聚磷酸激酶(polyphosphate kinase,PPK)是一类磷酸基团转移酶,能催化磷酸基团在ATP与多聚磷酸之间的转移反应,可用于生物催化过程中的ATP再生,基于PPK的ATP再生系统已成为生物催化领域的研究热点之一。该研究旨在利用序列驱动的宏基因组技术,从土壤宏基因组中挖掘新型的PPK基因。根据文献报道的PPK氨基酸序列保守区域,设计简并引物,以土壤宏基因组DNA为模板进行PCR扩增,筛选到一条来源于Serratia marcescens的PPK编码基因,该基因包含一个2 064 bp的开放阅读框,编码一个由687个氨基酸组成的蛋白(SmPPK)。多重序列比对发现SmPPK与Escherichia coli来源的PPK具有较高的序列一致性(87.9%)。系统发育树分析表明,SmPPK与Serratia nevei、Gibbsiella quercinecans等来源的PPK在同一分支上,同属于PPK1家族。同源建模结果显示,SmPPK由4个结构域组成典型的L型空间结构,其活性中心主要由His433、Asp468、His590和Glu621组成。将SmPPK基因与pET 28a连接后转化至E.coli BL21(DE3)中,经IPTG诱导,SDS-PAGE电泳显示在80 kDa处存在明显的蛋白表达条带,与理论分子质量一致。酶活性检测显示,SmPPK可以在多聚磷酸钠的存在下,实现ATP的合成,其最高产率为46.5%。利用SmPPK构建ATP再生系统与灵菌红素缩合酶PigC耦合,成功实现灵菌红素类似物的合成。该研究的开展为ATP依赖的生物催化过程提供了构建ATP再生系统的新酶源。
Polyphosphate kinase (PPK) is a class of phosphotransferase, which can catalyze the transfer reaction of phosphate groups between ATP and polyphosphates, and can be used for ATP regeneration in biocatalytic processes. The ATP regeneration system based on PPK has attracted extensive attention in the field of biocatalysis. The aim of this study is to utilize sequence driven metagenomic technology to explore novel PPK genes from soil metagenomes. A pair of degenerate primers was designed based on the conserved region of reported PPKs. PCR amplification was performed using soil metagenomic DNA as a template. A PPK encoding sequence from Serratia marcescens was obtained. The gene had a length of 2 064 bp, and encoded a protein (named SmPPK) composed of 687 amino acids. Multiple sequence alignment showed that SmPPK had high sequence identity (87.9%) with PPK from Escherichia coli. Phylogenetic tree analysis revealed that SmPPK and PPKs from Serratia nevei, Gibbsiella quercinecans were on the same branch and belonged to the PPK1 family. The homologous modeling results indicated that SmPPK showed a typical L-shaped structure with four domains. Its active centers were mainly composed of His433, Asp468, His590, and Glu621. The SmPPK gene was cloned into the vector pET28a and overexpressed in E. coli BL21(DE3). The SDS-PAGE showed a significant band at 80 kDa, which was consistent with the theoretical molecular weight. Enzyme activity detection showed that SmPPK can achieve ATP synthesis in the presence of sodium polyphosphate, with a maximum yield of 46.5%. The ATP regeneration system constructed by SmPPK was coupled with prodigiosin condensation enzyme PigC, and the synthesis of prodigiosin analogues was successfully realized. This study provides a new enzyme source for constructing ATP regeneration system for ATP dependent biocatalytic processes.
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