Abstract
The study sought to isolate asporopollenin-like biopolymer from Aspergillus niger (A. niger) spore. The spore exine (Sp-exine) from A. niger was isolated using four different methods. The highest isolation efficiency (9.32 %) was found for the method based on H2SO4 treatment. Optical microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images showed that the isolated Sp-exine had a spherical shape with a spun morphology and was highly uniform in size. The elemental compositions of the bodies of the Sp-exine materials, as well as their surfaces, were observed by means of CHN combustion analysis and SEM with energy-dispersive X-ray spectroscopy (SEM-EDX), respectively. The result demonstrates C, H and O to be the main structural elements in all the Sp-exine samples. Fourier-transform infrared (FTIR) spectroscopy coupled with solid state 13C nuclear magnetic resonance (solid state 13C NMR) spectroscopy and UV-Vis spectroscopy were used to compare the changes in the functional groups of the Sp-exine samples isolated using the different methods. A thermogravimetric analysis (TGA) study demonstrated all the Sp-exine samples showed high thermal stability. Among all tested methods, the treatment with H2SO4 and alcoholic potassium hydroxide exhibited the best results in removing cellular and other contents with minimal drastic effect on the structure and morphology and is proposed as the best method for isolating Sp-exine since it required minimal isolation time and showed good isolation efficiency.
Acknowledgements
One of the authors (Murari Lal Soni) wishes to express gratitude to the All India Institute of Medical Sciences, New Delhi, India and Indian Institute of Technology, Roorkee, India, for providing access to the TEM and the SEM facilities respectively, to the Microbial-type Culture Collection and Gene Bank, Chandigarh, India, for providing the A. niger fungi, and to SAIF-CDRI (Sophisticated Analytical Instrument Facility, CSIR-Central Drug Research Institute, Lucknow, India) for allowing access to the CHN facility. The authors also wish to thank Dr. (Mrs.) Sudha Srivastava, National Facility for High Field NMR (TIFR, Mumbai), for permitting access to the solid-state 13 C NMR facility. The authors also wish to thank Dr. Edward J. Button, Button and Associates, Virginia, USA, for language editing.
References
Adamson, R., Gregson, S., & Shaw, G. (2009). New applications of sporopollenin as a solid phase support for peptide synthesis and the use of sonic agitation. International Journal of Peptide and Protein Research, 22, 560–564. 10.1111/J.1399-3011.1983.tb02128.x.Search in Google Scholar
Amer, S. M., Tawashi, R., Samir Amer, M., Amer, M., & Tawashi, R. (1991). U.S. Patent No.5013552. Washington, D.C., USA: U.S. Patent and Trademark Office.Search in Google Scholar
Amer, S. M., & Tawashi, R. (1994). U.S. Patent No. 5275819. Washington, D.C., USA: U.S. Patent and Trademark Office.Search in Google Scholar
Ayar, A., & Ali Gürten, A. (2003). Determination of the rate control step of purin and pyrimidine bases adsorption on cobalt(II)-CDAE-sporopollenin. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 229, 149–155. 10.1016/j.colsurfa.2003.08.017.Search in Google Scholar
Barrier, S., Rigby, A. S., Diego-Taboada, A., Thomasson, M. J., Mackenzie, G., & Atkin, S. L. (2010). Sporopollenin exines: A novel natural taste masking material. LWT - Food Science and Technology, 43, 73–76. 10.1016/j.lwt.2009.07.001.Search in Google Scholar
Barrier, S., Diego-Taboada, A., Thomasson, M. J., Madden, L., Pointon, J. C., Wadhawan, J. D., Beckett, S. T., Atkin, S. L., & Mackenzie, G. (2011). Viability of plant spore exine capsules for microencapsulation. Journal of Materials Chemistry, 21, 975–981. 10.1039/c0jm02246b.Search in Google Scholar
Beckett, S., & Mackenzie, G. (2010). Using nature to preserve fish oils. Chemistry Review, 20, 10–14.Search in Google Scholar
Brooks, J., & Shaw, G. (1968), Chemical structure of the exine of pollen walls and a new function for carotenoids in nature. Nature, 219, 532–533. 10.1038/219532a0.Search in Google Scholar
Brooks, J., & Shaw, G. (1972). Geochemistry of sporopollenin. Chemical Geology, 10, 69–87. 10.1016/0009-2541(72)90078-2.Search in Google Scholar
Bubert, H., Lambert, J., Steuernagel, S., Ahlers, F., & Wiermann, R. (2002). Continuous decomposition of sporopollenin from pollen of Typha angustifolia L. by acidic methanoly-sis. Zeitschrift für Naturforschung C, 57, 1035–1041. 10.1515/znc-2002-11-1214.Search in Google Scholar
Burczyk, J., & Dworzanski, J. (1988). Comparison of sporopollenin-like algal resistant polymer from cell wall of Botryococcus, scenedesmus and lycopodium clavatum by GC-pyrolysis. Phytochemistry, 27, 2151–2153. 10.1016/0031-9422(88)80115-8.Search in Google Scholar
Diego-Taboada, A., Barrier, S., Thomasson, M., Atkin, S., & Mackenzie, G. (2007). Pollen: A novel encapsulation vehicle for drug delivery. Innovations in Pharmaceutical Technology, 2007, 63–68.Search in Google Scholar
Diego-Taboada, A., Maillet, L., Banoub, J. H., Lorch, M., Rigby, A. S., Boa, A. N., Atkin, S. L., & Mackenzie, G. (2013) . Protein free microcapsules obtained from plant spores as a model for drug delivery: ibuprofen encapsulation, release and taste masking. Journal of Materials Chemistry B, 2013, 707–713. 10.1039/c2tb00228k.Search in Google Scholar PubMed
Diego-Taboada, A., Beckett, S. T., Atkin, S. L., & Mackenzie, G. (2014) . Hollow pollen shells to enhance drug delivery. Pharmaceutics, 6, 80–96. 10.3390/pharmaceutics6010080.Search in Google Scholar PubMed PubMed Central
Domínguez, E., Mercado, J. A., Quesada, M. A., & Heredia, A. (1998). Isolation of intact pollen exine using anhydrous hydrogen fluoride. Grana, 37, 93–96. 10.1080/00173139809362649.Search in Google Scholar
Domínguez, E., Mercado, J. A., Quesada, M. A., & Heredia, A. (1999). Pollen sporopollenin: degradation and structural elucidation. Sexual Plant Reproduction, 12, 171–178. 10.1007/s004970050189.Search in Google Scholar
Erzengin, M., Ünlü, N., & Odabaşi, M. (2011). A novel adsorbent for protein chromatography: Supermacroporous monolithic cryogel embedded with Cu2+ -attached sporopollenin particles. Journal of Chromatography A, 1218, 484–490. 10.1016/j.chroma.2010.11.074.Search in Google Scholar
Espelie, K. E., Loewus, F. A., Pugmire, R. J., Woolfenden, W. R., Baldi, B. G., & Given, P. H. (1989). Structural analysis of lilium longiflorum sporopollenin by 13 C NMR spectroscopy. Phytochemistry, 28, 751–753. 10.1016/0031-9422(89)80108-6.Search in Google Scholar
Fraser, W. T., Scott, A. C., Forbes, A. E. S., Glasspool, I. J., Plotnick, R. E., Kenig, F., & Lomax, B. H. (2012). Evolutionary stasis of sporopollenin biochemistry revealed by unaltered Pennsylvanian spores. The New Phytologist, 196, 397–401. 10.1111/j.1469-8137.2012.04301.x.Search in Google Scholar PubMed
Geisert, M., Rose, T., Zahn, R. K., Shaw, G., & Brooks, J. (1988). In vitro polymerization of β-carotene into sporopollenin. Journal für Praktische Chemie, 330, 476–478. 10.1002/prac.19883300321.Search in Google Scholar
Gezici, O., Küçükosmanoglu, M., & Ayar, A. (2006). The adsorption behavior of crystal violet in functionalized sporopollenin-mediated column arrangements. Journal of Colloid and Interface Science, 304, 307–316. 10.1016/j.jcis.2006.09.048.Search in Google Scholar PubMed
Gode, F., & Pehlivan, E. (2007). Sorption of Cr(III) onto chelating b-DAEG-sporopollenin and CEP-sporopollenin resins. Bioresource Technology, 98, 904–911. 10.1016/j.biortech.2006.02.043.Search in Google Scholar PubMed
Gubbuk, I. H., Ozmen, M., & Maltas, E. (2012). Immobilization and characterization of hemoglobin on modified sporopollenin surfaces. International Journal of Biological Macro-molecules, 50, 1346–1352. 10.1016/j.ijbiomac.2012.04.009.Search in Google Scholar PubMed
Gunnison, D., & Alexander, M. (1975). Basis for the resistance of several algae to microbial decomposition. Applied Microbiology, 29, 729–738.10.1128/am.29.6.729-738.1975Search in Google Scholar PubMed PubMed Central
Hamad, S. A., Dyab, A. F. K., Stoyanov, S. D., & Paunov, V. N. (2011). Encapsulation of living cells into sporopollenin microcapsules. Journal of Materials Chemistry, 21, 18018. 10.1039/c1jm13719k.Search in Google Scholar
Hayatsu, R., Botto, R. E., McBeth, R. L., Scott, R. G., & Winans, R. E. (1988). Chemical alteration of a biological polymer ‘sporopollenin’ during coalification: origin, formation, and transformation of the coal maceral sporinite. Energy & Fuels, 2, 843–847. 10.1021/ef00012a019.Search in Google Scholar
Hemsley, A. R., Barrie, P. J., & Scott, A. C. (1995). 13 C solidstate n.m.r. spectroscopy of fossil sporopollenins. Variation in composition independent of diagenesis. Fuel, 74, 1009–1012. 10.1016/0016-2361(95)00028-4.Search in Google Scholar
Hesse, M., & Waha, M. (1989). A new look at the acetolysis method. Plant Systematics and Evolution, 163, 147–152. 10.1007/bf00936510.Search in Google Scholar
Jardine, P. E., Fraser, W. T., Lomax, B. H., & Gosling, W. D. (2015). The impact of oxidation on spore and pollen chemistry. Journal of Micropalaeontology, 34, 139–149. 10.1144/jmpaleo2014-022.Search in Google Scholar
Jungfermann, C., Ahlers, F., Grote, M., Gubatz, S., Steuernagel, S., Thom, I., Wetzels, G., Wiermann, R., Ungfermann, C., Ahlers, F., Grote, M., Gubatzl, S., Steuernagel, S., Thom, I. N. A., Wetzels, G., & Wiermann, R. (1997). Solution of sporopollenin and reaggregation of a sporopollenin-like material: A new approach in the sporopollenin research. Journal of Plant Physiology, 151, 513–519. 10.1016/s0176-1617(97)80224-6.Search in Google Scholar
Kadouri, A., Derenne, S., Largeau, C., Casadevall, E., & Berkaloff, C. (1988). Resistant biopolymer in the outer walls of Botryococcus braunii, B race. Phytochemistry, 27, 551– 557. 10.1016/0031-9422(88)83140-6.Search in Google Scholar
Kawase, M., & Takahashi, M. (1995). Chemical composition of sporopollenin in Magnolia grandiflora (Magnoliaceae) and Hibiscus syriacus (Malvaceae). Grana, 34, 242–245. 10.1080/00173139509429052.Search in Google Scholar
Kokinos, J. P., Eglinton, T. I., Goni, M. A., Boon, J.J., Martoglio, P. A., & Anderson, D. M. (1998). Characterization of a highly resistant biomacromolecular material in the cell wall of a marine dinoflagellate resting cyst. Organic Geochemistry, 28, 265–288. 10.1016/s0146-6380(97)00134-4.Search in Google Scholar
Koley, D., & Bard, A. J. (2010). Triton X-100 concentration effects on membrane permeability of a single HeLa cell by scanning electrochemical microscopy (SECM). Proceedings of the National Academy of Sciences of the United States of America, 107, 16783–16787. 10.1073/pnas.1011614107.Search in Google Scholar PubMed PubMed Central
Leeuw, J. W. D., Bergen, P. F. V., Aarssen, B. G. K. V., Gatellier, J. P. L. A., Damste, J. S. S., Collinson, M. E. (1991). Resistant biomacromolecules as major contributors to kerogen. Philosophical Transactions of the Royal Society B: Biological Sciences, 333, 329–337. 10.1098/rstb.1991.0082.Search in Google Scholar
Lorch, M., Thomasson, M. J., Diego-Taboada, A., Barrier, S., Atkin, S. L., Mackenzie, G., & Archibald, S. J. (2009). MRI contrast agent delivery using spore capsules: controlled release in blood plasma. Chemical Communications, 2009 6442–6444. 10.1039/b909551a.Search in Google Scholar PubMed
Maack, A. (2007). U.S. Patent No. 7182965. Washington, D.C., USA: U.S. Patent and Trademark Office.Search in Google Scholar
Mackenzie, G., & Shaw, G. (1980). Sporopollenin. A novel, naturally occurring support for solid phase peptide synthesis. International Journal of Peptide and Protein Research, 15, 298–300. 10.1111/j.1399-3011.1980.tb02580.x.Search in Google Scholar
Mackenzie, G., Boa, A. N., Diego-Taboada, A., Atkin, S. L., & Sathyapalan, T. (2015). Sporopollenin, the least known yet toughest natural biopolymer. Frontiers in Materials, 2, 1–5. 10.3389/fmats.2015.00066.Search in Google Scholar
Paknikar, K., & Kulkarni, P. (2011). U.S. Patent No. WO 2011058573 A1. Washington, D.C., USA: U.S. Patent and Trademark Office.Search in Google Scholar
Paunov, V. N., Mackenzie, G., & Stoyanov, S. D. (2007). Sporopollenin micro-reactors for in-situ preparation, encapsulation and targeted delivery of active components. Journal of Materials Chemistry, 17, 609. 10.1039/b615865j.Search in Google Scholar
Sargin, I., & Arslan, G. (2015). Chitosan/sporopollenin microcapsules: Preparation, characterisation and application in heavy metal removal. International Journal of Biological Macromolecules, 75, 230–238. 10.1016/j.ijbiomac.2015. 01.039.Search in Google Scholar
Sayin, S., Gubbuk, I. H., & Yilmaz, M. (2013). Preparation of calix[4]arene-based sporopollenin and examination of its dichromate sorption ability Journal of Inclusion Phenomena and Macrocyclic Chemistry, 75, 111–118. 10.1007/s10847-012-0152-6.Search in Google Scholar
Schraudolf, H., & Haag, R. (1985). The sporopollenin of the schizaeaceous fern Anemia phyllitidis. Naturwissenschaften, 72, 433–434. 10.1007/bf00404888.Search in Google Scholar
Shaw, G., & Yeadon, A. (1966). Chemical studies on the constitution of some pollen and spore membranes. Journal of the Chemical Society C: Organic, 5, 16–22. 10.1039/J39660000016.Search in Google Scholar
Shaw, G. (1970). The chemistry of sporopollenin. In P. R. Grant, M. Muir, P. van Gijzel, & G. Shaw (Eds.), Sporpoollenin (pp. 305–350). London, UK: Academic Press.Search in Google Scholar
Shaw, G., & Apperley, D. C. (1996). 13 C-NMR spectra of Lycopodium clavatum sporopollenin and oxidatively polymerised β-carotene. Grana, 35, 125–127. 10.1080/00173139609429483.Search in Google Scholar
Southworth, D. (1969). Ultraviolet absorption spectra of pollen and spore walls. Grana Palynologica, 9, 5–15. 10.1080/00173136909436421.Search in Google Scholar
Strohl, W.R., Larkin, J.M., Good, B.H., &Chapman, R. L. (1977). Isolation of sporopollenin from four myxobacteria. Canadian Journal of Microbiology, 23, 1080–1083. 10.1139/m77-162.Search in Google Scholar PubMed
Valdés, F., Catalá, L., Hernández, M. R., García-Quesada, J. C., & Marcilla, A. (2013). Thermogravimetry and Py-GC/MS techniques as fast qualitative methods for comparing the biochemical composition of Nannochloropsis oculata samples obtained under different culture conditions. Bioresource Technology, 131, 86–93. 10.1016/j.biortech.2012.12.133.Search in Google Scholar PubMed
Watson, J.S., Sephton, M.A., Sephton, S.V., Self, S., Fraser, W. T., Lomax, B. H., Gilmour, I., Wellman, C. H., & Beerling, D. J. (2007). Rapid determination of spore chemistry using thermochemolysis gas chromatography-mass spectrometry and micro-Fourier transform infrared spectroscopy. Photochemical & Photobiological Sciences, 6, 689–694. 10.1039/b617794h.Search in Google Scholar PubMed
Zetzsche, F., & Kälin, O. (1931). Untersuchungen über die Membran der Sporen und Pollen V. 4. Zur Autoxydation der Sporopollenine. Helvetica Chimica Acta, 14, 517–519. 10.1002/hlca.19310140151. (in German)Search in Google Scholar
Zetzsche, F. (1932). Kork und Cuticularsubstanzen. In G. Klein (Ed)., Spezielle Analyse, Zweiter Teil, Organische Stoffe II, Handbuch der Pflanzen analyse (pp. 205–239). Berlin, Germany: Springer-Verlag. (in German)Search in Google Scholar
Zimmermann, B., & Kohler, A. (2014). Infrared spectroscopy of pollen identifies plant species and genus as well as environmental conditions. PloS ONE, 9, e95417. 10.1371/journal.pone.0095417.Search in Google Scholar PubMed PubMed Central
Zimmermann, B., Tkalčec, Z., Mešić, A., & Kohler, A. (2015). Characterizing aeroallergens by infrared spectroscopy of fungal spores and pollen. PloS ONE, 10, e0124240. 10.1371/journal.pone.0124240.Search in Google Scholar PubMed PubMed Central
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