Biomacromolecules 2009, 10, 793–797
An Efficient Method for
793
15
N-Labeling of Chitin in Fungi
Helen R. Watson,† David C. Apperley,† David P. Dixon,†,‡ Robert Edwards,‡ and David R. W. Hodgson*,† Department of Chemistry and School of Biological and Biomedical Sciences, Science Laboratories, Durham University, South Road, Durham, DH1 3LE, United Kingdom Received November 7, 2008; Revised Manuscript Received January 30, 2009
To permit facile 15N solid-state NMR (ssNMR) analysis of the degree of acetylation (DA) of chitinous materials in fungi a method for the introduction of a 15N isotopic label has been developed. Using Penicillium chrysogenum as a model system, a series of 15N-based media were surveyed for their abilities to support mycelial growth, and a rich medium supplemented with (15NH4)2SO4 supported good growth. Uptake of label into chitin extracted from mycelia grown in the rich (15NH4)2SO4-based media was monitored by mass spectrometry, with ∼1 g/L of (15NH4)2SO4 leading to ∼65% incorporation. The labeled chitin was studied by ssNMR to determine its DA, and the 15N label permitted measurement of DA to within 0.5% with acquisition times of on the order of half an hour. Similar studies validated the method for DA measurements on chitin from cultures of Aspergillus niger and Mucor rouxii.
1. Introduction The degree of acetylation (DA) has significant bearing on the biotechnological uses of chitinous materials and is also an important factor in fungal physiology studies where its value has significant consequences on the viability of fungi.1-3 With these issues in mind we have developed a rapid, reliable approach toward the determination of this important parameter in chtinous materials extracted from fungal systems. Many spectroscopic and analytical methods that measure the levels of either acetylated or deacetylated material in a given sample have been investigated for the determination of DA, but purity can be a significant issue.4-9 Ratiometric methods where both acetylated and deacteylated forms can be measured simultaneously offer better solutions to this problem. IR spectroscopy10,11 and UV spectroscopy12-15 offer ratiometric methods, but these can be severely affected by the solubility of the analyte. Nuclear magnetic resonance (NMR) spectroscopy also offers ratiometric analysis, and the technique has gained the reputation as the “gold standard” for the measurement of DA in chitin science. Solution state 1H NMR spectroscopy offers good sensitivity, but once again, it is limited by solubility issues.16,17 Some progress toward overcoming this obstacle has been made through the use of deuterium chloride solutions and elevated temperatures for dissolution, but these conditions are likely to lead to some depolymerization and deacetylation.18,19 Solid state NMR spectroscopy (ssNMR) avoids solubility problems, but it is only available for 13C and 15N nuclides. The 13 C ssNMR method offers reasonable acquisition times and the technique has been used widely to benchmark other techniques for the measurement of DA.6,20,21 The ratiometric analysis can, however, be impeded by the presence of other oligosaccharides within the analyte whose NMR signals overlap with chitin and chitosan signals. 15N ssNMR spectroscopic analyses have been shown to give two very clear signals with one corresponding to acetylated material and one to deacetylated material.22,23 * To whom all correspondence should be addressed. Phone: +44 (0)191 334 2100. E-mail:
[email protected]. † Centre for Bioactive Chemistry, Department of Chemistry. ‡ School of Biological and Biomedical Sciences.
Importantly, the information on the acetylation state of the chitin analyte was not obscured by signals from other oligosaccharides (e.g., glucans). Thus, the technique offers a significant advantage over many methods as the chitin does not need to be pure. Unfortunately, the use of 15N NMR spectroscopy is limited by the low natural abundance (99.5% and M. rouxii showing a DA of 90.9 ( 0.5%.
4. Conclusions Figure 4. Deacetylation kinetics of chitin using the 15N CP MAS ssNMR technique. The experimentally measured DA data are presented as circles and reference to the left-hand axis. The DA data have been fitted to a simple exponential using the least-squares approach. Calculated ln (DA (%)) data are presented as triangles and reference to the right-hand axis. The ln (DA (%)) data have been fitted to a simple linear function.
the mass of labeled chitin was kept very high (approximately 1333 mL of hydroxide solution per g of chitin). While this experimental setup does not mimic the processes that are often used for the large-scale preparation of chitosan (relatively low ratio hydroxide solution relative to the mass of chitin), it avoids complicating factors such as the build up of high concentrations of acetate, which have been shown to inhibit deacetylation.33 Under our conditions, the hydrolysis process would be expected to approximate to a pseudo first order process with respect to the concentration of acetamide sites in chitin. Samples of material were withdrawn periodically and these were subjected to 15N CP MAS ssNMR analysis to estimate the DA. 15N CP MAS ssNMR spectra corresponding to each time point are presented in Figure 3. The 15N ssNMR signals in each spectrum were integrated to estimate the DA and the data were plotted (Figure 4) as a function of time to assess the kinetics of deacetylation and to validate the use of the labeling approach toward the determination of the DA. The kinetics appear to be first order on initial inspection, however, on closer scrutiny, a slight, systematic deviation from simple exponential behavior was observed. This was illustrated most clearly by taking natural logarithms of the
We have demonstrated a facile method for the cost-effective incorporation of 15N into the glucosamine units of chitin in P. chrysogenum, A. niger, and M. rouxii. We have used spectroscopic techniques in order to optimize label incorporation and performed a deacetylation study to prove the validity and accuracy of the 15N ssNMR approach toward the measurement of DA. We have found that the combined labeling-15N ssNMR strategy provided rapid DA measurements to within 0.5% accuracy (based on signal-to-noise estimations of the individual 15 N ssNMR spectra). Importantly, contaminating glucans (and other impurities) do not affect these measurements. Based on the results obtained with three fungal systems, we believe we have an accurate methodology for the determination of DA in chitin derived from fungal sources. Acknowledgment. We thank EPSRC, RTC North, and Angel Biotechnology Ltd. for financial support of this work in the form of a studentship to H.R.W. and Durham University Chemistry Department Analytical Services for their assistance throughout this project.
References and Notes (1) Baker, L. G.; Specht, C. A.; Donlin, M. J.; Lodge, J. K. Eukaryotic Cell 2007, 6 (5), 855–867. (2) Banks, I. R.; Specht, C. A.; Donlin, M. J.; Gerik, K. J.; Levitz, S. M.; Lodge, J. K. Eukaryotic Cell 2005, 4 (11), 1902–1912. (3) Deising, H.; Siegrist, J. FEMS Microbiol. Lett. 1995, 127 (3), 207– 211. (4) Neugebauer, W. A. Carbohydr. Res. 1989, 189, 363–367. (5) Maghami, G. G.; Roberts, G. A. F. Macromol. Chem. Phys. 1988, 189, 2239–2243.
15
N-Labeling of Chitin in Fungi
(6) Niola, F.; Basora, N.; Chornet, E.; Vidal, P. F. Carbohydr. Res. 1993, 238, 1–9. (7) Xu, J.; McCarthy, S. P.; Gross, R. A. Macromolecules 1996, 29, 3436– 3440. (8) Hu, X.; Du, Y.; Tang, Y.; Wang, Q.; Feng, T.; Yang, J.; Kennedy, J. F. Carbohydr. Polym. 2007, 70, 451–458. (9) Roberts, G. A. F. Chitin Chemistry; Macmillan: New York, 1992; pp 85-115. (10) Yamaguchi, Y.; Nge, T. T.; Takemura, A.; Hori, N.; Ono, H. Biomacromolecules 2005, 6 (4), 1941–1947. (11) Baxter, A.; Dillon, M.; Taylor, K. D. A.; Roberts, G. A. F. Int. J. Biol. Macromol. 1992, 14, 166–169. (12) da Silva, R. M. P.; Mano, J. F.; Reis, R. L. Macromol. Chem. Phys. 2008, 209 (14), 1463–1472. (13) Hein, S.; Ng, C. H.; Stevens, W. F.; Wang, K. J. Biomed. Mater. Res. 2008, 86B (2), 558–568. (14) Muzzarelli, R. A. A.; Rocchetti, R. Carbohydr. Polym. 1985, 5 (6), 461–472. (15) Melvik, J. E.; Brekka, G.; Dornish, M. In Chitin Handbook; Muzzarelli, R. A. A., Peter, M. G., Eds.; Atec: Grottammare, Italy, 1997; pp 121125. (16) Hirai, A.; Odani, H.; Nakajima, A. Polym. Bull. 1991, 26, 87–94. (17) Vårum, K. M.; Anthonsen, M. W.; Grasdalen, H.; Smidsrod, O. Carbohydr. Res. 1991, 211, 17–23. (18) Fernandez-Megia, E.; Novoa-Carballal, R.; Quinoa, E.; Riguera, R. Carbohydr. Polym. 2005, 61 (2), 155–161. (19) Lavertu, M.; Xia, Z.; Serreqi, A. N.; Berrada, M.; Rodrigues, A.; Wang, D.; Buschmann, M. D.; Gupta, A. J. Pharm. Biomed. Anal. 2003, 32, 1149–1158. (20) Cervera, M. F.; Heinamaki, J.; Rasanen, M.; Maunu, S. l.; Karjalainen, M.; Nieto Acosta, O. M.; Iraizoz Colarte, A.; Yliruusi, J. Carbohydr. Polym. 2004, 58, 401–408.
Biomacromolecules, Vol. 10, No. 4, 2009
797
(21) Brugnerotto, J.; Lizardi, J.; Goycoolea, F. M.; Arguelles-Monal, W.; Desbrieres, J.; Rinaudo, M. Polymer 2001, 42, 3569–3580. (22) Yu, G. E.; Morin, F. G.; Nobes, G. A. R.; Marchessault, R. H. Macromolecules 1999, 32, 518–520. (23) Heux, L.; Brugnerotto, J.; Desbrieres, J.; Versali, M.-F.; Rinaudo, M. Biomacromolecules 2000, 1, 746–751. (24) Christensen, B.; Thykaer, J.; Nielsen, J. Appl. Microbiol. Biotechnol. 2000, 54, 212–217. (25) Bridson, E. Y. The Oxoid Manual, 7th ed.; Unipath: Basingstoke, U.K., 1995. (26) de Vries, R. P.; Visser, J. Appl. EnViron. Microbiol. 1999, 65 (12), 5500–5503. (27) Park, H. G.; Jong, S. C. Mycoscience 2003, 44, 25–32. (28) Synowiecki, J.; Al-Khateeb, N. A. A. Q. Food Chem. 1997, 60 (4), 605–610. (29) van den Burg, H. A.; de Wit, P. J. G. M.; Vervoort, J. J. Biomol. NMR 2001, 20, 251–261. (30) Martin, F. FEBS Lett. 1985, 182 (2), 350–354. (31) Engelsberger, W. R.; Erban, A.; Kopka, J.; Schulze, W. X. Plant Methods 2006, 2, (14). (32) Leloir, L. F. Science 1971, 172, 1299–1303. (33) Lamarque, G.; Chaussard, G.; Domard, A. Biomacromolecules 2007, 8 (6), 1942–1950. (34) Methacanon, P.; Prasitsilp, M.; Pothsree, T.; Pattaraarchachai, J. Carbohydr. Polym. 2003, 52 (2), 119–123. (35) Galed, G.; Miralles, B.; Panos, I.; Santiago, A.; Heras, A. Carbohydr. Polym. 2005, 62, 316–320. (36) Rinaudo, M. Prog. Polym. Sci. 2006, 31, 603–632. (37) Zamani, A.; Edebo, L.; Sjostrom, B.; Taherzadeh, M. J. Biomacromolecules 2007, 8 (12), 3786–3790. (38) Wang, W. P.; Du, Y. M.; Wang, X. Y.; World, J. Microbiol. Biotechnol. 2008, 24 (11), 2717–2720.
BM8012814
