Exploring thermostable glycoside hydrolases: Amylases and beta-glucosidases

Pernilla Turner

Exploring thermostable glycoside hydrolases: Amylases and beta-glucosidases

Pernilla Turner

Forskningsoutput: Avhandling › Doktorsavhandling (sammanläggning)

Sammanfattning

Popular Abstract in Swedish
Arbetet i denna avhandling presenteras i 6 olika delarbeten (artiklar I-VI). Artiklarna I-III behandlar två olika enzymer som bryter ner cyklodextriner, vilka är ringformade molekyler med samma beståndsdelar som i stärkelse (dvs sammanlänkade glukosmolekyler). Dessa s.k. cyklodextrinaser kommer ursprungligen från två bakterier som har isolerats i varma källor på Island. I artiklarna presenteras karakterisering av enzymerna för den typ av substrat de använder, vilka temperaturer de är aktiva och stabila vid och andra biokemiska undersökningar (generell uppbyggnad av enzymerna, produktbildningsmönster, etc). En av artiklarna (II) beskriver optimeringen kring att framställa det ena enzymet genom att använda en värdorganism, Escherichia coli, som ofta används på laboratorier och även industriellt för storskalig produktion av enzymer. Generellt är de här enzymerna svåra att framställa och inte särskilt termostabila eller termoaktiva (optimala temperaturen för aktivitet var 57 grader).

Nästa del (artiklarna IV-VI) handlar om två andra enzymer som är förhållandevis lika varandra. De är verksamma på cellulosa där de bryter ned de minsta små bitarna (cello-oligosaccharider) efter att andra enzymer (cellulaser) har brutit ned längre kedjor till mindre delar. Dessa enzymer, som benämns beta-glukosidaser, kommer ursprungligen från en extremt termofil bakterie, Thermotoga neapolitana, som från början har hittats i en varm källa i havsbukten utanför Neapel i Italien. Artikel IV beskriver hur det ena beta-glukosidaset testas för att framställa ytaktiva ämnen som består av alkohol och socker, s.k. sockersurfaktanter, som bl a används inom läkemedelsindustrin för stabilisering av formuleringar. Jämfört med andra liknande enzymer så uppvisade beta-glukosidaset från Thermotoga högre selektivitet för den önskvärda reaktionen. I nästa artikel (V) undersöks enzymernas förmåga att klyva av ett socker från en antioxidant (quercetin) som extraherats ur lök. Det visade sig att de klarade den uppgiften mycket snabbare än i traditionella metoder som använder saltsyra som katalysator. Dessutom bryts inte själva antioxidanten ner, vilket ofta sker med saltsyra. I den sista artikeln (VI) presenteras kristallisering av det ena beta-glukosidaset. Kristallisering gör man för att kunna se hur enzymet ser ut 3-dimensionellt. När en viss typ av röntgenstrålning träffar proteinet får man ett mönster som ser olika ut beroende på hur aminosyrorna sitter i förhållande till varandra. Man kan sedan med matematiska metoder få fram ett 3-dimensionellt mönster av s.k. elektrontätheter, där de olika atomerna finns. Strukturen är inte helt färdig än eftersom aminosyrakedjan manuellt måste passas in i elektrontätheterna, varefter alla vinklar hos de olika kovalenta bindningarna mellan atomerna finjusteras. I den sista artikeln presenteras även resultat från en undersökning av vilka aminosyror som direkt deltar i den katalytiska reaktionen när substraten omvandlas. Några predikterade katalytiska aminosyror byttes ut till overksamma aminosyror, vilket medförde att enzymet förlorade sin katalytiska förmåga.

Avhandlingen visar att termostabila enzymer är väl lämpade för vissa processer som kräver höga temperaturer. Vanliga enzymer fungerar t ex inte i processer där extrahering av antioxidanter sker med trycksatt vatten med en temperatur över 100 grader. De testade enzymerna kan vara användbara i en mängd olika industriella processer. De kan även utvecklas med laboratorietekniker för att bli ännu stabilare.

Originalspråk	engelska
Kvalifikation	Doktor
Tilldelande institution
Handledare	Holst, Olle, handledare
Tilldelningsdatum	2007 feb. 1
Förlag	Department of Biotechnology, Lund University
ISBN (tryckt)	978-91-89627-51-2
Status	Published - 2007

Bibliografisk information

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>

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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>",

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publisher = "Department of Biotechnology, Lund University",

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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

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