Preparation And Enzymatic Characterization Of An Ancient Aminopeptidase

Applying as anti-oxidant,anti-glycation component,L-carnosine has been widely used in areas such as pharmaceutical and health care by taking advantage of its various physiological properties.Most of the current methods to obtain L-carnosine are still chemical synthesis methods,which are not only complex process,tedious reaction steps,but also often accompanied by a great environmental burden;In comparation,enzymatic synthesis of dipeptides has been revealed as a promising solution due to its gentle reaction conditions and environmental friendless.Aminopeptidases have been used as an effective strategy for peptide synthesis due to their broad substrate spectrum and mild enzyme-catalyzed reaction conditions,however,aminopeptidase has shortcomings such as long reaction times and low catalytic efficiency.In order to obtain aminopeptidases with better properties,reported aminopeptidase protein sequences are collected for the screen of potential novel aminopeptidase with the catalytic activity of L-carnosine synthetization by ancestral sequence reconstruction（ASR）technique,using ASR,the last universal common ancestor（LUCA-dmpA）was reconstructed according to the 56 protein sequences screened out from four peptidase families and further cloned in pET28a（+）vector and heterologous expressed in E.coli BL21（DE3）.The main study contents and findings are as follows.（1）In this study,a gene sequence with the function of carnosine synthesizing was attempted to be obtained by ASR technique.Using ASR,the last universal common ancestor（LUCA-dmpA）was reconstructed according to the 56 protein sequences screened out from four peptidase families and further.Purification results of the recombinant ancestral enzyme showed that the ancient aminopeptidase was expressed with three bands:42.1 kDa、31.1 kDa and 13.8 kDa.The crude enzymatic activity for carnosine synthesizing was 18 U·mL-1;the purified enzymatic activity was 42.50 U·mL-1,and a specific enzymatic activity was 10.24 U.mg₁.（2）Verification and mutation of the self-cleaved site of LUCA-DmpA.In order to investigate the self-cleaved site of LUCA-DmpA,a recombinant expression vector fused to a C-terminal His-tag was reconstructed for N-terminal sequencing and Western Blot.The results showed that the ancient aminopeptidase was self-cleaved at Gly237-Ser238 to exert its enzymatic activity.A total of five mutants of the ancient aminopeptidase were constructed based on the self-cleaved site,and the results showed that after mutating Gly237 to Ala and Ser23 8 to Thr,Cys,Ala,and Gly,respectively,none of the mutant’s enzymatic activities could be exerted,indicating that the Gly 237-Ser238 is essential for the ancient aminopeptidase.（3）The shake fermentation conditions of LUCA-DmpA were optimizd.The results showed that the optimal fermentation temperature,inducer addition time and the inducer addition concentration for LUCA-DmpA were 20℃,80 min and 0.10 mmol·L-1 IPTG,respectively;the optimal fermentation time was 32 hours.After exploring the optimum enzyme reaction conditions and optimizing the shake flask fermentation,the enzyme activity of crude ancient aminopeptidase was 4.90 times higher than original.（4）Characterization of the enzymatic properties for purified LUCA-DmpA.Purified with a Hi strap HP column,the enzymatic properties of this ancient aminopeptidase were characterized.Circular dichroism（CD）spectroscopy results showed that the ancestral enzyme was composed of α-helix（35.23%）,β-sheet（11.06%）,β-turn（23.67%）and random coil（32.03%）.The melting temperature（Tm）and denaturation enthalpy（AH）of LUCA-DmpA were 60.27℃ and 1306 kJ·mol-1.The enzyme was characterized with the optimal temperature and pH of 45℃ and 9.0,respectively.Notably,the LUCA-DmpA was suggested with remarkably pH tolerance based on the observation of more than 85%remaining enzymatic activity after the incubation at different pH buffers（pH=5-11）for 12 h.Also,rather than being improved or inhibited by mental ions,the enzymatic activity was found to be promoted by introducing organic solvent with a larger log P value.The kinetic parameters of LUCA-DmpA were also analyzed.The results showed that the Km,kcat and the kcat/Km of LUCA-DmpA were measured as 22.06±1.02 mmoI·L-1,76.53±2.68 s-1 and 3.47 s-1·L·mmol-1,respectively.（5）Preliminary investigation of LUCA-DmpA’s lipase activity:the ancient aminopeptidase possessed a weak esterase activity to hydrolyze short chain p-nitrophenol esters.