Enzymes, as crucial raw materials for diagnostic reagents, directly influence the quality of downstream products. A profound understanding of enzymatic properties is essential for new product development, process optimization, and product transportation and storage. Enzyme characterization is not only the foundation of theoretical research but also the key to practical applications.

However, currently, enzyme preparations on the market usually only provide basic enzymatic information, and the characterizations related to application performance are not comprehensive enough to offer strong references for application tests, thus increasing the cycle and cost of enzyme application development tests. Therefore, a systematic and comprehensive characterization of enzymatic properties is an important bridge connecting theoretical research and practical applications. It can not only guide the rational use of enzymes in downstream applications but also play a significant role in enhancing production efficiency and economic benefits.

Kinetic Parameters: The Yardstick of Catalytic Efficiency and Reaction Rate

Kinetic parameters are crucial indicators for evaluating enzyme catalytic performance, mainly including the maximum reaction rate (Vmax), Michaelis constant (KM), turnover number (Kcat), and catalytic efficiency (Kcat / KM). These parameters can be determined by the Lineweaver-Burk method.

In the research and development of biosensors, the characterization of enzyme kinetic parameters is of great significance for understanding the catalytic mechanism of enzymes, optimizing enzyme reaction conditions, and designing enzyme-based applications. A higher Vmax indicates a faster response time and higher signal output intensity. An enzyme with a low KM value has a higher affinity for the substrate and can effectively detect the target analyte even at low substrate concentrations, showing a more sensitive signal. An enzyme with a high Kcat / KM can provide a faster response time and higher sensitivity, enabling earlier and more accurate detection.

Figure caption: Influence of substrate concentration on the rate of enzyme-catalyzed reaction

Figure caption: Double reciprocal plot

For example, Yang Y et al. [1] showed that AnGDH with a lower Km value generated a larger current signal when reacting with 1 mM low-concentration glucose, thus enhancing the detection sensitivity. Therefore, kinetic parameters are not only the basis for studying enzyme functions but also a key tool for promoting innovation in biotechnology and industrial applications.

Substrate Spectrum: The Criterion of Enzyme Specificity and Broad-spectrum Activity

The substrate spectrum specifically refers to the types and ranges of substrates that an enzyme can catalyze. It reflects both the specificity and flexibility of the enzyme. In clinical medical applications, cholesterol esterase is mainly responsible for catalyzing the hydrolysis reaction of cholesterol esters, converting cholesterol from the cholesterol ester form in lipoproteins or cell membranes into free cholesterol. The hydrolysis ability of cholesterol esterase for cholesterol esters with different carbon chain lengths is particularly important for the early assessment of the risk of cardiovascular diseases.

To ensure the accuracy of application measurements, we simulated the actual application scenarios and measured the decomposition ability of the mainstream cholesterol esterase products on the market with cholesterol esters of different carbon chain lengths to cover the common cholesterol ester types in the human body. Through the evaluation of the substrate spectrum, it was found that there are certain differences in the decomposition ability of cholesterol esterase products from different manufacturers for cholesterol esters with different carbon chain lengths, which is a noteworthy point. When reagent developers select different esterase, due to the differences in the substrate spectrum, the measured values of the reagents may vary significantly. The characterization of the enzyme substrate spectrum can provide guidance for product development directions and clinical applications.

Stability: The Touchstone of Enzyme Performance Reliability

Stability is an important property for measuring the ability of an enzyme to withstand extreme conditions, including half-life, thermal acceleration stability, long-term stability, freeze-thaw stability, and open-bottle stability. Half-life and thermal acceleration stability mainly evaluate the ability of an enzyme to resist irreversible denaturation at high temperatures under thermal conditions, simulating the high-temperature environment in industrial applications. Freeze-thaw and open-bottle stability simulate the environment during transportation and use, comprehensively evaluating the stability performance of the enzyme under actual conditions. Long-term stability directly determines the product’s expiration date.

The Arrhenius equation has a wide range of applications in the chemical field. It can not only describe and predict the influence of temperature on the reaction rate of a chemical reaction but also calculate the apparent activation energy, reaction rate constant, etc. of the reaction. The document “EN 13640 Stability testing of in vitro diagnostic reagents” published by the European Committee for Standardization in 2022 [2] and the document “EP25-A Evaluation of stability of in vitro diagnostic reagents” released by the Clinical and Laboratory Standards Institute (CLSI) in 2009 [3] both recommend using this method to estimate the shelf life of in vitro diagnostic reagents. Leidy D. Ardila-Leal et al. [4] used the Arrhenius equation to estimate the stability of laccase rPOXA 1B at low temperatures. The half-lives at -32.55 ± 4.12 °C, 4.32 ± 1.22 °C, and 24.99 ± 0.25 °C were 230.8, 46.2, and 12.6 months, respectively.



We also used the Arrhenius model to estimate the expiration date of the product for reference. In the stability assessment of T7 RNA polymerase, using the Arrhenius curve model, by monitoring the stability of the product at 20, 25, 30, and 37 °C, taking 90% of the initial activity as the failure point, calculating the inactivation rate constant Kd at each temperature condition, and then drawing the relationship diagram of ln (Kd) and 1/T according to the Arrhenius equation. Through the linear regression relationship between ln (Kd) and 1/T, the activation energy Ea was calculated from the slope, and the pre-exponential factor A was calculated from the intercept. Using the calculated Ea and A and substituting them into the Arrhenius curve, the inactivation rate Kd at the required temperature could be predicted. According to this model, it was speculated that when stored at -20 °C, the effective activity preservation times of the three batches were 27.6 months, 30.6 months, and 31.8 months, respectively. At the same time, the long-term stability of the product was monitored, and there was no significant decrease in 18 months, providing effective data support for the product’s expiration date monitoring.

Activators and Inhibitors: The Regulators of Catalytic Potential

The activity of enzymes can be regulated by activators and inhibitors. Activators enhance catalytic efficiency by promoting the binding of enzymes to substrates or changing the conformation of enzymes. They include inorganic ions, such as certain metal ions, which can participate in various enzyme-catalyzed reactions; small organic molecules (such as NAD⁺/NADH, FAD/FADH₂, CoA, etc.) play a key role as cofactors in metabolism; proteins and other macromolecules (such as regulatory proteins and transcription factors) affect enzyme activity through regulatory mechanisms; and certain reducing agents and surfactants enhance enzyme activity by changing the conformation of enzymes.

Conversely, inhibitors reduce enzyme activity or even inactivate enzymes. There are two types: irreversible and reversible. Irreversible inhibitors form covalent bonds with enzymes, resulting in permanent inactivation. For example, heavy metal ions (such as mercury, silver, platinum, arsenic, etc.) bind to sulfhydryl enzymes. Reversible inhibitors do not form covalent bonds and can be divided into three categories. Competitive inhibitors usually have a structure similar to that of the substrate and compete for the binding site of the enzyme, increasing the KM value while keeping the Vmax unchanged. Non-competitive inhibitors bind to the non-active site of the enzyme, changing the shape of the enzyme, resulting in a decrease in Vmax while keeping the KM unchanged. Uncompetitive inhibitors only bind to the enzyme-substrate complex, causing both the KM and Vmax to decrease, but the Vmax/KM ratio remains unchanged.

The diagnostic enzyme raw materials will involve various types of metal ions, surfactants, and preservatives in the downstream application environment, and their tolerance is particularly important for the stability of the reagents and the accuracy of the diagnostic results.

Systematic and Comprehensive Enzyme Characterization

In addition to kinetic parameters, substrate spectrum, stability, activators, and inhibitors, we also comprehensively evaluated key indicators such as enzyme activity, specific activity of protein, optimal temperature, optimal pH value, temperature and pH tolerance, and contaminating enzymes. The enzyme products of HZYMES rely on a systematic and rigorous quality control and evaluation system to ensure excellent product quality and high consistency, providing solid and reliable data support to meet the diverse application needs of customers and helping customers achieve more remarkable achievements in scientific research exploration and industrial production.

Hzymes’ Enzyme Products

01 G3PO Glycerol-3-phosphate Oxidase

Glycerol-3-phosphate oxidase (G3PO) is an important metabolic enzyme widely used in scientific research and clinical detection. G3PO is often used in combination with lipoprotein lipase and glycerol kinase for the development of triglyceride detection kits (enzymatic method).

Product Basic Information

Core Performance Comparison Table

Excellent Thermal Stability

Wider pH Stability Range

Tolerant to Multiple Surfactants and Metal Ions

Application Test in Kit

02 Raw Material Product Information

References

1.Yang Y, Huang L, Wang J, et al. Expression, characterization and mutagenesis of an FAD-dependent glucose dehydrogenase from Aspergillus terreus [J]. Enzyme Microb Technol, 2015, 68:43-49.
2.European Committee for Standardization. EN 13640 Stability testing of in vitro diagnostic reagents[S]. Brussels: European Committee for Standardization, 2002.
3.CLSI. EP25-P Evaluation of stability of in vitro diagnostic reagents[S]. Wayne, PA: CLSI, 2009.
4.Ardila-Leal, Leidy D., et al. “Recombinant laccase rPOXA 1B real-time, accelerated and molecular dynamics stability study.” BMC biotechnology 21.1 (2021): 37.