Novel Protease-Resistant Exochitinase (Echi47) from Pig Fecal Environment DNA with Application Potentials in the Food and Feed Industries

• Published in: Journal of agricultural and food chemistry Vol. 63; no. 27; pp. 6262 - 6270
• Main Authors: Liu, Yuchun, Yan, Qiaojuan, Yang, Shaoqing, Jiang, Zhengqiang
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 15.07.2015; American Chemical Society, Books and Journals Division
• Subjects: Amino Acid Sequence; amino acid sequences; amino acids; Animals; chitin; Chitin - metabolism; chitinase; Chitinases - chemistry; Chitinases - genetics; Chitinases - isolation & purification; Chitinases - metabolism; chromosome walking; Cloning, Molecular; Coprinellus; DNA; Enzyme Stability; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Feces - chemistry; Feces - enzymology; feed industry; flavor; food additives; genes; genome walking; Molecular Sequence Data; open reading frames; pepsin; peptidase K; Peptide Hydrolases - chemistry; Sequence Alignment; Substrate Specificity; Sus scrofa; Swine; trypsin; chitinase; metagenome; exochitinase; N-acetylchitobiose; direct cloning and heteroexpression; protease resistance
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/acs.jafc.5b01457

A novel exochitinase gene (Echi47) was directly cloned from the pig fecal environment DNA using the genomic walking PCR technique and expressed in Escherichia coli BL21 (DE3). Echi47 has an open reading frame (ORF) of 1,161 bp encoding 386 amino acids. The amino acid sequence of Echi47 showed 36% identity with that of chitinase from Coprinellus congregatus. The recombinant exochitinase was purified with specific activity toward colloidal chitin of 6.84 U/mg. Echi47 was optimally active at pH 5.0 and 40 °C, respectively. When colloidal chitin was used as substrate, N-acetylchitobiose [(GlcNAc)2] was mostly produced at the initial stage, suggesting that it is an exochitinase. Echi47 exhibited excellent resistance to pepsin, trypsin, proteinase K, and flavor protease. Under simulated alimentary tract conditions, Echi47 was stable and active, releasing 21.1 mg of N-acetylchitooligosaccharides from 80 mg of colloidal chitin. These properties make Echi47 a potential additive in the food and feed industries.