| Author | Naing Naing Win |
| Call Number | AIT Diss. no.BP-01-01 |
| Subject(s) | Chitin
|
| Note | A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Technical Science, School of Environment, Resources and Development |
| Publisher | Asian Institute of Technology |
| Series Statement | Dissertation ; no. BP-01-01 |
| Abstract | The aim of this study is to convert natural chitin to high quality chitosan using the fungal enzyme chitin deacetylase (CDA). In order to select a high producing chitin deacetylase strain, a number of fungal strains (19 strains0 were collected form different national collections. Four candidates: Absidia coerulea, Absidia glauca, Colletotrichum lindemuthianum and Mucor rouxii were selected on the basis of their higher enzyme production for comparison of their enzymological properties. The optimum temperature and pH, the substrate specificity, thermostability and absence of acetic acid inhibition were investigated. The best enzyme producing strains are Absidia coerulea and Colletotrichum lindemuthianum. Absidia has the advantage that it can be produced easily with a maximum activity within 3 days. However, it cannot act on natural chitin. Clletotrichum has the advantage that it can be act on natural chitin and PDC but is less attractive due to its plant pathogenicity and its slow growth with maximum activity only after 5 days. Absidia coerulea was selected for further optimization of the fermentation conditions and medium compositions to improve CDA production. Conditions for fermentation were selected: glucose and yeast extract as carbon and nitrogen sources, fermentation temperature 30ฺC, pH 4.5. Chitin, especially partially deacetylated chitin (PDC, 58% degree of deacetylation), is able to induce Absidia CDA. Surfactant such as Tween 80 enhances the extracellular CDA production. In bioreactor experiments, it was confirmed that more CDA was produced at higher aeration rate and in the presence of partially deacetylated chitin (PDC) as inducer. In order to avoid the interference by other enzymes especially chitinase and chitosanase, partial purification of CDA was carried out using ion exchange and gel filtration chromatography. The apparent molecular weight of Absodoa CDA was determined by Sephadex G-100 to be 66 kDa. The optimum temperature, pH, thermostability, and metal ion inhibition were investigated. Chitin cannot be deacetylated by the enzyme. Partially deacetylated chitin can be deacetylated to a limited extent. In a kinetic study it was shown that this limited deacetylation has a biphasic nature, a fast process followed by a slow conversion. It was concluded that Absodoa CDA only acts on the N-acetyl glucosamine residues at the outer surface of chitin. The crystalline interior of the substrate is apparently not accessible for the enzyme. CDA from Colletotrichum can deacetylate chitin, but surprisingly only to a limited extent as like Absidia CDA. In order to obtain a chitin preparation that could be used more efficiently by Absidia CDA, natural chitin was exposed to various physical and chemical conditions such as heating, sonicating, grinding, derivatization and coupling to saccharides. But chitin remained too much crystallized to be accessible for the enzyme. Dissolution of chitin in specific solvents followed by fast precipitation in order to get microparticles was not successful either. After transfer to aqueous solution, no increase in enzymatic deacetylation was observed. However, in this way chitin with a very small particle size, called super fine (SF) chitin was obtained. This SF chitin, pretreated with 18% formic acid appeared to be a good substrate3 for fungal deacetylase. This was confirmed both by enzyme dependent deacetylation measured by acetate production as well as by isolation of the produced chitosan, followed by assay of its degree of deacetylation. In this way, enzymatic deacetylation from 10 to 90% was achieved. The formic acid treatment affects the molecular weight. Under the conditions used, the formic acid reduces the relative molecular weight of chitosan to 1.2 x 104 Daltons. It is concluded that nearly complete enzymatic deacetylation was demonstrated for medium size chitin. |
| Year | 2001 |
| Corresponding Series Added Entry | Asian Institute of Technology. Dissertation ; no. BP-00-01 |
| Type | Dissertation |
| School | School of Environment, Resources, and Development (SERD) |
| Department | Department of Food, Agriculture and Natural Resources (Former title: Department of Food Agriculture, and BioResources (DFAB)) |
| Academic Program/FoS | Bioprocess Technology (BP) |
| Chairperson(s) | Stevens, Willem F.; |
| Examination Committee(s) | Suwalee Chandrkrachang;Rakshit, Sudip Kumar;Udomchai Chinadit;Hirono, Shigehiro; |
| Scholarship Donor(s) | Hirono, Shigehiro; |
| Degree | Thesis (Ph.D.) - Asian Institute of Technology, 2001 |