Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming 650500, People's Republic of China.
J Biosci Bioeng. 2012 May;113(5):568-74. doi: 10.1016/j.jbiosc.2011.12.011. Epub 2012 Jan 23.
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.
从一个有超过 20 年历史的磷矿堆积场的矿渣中分离到一株斯氏假单胞菌（Sphingomonas sp.）JB13，其 16S rDNA（1343bp）与 Sphingomonas sp. ERB1-3（FJ948169）的相似度最高，为 97.2%，与其他已鉴定的斯氏假单胞菌菌株的相似度则低于 97%。克隆了一株甘露聚糖酶编码基因（1191bp），该基因编码一个 396 个氨基酸残基的多肽（ManAJB13），与 Rhodothermus marinus（YP_004824245）中假定的糖苷水解酶（GH）家族 26 内切-1,4-β-甘露聚糖酶的氨基酸序列相似度最高，为 56.2%，与已鉴定的来自 Cellvibrio japonicus（2VX5_A）的 GH 26 内切-1,4-β-甘露聚糖酶的相似度则为 44.2%。重组 ManAJB13（rManAJB13）在大肠杆菌 BL21（DE3）中表达。纯化的 rManAJB13 表现出低温活性酶的典型特征：最适温度为 40°C，在 20°C 时约有 55%的最大活性，在 10°C 时约有 20%的活性，在 45°C 时热稳定性较差（半衰期约为 15min）。与典型的嗜热和嗜中温同工酶相比，GH 26 内切-1,4-β-甘露聚糖酶的低温活性可能归因于更多的疏水性残基（AILFWV）和较少的极性残基（NCQSTY）。在 0-4.0M NaCl 浓度下，纯化的 rManAJB13 仍保持超过 85%的甘露聚糖酶活性。在 37°C 和 pH 6.5 下，用 1M 或 2M NaCl 处理酶，或用胰蛋白酶或蛋白酶 K 处理酶 1h 后，酶活性没有损失。以罗望子胶为底物时，K(m)、V(max)和 k(cat)值分别为 5.0mgml(-1)、277.8μmol min(-1)mg(-1)和 211.9s(-1)。
