Horseradish Peroxidase (HRP, EC 1.11.1.7) is a heme-containing glycoprotein derived from the roots of horseradish (Armoracia rusticana). As a classical biocatalytic tool enzyme, HRP is widely utilized in medical diagnostics, molecular biology, and environmental remediation due to its high catalytic activity, robust stability, and excellent labeling compatibility. This article provides a systematic overview of HRP's core characteristics, manufacturing processes, and application scenarios for life science professionals.

Part I: Core Characteristics of HRP

1.1 Structure and Catalytic Mechanism

HRP is a heme-containing glycoprotein with a molecular weight of approximately 40 kDa, comprising 308 amino acid residues and 8 N-glycosylation sites (carbohydrate content: 18-22%). The prosthetic group consists of heme and two Ca²⁺ ions, with heme serving as the catalytic core. Its primary function is to catalyze hydrogen peroxide-mediated oxidation of hydrogen donors, following the reaction: Donor + H₂O₂ → Oxidized donor + 2H₂O. The enzyme remains unconsumed during catalysis, exhibiting high efficiency and strong substrate specificity.

1.2 Key Characteristics and Purity Indicators

Core characteristics of HRP include: stability at room temperature for several weeks; retention of activity at 63°C for 15 minutes; superior specific enzyme activity; and specific recognition of H₂O₂ and particular hydrogen donors.

HRP exhibits maximum absorbance at 403 nm (Soret band) and 275 nm (protein band). The ratio of absorbance at 403 nm to 275 nm (A₄₀₃/A₂₇₅) serves as the critical purity indicator. This methodology was developed by German scientist Reinheit Zahl, and the ratio is commonly referred to as the RZ value (Reinheit Zahl). HRP purity grades are classified as follows:

• RZ > 3.0: High-purity grade for immunological assays
• RZ > 2.0: Clinical chemistry grade
• RZ > 1.0: General diagnostic test strips
• RZ > 0.6: Low-precision applications

HRP contains at least seven isoenzymes, with isoenzyme C1A (UniProt: P00433) being the most abundant, exhibiting optimal activity, and most widely applied.

Tinzyme HRP demonstrates 100% isoenzyme C1A content as verified by liquid chromatography analysis.

1.3 Product Forms and Storage Requirements

Commercial HRP is available in two primary formats: lyophilized powder and liquid enzyme preparation. Lyophilized powder offers extended shelf life and convenient transportation, requiring reconstitution in appropriate buffer prior to use. Liquid formulations are ready-to-use and require storage at 2-8°C. All HRP products must be protected from elevated temperatures and inhibitors such as cyanides and fluorides to prevent activity loss.

Part II: HRP Manufacturing Technologies

The primary objective of HRP production is to obtain high-purity, high-activity enzyme preparations. Current mainstream production methodologies are divided into natural extraction and heterologous recombinant expression, which complement each other to meet diverse manufacturing requirements.

2.1 Natural Extraction Method

The extraction process utilizes fresh horseradish roots as raw material, proceeding through washing and homogenization, low-temperature buffer extraction, ammonium sulfate/acetone fractional precipitation, dialysis desalting, zinc ion purification, and lyophilization to obtain finished HRP product. This method offers readily available raw materials, straightforward processing, and lower costs, suitable for small-scale production. However, it presents limitations including restricted yield, inconsistent purity, and susceptibility to agricultural cultivation conditions.

2.2 Recombinant Expression and Advanced Technologies

Heterologous recombinant expression represents the mainstream approach for large-scale production. The core workflow comprises: HRP gene cloning and optimization → host cell transformation (E. coli, Pichia pastoris, etc.) → fermentation culture → chromatographic purification → activity assessment. This methodology delivers high yield, consistent purity, and enables enzyme activity enhancement through genetic optimization. The primary limitations involve complex processing and higher initial capital investment.

Recombinant plant expression represents an advanced HRP production technology. This approach involves cloning the HRP gene into appropriate vectors and introducing the construct into plant tissues (e.g., leaves), enabling expression during plant growth. The core workflow: HRP gene cloning and optimization → gene vector construction → plant tissue transformation (e.g., leaves) → hydroponic cultivation → leaf harvest → tissue homogenization → chromatographic purification → activity assessment.

This method offers significant advantages including high yield, superior activity, exceptional purity, and scalability for expanded production.

Cell-free protein synthesis (CFPS) systems are increasingly adopted, achieving synchronous synthesis of HRP and prosthetic groups through in vitro modular assembly, further enhancing activity and yield while reducing production costs.

2.3 Quality Control Standards

Critical quality parameters for finished products include RZ value (purity), specific enzyme activity (catalytic efficiency), molecular weight, and contaminant protein content. Primary analytical methods encompass spectrophotometry (for RZ value and enzyme activity determination) and electrophoresis (for purity and molecular weight verification). Only products meeting established specifications are approved for commercial distribution.

Comparative testing demonstrates that Tinzyme HRP delivers superior performance with minimal batch-to-batch variation.

Part III: Comprehensive Application Areas

3.1 Medical and Clinical Diagnostics

HRP finds its most extensive applications in medical diagnostics, with core applications including:

Enzyme-Linked Immunosorbent Assay (ELISA): HRP-conjugated antibodies combined with TMB, OPD, or other chromogenic substrates enable quantitative detection of antigens/antibodies for infectious disease and tumor marker screening.

Immunohistochemistry (IHC) and Immunofluorescence (IF): HRP-labeled antibodies facilitate localization of target proteins in tissue sections, supporting pathological diagnosis.

Western Blot: HRP-conjugated secondary antibodies enable detection of target protein expression levels through chemiluminescence (luminol substrates) or chromogenic development.

Additionally, HRP serves as a superior alternative to colloidal gold in home diagnostic test strips, significantly enhancing detection sensitivity.

3.2 Research Applications

In molecular biology, HRP functions as a reporter enzyme for gene detection, nucleic acid hybridization, and gene cloning/expression analysis. In biochemistry, HRP serves as a model enzyme for investigating oxidation-reduction reaction mechanisms, providing foundational insights for novel enzyme development.

3.3 Environmental and Industrial Applications

Environmental remediation applications leverage HRP to catalyze degradation of phenolic and aromatic contaminants in industrial wastewater and soil. In food industry applications, HRP enables hydrogen peroxide residue detection and polyphenol analysis for food safety assurance. HRP is additionally employed in polymer synthesis and biosensor fabrication.

3.4 Application Guidelines

Appropriate substrate selection is essential for specific applications: TMB and DAB for routine chromogenic detection; luminol for high-sensitivity detection; Amplex Red for fluorescent detection. Contact with inhibitors including cyanides, fluorides, and azides must be avoided to prevent enzyme inactivation.

Part IV: Future Perspectives

As a classical biocatalytic tool enzyme, HRP maintains its prominent position after decades of widespread application. Future developments will focus on optimization of heterologous recombinant technologies and cell-free synthesis systems to reduce production costs and enable large-scale, high-quality manufacturing. Genetic engineering approaches will further optimize HRP structure and performance, expanding applications in precision medicine and next-generation biosensors, thereby sustaining its core value in biotechnology.

Tinzyme HRP Advantages:

• Manufactured through recombinant expression technology with 100% isoenzyme C1A content, delivering specific catalytic activity ideal for high-sensitivity diagnostic reagents (e.g., ELISA) and biosensor applications

• Produced via plant expression systems, completely free from exogenous microorganisms, viral contaminants, and animal-derived components, eliminating pathogenic risk and ensuring full compliance with FDA/EMA stringent regulatory requirements for in vitro diagnostic reagents

• Engineered through directed evolution to maintain activity across broad pH range (3-10) and elevated temperatures (≤60°C), accommodating demanding reaction conditions

Think of it as a complete toolkit for "extracting and reading human genetic fingerprints." Below is an overview of its core principles, components, and primary applications.

1. Core Principle: What is STR?

STR (Short Tandem Repeats) refers to short DNA sequences (typically 2–6 base pairs) that repeat at specific locations within the human genome.

• Repetitive Nature: These sequences connect end-to-end, much like train carriages.
• Polymorphism: The number of repeats at the same genetic locus usually varies between individuals. For example, you might have 10 repeats at a specific location, while another person has 15.
• Uniqueness: By simultaneously examining dozens of these loci, the resulting combination is virtually unique worldwide (except for identical twins).

2. What's Inside the Kit?

A standard STR Detection Kit typically contains the following key components:

3. Workflow

Step 1: DNA Extraction
DNA is extracted from biological samples such as blood, saliva, hair follicles, or semen stains.

Step 2: PCR Amplification
Using the kit on a thermal cycler, specific STR loci are amplified millions of times while fluorescent tags are incorporated into the fragments.

Step 3: Capillary Electrophoresis
Amplified products are loaded into a genetic analyzer, where fragments are separated based on length (i.e., repeat number).

Step 4: Data Analysis
Software captures fluorescent signals and generates peak profiles, ultimately producing numerical values for each locus (e.g., 12, 14)—this is the individual's STR profile.

4. Key Applications

Summary

The STR Detection Kit represents the gold standard in modern identity verification. It offers exceptional sensitivity (detectable from minimal sample amounts), high discriminatory power, and results that are easily standardized and compatible with database comparisons. Current mainstream kits can simultaneously analyze 20+ loci, delivering outstanding accuracy for reliable genetic identification.

1. Freeze/Thaw Cycle Test

This test evaluates the stability of dNTPs when subjected to repeated freezing and thawing, simulating frequent laboratory use.

2. Thermal Stability Test

This test measures the degradation of dNTPs when stored at room temperature (25℃) and elevated temperature (37℃) for 10 days.

3. Long-Term Stability Test

Data represents the purity maintained over a 5-year period under standard storage conditions.

Our dNTPs undergo rigorous performance testing to ensure they meet the highest laboratory standards:

Why Choose Mebep dNTPs?

• Exceptional Sensitivity in Long PCR: Our dNTPs excel in amplifying large fragments (up to 30Kb), maintaining high yields even at low template concentrations.
• Guaranteed Purity: Rigorous 16S rRNA testing ensures our products are free from bacterial DNA contamination, providing a clean background for your most sensitive assays.
• Precision in Low Copy Assays: Optimized for high-efficiency amplification of human genomic DNA, even when starting with template quantities as low as 0.01ng.
• Versatility for RT-PCR: Highly effective in Reverse Transcription PCR workflows, enabling the detection of specific markers like beta-actin from minimal total RNA input.

Technical Performance Data

Below are the results from our standard quality control assays, demonstrating the superior performance of Mebep dNTPs compared to industry competitors.

• Ladder Marker
  • DNA Ladders DNA Markers
  • Protein Markers
  • RNA Markers

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Stability at 4°C

Stability at 25°C

Stability at 37°C

Date: Mar 04, 2024

Storage Temperature: 25°C

Date: May 10, 2022

Mebep: DNK46 FastBeat Water DNA Kit (Bead Beating)

Step 1. Place the filter membrane

Step 2. Pour in water sample and start filtration

Step 3. Filtration completed

Step 4. Remove the filter membrane

Step 5. Roll the filter membrane inward and insert into a 5ml grinding tube

Step 6. Add grinding beads to the center of the grinding tube

Step 7. Proceed with subsequent grinding and extraction operations according to the DNK46 FastBeat Water DNA Kit (Bead Beating) protocol (as follows)

1. Add 980 μl Sodium Phosphate Buffer to sample in Bead Tube. Gentle vortex to mix. Add 120 μl MT Buffer.
   Note: Check MT Buffer. If MT Buffer is precipitated, heat solution to 60°C until dissolved before use.
2. Homogenize in the FastPrep® Instrument for 40 seconds at a speed setting of 6.0.
3. Centrifuge at 12,000 x g for 5minutes to pellet debris.
4. Transfer supernatant to a clean 2.0 ml centrifuge tube. Add 250μl PPS Solution and mix by shaking the tube by hand 10 times. Incubate at 4°C for 5 minutes.
5. Centrifuge tubes at 10,000 x g for 3 minute at room temperature. Avoiding pellet, transfer up to, but no more than, 900 μl of supernatant to a clean 2 ml centrifuge tube.
6. Add 300 μl of IRS Solution(1/3 volume) and vortex briefly. Incubate at 4°C for 5 minutes.
7. Centrifuge tubes at 10,000 x g for 1 minute at room temperature. Avoiding pellet, transfer the supernatant into a clean 5 ml centrifuge tube.
8. Add 1.5 volumes of PQ Solution to the cleared supernatant and mix by pipetting.
   Example: To 1100 μl lysate add 1650 μl PQ Solution. Reduce the amount of PQ Solution accordingly if less supernatant is recovered. A precipitate may form after the addition of ethanol but this will not affect the procedure.
   Note: Ensure ethanol has been added to PQ Solution.
   Note: It is important to pipet PQ Solution directly onto the cleared supernatant and to mix immediately.
9. Load approximately 700 μl mixture onto Spin Filter(sitting in collection tube) and centrifuge at 10,000 x g for 1 minute at room temperature. Discard flow through. Load another 700 μl and repeat until all remaining mixture is loaded on Spin Filter.
   Note: A total of 4-5 loads for each sample processed may be required.
10. Add 600 μl of Buffer WB to Spin Filter and centrifuge at 10,000 x g for 30 seconds at room temperature. Discard flow through. Repeat Step 10 with another 600 µl Buffer WB.
    Note: Ensure ethanol is added to Buffer WB.
11. Centrifuge Spin Filter at 13,000 x g for 2 minute at room temperature to dry the Spin Filter..
12. Carefully place Spin Filter in clean 1.5 ml centrifuge tube. Avoid splashing any Buffer WB onto Spin Filter. Add 100 µl of Elution Buffer (Optional: pre-warm the water to 70–90℃ will increase the DNA yield) to the center of the column membrane. Incubate at room temperature for 3-5 min, and centrifuge at 13,000 x g for 1 min to elute the DNA.
    Note: Use smaller volume(minimum 30µl) of Elution Buffer will obtain higher concentration.
    Optional: Put eluate back to the Spin Column to repeat elution once. This increases concentration of DNA about 10-15%

Product Code: PK01

We hereby declare that our Proteinase K (Product No. PK01) is manufactured under strict quality control standards and meets the following criteria:

• Animal-Free Composition: The product does not contain any ingredients derived from animal sources, nor does it contain any components contaminated by animal-derived substances.
• TSE/BSE Compliance: This product has not been exposed to animals affected by, or under quarantine for, Transmissible Spongiform Encephalopathy (TSE) or Bovine Spongiform Encephalopathy (BSE).
• Research Use Only: This product is designed exclusively for scientific research and laboratory experiments, specifically for protein degradation.

Disclaimer: This product is not intended for human or veterinary use, nor is it for use in food, cosmetics, or medicinal products. It is strictly for in vitro scientific research purposes.