Date Published: February 16, 2012
Author(s): Xu-Fen Zhu, Hai-Qin Tan, Chu Zhu, Li Liao, Xin-Qi Zhang, Min Wu.
A chitosanase gene, csn, was cloned from Penicillium sp. D-1 by inverse PCR. The cDNA sequence analysis revealed that csn had no intron. The deduced CSN protein consists of 250 amino acids including a 20-amino acid signal peptide, and shared 83.6% identity with the family 75 chitosanase from Talaromyces stipitatus (B8M2R4). The mature protein was overexpressed in Escherichia coli and purified with the affinity chromatography of Ni2+-NTA. The novel recombinant chitosanase showed maximal catalytic activity at pH 4.0 and 48°C. Moreover, the activity of CSN was stable over a broad pH range of 3.0-8.0, and the enzymatic activity was significantly enhanced by Mg2+ and Mn2+. The CSN could effectively hydrolyze colloidal chitosan and chitosan, while could not hydrolyze chitin and carboxymethylcellulose (CMC). Due to the particular acidophily, CSN has the potential application in the recycling of chitosan wastes.
Cellulose, chitin, and chitosan are all composed of β-1,4-linked glucopyranoses, and the difference is in functional groups at the C-2 positions of their constituent sugars, i.e., the hydroxyl, acetamido, and amino groups, respectively. Chitin is a linear homopolymer composed of β-(1,4)-N-acetyl-D-glucosamine (GlcNAc), while chitosan is a polycationic carbohydrate consisting of β-(1,4) linked D-glucosamine (GlcN) residues and derived from chitin by partial or complete deacetylation. Chitosan could be found only in fungi cell wall and insect cuticle of limited groups in nature. Chitosan and the products derived from its hydrolysis have attracted much attention because of their interesting biological properties, such as the antibacterial, antifungal and antitumor functions, and thus have been used in agriculture, food and pharmaceutical industries (Somashekar and Joseph 1996; Chiang et al. 2003).
Based on the phenotypic characteristics and 18S rRNA gene sequence analysis, the fungus was identified as a member of the genus Penicillium. Up to now, other reported Penicillium strains which could produce chitosanase were P. islandicum, P. spinulosum and P. chrysogenum (Fenton and Eveleigh, 1981; Ak et al., 1998; Rodríguez-Martín et al, 2010).
CMC: carboxymethylcellulose; GH: glycosyl hydrolase; csn: chitosanase; GlcNAc: N-acetyl-D-glucosamine; GlcN: D-glucosamine; IPTG: isopropyl-D-thiogalactopyranoside
The authors declare that they have no competing interests.