 2005. Two novel cyclodextrin-degrading enzymes isolated from thermophilic bacteria have similar domain structures but differ in oligomeric state and activity profile Journal of Bioscience and Bioengineering, vol 100 pp 380-390. Elsevier</div> <div class="article_info">Pernilla Turner, Olle Holst and Eva Nordberg Karlsson. 2005. Optimized expression of soluble cyclomaltodextrinase of thermophilic origin in Escherichia coli by using a soluble fusion-tag and by tuning of inducer concentration Protein Expression and Purification, vol 39 pp 54-60. Elsevier</div> <div class="article_info">Pernilla Turner, Carina Nilsson, David Svensson, Olle Holst, Lo Gorton and Eva Nordberg Karlsson. 2005. Monomeric and dimeric cyclomaltodextrinases reveal different modes of substrate degradation Biologia, Bratislava, vol 60 pp 79-87. Slovak Academic Press</div> <div class="article_info">Pernilla Turner, David Svensson, Patrick Adlercreutz and Eva Nordberg Karlsson. 2007. A novel variant of Thermotoga neapolitana beta-glucosidase B is an efficient catalyst for the synthesis of alkyl glucosides by transglycosylation Department of Biotechnology, Lund University, Sweden (submitted)</div> <div class="article_info">Charlotta Turner, Pernilla Turner, Gunilla Jacobson, Knut Almgren, Monica Waldebäck, Per Sjöberg, Eva Nordberg Karlsson and Karin E. Markides. 2006. Subcritical water extraction and beta-glucosidase-catalyzed hydrolysis of quercetin glycosides in onion waste Green Chemistry, vol 8 pp 949-959. Royal Society of Chemistry</div> <div class="article_info">Pernilla Turner, Anna Lundell, Erik Kanders, Christina Wennerberg, Derek Logan and Eva Nordberg Karlsson. 2007. Crystallization, preliminary X-ray diffraction analysis and mutation of active site residues of Thermotoga neapolitana beta-glucosidase B Department of Biotechnology, Lund University, Sweden (manuscript)</div>

PY - 2007

Y1 - 2007

N2 - Two putative cyclodextrinases (CDases) were amplified from two moderate thermophiles isolated in Iceland: Anoxybacillus flavithermus and Laceyella sacchari. They were cloned, expressed and characterized. The expression of the former was optimized in E. coli by using a solubility-enhancing tag, NusA, and tuning of inducer concentration in the expression strain Tuner(DE3). The CDases were optimally active at around 57 ºC and had a poor thermal stability. AfCda13 had significantly higher activity for cyclodextrins and formed a dimer in solution, whereas LsCda13 existed as a monomer, which likely lowered the activity for cyclodextrins. Two beta-glucosidases, members of glycoside hydrolase families 1 and 3 and originating from the extreme thermophile Thermotoga neapolitana were cloned and expressed in E. coli. The apparent thermal unfolding temperatures were around 90 ºC and 102 ºC for TnBgl3B and TnBgl1A, respectively. TnBgl3B hydrolyzed para-nitrophenyl-glucoside with a Vmax at 90 ºC of 93 ± 13 U/mg and a Km of 0.11 ± 0.03 mM. TnBgl3B was tested for its transglycosylating ability by forming hexyl- and octyl-beta-D-glucoside. The selectivity of alcoholysis compared to hydrolysis was higher than for other reported enzymes. Both beta-glucosidases were also tested in reactions cleaving off glucose from quercetin-glycosides extracted from onion by subcritical water. They both performed better than the commercially available almond beta-glucosidase. Predicted catalytic amino acids of TnBgl3B were replaced with non-catalytic glycines. The aspartate nucleophile is highly conserved in GH3 and the D242G mutant lost all activity. The acid/base residue is less conserved and two residues were mutated, Glu458 and Asp461. Mutant E458G had low but significant activity, while D461G lost almost all activity, which points to the latter residue as the catalytic acid/base of TnBgl3B. However, further analyses including structural information should confirm this. TnBgl3B was crystallized and the structure was solved by multiple wavelength anomalous diffraction using selenomethionyl crystals in addition to native crystals. X-ray diffraction data was collected to 2.4 Å resolution and the structure has been solved to 2.7 Å. Manual model building and refinement is in progress.

AB - Two putative cyclodextrinases (CDases) were amplified from two moderate thermophiles isolated in Iceland: Anoxybacillus flavithermus and Laceyella sacchari. They were cloned, expressed and characterized. The expression of the former was optimized in E. coli by using a solubility-enhancing tag, NusA, and tuning of inducer concentration in the expression strain Tuner(DE3). The CDases were optimally active at around 57 ºC and had a poor thermal stability. AfCda13 had significantly higher activity for cyclodextrins and formed a dimer in solution, whereas LsCda13 existed as a monomer, which likely lowered the activity for cyclodextrins. Two beta-glucosidases, members of glycoside hydrolase families 1 and 3 and originating from the extreme thermophile Thermotoga neapolitana were cloned and expressed in E. coli. The apparent thermal unfolding temperatures were around 90 ºC and 102 ºC for TnBgl3B and TnBgl1A, respectively. TnBgl3B hydrolyzed para-nitrophenyl-glucoside with a Vmax at 90 ºC of 93 ± 13 U/mg and a Km of 0.11 ± 0.03 mM. TnBgl3B was tested for its transglycosylating ability by forming hexyl- and octyl-beta-D-glucoside. The selectivity of alcoholysis compared to hydrolysis was higher than for other reported enzymes. Both beta-glucosidases were also tested in reactions cleaving off glucose from quercetin-glycosides extracted from onion by subcritical water. They both performed better than the commercially available almond beta-glucosidase. Predicted catalytic amino acids of TnBgl3B were replaced with non-catalytic glycines. The aspartate nucleophile is highly conserved in GH3 and the D242G mutant lost all activity. The acid/base residue is less conserved and two residues were mutated, Glu458 and Asp461. Mutant E458G had low but significant activity, while D461G lost almost all activity, which points to the latter residue as the catalytic acid/base of TnBgl3B. However, further analyses including structural information should confirm this. TnBgl3B was crystallized and the structure was solved by multiple wavelength anomalous diffraction using selenomethionyl crystals in addition to native crystals. X-ray diffraction data was collected to 2.4 Å resolution and the structure has been solved to 2.7 Å. Manual model building and refinement is in progress.

KW - Bioteknik

KW - Biotechnology

KW - Proteiner

KW - beta-glucosidase

KW - thermostability

KW - activity

KW - cyclodextrinase

KW - enzymology

KW - crystallization

KW - enzymologi

KW - expression

KW - glycoside hydrolase

KW - Proteins

KW - transglycosylation

M3 - Doctoral Thesis (compilation)

SN - 978-91-89627-51-2

PB - Department of Biotechnology, Lund University

ER -

Exploring thermostable glycoside hydrolases: Amylases and beta-glucosidases

Abstract

Bibliographical note

Subject classification (UKÄ)

Free keywords

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Cite this