Thesis (M. Sc.) - Asian Institute of Technology, 2002

A thesis submitted in partial fulfillment of the requirement for the degree of Master of Science, School of Environment, Resources and Development

Chitin, a heteropolymer of p (1-4)-linked N-acetyl glucosarnine and glucosamine, is extremely insoluble and crystalline material. The deacetyl N-groups of chitin exist as positively charged (protonated) in acidic condition. Therefore, it has potentiality to electrostatically adsorb negatively charged compounds. The exterior N-acetyl groups of chitin particles could be removed by an enzyme named chitin deacetylase (CDA) to increase the charged groups on the surface. The aim of this study was to use CDA treated chitin (CTC) as anionic exchange resin/adsorbent in bioseparation and purification of proteins and other compounds. Chitin (about 10% DD) was treated with CDA from Absidia coerulea and it was found that the overall increasing of DD was only about I%. In order to check whether the increased deacetylation is only on the exterior of chitin particle, orange II dye was adsorbed to chitin and CTC. The results suggested that orange II dye was adsorbed with chitin by both physical and electrostatic interaction. In addition, orange II entered into the interior of the particles because of its low molecular weight. Consequentl y, the purpose was fulfilled by adsorbing acidic proteins. Chitin materials adsorbed acidic proteins, ovalbumin and bovine serum albumin, in pH 5.8. On the other hand, chitin materials didn't adsorb basic proteins, like hemoglobin. The adsorption was mainly driven by ionic interaction. The protein adsorption took place according to accessible deacetyl groups of chitin materials. CTC adsorbed very higher amount of protein than non-treated one, although their DD difference was only l %. The results confirmed that CDA deacetylated only exterior acetyl groups and protein was adsorbed on the surface of the chitin particles. Chitin, CTC and partially deacety lated chitin (58% DD) were used in a typical ion exchange chromatographic column as resin. ln column, maximum protein adsorption capacity of CTC was about 6 times higher than untreated chitin. PDC formed gel in pH 5 and fluid flow tlu·ough the column was stopped. Conversely, chitin and CTC swelled in pH below 2, but fluid flow tlu·ough the column was not hampered in all pH range (7 to 1 ). The adsorbed protein was eluted easily from the column by I mM NaOH solution or salt solution. The overall results suggested that CTC could be a potential alternative a111on exchange res in/adsorbent for purification of proteins and other anionic compounds.