Lee Dae-Hee, Choi Su-Lim, Rha Eugene, Kim Soo Jin, Yeom Soo-Jin, Moon Jae-Hee, Lee Seung-Goo
Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea.
Biosystems and Bioengineering Program, Korea University of Science and Technology (UST), Daejeon, Korea.
BMC Biotechnol. 2015 Jan 31;15(1):1. doi: 10.1186/s12896-015-0115-2.
Alkaline phosphatase (AP) catalyzes the hydrolytic cleavage of phosphate monoesters under alkaline conditions and plays important roles in microbial ecology and molecular biology applications. Here, we report on the first isolation and biochemical characterization of a thermolabile AP from a metagenome.
The gene encoding a novel AP was isolated from a metagenomic library constructed with ocean-tidal flat sediments from the west coast of Korea. The metagenome-derived AP (mAP) gene composed of 1,824 nucleotides encodes a polypeptide with a calculated molecular mass of 64 kDa. The deduced amino acid sequence of mAP showed a high degree of similarity to other members of the AP family. Phylogenetic analysis revealed that the mAP is shown to be a member of a recently identified family of PhoX that is distinct from the well-studied classical PhoA family. When the open reading frame encoding mAP was cloned and expressed in recombinant Escherichia coli, the mature mAP was secreted to the periplasm and lacks an 81-amino-acid N-terminal Tat signal peptide. Mature mAP was purified to homogeneity as a monomeric enzyme with a molecular mass of 56 kDa. The purified mAP displayed typical features of a psychrophilic enzyme: high catalytic activity at low temperature and a remarkable thermal instability. The optimal temperature for the enzymatic activity of mAP was 37°C and complete thermal inactivation of the enzyme was observed at 65°C within 15 min. mAP was activated by Ca(2+) and exhibited maximal activity at pH 9.0. Except for phytic acid and glucose 1-phosphate, mAP showed phosphatase activity against various phosphorylated substrates indicating that it had low substrate specificity. In addition, the mAP was able to remove terminal phosphates from cohesive and blunt ends of linearized plasmid DNA, exhibiting comparable efficiency to commercially available APs that have been used in molecular biology.
The presented mAP enzyme is the first thermolabile AP found in cold-adapted marine metagenomes and may be useful for efficient dephosphorylation of linearized DNA.
碱性磷酸酶（AP）在碱性条件下催化磷酸单酯的水解裂解，在微生物生态学和分子生物学应用中发挥重要作用。在此，我们报道了从宏基因组中首次分离出一种热不稳定的AP及其生化特性。
从用韩国西海岸潮间带沉积物构建的宏基因组文库中分离出编码新型AP的基因。该宏基因组来源的AP（mAP）基因由1824个核苷酸组成，编码一个计算分子量为64 kDa的多肽。推导的mAP氨基酸序列与AP家族的其他成员高度相似。系统发育分析表明，mAP是最近鉴定的PhoX家族的成员，与研究充分的经典PhoA家族不同。当编码mAP的开放阅读框在重组大肠杆菌中克隆并表达时，成熟的mAP分泌到周质中，并且缺少一个81个氨基酸的N端Tat信号肽。成熟的mAP作为分子量为56 kDa的单体酶被纯化至同质。纯化的mAP表现出嗜冷酶的典型特征：在低温下具有高催化活性且热稳定性显著。mAP酶活性的最佳温度为37°C，在65°C下15分钟内观察到酶完全热失活。mAP被Ca(2+)激活，在pH 9.0时表现出最大活性。除了植酸和葡萄糖1-磷酸外，mAP对各种磷酸化底物表现出磷酸酶活性，表明其底物特异性较低。此外，mAP能够从线性化质粒DNA的粘性末端和平末端去除末端磷酸，其效率与分子生物学中使用的市售AP相当。
所呈现的mAP酶是在适应寒冷的海洋宏基因组中发现的首个热不稳定AP，可能有助于线性化DNA的高效去磷酸化。
