Laccase, Polyphenol Oxidase, Diphenol Oxidase, Multi-Copper Oxidase, 80498-15-3

Laccase, Polyphenol Oxidase, Diphenol Oxidase, Multi-Copper Oxidase, 80498-15-3

LACCASE (POLYPHENOL OXIDASE)

Laccase / Polyphenol Oxidase / p-Diphenol Oxidase / Multi-Copper Oxidase / EC 1.10.3.2

• CAS Number: 80498-15-3

• EC Number: 420-150-4

• Enzyme Class: EC 1.10.3.2

SECTION 1: PRODUCT DEFINITION AND CHEMICAL IDENTITY

Description:
Laccase is an oxidoreductase enzyme that oxidizes polyphenols and other aromatic compounds. Belonging to the multi-copper oxidase family, laccase contains four copper atoms per enzyme molecule. It removes electrons from its substrate and reduces molecular oxygen (O₂) to water (H₂O). Due to its broad substrate specificity, it can oxidize various compounds including phenols, polyphenols, aromatic amines, carboxylic acids, and steroid hormones. In the food and beverage industry, it is used to improve color, flavor, and stability through polyphenol oxidation in fruit juice, wine, and beer. It is also widely used in textile (denim stone-washing), paper (bio-bleaching), environmental (wastewater treatment), cosmetic (hair dyeing), and biotechnology (biosensor) fields.

SECTION 2: SYNONYMS AND OTHER NAMES

SECTION 3: ENZYMATIC STRUCTURE

3.1. Molecular Structure
Laccase is a globular glycoprotein consisting of three cupredoxin-like domains. It contains four copper atoms (Cu²⁺) per enzyme molecule, classified into three different types: Type I (T1), Type II (T2), and Type III (T3) copper centers. Type I copper serves as the electron acceptor in substrate oxidation. Type II and Type III copper form a trinuclear cluster responsible for molecular oxygen binding and reduction. The enzyme is a glycoprotein with carbohydrate content ranging from 10-80%. Molecular weight varies between 50,000-120,000 Da depending on the source.

3.2. Catalytic Mechanism
Laccase works as a one-electron oxidoreductase:

1. Substrate Oxidation: The substrate (e.g., polyphenol) binds to Type I Cu²⁺ in the enzyme's active site. It is oxidized by donating one electron, forming a substrate radical.

2. Electron Transfer: The electron is transferred from Type I copper to the trinuclear cluster (Type II and Type III copper) via the Cys-His pathway.

3. Oxygen Reduction: Electrons accumulated in the trinuclear cluster are transferred to molecular oxygen (O₂) bound to the active site. Oxygen is reduced to water (H₂O) in two steps (via a hydrogen peroxide intermediate).

3.3. Reaction Scheme
4 Substrate (reduced) + O₂ + 4 H⁺ → 4 Substrate (oxidized radical) + 2 H₂O

3.4. Basic Structural Properties

SECTION 4: PHYSICAL AND CHEMICAL PROPERTIES

SECTION 5: ENZYMATIC ACTIVITY AND REACTION

SECTION 6: REACTION MECHANISM

Laccase catalyzes the oxidation of substrates through a mechanism involving the following steps:

6.1. Substrate Binding
The substrate molecule (phenol, polyphenol, amine, etc.) binds to the Type I (T1) copper center in the enzyme's active site.

6.2. Electron Transfer and Radical Formation
One electron is transferred from the substrate to the Type I copper center. The substrate is oxidized, forming a radical. These radicals can later participate in polymerization or other chemical reactions.

6.3. Electron Transfer
The electron accumulated at Type I copper is transferred to the trinuclear cluster (Type II and Type III copper) via the Cys-His pathway.

6.4. Oxygen Reduction
Four electrons accumulated in the trinuclear cluster are transferred to molecular oxygen (O₂) bound to the active site. Oxygen is reduced in two steps:

• First Step: O₂ + 2 e⁻ + 2 H⁺ → H₂O₂ (hydrogen peroxide intermediate)

• Second Step: H₂O₂ + 2 e⁻ + 2 H⁺ → 2 H₂O

6.5. Overall Reaction
4 Substrate (reduced) + O₂ + 4 H⁺ → 4 Substrate (oxidized radical) + 2 H₂O

SECTION 7: REACTION EQUATIONS

7.1. Laccase Reaction (General)
4 Substrate (reduced) + O₂ + 4 H⁺ → 4 Substrate (oxidized radical) + 2 H₂O

7.2. Polyphenol Oxidation (Example)
4 Polyphenol + O₂ + 4 H⁺ → 4 Polyphenol Radicals + 2 H₂O

7.3. Oxygen Reduction (Intermediates)
O₂ + 2 e⁻ + 2 H⁺ → H₂O₂
H₂O₂ + 2 e⁻ + 2 H⁺ → 2 H₂O

7.4. Enzyme Inactivation (Thermal)
Laccase (active) → Laccase (inactive) (≥60°C, 10-15 minutes)

7.5. Enzyme Inhibition (Cyanide)
Laccase + CN⁻ → Laccase-CN⁻ Complex (inactive)

SECTION 8: LACCASE TYPES

SECTION 9: APPLICATIONS AND INDUSTRIAL USES

9.1. Beverage Industry (Fruit Juice, Wine, Beer)

Example Recipe – Wine Clarification:

• Wine (red/white): 1000 L

• Laccase: 5-50 g (depending on activity, total 5-500 g/ton)

• Silica sol or polyvinylpolypyrrolidone (PVPP) (optional)

• Application temperature: 30-40°C

• pH: 2.5-4.0

Dissolve laccase in a small amount of wine. Add to the wine and incubate at 30-40°C for 1-4 hours. Laccase oxidizes and polymerizes polyphenols. The polymerized phenols are removed by filtration or precipitation. Combination with silica sol or PVPP increases clarification efficiency.

9.2. Food Industry (Baking and Cereal Products)

9.3. Textile Industry (Denim Stone-Washing)

9.4. Paper and Pulp Industry

9.5. Environmental and Wastewater Treatment

9.6. Cosmetic and Personal Care

9.7. Biotechnology and Biosensors

9.8. Other Applications

SECTION 10: ALTERNATIVES AND COMPARISON

10.1. Laccase vs Peroxidase (EC 1.11.1.7)

10.2. Laccase vs Chemical Oxidation (H₂O₂, Chlorine)

10.3. Laccase vs Catalase (EC 1.11.1.6)

SECTION 11: TOXICOLOGY AND SAFETY

11.1. Acute Toxicity

11.2. Regulatory Status

SECTION 12: GHS CLASSIFICATION (Powder Form)

Signal Word: Danger

Hazard Pictograms: GHS07 (Exclamation mark), GHS08 (Health Hazard)

Hazard Statements (H-Codes):

• H334: May cause allergy or asthma symptoms if inhaled

Safety Statements (Historical):

SECTION 13: PRECAUTIONARY STATEMENTS (P-CODES)

SECTION 14: FIRST AID MEASURES

SECTION 15: FIREFIGHTING MEASURES

SECTION 16: ACCIDENTAL RELEASE MEASURES

SECTION 17: STORAGE AND SHELF LIFE

SECTION 18: PACKAGING OPTIONS

SECTION 19: TRANSPORT INFORMATION

SECTION 20: ENVIRONMENTAL INFORMATION

SECTION 21: REGULATORY STATUS

SECTION 22: FREQUENTLY ASKED QUESTIONS (FAQ)

Q1: Is laccase safe for food applications?
A1: Yes. Laccase is classified as GRAS (Generally Recognized As Safe) by the FDA and approved as a food enzyme by EFSA. It is widely used in wine, fruit juice, beer, and bakery products. However, the powder form may cause allergic reactions when inhaled, so appropriate personal protective equipment should be used.

Q2: What is the role of laccase in food and beverages?
A2: Laccase oxidizes polyphenols to provide various functions: clarification and color stabilization in fruit juice and wine, oxygen removal and shelf life extension in beer, dough strengthening and workability improvement in baking. Oxidation of polyphenols enables polymerization, and these polymers are removed by filtration.

Q3: What is the difference between laccase and peroxidase?
A3: Laccase uses molecular oxygen (O₂) as electron acceptor and does not require hydrogen peroxide (H₂O₂). Peroxidase uses H₂O₂ as electron acceptor. Laccase is more sustainable and environmentally friendly because it produces only water as a byproduct. Peroxidase is more expensive and may carry oxidative damage risk due to H₂O₂ use.

Q4: Is laccase suitable for vegan and halal products?
A4: Microbial (fungal) sourced laccases are suitable for vegan and halal certification. Plant-derived laccases are also vegan. Since the product is produced from microorganisms such as fungi or bacteria, it contains no animal ingredients. Relevant certificates can be obtained from manufacturers.

Q5: What is the shelf life of laccase?
A5: In unopened original packaging, stored at 2-8°C (refrigeration), a shelf life of 12-24 months is recommended. For long-term storage, -20°C is recommended. It is recommended to use within a short period after opening.

Q6: How should laccase be stored?
A6: Store in a cool (preferably 2-8°C), dry, well-ventilated area in tightly closed containers. For long-term storage, -20°C is recommended. Moisture, high temperature (>40°C), and direct sunlight cause activity loss. Contact with inhibitors such as cyanide and azide should be avoided.

Q7: What are the optimum working conditions for laccase?
A7: The optimum pH of laccase is between 4.0-6.0 (broad range: 3.0-6.5) and the optimum temperature is between 30-50°C (optimum: 40°C). The enzyme is active in the range of 10-60°C. It is stable in acidic conditions (pH 2.5-4.0). Cyanide (CN⁻) and azide (NaN₃) inhibit the enzyme.

Q8: Why is laccase used in wine production?
A8: In wine production, laccase provides clarification by removing unwanted phenolic compounds through polyphenol oxidation. It also improves wine color and aroma stability and prevents oxidative deterioration. When used in combination with silica sol or PVPP, it is more effective than traditional clarification methods. Recommended dosage: 5-500 g/ton wine.

Q9: How is laccase used in the textile industry?
A9: In the textile industry, laccase bleaches indigo dye on denim fabrics to provide stone-washing effects. This is an environmentally friendly alternative to traditional stone-washing methods. It is also used for bio-bleaching and fabric surface modification on cotton fabrics. Laccase offers a sustainable solution as an alternative to chlorine-based bleaches.

Q10: What are the effects of inhaling laccase powder?
A10: Laccase powder, when inhaled, can cause allergic reactions, asthma-like symptoms, and respiratory tract irritation (H334). Occupational asthma can develop upon repeated exposure to enzyme dust in food, textile, and biotechnology industries. Therefore, a dust mask (P2/P3) must always be used when handling the powder form, and local exhaust ventilation should be provided.

SECTION 23: QUICK REFERENCE TABLE

SECTION 24: CRITICAL WARNINGS AND BEST PRACTICES

CRITICAL WARNINGS:

1. Dust Control and Respiratory Sensitization: Laccase powder can cause occupational asthma upon repeated exposure (H334). Use local exhaust ventilation to prevent dust dispersion and always wear a dust mask (P2/P3). Liquid formulations are safer than powder forms.

2. Oxygen Requirement: Laccase requires molecular oxygen (O₂) as electron acceptor. The enzyme does not work under anaerobic conditions. Ensure sufficient oxygen presence in applications.

3. Activity Loss - Temperature: Laccase is rapidly and irreversibly inactivated above 60°C. Strictly maintain application temperatures within the optimum range (30-50°C).

4. Activity Loss - pH: Laccase loses stability below pH 3.0 or above pH 6.5. Maintain application pH within the optimum range (4.0-6.0). However, it is stable in acidic conditions (pH 2.5-4.0).

5. Activity Loss - Inhibitors: Cyanide (CN⁻), azide (NaN₃), EDTA, and halides (F⁻, Cl⁻) inhibit laccase. Avoid these substances in equipment that comes into contact with the enzyme.

6. Copper Content: Laccase contains four copper atoms per enzyme molecule. Due to the enzyme's metal content, ensure copper levels are within permissible limits in food applications.

7. Food Grade: Only FDA/EFSA-approved food-grade laccase meeting specifications should be used in food applications. Industrial grade enzymes may contain impurities and are not suitable for human consumption.

8. Substrate Specificity: Laccase can oxidize >200 different compounds. Before application, test the enzyme's efficacy on the target substrate.

BEST PRACTICE RECOMMENDATIONS:

• Storage: Store in a cool (2-8°C), dry, well-ventilated area in tightly closed containers. For long-term storage, -20°C is recommended. Use within a short period after opening.

• Handling: Use protective gloves, dust mask (P2/P3), and protective goggles. To prevent dust dispersion, preferably use liquid formulations. Prepare working solutions fresh for each application.

• Oxygen Supply: Provide sufficient aeration or oxygen to meet the enzyme's O₂ requirement. Monitor O₂ levels in closed systems.

• Dosage: Calculate the correct dosage according to enzyme activity (LAMU units). Check enzyme activity for each batch. Start with low dosage and adjust based on results.

• Enzyme Inactivation: Inactivate the enzyme by heat treatment (≥60°C, 10-15 minutes) if necessary. In food products, pasteurization or cooking processes will inactivate the enzyme.

• Quality Control: Perform activity testing for each batch. Maintain production and handling hygiene to prevent microbiological contamination. Monitor oxidation efficiency and clarification effectiveness for consistent product quality.

• Waste Management: Dispose of enzyme and contaminated materials in accordance with local regulations. Do not discharge into sewers. If released into wastewater, inactivate the enzyme by heat treatment before disposal.

LEGAL DISCLAIMER:

This Technical Data Sheet (TDS) is for informational purposes only and is prepared based on available technical data. The user is responsible for determining the suitability of the product for their specific application and for complying with all local, national, and international regulations. For complete safety, storage, handling, transport, waste, and regulatory compliance information, refer to the official Safety Data Sheet (SDS/MSDS) provided by the manufacturer/supplier. Laccase powder may cause sensitization when inhaled; appropriate protective equipment should be used. This document does not replace professional or medical advice.