Abstract
A xylanase gene xynAMG1 with a 1,116-bp open reading frame, encoding an endo-β-1,4-xylanase, was cloned from a chicken cecum metagenome. The translated XynAMG1 protein consisted of 372 amino acids including a putative signal peptide of 23 amino acids. The calculated molecular mass of the mature XynAMG1 was 40,013 Da, with a theoretical pI value of 5.76. The amino acid sequence of XynAMG1 showed 59% identity to endo-β-1,4-xylanase from Prevotella bryantii and Prevotella ruminicola and 58% identity to that from Prevotella copri. XynAMG1 has two conserved motifs, DVVNE and TEXD, containing two active site glutamates and an invariant asparagine, characteristic of GH10 family xylanase. The xynAMG1 gene without signal peptide sequence was cloned and fused with thioredoxin protein (Trx.Tag) in pET-32a plasmid and overexpressed in Escherichia coli Tuner™(DE3)pLysS. The purified mature XynAMG1 was highly salt-tolerant and stable and displayed higher than 96% of its catalytic activity in the reaction containing 1 to 4 M NaCl. It was only slightly affected by common organic solvents added in aqueous solution to up to 5 M. This chicken cecum metagenome-derived xylanase has potential applications in animal feed additives and industrial enzymatic processes requiring exposure to high concentrations of salt and organic solvents.
Highlights
Microbial enzymes have been recognized as a major source of various types of biocatalysts which can be successfully applied in different industrial processes [1]
We report the gene cloning, sequence and phylogenetic analyses, structural prediction, heterologous expression, and molecular and catalytic characterizations of a new glycosyl hydrolase family 10 (GH10) family endo-xylanase derived from a chicken cecum metagenome
An E. coli clone harboring DNA from chicken cecum metagenome was found to contain an open reading frame of 1,116 bp encoding a protein of 372 amino acids, sequence of which was related to xylanase family GH10 and it was named the XynAMG1 xylanase
Summary
Microbial enzymes have been recognized as a major source of various types of biocatalysts which can be successfully applied in different industrial processes [1]. To be practically useful and economically competitive, industrial enzymes must display high activity and stability under harsh conditions to help reduce the production costs [2]. Carbohydrases, enzymes that degrade polymeric carbohydrates, are currently employed in various industries including food and beverages, detergent, biofuel production, textile, paper and pulp, leather industries, and animal feed. Majority of the xylanases are confined either to glycosyl hydrolase family (GH10) or to family (GH11) based on similarities in their hydrophobic clusters and amino acid sequences of the catalytic domains. Some are found in other glycosyl hydrolase families including 5, 7, 8, 16, 26, 43, 52, and 62 [4, 8, 9]
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