Novel low-temperature-active, salt-tolerant and proteases-resistant endo-1,4-β-mannanase from a new Sphingomonas strain

Sphingomonas sp. JB13, isolated from slag of a >20-year-old phosphate rock-stacking site, showed the highest 16S rDNA (1343 bp) identity of 97.2% with Sphingomonas sp. ERB1-3 (FJ948169) and <97% identities with other identified Sphingomonas strains. A mannanase-coding gene (1191 bp) was cloned and encodes a 396-residue polypeptide (ManAJB13) showing the highest amino acid sequence identities of 56.2% with the putative glycosyl hydrolase (GH) family 26 endo-1,4-beta-mannanase from Rhodothermus marinus (YP_004824245), and 44.2% with the identified CH 26 endo-1,4-beta-mannanase from Cellvibrio japonicus (2VX5_A). The recombinant ManAJB13 (rManAJB13) was expressed in Escherichia coli BL21 (DE3). Purified rManAJB13 displayed the typical characteristics of low-temperature-active enzymes: showing apparent optimal at 40 degrees C, similar to 55% of the maximum activity at 20 degrees C and similar to 20% at 10 degrees C, and thermolability at 45 degrees C (similar to 15 min half-life). The potential mechanism for low-temperature-activity of GH 26 endo-1,4-beta-mannanases might be ascribed to the more hydrophobic residues (AILFWV) and less polar residues (NCQSTY) compared with typical thermophilic and mesophilic counterparts. The purified rManAJB13 exhibited >85% mannanase activity at the concentration of 0-4.0 M NaCl. No loss of enzyme activity was observed after incubating the enzyme with 1 M or 2 M NaCl, or trypsin or proteinase K at 37 degrees C and pH 6.5 for 1 h. The K-m, V-max, and k(cat) values were 5.0 mg ml(-1), 277.8 mu mol min(-1) mg(-1), and 211.9 s(-1), respectively, using locust bean gum as the substrate. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.