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ORGANIC SYNTHESIS OF MAIZE STARCHBASED POLYMER USING Rhizopus oryzae LIPASE, SCALE UP, AND ITS CHARACTERIZATION a
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Vinod Kumar , Sweta Yadav , Firdaus Jahan & Rajendra Kumar Saxena
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Department of Microbiology , University of Delhi South Campus , New Delhi , India Accepted author version posted online: 24 Aug 2013.Published online: 09 Dec 2013.
To cite this article: Vinod Kumar , Sweta Yadav , Firdaus Jahan & Rajendra Kumar Saxena (2014) ORGANIC SYNTHESIS OF MAIZE STARCH-BASED POLYMER USING Rhizopus oryzae LIPASE, SCALE UP, AND ITS CHARACTERIZATION, Preparative Biochemistry and Biotechnology, 44:4, 321-331, DOI: 10.1080/10826068.2013.803481 To link to this article: http://dx.doi.org/10.1080/10826068.2013.803481
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Preparative Biochemistry & Biotechnology, 44:321–331, 2014 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6068 print/1532-2297 online DOI: 10.1080/10826068.2013.803481
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ORGANIC SYNTHESIS OF MAIZE STARCH-BASED POLYMER USING Rhizopus oryzae LIPASE, SCALE UP, AND ITS CHARACTERIZATION
Vinod Kumar, Sweta Yadav, Firdaus Jahan, and Rajendra Kumar Saxena Department of Microbiology, University of Delhi South Campus, New Delhi, India
& The industrial utilization of native starches is limited because of their inherit nature, with characteristics such as water insolubility and their tendency to form unstable pastes and gels. In this investigation, a lipase produced from Rhizopus oryzae was used for modification of maize starch with palmitic acid at a reaction temperature of 45 C for 18 hr in the presence of dimethyl sulfoxide (DMSO). The synthesis of maize starch palmitate was confirmed by Fourier-transform infrared (FT-IR) and 1H-nuclear magnetic resonance (NMR) spectra with a higher degree of substitution (DS) of 1.68. Thermal gravimetric analysis (TGA) showed that the maize starch palmitate is more stable even up to 496 C as compared to unmodified maize starch (231.4 C). Maize starch palmitate possesses high degree of substitution and thermal properties and thus can be widely used in food and pharmaceutical industry. Keywords high degree of substitution (DS), lipase esterification, maize starch, structural characterization
INTRODUCTION Naturally abundant polysaccharides, in particular starch and cellulose, are increasingly being considered as renewable, potentially biodegradable, and widely used in the production of both food and industrial products.[1,2] Certain properties such as solubility, gelatinization temperature, viscosity, stability of the paste, moisture, emulsifiability, water retention, and film property are changed when starch undergoes modification.[3] Such modification of starch has biomedical applications, such as materials for bone fixation and replacements, and carriers for controlled release of drugs and other bioactive agents.[4,5] These modified starches are used in various industries as glues, adhesives, and auxiliaries of a wide range of rheological and functional purposes.[6] Address correspondence to Rajendra Kumar Saxena, Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi-110021, India. E-mail: [email protected]
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In the past few years, an enzymatic-based technique for an organic method of synthesis has been used both in academic and industrial laboratories. For the last 10 years, dedicated enzymatic synthesis at small scale in research laboratories has been commercially available and offers a number of advantages.[7] The enzymatic synthesis is an environmentally friendly method that occurs under milder conditions.[8] At present the big challenge in this area is to establish a reliable bioprocess method, where typical scaleup issues, for example, the limited depth, a reliable temperature control, and a suitable reactor design, are carefully considered.[9] Small-scale synthesis is traditionally conducted in batches, and the majority of chemical reactors also operate in this way, utilizing vessels generally below 100 mL in volume. Eventually, in certain industrial applications, the properties of natural polysaccharides are not appropriate for their specific uses and physical or chemical changes are employed to obtain these molecules with improved properties.[10] It has been reported that a small change in the structure of the polysaccharide can make extensive alterations in the physicochemical properties.[11] Although the addition of an ester group into a starch is an important chemical modification, it is a challenge to synthesize highly substituted starch derivatives, mainly because of the difficulties of the granular starch in the suitable medium.[12–14] Chemical modification of starch is often required to better suit its properties to specific applications. Chemical esterification of starch is usually done at high pH and high temperature.[15] Esterification of starch with long-chain fatty acids gives thermoplastic starches. By using a catalyst lipase from Rhizopus oryzae, this study aimed to achieve a biosynthesis of highly substituted maize starch palmitate and to test the product convertibility to batch culture. The esterification of starch with palmitic acid was carried out in a solvent system. The structure and physicochemical properties of starch palmitate were also studied. MATERIALS AND METHODS Microbial Culture Condition and Materials Rhizopus oryzae, obtained from our laboratory culture collection, was maintained on potato dextrose agar (PDA). Maize starch from our lab stock, purchased earlier, was used. Palmitic acid (99.9%) with dialysis tubing (12KD), was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Microorganism and Production Conditions Production of lipase was carried out in 2-L Erlenmeyer flasks containing 400 mL of optimized production medium (%wt=vol: soybean oil emulsified
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with 0.2% gum acacia, 1.50 mL; cane molasses, 1.0 g; maize starch, 1.0 g; soybean meal, 2.0 g; CaCl2 2H2O, 0.10 g; KH2PO4, 0.50 g; pH 9.0) inoculated with 1 107 spores=50 mL and incubated at 30 C, 200 rpm, for 96 hr. The purified enzyme was obtained after filtration and centrifugation of the culture broth and subsequent four fold concentration using 10-kD cellulose acetate membranes (Millipore). Subsequently, this concentrated retentate containing lipase was subjected to precipitation using salts, an established procedure for carrying out partial purification of lipases by Shu et al.[16] The concentrated, partially purified lipase was lyophilized and directly used for carrying out the reaction. Determination of Lipase Activity The lipase activity of both the immobilized and the free lipase was measured as per to the procedure described by Winkler and Stuckmann,[24] where 1 unit (U) of enzyme activity is defined as the amount of enzyme that liberates 1 mmol p-nitrophenol per minute under assay conditions. Esterification of Maize Starch Maize starch (200 mM) and palmitic acid (100 mM) were dissolved in dimethyl sulfoxide (DMSO), and 200 mg lyophilized lipase (3.6 IU=mg) was added and incubated in a shaker at 45 C for 24 hr. After the completion of the reaction, the enzyme was filtered off and fatty acid ester were precipitated by adding 10 mL ethanol and then dried in a hot air oven at 60 2 C. The precipitate was purified by using the procedure reported by Qiao et al.[16] and then characterized by 1H-nuclear magnetic resonance (NMR), Fourier-transform infrared (FT-IR), and thermogravimetric analysis (TGA). Degree of Substitution The degree of substitution (DS) was determined by hydrolyzing and release of fatty acids with 1 M NaOH and then titrating back with 0.5 M HCl. DS was calculated according to the modified method of Apostolos et al.[17] as in Eq. (1): DS ¼
n M1 M0 n ðM2 MH2 OÞ
ð1Þ
where n is palmitic molecules=glucose molecules; M0 is weight of sample, g; M1 is molecular weight of anhydrous glucose unit, 162; M2 is molecular weight of palmitic acid, 256.42; and MH2O is molecular weight of H2O.
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Characterization of Modified Starch Ester The purified maize starch palmitate was evaluated by Fourier-transfer infrared (FT-IR; Bomem MB Series), which showed the shift of the carbonyl of carboxylic acid group to the carbonyl of ester group. For FT-IR spectroscopic analysis, the sample was mixed with anhydrous potassium bromide powder. The mixture was filled in the disk and compressed at 9 kN for 2 min. A film was then made and analyzed in the beam of the FT-IR spectrophotometer. The infrared spectra were detected over 400–4000 cm1 using 8 scans and with resolution equal to 4 cm1. The thermostability of modified ester was determined by a thermogravimetric analyser (TGA; Perkin-Elmer Thermal Analysis, Germany) with a heating rate of 10 C=min from 50 to 600 C in air. Purified modified maize starch palmitate was subjected to 1 H-NMR analysis for analysis its structural features. 1H-NMR spectra were recorded at 400 MHz. The chemical shifts were reported in parts per million (ppm) relative to either tetramethylsilane or the deuterated solvent as an internal standard for 1H-NMR. Coupling constants (J values) are given in hertz. Scale-Up Esterification of Maize Starch Process optimization was initially carried out in 250-mL screwcapped flasks containing 200 mM of maize starch concentration in 50 mL reaction medium. It was thus felt necessary to validate the synthesis of maize starch-based polymer to larger volumes. Scale-up experiments were performed using 2 L of optimized reaction mixtures containing starch (200 g), palmitic acid (100 g), DMSO (2000 mL) solvent medium, and lipase in a 5-L round-bottom flask. Reaction was carried out at 45 C and 200 rpm agitation rate. Samples were withdrawn for 18 hr and analyzed for degree of substitution. RESULTS AND DISCUSSIONS Lipase Preparation for Esterification Culture-free fermentation broth obtained after centrifugation was subsequently fourfold concentrated using 10-kD cellulose acetate membranes (Millipore) and then treated with ammonium sulfate (60.0% saturation), and it stood overnight at 4 C. The precipitate was collected by centrifugation (10,000 rpm for 30 min), suspended in distilled water, and dialyzed against distilled water.[16] The lipase activity was determined by the modified procedure of Winker and Stuckmann.[18] The resultant concentrated partially purified lyophilized lipase (3.6 IU=mg) was used directly for esterification.
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Degree of Substitution of Modified Maize Starch Degree of substitution increases with reaction time. It is evident from results presented in Figure 1 that a high degree of substitution (DS) of 1.68 was observed after 18 hr of incubation time. However, further incubation of the reaction did not increase the DS. Furthermore, the elemental analysis of the products obtained by the maize starch modification was carried out in aqueous media, dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), and DMSO þ DMF (1:1, v=v) and the observations are presented in Figure 2. Results reveal that starch esters with high DS can be synthesized in solvent DMSO using lipase as the catalyst. In many cases the reported DS values lies well above 1.0, which means that more than one hydroxyl position is substituted on each anhydroglucose unit despite the enzymatic regioselectivity, which is normally strong (C-6 for lipases used in the present study).[19] Rajan et al.[20] reported DS values ranging from 0.98 to 1.55 for microwave-heated reactions and lower (0.45) for a reaction performed on gelatinized starch with hydrolyzed coconut oil spread throughout with the help of surfactant (Triton X-100). In another investigation Horchani et al.[21] reported complete acylation with oleic acid in a reaction of gelatinized starch (termed solvent free) catalyzed by immobilized Staphylococcus aureus lipase, under a combination of microwave heating with a high degree of substitution (2.86). Alissandratos et al.[18] also reported that starch modification by using oleic acid catalyzed by lipase achieved a degree of substitution (DS) of 0.01.
FIGURE 1 Effect of time on degree of substitution. Reaction condition: maize starch (200 mM), palmitic acid (100 mM); 5% (by weight of maize starch) R. oryzae lipase; DMSO solvent and 200 rpm at 45 C. Data are represented a mean standard deviation of triplicate observation.
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FIGURE 2 Comparison of degree of substitution (DS) in different solvents. Reaction condition: Maize starch:palmitic acid (1:0.5, m=m); 5% (weight by maize starch) lipase; and 200 rpm at 45 C. Data are represented a mean standard deviation of triplicate observation.
FT-IR Spectroscopy of Modified Maize Starches It was observed from the FT-IR spectral curve presented a peak at about 578.65 cm1. Another broad absorption peak at 3401.15 cm1 could be ascribed to hydroxyls, which showed that the surface had large numbers of hydroxyls. Further, on comparing the spectral curves of the maize starch and modified starch particles, another strong broad band due to hydroxyl bond stretching appears at 3000–3600 cm1 (Figure 3A), which is reduced on esterification (Figure 3B). The represent stretching vibration of CH2 groups, with the peak at 1717.95 cm1, was due to bending vibration of the carbonyl -C-O bond, adsorption. In the native starch spectrum, the characteristic peaks (958–1255 cm1) are attributed to CO bond stretching.[10] An absorption band at 1,023 cm1 was observed, which is probably due to the stretching of the C–OH bond, in the spectra of the starch, which is consistent with the earlier report by Marcazzan et al.[23] The ester groups impart plasticity, with increase in the size of fatty acid chain used.[14] The starch esters thus produced behave like thermoplastic materials. Thermogravimetric Analysis of Maize Starch Palmitate To evaluate the thermal stability, maize starch palmitate was analyzed by thermogravimetric analysis (TGA). The TGA curves of maize starch and maize starch palmitate are shown in Figure 4. The TGA curve shows that maize starch palmitate is more stable at 496.2 C compared to maize starch. A similar experiment was done by Thiebaud et al.[24] and found that the degradation of native starch starts at 280 C with a 30% of weight loss, and
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FIGURE 3 FT-IR spectra of (A) maize starch and (B) maize starch palmitate. Sample was mixed with dry potassium bromide; transmittance was collected at a wavenumber range of 4000–400 cm1 (color figure available online).
further heating to 600 C resulted in complete degradation of native starch. Rajan et al.[21] also reported that modified starch was more thermostable compared to native starch at 600 C with a high degree of substitution DS of 1.45. A similar result has also been reported by Rajan et al.[24] with fungal lipase. This increase in thermal stability with increasing DS was due to the low amount of remaining hydroxyl groups in starch molecules after modification. Thiebaud et al.[11] gave the region of the decomposition of modified starch due to the water molecule. There is also a beneficial influence of
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FIGURE 4 Thermogravimetric curves of (A) maize starch and (B) maize starch palmitate. The thermostability of maize starch and maize starch palmitate was determined by a thermogravimetric analyzer with a heating rate of 10 C=min from 50 to 600 C in air.
increasing carbon chain length of the ester on thermal stability, as it acts as a more efficient internal plasticizer. No decomposition was observed at 600 C in TGA by Rajan et al.[21] 1
H-NMR Spectroscopy of Modify Maize Starch
The 1H-NMR spectral signals were between 3.2 and 5.2 ppm (Figure 5A), which corresponds to the protons of the constituent repeating a-D-glucopyranosyl units, while the starch palmitate exhibited the characteristic peaks of palmitic acid protons in the region from 0.5 to 2.5 ppm (Figure 5B). The existence of a pair of downfield doublets at 1.25 and 1.3 ppm and 1.65 and 2.3 ppm confirmed the presence of protons in palmitic acid, and also the aliphatic protons were observed in the 0.5–2.4 ppm range. NMR for native maize starch was 1H-NMR (400 HZ, DMSO, d6): d 5.03 (d, 1, J ¼ 7.0, CH) 3.73 (t, 2, J ¼ 7.33, CH, CH) 3.65 (d, 1, J ¼ 7.0, OH), 3.58 (d, 1, J ¼ 7.0, OH), 3.02 (s, 1, J ¼ 7.33, CH); and for modified maize starch palmitate, 1H-NMR (400 HZ, DMSO, d6): d 5.03 (d, 1, J ¼ 7.0, CH), 3.73 (t, 2, J ¼ 7.33, CH, CH), 3.58 (s, 1, J ¼ 7.0, OH) 3.02 (s, 1, J ¼ 7.3, CH), 2.45 (m, 2, J ¼ 7.3, CH2), 1.53 (m, 2, J ¼ 7.0,
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FIGURE 5 1H-NMR spectra of (A) maize starch and (B) maize starch palmitate. 1H-NMR spectra were recorded with Bruker avance II 400 MHz spectrometer at 294.3 K, and the data processing was performed with top spin 1.3 standard software (color figure available online).
CH2), 1.29 (m, 20, J ¼ 7.3, CH2), 1.31(m, 2, J ¼ 7.0, CH2) 0.88 (m, 3, J ¼ 7.33, CH3). Scale-Up of Synthesis of Maize Starch Palmitate Process optimization was initially carried out in a 250-mL round-bottom flask containing 200 mM of maize starch. It was thus felt necessary to validate the synthesis of starch palmitate to larger volumes when the reaction was scaled up. It is clearly evident from Figure 6 that maize starch palmitate was successfully scaled up in reaction volume size (2 L) with a high degree of substitution (1.68). Maize starch palmitate synthesis could be scaled up to various reaction sizes with the same degree of substitution.
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FIGURE 6 Scale-up of the modification of maize starch. Reaction condition: maize starch (200 g), palmitic acid (100 g); 5% (by weight of maize starch) R. oryzae lipase; 2 L DMSO solvent and 200 rpm at 45 C. Data are represented a mean standard deviation of triplicate observation.
CONCLUSION Lipase obtained from Rhizopus oryzae was found to be a useful biocatalyst in starch modification. Esterification of maize starch with palmitic acid was carried out at 10% (w=w) lipase and 45 C for 18 hr, with a maximum degree of substitution (DS) of 1.68, and thermal degradation on set temperature increased from 231.2 C to 496.2 C and the ester peak was found in the region of 1717.95 cm1. Our scale-up approach is a part of the green chemistry and can be regarded as environmentally benign. The process is eco-friendly, as there are no toxic waste products and hence the starch esters can be used directly for various end uses. As a general conclusion it can be considered that the thermal properties of the starch-based polymers, together with their damping properties, might allow their use in several of the proposed biomedical applications. Starch-based polymers also combine a much cheaper price with a clearly better biocompatible behavior.
ACKNOWLEDGMENT The authors gratefully acknowledge financial support from the Ministry of New and Renewable Energy (MNRE). The authors thank Technology Based Incubator, UDSC, and New Delhi for providing the infrastructure support.
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REFERENCES 1. Jarowenko, W. Acetylated Starch and Miscellaneous Organic Esters. In Modified Starches: Properties and Uses; Wurzburg, O.B., Ed.; CRC Press, Boca Raton, FL, 1986; pp. 55–77. 2. Singh, J.; Kaur, L.; McCarthy, O.J. Factors Influencing the Physico-Chemical, Morphological, Thermal and Rheological Properties of Some Chemically Modified Starches for Food Applications— A Review. Food Hydrocolloids 2007, 21, 1–22. 3. Sousa, R.A.; Mano, J.F.; Reis, R.L.; Cunha, A.M.; Bevis, M.J. Mechanical Performance of Starch Based Bioactive Composites Moulded With Preferred Orientation for Potential Medical Applications. Polym. Eng. Sci. 2002, 42, 1032–1045. 4. Espigares, I.; Elvira, C.; Mano, J.F.; Vasquez, B.; Yoman, J.; Reis, R.L. New Partially Degradable and Bioactive Acrylic Bone Cements Based on Starch Blends and Ceramic Fillers. Biomaterials 2002, 23, 1883–1895. 5. Alissandratos, A.; Halling, P.J. Enzymatic Acylation of Starch. Bioresource Technol. 2012, 115, 41–47. 6. Miladinov, V.D.; Hanna, M.A. 1999. Physical and Molecular Properties of Starch Acetates Extruded With Water and Ethanol. Ind. Eng. Chem. Res. 1999, 10, 3892–3897. 7. Fernandez-Lafuente, R. Lipase From Thermomyces lanuginosus: Uses and Prospects as an Industrial Biocatalyst. J. Mol. Catal. B Enzym. 2010, 62, 197–212. 8. Gomes, M.E.; Ribeiro, A.S.; Malafaya, P.B.; Reis, R.L.; Cunha, A.M. A New Approach Based on Injection Moulding to Produce Biodegradable Starch-Based Polymeric Scaffolds: Morphology, Mechanical and Degradation Behaviour. Biomaterials 2001, 22, 883–889. 9. Pandey, A.; Benjamin, S.; Soccol, C.R.; Krieger, P.N.; Soccol, V.T. The Realm of Microbial Lipases in Biotechnology. Biotechnol. Appl. Biochem. 1999, 29, 119–131. 10. Zhao, W.X.; Zheng, W.W.; Li, J.H.; Lin, H.H. Synthesis and Characterization of Starch Fatty Acid Esters. Mod. Chem. Ind. 2007, 27, 281–283. 11. Thiebaud, S.; Aburto, J.; Alric, I.; Borredon, E.; Bikiaris, D.; Prinos, J.; Panayiotou, C. Properties of Fatty-Acid Esters of Starch and Their Blends With LDPE. J. Appl. Polym. Sci. 1997, 65, 705–721. 12. Reis, R.L.; Cunha, A.M. New Degradable Load-Bearing Biomaterials Composed of Reinforced Starch Based Blends. J. Appl. Med. Polym. 2000, 4, 1–5. 13. Gomes, M.E.; Ribeiro, A.S.; Malafaya, P.B.; Reis, R.L.; Cunha, A.M. A New Approach Based on Injection Moulding to Produce Biodegradable Starch-Based Polymeric Scaffolds: Morphology, Mechanical and Degradation Behaviour. Biomaterials 2001, 22, 883–889. 14. Rajan, A.; Abraham, T.E. Enzymatic Modification of Cassava Starch by Bacterial Lipase. Bioprocess Biosyst. Eng. 2006, 29, 65–71. 15. Caddick, S. Microwave Assisted Organic Reactions. Tetrahedron 1995, 51, 10403–10432. 16. Shu, Z.Y.; Yang, J.K.; Yan, Y.J. (2007). Purification and Characterization of a Lipase From Aspergillus niger F044. Chin. J. Biotech. 2007, 23, 96–100. 17. Qiao, L.; Gu, Q.M.; Cheng, H.N. Enzyme-Catalyzed Synthesis of Hydrophobically Modified Starch. Carbohyd. Polym. 2006, 66, 135–140. 18. Apostolos, A.; Nina, B.; Sabine, L.F.; Bernhard, H.; Peter, J.H. Lipase-Catalysed Acylation of Starch and Determination of the Degree of Substitution by Methanolysis and GC. BMC Biotechnol. 2010, 10, 1–25. 19. Winkler, U.K.; Stuckmann, M. Glycogen, Hyaluronidate and Some Other Polysaccharides Greatly Enhance the Formation of Exocellular Lipase by Serratia marcescens. J. Bacteriol. 1979, 138, 663–670. 20. Xin, J.Y.; Wang, Y.; Liu, T.; Lin, K.; Chang, L.; Xia, C.G. Biosysthesis of Corn Starch Palmitate by Lipase Novozym 435. Int. J. Mol. Sci. 2012, 13, 7226–7236. 21. Rajan, A.; Prasad, V.S.; Abraham, T.E. Enzymatic Esterification of Starch Using Recovered Coconut Oil. Int. J. Bio. Macromol. 2006, 39, 265–272. 22. Horchani, H.; Chaabouni, M.; Gargouri, Y.; Sayari, A. Solvent-Free Lipase Catalyzed Synthesis of Long-Chain Starch Esters Using Microwave Heating: Optimization by Response Surface Methodology. Carbohydr. Polym. 2010, 79, 466–474. 23. Alissandratos, A.; Baudendistel, N.; Hauer, B.; Baldenius, K.; Flitsch, S.; Halling, P. Biocompatible Functionalisation of Starch. Chem. Commun. 2011, 47, 683–685. 24. Marcazzan, M.; Vianello, F.; Scarpa, M.; Rigo, A. An ESR Assay for Alpha-Amylase Activity Toward Succinylated Starch, Amylose and Amylopectin. J. Biochem. Biophys. Methods 1999, 38, 191–202. 25. Rajan, A.; Sudha, J.D.; Abraham, T.E. Enzymatic Modification of Cassava Starch by Fungal Lipase. Ind. Crop. Prod. 2008, 27, 50–59.
Preparative Biochemistry and Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb20
ORGANIC SYNTHESIS OF MAIZE STARCHBASED POLYMER USING Rhizopus oryzae LIPASE, SCALE UP, AND ITS CHARACTERIZATION a
a
a
Vinod Kumar , Sweta Yadav , Firdaus Jahan & Rajendra Kumar Saxena
a
a
Click for updates
Department of Microbiology , University of Delhi South Campus , New Delhi , India Accepted author version posted online: 24 Aug 2013.Published online: 09 Dec 2013.
To cite this article: Vinod Kumar , Sweta Yadav , Firdaus Jahan & Rajendra Kumar Saxena (2014) ORGANIC SYNTHESIS OF MAIZE STARCH-BASED POLYMER USING Rhizopus oryzae LIPASE, SCALE UP, AND ITS CHARACTERIZATION, Preparative Biochemistry and Biotechnology, 44:4, 321-331, DOI: 10.1080/10826068.2013.803481 To link to this article: http://dx.doi.org/10.1080/10826068.2013.803481
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Preparative Biochemistry & Biotechnology, 44:321–331, 2014 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6068 print/1532-2297 online DOI: 10.1080/10826068.2013.803481
Downloaded by [University of Manitoba Libraries] at 11:49 15 February 2015
ORGANIC SYNTHESIS OF MAIZE STARCH-BASED POLYMER USING Rhizopus oryzae LIPASE, SCALE UP, AND ITS CHARACTERIZATION
Vinod Kumar, Sweta Yadav, Firdaus Jahan, and Rajendra Kumar Saxena Department of Microbiology, University of Delhi South Campus, New Delhi, India
& The industrial utilization of native starches is limited because of their inherit nature, with characteristics such as water insolubility and their tendency to form unstable pastes and gels. In this investigation, a lipase produced from Rhizopus oryzae was used for modification of maize starch with palmitic acid at a reaction temperature of 45 C for 18 hr in the presence of dimethyl sulfoxide (DMSO). The synthesis of maize starch palmitate was confirmed by Fourier-transform infrared (FT-IR) and 1H-nuclear magnetic resonance (NMR) spectra with a higher degree of substitution (DS) of 1.68. Thermal gravimetric analysis (TGA) showed that the maize starch palmitate is more stable even up to 496 C as compared to unmodified maize starch (231.4 C). Maize starch palmitate possesses high degree of substitution and thermal properties and thus can be widely used in food and pharmaceutical industry. Keywords high degree of substitution (DS), lipase esterification, maize starch, structural characterization
INTRODUCTION Naturally abundant polysaccharides, in particular starch and cellulose, are increasingly being considered as renewable, potentially biodegradable, and widely used in the production of both food and industrial products.[1,2] Certain properties such as solubility, gelatinization temperature, viscosity, stability of the paste, moisture, emulsifiability, water retention, and film property are changed when starch undergoes modification.[3] Such modification of starch has biomedical applications, such as materials for bone fixation and replacements, and carriers for controlled release of drugs and other bioactive agents.[4,5] These modified starches are used in various industries as glues, adhesives, and auxiliaries of a wide range of rheological and functional purposes.[6] Address correspondence to Rajendra Kumar Saxena, Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi-110021, India. E-mail: [email protected]
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In the past few years, an enzymatic-based technique for an organic method of synthesis has been used both in academic and industrial laboratories. For the last 10 years, dedicated enzymatic synthesis at small scale in research laboratories has been commercially available and offers a number of advantages.[7] The enzymatic synthesis is an environmentally friendly method that occurs under milder conditions.[8] At present the big challenge in this area is to establish a reliable bioprocess method, where typical scaleup issues, for example, the limited depth, a reliable temperature control, and a suitable reactor design, are carefully considered.[9] Small-scale synthesis is traditionally conducted in batches, and the majority of chemical reactors also operate in this way, utilizing vessels generally below 100 mL in volume. Eventually, in certain industrial applications, the properties of natural polysaccharides are not appropriate for their specific uses and physical or chemical changes are employed to obtain these molecules with improved properties.[10] It has been reported that a small change in the structure of the polysaccharide can make extensive alterations in the physicochemical properties.[11] Although the addition of an ester group into a starch is an important chemical modification, it is a challenge to synthesize highly substituted starch derivatives, mainly because of the difficulties of the granular starch in the suitable medium.[12–14] Chemical modification of starch is often required to better suit its properties to specific applications. Chemical esterification of starch is usually done at high pH and high temperature.[15] Esterification of starch with long-chain fatty acids gives thermoplastic starches. By using a catalyst lipase from Rhizopus oryzae, this study aimed to achieve a biosynthesis of highly substituted maize starch palmitate and to test the product convertibility to batch culture. The esterification of starch with palmitic acid was carried out in a solvent system. The structure and physicochemical properties of starch palmitate were also studied. MATERIALS AND METHODS Microbial Culture Condition and Materials Rhizopus oryzae, obtained from our laboratory culture collection, was maintained on potato dextrose agar (PDA). Maize starch from our lab stock, purchased earlier, was used. Palmitic acid (99.9%) with dialysis tubing (12KD), was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Microorganism and Production Conditions Production of lipase was carried out in 2-L Erlenmeyer flasks containing 400 mL of optimized production medium (%wt=vol: soybean oil emulsified
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with 0.2% gum acacia, 1.50 mL; cane molasses, 1.0 g; maize starch, 1.0 g; soybean meal, 2.0 g; CaCl2 2H2O, 0.10 g; KH2PO4, 0.50 g; pH 9.0) inoculated with 1 107 spores=50 mL and incubated at 30 C, 200 rpm, for 96 hr. The purified enzyme was obtained after filtration and centrifugation of the culture broth and subsequent four fold concentration using 10-kD cellulose acetate membranes (Millipore). Subsequently, this concentrated retentate containing lipase was subjected to precipitation using salts, an established procedure for carrying out partial purification of lipases by Shu et al.[16] The concentrated, partially purified lipase was lyophilized and directly used for carrying out the reaction. Determination of Lipase Activity The lipase activity of both the immobilized and the free lipase was measured as per to the procedure described by Winkler and Stuckmann,[24] where 1 unit (U) of enzyme activity is defined as the amount of enzyme that liberates 1 mmol p-nitrophenol per minute under assay conditions. Esterification of Maize Starch Maize starch (200 mM) and palmitic acid (100 mM) were dissolved in dimethyl sulfoxide (DMSO), and 200 mg lyophilized lipase (3.6 IU=mg) was added and incubated in a shaker at 45 C for 24 hr. After the completion of the reaction, the enzyme was filtered off and fatty acid ester were precipitated by adding 10 mL ethanol and then dried in a hot air oven at 60 2 C. The precipitate was purified by using the procedure reported by Qiao et al.[16] and then characterized by 1H-nuclear magnetic resonance (NMR), Fourier-transform infrared (FT-IR), and thermogravimetric analysis (TGA). Degree of Substitution The degree of substitution (DS) was determined by hydrolyzing and release of fatty acids with 1 M NaOH and then titrating back with 0.5 M HCl. DS was calculated according to the modified method of Apostolos et al.[17] as in Eq. (1): DS ¼
n M1 M0 n ðM2 MH2 OÞ
ð1Þ
where n is palmitic molecules=glucose molecules; M0 is weight of sample, g; M1 is molecular weight of anhydrous glucose unit, 162; M2 is molecular weight of palmitic acid, 256.42; and MH2O is molecular weight of H2O.
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Characterization of Modified Starch Ester The purified maize starch palmitate was evaluated by Fourier-transfer infrared (FT-IR; Bomem MB Series), which showed the shift of the carbonyl of carboxylic acid group to the carbonyl of ester group. For FT-IR spectroscopic analysis, the sample was mixed with anhydrous potassium bromide powder. The mixture was filled in the disk and compressed at 9 kN for 2 min. A film was then made and analyzed in the beam of the FT-IR spectrophotometer. The infrared spectra were detected over 400–4000 cm1 using 8 scans and with resolution equal to 4 cm1. The thermostability of modified ester was determined by a thermogravimetric analyser (TGA; Perkin-Elmer Thermal Analysis, Germany) with a heating rate of 10 C=min from 50 to 600 C in air. Purified modified maize starch palmitate was subjected to 1 H-NMR analysis for analysis its structural features. 1H-NMR spectra were recorded at 400 MHz. The chemical shifts were reported in parts per million (ppm) relative to either tetramethylsilane or the deuterated solvent as an internal standard for 1H-NMR. Coupling constants (J values) are given in hertz. Scale-Up Esterification of Maize Starch Process optimization was initially carried out in 250-mL screwcapped flasks containing 200 mM of maize starch concentration in 50 mL reaction medium. It was thus felt necessary to validate the synthesis of maize starch-based polymer to larger volumes. Scale-up experiments were performed using 2 L of optimized reaction mixtures containing starch (200 g), palmitic acid (100 g), DMSO (2000 mL) solvent medium, and lipase in a 5-L round-bottom flask. Reaction was carried out at 45 C and 200 rpm agitation rate. Samples were withdrawn for 18 hr and analyzed for degree of substitution. RESULTS AND DISCUSSIONS Lipase Preparation for Esterification Culture-free fermentation broth obtained after centrifugation was subsequently fourfold concentrated using 10-kD cellulose acetate membranes (Millipore) and then treated with ammonium sulfate (60.0% saturation), and it stood overnight at 4 C. The precipitate was collected by centrifugation (10,000 rpm for 30 min), suspended in distilled water, and dialyzed against distilled water.[16] The lipase activity was determined by the modified procedure of Winker and Stuckmann.[18] The resultant concentrated partially purified lyophilized lipase (3.6 IU=mg) was used directly for esterification.
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Degree of Substitution of Modified Maize Starch Degree of substitution increases with reaction time. It is evident from results presented in Figure 1 that a high degree of substitution (DS) of 1.68 was observed after 18 hr of incubation time. However, further incubation of the reaction did not increase the DS. Furthermore, the elemental analysis of the products obtained by the maize starch modification was carried out in aqueous media, dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), and DMSO þ DMF (1:1, v=v) and the observations are presented in Figure 2. Results reveal that starch esters with high DS can be synthesized in solvent DMSO using lipase as the catalyst. In many cases the reported DS values lies well above 1.0, which means that more than one hydroxyl position is substituted on each anhydroglucose unit despite the enzymatic regioselectivity, which is normally strong (C-6 for lipases used in the present study).[19] Rajan et al.[20] reported DS values ranging from 0.98 to 1.55 for microwave-heated reactions and lower (0.45) for a reaction performed on gelatinized starch with hydrolyzed coconut oil spread throughout with the help of surfactant (Triton X-100). In another investigation Horchani et al.[21] reported complete acylation with oleic acid in a reaction of gelatinized starch (termed solvent free) catalyzed by immobilized Staphylococcus aureus lipase, under a combination of microwave heating with a high degree of substitution (2.86). Alissandratos et al.[18] also reported that starch modification by using oleic acid catalyzed by lipase achieved a degree of substitution (DS) of 0.01.
FIGURE 1 Effect of time on degree of substitution. Reaction condition: maize starch (200 mM), palmitic acid (100 mM); 5% (by weight of maize starch) R. oryzae lipase; DMSO solvent and 200 rpm at 45 C. Data are represented a mean standard deviation of triplicate observation.
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FIGURE 2 Comparison of degree of substitution (DS) in different solvents. Reaction condition: Maize starch:palmitic acid (1:0.5, m=m); 5% (weight by maize starch) lipase; and 200 rpm at 45 C. Data are represented a mean standard deviation of triplicate observation.
FT-IR Spectroscopy of Modified Maize Starches It was observed from the FT-IR spectral curve presented a peak at about 578.65 cm1. Another broad absorption peak at 3401.15 cm1 could be ascribed to hydroxyls, which showed that the surface had large numbers of hydroxyls. Further, on comparing the spectral curves of the maize starch and modified starch particles, another strong broad band due to hydroxyl bond stretching appears at 3000–3600 cm1 (Figure 3A), which is reduced on esterification (Figure 3B). The represent stretching vibration of CH2 groups, with the peak at 1717.95 cm1, was due to bending vibration of the carbonyl -C-O bond, adsorption. In the native starch spectrum, the characteristic peaks (958–1255 cm1) are attributed to CO bond stretching.[10] An absorption band at 1,023 cm1 was observed, which is probably due to the stretching of the C–OH bond, in the spectra of the starch, which is consistent with the earlier report by Marcazzan et al.[23] The ester groups impart plasticity, with increase in the size of fatty acid chain used.[14] The starch esters thus produced behave like thermoplastic materials. Thermogravimetric Analysis of Maize Starch Palmitate To evaluate the thermal stability, maize starch palmitate was analyzed by thermogravimetric analysis (TGA). The TGA curves of maize starch and maize starch palmitate are shown in Figure 4. The TGA curve shows that maize starch palmitate is more stable at 496.2 C compared to maize starch. A similar experiment was done by Thiebaud et al.[24] and found that the degradation of native starch starts at 280 C with a 30% of weight loss, and
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FIGURE 3 FT-IR spectra of (A) maize starch and (B) maize starch palmitate. Sample was mixed with dry potassium bromide; transmittance was collected at a wavenumber range of 4000–400 cm1 (color figure available online).
further heating to 600 C resulted in complete degradation of native starch. Rajan et al.[21] also reported that modified starch was more thermostable compared to native starch at 600 C with a high degree of substitution DS of 1.45. A similar result has also been reported by Rajan et al.[24] with fungal lipase. This increase in thermal stability with increasing DS was due to the low amount of remaining hydroxyl groups in starch molecules after modification. Thiebaud et al.[11] gave the region of the decomposition of modified starch due to the water molecule. There is also a beneficial influence of
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FIGURE 4 Thermogravimetric curves of (A) maize starch and (B) maize starch palmitate. The thermostability of maize starch and maize starch palmitate was determined by a thermogravimetric analyzer with a heating rate of 10 C=min from 50 to 600 C in air.
increasing carbon chain length of the ester on thermal stability, as it acts as a more efficient internal plasticizer. No decomposition was observed at 600 C in TGA by Rajan et al.[21] 1
H-NMR Spectroscopy of Modify Maize Starch
The 1H-NMR spectral signals were between 3.2 and 5.2 ppm (Figure 5A), which corresponds to the protons of the constituent repeating a-D-glucopyranosyl units, while the starch palmitate exhibited the characteristic peaks of palmitic acid protons in the region from 0.5 to 2.5 ppm (Figure 5B). The existence of a pair of downfield doublets at 1.25 and 1.3 ppm and 1.65 and 2.3 ppm confirmed the presence of protons in palmitic acid, and also the aliphatic protons were observed in the 0.5–2.4 ppm range. NMR for native maize starch was 1H-NMR (400 HZ, DMSO, d6): d 5.03 (d, 1, J ¼ 7.0, CH) 3.73 (t, 2, J ¼ 7.33, CH, CH) 3.65 (d, 1, J ¼ 7.0, OH), 3.58 (d, 1, J ¼ 7.0, OH), 3.02 (s, 1, J ¼ 7.33, CH); and for modified maize starch palmitate, 1H-NMR (400 HZ, DMSO, d6): d 5.03 (d, 1, J ¼ 7.0, CH), 3.73 (t, 2, J ¼ 7.33, CH, CH), 3.58 (s, 1, J ¼ 7.0, OH) 3.02 (s, 1, J ¼ 7.3, CH), 2.45 (m, 2, J ¼ 7.3, CH2), 1.53 (m, 2, J ¼ 7.0,
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FIGURE 5 1H-NMR spectra of (A) maize starch and (B) maize starch palmitate. 1H-NMR spectra were recorded with Bruker avance II 400 MHz spectrometer at 294.3 K, and the data processing was performed with top spin 1.3 standard software (color figure available online).
CH2), 1.29 (m, 20, J ¼ 7.3, CH2), 1.31(m, 2, J ¼ 7.0, CH2) 0.88 (m, 3, J ¼ 7.33, CH3). Scale-Up of Synthesis of Maize Starch Palmitate Process optimization was initially carried out in a 250-mL round-bottom flask containing 200 mM of maize starch. It was thus felt necessary to validate the synthesis of starch palmitate to larger volumes when the reaction was scaled up. It is clearly evident from Figure 6 that maize starch palmitate was successfully scaled up in reaction volume size (2 L) with a high degree of substitution (1.68). Maize starch palmitate synthesis could be scaled up to various reaction sizes with the same degree of substitution.
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FIGURE 6 Scale-up of the modification of maize starch. Reaction condition: maize starch (200 g), palmitic acid (100 g); 5% (by weight of maize starch) R. oryzae lipase; 2 L DMSO solvent and 200 rpm at 45 C. Data are represented a mean standard deviation of triplicate observation.
CONCLUSION Lipase obtained from Rhizopus oryzae was found to be a useful biocatalyst in starch modification. Esterification of maize starch with palmitic acid was carried out at 10% (w=w) lipase and 45 C for 18 hr, with a maximum degree of substitution (DS) of 1.68, and thermal degradation on set temperature increased from 231.2 C to 496.2 C and the ester peak was found in the region of 1717.95 cm1. Our scale-up approach is a part of the green chemistry and can be regarded as environmentally benign. The process is eco-friendly, as there are no toxic waste products and hence the starch esters can be used directly for various end uses. As a general conclusion it can be considered that the thermal properties of the starch-based polymers, together with their damping properties, might allow their use in several of the proposed biomedical applications. Starch-based polymers also combine a much cheaper price with a clearly better biocompatible behavior.
ACKNOWLEDGMENT The authors gratefully acknowledge financial support from the Ministry of New and Renewable Energy (MNRE). The authors thank Technology Based Incubator, UDSC, and New Delhi for providing the infrastructure support.
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REFERENCES 1. Jarowenko, W. Acetylated Starch and Miscellaneous Organic Esters. In Modified Starches: Properties and Uses; Wurzburg, O.B., Ed.; CRC Press, Boca Raton, FL, 1986; pp. 55–77. 2. Singh, J.; Kaur, L.; McCarthy, O.J. Factors Influencing the Physico-Chemical, Morphological, Thermal and Rheological Properties of Some Chemically Modified Starches for Food Applications— A Review. Food Hydrocolloids 2007, 21, 1–22. 3. Sousa, R.A.; Mano, J.F.; Reis, R.L.; Cunha, A.M.; Bevis, M.J. Mechanical Performance of Starch Based Bioactive Composites Moulded With Preferred Orientation for Potential Medical Applications. Polym. Eng. Sci. 2002, 42, 1032–1045. 4. Espigares, I.; Elvira, C.; Mano, J.F.; Vasquez, B.; Yoman, J.; Reis, R.L. New Partially Degradable and Bioactive Acrylic Bone Cements Based on Starch Blends and Ceramic Fillers. Biomaterials 2002, 23, 1883–1895. 5. Alissandratos, A.; Halling, P.J. Enzymatic Acylation of Starch. Bioresource Technol. 2012, 115, 41–47. 6. Miladinov, V.D.; Hanna, M.A. 1999. Physical and Molecular Properties of Starch Acetates Extruded With Water and Ethanol. Ind. Eng. Chem. Res. 1999, 10, 3892–3897. 7. Fernandez-Lafuente, R. Lipase From Thermomyces lanuginosus: Uses and Prospects as an Industrial Biocatalyst. J. Mol. Catal. B Enzym. 2010, 62, 197–212. 8. Gomes, M.E.; Ribeiro, A.S.; Malafaya, P.B.; Reis, R.L.; Cunha, A.M. A New Approach Based on Injection Moulding to Produce Biodegradable Starch-Based Polymeric Scaffolds: Morphology, Mechanical and Degradation Behaviour. Biomaterials 2001, 22, 883–889. 9. Pandey, A.; Benjamin, S.; Soccol, C.R.; Krieger, P.N.; Soccol, V.T. The Realm of Microbial Lipases in Biotechnology. Biotechnol. Appl. Biochem. 1999, 29, 119–131. 10. Zhao, W.X.; Zheng, W.W.; Li, J.H.; Lin, H.H. Synthesis and Characterization of Starch Fatty Acid Esters. Mod. Chem. Ind. 2007, 27, 281–283. 11. Thiebaud, S.; Aburto, J.; Alric, I.; Borredon, E.; Bikiaris, D.; Prinos, J.; Panayiotou, C. Properties of Fatty-Acid Esters of Starch and Their Blends With LDPE. J. Appl. Polym. Sci. 1997, 65, 705–721. 12. Reis, R.L.; Cunha, A.M. New Degradable Load-Bearing Biomaterials Composed of Reinforced Starch Based Blends. J. Appl. Med. Polym. 2000, 4, 1–5. 13. Gomes, M.E.; Ribeiro, A.S.; Malafaya, P.B.; Reis, R.L.; Cunha, A.M. A New Approach Based on Injection Moulding to Produce Biodegradable Starch-Based Polymeric Scaffolds: Morphology, Mechanical and Degradation Behaviour. Biomaterials 2001, 22, 883–889. 14. Rajan, A.; Abraham, T.E. Enzymatic Modification of Cassava Starch by Bacterial Lipase. Bioprocess Biosyst. Eng. 2006, 29, 65–71. 15. Caddick, S. Microwave Assisted Organic Reactions. Tetrahedron 1995, 51, 10403–10432. 16. Shu, Z.Y.; Yang, J.K.; Yan, Y.J. (2007). Purification and Characterization of a Lipase From Aspergillus niger F044. Chin. J. Biotech. 2007, 23, 96–100. 17. Qiao, L.; Gu, Q.M.; Cheng, H.N. Enzyme-Catalyzed Synthesis of Hydrophobically Modified Starch. Carbohyd. Polym. 2006, 66, 135–140. 18. Apostolos, A.; Nina, B.; Sabine, L.F.; Bernhard, H.; Peter, J.H. Lipase-Catalysed Acylation of Starch and Determination of the Degree of Substitution by Methanolysis and GC. BMC Biotechnol. 2010, 10, 1–25. 19. Winkler, U.K.; Stuckmann, M. Glycogen, Hyaluronidate and Some Other Polysaccharides Greatly Enhance the Formation of Exocellular Lipase by Serratia marcescens. J. Bacteriol. 1979, 138, 663–670. 20. Xin, J.Y.; Wang, Y.; Liu, T.; Lin, K.; Chang, L.; Xia, C.G. Biosysthesis of Corn Starch Palmitate by Lipase Novozym 435. Int. J. Mol. Sci. 2012, 13, 7226–7236. 21. Rajan, A.; Prasad, V.S.; Abraham, T.E. Enzymatic Esterification of Starch Using Recovered Coconut Oil. Int. J. Bio. Macromol. 2006, 39, 265–272. 22. Horchani, H.; Chaabouni, M.; Gargouri, Y.; Sayari, A. Solvent-Free Lipase Catalyzed Synthesis of Long-Chain Starch Esters Using Microwave Heating: Optimization by Response Surface Methodology. Carbohydr. Polym. 2010, 79, 466–474. 23. Alissandratos, A.; Baudendistel, N.; Hauer, B.; Baldenius, K.; Flitsch, S.; Halling, P. Biocompatible Functionalisation of Starch. Chem. Commun. 2011, 47, 683–685. 24. Marcazzan, M.; Vianello, F.; Scarpa, M.; Rigo, A. An ESR Assay for Alpha-Amylase Activity Toward Succinylated Starch, Amylose and Amylopectin. J. Biochem. Biophys. Methods 1999, 38, 191–202. 25. Rajan, A.; Sudha, J.D.; Abraham, T.E. Enzymatic Modification of Cassava Starch by Fungal Lipase. Ind. Crop. Prod. 2008, 27, 50–59.
