Malays. Appl. Biol. (2006) 35(2): 59–65
CHARACTERIZATION OF CHITIN DEACETYLASE FROM FUNGUS ABSIDIA BUTLERI dr
NADARAJAH, K., LAU, B.Y.C., OTHMAN, O., HASIDAH, M.S. and WAN MOHTAR, W.Y.
School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia,
43600 Bangi, Selangor Darul Ehsan, Malaysia
ABSTRACT
The chitin deacetylase enzyme that catalyses the conversion of chitin to chitosan via the deacetylation of N-acetyl- glucosamine residues has been found in a few Zygomycetes and insects. Currently, chitosan is produced from chitin through harsh thermochemical procedure that has their disadvantages. The development of a controllable process using the enzymatic deacetylation of chitinous materials presents an alternative process that enables, in principle, the preparation of novel chitosan polymers and oligomers. Here we report the partial purification of chitin deacetylase (CDA; EC 3.5.1.41) from Absidia butleri dr, a local fungal isolate. The partially pure enzyme obtained will be used to further elucidate the properties of CDA. Homogenization of the mycelia to obtain the cell-free crude extract of CDA by grinding with acid washed-sand produced a lower yield (17.6 mU/mL) compared to the use of a homogenizer (21.6 mU/mL). However, the former was chosen in this study to obtain the crude CDA as the method gave less contaminating proteins. Through a combination of anion exchange and gel filtration chromatography, the crude CDA was isolated as two isoenzymes, designated CDA1 and CDA2. The isoenzymes have apparent native molecular masses of 97.6 kDa (CDA1) and 221.7 kDa (CDA2). The analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that CDA1 and CDA2 have yet to be purified to electrophoretic homogeneity. A modified method to determine the enzyme activity has also been described.
ABSTRAK
Kitin deasetilase memangkinkan penukaran kitin kepada kitosan melalui penyahasetilan residu N-asetil-glukosamina dan boleh dijumpai dalam beberapa spesis Zygomycetes dan serangga. Pada masa ini, kitosan dihasilkan daripada kitin melalui suatu proses termokimia rumit yang mempunyai beberapa kekurangan. Pembangunan suatu proses yang boleh dikawal dengan menggunakan kaedah penyahasetilan berenzim ke atas bahan-bahan berkitin memberi suatu proses alternatif yang membolehkan, secara teori, penyediaan polimer dan oligomer kitosan yang baru. Kajian ini melaporkan penulenan separa kitin deasetilase (CDA; EC 3.5.1.41) daripada kulat pencilan tempatan, iaitu, Absidia butleri dr. Enzim separa tulen yang diperolehi akan digunakan seterusnya untuk mengkaji ciri-ciri CDA dengan lebih lanjut. Penghomogenan miselium untuk mendapatkan ekstrak kasar CDA yang bebas-sel melalui pengisaran dengan pasir yang dicuci dengan asid didapati memberi hasil pulangan yang lebih rendah (7.6 mU/mL) berbanding dengan penggunaan alat penghomogenan (21.6 mU/mL). Walau bagaimanapun, kaedah penghomogenan dengan pasir yang dicuci dengan asid dipilih dalam kajian ini disebabkan kaedah ini dapat mengurangkan kontaminasi oleh protein. Melalui kombinasi kaedah kromatografi penukaran anion dan penurasan gel, ekstrak kasar CDA dipisahkan kepada dua isoenzim yang digelar CDA1 dan CDA2. Isoenzim-isoenzim tersebut mempunyai berat molekul natif sebanyak 97.6 kDa (CDA1) dan 221.7 kDa (CDA2) dan analisis dengan elektroforesis gel poliakrilamida-natrium dodesil sulfat (SDS-PAGE) menunjukkan bahawa CDA1 dan CDA2 belum lagi ditulenkan dengan sepenuhnya. Satu kaedah terubahsuai untuk penentuan aktiviti enzim juga akan dijelaskan.
Key words: Chitin, chitosan, Absidia butleri, chitin deacetylase
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