Advanced enzyme analysis technology
Enzymes-biocatalysts that accelerate intracellular chemical reactions play a vital role in every organism. The catalytic activity of these enzymes has been widely recognized, accelerating the reaction with high specificity and efficiency under mild conditions (Figure 1).
Figure 1. Catalytic activity of enzyme
In recent decades, the use of biocatalysts has expanded rapidly in many industrial processes. According to the types of reactions they catalyze, these enzymes are divided into six categories: hydrolase, oxidoreductase, transferase, lyase, isomerase and ligase / synthetase. They are used in the form of cell lysates, whole cells, or purified enzymes, either from their primary cells or using recombinant techniques in alternate host cells. Compared with traditional chemical catalysts, enzyme-based catalysis can provide significant advantages. Biocatalysis not only helps to speed up the manufacturing process, but also improves quality, reduces waste, optimizes product output, and ensures higher cost-effectiveness and profitability.
In the biomanufacturing industry, enzymes are rapidly becoming the preferred catalyst for the development of new drugs, vaccines and diagnostic kits. Their potential advantages include special specificity for recognized substrates, simplified drug synthesis routes, and opportunities for more sustainable and consumer-friendly approaches by replacing irritating chemicals commonly used in vaccine production. Despite the obvious advantages of biocatalysis, the ability to identify, acquire, test and optimize enzymes with characteristics suitable for specific applications remains challenging.
Advances in screening technology and bioinformatics have promoted the rapid discovery of new enzyme variants. With the support of computer modeling, the application of protein engineering also expands the opportunity to change the structure of enzymes in an attempt to improve their properties. Each candidate enzyme works best under a specific set of conditions that need to be carefully defined by the R&D laboratory. However, the traditional detection methods used to evaluate enzyme activity have great limitations. With the increasing demand for enzymes in biological / pharmaceutical production, accurate and simplified enzyme activity and enzyme kinetic analysis are needed.
The importance of enzyme analysis
Enzyme determination is a laboratory method to determine enzyme activity, which is very important for the study of enzyme kinetics and enzyme inhibition. Enzymes work by binding to their specific substrates and forming enzyme-substrate complexes. In a particular reaction, the substrate is converted into a product and then released1. Enzyme activity measures the rate of enzyme reaction, which is usually defined by the amount of substrate transformation (or product formation) in a given period of time.
Enzyme kinetics describes the rate of enzyme-catalyzed chemical reactions. The rate of reaction depends on the amount of enzyme used and the amount of substrate. Although the reaction rate is proportional to the concentration of the enzyme, the addition of more substrates will initially linearly increase the reaction rate, but the final reaction will reach the maximum speed. Most enzyme analyses are traditionally based on spectroscopic techniques, and the most commonly used types are absorbance and fluorescence. Because of its low cost and relative simplicity, spectrophotometry is still the most widely used method. This classic enzyme test involves tracking the reaction by measuring changes in the intensity of light absorbed or scattered by the solution. The high-throughput enzyme analysis based on photometry, metrology, 96-well, 384-well or even 1536-well microplate format replaces the traditional spectral technique. However, due to the influence of edge effect, the incubation temperature, temperature stability and precision of these methods are limited, which limits their scope of application.
The challenge of enzyme analysis
Determining enzyme activity is a sophisticated task and may be affected by many variables, such as temperature, pH, buffer type, or ionic strength (Figure 2). Ensuring that all these variables are fixed is essential for achieving repeatable results in enzyme analysis. For example, the accuracy of the results is highly dependent on temperature stability. As long as the temperature changes by one-degree, the enzyme activity will change by 4-8%. Adding samples, buffers and substrates in a specific order and time interval is also essential to obtain accurate and reliable results.
Figure 2. Examples of variables that may affect the measurement of enzyme activity
The main disadvantage of traditional spectral techniques is that all the steps involved are carried out manually, which may lead to inconsistent results. Depending on the type of enzyme, the process of each enzyme may take 30 to 60 minutes from the start of the sample to data collection, which severely limits throughput. These methods are applicable only when analyzing a small number of samples or performing routine tasks. Although the microplate format method provides a convenient alternative method for high-throughput analysis, it has some problems such as temperature stability, absorption correction and edge effect. Because the absorbance is measured vertically through the well on the microporous plate, several factors affect the path length of the liquid, thus affecting the absorbance. Therefore, in the photometric micropore plate measurement, the optical path length needs to be modified to calculate the enzyme activity, and the main reason for the "edge effect" phenomenon usually related to the 96-well plate is attributed to the increase in the evaporation rate in the circumferential hole compared with the central position.
For some enzyme analysis, it is necessary to stop the reaction at a specific time in order to prevent further production of products. For example, samples can be collected every five minutes within a predetermined time and then measured using high performance liquid chromatography (HPLC), and each analysis takes 30 minutes to complete. The development of a reliable method for enzyme analysis involves many steps and usually requires a lot of work (Figure 3). The development of the whole method is laborious and time-consuming, and even trained personnel take four to six weeks. This process begins with the determination of key method variables, which requires testing multiple groups of samples to determine the best experimental conditions. It is clear that a new approach using automated solutions is needed to overcome the key challenges associated with traditional enzyme analysis methods.
Figure 3. Six steps in the development of enzyme analysis methods.
Using discrete Analysis technique to simplify enzyme Analysis
The introduction of innovative dedicated discrete analyzers provides an opportunity to streamline and simplify enzyme analysis. These dedicated discrete analyzers (enzyme analyzers) can effectively analyze many different enzymes and measurement conditions simultaneously in one instrument, providing consistent and reliable performance. All necessary enzyme analysis steps are automated, which helps to control variables and make the whole method development easy and repeatable.
Compared with the traditional enzyme analysis methods, the automatic enzyme analyzer has several advantages. First, these instruments provide the opportunity for fully automated and reliable enzyme analysis of the key steps involved. In order to achieve consistent results, analysis should be carried out under well-defined conditions that can be replicated in other laboratories. The accurate temperature is controlled within±0.3℃, which eliminates the risk of overheating of the sample and provides an accurate incubation time. Excellent temperature control, coupled with no edge effect, can ensure the credibility of the results. Precise liquid treatment (addition and mixing of samples, buffers and substrates) can also improve repeatability between experiments without affecting yield2.
This technology can also increase productivity. In the instrument, samples are prepared and queued at the same time, providing one set of results after another. This makes it faster and easier to evaluate many different enzymes, substrates and methods2. Fully automated workflows also allow for simpler and faster overall method development. Once the testing workflow is established, the instrument can be left unattended, making time for laboratory staff to perform other important tasks. Because of the special software attached to the enzyme analyzer, the workflow is very simple, and there is almost no conversion time from one method to another. Data traceability is tracked by timestamped raw data and audit.
The flexible method parameters for each enzyme type-measurement wavelength, blank measurement, buffer addition, reagent addition, substrate addition, specific enzyme incubation temperature and time, and data collection duration-allow enzyme analysis to be developed and transferred from research and development to the quality control laboratory. Finally, different from spectrophotometry and microplate method, the special enzyme analyzer can provide a wide range of culture temperature from 25℃ to 60℃, which expands the potential application range3.
Conclusion
For many biological/pharmaceutical manufacturers, it is beneficial to use enzymes to catalyze reactions to replace chemicals. Biocatalysis follows the principle of green chemistry and provides an attractive choice for drug synthesis and vaccine development with low cost and sustainable manufacturing. The enzyme analyzer based on discrete analyzer technology provides significant advantages for enzyme research and analysis, including the combination of convenient efficiency with faster, easier and repeatable method development to produce better results. These instruments can help meet the growing demand for automation and digitization while overcoming the challenges associated with traditional enzyme analysis. The use of accurate and simplified enzyme activity and kinetic analysis will pave the way for the wide application of biocatalysis in the development of new drugs, vaccines and diagnostic kits.
References
1. H. Bisswanger, Perspectives in Science 1 (1–6) 41–55 (2014).
2. S.F. Xu, et al., Journal of Food Safety and Quality 8 (11) 4208–4211 (2017).
3. M.E. Peterson, et al., Biochem J. 402 (2) 331–337 (2007).