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

Abstract

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.

Figure 1

Reactions and side-reactions of starch ester analyses. a) Titration Analysis: 1 Hydrolysis of starch ester (circle denotes starch), 2 Saponification of acid, 3 Titration of residual hydroxide ions, 4 Titration of saponified acid, b) Transesterification/GC Analysis: 1 Methanolysis of starch ester and formation of methyl ester, 2 Hydrolysis of methoxide 3 Hydrolysis of methyl ester, 4 Deprotonation of acid to form acyl ion, no methyl ester synthesis.

Figure 2

pH curves from titration analysis. Curves from titration by 0.5 M HCl of (···):1.4 mL of 1.0 M NaOH, (—): Native starch treated with 1.4 mL 1.0 M NaOH, (---): Native starch and decanoic acid mixture treated with 1.4 mL 1.0 M NaOH.

Figure 3

Enzymatic acylation of starch. With lipolase, at 50°C for 60 minutes, mixed by overhead stirrer.

Figure 4

Progress of lipolase catalysed synthesis of tapioca starch decanoate. This used standard conditions with 1.5 mL H₂O per g starch during gelatinisation.

Figure 5

Change in obtained DS for different initial concentrations of tapioca starch/water mixtures. This shows the ratio of starch to water mixed prior to the gelatinisation stage. Water content of the starch, and water losses were negligible, but additional water is present during the enzyme reaction, because this is added as an aqueous solution (0.5 mL per g starch). Reaction time was 1 hr.

Figure 6

NMR Spectrum for starch decanoic acid ester and unmodified native starch. The large peak at 3.2 ppm can be attributed to water from the starch sample.