Cloning, Expression And The Product Biochemical Characterization Of A Novel Phosphoesterase Gene

Phosphoesterases are widely exist in organisms,and play an important role in the phosphorus metabolism. It regulates the phosphorylation of proteins in the cell signal transduction channels to coordinate physiological and biochemical function. Phosphoesterases from various species have different characterization which makes an extensive use in molecular biology, medical research and industrial applications. Based on the constructed metagenomic library from alkaline soil, we got a positive clone pGXA1009which possible contained a gene encoding phosphoesterase, after subcloning and sequences analysing,an open reading frame which encoded phosphoesterases to be named phop1.Bioinformatics analysis indicated that phopl was composed of1389base pairs,which encoded in463amino acid with a molecular mass of51451.79Da and an isoelectric point of6.26. In the current GenBank database,there is no similarity between phop1and other nucleotide sequenses in nucleotide level,and in the amino acid level, Phop1shows28%identities and45%similarities with the containing PHP domain phosphoesterase from Thermincola potens. Phop1has motifs which were similar with PHP family proteins’that containing phosphoesterase function via amino acid sequence alignment,then we predicted that Phopl would be a new member of PHP family proteins.The ORF of phopl was amplified and clonded into the expression vector pETBlue-2, then the recombinant plasmid was transformed into Rosetta-gamiTM2(DE3)placI to over expression, after purified by nickel column,we got an single band which molecular weight was consistent with the bioinformatics analysis in SDS-PAGE.Then the biochemical properties of Phop1were characterized with the substrate pNPP,and it showed that the optimum pH value and temperature was6.0and50℃respectively,the enzyme had an adaptability and thermostability in high temperature.On the optimum condition, enzymatic activity was1.21U/mg, from double reciprocal Lineweaver-B,Km was0.130mM, Vmax was35.2μmol/min, Na2HPO4was a liner competitive inhibitor and inhibition constant Ki was0.952mM, Mg2+andCo2+ions enhanced the enzymatic activity in0.5-2mM range, EDTA caused obvious inhibition. We had completed the functional and biochemical research of a novel gene which encoded phosphoesterase from uncultured microorganisms,this will make a foundation of molecular modification,as well as a deep utilized of a novel gene resources in phosphoesterases for future research.