Enzyme Aggregates Research Articles

Cross-linked cyclodextrin glucanotransferase aggregates from Bacillus lehensis G1 for cyclodextrin production: Molecular modeling, developmental, physicochemical, kinetic and thermodynamic properties

Type of cross-linking agents influence the stability and active cross-linked enzyme aggregates (CLEA) immobilization. The information of molecular interaction between enzyme-cross linker is not well explored thus screening wide numbers of cross-linker is crucial in CLEA development. This study combined the molecular modeling and experimental optimization to investigate the influences of different cross-linking agents in developing CLEA of cyclodextrin glucanotranferase G1 (CGTase G1) for cyclodextrins (CDs) synthesis. Seven types of cross-linkers were tested and CGTase G1 cross-linked with chitosan (CS-CGTG1-CLEA) displayed the highest activity recovery (84.6 ± 0.26%), aligning with its highest binding affinity, radius of gyration and flexibility through in-silico analysis towards CGTase G1. CS-CGTG1-CLEA was characterized and showed a longer half-life (30.06 ± 1.51 min) and retained a greater thermal stability (52.73 ± 0.93%) after 30 min incubation at optimal conditions compared to free enzyme (10.30 ± 1.34 min and 5.51 ± 2.10% respectively). CS-CGTG1-CLEA improved CDs production by 33% and yielded cumulative of 52.62 g/L CDs after five cycles for 2 h of reaction. This study reveals that abundant of hydroxyl group on chitosan interacted with CGTase G1 surface amino acid residues to form strong and stable CLEA thus can be a promising biocatalyst in CDs production.

Protein surface engineering and interaction studies of maltogenic amylase towards improved enzyme immobilisation

A combined strategy of computational, protein engineering and cross-linked enzyme aggregates (CLEAs) approaches was performed on Bacillus lehensis G1 maltogenic amylase (Mag1) to investigate the preferred amino acids and orientation of the cross-linker in constructing stable and efficient biocatalyst. From the computational analysis, Mag1 exhibited the highest binding affinity towards chitosan (−7.5 kcal/mol) and favours having interactions with aspartic acid whereas glutaraldehyde was the least favoured (−3.4 kcal/mol) and has preferences for lysine. A total of eight Mag1 variants were constructed with either Asp or Lys substitutions on different secondary structures surface. Mutant Mag1-mDh exhibited the highest recovery activity (82.3%) in comparison to other Mag1 variants. Mutants-CLEAs exhibited higher thermal stability (20–30% activity) at 80 °C whilst Mag1-CLEAs could only retain 9% of activity at the same temperature. Reusability analysis revealed that mutants-CLEAs can be recovered up to 8 cycles whereas Mag1-CLEAs activity could only be retained for up to 6 cycles. Thus, it is evident that amino acids on the enzyme's surface play a crucial role in the construction of highly stable, efficient and recyclable CLEAs. This demonstrates the necessity to determine the preferential amino acid by the cross-linkers in advance to facilitate CLEAs immobilisation for designing efficient biocatalysts.

Precipitation-based microscale enzyme reactors coupled with porous and adhesive elastomer for effective bacterial decontamination and membrane antifouling on-demand

Bacterial contamination of water environments can cause various troubles in various areas. As one of potential solutions, we develop enzyme-immobilized elastomer, and demonstrate the uses of enzyme reactions on-demand for effective microbial decontamination and antifouling. Asymmetrically-structured elastomer is prepared by combining two polydimethylsiloxane (PDMS) layers with different degrees of crosslinking: highly-crosslinked and lightly-crosslinked PDMS layers. At the surface of highly-crosslinked PDMS layer, porous structure with average diameter of 842 nm is formed by dissolving pre-packed and entrapped latex beads. Lightly-crosslinked PDMS on the other side, due to its adhesive nature, enables iterative attachments on various materials under either dry or wet condition. Glucose oxidase (GOx) is immobilized by using the pores at the surface of highly-crosslinked PDMS matrix via a ship-in-a-bottle protocol of precipitation-based microscale enzyme reactor (p-MER), which consists of GOx adsorption, precipitation and chemical crosslinking (EAPC). As a result, crosslinked enzyme aggregates (CLEAs) of GOx not only are well entrapped within many pores of highly-crosslinked PDMS layer (ship-in-bottle) but also cover the external surface of matrix, both of which are well connected together. Highly-interconnected network of CLEAs themselves effectively prevents enzyme leaching, which shows the 25% residual activity of GOx under shaking at 200 rpm for 156 days after 48% initial drop of loosely-bound p-MER after 4 days. In presence of glucose, the underwater attachment of biocatalytic elastomer demonstrates the generation of hydrogen peroxide via p-MER-catalyzed glucose oxidation, exhibiting effective biocidal activities against both gram-positive S. aureus and gram-negative E. coli. Adhesion-induced GOx-catalyzed reaction also alleviates the biofouling of membrane, suggesting its extendibility to various engineering systems being suffered by biofouling. This study of biocatalytic elastomer has demonstrated its new opportunities for the facile and on-demand enzyme-catalyzed reactions in various environmental applications, such as bactericidal treatment, water treatment/purification, and pollutant degradation.

Immobilization of carbonic anhydrase for CO2 capture and utilization.

Carbonic anhydrase (CA) is an excellent candidate for novel biocatalytic processes based on the capture and utilization of CO2. The setup of efficient methods for enzyme immobilization makes CA utilization in continuous bioreactors increasingly attractive and opens up new opportunities for the industrial use of CA. The development of efficient processes for CO2 capture and utilization (CCU) is one of the most challenging targets of modern chemical reaction engineering. In the general frame of CCU processes, the interest in the utilization of immobilized CA as a biocatalyst for augmentation of CO2 reactive absorption has grown consistently over the last decade. The present mini-review surveys and discusses key methodologies for CA immobilization aimed at the development of heterogeneous biocatalysts for CCU. Advantages and drawbacks of covalent attachment on fine granular solids, immobilization as cross-linked enzyme aggregates, and "in vivo" immobilization methods are presented. In particular, criteria for optimal selection of CA-biocatalyst and design of CO2 absorption units are presented and discussed to highlight the most effective solutions. Perspectives on biocatalytic CCU processes that can include the use of CA in an enzymatic reactive CO2 absorption step are eventually presented with a special focus on two examples of CO2 fixation pathways: hybrid enzyme-microalgae process and enzyme cascade for theproduction of carboxylic acids. KEY POINTS: • Covalent immobilization techniques applied to CA are effective for CO2 ERA. • Biocatalyst type and morphology must be selected considering CO2 ERA conditions. • Immobilized CA can offer novel routes to CO2 capture and direct utilization.

Cuproptosis: Cellular and molecular mechanisms underlying copper-induced cell death

Tsvetkov etal. (2022) discovered a new form of cell death triggered by targeted accumulation of Cu in mitochondria that drives lipoylated TCA cycle enzyme aggregation via direct Cu binding.

Synthesis and Characterization of Laccase Enzyme Aggregates From Trametes villosa for Simultaneous Elimination of Rifampicin and Isoniazid

Synthesis and Characterization of Laccase Enzyme Aggregates From Trametes villosa for Simultaneous Elimination of Rifampicin and Isoniazid

Controllably crosslinked dual enzymes enabled by genetic-encoded non-standard amino acid for efficiently enantioselective hydrogenation.

In traditional method for preparing crosslinked enzymes aggregates using glutaraldehyde, random linkage is inevitable, which often destroys the enzyme active sites and severely decreases the activity. To address this issue, using genetic encode expanding, nonstandard amino acids (NSAAs) were inserted into enzyme proteins at the preselected sites for crosslinking. When aldehyde ketone reductase (AKR), alcohol dehydrogenase (ADH) and glucose dehydrogenase (GDH) were utilized as model enzymes, their mutants containing p-azido-L-phenylalanine were bio-orthogonally crosslinked with diyne to form crosslinked dual enzymes (CLDEs) acting as a cascade biological oxidation and reduction system. Then, the resultant self-purified CLDEs were characterized using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM), etc. In the asymmetric synthesis of (S)-1-(2,6-dichloro-3-fluorophenyl) ethanol using CLDEs, high product yield (76.08%), ee value (99.99%) and reuse stability were achieved. The yield and ee value were 12.05 times and 1.39 times higher than those using traditional crosslinked enzyme aggregates, respectively. Thus, controllable insertion NSAAs in number and location can engender reasonable linkage and metal-free self-purification for target enzyme proteins. This facile and sustainable method could be further expanded to other dual and multienzyme systems for cascade biocatalysis.

Concurrent production of cellulase, xylanase, pectinase and immobilization by combined Cross-linked enzyme aggregate strategy- advancing tri-enzyme biocatalysis

In this study Bacillus sp. isolated from snail droppings displayed ability to coproduce cellulase, xylanase and pectinase by solid state fermentation on citrus peel. The enzymes were immobilized by cross-linking enzyme aggregate (CLEA). Parameters like cross-linking concentration/cross-linking time, pH, thermal stability and reusability were evaluated. Bacillus sp. expressed cellulase (2.563 ± 0.082 U/gds), xylanase (2.253 ± 0.101 U/gds) and pectinase (2.910 ± 0.097 U/gds) activity. Moisture content (75%), pH 5, particle size 348 nm, One (1 mL) mililiter inoculum/nitrogen were best for enzymes production. Optimum aggregation of enzyme complex with glutaraldehyde concentration (50 mM) and cross-linking time 3 h was achieved using three-phase portioning (TPP). Combined-CLEAs expressed thermal stability. Cellulase, xylanase and pectinase, retained 90% of initial activity at 1 h, over 65% after 3 h incubation at 50 °C; optimum activity was pH 6. After 4 cycles combined-CLEAs retained over 80% activity.

The Synthesis of (Magnetic) Crosslinked Enzyme Aggregates With Laccase, Cellulase, β-Galactosidase and Transglutaminase.

Immobilized enzymes have important aspects due to the fact that they possess higher stability, have the possibility to be easily removed from the reaction mixture, and are much easier to use when compared to free enzymes. In this research, the enzymes laccase, cellulase, β-galactosidase (β-gal), and transglutaminase (TGM) were immobilized by two different methods: crosslinked enzyme aggregates (CLEAs) and magnetic crosslinked enzyme aggregates (mCLEAs). The processes for CLEAs and mCLEAs preparation with different enzymes have been optimized, where the aim was to achieve the highest possible relative activity of the immobilized enzyme. The optimal conditions of the synthesis of CLEAs in mCLEAs are described, thus emphasizing the difference between the two types of immobilization based on different enzymes. This comparative study, which represents the synthesis of crosslinked enzyme aggregates using different enzymes, has not been performed so far. Moreover, the obtained activity of CLEAs and mCLEAs is presented, which is important for further use in different biocatalytic processes. Specifically, of a higher importance is the selection of enzymes involved in immobilization, as they belong to the three different most applicable enzymes (oxidoreductases, hydrolases, and transferases). The study confirmed that the resulting activity of the immobilized enzyme and the optimization of enzyme immobilization depended on the type of the enzyme. Moreover, the prepared CLEAs and mCLEAs were exposed to the supercritical carbon dioxide (scCO2) at different pressures to determine the effect of scCO2 on enzyme activity in immobilized form. Additionally, to demonstrate the reuse and stability of the immobilized enzyme, the stability and reusability tests of CLEAs and mCLEAs were performed. The catalytic performance of immobilized enzyme was tested, where the catalytic efficiency and long-term operational stability of mCLEAs were obviously superior to those of CLEAs. However, the higher activity observed for CLEAs compared to mCLEAs suggests a significant effect of magnetic nanoparticles in the stabilization of an enzyme crosslinked aggregate structure.

Cross-linked enzyme aggregates (CLEAs) of cellulase with improved catalytic activity, adaptability and reusability.

In this study, cross-linked cellulase aggregates (C-CLEAs) were synthesized by precipitation of cellulase with ammonium sulfate and then cross-linking with glutaraldehyde. The results revealed that the optimal pH of C-CLEAs shifted toward a more acidic environment by 2.0 pH units, and the optimal temperature shifted toward higher temperature by 20°C after immobilization. The half-life (t1/2) and inactivation energy (Ed) values of the C-CLEAs were 5.98 times and 1.93 times than that of free cellulase, respectively. Moreover, the C-CLEAs can also maintain about 65.22% of activity after 10 cycles and 63.03% of activity after storage for 56days at 4°C. Enzymatic hydrolysis of carboxymethylcellulose sodium and corncob in C-CLEAs system verified that the C-CLEAs performed better than free cellulase (P < 0.01).

Lipases: Sources of Acquisition, Ways of Production, and Recent Applications

Enzymes are extensively used in biotechnological processes in several areas of industry. They are sustainable and safe, and their specificity is another characteristic that improves the performance in the process. Among enzymes, lipase is relevant due to the ability to play different roles in the industry and the possibility of collecting them from microbial sources that are found in industrial residues. This can reduce the costs of enzyme production. In relation to that, lipase immobilization is an interesting process that allows the enzymes to be reused and improves enzyme robustness. Among them, the cross-linked enzyme aggregates (CLEAs) methodology is attractive due to its simplicity, low cost (given the absence of support), and greater interaction with the substrate. Thus, in this review, we discussed the potential of lipase. We reviewed the traditional and new sources of obtaining lipases, along with the ways of improving production, activity, and application in the industry.

Optimization of cephalosporin C acylase immobilization using crosslinked enzyme aggregates technique

Cephalosporin C acylase (CCA) is an essential enzyme for the one-step conversion of cephalosporin C into 7-aminocephalosporanic acid (7-ACA), an intermediate compound used to synthesize various semi-synthetic cephalosporin antibiotics. The industrial process prefers to use enzymes in immobilized form rather than soluble. A crosslinked enzyme aggregate (CLEAs) is a potential matrix-less enzyme immobilization technique to produce stable immobilized enzymes with high activity and low production costs. This study aimed to optimize the CCA immobilization using the CLEAs technique with Chitosan as a co-aggregate. The CCA lysate was obtained from harvesting CCA fermentation broth using a mutant strain of Escherichia coli through cell separation and lysis steps. Partially purified CCA by ammonium sulfate addition was conducted to obtain an active fraction of 20-60% saturation, followed by co-aggregation with Chitosan to form physical CCA aggregates. The aggregates were then immobilized by a crosslinking technique using glutaraldehyde to form CLEAs-CCA. Optimization of the immobilization process was carried out by Response Surface Methodology in three steps, (i) screening of the influencing factors, (ii) determining the level of the significant factors, and (iii) optimizing the immobilization condition. The CLEAs-CCA activity was used as a response parameter. Under optimum conditions, CLEAs-CCA activity obtained was 85.91 Ug-1.

Application of undecanedicarboxylic acid to prepare cross-linked enzymes (CLEs) of Rhizomucor miehei lipase (RML); Selective enrichment of polyunsaturated fatty acids

We have introduced 1,11-undecanedicarboxylic acid as a new cross-linker to prepare cross-linked preparation of Rhizomucor miehei lipase (RML) from its soluble form by applying an isocyanide-based multi-component reaction. This one-step cross-linking of soluble enzyme allowed skipping the prior step of enzyme aggregation or crystallization which is inevitable in the traditional methods of cross-linking. High immobilization yields were achieved up to 92% in an extremely mild condition. The addition of bovine serum albumin (BSA) as a feeder protein affected the final properties of RML by producing 2.5-fold higher specific activity compared to the specific activity of free RML. Furthermore, dynamic light scattering analysis (DLS) showed that by applying BSA, the cross-linked enzymes (CLEs) particle sizes of 1.75–3.91 µm were obtained. Considerable improvement in the thermal and co-solvent stability was observed, in particular for the derivative obtained by immobilization in the presence of BSA. This derivative remained considerably active at 60 °C by keeping 79% of its initial activity after 2 h of incubation and retained 75% and 86% of its activity after 24 h incubation in the presence of 20% of methanol and ethanol, respectively. The cross-linked preparations were then applied for the selective hydrolysis of the essential omega-3 fatty acids, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), from fish oil. The results showed that the immobilized derivatives tend to preferentially hydrolyze EPA.

Insolubilization of Tramates versicolor laccase as cross-linked enzyme aggregates for the remediation of trace organic contaminants from municipal wastewater

The novelty of this study deals with the biocatalytic treatment of trace organic contaminants (TrOCs) from municipal wastewater by insolubilized laccase. Laccase from Trametes versicolor was aggregated by three-phase partitioning technique followed by cross-linking with glutaraldehyde to produce insolubilized laccase as cross-linked enzyme aggregates (CLEAs). The optimal conditions for CLEAs preparation include ammonium sulphate concentration of 83% (w/v), crude to t-butanol ratio of 1.00: 1.05 (v/v), pH 5.3, and glutaraldehyde concentration of 20 mM obtained via statistical design. The efficiency of insolubilization of the CLEAs laccase based on the kcat/km ratio was approximately 4.8-fold greater than that of free laccase. The developed CLEAs showed greater resistance to product inhibition mediated by ABTS than the free enzyme and exhibited excellent catalytic activity even after the tenth successive cycle. Further, free laccase and the synthesized CLEAs laccase were utilized to treat five analgesics, two NSAIDS, three antibiotics, two antilipemics, and three pesticides in the municipal wastewater. Under the batch process with operating conditions of pH 7.0 and 20 °C, 1000 U/L of CLEAs, laccase removed 11 TrOCs in the range of about 20–99%. However, the inactivated CLEAs only adsorbed 2–25% of TrOCs. It was observed that acetaminophen, mefenamic acid, trimethoprim, and metolachlor depicted almost complete removal with CLEAs laccase. The performance of CLEAs laccase in a perfusion basket reactor was tested for the removal of TrOCs from municipal wastewater.

Preparation of enantiopure pregabalin intermediate using cross linked enzyme aggregates (CLEAs) in basket reactor

Route to the synthesis of enantiomerically pure ethyl (S)-3-cyano-5-methylhexanoate, (S)-5, a key chiral intermediate for Pregabalin has been improved. The racemic β-cyano diester, 3 was prepared in 98% purity via gelatine catalysed Knoevenagel condensation of diethylmalonate with isovaleraldehyde followed by hydrocyantion of α,β-unsaturated diester 14 using acetone cyanohydrin and K2CO3. Racemic diethyl 2-(1-cyano-3-methylbutyl)malonate, rac-3, has been resolved using lipase from Thermomyces lanuginosus immobilised in form of crosslinked enzyme aggregates, CLEAs. The CLEAs were made by employing commercial soymilk as an additional protein source and a reaction was carried out in a moving basket reactor. The immobilised enzyme was found to be stable in many organic solvents and temperature up to 50 °C. The resolution reaction was studied in a basket reactor at 50% substrate loading in calcium acetate buffer, pH 7.5 at 30 °C by using 20% w/w enzyme loading. The apparent kinetic parameters were V max,app = (8.74 ± 0.43) mM/h/g and K m,app = (1.5 ± 0.07) M (correlation coeff. r = 0.98). The desired ethyl (S)-3-cyano-5-methylhexanoate, (S)-5 is obtained in 90-92% theoretical yield and e.e > 99%. The advantages of this improved process are mild reaction conditions; an alternate method for hydrocyanation step avoiding the use of highly toxic potassium cyanide at large scale operation and an immobilised enzyme that can be reused for at least 11 recycles.

Ultrasound-Assisted Intensification of β-Glucosidase Enzyme Activity in Free and Immobilized Forms

The focus of the current work was to enhance the productivity and activity of enzymes based on immobilization and application of ultrasound. β-Glucosidase was immobilized using a cross-linking method that was a carrier-free approach. Parameters such as the ammonium sulfate concentration (60–90%), glutaraldehyde concentration (0.5–1.5%), and use of bovine serum albumin (BSA) were optimized during the preparation of cross-linked enzyme aggregates (CLEAs). The optimum temperature (50 °C) and pH (5) were observed to be the same for both the soluble and immobilized enzymes in CLEAs. Use of 1% glutaraldehyde concentration gave a recovery of 30%, but after adding BSA (0.3 mM) the recovery enhanced to 57%. The effect of ultrasonic treatment on enzyme activity was analyzed using an ultrasonic bath with maximum activity observed at a 30% duty cycle, a 50 W power, and a treatment time of 20 min with an actual value of 9.7 U/mL, which was higher as compared to that of the untreated enzyme (8.86 U/mL). The enzyme immobilized after pretreatment with ultrasound allowed an activity recovery of 82% that was significantly higher compared to that of the untreated immobilized enzyme. The immobilized enzyme prepared with a pretreated enzyme exhibited higher reusability and thermal stability as compared to the enzyme immobilized with the untreated enzyme. Overall, an effective method for immobilization of enzymes along with the use of ultrasound for intensification was demonstrated.

Co-Cross-Linked Aggregates of Baeyer–Villiger Monooxygenases and Formate Dehydrogenase for Repeated Use in Asymmetric Biooxidation

Baeyer–Villiger monooxygenases (BVMOs) are versatile biocatalysts, but their applications are hindered by their poor stability and cofactor dependence. In this study, cross-linked enzyme aggregate (CLEA) technology was adopted to coimmobilize BVMO and its accessory cofactor-regeneration enzyme. Combi-CLEAs of a pyrmetazole monooxygenase from Acinetobacter calcoaceticus (AcPSMO) and a formate dehydrogenase from Burkholderia stabili (BstFDH) were prepared for the synthesis of (S)-omeprazole. After optimization, AcPSMO and BstFDH were coprecipitated with an activity ratio of 1:6 using ammonium sulfate and then cross-linked with glutaraldehyde (0.12% w/v). The activity recoveries of AcPSMO and BstFDH in the prepared combi-CLEAs were 43% and 38%, respectively. Compared with the free enzymes AcPSMO and BstFDH, the thermostabilities of AcPSMO and BstFDH in combi-CLEAs were improved by 2.5- and 1.6-fold, respectively. Both enzymes were more stable against alkaline buffer after being immobilized. The combi-CLEAs could be reused for seven cycles in the biooxidative synthesis of (S)-omeprazole without significant activity loss, indicating the excellent operational stability and reusability in repeated reactions for the enzymatic synthesis of (S)-omeprazole. Another two combi-CLEAs prepared under the same conditions, TmCHMO–BstFDH and RpBVMO–BstFDH, can be reused for at least 15 consecutive batches for the cyclohexanone mono-oxygenation reaction, which indicates the promising potential for coimmobilization of BVMOs and FDH with CLEA methodology.

Nonlinear Analytics for Electrochemical Biosensor Design Using Enzyme Aggregates and Delayed Mass Action

The paper is devoted to the extension of Brown’s model of enzyme kinetics to the case with distributed delays. Firstly, we construct a multi-substrate multi-inhibitor model using discrete and distributed delays. Furthermore, we consider simplified models including one substrate and one inhibitor, for which an experimental study has been performed. The algorithm of parameter identifications was developed which was tested on the experimental data of solution conductivity. Both the model and Kohlrausch’s law parameters are obtained as a result of the optimization procedure. Comparison of plots constructed with the help of the estimated parameters has shown that in such case the model with distributed delays is more chemically adequate in comparison with the discrete one. The methods of generalization of the results to the multi-substrate multi-inhibitor cases are discussed.

Combined action of chemical chaperones on stability, aggregation and oligomeric state of muscle glycogen phosphorylase b

Chemical chaperones are a class of small molecules, which enhance protein stability, folding, inhibit protein aggregation, and are used for long-term storage of therapeutic proteins. The combined action of chemical chaperones trehalose, betaine and lysine on stability, aggregation and oligomeric state of muscle glycogen phosphorylase b (Phb) has been studied. Dynamic light scattering data indicate that the affinity of trehalose to Phb increased in the presence of betaine or lysine at both stages (stage of nucleation and aggregate growth) of enzyme aggregation at 48 °C, in contrast, the affinity of betaine to the enzyme in the presence of lysine remained practically unchanged. According to differential scanning calorimetry and analytical ultracentrifugation data, the mixture of trehalose and betaine stabilized Phb stronger than either of them in total. Moreover, the destabilizing effect of lysine on the enzyme was almost completely compensated by trehalose and only partially by betaine. The main protective effect of the mixtures of osmolytes and lysine is associated with their influence on the dissociation/denaturation stage, which is the rate-limiting one of Phb aggregation. Thus, a pair of chaperones affects the stability, oligomeric state, and aggregation of Phb differently than individual chaperones.

SpyTag/Catcher chemistry induces the formation of active inclusion bodies in E. coli

SpyTag/Catcher chemistry is usually applied to engineer robust enzymes via head-to-tail cyclization using spontaneous intramolecular isopeptide bond formation. However, the SpyTag/Catcher induced intercellular protein assembly in vivo cannot be ignored. It was found that some active inclusion bodies had generated to different proportions in the expression of six SpyTag/Catcher labeled proteins (CatIBs-STCProtein). Some factors that may affect the formation of CatIBs-STCProtein were discussed, and the subunit quantities were found to be strongly positively related to the formation of protein aggregates. Approximately 85.44% of the activity of the octameric protein leucine dehydrogenase (LDH) was expressed in aggregates, while the activity of the monomeric protein green fluorescence protein (GFP) in aggregates was 12.51%. The results indicated that SpyTag/Catcher can be used to form protein aggregates in E. coli. To facilitate the advantages of CatIBs-STCProtein, we took the CatIBs-STCLDH as an example and further chemically cross-linked with glutaraldehyde to obtain novel cross-linked enzyme aggregates (CLEAs-CatIBs-STCLDH). CLEAs-CatIBs-STCLDH had good thermal stability and organic solvents stability, and its activity remained 51.03% after incubation at 60 °C for 100 mins. Moreover, the crosslinked CatIBs-STCLDH also showed superior stability over traditional CLEAs, and its activity remained 98.70% after 10 cycles of catalysis.