Chitin deacetylases (CDs) are a family of enzymes that catalyze the deacetylation of chitin. This study investigates the use of CDs for the preparation of positively charged chitin nanocrystals by enzymatic surface deacetylation. Chitin nanocrystals were prepared by hydrolysis of milled chitin with hydrochloric acid. CDs from two fungi, namely Colletotrichum lindemuthianum and Alternaria alternata, were obtained by recombinant methods. A putative CD gene sequence (AaCD1) was selected from the Alternaria genome database and used to design primers for amplification. The total RNA from mycelial cultures of A. alternata was converted into cDNA and used as template to amplify the gene of interest. Amplified cDNAs from A. alternata and the CD gene from C. lindemuthianum (AY633657.1) were cloned into pET vectors. Proteins were expressed in the BL 21 strain of E. coli, purified with Ni column affinity chromatography, and analyzed on SDS-PAGE. The molecular weights of the expressed proteins were 28 kDa for C. lindemuthianum and 60 kDa for A. alternata. For surface deacetylation, chitin nanocrystals were added to CD solutions and incubated with shaking for 24 h. The activity of the enzymes was evaluated with acetate assays. The physicochemical properties of the nanocrystals before and after incubation were determined by conductometric titration, atomic force microscopy, zeta potential measurement, and dynamic light scattering.

Gene cloning and expression of fungal chitin deacetylase for the surface deacetylation of chitin nanocrystals

243rd National Meeting (2012)

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