Contribution
In the field of enzymology, the impact of metal ions on enzyme activity has long been a core research topic. With the continuous innovation of research technologies, this field has witnessed abundant achievements in recent years. Metal ions play a crucial role in numerous enzymatic reaction processes, and the regulatory mechanisms governing enzyme activity are intricate [1][2][3].
From the perspective of metal ions as components of enzymes, studies have shown that in some metalloenzymes, metals form stable bonds with enzymes, directly participating in the construction of active centers and exerting a decisive influence on enzyme activity[10]. For instance, a 2023 study on natural product biosynthetic enzymes revealed that during the biosynthesis of the complex polycyclic antitumor natural products eupenifeldin and pycnidione, the biosynthetic enzyme EupfF and its homologous protein PycR1 chelate with calcium ions. These calcium ions not only possess catalytic functions but also are essential for maintaining the specific structure of the enzymes. Their synergy significantly enhances the efficiency of the enzyme-catalyzed tandem (4 + 2) cycloaddition reaction[15].
In terms of metal ions acting as enzyme activators, numerous experiments have confirmed that various metal ions can regulate enzyme activity[6][7]. Taking lipase as an example, a 2025 study employed spectroscopic methods and molecular dynamics to investigate the interaction between different metal ions and lipase. It was found that Zn²⁺, Mg²⁺, and other ions can alter the structural conformation of lipase to a certain extent, thereby affecting its activity [13]. Another study conducted in the same year on dimethyl sulfoxide (DMSO)-dependent RNA-cleaving deoxyribozymes (RCDs) pointed out that divalent metal ions are crucial for the catalytic activity of RCDs. Different divalent metal ions exhibit varying degrees of activity-promoting effects, among which Zn²⁺ and Mg²⁺ stand out [14].
Furthermore, the inhibitory effect of heavy metal ions on enzyme activity has also been extensively explored[8][9]. Studies have indicated that introducing high concentrations of heavy metal ions (such as Hg²⁺ and Ag⁺) into specific enzymatic reaction systems causes these ions to bind to key groups in enzyme proteins, altering the spatial structure of the enzymes and resulting in the loss of enzyme activity. This inhibitory effect is often irreversible[4].
Additionally, the influence of metal ions on enzyme activity is closely related to their concentration[11][12]. A 2023 study on the hydrolysis of wheat bran by xylanase found that Mn²⁺ can enhance the ability of xylanase to hydrolyze wheat bran for the production of feruloyl oligosaccharides. When the concentration of Mn²⁺ reached 4 mM, the yield of the product was 2.8 times higher than that without the addition of Mn²⁺. Molecular dynamics simulation analysis revealed that Mn²⁺ can induce structural changes in the active site of xylanase, expand the substrate-binding pocket, and help stabilize the enzyme-substrate complex[16].
In summary, in recent years, research on the mechanism of metal ions' impact on enzyme activity has been continuously deepened. Analyses have been conducted from multiple perspectives, including enzyme structural composition, the microenvironment of active centers, the interaction between enzymes and substrates, and reaction kinetics. This has laid a solid theoretical foundation for further understanding the catalytic nature of enzymes, precisely regulating enzyme activity, and expanding the application of enzymes in industrial, pharmaceutical, and other fields[5].
Analysis of Experimental Cases
Overview:
The β-agarase AgaA(BBa_K5216000) studied by the OTIA-Hangzhou 2024 team can specifically recognize and hydrolyze the β-1,4-glycosidic bonds in agar, catalyzing the production of neoagarooligosaccharides (NAOS). This product exhibits strong antioxidant properties, can effectively scavenge free radicals in the skin, and holds excellent application potential. To further improve the catalytic efficiency of AgaA, this study selected seven common metal ions and systematically evaluated their effects on the activity of this enzyme. The experimental results showed that the introduction of metal ions can significantly enhance the activity of AgaA, thereby improving its hydrolysis efficiency and the yield of NAOS.
Experience and Results
Through the screening of seven metal ions, it was found that magnesium ions (Mg²⁺) have a significant enhancing effect on the activity of AgaA. Further concentration optimization experiments showed that adding 10 mM Mg²⁺ to the enzyme reaction system achieves the optimal promoting effect, effectively improving the hydrolysis efficiency of the enzyme.
Plasmid Acquisition
We obtained the AgaA_pET-32a(+) plasmid from the OTIA-Hangzhou 2024 team and introduced it into Rosetta competent Escherichia coli (E. coli) using the heat shock transformation method. As shown in Figure 1A, the transformed expression strains formed clear single colonies on the LB solid medium, and these single colonies can be used for subsequent experiments to verify the success of plasmid transformation.
After selecting the single colonies, they were cultured under suitable conditions (37°C, 220 rpm) for approximately 4 hours. Subsequently, the cultured strains were used as templates for PCR verification. The verification results in Figure 1B showed that the three selected single colonies all amplified specific bands consistent with the positive control, indicating that the AgaA_pET-32a(+) plasmid was successfully transferred into Rosetta E. coli. Based on this result, any one of the positive strains can be selected for expanded culture to obtain the target protein AgaA.
(A) Transformation results of E. coli Rosetta. (B) PCR results of E. coli Rosetta. M: DNA molecular weight marker; 1: Positive control; 2–4: PCR results of positive clones.
Induced Expression of AgaA Protein
4 mL of overnight-cultured seed solution (E. coli Rosetta AgaA_pET-32a(+)) was added to 200 mL of LB liquid medium containing ampicillin, and cultured at 37°C with 200 rpm shaking until the OD600 reached approximately 0.5. Then, 100 µL of 1 M IPTG (final concentration: 0.5 mM) was added, and the culture was continued at 16°C with 200 rpm shaking for 18 hours. The cultured strains were collected, subjected to ultrasonic disruption, and after centrifugation, soluble proteins and inclusion bodies were collected. 10% SDS-PAGE (Fig. 2) showed that the AgaA protein was successfully expressed and mainly existed in the form of soluble protein. Collecting the crude enzyme solution of AgaA prepares for the next step of enzyme activity testing.
M: Protein molecular weight marker; 1: Before IPTG induction; 2: Precipitate after ultrasonic disruption of IPTG-induced cells; 3: Supernatant after ultrasonic disruption of IPTG-induced cells.
Enzyme Activity Determination
To investigate the effects of different metal ions on AgaA enzyme activity, the experiment was conducted as follows:
First, using 20 mM Tris-HCl buffer (pH 7.0) as the solvent, 5 mL solutions of NaCl, KCl, CuSO₄, CaCl₂, MgCl₂, FeSO₄, and FeCl₃ were prepared, each with a concentration of 50 mM.
Reaction system setup: 50 µL of 2% agarose solution (substrate) was added to each of multiple 2 mL EP tubes, followed by the addition of 50 µL of each of the above metal ion solutions; for the control group, 50 µL of Tris-HCl buffer (without metal ions) was added. 400 µL of AgaA enzyme solution was added to each tube to initiate the reaction, which was incubated at 35°C for 30 minutes.
Reaction termination and detection: All samples were heated in a 95°C water bath for 10 minutes to inactivate the enzyme. 200 µL of the reaction solution was transferred to a new 2 mL EP tube, 200 µL of DNS reagent was added, and the mixture was heated in a 95°C water bath for 5 minutes. After cooling, 1 mL of ddH₂O was added to each tube. A microplate reader was used to measure the absorbance at a wavelength of 540 nm. The effect of different metal ions on AgaA enzyme activity was evaluated by comparing the absorbance differences among the groups. Taking the enzyme activity of the control group as 100%, the relative enzyme activity of each experimental group was calculated.
As shown in Fig. 3, the experimental results demonstrated significant differences in the effects of different metal ions on AgaA enzyme activity: the enzyme activity of the groups treated with Na⁺, K⁺, and Ca²⁺ showed no significant difference from that of the control group, exerting almost no regulatory effect on AgaA enzyme activity; Cu²⁺ and Fe³⁺ completely inhibited AgaA enzyme activity, while Fe²⁺ exhibited partial inhibition, reducing the enzyme activity to 79.3% of that of the control group; in contrast, Mg²⁺ had a significant activating effect on AgaA enzyme activity, increasing it to 115.6% of that of the control group. Based on this, the mechanism and regulatory law of Mg²⁺ promoting AgaA enzyme activity can be the focus of subsequent research.
Effects of Different Concentrations of Mg²⁺ on AgaA Enzyme Activity
To explore the effects of different concentrations of Mg²⁺ on AgaA enzyme activity, the experiment was carried out as follows:
First, using 20 mM Tris-HCl buffer (pH 7.0) as the solvent, a series of MgCl₂ solutions with concentrations of 0, 0.5, 1, 5, 10, 20, 50, 100, and 150 mM were prepared.
Reaction system setup: 50 μL of 2% agarose solution was added to each of multiple 2 mL EP tubes, followed by the addition of 50 μL of MgCl₂ solutions with the above different concentrations; for the control group, 50 μL of Tris-HCl buffer (without Mg²⁺) was added. 400 μL of AgaA enzyme solution was added to each tube to start the reaction, which was incubated at 35°C for 30 minutes.
Reaction termination and detection: All samples were heated in a 95°C water bath for 10 minutes to inactivate the enzyme. 200 μL of the reaction solution was taken into a new 2 mL EP tube, 200 μL of DNS reagent was added, and the mixture was heated in a 95°C water bath for 5 minutes. After cooling, 1 mL of ddH₂O was added to each tube. A microplate reader was used to measure the absorbance at 540 nm. The effect of different concentrations of Mg²⁺ on AgaA enzyme activity was evaluated by comparing the absorbance differences among the groups. With the enzyme activity of the control group set as 100%, the relative enzyme activity of each experimental group was calculated.
As shown in the figure, the experimental results indicated an obvious concentration-dependent pattern in the effects of different concentrations of Mg²⁺ on AgaA enzyme activity: when the concentration of Mg²⁺ was 0 mM (control group), the enzyme activity was at the baseline level; as the concentration increased, within the range of 0.5–10 mM, the activity of AgaA showed an upward trend. When the concentration reached 10 mM, the enzyme activity was enhanced most significantly, reaching approximately 139.0% of that of the control group; when the concentration of Mg²⁺ exceeded 10 mM, the enzyme activity began to decrease, dropping to approximately 68.3% at 50 mM. When the concentration further increased to 100 mM and 150 mM, the enzyme activity decreased sharply, reaching only about 20% of that of the control group. This indicates that an appropriate concentration of Mg²⁺ has an activating effect on AgaA enzyme activity, while an excessively high concentration inhibits its activity.
The β-agarase (AgaA) studied by the OTIA-Hangzhou 2024 team can specifically hydrolyze the β-1,4-glycosidic bonds in agar to produce NAOS, which has antioxidant properties and the ability to scavenge free radicals in the skin. To improve its catalytic efficiency, this study evaluated the effects of seven common metal ions on enzyme activity and found that magnesium ions (Mg²⁺) can significantly enhance the activity of AgaA; further concentration optimization experiments showed that adding 10 mM Mg²⁺ to the enzyme reaction system achieves the optimal promoting effect, effectively improving the hydrolysis efficiency of AgaA and the yield of NAOS.
Summary
Experiments on AgaA further verified that different metal ions have significantly different effects on its activity, with 10 mM Mg²⁺ showing the optimal activation effect. This mechanism provides a clear direction for the regulation of enzyme activity. In the future, it can be extended to applications such as improving the catalytic efficiency of industrial enzymes and optimizing enzyme preparations in the medical field, laying a foundation for technological breakthroughs and product upgrades in related fields.
References
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