Efficient biosynthesis of γ-aminobutyric acid by rationally engineering the catalytic pH range of a glutamate decarboxylase from Lactobacillus plantarum / 生物工程学报

γ-aminobutyric acid can be produced by a one-step enzymatic reaction catalyzed by glutamic acid decarboxylase. The reaction system is simple and environmentally friendly. However, the majority of GAD enzymes catalyze the reaction under acidic pH at a relatively narrow range. Thus, inorganic salts are usually needed to maintain the optimal catalytic environment, which adds additional components to the reaction system. In addition, the pH of solution will gradually rise along with the production of γ-aminobutyric acid, which is not conducive for GAD to function continuously. In this study, we cloned the glutamate decarboxylase LpGAD from a Lactobacillus plantarum capable of efficiently producing γ-aminobutyric acid, and rationally engineered the catalytic pH range of LpGAD based on surface charge. A triple point mutant LpGADS24R/D88R/Y309K was obtained from different combinations of 9 point mutations. The enzyme activity at pH 6.0 was 1.68 times of that of the wild type, suggesting the catalytic pH range of the mutant was widened, and the possible mechanism underpinning this increase was discussed through kinetic simulation. Furthermore, we overexpressed the Lpgad and LpgadS24R/D88R/Y309K genes in Corynebacterium glutamicum E01 and optimized the transformation conditions. An optimized whole cell transformation process was conducted under 40 ℃, cell mass (OD600) 20, 100 g/L l-glutamic acid substrate and 100 μmol/L pyridoxal 5-phosphate. The γ-aminobutyric acid titer of the recombinant strain reached 402.8 g/L in a fed-batch reaction carried out in a 5 L fermenter without adjusting pH, which was 1.63 times higher than that of the control. This study expanded the catalytic pH range of and increased the enzyme activity of LpGAD. The improved production efficiency of γ-aminobutyric acid may facilitate its large-scale production.

Research progress on application of multi-enzyme-catalyzed cascade reactions in enzymatic synthesis of natural products / 中国中药杂志

As a biocatalyst, enzyme has the advantages of high catalytic efficiency, strong reaction selectivity, specific target products, mild reaction conditions, and environmental friendliness, and serves as an important tool for the synthesis of complex organic molecules. With the continuous development of gene sequencing technology, molecular biology, genetic manipulation, and other technologies, the diversity of enzymes increases steadily and the reactions that can be catalyzed are also gradually diversified. In the process of enzyme-catalyzed synthesis, the majority of common enzymatic reactions can be achieved by single enzyme catalysis, while many complex reactions often require the participation of two or more enzymes. Therefore, the combination of multiple enzymes together to construct the multi-enzyme cascade reactions has become a research hotspot in the field of biochemistry. Nowadays, the biosynthetic pathways of more natural products with complex structures have been clarified, and secondary metabolic enzymes with novel catalytic activities have been identified, discovered, and combined in enzymatic synthesis of natural/unnatural molecules with diverse structures. This study summarized a series of examples of multi-enzyme-catalyzed cascades and highlighted the application of cascade catalysis methods in the synthesis of carbohydrates, nucleosides, flavonoids, terpenes, alkaloids, and chiral molecules. Furthermore, the existing problems and solutions of multi-enzyme-catalyzed cascade method were discussed, and the future development direction was prospected.

Advances in enzyme immobilization based on hierarchical porous metal-organic frameworks / 生物工程学报

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.

Preparation and catalytic properties of catalase-inorganic hybrid nanoflowers / 生物工程学报

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 Km of the free KatA to the substrate hydrogen peroxide was (8.80±0.42) mmol/L, and the kcat/Km was (13 151.53± 299.19) L/(mmol·s). The Km of the KatA/Ca3(PO4)2 hybrid nanoflowers was (32.75±2.96) mmol/L, and the kcat/Km 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.

Improving the activity of creatinase from Alcaligenes sp. KS-85 through semi-rational design / 生物工程学报

Creatinine levels in biological fluids are important indicators for the clinical evaluation of renal function. Creatinase (CRE, EC3.5.3.3) is one of the key enzymes in the enzymatic measurement of creatinine concentration, and it is also the rate-limiting enzyme in the whole enzymatic cascade system. The poor catalytic activity of CRE severely limits its clinical and industrial applications. To address this issue, a semi-rational design is applied to increase the activity of a creatinase from Alcaligenes sp. KS-85 (Al-CRE). By high-throughput screen of saturation mutagenesis libraries on the selected hotspot mutations, multiple variant enzymes with increased activity are obtained. The five-point best variant enzyme (I304L/F395V/K351V/Y63S/Q88A) were further obtained by recombine the improved mutations sites that to showed a 2.18-fold increased specific activity. Additionally, structure analysis is conducted to understand the mechanism of the activity change. This study paves the way for a better practical application of creatinase and may help further understand its catalytic mechanism.

Molecular engineering and immobilization of lysine decarboxylase for synthesis of 1, 5-diaminopentane: a review / 生物工程学报

1, 5-diaminopentane, also known as cadaverine, is an important raw material for the production of biopolyamide. It can be polymerized with dicarboxylic acid to produce biopolyamide PA5X whose performances are comparable to that of the petroleum-based polyamide materials. Notably, biopolyamide uses renewable resources such as starch, cellulose and vegetable oil as substrate. The production process does not cause pollution to the environment, which is in line with the green and sustainable development strategy. The biosynthesis of 1, 5-diaminopentane mainly includes two methods: the de novo microbial synthesis and the whole cell catalysis. Lysine decarboxylase as the key enzyme for 1, 5-diaminopentane production, mainly includes an inducible lysine decarboxylase CadA and a constituent lysine decarboxylase LdcC. Lysine decarboxylase is a folded type Ⅰ pyridoxal-5' phosphate (PLP) dependent enzyme, which displays low activity and unstable structure, and is susceptible to deactivation by environmental factors in practical applications. Therefore, improving the catalytic activity and stability of lysine decarboxylase has become a research focus in this field, and molecular engineering and immobilization are the mainly approaches. Here, the mechanism, molecular engineering and immobilization strategies of lysine decarboxylase were reviewed, and the further strategies for improving its activity and stability were also prospected, with the aim to achieve efficient production of 1, 5-diaminopentane.

Catalysis at gold-ligand interfaces: a frustrated Lewis pair mechanism for selective reduction reaction / Catalise na interface ouro-ligante: pares de Lewis frustrados aplicados em reações de redução seletivas

The interaction of the organic ligands with metal nanoparticle has a very important role for applications in catalysis, as well as other processes involving ligands that can activate or poison the surface of metal nanoparticles. Very little has been studied so far on the role of organic ligands used either in the preparation of nanoparticles for applications in catalysis or addition in the reaction to activate the catalyst. In this thesis, we have studied strategies for the synthesis of metal nanoparticles, their use as components for the preparation of supported catalysts and activation and deactivation processes involving the ligands used as stabilizers or purposely added to the reaction medium or support for stimulate new reactivity and selectivity in reactions of industrial interest, such as hydrogenation. Here, the concept of frustrated Lewis pairs (FLPs) has been expanded to surface-FLP analogous formed by combining gold nanoparticles (NPs) and Lewis bases, such as amines or phosphines, creating a new channel for the heterolytic cleavage of H2, and thereby performing selective hydrogenation reactions with gold. A first approach to improve the catalytic activity of gold nanoparticles was to analyze the effect of nitrogen-containing bases. The starting inactive gold nanoparticles became highly active for the selective hydrogenation of alkyne into cis-alkenes. The hydrogenation proceeded smoothly and fully selective using H2 as the hydrogen source and under relatively mild conditions (80 °C, 6 bar H2). Our studies also have revealed that the presence of capping ligands blocks the adsorption of the amine to the gold surface, avoiding the FLPs interface and thereby leading to low catalytic activity. When the capping ligands were removed from the catalyst surface and an amine ligand was added, the FLPs interface is recovered and an enhanced catalytic activity was observed. Furthermore, we have demonstrated the successful use of simple organophosphorus ligands to boost the catalytic activity of Au NPs for a range of important reduction reactions, namely, epoxides, N-oxides, sulfoxides, and alkynes. Furthermore, the choice of phosphorus-containing ligands resulted in a decrease in the amount necessary to reach high conversion and selectivity in comparison with our previous study with N-containing ligands. The ligand-to-metal ratio decreased from 100 (amine/Au) to 1 (phosphite/Au). The synthesis of gold nanoparticles supported on N-doped carbon supports was used as an alternative method for the synthesis of a heterogeneous active gold catalyst for selective hydrogenations. The main advantage with respect to previous studies was to avoid the addition of external ligands, in large excess, for the activation of gold surfaces via FLP, making the whole process environmentally and economically attractive

A interação dos ligantes orgânicos com nanopartículas de metal certamente tem um papel muito importante para aplicações em catálise, bem como outros processos envolvendo ligantes que podem ativar ou envenenar a superfície de nanopartículas metálicas. Até agora, muito pouco foi estudado sobre o papel dos ligantes orgânicos utilizados na preparação de nanopartículas para aplicações em catálise ou adição na reação para ativar o catalisador. Nesta tese, foram estudadas estratégias para a síntese de nanopartículas metálicas, seu uso como componentes para a preparação de catalisadores suportados e processos de ativação e desativação envolvendo ligantes empregados como estabilizantes ou propositalmente adicionados ao meio de reação ou suporte para estimular novas reatividades e seletividade em reações de interesse industrial, como reações de hidrogenação. Aqui, o conceito de pares de Lewis frustrados (FLPs) foi expandido para o seu análogo de superfície formado pela combinação de nanopartículas (NPs) de ouro e bases de Lewis, como aminas ou fosfinas, criando um novo canal para a clivagem heterolítica de H2 e, assim, realizando reações seletivas de hidrogenação com ouro. Uma primeira abordagem para melhorar a atividade catalítica das nanopartículas de ouro foi analisar o efeito de bases contendo nitrogênio. As nanopartículas de ouro inicialmente inativas tornaram-se altamente ativas para a hidrogenação seletiva de alquino em cis-alquenos. A hidrogenação prosseguiu foi factível e totalmente seletiva usando H2 como fonte de hidrogênio e sob condições relativamente amenas (80 °C, 6 bar de H2). Nossos estudos também revelaram que a presença de estabilizantes pode bloquear a adsorção da base na superfície do ouro, impedindo a formação da interface FLPs e, portanto, levando a baixa atividade catalítica. Quando os estabilizantes foram removidos da superfície do catalisador e um ligante foi adicionado, o FLPs é formado sendo a atividade catalítica aprimorada. Além disso, demonstramos o uso bem-sucedido de ligantes organofosforados atuando como ativadores de Au NPs em uma série de importantes reações de redução, como, epóxidos, N-óxidos, sulfóxidos e alquinos. Além disso, a escolha do ligante fosforado resultou em uma diminuição na quantidade necessária para alcançar alta conversão mantendo a seletividade inalterada. A relação ligante/metal diminuiu de 100/1 (amina/Au) para 1/1 (fosfito/Au). A síntese de nanopartículas de ouro suportadas em carbono dopado com nitrogênio foi utilizada como método alternativo para a síntese de um catalisador heterogêneo de ouro ativo para hidrogenações seletivas. A principal vantagem em relação aos estudos anteriores foi evitar a adição de ligantes externos, em grande excesso, para a ativação de superfícies de ouro via FLP, tornando todo o processo ambiental e economicamente atraente

Formate dehydrogenase and its application in biomanufacturing of chiral chemicals / 生物工程学报

The redox biosynthesis system has important applications in green biomanufacturing of chiral compounds. Formate dehydrogenase (FDH) catalyzes the oxidation of formate into carbon dioxide, which is associated with the reduction of NAD(P)+ into NAD(P)H. Due to this property, FDH is used as a crucial enzyme in the redox biosynthesis system for cofactor regeneration. Nevertheless, the application of natural FDH in industrial production is hampered by low catalytic efficiency, poor stability, and inefficient coenzyme utilization. This review summarized the structural characteristics and catalytic mechanism of FDH, as well as the advances in protein engineering of FDHs toward improved enzyme activity, catalytic efficiency, stability and coenzyme preference. The applications of using FDH as a coenzyme regeneration system for green biomanufacturing of chiral compounds were summarized.

Advances of enzymes in the applications of disease treatment and drug preparation / 生物工程学报

The development of biotechnology and the in-depth research on disease mechanisms have led to increased application of enzymes in the treatment of diseases. In addition, enzymes have shown great potential in drug manufacturing, particularly in production of non-natural organic compounds, due to the advantages of mild reaction conditions, high catalytic efficiency, high specificity, high selectivity and few side reactions. Moreover, the application of genetic engineering, chemical modification of enzymes and immobilization technologies have further improved the function of enzymes. This review summarized the advances of using enzymes as drugs for disease treatment or as catalysts for drug manufacturing, followed by discussing challenges, potential solutions and future perspectives on the application of enzymes in the medical and pharmaceutical field.

Catalytic mechanism, molecular engineering and applications of threonine aldolases / 生物工程学报

Threonine aldolases catalyze the aldol condensation of aldehydes with glycine to furnish β-hydroxy-α-amino acid with two stereogenic centers in a single reaction. This is one of the most promising green methods for the synthesis of optically pure β-hydroxy-α-amino acid with high atomic economy and less negative environmental impact. Several threonine aldolases from different origins have been identified and characterized. The insufficient -carbon stereoselectivity and the challenges of balancing kinetic versus thermodynamic control to achieve the optimal optical purity and yield hampered the application of threonine aldolases. This review summarizes the recent advances in discovery, catalytic mechanism, high-throughput screening, molecular engineering and applications of threonine aldolases, with the aim to provide some insights for further research in this field.

Application of immobilized glycosidase in the synthesis of glycoside compounds / 生物工程学报

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.

Advances in anti-adversity of biological composites based on metal-organic frameworks / 生物工程学报

Metal-organic frameworks (MOFs) are formed by self-assembly of metal ions or clusters with organic ligands, and are widely used in the fields of catalysis, sensing, energy and biomedicine. Recently, biological composites based on MOFs have attracted increasing attention. MOFs can be used as a platform for encapsulating bioactive substances due to the advantages such as large pore capacity, large specific surface area and diverse structure composition. These features can protect bioactive substances from adverse conditions, e.g. high temperature, high pressure, and organic solvents, thus improving the anti-adversity of bioactive substances. This review summarizes the advances of using MOFs as protective coatings to improve the anti-adversity of different bioactive substances, and introduces the synthesis strategy of MOFs-based biological composites, with the aim to promote the practical application of MOFs-based biological composites.

Bio-based molecules for biosynthesis of nano-metallic materials / 生物工程学报

Nano-metallic materials are playing an important role in the application of medicine, catalysis, antibacterial and anti-toxin due to their obvious advantages, including nanocrystalline strengthening effect, high photo-absorptivity, high surface energy and single magnetic region performance. In recent years, with the increasing consumption of global petrochemical resources and the aggravation of environmental pollution, nanomaterials based on bio-based molecules have aroused great concern. Bio-based molecules refer to small molecules and macromolecules directly or indirectly derived from biomass. They usually have good biocompatibility, low toxicity, degradability, wide source and low price. Besides, most bio-based molecules have unique physical, chemical properties and physiological activity, such as optical activity, acid/alkali amphoteric property, hydrophilic property and easy coordination with metal ions. Thus, the corresponding nano-materials based on bio-based molecules also have unique functions, such as anti-inflammatory, anti-cancer, anti-oxidation, antiviral fall blood sugar and blood fat etc. In this paper, we give a comprehensive overview of the preparation and application of nano-metallic materials based on bio-based molecules in recent years.

Degradation of Caffeine by Heterogeneous Photocatalysis Using ZnO with Fe and Ag

Abstract The organic compound caffeine when detected in environmental matrices such as surface waters and groundwater is considered as an emerging contaminant, in which its effects are still unknown. Therefore, in the present research, zinc oxide-based catalysts impregnated with iron and silver were prepared for the reaction of caffeine degradation by heterogeneous photocatalysis. The wet impregnation method with excess solvent was applied to the preparation of the materials, later they were characterized by adsorption of N2, X-ray diffraction and photoacoustic spectroscopy. Then, the photodegradation, photolysis and adsorption tests were carried out, in which it was observed that only the presence of the radiation or photocatalysts could not sufficiently degrade the caffeine, however when combined radiation with all the catalysts studied here presented degradation above 70% at the end of 300 minutes of the reaction, and the best catalyst studied was that containing 8% Ag in non-calcined ZnO. Thus, these results point out that the methodology employed in this research, both for the preparation of the catalysts and in the process of the photocatalysis reaction, was efficient in the degradation of the emerging contaminant, caffeine, which could later be used for a mixture of other contaminants.

Photocatalytic Degradation of Textile dye Orange-122 Via Electrospray Mass Spectrometry

Abstract This work reports the study of the potential application of Zn/TiO2 catalysts, obtained by the sol-gel method, in processes of environmental decontamination through the reactions of photodegradation of textile dye, followed by electrospray mass spectrometry. The catalysts synthesis was performed according to a 2² factorial design with repetition at the central point. The characterization techniques used were: N2 adsorption measurements (BET method), scanning electron microscopy with energy dispersive X-ray (MEV/EDS), X-ray diffraction and point of zero charge (PZC). The photocatalytic tests were performed in batch in the presence of sunlight, and to evaluate the degradation kinetics study, a rapid direct injection electrospray mass spectrometry (DI-ESI-MS) method has been developed. By the photocatalytic tests, the calcination temperature of 400 °C has shown the best results of discoloration for the reactive Orange-122 dye (99.76%) in a reaction time of 2h. The discoloration kinetics were a pseudo-first order, and a statistical analysis was performed to investigate the effects of the variables and to optimize the conditions of discoloration to the dye. After the reactional time of 2 h, an ion of m/z 441.5 was detected by ESI-MS, indicating that the photocatalytic process was effective for the degradation of the dye to secondary compounds.

Recent progress in 2-haloacid dehalogenases / 生物工程学报

2-Haloacid dehalogenases (EC 3.8.1.X) catalyze the hydrolytic dehalogenation of 2-haloacids, releasing halogen ions and producing corresponding 2-hydroxyacids. The enzymes not only degrade xenobiotic halogenated pollutants, but also show wide substrate profile and astonishing efficiency for enantiomer resolution, making them valuable in environmental protection and the green synthesis of optically pure chiral compounds. A variety of 2-haloacid dehalogenases have been biochemically characterized so far. Further studies have been made in protein crystal structures and catalytic mechanisms. Here, we review the recent progresses of 2-haloacid dehalogenases in their source, protein structures, reaction mechanisms, catalytic properties and application. We also suggest further research directions for 2-haloacid dehalogenase.

Expression and characterization of a novel cytochrome P450 enzyme from Variovorax paradoxus S110 / 生物工程学报

Cytochrome P450 monooxygenases as powerful biocatalysts catalyze a wide range of chemical reactions to facilitate exogenous substances metabolism and biosynthesis of natural products. In order to explore new catalytic reactions and increase the number of P450 biocatalysts used in synthetic biology, a new self-sufficient cytochrome P450 monooxygenase (P450(VpMO)), belongs to CYP116B class, was mined from Variovorax paradoxus S110 genome and expressed in Escherichia coli. Based on characterization of the enzymatic properties, it shows that the optimal pH and temperature for P450(VpMO) reaction activity are 8.0 and 45 °C, respectively. P450(VpMO) is relatively stable at temperatures below 35 °C. The Km and kcat of P450(VpMO) toward 4-Methoxyacetophenone are 0.458 mmol/L and 2.438 min⁻¹, respectively. Importantly, P450(VpMO) was able to catalyze the demethylation reaction for a range of substrates containing methoxy group. Its demethylation reactivity is reasonably better than other P450s belongs to CYP116B class, particularly, for 4-methoxyacetophenone with a great conversion efficiency at 91%, showing that P450(VpMO) could be used as a great biocatalyst candidate for further analysis.

Structural characteristics and catalytic cycle of dihydroorotate dehydrogenase-a review / 生物工程学报

Dihydroorotate dehydrogenase is a flavin-dependent mitochondrial enzyme to catalyze the fourth step of the de novo synthesis of pyrimidine and to oxidize dihydroorotate to orotate. By selectively inhibiting dihydroorotate dehydrogenase, thereby inhibiting pyrimidine synthesis, the enzyme has been developed for the treatment of cancer, autoimmune diseases, bacterial or viral infections, parasitic diseases and so on. The development of inhibitory drugs requires a detailed understanding of the structural characteristics and catalytic cycle mechanism of dihydroorotate dehydrogenase. Therefore, this paper reviews these two aspects, and indicates perspectives of these inhibitors in clinical application.

Selenoprotein thioredoxin reductase mediated menadione reduction: catalytic properties & inhibition effects / 生物工程学报

Thioredoxin reductase (TrxR) is one class of the most important antioxidant selenoproteins and is involved in regulating tumor genesis and progression. It has been reported that naphthoquinones can target and inhibit TrxR1 activity therefore produce reactive oxygen species (ROS) mediated by TrxR1, resulting into cellular redox imbalance and making the naphthoquinone compounds to become potential antitumor chemotherapy drugs. The purpose of this work is to explore the interaction between TrxR1 and menadione using biochemical and mass-spectrometric (MS) analyses, to further reveal the detailed mechanisms of TrxR1-mediated naphthoquinone reduction and inhibition of TrxR1 by naphthoquinone compounds. Using the site-directed mutagenesis and recombinantly expressed TrxR1 variants, we measured the steady-state kinetic parameters of menadione reduction mediated by TrxR1 and its variants, performed the inhibition analysis of menadione on TrxR1 activity, and eventually identified the interaction between menadione and TrxR1 through MS analysis. We found that Sec-to-Cys mutation at residue of 498 significantly enhanced the efficiency of TrxR1-mediated menadione reduction, though the Sec⁴⁹⁸ is capable to catalyze the menadione reduction, indicating that TrxR1-mediated menadione reduction is dominantly in a Se-independent manner. Mutation experiments showed that Cys⁴⁹⁸ is mainly responsible for menadione catalysis in comparison to Cys⁴⁹⁷, while the N-terminal Cys⁶⁴ is slightly stronger than Cys⁵⁹ regarding the menadione reduction. LC-MS results detected that TrxR1 was arylated with one molecule of menadione, suggesting that menadione irreversibly modified the hyper-reactive Sec residue at the C-terminus of selenoprotein TrxR1. This study revealed that TrxR1 catalyzes the reduction of menadione in a Se-independent manner meanwhile its activity is irreversibly inhibited by menadione. Hereby it will be useful for the research and development of naphthoquinone anticancer drugs targeting TrxR1.

Research progress of Pictet-Spenglerases / 生物工程学报

Pictet-Spenglerases (P-Sases) catalyze the Pictet-Spengler (P-S) reactions and exhibit high stereoselectivity and regioselectivity under mild conditions. The typical P-S reaction refers to the condensation and recyclization of β-arylethylamine with aldehyde or ketone under acidic conditions to form tetrahydroisoquinoline and β-carboline alkaloid derivatives. The related enzymatic products of P-Sases are the backbones of various bioactive compounds, including clinical drugs: morphine, noscapine, quinine, berberine, ajmaline, morphine. Furthermore, the activity of P-Sases in stereoselective and regioselective catalysis is also valuable for chemoenzymatic synthesis. Therefore, this review summarizes the research progress in the discovery, functional identification, biological characteristics and catalytic applications of P-Sases, which provide the useful theoretical reference in future P-Sases research and development.