A novel β-mannanase from Pantoea agglomerans A021: gene cloning, expression, purification and characterization

This paper describes the construction of a genomic library from the bacterium Pantoea agglomerans A021 and the subsequent cloning and expression of a novel mannanase gene (man26P). The gene consists of 1,047 bp and encodes a peptide (Man26P) of 348 amino acids with a calculated molecular mass of 38.5 kDa. Man26P is 63% identical with mannanase from Pectobacterium carotovorum at protein level and considered to be a member of the glycoside hydrolase family 26 (GH26). Man26P was expressed efficiently in E.coli BL21 (DE3) after induction with isopropylthiogalactoside (IPTG) and purified with a GST Bind Purification Kit. Maximum activity of purified Man26P was 514 U mg−1, which was seen at pH 6.0 and a temperature of 55°C. Man26P was stable on exposure to buffers ranging from pH 4.0–10.0, and tolerant of temperature below 60°C. Zn2+, Mg2+ and Co2+ enhanced the activity, while Mn2+, Cu2+ and Hg+ had a negative effect. β-mercaptoethanol (1%) increased the activity twofold, while SDS (1%) inhibited it significantly. The enzyme showed optimal activity in a NaCl solution. The properties make it a candidate for various industrial applications.