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Substrate analysis and molecular cloning of the extracellular alkaline phosphatase of Streptomyces griseus

机译：链霉菌细胞外碱性磷酸酶的底物分析及分子克隆

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Streptomyces species secrete large amounts of alkaline phosphatase (AP) enzymes that have not been characterized so far. An AP has been purified to homogeneity from cultures of Streptomyces griseus IMRU 3570. The enzyme has a monomer size of 62?kDa and is processed in the culture to a 33?kDa protein as shown by immunoblotting. The enzyme was purified by ammonium sulfate precipitation, CM-Sephadex cationic exchange, chromatofocusing and HPLC Sphaerogel 3000SW filtration. The pure enzyme uses a variety of organic phosphorylated compounds as substrates. The N-terminal end of the mature protein was found to be RLREDPFTLGVASGDPHP. The gene phoA has been cloned using as probe an oligomer based on the N-terminal sequence of the S. griseus AP. phoA encodes a protein of 62678 Da with low homology to the AP of Escherichia coli. The phoA gene was found to be homologous to three alkaline-phosphatase-encoding genes previously identified in the Streptomyces coelicolor genome. On the basis of the optimal pH, substrate specificity and differences in amino acid sequence of motifs defining the active centre of APs, the S. griseus AP uses a wide range of organic phosphate substrates and is different from the phosphatases of Gram-negative bacteria.
机译：Streptomyces物种分泌大量尚未表征到目前为止的碱性磷酸酶（AP）酶。将AP从链霉菌综合症3570的培养物中纯化至均匀性。酶的单体尺寸为62Ω·kda，并在培养物中加工至33μk蛋白质，如免疫印迹所示。通过硫酸铵沉淀，CM-Sephadex阳离子交换，染色粒子壳和HPLC Sphaerogel 3000SW过滤纯化酶。纯酶使用各种有机磷酸化化合物作为基材。发现成熟蛋白的N-末端是RLREDPFTLGVASGDPHP。基于探针是基于S.Griseus AP的N-末端序列的探针，基因肽已克隆。 PHOA编码62678AD的蛋白质，其与大肠杆菌的AP具有低同源性。发现PhOA基因对三种碱性磷酸酶编码基因同源，以前在链霉菌基因溶胶基因组中鉴定出来。基于定义APS活性中心的基序的最佳pH值，底氨酸特异性和氨基酸序列的差异，S.Griseus AP使用宽范围的有机磷酸盐基材并与革兰氏阴性细菌的磷酸酶不同。

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