Elsevier

Tetrahedron: Asymmetry

Volume 14, Issue 6, 21 March 2003, Pages 667-673
Tetrahedron: Asymmetry

Synthesis of sucrose laurate using a new alkaline protease

https://doi.org/10.1016/S0957-4166(03)00086-7Get rights and content

Abstract

Sucrose laurate esters were synthesized from sucrose and vinyl laurate in organic solvents using an alkaline protease from a new alkalophilic strain, Bacillus pseudofirmus AL-89. Maximum synthetic activity was observed in the presence of 7.5% v/v water and in the pH range of 7–10. With protease AL-89 esterification occurred predominantly at the 2-O-position while subtilisin A-catalyzed monoester formation predominantly at the 1′-O position. In the absence of enzyme, buffer salts catalyzed non-specific reactions, resulting in the formation of a number of esters. Non-specific catalysis was also observed upon inhibition of the enzyme using a serine protease inhibitor or upon deactivation of the enzyme at pH above 10.

2-O-lauroyl sucrose was synthesized in a protease (AL-89) catalyzed transesterification process in DMF:pyridine, 1:1 (v/v).

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Introduction

Sugar esters find important applications in a variety of industrial processes including their use as biodegradable surfactants in the food and cosmetic industry, as antitumoral agents and plant growth inhibitors.1., 2. Currently sucrose esters are synthesized non-enzymatically at high temperatures, resulting in low specificity and side reactions with the formation of colored derivatives.3 In recent years, hydrolytic enzymes have received increasing attention for specific and regioselective acylation of carbohydrates in organic solvents.4., 5., 6., 7. In addition to their selectivity, enzyme-catalyzed reactions are advantageous as they can be performed under milder conditions than non-enzymatic reactions, potentially lowering the cost of ester production.

The two main problems encountered with the enzymatic acylation of carbohydrates in organic solvents are the stability of the biocatalyst and the solubility of the sugar substrate.6., 7. While better sugar solubility is obtained in water miscible organic solvents, such as dimethylsulfoxide (DMSO) and dimethylformamide (DMF), most enzymes are denatured in the presence of these polar solvents.7., 8.

Lipases and serine proteases are commonly used for the synthesis of sugar fatty acid esters and the enzymes catalyze reactions by a similar mechanism.9 To date only a handful of commercially available lipases and serine proteases have been used for the synthesis of sugar esters.10 The serine protease, subtilisin is known to synthesize sucrose esters predominantly at the 1′-O-position in DMF and pyridine11., 12., 13. and the metalloprotease, thermolysin has recently been shown to catalyze the acylation of sucrose at the 2-O-position in DMSO.14 Thus, enzymes from different sources have different properties, including stability and selectivity in the presence of chemical denaturants. This shows the need to search for new and better biocatalysts that can withstand the reaction conditions required for efficient industrial productions. Herein, we report on the use of an alkaline protease from Bacillus pseudofirmus AL-89, recently isolated from an alkaline soda lake in Ethiopia15 for sucrose ester synthesis in organic solvents.

Section snippets

Effect of water

Protease AL-89 catalyzed the synthesis of sucrose esters using vinyl laurate as the acyl donor in DMF. No ester synthesis was detected in the absence of water showing that the enzyme needed to be hydrated to catalyze the reaction. Maximum synthetic activity was observed in the presence of 7.5% v/v water (Fig. 1). Following the progress of the reaction in the presence of 10% v/v water by TLC, showed that ester formation reached a maximum at 12 h and decreased markedly at 24 h, resulting in a

Discussion

Protease AL-89 adjusted at pH 10 catalyzed the synthesis of sucrose laurate esters using vinyl laurate as the acyl donor in three hydrophilic solvents. Ester synthesis was observed only when water was present in the reaction medium. In organic solvents the enzyme conformation is believed to be very rigid, leading to significant reduction in activity.16 Hydration is thought to reduce the rigidity of the enzyme by forming multiple hydrogen bonds with the amino acid residues, thus increasing the

Materials

Celite, silica gel, Amberlite XAD 7, sucrose, molecular sieves (3 Å, 8–12 mesh), phenyl methylsulfonyl fluoride (PMSF) and organic solvents were from Sigma, St. Louis, MO, USA. Accurel EP100 was from Akzo Nobel, Obernburg, Germany. Silica gel 60 and thin-layer chromatography (TLC) plates were from Merck (Darmstadt, Germany). Lauric acid and vinyl laurate were from Fluka (Buchs, Switzerland). The organic solvents and vinyl laurate were stored over molecular sieves.

Enzyme sources

Subtilisin A was a kind gift

Acknowledgements

The present work was financed by Aalborg University. The authors wish to thank Professor P. Halling (University of Strathclyde, Glasgow, UK) for valuable suggestions and Associate professor Dr. M. Wiebe (Aalborg University, Denmark) for valuable suggestions and critical reading of the manuscript.

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      Biocatalysts has also been employed for the production of SEs, with the main advantage of high specificity and selectivity in the degree of substitutions in the products formed (Fig. 2b). Lipases extracted from Thermomyces lanuginosus (Ferrer et al., 2005) and Candida antarctica B (Neta et al., 2012; Zhao et al., 2015), in addition to alkaline proteases from Bacillus pseudofirmus AL-89 (Pedersen et al., 2003; Potier et al., 2001), have been reported as biocatalysts in the production of SEs with high selectivity for OH-6 and OH-6′ hydroxyl in sucrose by lipases, and high selectivity for OH-1′ hydroxyl in sucrose by proteases. However, the use of biocatalysts is restricted by a narrow temperature range due to enzyme protein denaturation.

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