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Enzymatic Synthesis of Alkyl Glycosides. New Applications for Glycoside Hydrolases

Svensson, David LU (2009)
Abstract
The environmental problems caused by human interventions, such as the consumption of non-renewable resources and the emission of greenhouse gases are well-recognised. Calls have been made for immediate measures to develop more sustainable society worldwide. Attention has also been devoted to the quest for sustainability within the field of chemistry. The concept of “green chemistry” is being introduced into the production of chemicals in order to reduce the impact on the environment through replacing petroleum-based resources with renewable feedstock and by making chemical processes more efficient.

Surfactants constitute one of the most abundant groups of chemicals used in everyday life. Most the products on the market contain... (More)
The environmental problems caused by human interventions, such as the consumption of non-renewable resources and the emission of greenhouse gases are well-recognised. Calls have been made for immediate measures to develop more sustainable society worldwide. Attention has also been devoted to the quest for sustainability within the field of chemistry. The concept of “green chemistry” is being introduced into the production of chemicals in order to reduce the impact on the environment through replacing petroleum-based resources with renewable feedstock and by making chemical processes more efficient.

Surfactants constitute one of the most abundant groups of chemicals used in everyday life. Most the products on the market contain some kind of surfactant, which acts as an emulsifier, a texture enhancer, detergent, softener or stabiliser. In this research the focus was on developing a route for the synthesis of alkyl glycosides with oligomeric head groups that can be used as excipient in pharmaceuticals or as emulsifiers in cosmetics and personal hygiene products. Alkyl glycosides are bio-based nonionic surfactants that have been shown to be completely biodegradable and to have low toxicity. Today, these types of alkyl glycosides are produced on a small scale using traditional organic chemistry using a protection/deprotection strategy, sometimes involving activated substrates. This leads to the considerable amounts of toxic waste such as catalysts, hazardous solvents and chemicals. Enzymes, on the other hand, are well adapted to perform these stereospecific reactions. In this work two different model reactions have been studied: (I) the synthesis of alkyl glycosides by alcoholysis catalysed by different glycoside hydrolases, and (II) the synthesis of alkyl glycosides with oligomeric head groups catalysed by CGTase.

In the first part of this work, the aim of the synthesis of alkyl glycosides using the alcoholysis strategy was to find alternative enzymes and means of adding one or several glucose units to an alcohol, to produce suitable material for the extension of the sugar part by CGTase. In the first study, an α-amylase from Aspergillus oryzae was used to catalyse the transglycosylation of different alcohols (nucleophile/acceptor) using starch as the glycosyl donor. It was found that in the degradation of starch, the alcohols were able to compete with water as the nucleophile, producing a variety of alkyl glycosides. Methanol was found to be the best acceptor among the alcohols, generating 3.6 mM of methyl glycosides (methyl maltoside and methyl maltotrioside) in the presence of 45 g/l soluble starch and 30 % (v/v) methanol. With larger alcohols, such as ethanol, propanol and butanol, alkyl maltosides and alkyl maltotetraoside were formed, and the maltoside, maltotrioside and maltopentaoside of benzyl alcohol were also detected. No products were seen when using hexanol or octanol.

In the next study, a novel thermostable β-glucosidase from Thermotoga neapolitana, belonging to glycoside hydrolase family 3 (TnBgl3B), was investigated, which showed promising performance in the synthesis of alkyl glycosides. TnBgl3B was able to efficiently catalyse the transglycosylation between p-nitrophenyl-β-glucopyranoside (pNPG) (the donor) and hexanol and octanol, producing hexyl glucoside and octyl glucoside, respectively. Interestingly, the enzyme was found to have an apparent optimum for hydrolysis at 90 °C, while a more favourable alcoholysis/hydrolysis ratio was observed at 60 °C. The selectivity towards alcoholysis was shown to be influenced by the water content and pH, giving optimal reaction conditions at 16 % (v/v) water, and a pH of 5.8 at a temperature of 60 °C in the synthesis of hexyl glucoside. Under these conditions, a total initial rate of 153 µmol min-1mg-1, a yield of 80.3 % and an alcoholysis/hydrolysis ratio of 5.1 were obtained at full conversion of pNPG after 180 minutes’ reaction. When octanol was used, the initial reaction rate was 5 times slower, and the alcoholysis/hydrolysis ratio by 6.3 times. This is probably the result of the 17 times lower solubility of octanol in water compared with hexanol.

In the second part of this work the synthesis of alkyl glycosides with oligomeric head groups, catalysed by CGTase, was investigated using primarily the model coupling reaction between alpha-cyclodextrin (α-CD) and dodecyl-β-(1,4)-maltoside (DDM) to produce dodecyl-β-(1,4)-maltooctaoside (DDMO). Due to the novelty of the enzymatic route, it was necessary to investigate the effects of substrate specificity, enzyme origin and reaction parameters. It was found that CGTase was highly dependent on the presence of an efficient donor such as α-CD. However, the acceptor specificity was somewhat promiscuous, accepting alkyl chains at least up to C16. The enzymes prefer alkyl maltoside, but are willing to accept alkyl glycosides with at least one glucose unit, although the reaction rate is reduced. Since the reaction is under kinetic control, the choice of enzyme is important to maximise the yield of the pure coupling product (DDMO) since it is dependent on the enzyme selectivity. When catalysing the model reaction, the CGTase from Bacillus macerans (BmCGTase) favoured the coupling reaction whereas the Thermoanaerobacter CGTase (TsCGTase) catalysed predominantly disproportionation. Moreover, a high donor-to-acceptor ratio favoured the product yield, and yields up to 80 % were obtained.

To further prove the applicability of CGTase in synthesising alkyl glycosides, the acceptor specificity of BmCGTase was further investigated using a technical grade alkyl polyglycoside (APG), which mainly contained glucosides of C12 and C14 of both binding isomers (α- and β-1,4). The main products were confirmed to be those of alkyl glucopyranosides extended by either 6 or 12 glucose residues, being the primary and secondary coupling products, respectively. Thus APG can be used as an acceptor in the CGTase-catalysed extension of alkyl glycosides, making this enzymatic route more economically feasible.

It was further demonstrated that when using immobilised BmCGTase in a packed-bed reactor, the product distribution could easily be controlled simply by altering the flow rate over the reactor. However, due to the propensity of the reaction mixture to precipitate, a high temperature (≥55 °C) had to be applied. Consequently, due to the poor enzyme stability (half-life 132 min at 60 °C and 18 min at 70 °C), the potential of immobilisation could not fully be utilised.

The work presented in this thesis gives an idea of the inherent advantages of the enzymatic synthesis of specialty chemicals. The route developed is superior to previously described organic chemical methods, in that it allows the single-step addition of six glucose residues without the use of toxic catalysts or organic solvents. (Less)
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author
supervisor
opponent
  • Dr. Clapés, Pere, Catalonia Institute for Advanced Chemistry, Barcelona, Spain
organization
publishing date
type
Thesis
publication status
published
subject
keywords
CGTase, cyclodextrin glycosyltransferase, alkyl glycoside, enzyme stabliity, enzymatic synthesis, enzyme immobilisation, surfactant, glycosylation
pages
183 pages
defense location
Lecture hall A, Center for Chemistry and Chemical Engineering, Sölvegatan 39, Lund University Faculty of Engineering
defense date
2009-09-24 09:15
external identifiers
  • other:ISRN LUTKDH/TKBT--09/1115--SE
ISBN
978-91-89627-61-1
language
English
LU publication?
yes
id
ebc244a1-b272-455e-a55b-cb0e61d4278e (old id 1468355)
date added to LUP
2009-08-28 14:36:04
date last changed
2016-09-19 08:45:17
@phdthesis{ebc244a1-b272-455e-a55b-cb0e61d4278e,
  abstract     = {The environmental problems caused by human interventions, such as the consumption of non-renewable resources and the emission of greenhouse gases are well-recognised. Calls have been made for immediate measures to develop more sustainable society worldwide. Attention has also been devoted to the quest for sustainability within the field of chemistry. The concept of “green chemistry” is being introduced into the production of chemicals in order to reduce the impact on the environment through replacing petroleum-based resources with renewable feedstock and by making chemical processes more efficient. <br/><br>
Surfactants constitute one of the most abundant groups of chemicals used in everyday life. Most the products on the market contain some kind of surfactant, which acts as an emulsifier, a texture enhancer, detergent, softener or stabiliser. In this research the focus was on developing a route for the synthesis of alkyl glycosides with oligomeric head groups that can be used as excipient in pharmaceuticals or as emulsifiers in cosmetics and personal hygiene products. Alkyl glycosides are bio-based nonionic surfactants that have been shown to be completely biodegradable and to have low toxicity. Today, these types of alkyl glycosides are produced on a small scale using traditional organic chemistry using a protection/deprotection strategy, sometimes involving activated substrates. This leads to the considerable amounts of toxic waste such as catalysts, hazardous solvents and chemicals. Enzymes, on the other hand, are well adapted to perform these stereospecific reactions. In this work two different model reactions have been studied: (I) the synthesis of alkyl glycosides by alcoholysis catalysed by different glycoside hydrolases, and (II) the synthesis of alkyl glycosides with oligomeric head groups catalysed by CGTase. <br/><br>
In the first part of this work, the aim of the synthesis of alkyl glycosides using the alcoholysis strategy was to find alternative enzymes and means of adding one or several glucose units to an alcohol, to produce suitable material for the extension of the sugar part by CGTase. In the first study, an α-amylase from Aspergillus oryzae was used to catalyse the transglycosylation of different alcohols (nucleophile/acceptor) using starch as the glycosyl donor. It was found that in the degradation of starch, the alcohols were able to compete with water as the nucleophile, producing a variety of alkyl glycosides. Methanol was found to be the best acceptor among the alcohols, generating 3.6 mM of methyl glycosides (methyl maltoside and methyl maltotrioside) in the presence of 45 g/l soluble starch and 30 % (v/v) methanol. With larger alcohols, such as ethanol, propanol and butanol, alkyl maltosides and alkyl maltotetraoside were formed, and the maltoside, maltotrioside and maltopentaoside of benzyl alcohol were also detected. No products were seen when using hexanol or octanol. <br/><br>
In the next study, a novel thermostable β-glucosidase from Thermotoga neapolitana, belonging to glycoside hydrolase family 3 (TnBgl3B), was investigated, which showed promising performance in the synthesis of alkyl glycosides. TnBgl3B was able to efficiently catalyse the transglycosylation between p-nitrophenyl-β-glucopyranoside (pNPG) (the donor) and hexanol and octanol, producing hexyl glucoside and octyl glucoside, respectively. Interestingly, the enzyme was found to have an apparent optimum for hydrolysis at 90 °C, while a more favourable alcoholysis/hydrolysis ratio was observed at 60 °C. The selectivity towards alcoholysis was shown to be influenced by the water content and pH, giving optimal reaction conditions at 16 % (v/v) water, and a pH of 5.8 at a temperature of 60 °C in the synthesis of hexyl glucoside. Under these conditions, a total initial rate of 153 µmol min-1mg-1, a yield of 80.3 % and an alcoholysis/hydrolysis ratio of 5.1 were obtained at full conversion of pNPG after 180 minutes’ reaction. When octanol was used, the initial reaction rate was 5 times slower, and the alcoholysis/hydrolysis ratio by 6.3 times. This is probably the result of the 17 times lower solubility of octanol in water compared with hexanol. <br/><br>
In the second part of this work the synthesis of alkyl glycosides with oligomeric head groups, catalysed by CGTase, was investigated using primarily the model coupling reaction between alpha-cyclodextrin (α-CD) and dodecyl-β-(1,4)-maltoside (DDM) to produce dodecyl-β-(1,4)-maltooctaoside (DDMO). Due to the novelty of the enzymatic route, it was necessary to investigate the effects of substrate specificity, enzyme origin and reaction parameters. It was found that CGTase was highly dependent on the presence of an efficient donor such as α-CD. However, the acceptor specificity was somewhat promiscuous, accepting alkyl chains at least up to C16. The enzymes prefer alkyl maltoside, but are willing to accept alkyl glycosides with at least one glucose unit, although the reaction rate is reduced. Since the reaction is under kinetic control, the choice of enzyme is important to maximise the yield of the pure coupling product (DDMO) since it is dependent on the enzyme selectivity. When catalysing the model reaction, the CGTase from Bacillus macerans (BmCGTase) favoured the coupling reaction whereas the Thermoanaerobacter CGTase (TsCGTase) catalysed predominantly disproportionation. Moreover, a high donor-to-acceptor ratio favoured the product yield, and yields up to 80 % were obtained. <br/><br>
To further prove the applicability of CGTase in synthesising alkyl glycosides, the acceptor specificity of BmCGTase was further investigated using a technical grade alkyl polyglycoside (APG), which mainly contained glucosides of C12 and C14 of both binding isomers (α- and β-1,4). The main products were confirmed to be those of alkyl glucopyranosides extended by either 6 or 12 glucose residues, being the primary and secondary coupling products, respectively. Thus APG can be used as an acceptor in the CGTase-catalysed extension of alkyl glycosides, making this enzymatic route more economically feasible. <br/><br>
It was further demonstrated that when using immobilised BmCGTase in a packed-bed reactor, the product distribution could easily be controlled simply by altering the flow rate over the reactor. However, due to the propensity of the reaction mixture to precipitate, a high temperature (≥55 °C) had to be applied. Consequently, due to the poor enzyme stability (half-life 132 min at 60 °C and 18 min at 70 °C), the potential of immobilisation could not fully be utilised.<br/><br>
The work presented in this thesis gives an idea of the inherent advantages of the enzymatic synthesis of specialty chemicals. The route developed is superior to previously described organic chemical methods, in that it allows the single-step addition of six glucose residues without the use of toxic catalysts or organic solvents.},
  author       = {Svensson, David},
  isbn         = {978-91-89627-61-1},
  keyword      = {CGTase,cyclodextrin glycosyltransferase,alkyl glycoside,enzyme stabliity,enzymatic synthesis,enzyme immobilisation,surfactant,glycosylation},
  language     = {eng},
  pages        = {183},
  school       = {Lund University},
  title        = {Enzymatic Synthesis of Alkyl Glycosides. New Applications for Glycoside Hydrolases},
  year         = {2009},
}