Cloning And Biological Activities Of Two Novel Angiotensin Converting Enzymes In Lysobacter Sp.

Angiotensin converting enzyme（ACE）is a key biocatalyst of the renin-angiotensin-aldosterone system（RASS）,which can hydrolyze angiotensin I to angiotensin II and release bradykinin.Dipeptidyl carboxypeptidase II is an important ACE in mammalian.Dipeptidyl carboxypeptidase II hydrolyzes angiotensin I to angiotensin II and releases Phe-Arg and Ser-Pro from the C-terminus bradykinin,but the enzyme dose not cleave amide-bonds.In this study,two gene sequences showing moderate similarity to mammalian ACE were observed in the genome of Lysobacter type species.The strain Lysobacter sp.CW239 from our laboratory was selected as experimental material and the ACE gene sequences were cloned.The obtained ACE-like genes were verified by prokaryotic expression and the enzymatic activity determination.On the basis of the purified recombinant proteins,the biological activities of the two ACEs was studied.The main results were as follows:1.Dipeptidyl carboxypeptidase II genes were observed based on the genomic analysis of Lysobacter type species,and two dipeptidyl carboxypeptidase II genes named as 239cp1 and 239cp2 were cloned from Lysobacter sp.CW239.The gene sequence of 239cp1 was 2175 bp which encoded 724 amino acids with a molecular weight of about 80.4 k Da and an isoelectric point of 5.21.The gene sequence of239cp2 was 2202 bp which encoded 733 amino acids with a molecular weight of about 80.5 k Da and an isoelectric point of 5.23.The signal peptide of two ACE proteins was predicted by the Signal P4.1 program.The results showed that no signal peptide was found in 231cp1 and 239cp2,indicating that the proteins expressed by231cp1 and 239cp2 had no transmembrane structure.At the same time,six representative ACEs from other species were selected by Blast searches in the NCBI database,and the multiple sequence alignment was analyzed by CLUSTAL＿X and Bio Edit.The results indicated that the selected enzymes showed three similar conservative regions and the two enzymes from strain CW239 showed the same structure as ACE.In the second conserved region,the putative catalytic sites containing two zinc-binding residues and one essential glutamic acid residue were observed for the two obtained proteins.2.Two ACE genes were cloned into the PET-32a（+）vector,and then were transformed into E.coli BL21（DE3）for overexpression.The expression results showed that the optimal condition for 239cp1 was at 28℃with 0.2 mmol/L IPTG induction for 4 h,and a large amount of soluble protein in supernatant could be obtained.The molecular weight of the recombinant 239cp1 was about 97 k Da.While the optimal condition for 239cp2 was at 16℃with 0.2 mmol/L IPTG induction for 4h,and a large amount of soluble protein in supernatant could be obtained.The molecular weight of recombinant 239cp2 was about 97 k Da.The activity of the two recombinant ACEs was determined by standard methods using spectrophotometry.The result showed that both fusion proteins showed the biological activity of angiotensin I hydrolyzation.LC-MS/MS analysis indicated that the hippuric acid（HA）was produced from Hip-His-Leu（HHL）by 239cp1 or 239cp2 hydrolyzation.Therefore,both ACE-like enzymes in this study showed the ACE activity.3.The optimum temperature and p H of 239cp1 is 37℃and 7.0,respectively.The optimum temperature and p H of 239cp2 is 30℃and 6.0,respectively.The two ACEs were stable at 4℃,the optimal store p H was between 5.0-9.0.The enzymatic activity of the two ACEs was inhibited by Na⁺and was enhanced by K⁺.The stability of the two ACEs was better in the 1 M Na Cl,but their enzymatic activity was inhibited obviously by 0.1 M Cu²⁺,1 M Cu²⁺,0.1 M Zn²⁺or 1 M Zn²⁺.4.The Km,Vmax,Kcat and Kcat/Km of 239cp1 were 6.385 m M,525.125μmol·min^-1·mg^-1,703.67 s^-1,and 110.203 s^-1·m M^-1,respectively;the Km,Vmax,Kcat and Kcat/Km of 239cp2 were 6.413 m M,381.375μmol·min^-1·mg^-1,511.68 s^-1,and79.788 s^-1·m M^-1,respectively,which was in contrast with that of the commercial ACE（A6778）(The Km,Vmax,Kcat and Kcat/Km of A6778 were 3.149 m M,63.2μmol·min^-1·mg^-1,147.47 s^-1,and 46.83 s^-1·m M^-1,respectively).No significant difference was observed among the Kms of 239cp1,239cp2 and ACE（A6778）.The Km of ACE（A6778）was the smallest ACE（A6778）,indicating that ACE（A6778）had the best affinity to substrate HHL.From the result of Kcat/Km,the difference was obvious in the three ACEs,of which the Kcat/Km of 239cp1 was the largest,indicating that 239cp1 the has the best catalytic efficiency.5.The influences of different metal ions on the two ACE enzymes were evaluated by the kinetics analysis of Kcat and Km.Other than 1 M Ca²⁺,the tested metal ions significantly increased the Kcat of 239cp1,while,all metal ions also significantly increased the Km of 239cp1.The Kcat/Km analysis indicated that the catalytic efficiency of 239cp1 was reduced by the addition of various metal ions.Compared to 239cp1,all metal ions significantly increased the Kcat of 239cp2,and the addition of 1M Ca²⁺resulted in the largest increase in the amount of Kcat in239cp2.Similar to 239cp1,all the metal ions significantly increased the Km of239cp2.The Kcat/Km analysis showed that the catalytic efficiency of 239cp2 was also reduced by the addition of various metal ions.