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Thermostable glycosidases and glycosynthases as biocatalysts in green chemistry applications

Pozzo, Tania LU (2012)
Abstract (Swedish)
Popular Abstract in Swedish

Idag baserat på konceptet grön kemi - börjar man att utveckla nya miljövänliga processer i produktionen av kemikalier, genom att använda förnyelsebara råvaror och minska giftigt avfall. I denna avhandling används enzymer för att bilda kemikalier som en del i utvecklingen av nya miljöprocesser. Enzymer är proteiner som påskyndar (katalyserar) kemiska reaktioner för olika funktioner i celler, varav en mycket viktig sådan är att skapa energi genom att bryta ned kolhydrater. De enzymer som används i denna studie kommer från två bakterier som lever i extremt varma ekosystem (termofiler).

T. neapolitana som återfinns i underjordiska varma källor utanför Neapels kust i Italien och R. marinus... (More)
Popular Abstract in Swedish

Idag baserat på konceptet grön kemi - börjar man att utveckla nya miljövänliga processer i produktionen av kemikalier, genom att använda förnyelsebara råvaror och minska giftigt avfall. I denna avhandling används enzymer för att bilda kemikalier som en del i utvecklingen av nya miljöprocesser. Enzymer är proteiner som påskyndar (katalyserar) kemiska reaktioner för olika funktioner i celler, varav en mycket viktig sådan är att skapa energi genom att bryta ned kolhydrater. De enzymer som används i denna studie kommer från två bakterier som lever i extremt varma ekosystem (termofiler).

T. neapolitana som återfinns i underjordiska varma källor utanför Neapels kust i Italien och R. marinus som först isolerades från heta källor på Island. Fördelen med att använda enzymer från termofila bakterier är att de bibehåller förmågan att fungera effektivt vid såväl höga temperaturer och tryck som vid svåra kemiska förhållanden (surt eller basiskt). Dessa egenskaper gör dem idealiska för användning vid produktion av kemikalier på en industriell nivå.

Tack vare användandet av molekylärbiologiska tekniker kan dessa enzymer produceras i stora mängder och på ett kontrollerat sätt i ett laboratorium genom att den genetiska kod som skapar enzymet förs in i en annan bakterie som kallas E. coli som kan odlas i lägre (normala) temperaturer. Således har genom arbetet för denna avhandling tre termostabila enzymer som tillhör gruppen glykosidaser producerats i laboratoriet - TnBgl3B och TnBgl1A från T. neapolitana och RmCel12A från R. marinus. Den första delen av avhandlingen fokuserar på studier av enzymaktivitet under nedbrytningen av kolhydrater. För att förstå mer om denna funktion gjordes strukturella studier.

En tredimensionell struktur skapas genom att kristallisera enzymet följt av bestrålning med röntgenstrålar som ger oss positionen för varje atom i enzymet. Detta visade att TnBgl3B var den första strukturen i sin familj av enzymer som rapporteras ha tre domäner, och utöver detta så upptäcktes en förklaring till enzymets interaktion med socker.

I den andra delen av avhandlingen, baserat på den information som den tredimensionella strukturen gav, infördes specifika mutationer med molekylärbiologisk teknik som förändrade den naturliga funktionen hos enzymet så att den skapar nya ämnen (syntes) istället för att bryta ned dem. Denna nya skräddarsydda ändring av enzymets egenskaper kan utnyttjas för att skapa nya kemikalier, syntetiserade (skapade) av sockerarter (som är förnybara råvaror). De nya kemikalierna som skapas kan användas till biologiskt nedbrytbara personliga hygienprodukter (alkylglykosider), som konserveringsmedel (antioxidanter), samt inom flera andra områden som till exempel inom nutrition och produktion av prebiotika (socker av specifik längd och sammansättning) (Less)
Abstract
The constant need to develop environmental friendly processes using renewable raw materials and to reduce harmful waste production motivates the present investigation focusing on biocatalyst development for enzymatic synthesis, a biotechnological approach in the field of green chemistry.

At first, structural functional experiments were made for a β-glucosidase from T. neapolitana (TnBgl3B). As a result the first three-domain thermostable member of a glycoside hydrolase (GH) from family 3 was reported, giving insights on substrate specificity and some structural explanations for its use in synthesis of alkyl glucosides. Moreover, efficiency in hexyl glucoside synthesis was compared between two thermostable β-glucosidases from T.... (More)
The constant need to develop environmental friendly processes using renewable raw materials and to reduce harmful waste production motivates the present investigation focusing on biocatalyst development for enzymatic synthesis, a biotechnological approach in the field of green chemistry.

At first, structural functional experiments were made for a β-glucosidase from T. neapolitana (TnBgl3B). As a result the first three-domain thermostable member of a glycoside hydrolase (GH) from family 3 was reported, giving insights on substrate specificity and some structural explanations for its use in synthesis of alkyl glucosides. Moreover, efficiency in hexyl glucoside synthesis was compared between two thermostable β-glucosidases from T. neapolitana, belonging to family 1 (GH1) and 3 (GH3), TnBgl1A and TnBgl3B, respectively. For this purpose, a novel direct screening method in 96-well format was developed, using glucose, a cheap substrate from renewables, together with hexanol for reverse hydrolysis in a two-phase system.

The GH1 β-glucosidase TnBgl1A was, in a following study, used as biocatalyst in hydrolysis reactions, connected to flavonoid extractions from onion waste using pressurized hot water, to obtain a uniform deglycosylated product. Structural homology models and mutagenesis around the aglycone in the active site, was in this case used to identify important residues that led to increased hydrolytic activity towards flavonoid 3-glucosides.

The second part of the project focused on thermostable glycosynthases, which were engineered and tested using oligosaccharide synthesis as main model reaction. Two methodologies were used to obtain synthesis products, one using fluorinated sugar as donor, and the second using an exogenous nucleophile. The product specificity was shown to be towards synthesis of β-1,3-linkages for both TnBgl1A and TnBgl3B.

Structural examination of theTnBgl1A glycosynthase, after docking with the substrate, showed flexibility at the active site with catalytic residues located on loops, a statement verified using molecular dynamics.

The construction of a glycosynthase from TnBgl3B required an extra mutation next to the nucleophile. A residue that stabilized the product at the +1 subsite was also identified, together with one more hydrophobic residue located on the loop from the second domain, suggested to be important for the accommodation of acceptor molecules.

A natural product (antioxidant), such as the flavonoid quercetin-3-glucoside was also used as acceptor molecule in glycosynthase reactions at high temperature (70° C), in an attempt to look at an alternative acceptor and to expand the glycosynthase application to selective glycosylation of antioxidants. This assay used the exo-glycosynthases from TnBgl1A and TnBgl3B plus a new endo-glycosynthase constructed of a cellulase belonging to GH12 from R. marinus (RmCel12A). The main product of the thermostable glycosynthases was quercetin-3,4’-diglucoside when quercetin-3-glucoside was used as acceptor. Hence the 4’-hydroxyl of the acceptor was selected in the transglycosylation reactions, when the molecule was fitted in the active site.

In conclusion, thermostable glycoside hydrolases and glycosynthases can be useful biocatalysts for synthesising chemicals from renewable resources, such as alkyl glucosides, oligosaccharides or flavonoids with modified glycosylation. Thanks to the structural and functional information, the biocatalysts can be developed to harbour new or altered activities relevant for use to process renewable resources, such as in the modification of flavonoids combined with extraction form onion waste. Structural data also aided the construction of new glycosynthases coming from less investigated families like GH3. New interesting applications in antioxidant stabilization were assayed using thermostable glycosynthase for the glucosylation of flavonoids. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Dr. Moracci, Marco, Institute of Protein Biochemistry, Consiglio Nazionale delle Ricerche, Naples, Italy
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Green chemistry, 3D sturcture, Glycosyl hydrolases, Glycosynthases, Alkyl glucosides, Oligosaccharides, Flavonoids
pages
166 pages
defense location
Lecture Hall K:C, Center of Chemistry and Chemical Engineering, Sölvegatan 39, Lund University Faculty of Engineering.
defense date
2012-12-19 13:30
ISBN
978-91-89627-91-8
language
English
LU publication?
yes
id
310a957d-108c-4a73-9cee-35c35b8053ad (old id 3193948)
date added to LUP
2012-11-26 11:15:04
date last changed
2016-09-19 08:45:18
@misc{310a957d-108c-4a73-9cee-35c35b8053ad,
  abstract     = {The constant need to develop environmental friendly processes using renewable raw materials and to reduce harmful waste production motivates the present investigation focusing on biocatalyst development for enzymatic synthesis, a biotechnological approach in the field of green chemistry. <br/><br>
At first, structural functional experiments were made for a β-glucosidase from T. neapolitana (TnBgl3B). As a result the first three-domain thermostable member of a glycoside hydrolase (GH) from family 3 was reported, giving insights on substrate specificity and some structural explanations for its use in synthesis of alkyl glucosides. Moreover, efficiency in hexyl glucoside synthesis was compared between two thermostable β-glucosidases from T. neapolitana, belonging to family 1 (GH1) and 3 (GH3), TnBgl1A and TnBgl3B, respectively. For this purpose, a novel direct screening method in 96-well format was developed, using glucose, a cheap substrate from renewables, together with hexanol for reverse hydrolysis in a two-phase system. <br/><br>
The GH1 β-glucosidase TnBgl1A was, in a following study, used as biocatalyst in hydrolysis reactions, connected to flavonoid extractions from onion waste using pressurized hot water, to obtain a uniform deglycosylated product. Structural homology models and mutagenesis around the aglycone in the active site, was in this case used to identify important residues that led to increased hydrolytic activity towards flavonoid 3-glucosides.<br/><br>
The second part of the project focused on thermostable glycosynthases, which were engineered and tested using oligosaccharide synthesis as main model reaction. Two methodologies were used to obtain synthesis products, one using fluorinated sugar as donor, and the second using an exogenous nucleophile. The product specificity was shown to be towards synthesis of β-1,3-linkages for both TnBgl1A and TnBgl3B. <br/><br>
Structural examination of theTnBgl1A glycosynthase, after docking with the substrate, showed flexibility at the active site with catalytic residues located on loops, a statement verified using molecular dynamics. <br/><br>
The construction of a glycosynthase from TnBgl3B required an extra mutation next to the nucleophile. A residue that stabilized the product at the +1 subsite was also identified, together with one more hydrophobic residue located on the loop from the second domain, suggested to be important for the accommodation of acceptor molecules.<br/><br>
A natural product (antioxidant), such as the flavonoid quercetin-3-glucoside was also used as acceptor molecule in glycosynthase reactions at high temperature (70° C), in an attempt to look at an alternative acceptor and to expand the glycosynthase application to selective glycosylation of antioxidants. This assay used the exo-glycosynthases from TnBgl1A and TnBgl3B plus a new endo-glycosynthase constructed of a cellulase belonging to GH12 from R. marinus (RmCel12A). The main product of the thermostable glycosynthases was quercetin-3,4’-diglucoside when quercetin-3-glucoside was used as acceptor. Hence the 4’-hydroxyl of the acceptor was selected in the transglycosylation reactions, when the molecule was fitted in the active site.<br/><br>
In conclusion, thermostable glycoside hydrolases and glycosynthases can be useful biocatalysts for synthesising chemicals from renewable resources, such as alkyl glucosides, oligosaccharides or flavonoids with modified glycosylation. Thanks to the structural and functional information, the biocatalysts can be developed to harbour new or altered activities relevant for use to process renewable resources, such as in the modification of flavonoids combined with extraction form onion waste. Structural data also aided the construction of new glycosynthases coming from less investigated families like GH3. New interesting applications in antioxidant stabilization were assayed using thermostable glycosynthase for the glucosylation of flavonoids.},
  author       = {Pozzo, Tania},
  isbn         = {978-91-89627-91-8},
  keyword      = {Green chemistry,3D sturcture,Glycosyl hydrolases,Glycosynthases,Alkyl glucosides,Oligosaccharides,Flavonoids},
  language     = {eng},
  pages        = {166},
  title        = {Thermostable glycosidases and glycosynthases as biocatalysts in green chemistry applications},
  year         = {2012},
}