The stability of L-leucine in industrial enzyme preparations

The widespread application of industrial enzymes in food processing, biocatalysis, detergent, and daily chemical industries imposes stringent requirements on their stability under complex operating conditions (e.g., high temperature, extreme pH, and high-salt environments). As a hydrophobic branched-chain amino acid, L-leucine exhibits significant advantages in regulating the spatial conformation of enzyme proteins and enhancing enzyme stability due to its unique molecular structural characteristics (containing an isopropyl side chain). This article systematically reviews the research progress of L-leucine in stabilizing industrial enzymes from the aspects of regulatory mechanisms, influencing factors, research methods, and industrial applications, providing theoretical support and technical reference for the performance optimization of enzyme preparations.

I. Core Mechanisms of L-Leucine Regulating the Stability of Industrial Enzymes

The stability-enhancing effect of L-leucine on industrial enzymes essentially modifies the structural stability of enzyme proteins through intermolecular interactions, reducing conformational damage and activity loss under extreme conditions. The core mechanisms include the following four aspects:

1. Strengthening Hydrophobic Interactions of Enzyme Proteins

The stability of the spatial conformation of enzyme proteins depends on secondary bonds such as hydrophobic interactions, hydrogen bonds, and disulfide bonds within the molecule. The side chain of L-leucine is a non-polar isopropyl group with strong hydrophobicity, and its mechanism of action is mainly reflected in:

Embedding in the Hydrophobic Core Region of Enzyme Proteins: L-leucine can bind to hydrophobic sites in the amino acid sequence of enzyme proteins through hydrophobic interactions, filling the cavities of the hydrophobic core, enhancing the dense packing of the hydrophobic region, and reducing conformational unfolding and denaturation of enzyme proteins under high temperature or in the presence of denaturants.

Promoting the Formation of Hydrophobic Networks: In the hydrophobic regions on the surface of enzyme molecules or at subunit interfaces, L-leucine can act as a "bridge" to connect adjacent hydrophobic groups, forming a more stable hydrophobic network, strengthening the interactions between enzyme subunits, and reducing the risk of depolymerization of multimeric enzymes.

For example, in α-amylase, L-leucine can form hydrophobic clusters with hydrophobic residues (e.g., alanine, valine) around the enzyme's active center, maintaining the stability of the active center conformation and reducing activity loss at high temperatures.

2. Protecting the Active Center Structure of Enzymes

The catalytic activity of enzymes relies on the specific spatial arrangement of amino acid residues in the active center. L-leucine protects the active center through two ways:

Steric Hindrance Effect: The isopropyl side chain of L-leucine has a large volume. When bound near the enzyme's active center, it can form a steric barrier, preventing denaturants (e.g., oxidants, heavy metal ions) in the external environment from binding to key residues (e.g., serine, histidine) of the active center, and reducing chemical modification of the active center.

Maintaining the Microenvironment of the Active Center: The hydrophobic nature of L-leucine can regulate the polar microenvironment around the active center, stabilize the conformation of the active center bound to substrates, and reduce the damage of water molecules to the active center, thereby improving the catalytic stability of enzymes in aqueous solutions.

3. Inhibiting Aggregation and Degradation of Enzyme Proteins

Industrial enzymes are prone to aggregation, precipitation, or protease degradation due to conformational changes during storage or application. L-leucine can inhibit this process through the following pathways:

Preventing Aggregation: L-leucine can adsorb on the hydrophobic surface of enzyme proteins, reduce the hydrophobic interactions between protein molecules, and decrease the occurrence of irreversible aggregation. Meanwhile, its branched-chain structure can increase the steric hindrance on the surface of enzyme molecules, hindering the close contact and aggregation of protein molecules.

Resisting Protease Degradation: Some industrial enzymes (e.g., proteases, cellulases) may be degraded by proteases in the system during application. L-leucine can bind to the degradation sites of enzyme proteins, mask the recognition sequences of proteases, or reduce the exposure of degradation sites by stabilizing the conformation of enzyme proteins, thereby improving the anti-degradation ability of enzymes.

4. Regulating the Solvation Layer Structure of Enzymes

In aqueous or organic phase reaction systems, a solvation layer is formed on the surface of enzyme proteins, and the stability of its structure directly affects the catalytic performance of enzymes. The hydrophobic side chain of L-leucine can regulate the solvation layer on the surface of enzyme proteins:

Reducing the Damage of Water Molecules to Enzyme Structure: In aqueous solutions, L-leucine can repel some bound water on the surface of enzyme proteins, reduce the disturbance of water molecules to the hydrogen bond network, and maintain the native conformation of enzyme proteins.

Enhancing the Stability of Enzymes in Organic Phases: In non-aqueous phase catalytic systems, L-leucine can improve the compatibility between enzyme proteins and organic solvents, reduce the denaturation effect of organic solvents on enzyme structure, and enhance the catalytic activity and stability of enzymes in organic phases.

II. Key Factors Influencing the Stabilization Effect of L-Leucine

The stability-enhancing effect of L-leucine on industrial enzymes is not fixed, but is affected by various factors such as enzyme type, reaction system conditions, and L-leucine addition method. The core influencing factors are as follows:

1. Enzyme Type and Structural Characteristics

Different industrial enzymes have significant differences in amino acid sequence, spatial conformation, and active center structure, leading to different responsiveness to L-leucine:

Hydrophobic Enzyme Proteins: For enzymes with high intrinsic hydrophobic residue content (e.g., lipases, proteases), the hydrophobic interactions of L-leucine are more likely to exert, resulting in more significant stabilization effects.

Hydrophilic Enzyme Proteins: For highly hydrophilic enzymes (e.g., glucoamylase, amylase), L-leucine can only exert stabilization effects through binding to specific sites, and its effect depends on the number and distribution of hydrophobic sites on the enzyme molecule surface.

Multimeric Enzymes and Monomeric Enzymes: The interactions between subunits are crucial for the stability of multimeric enzymes (e.g., lactate dehydrogenase), and L-leucine can improve stability by strengthening hydrophobic interactions between subunits. In contrast, the stability of monomeric enzymes (e.g., lysozyme) mainly relies on intramolecular secondary bonds, and the stabilization effect of L-leucine is relatively weak.

2. Addition Concentration and Method of L-Leucine

Concentration Effect: There is an optimal concentration range for L-leucine. A concentration that is too low cannot form effective hydrophobic networks and steric barriers, while an excessively high concentration may lead to overly strong intermolecular interactions, which in turn induces enzyme protein aggregation. Generally, the optimal addition concentration of L-leucine in industrial enzyme preparations is 0.1~10 mmol/L, which needs to be optimized according to the enzyme type and application scenario.

Addition Method: Direct addition of free L-leucine is simple and convenient, but it is prone to loss in high-temperature or high-salt systems. Covalent binding of L-leucine to enzyme proteins through chemical modification (e.g., amidation, esterification) can improve the binding stability between L-leucine and enzymes and prolong the action time. However, this may affect the catalytic activity of enzymes, so it is necessary to balance the modification degree and activity retention rate.

3. Environmental Conditions of the Reaction System

The application scenarios of industrial enzymes are often accompanied by extreme environments, which can affect the interaction between L-leucine and enzymes, thereby influencing the stabilization effect:

Temperature: Within the appropriate temperature range (e.g., 25~50℃), L-leucine can stabilize the enzyme structure through hydrophobic interactions. However, at high temperatures (e.g., >60℃), the conformation of enzyme proteins changes drastically, and the stabilization effect of L-leucine may be weakened, requiring combination with other stabilizers (e.g., polyols, trehalose).

pH Value: Extreme pH (pH < 3 or pH > 10) can destroy the hydrogen bonds and ionic bonds of enzyme proteins, affect the binding sites between L-leucine and enzymes, and reduce the stabilization effect. L-leucine exhibits the best stabilization effect under neutral to weakly acidic conditions (pH 5~8), which matches the optimal pH range of most industrial enzymes.

Ionic Strength and Metal Ions: A high-salt environment (e.g., >1 mol/L NaCl) can compress the electric double layer of enzyme proteins, enhance intermolecular hydrophobic interactions, and may produce a synergistic stabilization effect with L-leucine. Some metal ions (e.g., Ca²⁺, Mg²⁺) can form complexes with L-leucine and enzyme proteins, further strengthening the enzyme structure stability. However, heavy metal ions (e.g., Cu²⁺, Fe³⁺) can destroy the active center of enzymes, offsetting the stabilization effect of L-leucine.

4. Synergistic Effect of Other Additives

Industrial enzyme preparations usually contain a variety of additives, and the synergistic or antagonistic effects between these substances and L-leucine will affect the final stabilization effect:

Synergistic Effect: Polyols (e.g., glycerol, sorbitol) and sugars (e.g., trehalose, sucrose) can jointly regulate the solvation layer of enzyme proteins with L-leucine, enhancing the stability of hydrophobic interactions and hydrogen bond networks. Antioxidants (e.g., vitamin C, glutathione) can reduce the oxidative damage of enzyme proteins, forming a synergistic mechanism of "structural stabilization + oxidative protection" with L-leucine.

Antagonistic Effect: Some denaturants (e.g., urea, guanidine hydrochloride) can destroy the hydrophobic core and hydrogen bonds of enzyme proteins, directly weakening the stabilization effect of L-leucine. High-concentration surfactants (e.g., SDS) may competitively bind to the hydrophobic sites of enzyme proteins, reducing the binding efficiency of L-leucine.

III. Research on the Effect of L-Leucine on the Stability of Typical Industrial Enzymes

Different industrial enzymes have significant differences in structure and application scenarios, and the stability-enhancing effect of L-leucine is also targeted. The following are the research progress of several types of typical industrial enzymes:

1. Amylases (α-Amylase, Glucoamylase)

Amylases are widely used in food processing (e.g., starch saccharification, beer brewing) and biofuel production, requiring tolerance to high temperatures (50~90℃) and acidic conditions. Studies have shown that:

Adding 5 mmol/L L-leucine to α-amylase can extend the enzyme's half-life at 70℃ from 20 min to 65 min, with the activity retention rate increased by 40%. The mechanism is that L-leucine binds to the hydrophobic domain of the enzyme, inhibiting conformational unfolding and aggregation at high temperatures.

For glucoamylase under pH 4.0 and 60℃, the addition of L-leucine increases the enzyme activity retention rate from 55% to 82% within 12 h, while reducing the generation of enzyme protein degradation products. This indicates that L-leucine can inhibit the hydrolytic degradation of glucoamylase by protecting its spatial structure.

2. Proteases (Alkaline Protease, Neutral Protease)

Proteases are widely used in detergent, leather processing, food fermentation, and other fields. The main stability challenges they face are high temperature, extreme pH, and autodegradation. Relevant studies have shown that:

For alkaline protease under pH 10.0 and 50℃, adding 2 mmol/L L-leucine can reduce the enzyme's autodegradation rate by 35% and improve the storage stability (25℃, 6 months) in laundry detergent systems by 50%. The reason is that L-leucine masks the autodegradation sites of proteases and strengthens the hydrophobic interactions between enzyme molecules.

For neutral protease in organic phases (e.g., n-hexane), the synergistic effect of L-leucine and glycerol can increase the enzyme's catalytic activity retention rate from 30% to 75%, and maintain more than 60% activity after 5 repeated uses. This indicates that L-leucine can improve the compatibility and structural stability of enzymes in organic phases.

3. Cellulases and Hemicellulases

Cellulases and hemicellulases are used in biomass conversion, textile desizing, and other fields, requiring maintenance of activity under high temperature, high salt, or acidic conditions. Studies have found that:

For cellulase under 50℃ and pH 5.0, adding 10 mmol/L L-leucine can extend the enzyme's half-life in the lignocellulosic hydrolysis system by 2.5 times and increase the hydrolysis efficiency by 30%. L-leucine enhances the binding ability between enzymes and substrates by stabilizing the catalytic domain and cellulose-binding domain of cellulase.

For xylanase (a type of hemicellulase) under 80℃ and pH 4.5, the synergistic effect of L-leucine and Ca²⁺ can increase the enzyme activity retention rate from 28% to 68%. The mechanism is that L-leucine strengthens the hydrophobic core of the enzyme, and Ca²⁺ stabilizes the active center conformation, forming a synergistic stabilization effect.

4. Lipases

Lipases are used in food processing, detergents, biocatalytic synthesis, and other fields, requiring tolerance to organic phases, high temperatures, and extreme pH. Relevant studies have shown that:

For lipase in organic phases (e.g., ethanol-water system, volume ratio 50:50), adding 3 mmol/L L-leucine can increase the enzyme's catalytic activity by 45% and improve the storage stability (4℃, 3 months) by 60%. L-leucine reduces the denaturation effect of organic solvents on enzyme structure by regulating the hydrophobicity of the enzyme protein surface.

For thermophilic lipase at 90℃, the addition of L-leucine extends the enzyme's half-life from 15 min to 45 min, with the activity retention rate increased by 55%, indicating that L-leucine can effectively inhibit the conformational unfolding and aggregation of lipase at high temperatures.

IV. Research Methods and Evaluation Indicators for L-Leucine-Stabilized Industrial Enzymes

1. Main Research Methods

(1) In Vitro Stability Evaluation Methods

Thermal Stability Test: Incubate enzyme preparations added with L-leucine at different temperatures (e.g., 40~90℃) for different times (e.g., 0~120 min), determine the residual enzyme activity, calculate the enzyme's half-life (t₁/₂) and activity retention rate, and evaluate the thermal stabilization effect.

pH Stability Test: Adjust the pH value of enzyme preparations (e.g., 2~12), incubate at an appropriate temperature, determine the enzyme activity under the optimal pH, and compare the activity retention rate under different pH conditions.

Storage Stability Test: Store enzyme preparations at room temperature (25℃), low temperature (4℃), or accelerated conditions (e.g., 40℃) for different times (e.g., 0~6 months), regularly determine the enzyme activity, and evaluate the long-term storage stability.

Anti-Degradation and Anti-Aggregation Tests: Analyze the degradation of enzyme proteins by SDS-PAGE electrophoresis, and determine the particle size distribution of enzyme proteins by dynamic light scattering (DLS) to evaluate the inhibitory effect of L-leucine on enzyme aggregation.

(2) Structural Characterization Methods

Spectroscopic Methods: Circular dichroism (CD) is used to analyze changes in the secondary structure (α-helix, β-sheet, random coil) of enzyme proteins; fluorescence spectroscopy is used to detect changes in the tertiary structure and active center microenvironment of enzyme proteins, evaluating the effect of L-leucine on enzyme conformation.

Chromatographic and Mass Spectrometric Methods: Gel filtration chromatography (GFC) is used to analyze the aggregation state and molecular weight distribution of enzyme proteins; high-performance liquid chromatography (HPLC) is used to determine the purity and degradation products of enzyme proteins; mass spectrometry (MS) is used to identify the binding sites between L-leucine and enzyme proteins.

X-ray Crystallography and Cryo-Electron Microscopy: Used to resolve the three-dimensional structure of enzyme proteins bound to L-leucine, clarifying the interaction mode between them (e.g., hydrophobic interactions, hydrogen bond binding sites).

(3) Industrial Application Simulation Tests

Simulated Operating Condition Tests: In systems simulating industrial application scenarios (e.g., laundry detergent systems, starch saccharification systems, biomass hydrolysis systems), after adding L-leucine, determine the catalytic efficiency, reaction conversion rate, and repeated use stability of enzymes.

Product Performance Tests: Apply enzyme preparations added with L-leucine to actual production (e.g., food processing, detergent production), and evaluate the product quality indicators (e.g., starch saccharification rate, detergency) and the use cost of enzyme preparations.

2. Core Evaluation Indicators

Enzyme Activity Retention Rate: The ratio of residual enzyme activity to initial activity after incubation or storage, reflecting the protective effect of L-leucine on enzyme activity.

Enzyme Half-Life (t₁/₂): The time required for enzyme activity to decrease to 50% of the initial value, which is a key quantitative indicator for evaluating enzyme stability.

Aggregation Rate: The proportion of aggregated enzyme protein particles to total protein determined by DLS or GFC, reflecting the inhibitory effect of L-leucine on enzyme aggregation.

Degree of Conformational Change: The magnitude of changes in the secondary/tertiary structure of enzyme proteins determined by CD, fluorescence spectroscopy, etc., evaluating the stabilizing effect of L-leucine on enzyme structure.

Industrial Application Efficiency: In actual production, the catalytic conversion rate, number of repeated uses, and use cost of enzyme preparations are the final indicators for evaluating the stabilization effect of L-leucine.

V. Industrial Application Challenges and Future Research Directions

1. Existing Challenges

Despite the significant potential of L-leucine in improving the stability of industrial enzymes, it still faces the following problems in practical applications:

Insufficient Specificity: The stabilization effect of L-leucine varies greatly among different enzymes, lacking universality. It is necessary to optimize the addition concentration and method for specific enzyme types.

Unclear Mechanism of Action: For complex industrial enzymes (e.g., multimeric enzymes, compound enzyme preparations), the binding sites and synergistic mechanism of L-leucine have not been fully clarified, limiting its directional optimization.

Cost and Process Issues: The production cost of high-purity L-leucine is relatively high, and it is necessary to balance the stabilization effect and cost in large-scale industrial applications. Although chemical modification can improve stability, the process is complex and may affect enzyme activity and product safety.

Limited Adaptability to Extreme Operating Conditions: Under extreme operating conditions such as ultra-high temperature (>100℃), strong acid-base (pH < 2 or pH > 11), and high-concentration organic solvents, the stabilization effect of L-leucine still needs to be further strengthened.

2. Future Research Directions

(1) Mechanism Deepening and Directional Design

Use bioinformatics methods such as molecular dynamics simulation and homology modeling to predict the binding sites between L-leucine and enzyme proteins, clarify its stabilization mechanism, and provide a theoretical basis for the directional design of high-efficiency stabilizers.

Modify the hydrophobic sites of enzyme proteins through protein engineering technologies (e.g., site-directed mutagenesis) to enhance their interaction with L-leucine, improving the specificity and universality of the stabilization effect.

(2) Development of Composite Stabilizer Systems

Construct composite stabilizer systems of "L-leucine + polyols/sugars/metal ions" to improve the stability of enzymes under extreme operating conditions through synergistic effects.

Develop intelligent responsive stabilizers by combining L-leucine with temperature-sensitive, pH-sensitive, or other responsive materials, enabling the stabilizer to exert its effect only under specific operating conditions and reducing the impact on enzyme catalytic activity.

(3) Optimization of Low-Cost and Green Processes

Develop low-cost L-leucine production processes (e.g., microbial fermentation) to reduce the cost of industrial applications.

Explore physical modification methods (e.g., ultrasound, microwave-assisted combination) to replace chemical modification, improving the binding stability between L-leucine and enzymes while avoiding chemical reagent residues and enhancing product safety.

(4) Expanding Application Scenarios and Adapting to Compound Enzyme Preparations

Study the stabilization effect of L-leucine in novel industrial enzymes (e.g., extremozymes, synthases) to expand its application range.

For compound enzyme preparations (e.g., cellulase-hemicellulase composite systems, amylase-protease composite systems), optimize the addition scheme of L-leucine to solve the synergistic stabilization problem between different enzymes.

As a natural hydrophobic amino acid, L-leucine can significantly improve the stability of industrial enzymes under complex operating conditions such as high temperature, extreme pH, and organic phases through mechanisms such as strengthening hydrophobic interactions of enzyme proteins, protecting active center structures, and inhibiting aggregation and degradation. Its stabilization effect is affected by factors such as enzyme type, addition concentration, and environmental conditions, and targeted optimization is required to achieve the best effect. At present, L-leucine has shown good application potential in typical industrial enzymes such as amylases, proteases, and cellulases, but further research is still needed in terms of specificity, mechanism of action, and cost control. In the future, with the deepening of mechanism research, the development of composite systems, and process optimization, L-leucine is expected to become a core additive for the performance optimization of industrial enzyme preparations, providing technical support for the efficient development of fields such as biocatalysis, food processing, and biomass conversion.