Nanozymes are a distinct category of nanomaterials that exhibit catalytic properties resembling those of enzymes such as peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Nanozymes derived from Chinese herbal medicines exhibit the catalytic functions of their enzyme mimics, while retaining the specific medicinal properties of the herb (termed "herbzymes"). These nanozymes can be categorized into three main groups based on their method of synthesis: herb carbon dot nanozymes, polyphenol-metal nanozymes, and herb extract nanozymes. The reported catalytic activities of herbzymes include POD, SOD, CAT, and GPx. This review presents an overview of the catalytic activities and potential applications of nanozymes, introduces the novel concept of herbzymes, provides a comprehensive review of their classification and synthesis, and discusses recent advances in their biomedical applications. Furthermore, we also discuss the significance of research into herbzymes, including the primary challenges faced and future development directions.
Nanostructures/chemistry* ; Humans ; Herbal Medicine/methods* ; Superoxide Dismutase/chemistry* ; Catalase/chemistry* ; Drugs, Chinese Herbal/chemistry* ; Catalysis ; Glutathione Peroxidase/chemistry*

As important biocatalysts, nitrilases can efficiently convert nitrile groups into acids and ammonia in a mild and eco-friendly manner, being widely used in the synthesis of important pharmaceutical intermediates. Early studies reported that nitrilases only had the hydrolysis activity of catalyzing the formation of corresponding carboxylic acid products from nitriles, showing catalytic specificity. However, recent studies have shown that some nitrilases exhibit the hydration activity for catalyzing the formation of amides from nitriles, showing catalytic promiscuity. The catalytic promiscuity of nitrilases has dual effects. On the one hand, the presence of amide by-products increases the difficulties and costs of subsequent separation and purification of carboxylic acid products. On the other hand, however, if the catalytic reaction pathways of nitrilases can be precisely regulated to reshape enzyme functions, the reactions catalyzed by nitrilases can be broadened to provide new ideas for the biosynthesis of high-value amides, which is crucial for the development of artificial enzymes and biocatalysis. This review summarized the research progress in the catalytic promiscuity of nitrilases and discussed the key regulatory factors that may affect the catalytic promiscuity of nitrilases from the evolutionary origin, catalytic domains, and catalytic mechanisms, hoping to provide reference and inspiration for the application of nitrilases in biocatalysis.
Aminohydrolases/chemistry* ; Biocatalysis ; Nitriles/chemistry* ; Substrate Specificity ; Catalytic Domain ; Catalysis

2-substituted quinolines are the building blocks for the synthesis of natural products and pharmaceuticals. In comparison with classical methods, dehydroaromatization of 2-substituted-1,2,3,4-tetrahydroquinolines has emerged in recent years as an efficient and straightforward method to synthesize quinolines due to its high atom economy and sustainability. However, existing chemical methods need transition metal catalysts and harsh reaction conditions. Biocatalysis with high efficiency, high selectivity, and mild reaction conditions has become an important method of organic synthesis. We mined a strain Pseudomonas monteilii ZMU-T06 capable of producing monoamine oxidase for the dehydroaromatization of 2-substituted-1,2,3,4-tetrahydroquinolines to synthesize 2-substituted quinolines (8 substrates, yields of 45.7%-48.4%) and then hypothesized the catalytic mechanism, providing a new method for green synthesis of 2-substituted quinolines.
Quinolines/chemistry* ; Pseudomonas/classification* ; Oxidation-Reduction ; Monoamine Oxidase/biosynthesis* ; Biocatalysis

Glucose oxidase (GOD) is an oxygen-consuming dehydrogenase that can catalyze the production of gluconic acid hydrogen peroxide from glucose, and its specific mechanism of action makes it promising for applications, while the low catalytic activity and poor thermostability have become the main factors limiting the industrial application of this enzyme. In this study, we used the glucose oxidase AtGOD reported with the best thermostability as the source sequence for phylogenetic analysis to obtain the GOD with excellent performance. Six genes were screened and successfully synthesized for functional validation. Among them, the glucose oxidase AhGODB derived from Aspergillus heteromorphus was expressed in Pichia pastoris and showed better thermostability and catalytic activity, with an optimal temperature of 40 ℃, a specific activity of 112.2 U/mg, and a relative activity of 47% after 5 min of treatment at 70 ℃. To improve its activity and thermal stability, we constructed several mutants by directed evolution combined with rational design. Compared with the original enzyme, the mutant T72R/A153P showcased the optimum temperature increasing from 40 to 50 ℃, the specific activity increasing from 112.2 U/mg to 166.1 U/mg, and the relative activity after treatment at 70 ℃ for 30 min increasing from 0% to 33%. In conclusion, the glucose oxidase mutants obtained in this study have improved catalytic activity and thermostability, and have potential for application.
Glucose Oxidase/chemistry* ; Enzyme Stability ; Aspergillus/genetics* ; Pichia/metabolism* ; Temperature ; Catalysis ; Fungal Proteins/metabolism* ; Hot Temperature

As a biocatalyst, laccase has been widely studied and applied in the papermaking industry. However, the low catalytic efficiency and poor stability of natural laccase limit its application in the pulping process. To develop the laccase with high activity and strong tolerance, we carried out directed evolution for modification of the laccase derived from Bacillus pumilus and screened out the mutants F282L/F306L and Q275P from the random mutant library by high-throughput screening. The specific activities of F282L/F306L and Q275P were 280.87 U/mg and 453.94 U/mg, respectively, which were 1.42 times and 2.30 times that of the wild-type laccase. Q275P demonstrated significantly improved thermal stability, with the relative activity 20% higher than that of the wild-type laccase after incubation at 40 ℃, 50 ℃, and 70 ℃ for 4 h. F282L/F306L and Q275P showed greater tolerance to metal ions and organic solvents than the wild-type laccase. The K_m value of the wild-type laccase was 374.97 μmo/L, and those of F282L/F306L and Q275P were reduced to 318.96 μmo/L and 360.71 μmo/L, respectively, which suggested that the substrate affinity of laccase was improved after mutation. The k_cat values of F282L/F306L and Q275P for the substrate ABTS were 574.00 s^-1 and 898.03 s^-1, respectively, which were 1.1 times and 1.7 times that of the wild-type laccase, indicating the improved catalytic efficiency. Q275P demonstrated better performance than the wild-type laccase in pulping, as manifested by the reduction of 0.82 in the Kappa number and the increases of 2.00% ISO, 7.8%, and 7.2% in whiteness, tensile index, and breaking length, respectively. This work lays a foundation for improving the adaptation of laccase to the environment of the papermaking industry.
Laccase/chemistry* ; Directed Molecular Evolution ; Enzyme Stability ; Bacillus pumilus/genetics* ; Mutation ; Biocatalysis ; Catalysis

As the chip of synthetic biology, enzymes play a vital role in the bio-manufacturing industry. The development of diverse functional enzymes can provide a rich toolbox for the development of synthetic biology. This article reports the construction of an artificial enzyme with the introduction of a non-natural cofactor. By introducing the 4-dimethylaminopyridine (DMAP) cofactor into the optimal protein skeleton via covalent bonds based on a click-chemistry strategy, we successfully constructed a novel artificial enzyme with the DMAP cofactor as the catalytic center. The artificial enzyme successfully catalyzed an unnatural asymmetric Morita-Baylis- Hillman (MBH) reaction between cycloketenone and p-nitrobenzaldehyde, with a conversion rate of 90% and enantioselectivity (e.e.) of 38%. This study not only provides an effective strategy for the design of new artificial enzymes but also establishes a theoretical basis for the development of unnatural biocatalytic MBH reactions.
Biocatalysis ; 4-Aminopyridine/chemistry* ; Enzymes/metabolism* ; Coenzymes/chemistry* ; Benzaldehydes/chemistry* ; Protein Engineering/methods* ; Click Chemistry

β-hydroxy-β-methylbutyric acid (HMB) is widely applied in sports nutrition, disease prevention and other fields. However, chemical synthesis methods, limited by toxic reagents and violent reactions, can hardly meet the demands of green production. The biosynthesis method mainly utilizes enzymatic catalysis or metabolic engineering techniques for synthesis, and has the advantages of high efficiency, low cost, and sustainability. Therefore, the production of HMB by the biosynthesis method has a good application prospect. In this research, a biosynthesis-based production strategy for HMB was developed. By using L-leucine as the substrate and constructing a dual-enzyme co-expression system, we established an efficient catalytic process. At first, the enzymatic properties of L-amino acid deaminase (PvL-AAD) from Proteus vulgaris and 4-hydroxyphenylpyruvate dioxygenase (Rn4-HPPD) from Rattus norvegicus were characterized. Rn4-HPPD had low relative activity and required an acidic environment for catalysis. Based on the surface charge modification strategy of the enzyme protein, site-directed mutagenesis and combinatorial mutagenesis were conducted on 10 sites of Rn4-HPPD. A double mutant Rn4-HPPD^H18R/N302R was thus obtained, with the enzyme activities being 2.00 times and 2.39 times that of the wild type at pH 5.5 and pH 6.5, respectively. Subsequently, the expression of the two enzymes in Escherichia coli was optimized. After the optimal expression ratio of the two enzymes was determined as 1:3 and under the conditions of OD₆₀₀ of 70, pH 6.0, 35 ℃, Fe²⁺ concentration of 1.5 mmol/L, and feeding of the substrate in batches in a 5 L fermenter, the maximum yield of HMB reached 8.60 g/L. This study not only enhances the optimal pH and activity of Rn4-HPPD but also provides new approaches for the efficient microbial synthesis of HMB.
Escherichia coli/metabolism* ; Valerates/metabolism* ; Recombinant Proteins/biosynthesis* ; Animals ; Metabolic Engineering/methods* ; Rats ; Catalysis

Ghrelin, a hormone mainly produced and released by the stomach, has numerous functions, including releasing growth hormones, regulating appetite, and processing sugar and lipids. Researchers have made great efforts to study the relationship between ghrelin and metabolic diseases. It is believed that human butyrylcholinesterase (hBChE) could hydrolyze ghrelin to the inactive form (desacyl-ghrelin). However, the low catalytic activity of wild hBChE against ghrelin hinders the clinical application. Recently, a soluble catalytically active hBChE mutant was successfully expressed in Escherichia coli for the first time. We then adopted HotSpot Wizard 3.0 to analyze the mutant structure and rationally selected 10 mutants. Furthermore, we determined the catalytic activities of the mutants against several substrates and the thermostability of these mutants. The results showed that the mutants E197D and A199S improved catalytic activity against ghrelin by 4.6 times and 3.5 times, respectively. The findings provide clues for treating endocrine diseases with the agents for regulating ghrelin.
Ghrelin/genetics* ; Butyrylcholinesterase/genetics* ; Humans ; Escherichia coli/metabolism* ; Mutation ; Catalysis ; Recombinant Proteins/metabolism*

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

Polyethylene terephthalate (PET) is one of the most widely used synthetic polyester. It poses serious threat to terrestrial, aquatic ecosystems and human health since it is difficult to be broken down and deposited in the environment. The biodegradation based on enzymatic catalysis offers a sustainable method for recycling PET. A number of PET hydrolases have been discovered in the last 20 years, and protein engineering has increased their degradation capabilities. However, no PET hydrolases that are practical for widespread industrial use have been identified. Screening of PET hydrolase using conventional detection techniques is laborious and inefficient process. Effective detection techniques are required to promote the commercialization of PET hydrolases. Using efficient detection techniques to screen potent industrial enzymes is essential for supporting the widespread industrial implementation of PET hydrolases. To define PET hydrolase, scientists have created a number of analytical techniques recently. The detection techniques that can be used to screen PET hydrolase, including high performance liquid chromatography, ultraviolet absorption spectrometric, and fluorescence activated droplet sorting method, are summarized in this study along with their potential applications.
Humans ; Polyethylene Terephthalates ; Ecosystem ; Biodegradation, Environmental ; Catalysis ; Hydrolases