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Exploring thermostable glycoside hydrolases: Amylases and beta-glucosidases

Turner, Pernilla LU (2007)
Abstract (Swedish)
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... (More)
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. (Less)
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... (More)
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. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Professor Svensson, Birte, Technical University of Denmark
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Bioteknik, Biotechnology, Proteiner, beta-glucosidase, thermostability, activity, cyclodextrinase, enzymology, crystallization, enzymologi, expression, glycoside hydrolase, Proteins, transglycosylation
pages
171 pages
publisher
Department of Biotechnology, Lund University
defense location
Hörsal A, Kemicentrum, Getingevägen 60, Lunds Tekniska Högskola
defense date
2007-02-01 13:15
external identifiers
  • Other:ISRN: LUTKDH/TKBT--07/1104--SE
ISBN
978-91-89627-51-2
language
English
LU publication?
yes
id
df445a2a-3f1e-4b6d-95e0-17e9e5fe8ae6 (old id 547887)
date added to LUP
2007-10-10 10:56:36
date last changed
2016-09-19 08:45:07
@misc{df445a2a-3f1e-4b6d-95e0-17e9e5fe8ae6,
  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.<br/><br>
<br/><br>
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.<br/><br>
<br/><br>
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.<br/><br>
<br/><br>
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.<br/><br>
<br/><br>
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.},
  author       = {Turner, Pernilla},
  isbn         = {978-91-89627-51-2},
  keyword      = {Bioteknik,Biotechnology,Proteiner,beta-glucosidase,thermostability,activity,cyclodextrinase,enzymology,crystallization,enzymologi,expression,glycoside hydrolase,Proteins,transglycosylation},
  language     = {eng},
  pages        = {171},
  publisher    = {ARRAY(0xb58d578)},
  title        = {Exploring thermostable glycoside hydrolases: Amylases and beta-glucosidases},
  year         = {2007},
}