title: Novel low-temperature-active, salt-tolerant and proteases-resistant endo-1,4-β-mannanase from a new Sphingomonas strain.
abstract: Sphingomonas sp. JB13, isolated from slag of a >20-year-old phosphate rock-stacking site, showed the highest 16S rDNA (1343bp) identity of 97.2% with Sphingomonas sp. ERB1-3 (FJ948169) and <97% identities with other identified Sphingomonas strains. A mannanase-coding gene (1191bp) 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-β-mannanase from Rhodothermus marinus (YP_004824245), and 44.2% with the identified GH 26 endo-1,4-β-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°C, ~55% of the maximum activity at 20°C and ~20% at 10°C, and thermolability at 45°C (~15min half-life). The potential mechanism for low-temperature-activity of GH 26 endo-1,4-β-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 0-4.0M NaCl. No loss of enzyme activity was observed after incubating the enzyme with 1M or 2M NaCl, or trypsin or proteinase K at 37°C and pH 6.5 for 1h. The K(m), V(max) and k(cat) values were 5.0mgml(-1), 277.8μmol min(-1)mg(-1), and 211.9s(-1), respectively, using locust bean gum as the substrate.
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming 650500, People's Republic of China. junpeizhou@126.com
authors.forename: Junpei
authors.initials: J
authors.lastname: Zhou
authors.forename: Rui
authors.initials: R
authors.lastname: Zhang
authors.forename: Yajie
authors.initials: Y
authors.lastname: Gao
authors.forename: Junjun
authors.initials: J
authors.lastname: Li
authors.forename: Xianghua
authors.initials: X
authors.lastname: Tang
authors.forename: Yuelin
authors.initials: Y
authors.lastname: Mu
authors.forename: Feng
authors.initials: F
authors.lastname: Wang
authors.forename: Chao
authors.initials: C
authors.lastname: Li
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Dong
authors.forename: Zunxi
authors.initials: Z
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D003080:Cold Temperature; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005685:Galactans / Q000378:metabolism; D020869:Gene Expression Profiling; D008351:Mannans / Q000378:metabolism; D008969:Molecular Sequence Data; D053149:Plant Gums / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012965:Sodium Chloride / Q000494:pharmacology; D020578:Sphingomonas / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D044902:beta-Mannosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D005685:Galactans; D008351:Mannans; D053149:Plant Gums; D012336:RNA, Ribosomal, 16S; D011994:Recombinant Proteins; D012965:Sodium Chloride; D044902:beta-Mannosidase; C017471:locust bean gum