Principles, Methods, and Applied Practice of Enzyme Activity Assays

Enzyme activity reflects the rate capability by which an enzyme catalyzes the conversion of substrate to product, and is a key parameter for characterizing metabolic state, evaluating pathological changes, describing the intensity of biological processes, and optimizing industrial biocatalytic systems. For different sample types and analytical objectives, enzyme activity is commonly quantified by monitoring substrate consumption, product formation, or changes in optical/electrochemical properties of the reaction system, with reaction-condition control, identification of the linear range, and correction of matrix interference serving as the core safeguards for data reliability. Establishing fit-for-purpose assay methodologies and standardized quality-control workflows helps improve sensitivity, specificity, and result comparability, thereby supporting multi-scenario applications in life science research, clinical testing, industrial process control, and food and environmental monitoring.

Keywords: enzyme activity; kinetics; spectrophotometry; fluorescence; electrochemistry; chromatography; biosensors; quality control

I. Background and Significance

Enzymes are highly efficient and specific catalytic molecules in biological systems. Changes in enzyme activity often precede changes in endpoint concentrations, and can therefore reflect physiological metabolism, signaling regulation, and stress responses with higher sensitivity. In life science research, enzyme activity is used to resolve pathway bottlenecks and regulatory mechanisms; in clinical testing, abnormal activity of specific enzymes can serve as an important clue for disease screening and therapeutic evaluation; in industrial production, enzyme activity is a core metric for fermentation and enzyme-preparation quality control, process optimization, and scale-up verification. Consequently, the choice of enzyme activity assay technology and the quality of implementation directly affect the reliability of scientific conclusions and the validity of process decisions.

II. Core Principles of Enzyme Activity Measurement

The essence of enzyme activity is the ability of an enzyme, under defined conditions, to catalyze the conversion of a specific substrate to product. The core quantitative logic is to monitor, under controllable conditions during the enzymatic reaction: the rate of decrease in substrate concentration, the rate of increase in product concentration, or the rate of change in physicochemical signals associated with reaction progress (absorbance, fluorescence intensity, luminescence intensity, current/potential, etc.), and then convert these rates into enzyme activity.

Common units include:

1. U (International Unit): the amount of enzyme that catalyzes the conversion of 1 μmol of substrate (or formation of 1 μmol of product) in 1 min under specified conditions;
2. kat (katal): the amount of enzyme that catalyzes the conversion of 1 mol of substrate in 1 s.

To obtain comparable and traceable data, measurement should satisfy three key conditions:

1. Reaction conditions are constant (temperature, pH, ionic strength, cofactors, etc.);
2. The reaction is within the initial-rate linear region (rate is approximately proportional to enzyme amount);
3. Bias sources such as substrate depletion, product inhibition, matrix interference, and non-specific reactions are avoided or corrected.

III. Mainstream Enzyme Activity Assay Technologies and Their Characteristics

3.1 Spectroscopic Methods: The Mainstream System for Routine Laboratory Testing

(1) UV–Visible spectrophotometry

The principle is to monitor the time-dependent change in absorbance of the substrate/product at a characteristic wavelength, and to convert reaction rate and enzyme activity using the Beer–Lambert law. A typical example is the dehydrogenase system in which NADH exhibits a characteristic absorption at 340 nm: NADH generation or consumption during the reaction causes absorbance changes that can be used to calculate the initial rate.

1. Advantages: widely available instruments, simple operation, good repeatability, suitable for process monitoring;
2. Limitations: more suitable for systems where substrate or product has a distinct absorption peak; color/turbidity in complex samples can elevate background.

(2) Fluorescence assays

The principle is to use fluorescent substrates/probes that produce changes in fluorescence signals (increase, decrease, or wavelength shift) after enzymatic catalysis, and to characterize enzyme activity via the rate of fluorescence change. This is commonly used for low-abundance enzymes, micro-volume systems, or high-throughput screening.

1. Advantages: high sensitivity; limits of detection are often superior to colorimetric systems;
2. Limitations: inner-filter effects, photobleaching, sample autofluorescence, and fluorescence quenching require evaluation and correction.

(3) Chemiluminescence assays

The principle is that enzyme catalysis triggers chemiluminescent substrate systems to generate light emission, with luminescence intensity correlated with enzyme activity. This is commonly used in scenarios requiring extremely high sensitivity or in trace biomarker systems.

1. Advantages: high sensitivity, low background, no external excitation light required;
2. Limitations: substrate stability and reaction time window strongly affect results; strict timing and consistent mixing are required.

3.2 Electrochemical Methods: An Important Direction for Micro-Volume, Rapid, and Portable Testing

Electrochemical methods quantify enzyme activity by monitoring changes in current, potential, or conductivity caused by the production/consumption of electroactive species generated in enzymatic reactions (e.g., H₂O₂, electron-transfer mediators).

(1) Typical detection modes

1. Amperometry: measure current changes at a fixed potential; commonly used for quantifying redox products;
2. Potentiometry: monitor potential changes; suitable for systems with pronounced ionic/charge changes;
3. Impedance: detect changes in interfacial electrochemical properties; can be combined with immobilized systems.

(2) Method characteristics

1. Advantages: fast response, high sensitivity, easy miniaturization and field deployment;
2. Limitations: electrode materials and surface states must be consistent; ionic strength and pH can strongly affect signals; electrode fouling and drift must be controlled via calibration and QC.

3.3 Chromatographic Methods: A High-Specificity Route for Precise Quantification

Chromatography separates substrates and products and quantifies their concentration changes to calculate enzyme activity. It is especially suitable for situations where structurally similar substrates/products coexist, multi-substrate/multi-product systems, or complex-matrix samples.

(1) Liquid chromatography (HPLC/UPLC)

Can be used to quantify sugars, amino acids, nucleotides, and various small-molecule products. Coupling with UV/fluorescence/mass spectrometric detectors can improve sensitivity and specificity.

(2) Gas chromatography (GC)

Suitable for volatile substrates/products; non-volatile samples usually require derivatization prior to analysis.

1. Advantages: strong separation capability, high specificity, reliable quantification;
2. Limitations: relatively complex workflow, longer turnaround time, higher requirements for instrumentation and method development; more suitable for confirmatory analysis and method validation.

3.4 Biosensor Methods: A Key Form Factor for Real-Time and Engineered Applications

Biosensors couple enzymatic recognition elements with signal transduction units to enable rapid, real-time, or continuous monitoring.

(1) Structural components

1. Recognition element: immobilized enzyme or enzymatic reaction system;
2. Transduction element: electrochemical, optical, piezoelectric, etc.;
3. Amplification and processing: signal amplification, filtering, and algorithmic correction.

(2) Method characteristics

1. Advantages: rapid detection, deployable online/on-site, easy integration with microfluidics and portable devices;
2. Limitations: immobilized enzyme stability, sensor lifetime, and inter-batch consistency are key bottlenecks, requiring systematic stability evaluation and calibration strategies.

IV. Key Influencing Factors and Quality Control

4.1 Control of Reaction-Condition Parameters

1. Temperature: enzyme activity is temperature-dependent; above the optimum temperature, conformational damage may cause inactivation. Use thermostatic devices and record temperature deviations.
2. pH: ionization states at the active site determine catalytic efficiency. Use buffering systems to maintain pH stability, and avoid buffer components that participate in side reactions or interfere with readouts.
3. Substrate concentration: ensure the reaction remains within the initial-rate linear region and is preferably close to substrate saturation; in practice, substrate concentration is often set to ~3–10×Km and the linear region confirmed by preliminary experiments.
4. Ionic strength and cofactors: metal ions, salt concentration, and coenzymes (e.g., NAD(P)H) can markedly affect activity; fix concentrations and include omission controls to verify dependence.

4.2 Sample and Reagent Factors

1. Sample preprocessing: complex samples may contain inhibitors/activators and exhibit turbidity/color interference. Strategies such as centrifugation, filtration, dialysis, desalting, or dilution can be used, and matrix effects evaluated via recovery or standard-addition approaches.
2. Enzyme stability: some enzymes lose activity readily ex vivo; operate at low temperature and minimize freeze–thaw cycles. If needed, add stabilizers (e.g., glycerol, inert proteins) and clarify their effects on assay signals.
3. Reagent purity and batch consistency: impurities in substrates, coenzymes, and buffer components can introduce non-specific reactions or background drift. Establish batch verification and incoming inspection mechanisms.

4.3 Data Processing and Decision Rules

1. Linear region: calculate initial rate using the linear-fitted region of the time–signal curve; avoid late-stage reaction data.
2. Blanks and controls: include at least a reagent blank, a sample blank (no substrate or no reaction initiation), and a positive control; for complex matrices, consider standard addition or spike recovery.
3. Repeatability: set technical replicates and within-batch QC materials; define outlier rules and retest conditions to ensure traceability.

V. Selection Criteria for Reagents and Consumables

5.1 Selection of Enzyme Activity Assay Kits

(1) Methodological compatibility

1. Colorimetric/UV micro-assays: suitable for routine quantification and process monitoring;
2. Fluorescence/luminescence systems: suitable for micro-volume, low-abundance targets, and high-throughput screening;
3. Mechanism-specific systems (e.g., tetrazolium salt color development): suitable for scenarios requiring wider dynamic range or stronger signal amplification.

(2) Compatibility with sample type

Different matrices (cell lysates, tissue homogenates, biofluids, food extracts, environmental samples, etc.) differ substantially. Prefer systems that explicitly specify compatible matrices and provide matrix-correction strategies; when necessary, verify suitability via spike-recovery experiments.

(3) Quality grade and batch management

Prefer high-purity grades and stable formulations suitable for bioactivity analysis; pay attention to batch consistency of critical components and in-shelf-life stability, and perform bridging validation when changing lots.

Product list

Catalog No.	Product Name	Grade and Purity	Assay Type	Scope	Typical Application Direction
A1501205	Pyruvate Kinase (PK) Activity Assay Kit (UV Micro Method)	BioReagent	UV micro-assay	Glycolysis-related enzyme activity evaluation; suitable for research comparisons across treatments	Glycolysis / energy metabolism
A1501274	Acetokinase (ACK) Activity Assay Kit (UV Colorimetric Method)	BioReagent	UV standard assay	ACK activity evaluation; suitable for comparing activity across samples/conditions	Microbial metabolism / metabolic engineering
A1501276	Acetokinase (ACK) Activity Assay Kit (UV Micro Method)	BioReagent	UV micro-assay	ACK activity evaluation; suitable for limited sample amount or multi-sample comparisons	Microbial metabolism / metabolic engineering
A1506759	Acetylcholinesterase (AchE) activity detection kit (DTNB, microcalorimetry)	BioReagent	DTNB, micro-assay	Cholinesterase-related activity evaluation; inhibition-effect studies or pre/post comparisons	Neuroenzymology / inhibitor screening
C1372489	Caspase 3/7 Activity Assay Kit	BioReagent	Activity assay kit (refer to manual)	Caspase-3/7 activity trend evaluation in apoptosis studies; group comparisons	Apoptosis / pharmacological evaluation
C1373312	Caspase 3 Activity Assay Kit	BioReagent; Suitable for Analysis; Colorimetry	Colorimetric	Comparative analysis of Caspase-3 activity; relative quantification and trend assessment	Apoptosis / mechanistic research
C1492383	Caspase 2 Activity Assay Kit	BioReagent	Activity assay kit (refer to manual)	Caspase-2 activity evaluation; comparisons under stress/apoptosis conditions	Stress response / apoptosis
C1492387	Caspase 6 Activity Assay Kit	BioReagent	Activity assay kit (refer to manual)	Caspase-6 activity evaluation; mechanism studies or condition comparisons	Apoptosis / mechanistic research
C1492389	Caspase 9 Activity Assay Kit	BioReagent	Activity assay kit (refer to manual)	Caspase-9 activity trend evaluation; comparisons for mitochondrial pathway	Mitochondrial apoptosis pathway
C1501293	Creatin Kinase (CK) Activity Assay Kit (UV Colorimetric Method)	BioReagent	UV standard assay	CK activity evaluation; suitable for energy metabolism studies or cross-sample comparisons	Energy metabolism / muscle-related research
C1505413	Chloroplast 3-Phosphoglycerate Kinase (chl PGK) Activity Assay Kit (UV Colorimetric Method)	BioReagent	UV colorimetric	Plant/chloroplast enzyme activity evaluation; sample/condition comparisons	Photosynthetic metabolism / plant physiology
C1505414	Chloroplast 3-Phosphoglycerate Kinase (chl PGK) Activity Assay Kit (UV Micro Method)	BioReagent	UV micro-assay	Plant/chloroplast enzyme activity evaluation; suitable for small sample volume or multi-sample comparisons	Photosynthetic metabolism / plant physiology
C1505460	Catalase (CAT) Activity Assay Kit (UV Colorimetric Method)	BioReagent	UV colorimetric	CAT activity evaluation; comparisons across samples in oxidative-stress studies	Oxidative stress / antioxidation
C1505482	Catalase (CAT) Activity Assay Kit (AHM, Colorimetric Method)	BioReagent	Ammonium molybdate, colorimetric	CAT activity evaluation; suitable for visible-light colorimetry comparisons	Oxidative stress / antioxidation
C1505488	Catalase (CAT) Activity Assay Kit (AHM, Micro Method)	BioReagent	Ammonium molybdate, micro-assay	CAT activity evaluation; suitable for limited sample amount or multi-condition comparisons	Oxidative stress / antioxidation
C1505940	Ca²⁺/Mg²⁺-ATPase Activity Assay Kit (AHM, Micro Method)	BioReagent	Ammonium molybdate, micro-assay	Ca²⁺/Mg²⁺-dependent ATPase activity evaluation; condition-based comparisons	Membrane protein function / ion pumps
C1507302	Cellulase (CL) Activity Assay Kit (DNS, Micro Method)	BioReagent	DNS, micro-assay	Cellulase activity evaluation; enzyme preparation comparison or condition optimization	Biomass degradation / industrial enzymology
C1507307	Cellulase (CL) Activity Assay Kit (DNS, Colorimetric Method)	BioReagent	DNS, colorimetric	Cellulase activity evaluation; routine colorimetric comparisons	Biomass degradation / industrial enzymology
D1373349	L-Lactate Dehydrogenase Assay Kit (WST-8)	BioReagent; Suitable for Analysis; Colorimetry	WST-8 method	L-LDH activity evaluation; trend and condition comparisons	Energy metabolism / lactate metabolism
D1505517	D-Lactate Dehydrogenase (D-LDH) Activity Assay Kit (DNPH, Micro Method)	BioReagent	DNPH, micro-assay	D-LDH activity evaluation; microbial metabolism or fermentation-related sample comparisons	Microbial metabolism / fermentation research
F1505396	Fructokinase (FRK) Activity Assay Kit (Micro Method)	BioReagent	Micro-assay	FRK activity evaluation; multi-sample comparisons and condition optimization	Sugar metabolism / fructose metabolism
F1505403	Fructokinase (FRK) Activity Assay Kit (UV Colorimetric Method)	BioReagent	UV colorimetric	FRK activity evaluation; comparisons under UV readout	Sugar metabolism / fructose metabolism
G1501772	α-Amylase (α-AL) Activity Assay Kit (DNS, Micro Method)	BioReagent	DNS, micro-assay	α-Amylase activity evaluation; hydrolytic capacity comparison or condition screening	Carbohydrate hydrolysis / enzyme preparation evaluation
G1501773	Glutathione S-transferase (GST) detection kit (CDNB, microcalorimetry)	BioReagent	CDNB, micro-assay	GST activity evaluation; condition comparisons in detoxification studies	Detox metabolism / pharmacology-toxicology
G1505323	Glucokinase (GK) Activity Assay Kit (Micro Method)	BioReagent	Micro-assay	GK activity evaluation; multi-sample comparisons or treatment-difference analysis	Sugar metabolism / energy metabolism
G1505393	Glucokinase (GK) Activity Assay Kit (UV Colorimetric Method)	BioReagent	UV colorimetric	GK activity evaluation; comparisons under UV readout	Sugar metabolism / energy metabolism
G1505754	Glutathione Peroxidase (GSH-Px) Activity Assay Kit (DTNB, Colorimetric Method)	BioReagent	DTNB, colorimetric	GSH-Px activity evaluation; sample comparisons under oxidative stress	Oxidative stress / antioxidation
G1505763	Glutathione Peroxidase (GSH-Px) Activity Assay Kit (DTNB, Micro Method)	BioReagent	DTNB, micro-assay	GSH-Px activity evaluation; suitable for limited sample amount or multi-sample comparisons	Oxidative stress / antioxidation
G1505936	Glutamic Dehydrogenase (GDH) Activity Assay Kit (UV Micro Method)	BioReagent	UV micro-assay	GDH activity evaluation; comparisons in amino-acid metabolism studies	Amino acid metabolism / nitrogen metabolism
G1505937	Glutamate Synthase (GOGAT) Activity Assay Kit (UV Micro Method)	BioReagent	UV micro-assay	GOGAT activity evaluation; comparisons in nitrogen assimilation research	Plant nitrogen assimilation / nitrogen metabolism
G1506100	Glutathione S-Transferase (GST) Activity Assay Kit (Micro Assay)	BioReagent	Micro-assay	GST activity evaluation; multi-sample screening and condition comparisons	Detox metabolism / pharmacology-toxicology
G1506769	Glutathione Reductases (GR) Activity Assay Kit (UV Micro Method)	BioReagent	UV micro-assay	GR activity evaluation; condition comparisons in antioxidative systems	Oxidative stress / antioxidation
G1506775	Glucose-6-Phosphate Dehydrogenase (G6PDH) Activity Assay Kit (UV Micro Method)	BioReagent	UV micro-assay	G6PDH activity evaluation; comparisons in PPP-related studies	PPP / redox support
H1501177	Hexokinase (HK) Activity Assay Kit (WST-8, Micro Method)	BioReagent	WST-8, micro-assay	HK activity evaluation; multi-sample comparisons and condition screening	Sugar metabolism / energy metabolism
H1501279	Hexokinase (HK) Activity Assay Kit (UV Micro Method)	BioReagent	UV micro-assay	HK activity evaluation; suitable for limited sample amount or multi-condition comparisons	Sugar metabolism / energy metabolism
H1501281	Hexokinase (HK) Activity Assay Kit (UV Colorimetric Method)	BioReagent	UV standard assay	HK activity evaluation; routine spectrophotometric comparisons	Sugar metabolism / energy metabolism
L1501762	Lactate Dehydrogenase (LDH) Cytotoxicity Assay Kit (WST-8, Micro Method)	BioReagent	WST-8, micro-assay	Trend evaluation of cell damage/toxicity (LDH-related); group comparisons	Cytotoxicity / pharmacology
L1501786	Lactate Dehydrogenase (LDH) Cytotoxicity Assay Kit (DNPH, Micro Method)	BioReagent	DNPH, micro-assay	Trend comparison of cell damage across treatments	Cytotoxicity / pharmacology
N1501164	NAD Kinase (NADK) Activity Assay Kit (UV Micro Method)	BioReagent	UV micro-assay	NADK activity evaluation; comparisons in cofactor-metabolism studies	Cofactor metabolism / redox
N1501270	NAD Kinase (NADK) Activity Assay Kit (UV Colorimetric Method)	BioReagent	UV standard assay	NADK activity evaluation; routine enzymology comparisons	Cofactor metabolism / redox
N1505953	Na⁺/K⁺-ATPase Activity Assay Kit (AHM, Micro Method)	BioReagent	Ammonium molybdate, micro-assay	Na⁺/K⁺-ATPase activity evaluation; membrane pump function comparisons	Membrane protein function / ion pumps
P1501170	Phosphofructokinase (PFK) Activity Assay Kit (UV Micro Method)	BioReagent	UV micro-assay	PFK activity evaluation; comparisons in glycolysis research	Glycolysis / energy metabolism
P1501278	Phosphoenol Pyruvate Carboxykinase (PEPCK) Activity Assay Kit (UV Colorimetric Method)	BioReagent	UV standard assay	PEPCK activity evaluation; comparisons in metabolic regulation research	Gluconeogenesis / metabolic regulation
P1501308	Pyruvate Kinase (PK) Activity Assay Kit (UV Colorimetric Method)	BioReagent	UV standard assay	PK activity evaluation; sample/condition comparisons	Glycolysis / energy metabolism
P1501322	Phosphoenol Pyruvate Carboxykinase (PEPCK) Activity Assay Kit (UV Micro Method)	BioReagent	UV micro-assay	PEPCK activity evaluation; suitable for limited sample amount or multi-sample comparisons	Gluconeogenesis / metabolic regulation
P1501774	Plant Polyphenol Oxidase (PPO) Activity Assay Kit (Catechol, Micro Method)	BioReagent	Catechol, micro-assay	PPO activity evaluation; comparisons in browning/stress-related research	Plant physiology / quality & stress tolerance
P1501935	Pyruvate Phosphate Dikinase (PPDK) Activity Assay Kit (UV Micro Method)	BioReagent	UV micro-assay	PPDK activity evaluation; trend comparisons in plant metabolism	Photosynthetic metabolism / metabolic regulation
P1501938	Pyruvate Phosphate Dikinase (PPDK) Activity Assay Kit (UV Colorimetric Method)	BioReagent	UV standard assay	PPDK activity evaluation; routine spectrophotometric comparisons	Photosynthetic metabolism / metabolic regulation
P1505406	3-Phosphoglycerate Kinase (PGK) Activity Assay Kit (UV Micro Method)	BioReagent	UV micro-assay	PGK activity evaluation; comparisons in metabolic pathway research	Sugar metabolism / energy metabolism
P1505407	3-Phosphoglycerate Kinase (PGK) Activity Assay Kit (UV Colorimetric Method)	BioReagent	UV colorimetric	PGK activity evaluation; comparisons under UV readout	Sugar metabolism / energy metabolism
P1505848	Peroxidase (POD) Activity Assay Kit (Guaiacol, Micro Method)	BioReagent	Guaiacol, micro-assay	POD activity evaluation; trend comparisons in oxidative stress/plant studies	Oxidative stress / plant stress tolerance
P1505878	Plant Root Activity Assay Kit (TTC, Colorimetric Method)	BioReagent	TTC, colorimetric	Root vigor trend evaluation; pre/post or cross-sample comparisons	Plant physiology / root phenotyping
S1505477	Soil Catalase (S-CAT) Activity Assay Kit (UV Micro Method)	BioReagent	UV micro-assay	Soil/environmental enzyme activity evaluation; soil-to-soil or treatment comparisons	Environmental / soil enzymology
T1505644	Total Superoxide Dismutase (T-SOD) Activity Assay Kit (WST-8, Micro Method)	BioReagent	WST-8, micro-assay	Total SOD activity evaluation; trend comparisons in oxidative stress studies	Oxidative stress / antioxidation

5.2 Substrate and Probe Selection

(1) Peptide substrates and reporter groups (e.g., AMC/AFC/pNA)

1. Specificity: substrate sequences should match the recognition sites of the target protease to avoid cross-reactivity that elevates “apparent activity”;
2. Purity and background: high-purity substrates reduce background from impurity peptides; select fluorescent or colorimetric reporter groups according to the instrument platform and evaluate inner-filter effects and baseline absorbance.

Product list

Catalog No.	Product Name	Grade and Purity	Signal Type	Scope	Typical Application Direction	Use Notes
A1456330	Ala-Ala-Phe-AMC, TFA salt	BioReagent; ≥98%	Fluorescence (AMC)	Protease/peptidase substrate screening and activity evaluation; condition comparisons; inhibition observation	Protease activity / substrate screening	TFA salt; suitable for fluorescence readers
A1456348	Ac-LEHD-AMC, TFA salt	BioReagent; ≥98%	Fluorescence (AMC)	Caspase-related activity evaluation; trend comparisons in apoptosis/inflammation studies	Apoptosis (caspase-related) / inhibitor studies	TFA salt; suitable for fluorescence readers
A1456354	Ac-WEHD-AMC, TFA salt	BioReagent; ≥98%	Fluorescence (AMC)	Caspase-related activity evaluation; sample comparisons or inhibitor prescreening	Inflammation (caspase-related) / inhibitor screening	TFA salt; suitable for fluorescence readers
A769903	Ac-DEVD-AFC	BioReagent; ≥98%	Fluorescence (AFC)	Caspase-related activity evaluation; treatment-condition comparisons	Apoptosis (Caspase-3/7-related)	Suitable for fluorescence readers
C743619	Caspase 2 substrate (chromogenic)	BioReagent; ≥98%(HPLC)	Colorimetric (pNA)	Caspase-related activity evaluation; trend analysis under visible-light readout	Apoptosis / stress (Caspase-2-related)	Chromogenic substrate; suitable for spectrophotometers/plate readers
G1456329	Gly-Phe-AMC, TFA salt	BioReagent; ≥98%	Fluorescence (AMC)	Protease/peptidase substrate screening and activity evaluation; comparisons across systems/conditions	Protease activity / substrate screening	TFA salt; suitable for fluorescence readers
G1456346	Gly-Phe-AFC，TFA salt	≥98%	Fluorescence (AFC)	Protease/peptidase substrate screening and activity evaluation; enzymology and inhibition observation	Protease activity / substrate screening	TFA salt; suitable for fluorescence readers
Z647630	Z-DEVD-AFC	BioReagent; ≥98%	Fluorescence (AFC)	Caspase-related activity evaluation; trend comparisons in apoptosis studies	Apoptosis (Caspase-3/7-related) / inhibitor screening	Suitable for fluorescence readers
Z658857	Z-DEVD-AMC	BioReagent; ≥98%	Fluorescence (AMC)	Caspase-related activity evaluation; sample comparisons and condition screening	Apoptosis (Caspase-3/7-related)	Suitable for fluorescence readers

(2) Non-peptide substrates/probes

Suitable for glycosidases, oxidases, and related enzyme systems. Key points to evaluate include:

1. probe stability;
2. interference from reaction by-products (including non-specific side reactions);
3. the linearity between product signal output and reaction rate (within the initial-rate window).

Product list

Catalog No.	Product Name	Grade and Purity	Scope	Typical Application Direction
D275740	D-Luciferin-6-O-β-D-galactopyranoside	≥98%	Substrate conversion/enzyme activity evaluation related to β-galactosidase; applicable to reporter systems or trend comparisons in assay optimization	Reporter genes / enzyme activity / substrate screening

5.3 Auxiliary Enzymes and Buffer Systems

(1) Auxiliary enzymes / recombinant proteins

Auxiliary enzymes used for coupled reactions, signal amplification, or system calibration should be evaluated for: specific activity, contaminating enzyme activities, potential effects of tag types on activity, and storage and repeated freeze–thaw stability.

Product list

Catalog No.	Product Name	Grade and Purity	Scope	Typical Application Direction	Key Use Points
A156186	Recombinant Taq DNA Polymerase Protein	EnzymoPure™; His-Tag; ≥95%; See COA	Enzymology applications in DNA amplification-related reactions; routine amplification and condition optimization	Molecular biology amplification / method development and optimization	Reaction system and cycling conditions can be optimized by template complexity and target length; refer to COA/manual
H1508159	Horseradish Peroxidase (HRP)	Bioactive; ActiBioPure™; Native; High Performance; EnzymoPure™; ≥300U/mg enzyme powder， Rz≥3; from Horseradish	Peroxidase-catalyzed chromogenic/luminescent systems; signal amplification and labeling assays	Immunoassays / protein labeling and chromogenic systems	Applicable to common HRP substrate systems; tune substrate and reaction time by required signal
R1507818	Horseradish Peroxidase (HRP)	Bioactive; Recombinant; ActiBioPure™; High Performance; EnzymoPure™; ≥250U/mg enzyme powder， Rz ≥3; expressed in Nicotiana benthamiana	Peroxidase-catalyzed chromogenic/luminescent systems; detection/labeling applications	Immunoassays / protein labeling and chromogenic systems	Optimize dilution and reaction conditions to match the target system; consult manual and assay conditions
R1507819	Horseradish Peroxidase (HRP)	Bioactive; Recombinant; ActiBioPure™; High Performance; EnzymoPure™; ≥150U/mg enzyme powder， Rz ≥2; expressed in Nicotiana benthamiana	Peroxidase-catalyzed chromogenic/luminescent systems; routine detection and method development	Immunoassays / protein labeling and chromogenic systems	Suitable for common HRP substrate systems; adjust enzyme amount/substrate concentration to achieve an appropriate SNR
rp176272	Recombinant Sumo Protease Protein	Carrier Free; Bioactive; ActiBioPure™; Azide Free; High Performance; His-Tag; ≥90%(SDS-PAGE)	Enzymology applications for fusion-protein tag removal/cleavage; applicable to purification workflows	Protein expression & purification / tag removal / process development	Optimize cleavage ratio, temperature, and time by substrate protein and tag construct; azide-free for azide-sensitive systems

(2) Buffers and supporting solutions

1. pH compatibility: match the optimal pH of the target enzyme;
2. System compatibility: electrochemical assays should avoid components that affect electrode signals; high-sensitivity fluorescence systems should consider fluorescent impurities;
3. Purity and sterility: select buffer systems with low impurities and low endogenous enzyme contamination; use sterile filtration and cold storage when necessary.

Product list

Catalog No.	Product Name	Grade and Purity	Scope	Typical Application Direction	Key Use Points
T476217	TNT Buffer (10×)	sterile-filtered; BioReagent; Suitable for molecular biology; 10×	Preparation and maintenance of common buffer systems in molecular biology experiments; applicable to routine nucleic acid/protein workflows (subject to protocol)	General molecular biology buffers / workflow support	10× concentrate, typically diluted to working concentration; sterile filtration is suitable for contamination-sensitive workflows; adjust per manual and assay system

VI. Applied Practice and Summary

6.1 Life Science Research

• Used to analyze metabolic pathways and signal transduction. For example, kinase/phosphatase activity reflects pathway activation status, and nuclease activity is used to evaluate nucleic acid processing and gene-editing system performance; in drug screening, high-throughput fluorescence/luminescence systems can rapidly assess enzyme inhibition or activation effects.

6.2 Clinical Testing

• Activities of many serum enzymes correlate with tissue injury and can be used for auxiliary diagnosis, disease-course monitoring, and prognosis evaluation. Clinical scenarios emphasize methodological standardization, establishment of reference intervals, inter-batch consistency, and completeness of QC rules.

6.3 Industrial Production and Process Control

• In fermentation, enzyme preparation manufacturing, and food processing, enzyme activity is used to monitor key process nodes, evaluate batch quality, and optimize scale-up parameters; online or nearline electrochemical/sensor systems can improve process response speed and reduce delays from sampling and off-site testing.

6.4 Food and Environmental Monitoring

• Enzyme activity in foods can reflect processing adequacy and quality-change trends; in environmental samples, specific enzyme activities can serve as sensitive indicators of ecological process intensity and pollution stress. However, matrix-interference correction and recovery verification must be strengthened to ensure cross-region and cross-time comparability.
• Enzyme activity assays have formed a multi-technology spectrum, with spectroscopic methods as the routine backbone, electrochemistry and biosensors supporting rapid and field-deployable testing, and chromatography supporting high-specificity confirmatory quantification. Regardless of the chosen route, the key to data reliability lies in: control of reaction conditions and the linear region, evaluation and correction of matrix interference, and an executable QC system (blanks, controls, recovery, and within-/between-batch QC). Looking forward, enzyme activity assays will further develop toward high throughput, miniaturization, automation, and intelligence, and—with coordinated optimization of standardized methodologies and supporting reagents/consumables—will improve cross-platform and cross-scenario consistency, expanding application depth and breadth in point-of-care clinical testing, online industrial monitoring, and rapid environmental assessment.

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