Enzyme immobilization is the core technology of biocatalysis. Over the past few decades, enzyme immobilization research mainly focused on single enzyme immobilization. In recent years, multi-enzyme immobilization attracts more and more attention as it could increase the local concentration of reaction and improve the reaction yield. In this review, a summary of the recent progress, together with our research, is presented. Special emphasis is placed on four methods in multi-enzymes co-immobilization, namely, the nonspecific covalent co-immobilization, the nonspecific non-covalent co-immobilization, the non-covalent encapsulation co-immobilized and the site specificity co-immobilized. Finally, some industrial uses of immobilized multi-enzymes were addressed and the application prospect of multi-enzyme immobilization was highlighted.
Biocatalysis ; Enzymes, Immobilized

This study was performed to prepare immobilized β-glucosidase and snailase, then optimize and compare the process conditions for conversion of icariin. Immobilized β-glucosidase and snailase were prepared using crosslink-embedding method. The best conditions of the preparation process were optimized by single factor analysis and the properties of immobilized β-glucosidase and snailase were investigated. The reaction conditions including temperature, pH, substrate ratio, substrate concentration, reaction time and reusing times of the conversion of icariin using immobilized β-glucosidase or snailase were optimized. Immobilized β-glucosidase and snailase exhibited better heat stabilities and could remain about 60% activity after storage at 4 degrees C for 4 weeks. The optimized conditions for the conversion of icariin were as follows, the temperature of 50 degrees C, pH of 5.0, enzyme and substrate ratio of 1 : 1, substrate concentration of 0.1 mg x mL(-1), reaction time of 6 h for β-glucosidase and 2 h for snailase, respectively. In 5 experiments, the average conversion ratio of immobilized β-glucosidase and snailase was 70.76% and 74.97%. The results suggest an effect of promoted stabilities, prolonged lifetimes in both β-glucosidase and snailase after immobilization. The immobilized β-glucosidase and snailase exhibited a higher conversion rate and reusability compared to the free β-glucosidase and snailase. Moreover, the conversion rate of immobilized snailase was higher than that of immobilized β-glucosidase. The process of icariin conversion using immobilized β-glucosidase and snailase was moderate and feasible, which suggests that immobilized enzymes may hold a promise for industrial usage.
Enzymes, Immobilized ; chemistry ; Flavonoids ; chemistry ; Hydrolysis ; Temperature ; beta-Glucosidase ; chemistry

The synthesis of CCK-4 (H-Trp-Met-Asp-Phe-NH2) by using enzymes exclusively was described. As protection group for the amino group we used the Phenylacetyl group (Phac) which had been cleaved at the end of the synthesis with Penicillin G Amidase (PGA) without affecting the peptide bonds. Thus, beginning with Phac-Trp-OH we had successfully synthesized the target peptide with following 4 enzymes, alpha-Chymotrypsin, Papain, Thermolysin and PGA in four reaction steps. All reactions were carried out in aqueous buffer in reasonable yields (> 65%). FAB-MS or FD-MS verified the correct molecular mass of all peptides.
Chymotrypsin ; Enzymes, Immobilized ; Papain ; Peptides ; Tetragastrin ; chemical synthesis ; Thermolysin

The synthesis of CCK-4 (H-Trp-Met-Asp-Phe-NH2) by using enzymes exclusively was described. As protection group for the amino group we used the Phenylacetyl group (Phac) which had been cleaved at the end of the synthesis with Penicillin G Amidase (PGA) without affecting the peptide bonds. Thus, beginning with Phac-Trp-OH we had successfully synthesized the target peptide with following 4 enzymes, alpha-Chymotrypsin, Papain, Thermolysin and PGA in four reaction steps. All reactions were carried out in aqueous buffer in reasonable yields (> 65%). FAB-MS or FD-MS verified the correct molecular mass of all peptides.
Chymotrypsin ; Enzymes, Immobilized ; Papain ; Peptides ; Tetragastrin/*chemical synthesis ; Thermolysin

Glycoside compounds are widely used in medicine, food, surfactant, and cosmetics. The glycosidase-catalyzed synthesis of glycoside can be operated at mild reaction conditions with low material cost. The glycosidase-catalyzed processes include reverse hydrolysis and transglycosylation, appropriately reducing the water activity in both processes may effectively improve the catalytic efficiency of glucosidase. However, glucosidase is prone to be deactivated at low water activity. Thus, glucosidase was immobilized to maintain its activity in the low water activity environment, and even in neat organic solvent system. This article summarizes the advances in glycosidase immobilization in the past 30 years, including single or comprehensive immobilization techniques, and immobilization techniques combined with genetic engineering, with the aim to provide a reference for the synthesis of glycosides using immobilized glycosidases.
Catalysis ; Enzymes, Immobilized ; Glycoside Hydrolases/genetics* ; Glycosides/biosynthesis* ; Hydrolysis

Enzyme separation, purification, immobilization, and catalytic performance improvement have been the research hotspots and frontiers as well as the challenges in the field of biocatalysis. Thus, the development of novel methods for enzyme purification, immobilization, and improvement of their catalytic performance and storage are of great significance. Herein, ferritin was fused with the lichenase gene to achieve the purpose. The results showed that the fused gene was highly expressed in the cells of host strains, and that the resulted fusion proteins could self-aggregate into carrier-free active immobilized enzymes in vivo. Through low-speed centrifugation, the purity of the enzymes was up to > 90%, and the activity recovery was 61.1%. The activity of the enzymes after storage for 608 h was higher than the initial activity. After being used for 10 cycles, it still maintained 50.0% of the original activity. The insoluble active lichenase aggregates could spontaneously dissolve back into the buffer and formed the soluble polymeric lichenases with the diameter of about 12 nm. The specific activity of them was 12.09 times that of the free lichenase, while the catalytic efficiency was 7.11 times and the half-life at 50 ℃ was improved 11.09 folds. The results prove that the ferritin can be a versatile tag to trigger target enzyme self-aggregation and oligomerization in vivo, which can simplify the preparation of the target enzymes, improve their catalysis performance, and facilitate their storage.
Biocatalysis ; Enzymes, Immobilized/metabolism* ; Ferritins/metabolism* ; Glycoside Hydrolases/metabolism*

Catalase is widely used in the food, medical, and textile industries. It possesses exceptional properties including high catalytic efficiency, high specificity, and environmental friendliness. Free catalase cannot be recycled and reused in industry, resulting in a costly industrial biotransformation process if catalase is used as a core ingredient. Developing a simple, mild, cost-effective, and environmentally friendly approach to immobilize catalase is anticipated to improve its utilization efficiency and enzymatic performance. In this study, the catalase KatA derived from Bacillus subtilis 168 was expressed in Escherichia coli. Following separation and purification, the purified enzyme was prepared as an immobilized enzyme in the form of enzyme-inorganic hybrid nanoflowers, and the enzymatic properties were investigated. The results indicated that the purified KatA was obtained through a three-step procedure that included ethanol precipitation, DEAE anion exchange chromatography, and hydrophobic chromatography. Then, by optimizing the process parameters, a novel KatA/Ca₃(PO₄)₂ hybrid nanoflower was developed. The optimum reaction temperature of the free KatA was determined to be 35 ℃, the optimum reaction temperature of KatA/Ca₃(PO₄)₂ hybrid nanoflowers was 30-35 ℃, and the optimum reaction pH of both was 11.0. The free KatA and KatA/Ca₃(PO₄)₂ hybrid nanoflowers exhibited excellent stability at pH 4.0-11.0 and 25-50 ℃. The KatA/Ca₃(PO₄)₂ hybrid nanoflowers demonstrated increased storage stability than that of the free KatA, maintaining 82% of the original enzymatic activity after 14 d of storage at 4 ℃, whereas the free KatA has only 50% of the original enzymatic activity. In addition, after 5 catalytic reactions, the nanoflower still maintained 55% of its initial enzymatic activity, indicating that it has good operational stability. The K_m of the free KatA to the substrate hydrogen peroxide was (8.80±0.42) mmol/L, and the k_cat/K_m was (13 151.53± 299.19) L/(mmol·s). The K_m of the KatA/Ca₃(PO₄)₂ hybrid nanoflowers was (32.75±2.96) mmol/L, and the k_cat/K_m was (4 550.67±107.51) L/(mmol·s). Compared to the free KatA, the affinity of KatA/Ca₃(PO₄)₂ hybrid nanoflowers to the substrate hydrogen peroxide was decreased, and the catalytic efficiency was also decreased. In summary, this study developed KatA/Ca₃(PO₄)₂ hybrid nanoflowers using Ca²⁺ as a self-assembly inducer, which enhanced the enzymatic properties and will facilitate the environmentally friendly preparation and widespread application of immobilized catalase.
Catalase ; Nanostructures/chemistry* ; Hydrogen Peroxide/metabolism* ; Enzymes, Immobilized/chemistry* ; Catalysis

As an excellent hosting matrices for enzyme immobilization, metal-organic framework (MOFs) provides superior physical and chemical protection for biocatalytic reactions. In recent years, the hierarchical porous metal-organic frameworks (HP-MOFs) have shown great potential in enzyme immobilization due to their flexible structural advantages. To date, a variety of HP-MOFs with intrinsic or defective porous have been developed for the immobilization of enzymes. The catalytic activity, stability and reusability of enzyme@HP-MOFs composites are significantly enhanced. This review systematically summarized the strategies for developing enzyme@HP-MOFs composites. In addition, the latest applications of enzyme@HP-MOFs composites in catalytic synthesis, biosensing and biomedicine were described. Moreover, the challenges and opportunities in this field were discussed and envisioned.
Metal-Organic Frameworks/chemistry* ; Porosity ; Enzymes, Immobilized/chemistry* ; Biocatalysis ; Catalysis

Enzyme-catalyzed CO₂ reduction to value-added commodities is important for alleviating the global environmental issues and energy crises due to high selectivity and mild conditions. Owing to high energy density, formic acid or methanol produced from CO₂ using formate dehydrogenase (FDH) or multi-enzyme cascades are promising target chemicals for CO₂ utilization. However, the low activity, poor stability and low reusability of key enzymes involved in such process hampered its large-scale application. Enzyme immobilization provides an effective solution to these problems and significant progress have been made in immobilization carriers. Moreover, integration of enzyme immobilization with other catalysis techniques have been explored extensively. This review summarized the recent advances in the immobilization of enzymes using membranes, inorganic materials, metal-organic frameworks, covalent organic frameworks and other carriers, and illustrated the characteristics and advantages of different immobilization materials and immobilization methods. The synergistic effects and applications of immobilized enzymes and electrocatalytic or photocatalytic coupling reaction systems for CO₂ reduction were further summarized. Finally, the current challenges of enzyme immobilization technology and coupling reaction systems were pointed out and their development prospects were presented.
Enzymes, Immobilized ; Carbon Dioxide ; Catalysis ; Formate Dehydrogenases ; Metal-Organic Frameworks

Industrial enzymes play dual roles for the production of chemicals and biochemicals, one is to act as direct catalyst for the reaction, the other is to participate in the fermentation process to convert substrates to fermentable sugars or to make it more efficient. The review briefs the applications of industrial enzymes for chemical productions, with emphasis on direct conversion of starch and their roles in bioethanol production process, also analyzes the benefits by using new enzymes and prospects for future development.
Biocatalysis ; Biochemistry ; Chemical Industry ; Enzymes ; chemistry ; metabolism ; Enzymes, Immobilized ; Ethanol ; chemistry ; Fermentation