| Life on earth exhibits an enormous adaptive capacity. In the pastmore than 40 years, extremophiles have been found in hot spring,volcanic areas, the deep sea and so on, all places earlier regarded astoo severe to support life. More kinds of extremophiles have beenfound now, including thermophiles, hyperthermophiles, psychrophiles,halophiles, acidophiles, alkaliphiles et al. The discovery of newextremophilic microorganisms and their enzymes has had a greatimpact on the field of biocatalysis. Currently, thermophilic enzymes(including hyperthermophilic enzymes)have been gained widelyattention because they often have high thermostability and are resistantto organic solvent or denaturant when comparing to mesophilicenzymes. Therefore, they are attractive in many biotechnology fields.Aeropyrum pernix K1 is the first reported strictly aerobichyperthermophilic archaeon, which grows optimally in a temperaturerange of between 90 and 98 ℃. APE 1547 has been cloned from it andexpressed in E. Coil by Dr. Gao in our group. Its properties of esterasehave been determined in detail and three-dimensional structure hasbeen solved by Gao and his colleagues.This dissertation was the extension of Gao's. Based on hisdissertation, the enzyme was presumed to be most like the acylaminoacid-releasing enzyme (AARE) by the crystal structure and sequencealignment. Therefore, its character of AARE, the effect of amino acidsand metal cations were examined. The mechanism of how calciumcation affecting the activity of the enzyme was study by fluorescenceand circular dichroism simply. From these results, the enzyme wasconsidered to be a bifunctional enzyme like other enzymes reported. Ithad the activity of esterase and acylamino acid-releasing enzyme.The enzyme was obtained from engineering bacteriumconstructed by Dr. Gao and purified and determined according to theprocedures described in the Gao's dissertation. The optimal AAREactivity of APE1547 was detected at 90 ℃, like its esterase activity.The optimal pH for the substrate Ac-Leu-pNA was 8.0 which was thesame to its esterase and for the peptide substrates was 7.25. The pHdiscrepancy may be caused by the distinctness in enzyme assaymethod of these substrates. Among the small peptides, f-Met-Ala wasthe optimum substrate and kinetic parameters of AARE were 0.88 ± 0.02mM, 1.959 ± 0.425 S-1 for Km and kcat respectively. The effect ofmetal cations on the enzyme was very complex: (1) calcium,magnesium and barium ions had stimulative effect of AARE;(2) zinc,manganese, sodium ions inhibited the activity of AARE and (3) copperion resulted in complete inactivation of enzyme. Amino acids had nosignificant inhibition to the activity of AARE and had no effect ofesterase activity. The performance of these amino acids was much likeproduct inhibition rather than inhibitor inhibition. The results maydemonstrate that the enzyme was an AARE, although it had esteraseactivity. Like the thermostability of esterase, The AARE also had highthermostability and protein concentration-dependent. When theenzyme (0.8mg/ml) was incubated at 90℃ for 36h, it still could retainapproximately 50% of its original activity. Even though the enzymeconcentration was low to 0.2mg/ml, the half-life was 16 h. It was easyto understand because the enzyme from the hyperthermophilicarchaeon that grows optimally in high temperature.Calcium cation had bi-modal effect of AARE, it could activateenzyme activity when the ion concentration was lower than 0.2 M, andwhen the concentration of calcium ion was 0.1 M, and the enzyme hadthe highest activity. But when the concentration was higher than 0.2 M,the enzyme activity was inhibited. The intrinsic fluorescence of theenzyme was found could be quenched by addition of the calciumcation. Recurring to the equation Stern-V?lmer, the calcium cation wasbound to the enzyme and the mechanism of fluorescence quenchingbelong to static quenching. From the calculation of fluorescencequenching results, association constant was 1.24x103 M-1, one enzymemolecule may be bound by one calcium cation. The relationshipbetween concentration-dependent variety in the enzyme activity andthe change in the secondary structure was studied by circulardichroism spectroscopy. The activity enhanced by addition of calciumcation could result from the increase of β-turn and decrease of randomcoil. However, change of α-helix content doesn't seem to associatewith the enzyme activity. But where was the calcium cation in theenzyme molecule and how to interact with it leading to the change ofenzyme activity should need to do further study.
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