Lipase-catalysed acylation of starch and determination of the degree of substitution by methanolysis and GC

Background: Natural polysaccharides such as starch are becoming increasingly interesting as renewable starting materials for the synthesis of biodegradable polymers using chemical or enzymatic methods. Given the complexity of polysaccharides, the analysis of reaction products is challenging. Results: Esterification of starch with fatty acids has traditionally been monitored by saponification and back-titration, but in our experience this method is unreliable. Here we report a novel GC-based method for the fast and reliable quantitative determination of esterification. The method was used to monitor the enzymatic esterification of different starches with decanoic acid, using lipase from Thermomyces lanuginosus. The reaction showed a pronounced optimal water content of 1.25 mL per g starch, where a degree of substitution (DS) of 0.018 was obtained. Incomplete gelatinization probably accounts for lower conversion with less water. Conclusions: Lipase-catalysed esterification of starch is feasible in aqueous gel systems, but attention to analytical methods is important to obtain correct DS values

Enzymatic acylation of starch

This thesis was previously held under moratorium from 04/10/10 to 04/10/12.The possibility of enzyme catalysed synthesis of starch esters, is an extremely interesting prospect, as a new route for synthesis of biodegradable polymers from renewable material, through mild processes. Starch esters have traditionally been analysed by saponification and back-titration, however this method was found to be unreliable. Quantification through integration of 1H-NMR peaks, was also found problematic for low degrees of substitution (DS, mol acyl group per mol anhydroglucose). Therefore an analytical scheme employing alkaline methanolysis of starch esters and analysis of the resulting methyl esters by GC/FID is presented. This technique is employed for starch esters with fatty acids of medium chain length and can be suitably modified to accommodate other chain lengths. Lipase catalysed synthesis of starch esters with decanoic acid was performed in concentrated aqueous systems, where the starch is gelatinised prior to enzymatic reaction. The reaction showed a pronounced optimal water content (1.25 mL of water for gelatinisation per g of starch), in which the equilibrium was sufficiently shifted, in order to produce starch acylated to a useful extent (0.018 DS). Use of fatty acids with other groups on their alkyl chains, as substrates, led to successful synthesis of starch 10-undecynoate. The triple bond in the -position of the fatty acid may act as a grafting site for further modification, with azide containing molecules, through "click" reactions (dipolar cycloaddition). Investigation of other reaction systems for enzymatic starch acylation, revealed nonenzymatic transesterification of vinyl decanoate with starch in DMSO. On the other hand, the ionic liquid 1-ethyl-3-methylimidazolium dicyanamide ([EMIM][DCA]), was a suitable medium for lipase catalysed synthesis of starch esters with decanoic acid (0.016 DS). Treatment with -amylase resulted in decreased hydrolysis for the modified starches, as the acyl groups present inhibited the action of the enzyme. The presence of approximately one acyl group for every one hundred glucose units is sufficient to decrease extent of hydrolysis by 11%.The possibility of enzyme catalysed synthesis of starch esters, is an extremely interesting prospect, as a new route for synthesis of biodegradable polymers from renewable material, through mild processes. Starch esters have traditionally been analysed by saponification and back-titration, however this method was found to be unreliable. Quantification through integration of 1H-NMR peaks, was also found problematic for low degrees of substitution (DS, mol acyl group per mol anhydroglucose). Therefore an analytical scheme employing alkaline methanolysis of starch esters and analysis of the resulting methyl esters by GC/FID is presented. This technique is employed for starch esters with fatty acids of medium chain length and can be suitably modified to accommodate other chain lengths. Lipase catalysed synthesis of starch esters with decanoic acid was performed in concentrated aqueous systems, where the starch is gelatinised prior to enzymatic reaction. The reaction showed a pronounced optimal water content (1.25 mL of water for gelatinisation per g of starch), in which the equilibrium was sufficiently shifted, in order to produce starch acylated to a useful extent (0.018 DS). Use of fatty acids with other groups on their alkyl chains, as substrates, led to successful synthesis of starch 10-undecynoate. The triple bond in the -position of the fatty acid may act as a grafting site for further modification, with azide containing molecules, through "click" reactions (dipolar cycloaddition). Investigation of other reaction systems for enzymatic starch acylation, revealed nonenzymatic transesterification of vinyl decanoate with starch in DMSO. On the other hand, the ionic liquid 1-ethyl-3-methylimidazolium dicyanamide ([EMIM][DCA]), was a suitable medium for lipase catalysed synthesis of starch esters with decanoic acid (0.016 DS). Treatment with -amylase resulted in decreased hydrolysis for the modified starches, as the acyl groups present inhibited the action of the enzyme. The presence of approximately one acyl group for every one hundred glucose units is sufficient to decrease extent of hydrolysis by 11%

Enzymatic acylation of starch

Starch a cheap, abundant and renewable natural material has been chemically modified for many years. The popular modification acylation has been used to adjust rheological properties as well as deliver polymers with internal plasticizers and other potential uses. However the harsh reaction conditions required to produce these esters may limit their use, especially in sensitive applications (foods, pharmaceuticals, etc.). The use of enzymes to catalyse acylation may provide a suitable alternative due to high selectivities and mild reaction conditions. Traditional hydrolase-catalysed synthesis in non-aqueous apolar media is hard due to lack of polysaccharide solubility. However, acylated starch derivatives have recently been successfully produced in other non-conventional systems: (a) surfactant-solubilised subtilisin and suspended amylose in organic media; (b) starch nanoparticles dispersed in organic medium with immobilised lipase; (c) aqueous starch gels with lipase and dispersed fatty acids. We attempt a systematic review that draws parallels between the seemingly unrelated approaches described

Biocatalysis for the application of CO2 as a chemical feedstock

Biocatalysts, capable of efficiently transforming CO2 into other more reduced forms of carbon, offer sustainable alternatives to current oxidative technologies that rely on diminishing natural fossil-fuel deposits. Enzymes that catalyse CO2 fixation steps in carbon assimilation pathways are promising catalysts for the sustainable transformation of this safe and renewable feedstock into central metabolites. These may be further converted into a wide range of fuels and commodity chemicals, through the multitude of known enzymatic reactions. The required reducing equivalents for the net carbon reductions may be drawn from solar energy, electricity or chemical oxidation, and delivered in vitro or through cellular mechanisms, while enzyme catalysis lowers the activation barriers of the CO2 transformations to make them more energy efficient. The development of technologies that treat CO2-transforming enzymes and other cellular components as modules that may be assembled into synthetic reaction circuits will facilitate the use of CO2 as a renewable chemical feedstock, greatly enabling a sustainable carbon bio-economy

Biocatalysis for the application of CO2 as a chemical feedstock

Biocatalysts, capable of efficiently transforming CO2 into other more reduced forms of carbon, offer sustainable alternatives to current oxidative technologies that rely on diminishing natural fossil-fuel deposits. Enzymes that catalyse CO2 fixation steps in carbon assimilation pathways are promising catalysts for the sustainable transformation of this safe and renewable feedstock into central metabolites. These may be further converted into a wide range of fuels and commodity chemicals, through the multitude of known enzymatic reactions. The required reducing equivalents for the net carbon reductions may be drawn from solar energy, electricity or chemical oxidation, and delivered in vitro or through cellular mechanisms, while enzyme catalysis lowers the activation barriers of the CO2 transformations to make them more energy efficient. The development of technologies that treat CO2-transforming enzymes and other cellular components as modules that may be assembled into synthetic reaction circuits will facilitate the use of CO2 as a renewable chemical feedstock, greatly enabling a sustainable carbon bio-economy

Recombinant cell-lysate-catalysed synthesis of uridine-5'-triphosphate from nucleobase and ribose, and without addition of ATP

Nucleoside triphosphates (NTPs) are important synthetic targets with diverse applications in therapeutics and diagnostics. Enzymatic routes to NTPs from simple building blocks are attractive, however the cost and complexity of assembling the requisite mixtures of multiple enzymes hinders application. Here, we describe the use of an engineered E. coli cell-free lysate as an efficient readily-prepared multi-enzyme biocatalyst for the production of uridine triphosphate (UTP) from free ribose and nucleobase. Endogenous lysate enzymes are able to support the nucleobase ribosylation and nucleotide phosphorylation steps, while uridine phosphorylation and the production of ribose phosphates (ribose 1-phosphate, ribose 5-phosphate and phosphoribosyl pyrophosphate) require recombinant enrichment of endogenous activities. Co-expression vectors encoding all required recombinant enzymes were employed for host cell transformation, such that a cell-free lysate with all necessary activities was obtained from a single bacterial culture. ATP required as phosphorylation cofactor was recycled by endogenous lysate enzymes using cheap, readily-prepared acetyl phosphate. Surprisingly, acetyl phosphate initiated spontaneous generation of ATP in the lysate, most likely from the breakdown of endogenous pools of adenosine-containing starting materials (e.g. adenosine cofactors, ribonucleic acids). The sub-stoichiometric amount of ATP produced and recycled in this way was enough to support all ATP-dependent steps without addition of any exogenous cofactor or auxiliary enzyme. Using this approach, equimolar solutions of orotic acid and ribose are transformed near quantitatively into 1.4 g L−1 UTP within 2.5 h, using a low-cost, readily-generated biocatalytic preparation.The authors acknowledge funding from the Australian Research Council (grant number DP150101425)

Biocompatible functionalisation of starch

The enzyme catalysed esterification of starch and fatty acids with terminal triple bonds is described. This material can be used as an acceptor for azide containing molecules, through azide/alkyne cycloaddition. The potential is illustrated by the production of fluorescently-labelled starch, and a biotinylated derivative which can bind streptavidin

One-Pot Multienzymatic Transformation of NH 3 , CO 2 , and Ornithine into the Organic Nitrogen Plant Fertilizer Citrulline Using a Single Recombinant Lysate of E. coli

Biocatalytic transformation of carbamate formed readily from CO2 and NH3 provides attractive green routes for mitigation of these important environmental pollutants. Accordingly, a coupled-enzyme system was developed for the one-pot production of citrulline through carbamoylation of ornithine in aqueous solutions of CO2 and NH3. Hyperthermophilic ornithine carbamoyltransferases are produced recombinantly in E. coli with carbamate kinases known to have a propensity for carbamoyl phosphate synthesis. Importantly, in vitro biocatalysis is carried out by E. coli cell lysate prepared through coexpression of the required recombinant enzymes in a single bacterial culture, greatly reducing limitations normally associated with protein production and purification. Acetate kinase that is endogenous in the lysate also recycles the required ATP cofactor, which would otherwise have been required in costly stoichiometric amounts. Recombinant lysates catalyze the production of carbamoyl phosphate with substoichiometric ATP (>300 turnovers) as well as its in situ reaction with ornithine to give citrulline in high yield (>95%) and g L–1 h–1 titers. The system is active over a wide range of NH3 concentrations (2.5 mM – 2 M), and >90% conversions of NH3 may be reached within 1.5 h. Aqueous NH3 used to sequester CO2 gas (10% v/v) may be directly used as the biocatalyst feedstock. In preliminary studies, citrulline is found to be an effective organic nitrogen fertilizer of the wheat grass Brachypodium distachyon. Therefore, lysates described here constitute a cost-effective biocatalytic platform for one-pot production of a promising organic nitrogen fertilizer, under mild reaction conditions, from environmental pollutants as feedstock.The authors acknowledge financial support by the Grains Research and Development Corporation (GRDC), the Australian Research Council (ARC), the Australian National University (ANU), and CSIRO and the advice and assistance of Dr Hideki Onagi in this work