Immobilised Enzymes

Enhancing Efficiency in Wastewater Treatment

Introduction

As global water reuse and pollution control become critical environmental challenges, the wastewater sector is seeking sustainable, efficient solutions to treat complex organic pollutants. Traditional methods—like chemical oxidation, activated sludge, and advanced oxidation processes—though effective, can be energy-intensive, costly, and generate secondary pollutants or excessive sludge.

A promising alternative is the use of oxidoreductase enzymes, particularly when immobilised on solid supports to enhance their activity, stability, and reusability. These biocatalysts can achieve more than 90 % removal of dyes, phenols, pharmaceuticals, bisphenols, endocrine disruptors, and other organic contaminants, while operating under milder conditions and generating less waste (e.g., sludge). This article examines how immobilisation transforms oxidoreductases into robust tools for modern wastewater treatment, exploring methods, case studies, challenges, and future trends.

What are Oxidoreductases—and Why Immobilise Them?

Oxidoreductases are a class of enzymes that drive oxidation–reduction reactions, transferring electrons from one molecule to another. Common examples in wastewater treatment include:

• Laccases (multi‑copper oxidases)
• Peroxidases (e.g., horseradish peroxidase, manganese peroxidase)
• Tyrosinases

These enzymes efficiently degrade aromatic and recalcitrant pollutants—such as phenolic compounds and dyes—into less toxic molecules or polymers that are easier to remove biologically (link.springer.com, pmc.ncbi.nlm.nih.gov).

However, free enzymes in solution often suffer from:

1. Low stability under variable pH, high temperature, heavy metals, and organic solvents
2. Difficulty in recovery and reuse due to wash-out
3. Dependence on expensive cofactors (e.g., NADH, heme)

Immobilisation—anchoring enzymes onto supports or cross-linking them—addresses these issues. It enhances:

• Stability against denaturation and inhibitors
• Recovery and reusability
• Compatibility with continuous-flow systems like fixed-bed reactors (ncbi.nlm.nih.gov, pmc.ncbi.nlm.nih.gov)

How Immobilisation is Achieved

Three main strategies are used to immobilise oxidoreductases:

1. Support-based immobilisation

Enzymes are bound—via covalent bonds, adsorption, ionic interactions, or entrapment—to carriers such as:

• Mesoporous silica
• Activated carbon
• Polymeric gels (e.g., alginate, chitosan, PEG)
• Membranes
• Metal–Organic Frameworks (MOFs) (ncbi.nlm.nih.gov, en.wikipedia.org, mdpi.com)

2. Entrapment/Encapsulation

Enzymes are physically enclosed within hydrogels or matrixes (e.g., cellulose, alginate), allowing substrates and products to diffuse while retaining the enzyme .

3. Carrier-free approaches

This involves cross-linking enzyme molecules directly—forming Cross-Linked Enzyme Aggregates (CLEAs) or Cross-Linked Enzyme Crystals (CLECs)—eliminating the need for carriers (en.wikipedia.org). This maximises catalytic concentration and simplifies recovery.

Biological and Engineering Benefits of Immobilisation

1. Enhanced Catalytic Efficiency

Immobilised enzymes often maintain > 90 % pollutant removal under optimised conditions (link.springer.com, link.springer.com).
Examples include:

• Dye decolourisation: Laccase nanotube-based reactors delivered 74–96 % removal with 90 % retained activity after 10 cycles (pmc.ncbi.nlm.nih.gov).
• Bisphenol A degradation: Cross-linked laccase reported > 99 % BPA removal (pmc.ncbi.nlm.nih.gov).

2. Greater Stability and Reusability

Compared with soluble enzymes, immobilised variants resist temperature, pH fluctuations, heavy metal poisoning, and keep activity across multiple cycles:

• Laccase immobilised on silica beads retained 65 % activity after 14 days, versus 5 % for free enzyme (link.springer.com).
• Magnetic laccase CLEAs sustained activity across 112 reuses (link.springer.com).
• Bead-immobilised laccase retained 94 % activity over 28 cycles; 35th cycle saw only 6 % loss (frontiersin.org).

3. Improved Process Integration

Immobilised enzymes can be incorporated into packed-bed, fluidised-bed, membrane, or nanoparticle‑suspension reactors, enabling continuous flow, minimised washout, and easier recovery (pmc.ncbi.nlm.nih.gov).

4. Lower Operating Costs and Environmental Impact

Optimised immobilisation enhances enzyme lifetime—driving down costs. As catalysts, enzymes work at ambient pressure and temperature, avoiding toxic chemicals and secondary pollution (pmc.ncbi.nlm.nih.gov). CLEAs reduce reliance on carrier materials and simplify separation.

Case Studies in Detail

1. Review by Toor & Nghiem (2021)

A comprehensive review revealed immobilised laccase, tyrosinase, and peroxidase systems consistently achieved > 90 % removal of dyes, phenols, pharmaceuticals, estrogens, and bisphenols in lab settings. They highlighted superior stability and recyclability of immobilised enzymes over free variants (link.springer.com).

2. Laccase-Embedded Membranes (Zdarta et al.)

Immobilised on cellulose‑based membranes, laccase displayed:

•  80 % immobilisation yield

•  90 % initial activity recovery

•  75 % activity retention after 10 treatment cycles and 20 days’ storage

• Removal rates: > 50 % for 2,4‑D and tetracycline, > 40 % for 17α‑ethynylestradiol and ketoprofen methyl ester (pubmed.ncbi.nlm.nih.gov, mdpi.com).

3. Nanotube-Based Dye Reactors

Lactube reactors featuring cross-linked laccase nanotubes achieved 74–96 % dye decolourisation, maintaining 90 % efficiency across 10 batches (pmc.ncbi.nlm.nih.gov).

4. Bead vs Silica-Graphene Composite Laccase

Floating beads and silica-graphene composites immobilised laccase achieved > 95 % removal of estrogens (E2, E1, EE2) over > 20–30 cycles. Beads retained 94 % activity over 28 cycles, losing only 6 % by cycle 35 (frontiersin.org).

5. MOF‑Supported Oxidoreductases

Metal–Organic Frameworks (MOFs) have emerged as robust enzyme supports:

• Laccase immobilised on Fe₃O₄-NH₂@MIL-101(Cr) removed 87 % of 2,4‑D in 12 h and remained recyclable via magnetic separation (pmc.ncbi.nlm.nih.gov).
• HRP encapsulated in Zr‑MOF retained 70 % activity after 12 cycles and removed 2,4‑D plus isoproturon (pmc.ncbi.nlm.nih.gov).

Engineering and Process Considerations

1. Support/Method Selection

Supports must balance:

• High enzyme loading
• Good mass transfer
• Mechanical and chemical stability
• Minimal diffusion limitation

Carrier-free CLEAs offer dense enzyme matrices but may form large aggregates. MOFs and membranes offer tunable porosity and favourable transfer dynamics (pmc.ncbi.nlm.nih.gov, mdpi.com).

2. Balancing Enzyme Activity and Stability

Immobilisation can sometimes reduce substrate affinity or active-site accessibility. Reactor design must consider diffusion, pore size, cross-linking density, and microenvironment optimisation .

3. Operational Format

Options include:

• Batch reactors
• Packed-bed columns
• Membrane bioreactors
• Fluidised carriers or nanoparticle suspensions

Each design involves trade-offs in residence time, fouling potential, and recovery efficiency (link.springer.com, pmc.ncbi.nlm.nih.gov).

4. Cofactor Regeneration Strategies

Many oxidoreductases need cofactors. Immobilisation enables co‑immobilisation in enzyme cascades (e.g., glucose oxidase + peroxidase) or embedding cofactor-recycling enzymes into the matrix .

Real‑World Challenges

• Cost of enzymes & supports: Although immobilisation extends life, upfront costs remain high. Research aims to use low-cost carriers like waste biomass or repurposed polymers .
• Long-term durability: Over months or years, fouling, microbial colonisation, or support degradation may occur. Field trials are scarce.
• Scale‑up: Transition from lab batches to continuous, volume treatment is technically demanding.
• Regulation & Trust: Enzyme-based treatments must meet regulatory discharge norms and gain operator acceptance alongside conventional systems.

Future Advances

1. Smart & Composite Materials

Enzyme‑MOF hybrids, magnetic carriers, nanostructured supports, and functional hydrogels improve stability, recovery, and mass transfer .

2. Carrier‑free Systems

CLEAs and CLECs maximise enzyme concentrations, reduce cost, and simplify back purification (en.wikipedia.org).

3. Multi‑enzyme Cascades

Entrapping multiple enzymes (e.g., oxidase + peroxidase) enables sequential pollutant breakdown in a single support .

4. Protein Engineering

Genetic and directed evolution methods can produce oxidoreductases with enhanced stability, activity, and solvent tolerance—ideal for immobilisation .

5. Membrane Bioreactors

Embedding enzymes in membranes adds bioactive treatment to filtration, combining physical and biochemical degradation .

Looking Ahead: Path to Commercial Adoption

• Pilot studies need scaling demonstration across municipal and industrial plants.
• Cost‑benefit analysis must account for capital cost, enzyme lifetime, sludge reduction, and compliance benefits.
• Automation & Monitoring will be essential for stable, long-term operation.
• Regulatory acceptance through standardised testing protocols, certification, and national guidelines.

Conclusion

Immobilised oxidoreductases are a powerful, sustainable solution for modern wastewater treatment:

• Achieving > 90 % removal of dyes, phenols, pharmaceuticals, and bisphenols.
• Increasing enzyme stability and recyclability via supports or CLEAs.
• Enabling continuous-flow operation, cofactor recycling, and combo treatments with minimal sludge.
• Lowering energy usage and environmental impact.

While challenges related to scale, cost, and durability remain, ongoing advances in materials science, bioengineering, and reactor design strongly suggest that immobilised enzyme systems will play a key role in future water treatment strategies. Their adaptability, eco-friendliness, and catalytic efficiency make them well-suited to meet increasingly ambitious sustainability and pollution-removal goals.

Selected References

• Toor, G. & Nghiem, L. (2021). Enhanced Wastewater Treatment by Immobilised Enzymes. Current Pollution Reports, 7, 167–179. (link.springer.com)
• Z.darta et al. (2022). Membrane‑Immobilised Oxidoreductases in Micropollutant Removal. MDPI International Journal of Molecular Sciences, 23(22), 14086. (mdpi.com)
• Sigurdardóttir, et al. (2022). Recyclability of immobilised laccase beads. Frontiers in Bioengineering & Biotechnology. (frontiersin.org)
• Yang, P., et al. (2023). MOFs for Oxidoreductase Immobilisation. Materials, 16(19), 6572. (mdpi.com)
• Gao, et al. (2020). Enzyme‑MOF Biocatalyst for Dye Degradation. PMC.
• Sheldon, R.A. (2003). Cross‑Linked Enzyme Aggregates. Wikipedia. (en.wikipedia.org)