Cunninghamella elegans

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Cunninghamella elegans
Scientific classification
Kingdom: Fungi
Subphylum: Mucoromycotina
Order: Mucorales
Family: Cunninghamellaceae
Genus: Cunninghamella
Species: C. elegans
Binomial name
Cunninghamella elegans
Lendner (1907)[1]
Synonyms

Cunninghamella echinulata var. elegans (Lendner) Lunn & Shipton[2]

Cunninghamella elegans is a species of fungus in the genus Cunninghamella found in soil.[3]

It can be grown in Sabouraud dextrose broth, a liquid medium used for cultivation of yeasts and molds from liquid which are normally sterile.

As opposed to C. bertholletiae, it is not a human pathogen,[4] with the exception of two documented patients.[5]

Description[edit]

C. elegans is a filamentous fungus that produces purely gray colonies.[6]

Electron microscopy studies show that the conidia are covered with spines.[7]

Use as a fungal organism capable of xenobiotics metabolism[edit]

Cunninghamella elegans is able to degrade xenobiotics.[8] It has a variety of enzymes of phases I (modification enzymes acting to introduce reactive and polar groups into their substrates) and II (conjugation enzymes) of the xenobiotic metabolism, as do mammals. Cytochrome P450 monooxygenase, aryl sulfotransferase, glutathione S-transferase, UDP-glucuronosyltransferase, UDP-glucosyltransferase activities have been detected in cytosolic or microsomal fractions.[9]

Cytochrome P-450 and cytochrome P-450 reductase in C. elegans are part of the phase I enzymes. They are induced by the corticosteroid cortexolone and by phenanthrene.[10] C. elegans also possesses a lanosterol 14-alpha demethylase, another enzyme in the cytochrome P450 family.[11]

C. elegans also possesses a glutathione S-transferase.[12]

Use as a fungal model organism of mammalian drug metabolism[edit]

Cunninghamella elegans is a microbial model of mammalian drug metabolism.[13][14][15][16] The use of this fungus could reduce the over-all need for laboratory animals.[17]

C. elegans is able to transform the tricyclic antidepressants amitriptyline[18] and doxepin,[19] the tetracyclic antidepressant mirtazapine,[20] the muscle relaxant cyclobenzaprine,[21] the typical antipsychotic chlorpromazine as well as the antihistamine and anticholinergic methdilazine[22] and azatadine. It is also able to transform the antihistamines brompheniramine, chlorpheniramine and pheniramine.[23]

It forms a glucoside with the diuretic furosemide.[16]

The transformation of oral contraceptive mestranol by C. elegans yields two hydroxylated metabolites, 6beta-hydroxymestranol and 6beta,12beta-dihydroxymestranol.[24]

Metabolism of polycyclic aromatic hydrocarbons[edit]

The phase I cytochrome P450 enzyme systems of C. elegans has been implicated in the neutralization of numerous polycyclic aromatic hydrocarbons (PAH).[6]

It can degrade molecules such as anthracene, 7-methylbenz[a]anthracene and 7-hydroxymethylbenz[a]anthracene,[25] phenanthrene,[26] acenaphthene,[27] 1- and 2-methylnaphthalene,[28] naphthalene,[29] fluorene[30] or benzo(a)pyrene.[31]

In the case of phenanthrene, C. elegans produces a glucoside conjugate of 1-hydroxyphenanthrene (phenanthrene 1-O-beta-glucose).[32]

Metabolism of pesticides[edit]

C. elegans is also able to degrade the herbicides alachlor,[33] metolachlor[34] and isoproturon[35] as well as the fungicide mepanipyrim.[3]

Metabolism of phenolics[edit]

Cunninghamella elegans can be used to study the metabolism of phenols. This type of molecules already have reactive and polar groups comprised within their structure therefore phases I enzymes are less active than phase II (conjugation) enzymes.

Metabolism of flavonoids[edit]

Flavonols

In flavonols, an hydroxyl group is available in the 3- position allowing the glycosylation at that position. The biotransformation of quercetin yields three metabolites, including quercetin 3-O-β-D-glucopyranoside, kaempferol 3-O-β-D-glucopyranoside and isorhamnetin 3-O-β-D-glucopyranoside. Glucosylation and O-methylation are involved in the process.[36]

Flavones

In flavones, there is no hydroxyl group available at the 3- position. Conjugation, in the form of sulfation occurs at the 7- or 4'- positions. Apigenin and chrysin are also transformed by C. elegans and produce apigenin 7-sulfate, apigenin 7,4′-disulfate, chrysin 7-sulfate.[37]
Sulfation also occurs on naringenin and produces naringenin-7-sulfate.[38]

Glucosylation may nevertheless occur but in 3'- position, as happens during the microbial transformation of psiadiarabin and its 6-desmethoxy analogue, 5,3′ dihydroxy-7,2′,4′,5′-tetramethoxyflavone, by Cunninghamella elegans NRRL 1392 that gives the 3′-glucoside conjugates of the two flavones.[39]

flavanones

As in flavones, there is no hydroxyl groups available at the 3- position for glycosylation in flavanones. Therefore, sulfation occurs at the 7- position. In compounds like 7-methoxylated flavanones like 7-O-methylnaringenin (sakuranetin), demethylation followed by sulfation occur.[40]

Metabolism of synthetic phenolics[edit]

It is also able to degrade synthetic phenolic compounds like bisphenol A.[41]

Metabolism of heterocyclic organic compounds[edit]

C. elegans can transform the nitrogen containing compound phthalazine[42] It is also able to oxidize the organosulfur compound dibenzothiophene.[43]

Uses in biotechnology[edit]

Methods for efficient C. elegans genomic DNA isolation and transformation have been developed.[44]

The cytochrome P450 of C. elegans has been cloned in Escherichia coli[45] as well as an enolase.[46]

Use in bioconversion[edit]

Techniques employed[edit]

Cunninghamella elegans can be grown in stirred tank batch bioreactor.[47] Protoplasts cultures have been used.[48]

Examples of uses[edit]

C. elegans can be used for phenanthrene bioconversion[47] or for steroid transformation.[48] It has been used to produce isoapocodeine from 10,11-dimethoxyaporphine,[49] triptoquinone from the synthetic abietane diterpene triptophenolide[50] or for the rational and economical bioconversion of antimalarial drug artemisinin to 7beta-hydroxyartemisinin.[51]

Environmental biotechnology[edit]

Cunninghamella elegans has been used in environmental biotechnology for the treatment of textile wastewaters,[52] for instance those discoloured by azo dyes[53] or malachite green.[54]

Chitin[55] and chitosan isolated from C. elegans can be used for heavy metal biosorption.[56] Production can be made on yam bean (Pachyrhizus erosus L. Urban) medium.[57]

Strains[edit]

Cunninghamella elegans ATCC 9245[36]
Cunninghamella elegans ATCC 36112[6]
Cunninghamella elegans ATCC 26269[6]
Cunninghamella elegans NRRL 1393[6]
Cunninghamella elegans IFM 46109[56]
Cunninghamella elegans UCP 542[53]

References[edit]

  1. ^ Lendner A. (1907). "Sur quelques Mucorinées". Bulletin de l´Herbier Boissier (in French) 7 (3): 249–51. 
  2. ^ Weitzmann I. (1984). "The case for Cunninghamella elegans, C. bertholletiae and C. echinulata as separate species". The Transactions of the British Mycological Society 83 (3): 527–529. doi:10.1016/S0007-1536(84)80056-X. 
  3. ^ a b Zhu, Y. Z.; Keum, Y. S.; Yang, L.; Lee, H.; Park, H.; Kim, J. H. (2010). "Metabolism of a Fungicide Mepanipyrim by Soil FungusCunninghamella elegansATCC36112". Journal of Agricultural and Food Chemistry 58 (23): 12379. doi:10.1021/jf102980y.  edit
  4. ^ Weitzman, I.; Crist, M. Y. (1979). "Studies with clinical isolates of Cunninghamella. I. Mating behavior". Mycologia 71 (5): 1024–1033. doi:10.2307/3759290. PMID 545137. JSTOR 3759290.  edit
  5. ^ Kwon-Chung, K. J.; Young, R. C.; Orlando, M. (1975). "Pulmonary mucormycosis caused by Cunninghamella elegans in a patient with chronic myelogenous leukemia". American journal of clinical pathology 64 (4): 544–548. PMID 1060379.  edit
  6. ^ a b c d e Asha S, Vidyavathi M (2009). "Cunninghamella - a microbial model for drug metabolism studies - a review". Biotechnol. Adv. 27 (1): 16–29. doi:10.1016/j.biotechadv.2008.07.005. PMID 18775773. Retrieved 2009-03-17. 
  7. ^ Hawker, L. E.; Thomas, B.; Beckett, A. (1970). "An Electron Microscope Study of Structure and Germination of Conidia of Cunninghamella elegans Lendner". Microbiology 60 (2): 181. doi:10.1099/00221287-60-2-181.  edit
  8. ^ Wackett, L. P.; Gibson, D. T. (1982). "Metabolism of xenobiotic compounds by enzymes in cell extracts of the fungus Cunninghamella elegans". The Biochemical journal 205 (1): 117–122. PMC 1158453. PMID 6812568.  edit
  9. ^ Zhang, D.; Yang, Y.; Leakey, J. E. A.; Cerniglia, C. E. (1996). "Phase I and phase II enzymes produced byCunninghamella elegansfor the metabolism of xenobiotics". FEMS Microbiology Letters 138 (2–3): 221–226. doi:10.1111/j.1574-6968.1996.tb08161.x. PMID 9026450.  edit
  10. ^ Lisowska, K.; Szemraj, J.; Rã³å¼Alska, S.; Då‚Ugoå„Ski, J. (2006). "The expression of cytochrome P-450 and cytochrome P-450 reductase genes in the simultaneous transformation of corticosteroids and phenanthrene byCunninghamella elegans". FEMS Microbiology Letters 261 (2): 175–180. doi:10.1111/j.1574-6968.2006.00339.x. PMID 16907717.  edit
  11. ^ Lanosterol 14-alpha demethylase from Cunninghamella elegans on www.uniprot.org
  12. ^ Cha, C. J.; Kim, S. J.; Kim, Y. H.; Stingley, R.; Cerniglia, C. E. (2002). "Molecular cloning, expression and characterization of a novel class glutathione S-transferase from the fungus Cunninghamella elegans". Biochemical Journal 368 (2): 589. doi:10.1042/BJ20020400.  edit
  13. ^ Kristian Björnstad, Anders Helander, Peter Hultén & Olof Beck (2009). "Bioanalytical investigation of asarone in connection with Acorus calamus oil intoxications". Journal of Analytical Toxicology 33 (9): 604–609. doi:10.1093/jat/33.9.604. PMID 20040135. 
  14. ^ Joanna D. Moody, Donglu Zhang, Thomas M. Heinze and Carl E. Cerniglia (2000). "Transformation of amoxapine by Cunninghamella elegans". Applied and Environmental Microbiology 66 (8): 3646–3649. doi:10.1128/AEM.66.8.3646-3649.2000. PMC 92200. PMID 10919836. 
  15. ^ A. Jaworski, L. Sedlaczek, J. Dlugoński & Ewa Zajaczkowska (1985). "Inducible nature of the steroid 11-hydroxylases in spores of Cunninghamella elegans (Lendner)". Journal of Basic Microbiology 25 (7): 423–427. doi:10.1002/jobm.3620250703. 
  16. ^ a b Hezari, M.; Davis, P. J. (1993). "Microbial models of mammalian metabolism. Furosemide glucoside formation using the fungus Cunninghamella elegans". Drug metabolism and disposition: the biological fate of chemicals 21 (2): 259–267. PMID 8097695.  edit
  17. ^ Sharma, KK; Mehta, T; Joshi, V; Mehta, N; Rathor, AK; Mediratta, KD; Sharma, PK (2011). "Substitute of animals in drug research: An approach towards fulfillment of 4R′s". Indian Journal of Pharmaceutical Sciences 73 (1): 1–6. doi:10.4103/0250-474X.89750. PMC 3224398. PMID 22131615. 
  18. ^ Zhang, D.; Evans, F. E.; Freeman, J. P.; Duhart Jr, B.; Cerniglia, C. E. (1995). "Biotransformation of amitriptyline by Cunninghamella elegans". Drug metabolism and disposition: the biological fate of chemicals 23 (12): 1417–1425. PMID 8689954.  edit
  19. ^ Moody, J. D.; Freeman, J. P.; Cerniglia, C. E. (1999). "Biotransformation of doxepin by Cunninghamella elegans". Drug metabolism and disposition: the biological fate of chemicals 27 (10): 1157–1164. PMID 10497142.  edit
  20. ^ Moody, J. D.; Freeman, J. P.; Fu, P. P.; Cerniglia, C. E. (2002). "Biotransformation of Mirtazapine by Cunninghamella Elegans". Drug Metabolism and Disposition 30 (11): 1274–1279. doi:10.1124/dmd.30.11.1274. PMID 12386135.  edit
  21. ^ Zhang, D.; Evans, F. E.; Freeman, J. P.; Yang, Y.; Deck, J.; Cerniglia, C. E. (1996). "Formation of mammalian metabolites of cyclobenzaprine by the fungus, Cunninghamella elegans". Chemico-Biological Interactions 102 (2): 79–92. doi:10.1016/S0009-2797(96)03736-2. PMID 8950223.  edit
  22. ^ Zhang, D.; Freeman, J. P.; Sutherland, J. B.; Walker, A. E.; Yang, Y.; Cerniglia, C. E. (1996). "Biotransformation of chlorpromazine and methdilazine by Cunninghamella elegans". Applied and environmental microbiology 62 (3): 798–803. PMC 167846. PMID 8975609.  edit
  23. ^ Hansen, E. B.; Cho, B. P.; Korfmacher, W. A.; Cerniglia, C. E. (1995). "Fungal transformations of antihistamines: Metabolism of brompheniramine, chlorpheniramine, and pheniramine toN-oxide andN-demethylated metabolites by the fungusCunninghamella elegans". Xenobiotica 25 (10): 1081–1092. doi:10.3109/00498259509061908. PMID 8578764.  edit
  24. ^ Choudhary, M. I.; Musharraf, S. G.; Siddiqui, Z. A.; Khan, N. T.; Ali, R. A.; Ur-Rahman, A. (2005). "Microbial transformation of mestranol by Cunninghamella elegans". Chemical & pharmaceutical bulletin 53 (8): 1011–1013. doi:10.1248/cpb.53.1011. PMID 16079537.  edit
  25. ^ Cerniglia, C. E.; Fu, P. P.; Yang, S. K. (1982). "Metabolism of 7-methylbenzaanthracene and 7-hydroxymethylbenzaanthracene by Cunninghamella elegans". Applied and environmental microbiology 44 (3): 682–689. PMC 242076. PMID 7138006.  edit
  26. ^ Cerniglia, C. E.; Yang, S. K. (1984). "Stereoselective metabolism of anthracene and phenanthrene by the fungus Cunninghamella elegans". Applied and environmental microbiology 47 (1): 119–124. PMC 239622. PMID 6696409.  edit
  27. ^ Pothuluri, J. V.; Freeman, J. P.; Evans, F. E.; Cerniglia, C. E. (1992). "Fungal metabolism of acenaphthene by Cunninghamella elegans". Applied and environmental microbiology 58 (11): 3654–3659. PMC 183157. PMID 1482186.  edit
  28. ^ Cerniglia, C. E.; Lambert, K. J.; Miller, D. W.; Freeman, J. P. (1984). "Transformation of 1- and 2-methylnaphthalene by Cunninghamella elegans". Applied and environmental microbiology 47 (1): 111–118. PMC 239621. PMID 6696408.  edit
  29. ^ Cerniglia, C. E.; Gibson, D. T. (1977). "Metabolism of naphthalene by Cunninghamella elegans". Applied and environmental microbiology 34 (4): 363–370. PMC 242664. PMID 921262.  edit
  30. ^ Pothuluri, J. V.; Freeman, J. P.; Evans, F. E.; Cerniglia, C. E. (1993). "Biotransformation of fluorene by the fungus Cunninghamella elegans". Applied and environmental microbiology 59 (6): 1977–1980. PMC 182201. PMID 8328814.  edit
  31. ^ Cerniglia, C. E.; Mahaffey, W.; Gibson, D. T. (1980). "Fungal oxidation of benzo\a]pyrene: Formation of (−)-trans-7,8-dihydroxy-7,8-dihydrobenzo\a]pyrene by Cunninghamella elegans". Biochemical and Biophysical Research Communications 94 (1): 226–232. doi:10.1016/S0006-291X(80)80210-5. PMID 7190014.  edit
  32. ^ Cerniglia, C. E.; Campbell, W. L.; Freeman, J. P.; Evans, F. E. (1989). "Identification of a novel metabolite in phenanthrene metabolism by the fungus Cunninghamella elegans". Applied and environmental microbiology 55 (9): 2275–2279. PMC 203068. PMID 2802607.  edit
  33. ^ Pothuluri, J. V.; Freeman, J. P.; Evans, F. E.; Moorman, T. B.; Cerniglia, C. E. (1993). "Metabolism of alachlor by the fungus Cunninghamella elegans". Journal of Agricultural and Food Chemistry 41 (3): 483. doi:10.1021/jf00027a026.  edit
  34. ^ Jairaj V. Pothuluri, Frederick E. Evans, Doerge, D.R., Churchwell, M.I. and Carl E. Cerniglia (1997). "Metabolism of metolachlor by the fungus Cunninghamella elegans". Arch. Environ. Contam. Toxicol. 32 (2): 117–125. doi:10.1007/s002449900163. PMID 9069185. 
  35. ^ Hangler, M.; Jensen, B.; Rønhede, S.; Sørensen, S. R. (2007). "Inducible hydroxylation and demethylation of the herbicide isoproturon by Cunninghamella elegans". FEMS Microbiology Letters 268 (2): 254–260. doi:10.1111/j.1574-6968.2006.00599.x. PMID 17328751.  edit
  36. ^ a b Zi, J.; Valiente, J.; Zeng, J.; Zhan, J. (2011). "Metabolism of quercetin by Cunninghamella elegans ATCC 9245". Journal of Bioscience and Bioengineering 112 (4): 360–362. doi:10.1016/j.jbiosc.2011.06.006. PMID 21742550.  edit
  37. ^ Ibrahim, A. R. S. (2005). "Biotransformation of Chrysin and Apigenin by Cunninghamella elegans". Chemical & Pharmaceutical Bulletin 53 (6): 671–672. doi:10.1248/cpb.53.671. PMID 15930780.  edit
  38. ^ Abdel-Rahim S. Ibrahim (2000). "Sulfation of naringenin by Cunninghamella elegans". Phytochemistry 53 (2): 209–212. doi:10.1016/S0031-9422(99)00487-2. PMID 10680173. 
  39. ^ Ibrahim, A. R.; Galal, A. M.; Mossa, J. S.; El-Feraly, F. S. (1997). "Glucose-conjugation of the flavones of Psiadia arabica by Cunninghamella elegans". Phytochemistry 46 (7): 1193–1195. doi:10.1016/s0031-9422(97)00421-4. PMID 9423290.  edit
  40. ^ Ibrahim, A. R.; Galal, A. M.; Ahmed, M. S.; Mossa, G. S. (2003). "O-demethylation and sulfation of 7-methoxylated flavanones by Cunninghamella elegans". Chemical & pharmaceutical bulletin 51 (2): 203–206. doi:10.1248/cpb.51.203. PMID 12576658. INIST:14569933.  edit
  41. ^ Keum, Y. S.; Lee, H. R.; Park, H. W.; Kim, J. H. (2010). "Biodegradation of bisphenol a and its halogenated analogues by Cunninghamella elegans ATCC36112". Biodegradation 21 (6): 989–997. doi:10.1007/s10532-010-9358-8. PMID 20455075.  edit
  42. ^ Biotransformation of Phthalazine by Fusarium moniliforme and Cunninghamela elegans. John B. Sutherland, James P. Freeman, Anna J. Williams and Joanna Deck, Mycologia, Jan. - Feb., 1999, Vol. 91, No., pages 114-116 JSTOR 3761198
  43. ^ Crawford, D. L.; Gupta, R. K. (1990). "Oxidation of dibenzothiophene byCunninghamella elegans". Current Microbiology 21 (4): 229. doi:10.1007/BF02092161.  edit
  44. ^ Zhang, D.; Yang, Y.; Castlebury, L. A.; Cerniglia, C. E. (1996). "A method for the large scale isolation of high transformation efficiency fungal genomic DNA". FEMS Microbiology Letters 145 (2): 261–265. doi:10.1111/j.1574-6968.1996.tb08587.x. PMID 8961565.  edit
  45. ^ Wang, R. F.; Cao, W. W.; Khan, A. A.; Cerniglia, C. E. (2000). "Cloning, sequencing, and expression inEscherichia coliof a cytochrome P450 gene fromCunninghamella elegans". FEMS Microbiology Letters 188 (1): 55–61. doi:10.1111/j.1574-6968.2000.tb09168.x. PMID 10867234.  edit
  46. ^ Wang, R. F.; Khan, A. A.; Cao, W. W.; Cerniglia, C. E. (2000). "Cloning, sequencing, and expression of the gene encoding enolase from Cunninghamella elegans". Mycological Research 104 (2): 175. doi:10.1017/S0953756299001112.  edit
  47. ^ a b Lisowska, K.; Bizukojc, M.; Długoński, J. (2006). "An unstructured model for studies on phenanthrene bioconversion by filamentous fungus Cunninghamella elegans". Enzyme and Microbial Technology 39 (7): 1464. doi:10.1016/j.enzmictec.2006.03.039.  edit
  48. ^ a b Długoński, J.; Sedlaczek, L.; Jaworski, A. (1984). "Protoplast release from fungi capable of steroid transformation". Canadian Journal of Microbiology 30 (1): 57–62. doi:10.1139/m84-010. PMID 6713303.  edit (French)
  49. ^ Smith, R. V.; Davis, P. J. (1978). "Regiospecific synthesis of isoapocodeine from 10,11-dimethoxyaporphine by using Cunninghamella elegans". Applied and environmental microbiology 35 (4): 738–742. PMC 242915. PMID 25623.  edit
  50. ^ TMilanova, R.; Stoynov, N.; Moore, M. (1996). "The optimization of triptoquinone production by Cunninghamella elegans using factorial design". Enzyme and Microbial Technology 19 (2): 86. doi:10.1016/0141-0229(95)00184-0.  edit
  51. ^ Parshikov, I. A.; Muraleedharan, K. M.; Avery, M. A.; Williamson, J. S. (2004). "Transformation of artemisinin by Cunninghamella elegans". Applied Microbiology and Biotechnology 64 (6): 782–786. doi:10.1007/s00253-003-1524-z. PMID 14735322.  edit
  52. ^ Tigini, V.; Prigione, V.; Donelli, I.; Anastasi, A.; Freddi, G.; Giansanti, P.; Mangiavillano, A.; Varese, G. C. (2010). "Cunninghamella elegans biomass optimisation for textile wastewater biosorption treatment: An analytical and ecotoxicological approach". Applied Microbiology and Biotechnology 90 (1): 343–352. doi:10.1007/s00253-010-3010-8. PMID 21127858.  edit
  53. ^ a b Ambrósio, S.; Campos-Takaki, G. M. (2004). "Decolorization of reactive azo dyes by Cunninghamella elegans UCP 542 under co-metabolic conditions". Bioresource Technology 91 (1): 69–75. doi:10.1016/S0960-8524(03)00153-6. PMID 14585623.  edit
  54. ^ Cha, C. -J.; Doerge, D. R.; Cerniglia, C. E. (2001). "Biotransformation of Malachite Green by the Fungus Cunninghamella elegans". Applied and Environmental Microbiology 67 (9): 4358–4360. doi:10.1128/AEM.67.9.4358-4360.2001. PMC 93171. PMID 11526047.  edit
  55. ^ Andrade, V. S.; Neto, B. B.; Souza, W.; Campos-Takaki, G. M. (2000). "A factorial design analysis of chitin production byCunninghamella elegans". Canadian Journal of Microbiology 46 (11): 1042–1045. doi:10.1139/w00-086. PMID 11109493.  edit
  56. ^ a b Franco, L. D. O.; Maia, R. D. C. S. C.; Porto, A. L. C. F.; Messias, A. S.; Fukushima, K.; Campos-Takaki, G. M. D. (2004). "Heavy metal biosorption by chitin and chitosan isolated from Cunninghamella elegans (IFM 46109)". Brazilian Journal of Microbiology 35 (3): 243. doi:10.1590/S1517-83822004000200013.  edit
  57. ^ Montenegro Stamford, T. C.; Montenegro Stamford, T. L.; Pereira Stamford, N.; De Barros Neto, B.; De Campos-Takaki, G. M. (2007). "Growth of Cunninghamella elegans UCP 542 and production of chitin and chitosan using yam bean medium". Electronic Journal of Biotechnology 10: 0. doi:10.2225/vol10-issue1-fulltext-1.  edit

External links[edit]