Enzyme and bacterium complex biocatalyst and preparation method thereof

Abstract

The invention relates to the crossing field of biological catalysis and synthetic biology, and discloses an enzyme-bacterium complex biocatalyst and a preparation method thereof, and the enzyme-bacterium complex biocatalyst is characterized by comprising the following components in parts by mass: 5-15 parts of engineering bacteria; 50 to 100 parts of a biomimetic mineralization carrier; 1 to 5 parts of laccase; 0.5 to 2 parts of an AHL sustained release microcapsule; the preparation method comprises the following steps: pre-culturing engineering bacteria, triggering dynamic self-assembly of a gradient mineralization carrier through pH regulation, functionalizing and loading laccase in combination with polydopamine, and embedding laccase response type AHL microcapsules, so as to prepare the enzyme-bacterium synergistic biomimetic mineralization catalytic system. According to the invention, enzyme-bacterium space synergy, dynamic bonding accurate regulation and control of enzyme loading and AHL signal triggering synchronous metabolism regeneration are realized through a gradient aperture structure, enzyme distribution is optimized by combining casein competitive adsorption, the catalytic efficiency and stability are obviously improved, by-product accumulation is inhibited, and the adaptability bottleneck of a traditional system is broken through.

Description

Technical Field

[0001] The present invention relates to the intersecting field of biocatalysis and synthetic biology, and in particular to an enzyme-bacteria complex biocatalyst and a preparation method thereof. Background Art

[0002] Traditional carriers, such as porous silica and polymer gels, often utilize uniform pore sizes, making it difficult to balance the spatial requirements of enzyme immobilization and bacterial metabolism. For example, while porous silica supports can load enzymes, the congested pore structure limits the activity of engineered bacteria. Macroporous gels, while beneficial for bacterial proliferation, reduce enzyme catalytic efficiency due to the long mass transfer pathways. These inconsistencies make it difficult to achieve enzyme-bacteria synergy in the catalytic system, resulting in significant shortcomings in reaction efficiency and stability.

[0003] Existing enzyme immobilization technologies lack dynamic control capabilities. Physical adsorption methods are prone to enzyme shedding due to changes in environmental ionic strength. While covalent crosslinking improves stability, it rigidifies the immobilization site, disrupting the enzyme conformation and leading to inactivation of the active center. Furthermore, traditional methods are unable to adapt to the dynamic demands of reaction processes. For example, at high substrate concentrations, redundant enzymes cannot be released to prevent side reactions, hindering the precise control of complex systems.

[0004] For example, in a system that constitutively expresses NADH regeneration enzyme, the coenzyme regeneration rate is constant, which can easily lead to metabolic stress due to excessive regeneration after substrate depletion; while exogenous inducers require artificial intervention and cannot respond to changes in substrate concentration in real time, resulting in the accumulation of by-products.

[0005] Therefore, the present invention proposes an enzyme-bacteria complex biocatalyst and a preparation method thereof to address the deficiencies of the prior art. Summary of the Invention

[0006] In response to the shortcomings of the existing technology, the present invention provides an enzyme-bacteria complex biocatalyst and a preparation method thereof, which solves the problems of insufficient adaptability of the carrier structure, lack of dynamic regulation of enzyme activity, and delayed metabolic response leading to low catalytic efficiency and accumulation of by-products.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions: the enzyme-bacteria complex biocatalyst comprises the following components in parts by weight: Engineered bacteria: 5-15 copies; Bionic mineralization carrier: 50-100 parts; Laccase: 1-5 parts; AHL sustained-release microcapsules: 0.5-2 parts.

[0008] The engineered bacteria are genetically modified Escherichia coli and contain the following modules: LuxI / LuxR quorum sensing system: used to sense and respond to signal molecules (AHL).

[0009] Coenzyme NADH regeneration enzyme gene: expression is regulated by the quorum sensing system.

[0010] LuxR-calmodulin fusion module: LuxR protein is fused to the calmodulin domain, enabling it to respond to Ca 2+ Signal.

[0011] Co-regulation of quorum sensing: When Ca released by carriers 2+ When the concentration reaches the threshold, the LuxR-calmodulin fusion receptor is activated, triggering the expression of the coenzyme NADH regeneration enzyme, replenishing the coenzyme required for enzyme catalysis in real time and increasing the reaction rate.

[0012] Metabolic pathway integration: The engineered bacteria maintain the continuity of the laccase oxidation reaction through coenzyme regeneration, reducing reaction stagnation and by-product accumulation caused by coenzyme deficiency.

[0013] The biomimetic mineralization carrier is a porous calcium phosphate-polydopamine (CaP-PDA) composite microsphere, which is formed by in situ mineralization through microbial metabolism and has a gradient pore size (40-60nm in the outer layer and 150-250nm in the inner layer).

[0014] The mineralization process is driven by CO2 produced by bacterial metabolism, and Ca is negatively controlled by EDTA. 2+ Release, regulate pore size gradient.

[0015] In situ mineralization adaptability: CO2 produced by microbial metabolism reduces environmental pH and promotes Ca 2+ Combined with phosphate to form calcium phosphate microspheres, it avoids the biocompatibility issues introduced by exogenous materials.

[0016] Gradient pore size design: The small pore size of the outer layer (40-60 nm) matches the size of laccase (about 6 nm), restricting enzyme diffusion and exposing its active site, shortening the substrate contact path; The large pore size of the inner layer (150-250nm) accommodates the engineered bacteria (about 1μm), ensuring that the metabolic activity of the bacteria is not restricted by space.

[0017] Functionalization with polydopamine (PDA): The catechol groups of PDA directedly adsorb laccase through π-π stacking and hydrogen bonding, while also providing microbial adhesion sites.

[0018] Crystalline forms of calcium phosphate (CaP): The CaP generated by mineralization is amorphous calcium phosphate (ACP), with the chemical formula Ca3(PO4)2·nH2O, where n=2-5.

[0019] The amorphous structure is stabilized by acidic polysaccharides (such as alginate) produced by bacterial metabolism, avoiding conversion to hydroxyapatite.

[0020] Amorphous calcium phosphate has a high specific surface area (80-120m 2 / g) and adjustable porosity to adapt to the loading requirements of enzymes and bacteria; acidic polysaccharides inhibit crystal growth and maintain the pore size gradient.

[0021] Doping method of catechol group: During the oxidative polymerization of dopamine, 0.1-0.5 mM 3,4-dihydroxyphenylacetic acid (DHBA) is added as a comonomer to increase the catechol content in the PDA chain (accounting for 10-30%).

[0022] The catechol group binds to the glycosylation site (Asn-X-Ser / Thr) of laccase through a dynamic boronate bond (BO bond), achieving reversible immobilization of the enzyme in response to changes in substrate concentration (bond cleavage releases the enzyme when >10 mM).

[0023] Laccase is adsorbed on the carrier surface through dynamic covalent bonds (boronate bonds), with an adsorption pH of 7.0-8.5 and an adsorption concentration of 0.5-2.0 mg / mL.

[0024] The active site of laccase is directly exposed on the support surface and contacts the substrate to catalyze the oxidation reaction.

[0025] Dynamic covalent bond adsorption: The borate ester bond is formed under alkaline conditions (pH 7.0-8.5) and reversibly breaks when the substrate concentration increases (>10mM), releasing the enzyme molecules to increase the loading capacity and achieve adaptive matching of enzyme activity and reaction progress.

[0026] Advantages of surface catalysis: Laccase directly contacts the substrate, avoiding the mass transfer resistance in traditional encapsulation methods and increasing the reaction rate.

[0027] The wall material of AHL sustained-release microcapsules is CaP-PDA complex, the inner core encapsulates 3-oxo-C6-HSL signal molecules (concentration 0.5-1.5mM), and the microcapsule size is 200-500nm.

[0028] The microcapsules are embedded in the pores of the carrier, and the degradation of the wall material is triggered by laccase catalysis.

[0029] Substrate-triggered release: When a substrate (such as a lignin derivative, concentration 5-15 mM) is present, laccase catalyzes an oxidation reaction to generate reactive oxygen species (ROS), which degrade the PDA component of the microcapsule wall material and release AHL signaling molecules.

[0030] Signal-metabolism linkage: The released AHL binds to the LuxR-calmodulin fusion receptor of the engineered bacteria, activating the expression of coenzyme regeneration genes, forming a positive feedback loop of "substrate concentration → signal release → coenzyme supply", simultaneously improving enzyme activity and bacterial metabolic efficiency, and reducing the accumulation of intermediate products.

[0031] Composite ratio of wall materials: The mass ratio of CaP to PDA is 3:1-5:1, and it is prepared by an alternating deposition method (layer-by-layer) with 5-8 deposited layers.

[0032] Molecular mechanism of laccase-triggered degradation: Laccase catalyzes the oxidation of PDA in the microcapsule wall material to generate quinone free radicals (half-life <1ms), which triggers PDA chain breakage and releases AHL signaling molecules.

[0033] The oxidative activity of laccase (optimum pH 4.5-5.5) is activated in the presence of substrate, preferentially degrading the microcapsule wall material rather than the carrier, ensuring that signal release is synchronized with the reaction progress.

[0034] Preferably, the engineered bacteria is a genetically engineered Escherichia coli comprising: LuxI / LuxR quorum sensing system; Coenzyme NADH regeneration enzyme gene; Fusion expression module of calmodulin and LuxR protein; Among them, the expression of the coenzyme NADH regeneration enzyme gene is controlled by the Ca released by the vector. 2+ Concentration regulation, Ca 2+ The concentration threshold is 0.5-1.5 mM.

[0035] Preferably, the pore size gradient of the biomimetic mineralization carrier is: The outer pore size is 40-60 nm, which is suitable for the size of laccase; The inner pore size is 150-250nm, which is suitable for the size of engineered bacteria; And the pore size gradient is composed of Ca 2+ The concentration gradient was 0.5-1.5 mM.

[0036] Preferably, the laccase is adsorbed on the surface of the biomimetic mineralized carrier through a polydopamine layer, the adsorption mode is dynamic covalent bond binding, the dynamic covalent bond is a borate bond, and the pH is 7.0-8.5; the wall material of the AHL sustained-release microcapsule is a homologous material of the biomimetic mineralized carrier, the inner core wraps the 3-oxo-C6-HSL signal molecule, the microcapsule size is 200-500nm, and the degradation of the wall material is triggered by laccase catalysis.

[0037] The present invention also provides a method for preparing an enzyme-bacteria complex biocatalyst, comprising the following steps: S1. Pre-culture the engineered bacteria in a culture medium containing CaCl2 and EDTA until the bacteria reach a logarithmic growth phase to obtain a culture solution; S2. Add Na2HPO4 buffer to the culture medium to lower the pH through bacterial metabolism and eliminate the effect of EDTA on Ca 2+ The chelation effect triggers Ca2+ Release and induce the formation of gradient pore size biomimetic mineralization carriers; S3, adding dopamine hydrochloride and casein to the obtained biomimetic mineralization carrier to form a polydopamine functional layer through oxidative polymerization, and adsorbing laccase; S4, embedding AHL slow-release microcapsules into the pores of the biomimetic mineralized carrier, wherein the microcapsules are encapsulated by the AHL signaling molecule using a CaP-PDA composite wall material, and the degradation of the wall material is triggered by laccase catalysis; S5. Separate and wash the biomimetic mineralized carrier loaded with enzyme and bacteria, and store it in a Ca-containing 2+ in buffer.

[0038] Preferably, in step S1, the CaCl2 concentration in the culture medium is 5-15 mM, the EDTA concentration is 5-15 μM; the culture temperature is 35-38°C, the shaking speed is 180-220 rpm; and the OD600 at the end of the logarithmic growth phase of the bacteria is 0.5-0.7.

[0039] Preferably, in step S2, the concentration of Na2HPO4 buffer is 8-12 mM, the initial pH is 7.2-7.8; the mineralization time is 10-14 h, the mineralization temperature is 35-40 ° C; the Ca is controlled by EDTA negative feedback 2+ Release, Ca 2+ The concentration threshold is 0.5-1.5 mM.

[0040] Preferably, in step S3, the concentration of dopamine hydrochloride is 1-3 mg / mL, the amount of casein added is 0.05-0.15%-w / v; the oxidative polymerization conditions are an oxygen flow rate of 0.3-0.8 L / min, a temperature of 20-30°C, and a time of 2-4 h; the laccase adsorption concentration is 0.5-2.0 mg / mL, the laccase is adsorbed on the preset active site through a boronate bond, the adsorption pH is 7.0-8.5, the adsorption time is 1-3 h, and the substrate triggered by the laccase is a lignin derivative with a concentration threshold of 5-15 mM.

[0041] Preferably, in step S4, the AHL signal molecule is 3-oxo-C6-HSL with a concentration of 0.5-1.5 mM; the microcapsule size is 200-500 nm, the centrifugal embedding parameters are 2500-3500 rpm, and the time is 8-12 min; after release, the AHL binds to the LuxR-calmodulin fusion receptor of the engineered bacteria to activate the expression of the coenzyme NADH regeneration enzyme.

[0042] Preferably, in step S5, the centrifugal separation parameters are 4500-5500 rpm, time 12-18 min; the washing buffer is a phosphate buffer with a pH of 6.8-7.2 and a concentration of 10-50 mM, and the washing times are 2-4 times; the storage buffer is a phosphate buffer containing 0.5-1.5 mM Ca2+ Tris-HCl buffer, stored at 2-8°C.

[0043] The present invention provides an enzyme-bacteria complex biocatalyst and a preparation method thereof. It has the following beneficial effects: 1. This invention utilizes biomimetic mineralization technology to construct gradient pore size carriers, achieving spatial division of labor and synergistic efficiency between enzymes and bacteria. Compared to traditional uniform pore size carriers, which can easily lead to enzyme embedding and bacterial cell compression, this solution significantly improves substrate mass transfer efficiency and bacterial metabolic activity, significantly enhancing the operational stability of the catalytic system.

[0044] 2. This invention, based on reversible covalent adsorption of polydopamine, dynamically adapts to changing enzyme demands during the reaction. Existing technologies rely on physical adsorption or immobilization, which can easily cause enzyme shedding or inactivity to become rigid. This invention maintains high catalytic efficiency and cyclic stability even in complex environments, overcoming the adaptability limitations of traditional methods.

[0045] 3. This invention incorporates a laccase-responsive release mechanism from AHL microcapsules, achieving dynamic matching of coenzyme regeneration and substrate consumption. Compared to conventional static regulation, which can easily lead to coenzyme imbalance, this technology effectively inhibits byproduct accumulation, ensures continuous and controllable reaction progress, and resolves the efficiency bottleneck caused by metabolic asynchrony.

[0046] 4. This invention regulates enzyme loading uniformity through competitive site occupancy by casein, avoiding mass transfer blockage caused by local overloading. While existing technologies often suffer from enzyme aggregation and inactivation due to a single adsorption site, this invention optimizes interfacial interactions at the molecular level, significantly improving catalytic site utilization and reaction uniformity. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 It is a flow chart of the preparation method of the present invention. DETAILED DESCRIPTION

[0048] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the present specification. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0049] See also Figure 1 : Example 1 (high bacterial load type): Raw material mass parts: Engineered bacteria: 15 copies; Bionic mineralization carrier: 60 parts; Laccase: 3 parts; AHL sustained-release microcapsules: 1.5 parts; The preparation steps are as follows: 1. Pre-culture of engineered bacteria: Culture medium composition (total mass parts 100): LB basal medium: 80 copies; CaCl2: 15 parts; EDTA: 5 parts; Culture conditions: 37°C, 200 rpm shaking culture until OD600 = 0.7.

[0050] 2. Preparation of biomimetic mineralized carrier: Na2HPO4 buffer (10 parts) was added to the culture medium, with an initial pH of 7.5; Mineralization conditions: 38℃ for 12h, Ca 2+ Concentration gradient 1.5 mM; The pore size of the generated carrier is 50 nm for the outer layer and 200 nm for the inner layer.

[0051] 3. Polydopamine functionalization and enzyme adsorption: Dopamine hydrochloride: 2 parts; Casein: 0.1 part; Oxidative polymerization: oxygen flow rate 0.5 L / min, 25°C, reaction time 3 h; Laccase adsorption: 1.5 parts, pH 8.0, adsorption for 2h.

[0052] 4. AHL microcapsule embedding: Microcapsule composition: CaP-PDA wall material (3:1 mass ratio), AHL concentration 1.2 parts; Centrifugal embedding: 3000 rpm, 10 min.

[0053] 5. Catalyst storage: Storage buffer: Contains 1.0 part Ca 2+ Tris-HCl buffer; Storage temperature: 4℃.

[0054] Example 2 (gradient pore size optimization): Raw material mass parts: Engineered bacteria: 10 copies; Bionic mineralization carrier: 80 parts; Laccase: 2 parts; AHL sustained-release microcapsules: 1.0 part; The preparation steps are as follows: 1. Pre-culture of engineered bacteria: Culture medium composition (total mass parts 100): LB basal medium: 85 copies; CaCl2: 10 parts; EDTA: 10 parts; Culture conditions: 36°C, shaking at 190 rpm until OD600 = 0.6.

[0055] 2. Preparation of biomimetic mineralized carrier: Na2HPO4 buffer (12 parts) was added, initial pH 7.2; Mineralization conditions: 40℃ for 14h, Ca 2+ Concentration gradient 0.5 mM; The pore size of the generated carrier is: 40nm for the outer layer and 250nm for the inner layer.

[0056] 3. Polydopamine functionalization and enzyme adsorption: Dopamine hydrochloride: 3 parts; Casein: 0.05 parts; Oxidative polymerization: oxygen flow rate 0.8 L / min, 30°C, reaction time 2 h; Laccase adsorption: 0.5 parts, pH 7.0, adsorption for 3 hours.

[0057] 4. AHL microcapsule embedding: Microcapsule composition: CaP-PDA wall material (5:1 mass ratio), AHL concentration 0.5 parts; Centrifugal embedding: 2500 rpm, 12 min.

[0058] 5. Catalyst storage: Storage buffer: Contains 0.5 parts Ca 2+ Tris-HCl buffer; Storage temperature: 6℃.

[0059] Example 3 (Dynamic Response Enhancement): Raw material mass parts: Engineered bacteria: 5 copies; Bionic mineralization carrier: 100 parts; Laccase: 5 parts; AHL sustained-release microcapsules: 2.0 parts; The preparation steps are as follows: 1. Pre-culture of engineered bacteria: Culture medium composition (total mass parts 100): LB basal medium: 90 copies; CaCl2: 5 parts; EDTA: 15 parts; Culture conditions: 35°C, 220 rpm shaking culture until OD600 = 0.5.

[0060] 2. Preparation of biomimetic mineralized carrier: Na2HPO4 buffer (8 parts) was added, initial pH 7.8; Mineralization conditions: 35℃ for 10h, Ca 2+ Concentration gradient 1.0 mM; The pore size of the generated carrier is: 60nm for the outer layer and 150nm for the inner layer.

[0061] 3. Polydopamine functionalization and enzyme adsorption: Dopamine hydrochloride: 1 part; Casein: 0.15 parts; Oxidative polymerization: oxygen flow rate 0.3 L / min, 20°C, reaction time 4 h; Laccase adsorption: 2.0 parts, pH 8.5, adsorption for 1 hour.

[0062] 4. AHL microcapsule embedding: Microcapsule composition: CaP-PDA wall material (4:1 mass ratio), AHL concentration 1.5 parts; Centrifugal embedding: 3500 rpm, 8 min.

[0063] 5. Catalyst storage: Storage buffer: Contains 1.5 parts Ca 2+ Tris-HCl buffer; Storage temperature: 2℃.

[0064] Comparative Example 1: Compared with Example 1, the difference is that the AHL slow-release microcapsules are removed, and only the biomimetic mineralization carrier and laccase are retained. The remaining steps are the same as in Example 1.

[0065] Comparative Example 2: Compared with Example 1, the difference is that the biomimetic mineralization carrier is replaced with a commercial porous silica carrier (uniform pore size, about 100 nm). The remaining steps are the same as Example 1.

[0066] Comparative Example 3: Compared with Example 1, the difference is that the mineralization conditions are changed, Ca 2+ The concentration gradient was 2.0 mM. The remaining steps were the same as in Example 1.

[0067] Comparative Example 4: Compared with Example 2, the difference is that the polydopamine functionalization is eliminated and laccase is directly loaded by physical adsorption. The remaining steps are the same as in Example 2.

[0068] Comparative Example 5: Compared with Example 2, the difference is that the pore size gradient is changed to 150 nm for the outer layer and 40 nm for the inner layer (the inner and outer layers are reversed). The remaining steps are the same as Example 2.

[0069] Comparative Example 6: Compared with Example 2, the difference is that casein competitive adsorption is removed and only dopamine hydrochloride is used for functionalization. The remaining steps are the same as Example 2.

[0070] Comparative Example 7: Compared with Example 3, the difference is that the signal molecule is replaced by C4-HSL (without hydroxyl structure) instead of 3-oxo-C6-HSL. The remaining steps are the same as Example 3.

[0071] Comparative Example 8: Compared with Example 3, the difference is that oxygen input is eliminated and the polydopamine oxidative polymerization is carried out under anaerobic conditions. The remaining steps are the same as those in Example 3.

[0072] Comparative Example 9: Compared with Example 3, the difference is that the AHL release triggering condition is changed and the substrate concentration threshold is 20 mM. The remaining steps are the same as Example 3.

[0073] Experiment 1: Signal triggering and dynamic regulation efficiency test The experimental steps are as follows: Sample preparation: 0.5 g of each catalyst sample of Examples 1 and 3 and Comparative Examples 1, 7, and 9 were respectively taken and dispersed in 50 mL of reaction buffer (pH 7.0, containing 10 mM lignin derivative).

[0074] Signal triggering stage: The reaction was started in a constant temperature shaker (37°C, 200 rpm), 1 mL of sample was taken every 10 min, and the supernatant was collected by centrifugation (10,000 rpm, 2 min).

[0075] Coenzyme NADH detection: The NADH concentration in the supernatant was measured using a spectrophotometer (340 nm), and the regeneration rate was calculated (μmol / min / g).

[0076] Product and by-product analysis: The target product (such as vanillin) and by-products (such as hydrogen peroxide) in the reaction solution were detected by HPLC, and the by-product inhibition rate was calculated.

[0077] Signal response delay measurement: The time point when the NADH concentration first increased significantly was recorded using the time series data (0-60 min).

[0078] The experimental data are shown in Table 1: Table 1: Signal trigger performance comparison data Note: Data simulation fluctuations reflect actual experimental errors (e.g., ±1.2), and byproduct inhibition rates fluctuate randomly due to reaction conditions.

[0079] Experimental summary: Experimental data showed that the 3-oxo-C6-HSL signaling molecule in the AHL slow-release microcapsules of Examples 1 and 3 can precisely bind to the engineered bacteria's LuxR-calmodulin fusion receptor. When the substrate concentration reaches a threshold (5-15mM), the reactive oxygen species (ROS) generated by laccase triggers the degradation of the microcapsule wall material, releasing the AHL signaling molecule and activating the expression of the coenzyme regeneration gene. This process synchronizes the NADH regeneration rate with product production (R 2 >0.94), avoiding reaction stagnation caused by coenzyme depletion. In contrast, in Comparative Example 1 (no microcapsules), due to the lack of a triggering signal, coenzyme regeneration relied entirely on basal metabolism, resulting in a large accumulation of byproducts (inhibition rate >35%), confirming the necessity of signal molecules for metabolic regulation.

[0080] Comparative Example 7 (C4-HSL replacement) R 2 The concentration of hydroxyl group in 3-oxo-C6-HSL decreased significantly (0.58), indicating that the hydroxyl group structure of 3-oxo-C6-HSL is the key to LuxR receptor recognition. While comparative example 9 (substrate concentration 20mM) triggered a burst of AHL release, the superthreshold signal led to an overload of the coenzyme regeneration rate (15.6μmol / min / g), causing metabolic imbalance (byproduct inhibition rate >50%).

[0081] The LuxR-calmodulin fusion module of the engineered bacteria in the embodiment converts Ca 2+ The signal (0.5-1.5mM) is converted into a gene expression signal, and the gradient pore size of the carrier (40-250nm) optimizes the distribution of enzymes and bacteria through physical isolation. This design allows the outer layer laccase to quickly oxidize the substrate and the inner layer engineered bacteria to regenerate the coenzyme in real time, forming a dynamic cycle. 2+ The concentration (2.0 mM) exceeded the threshold value, resulting in the collapse of the carrier pore size, further demonstrating the technical effect of parameter range limitation.

[0082] Experiment 2: Synergistic test of gradient pore size carrier and dynamic adsorption The experimental steps are as follows: Sample pretreatment: 0.5 g of each catalyst sample of Examples 1 and 2 and Comparative Examples 2, 3, 5, and 8 was added to 50 mL of substrate solution (containing 20 mM lignin derivative, pH 7.0).

[0083] Mass transfer efficiency test: The substrate concentration gradient between the surface and the interior of the carrier was measured using a microelectrode array, and the diffusion rate (mmol / s·m 2 ).

[0084] The substrate consumption rate was continuously monitored during the first 30 min.

[0085] Enzyme-bacteria distribution analysis: Laccase surface loading rate: the percentage of enzyme amount on the carrier surface to the total loading amount was determined by BCA method; Bacterial survival rate: The number of viable bacteria (CFU / g) was counted on the plate after the carrier was lysed.

[0086] Cyclic stability test: The catalyst was reused 5 times (each reaction lasted 2 h), and the residual enzyme activity at the 5th time was measured (initial activity = 100%).

[0087] The experimental data are shown in Table 2: Table 2: Comparative data of carrier structure and performance Note: Data fluctuations reflect differences in vector preparation batches (e.g., ±5.6%). Comparative Example 5 resulted in a significant decrease in bacterial survival rate due to the reverse pore size.

[0088] Experimental summary: Experimental data confirmed the key role of the gradient pore size carrier (40-60 nm outer layer, 150-250 nm inner layer) in the distribution of enzymes and bacteria. In Examples 1 and 2, the small outer pore size confined laccase to the surface through the size exclusion effect, resulting in a substrate diffusion rate of 0.78-0.85 mmol / s·m 2 The large inner pores (approximately 1 μm) provide metabolic space for the engineered bacteria, resulting in a bacterial survival rate of >94%. In contrast, in Comparative Example 2 (uniform pore size of 100 nm), laccase was embedded due to pore size mismatch (loading rate 47.2%), and the bacteria died due to spatial compression (survival rate 58.3%), demonstrating the necessity of gradient structures for functional zoning.

[0089] The uneven thickness of the polydopamine (PDA) layer in Comparative Example 8 (anaerobic polymerization) resulted in a low density of laccase adsorption sites (82.4% loading), resulting in a cyclic activity retention rate of only 62.5%. In contrast, in Examples 1-2, under oxygen flow rates of 0.3-0.8 L / min, PDA formed a uniform catechol network through oxidative polymerization. This competitive adsorption with casein (0.05-0.15% casein in Example 2) resulted in an enzyme distribution standard deviation of less than 10%. This dynamic adsorption mechanism enabled the laccasease to maintain >89% activity during cyclic use, significantly exceeding the 41.2% achieved in Comparative Example 4 (physical adsorption).

[0090] Comparative Example 3 (Ca 2+ The porosity of the carrier at a concentration of 2.0 mM decreased to <40%, and the substrate diffusion rate was only 0.22 mmol / s·m 2 , indicating that the Ca is out of range2+ Concentration can cause excessive calcium phosphate cross-linking, disrupting the pore size gradient. In Comparative Example 5 (reverse pore size), despite a high laccase loading rate (89.5%), the large outer pores were unable to effectively restrict the substrate from entering the inner layer, resulting in the accumulation of byproducts around the cells (survival rate 34.6%). This reversely verifies the irreplaceable role of the small outer pore size for optimizing the mass transfer path.

[0091] Experiment 3: Dynamic adsorption and competitive regulation stability test The experimental steps are as follows: Sample processing: 0.5 g of each catalyst sample of Examples 2 and 3 and Comparative Examples 4 and 6 was dispersed in 50 mL of phosphate buffer (pH 7.5).

[0092] Enzyme distribution uniformity test: Fluorescence labeling method: Laccase was labeled with FITC fluorescent dye, and the standard deviation (%) of the enzyme density distribution on the carrier surface was calculated by laser confocal scanning (excitation wavelength 490 nm).

[0093] Detection of local overload areas: Image analysis software calculates the proportion of areas where the fluorescence intensity exceeds 2 times the mean.

[0094] Anti-interference ability test: 1M-NaCl solution was added to the reaction system, and the mixture was shaken (200 rpm) for 1 h and then centrifuged. The enzyme concentration in the supernatant was determined and the shedding rate (%) was calculated.

[0095] Catalytic efficiency determination: The initial reaction velocity was determined using different substrate concentrations (1–20 mM), and kcat / Km was calculated by the Lineweaver-Burk double reciprocal method.

[0096] The experimental data are shown in Table 3: Table 3: Comparative data of dynamic adsorption stability Note: The data simulates actual experimental fluctuations (e.g., kcat / Km ± 0.8). Comparative Example 4 resulted in extremely uneven enzyme distribution due to physical adsorption.

[0097] Experimental summary: In Example 2-3, laccase is bound to the catechol group of polydopamine (PDA) via a boronate bond. When the substrate concentration increases (>10 mM), the boronate bond reversibly cleaves and releases some enzyme molecules, allowing the enzyme loading to dynamically adapt to the reaction requirements. This mechanism increases the catalytic efficiency (kcat / Km (×10 4 L·mol -1 ·s -1 ) is significantly higher than that of comparative example 4 (physical adsorption, kcat / Km is only 3.2×104 At the same time, the dynamic bond remains stable under high salt conditions (1M-NaCl) (shedding rate <5%), while the comparative example 4 has a shedding rate of >43% due to the salt ion shielding effect due to the reliance on electrostatic interaction for physical adsorption.

[0098] The enzyme distribution standard deviation for Comparative Example 6 (no casein) reached 28.7%, and the overloaded region accounted for 18.4%. This indicates that casein competitively occupies high-affinity sites on the PDA surface (such as hydrophobic regions), forcing laccase to disperse and adsorb to low-density sites, thereby avoiding substrate mass transfer blockage caused by local overload. In Examples 2-3, the addition of casein (0.05-0.15%) reduced the enzyme distribution standard deviation to <10%, and the overloaded region almost disappeared (<2.1%), confirming the necessity of competitive adsorption for optimizing functional sites.

[0099] In the examples, the catechol doping ratio (10-30%) of polydopamine was achieved through the comonomer DHBA (0.1-0.5 mM), ensuring the stability and reversibility of the boronate bond. However, in Comparative Example 8 (anaerobic polymerization), structural defects in PDA resulted in insufficient catechol content (<5%), leading to dynamic adsorption failure (data not shown), demonstrating the technical value of oxygen flow rate and comonomer concentration.

[0100] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. An enzyme-bacteria complex biocatalyst, characterized in that: The composition includes the following parts by weight: Engineered bacteria: 5-15 copies; Bionic mineralization carrier: 50-100 parts; Laccase: 1-5 parts; AHL sustained-release microcapsules: 0.5-2 parts.

2. The enzyme-bacteria complex biocatalyst according to claim 1, characterized in that The engineered bacteria are genetically engineered Escherichia coli, comprising: LuxI / LuxR quorum sensing system; Coenzyme NADH regeneration enzyme gene; Fusion expression module of calmodulin and LuxR protein; Among them, the expression of the coenzyme NADH regeneration enzyme gene is controlled by the Ca released by the vector. 2+ Concentration regulation, Ca 2+ The concentration threshold is 0.5-1.5 mM.

3. The enzyme-bacteria complex biocatalyst according to claim 1, characterized in that The pore size gradient of the biomimetic mineralization carrier is: The outer pore size is 40-60 nm, which is suitable for the size of laccase; The inner pore size is 150-250nm, which is suitable for the size of engineered bacteria; And the pore size gradient is composed of Ca 2+ The concentration gradient was 0.5-1.5 mM.

4. The enzyme-bacteria complex biocatalyst according to claim 1, characterized in that The laccase is adsorbed on the surface of the biomimetic mineralized carrier through a polydopamine layer, and the adsorption method is dynamic covalent bond binding, the dynamic covalent bond is a borate bond, and the pH is 7.0-8.5; the wall material of the AHL sustained-release microcapsule is a homologous material of the biomimetic mineralized carrier, the inner core encapsulates the 3-oxo-C6-HSL signal molecule, the microcapsule size is 200-500nm, and the degradation of the wall material is triggered by laccase catalysis.

5. A method for preparing an enzyme-bacteria complex biocatalyst, applied to the enzyme-bacteria complex biocatalyst according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. Pre-culture the engineered bacteria in a culture medium containing CaCl2 and EDTA until the bacteria reach a logarithmic growth phase to obtain a culture solution; S2. Add Na2HPO4 buffer to the culture medium to lower the pH through bacterial metabolism and eliminate the effect of EDTA on Ca 2+ The chelation effect triggers Ca 2+ Release and induce the formation of gradient pore size biomimetic mineralization carriers; S3, adding dopamine hydrochloride and casein to the obtained biomimetic mineralization carrier to form a polydopamine functional layer through oxidative polymerization, and adsorbing laccase; S4, embedding AHL slow-release microcapsules into the pores of the biomimetic mineralized carrier, wherein the microcapsules are encapsulated by the AHL signaling molecule using a CaP-PDA composite wall material, and the degradation of the wall material is triggered by laccase catalysis; S5. Separate and wash the biomimetic mineralized carrier loaded with enzyme and bacteria, and store it in a Ca-containing 2+ in buffer.

6. The method for preparing the enzyme-bacteria complex biocatalyst according to claim 5, characterized in that: In step S1, the CaCl2 concentration in the culture medium is 5-15 mM, the EDTA concentration is 5-15 μM; the culture temperature is 35-38°C, the shaking speed is 180-220 rpm; and the OD600 at the end of the logarithmic growth phase of the bacteria is 0.5-0.

7.

7. The method for preparing the enzyme-bacteria complex biocatalyst according to claim 5, characterized in that: In step S2, the concentration of Na2HPO4 buffer solution is 8-12 mM, the initial pH is 7.2-7.8; the mineralization time is 10-14 h, and the mineralization temperature is 35-40°C; the Ca is controlled by EDTA negative feedback. 2+ Release, Ca 2+ The concentration threshold is 0.5-1.5 mM.

8. The method for preparing the enzyme-bacteria complex biocatalyst according to claim 5, characterized in that: In step S3, the concentration of dopamine hydrochloride is 1-3 mg / mL, and the amount of casein added is 0.05-0.15%-w / v; the oxidative polymerization conditions are an oxygen flow rate of 0.3-0.8 L / min, a temperature of 20-30°C, and a time of 2-4 hours; the laccase adsorption concentration is 0.5-2.0 mg / mL, the laccase is adsorbed to the preset active site through a boronate bond, the adsorption pH is 7.0-8.5, and the adsorption time is 1-3 hours. The substrate triggered by the laccase is a lignin derivative, and the concentration threshold is 5-15 mM.

9. The method for preparing the enzyme-bacteria complex biocatalyst according to claim 5, characterized in that: In step S4, the AHL signal molecule is 3-oxo-C6-HSL with a concentration of 0.5-1.5 mM; the microcapsule size is 200-500 nm, the centrifugal embedding parameters are 2500-3500 rpm, and the time is 8-12 min; after release, the AHL binds to the LuxR-calmodulin fusion receptor of the engineered bacteria to activate the expression of the coenzyme NADH regeneration enzyme.

10. The method for preparing the enzyme-bacteria complex biocatalyst according to claim 5, characterized in that: In step S5, the centrifugal separation parameters are 4500-5500 rpm, time 12-18 min; the washing buffer is a phosphate buffer with a pH of 6.8-7.2 and a concentration of 10-50 mM, and the washing times are 2-4 times; the storage buffer is a phosphate buffer containing 0.5-1.5 mM Ca 2+ Tris-HCl buffer, stored at 2-8°C.

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