Chitin Lab Report

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Chitin is the second most abundant natural polysaccharide consisting of β(1→ 4)-linked N-acetyl-D-glucosamine (GlcNAc) units in a linear form. Chitin is insoluble in water and mainly exists in two crystalline (α- and β-) forms. The α-chitin consists of sheets of tightly packed alternating parallel and antiparallel chains (1) and is abundant in the exoskeleton of arthropods, insects, fungi, and yeast cell walls. The chains are arranged in parallel orientation in β-chitin (2), which occurs less frequently in nature and is often extracted from squid pens. The insolubility of chitin is a major limitation in eliciting biological activities.

The polymeric chitin and its breakdown products like chitooligosaccharides (CHOS)5 are of increasing interest
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(22). The amino acid sequence of SpChiD (NCBI RefSeq, YP_001478954) was obtained from NCBI database (www.ncbi.nlm.nih.gov). A BLAST search of the amino acid sequence against Protein Data Bank (PDB) was performed to identify the three-dimensional structures that could share high sequence homology. The three-dimensional coordinates of the basic structure were obtained from the PDB. We built the three-dimensional structure of the SpChiD protein using the MODELLER (23) implemented in Discovery Studio 2.5 (Accelrys Inc.). The structural validation of the generated three-dimensional models was performed using Verify_3D (24, 25) and Ramachandran plot (26).

Selection of Amino Acid Residues for Point Mutations
The amino acid residues that might influence hydrolytic and TG activities of Sp ChiD were selected based on the sequence alignment and the crystal structures of the homologous proteins (1E15, 1ITX, 1E9L, 1HKM, 1EDQ, and 1D2K). We considered the hydrophobic aromatic residues of chitinases that are mainly involved in catalytic interactions with chitin. Using the three-dimensional model, a few amino acid residues were selected for point mutations based on their probable involvement in hydrolytic and TG activities of SpChiD.

Bacterial Strains, Plasmids, Culture Conditions, Biochemicals, and

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