Enzymatic modification of cassava starch by fungal lipase
Introduction
Starch is an abundant natural polysaccharide that is inexpensive, renewable, and fully biodegradable. It has been widely studied for many years in the field of materials (Doane, 1992, Swanson et al., 1993a, Swanson et al., 1993b, Shogren et al., 1993). It is composed of two kinds of molecules—amylose and amylopectin. Starch was modified to expand its usefulness for a myriad of industrial applications.
Depending on the nature of the substituents and on the degree of substitution (DS), the properties of modified starch can be varied in an extensive way (Light, 1990). Starch shows various characteristics like solubility, stability of paste, moisture, emulsifiability, viscosity, water retention, and film property after modifying (or processing). Modification of the starch OH groups by esterification to form appropriate degree of substitution (1.5–3.0 DS) and with high percentage degree of substitution (50–95%) imparts thermoplasticity and water resistance to the starch ester over the unmodified starch. Enzymes like lipases and proteases have been used for the esterification of simple sugars. The sugar esters produced from short chain fatty acids have applications as flavouring agents in food industry and also the methyl and ethyl esters of long chain acids have been used to enrich diesel fuels (Vulfson, 1994). Esterification of five positional isomers of fatty acids (different chain lengths) in n-butanol was studied by Lie et al. (1998) using eight different lipases. Starch esters are used in many branches of industry as glues, adhesives, and auxiliaries of a wide range of rheological and functional properties.
In recent years, a number of authors (Thiebaud et al., 1997, Aburto et al., 1997, Aburto et al., 1999a, Aburto et al., 1999b, Fang et al., 2002) have reported the preparation of esterified starches of higher degrees of substitution, in presence of organic solvents so as to provide suitable reaction conditions. Such procedures rely on the use of sophisticated experimental techniques, solvents, or systems of solvents, to ultimately achieve homogeneous modification of the chosen starch. However, these techniques are expensive, typically use toxic solvents and, we believe, are not viable for the large-scale industrial production of modified starches.
The idea of applying microwave ovens instead of rotating roasters for esterification of starch seems promising. Microwave radiation (2450 MHz) does not activate specific bonds on molecules and consequently this form of heating will not lead to any kinetic differences compared to other forms of heating (Caddick, 1995). The rate of temperature rise depends on the moisture content of microwave-irradiated starches (Lewandowicz et al., 1997).
The homogeneous modification of starch relies upon the destructurisation of the semi-crystalline starch granules and the effective dispersion of their component amylose and amylopectin polymers. In this way, the reactive sites (hydroxyl groups) of the polymers become accessible to electrophilic reactants. Starch is effectively destructured by gelatinization in hot water, the precise temperature (which can vary extensively) depending on the source of starch or, more correctly on the branch chain length of the amylopectin component. High amylose starches (typical amylose:amylopectin ratio 70:30) gelatinize at around 160–170 °C, while wheat starch (amylose:amylopectin ratio 28:72) gelatinizes in the range of 52–65 °C (Whistler and BeMiller, 1997).
Starch esters produced are used in various biodegradable plastic materials, which are moldable. Esterification of starch with long chain fatty acid gives thermoplastic starch which has got wide use in plastic industry, pharmaceutical industries, and in biomedical applications such as materials for bone fixation and replacements, carriers for controlled release of drugs and other bioactive agents. They also have extensive applications in papermaking, textile and other fields. Starch/synthetic polymer blends have been used for distinct biomedical applications. These include starch-based biomaterials as scaffolds for the tissue engineering of bone and cartilage (Gomes et al., 2001) materials for bone fixation and replacement as well as for filling bone defects (Reis and Cunha, 2000, Sousa et al., 2002), carriers for the controlled release of drugs and other bioactive agents (Malafaya et al., 2001) and new hydrogels and partially degradable bone cements (Espigares et al., 2002). Starch esters have various industrial applications, for making thermoplastics, glues, adhesives, etc.
The aim of this work was to examine the possibility of enzymatic esterification of various starches by application of microwave instead of rotating roasters or extrusion cookers to obtain starch esters of C-12 to C-18 fatty acids with a structure and functionality similar to those of industrial products.
Section snippets
Starch
Cassava starch was procured commercially and purified by washing with distilled water repeatedly and settling and then drying in a cross-flow drier at 50 ± 5 °C to a moisture content of 12%, purity 99%.
Enzyme
Lipase AYS obtained from Candida rugosa (Amano, Japan) had about 6–8% enzyme protein along with solid fillers, which may be both stabilizers and bulking agents. Enzyme activity was 164 U/ml. The extraction of the enzymes from the unwanted filler materials was done with phosphate buffer (0.1 M, pH 7)
Thermostability and microwave oven stability of lipase
The optimum activity was found at 40 °C. After 60 °C there was a rapid decrease in enzyme activity over a time. At 70 °C there was a substantial decrease in enzyme activity over time. Half-life was attained in 30 min time. Microwave heating reduced the stability of the enzyme as an increase in time. The half-life was obtained within 1 min 15 s. This is due to the denaturation caused by in situ generation of heat due to the vibration of water molecules, if the exposure is above 30 s. Results are given
Conclusion
Lipase obtained from C. rugosa was found to be a useful biocatalyst in the esterification of starch. Liquid state esterification of cassava starch with palmitic acid using lipase for 4 h at 70 °C in DMSO gave a degree of substitution of 1.05 (65.86% DS). Esterification of cassava starch with hydrolysed coconut oil using microwave energy gave a DS of 1.1 (55.28%) while palmitic acid as acyl donor gave a DS of 0.33 (27.24% DS). The degree of substitution was found to be higher in microwave oven
Acknowledgement
The authors are grateful to Director, RRL (CSIR) for providing the necessary facilities to carry out the work.
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