Cloning, Expressing And Further Characterization Of Thermostable Acylpeptide Releasing Enzyme ST0779

There are many spices of microorganisms lived in the harsh environments on earth, most of which are extremophile archaea, which include the thermophiles, hyperthermophiles, psychrophiles, haophiles, acidophiles, alkaliphiles, et al. The extremophiles live in such environments as hot springs, volcanic crater and deep sea hydrothermal vents. And current theory and circumstantial evidence suggest that hyperthermophiles were the first life-forms arisen on Earth. As a prerequisite for their survival in the harsh living condition, thermophiles not only possess their specific heat-resistant molecular structure and mechanism but also contain enzymes that can function at high temperature. Owning to their thermostability associated with a higher resistance to chemical denaturants and great enzymatic activity at high temperatures in water or organic solvents, hyperthermophilic and thermophilic enzymes in archaea cells can therefore offer major biotechnological advantages over the mesophilic enzyme, for this reason, thermostable enzymes are focused on day after day.Acylpeptide hydrolase (APH) is a member of the prolyl olgopeptidase (POP) family belonged to serine protease. The POP family includes prolyl olgopeptidase, dipeptidyl pepiase IV, oligopeptidase B, APHs and glutamyl endopeptidase, classified according to their sequence homology and biochemical characteristic. The reason for the wide distribution and physiological roles of the POP family enzymes has been clarified. Some enzymes from this family are concerned with degradation of biologically active peptides, so most of them are targets to the medicine, and are focused on by some researchers in pharmacology. APHs may catalyze the NH2-terminal hydrolysis of Na-acylpeptides to release Na-acylated amino acids, so it could process and pick out specific peptides; thermophilic APHs have comprehensive application on protein sequencing analysis and small peptide synthesis.The complete genomic sequence of an thermoacidophilic crenarchaeon, Sulfolobus Tokodaii which optimally grows under aerobic condtions at 80℃, pH 2-3 and was previously isolated from the Beppu Hot Springs in the geothermal area of Kyushu Island (Inatomi et al. 1983, Japan), has been determined by the whole genome shotgun method. Here we report the characterization an APH expressed from the putative APH gene (ST0779, encoding 583 amino acids) selected from the total genome analysis of the strain. The putative molecular mass and the isoelectric point of the ST0779 are 65,881Da and 6.74, respectively. All the five cysteines in the protein sequence form no disulfide bond. The observed structure domain and result from BLAST on NCBI indicats that the production of ST0779 belongs to proly oligopeptide family, and have high degree sequence homology with APHs from other spices which contain the propeller domain,α/βhydrolytic catalytic domain and conservative sequence GXSXG. Qualitative predict indicates that the amino acid in both of N- and C-terminal in this protein process strong hydrophilicity, the catalytic triad consisted of Ser439, Asp523, His555 and residules around them site in the flexible region, which is the reason of their high-efficiency catalyzing ability.Our group obtained the gene ST0779 from the thermophiles Sulfolobus Tokodaii and constructed the recombinant plasmid using the gene of ST0779 and pET28a, which was cloned and expressed in E. coli cells. The target protein could be optimally overexpressed within the E.coli under 37℃for 8h with 0.1mM IPTG, but some of the protein were expressed as inclusion bodies. Luckily, it was further found that under a condition of 30℃, 8h and 0.1mM IPTG, as well as using BLP as the host, the product of ST0779 would totally stay soluble and twice fold abundant to that in BL21. Because of the thermostability of the target protein and the His Tags on the pET28a , it was easy for us to use simple but high efficient method to purify the target protein: the crude enzyme was heated in 70℃for 30min, after which the target protein could be above 40% among the whole protein. Pure target protein could be able to get through the Ni2+ Affnity colume chromatography and Hitrap Q Sepharose, and the purification fold is 27. For each step above, the enzyme activity was detected.APH-like activity and features of the recombination ST0779 expression product shows a maximum activity at a temperature of 70℃at pH 8.0 when using Ac-Ala3 as the substrate. The half-life period of the enzyme(concentration 0.2mg/mL) at 90℃is about 16h, which indicates the enzyme exhibit favorable thermoability as expected from the property of the organism, and it is stable within the neutral or slight alkali environment , showing 85% activity remained after being treated at 25℃for 24h in pH 6.0-10.0 the enzyme exhibits really high catalytic efficiency for the acylpeptide, and the optimal substrate is Ac-Ala3(Km=3.12*10-4 M, kcat=8.64*104 s-1), but the activity for the f-Met-Ala only reaches about 40% of the Ac-Ala3's. The enzyme possess lower specificity for peptide, it could hydrolyze dipeptide and other small peptide without blocked in N-terminal, especially for Ala4 the activity could reach 60% of the optimal substrate , while no detected activity on (D-Ala)2. The effect of metal ions on the enzyme are ever so slight that only Kalium ion could inhibit the activity of ST0779 for 4%. Non-polar surfactants such as Tween 20,Triton X-100,Tween 80 and metal chelating agent EDTA also exhibit some slight inhibited effects, as 10% treated by the EDTA. Negative ion surfactant completely deactivate the protein. Meanwhile the enzyme could hydrolyzes ester whose alkyl chain length is below 4, the specific activity for p-nitrophenyl propionate could reach 1871mU/mg. besides, a lipase activity is also detected when using the tributyrin as the substrate. With the results above, we speculate that the acylpeptide releasing-enzyme ST0779 possesses the selectivity for the first residue in the N terminal of the substrate, and the catalysis reaction is corresponding with characteristics of the POP family, namely, the substrate with large steric hindrance could not access the catalytic center.In conclusion, we have isolated the S.tokodaii gene ST0779 and identified that it codes a thermostable APH. Revealed features of the enzyme represent important prerequisites for biotechnological applications, as high temperatures is required for the solubilization of both reagents and products. We are hoping these results will contribute to further application of the enzyme, such as protein sequencing analysis, production of useful synthesized compounds and the molecular reconstruction toward the esterase/lipase.