Cloning and expression of Cel8A from Klebsiella pneumoniae in Escherichia coli and comparison to cel gene of Cellulomonas uda

Klebsiella pneumoniae XM-4 is first isolated from a bacterial consortium. According to the 16S rRNA sequence, it has only 75% sequence similarity to Cellulomonas sp. However, a 999-bp open reading frame of K. pneumoniae, which encodes a putative endo-glucanase of 333 amino acids belonging to GH8 family and designated as Cel8A, is found closely to Cellulomonas uda with 95% similarity in amino acid sequence. This implies that the biological evolution between both strains has occurred. The cel8A is constructed in vectors of pET-22b(+) and pET-32a(+) with or without containing a signal peptide, and then cloned and expressed in Escherichia coli. The best endo-glucanase production of 25.4 mg/l is then obtainable by applying pET-32a(+) without the signal peptide. The recombinant CelA8 has an optimal specific activity of 62.4 U/mg against CMC or 23.3 U/mg against β-d-glucan at 55 °C and pH 5.0, indicating good potential for the industrial application.

► Cel8A from Klebsiella pneumoniae XM-4 has firstly been cloned and expressed in Escherichia coli.
► The highest recombinant protein was obtained by used pET-32a(+) without signal peptide to cel8A.
► 95% similarity of Cel8A to Cellulomonas uda showed the biological evolution between both strains.