Improved Enzyme Activity Research Articles

Creation of catalytic activity-improved hyperthermophilic PQQ-dependent aldose sugar dehydrogenase and its efficient use for high performance electro-device

PQQ-dependent aldose sugar dehydrogenase (PQQ-ASD) from the hyperthermophilic archaeon Pyrobaculum aerophilum (PaeASD) has great potential as an element for durable bioelectrodevices owing to its exceptional stability against high temperatures and across a broad pH spectrum. However, its application is constrained by low electric current output of the enzyme-immobilized electrodes, which is attributable to its low catalytic activity. A directed evolutionary approach was performed on PaeASD to improve enzyme activity, resulting in the identification of a PaeASD s24 mutant containing six amino acid substitutions, which exhibited a 16-fold higher specific activity than that of wild type. Although each single amino acid mutant among these substitutions exhibited lower enzyme activity than PaeASD s24, the double mutant R64Q/D350N showed enzyme activity comparable to that of PaeASD s24. These amino acids located in the vicinity of coenzyme PQQ within the PaeASD molecule are also highly conserved with those of PQQ-ASDs reported to date. Thus, these amino acids play crucial roles in the catalytic activity of PQQ-ASD. Furthermore, the Km value for d-glucose of PaeASD s24-immobilized electrode decreased to approximately 1/3 that of the wild-type-immobilized electrode. These results indicate that the PaeASD s24 mutant is an excellent catalyst for potential bioelectrodevice applications.

All‐Atom Protein Sequence Design Based on Geometric Deep Learning

AbstractDesigning sequences for specific protein backbones is a key step in creating new functional proteins. Here, we introduce GeoSeqBuilder, a deep learning framework that integrates protein sequence generation with side chain conformation prediction to produce the complete all‐atom structures for designed sequences. GeoSeqBuilder uses spatial geometric features from protein backbones and explicitly includes three‐body interactions of neighboring residues. GeoSeqBuilder achieves native residue type recovery rate of 51.6 %, comparable to ProteinMPNN and other leading methods, while accurately predicting side chain conformations. We first used GeoSeqBuilder to design sequences for thioredoxin and a hallucinated three‐helical bundle protein. All the 15 tested sequences expressed as soluble monomeric proteins with high thermal stability, and the 2 high‐resolution crystal structures solved closely match the designed models. The generated protein sequences exhibit low similarity (minimum 23 %) to the original sequences, with significantly altered hydrophobic cores. We further redesigned the hydrophobic core of glutathione peroxidase 4, and 3 of the 5 designs showed improved enzyme activity. Although further testing is needed, the high experimental success rate in our testing demonstrates that GeoSeqBuilder is a powerful tool for designing novel sequences for predefined protein structures with atomic details. GeoSeqBuilder is available at https://github.com/PKUliujl/GeoSeqBuilder.

All-Atom Protein Sequence Design Based on Geometric Deep Learning.

Designing sequences for specific protein backbones is a key step in creating new functional proteins. Here, we introduce GeoSeqBuilder, a deep learning framework that integrates protein sequence generation with side chain conformation prediction to produce the complete all-atom structures for designed sequences. GeoSeqBuilder uses spatial geometric features from protein backbones and explicitly includes three-body interactions of neighboring residues. GeoSeqBuilder achieves native residue type recovery rate of 51.6 %, comparable to ProteinMPNN and other leading methods, while accurately predicting side chain conformations. We first used GeoSeqBuilder to design sequences for thioredoxin and a hallucinated three-helical bundle protein. All the 15 tested sequences expressed as soluble monomeric proteins with high thermal stability, and the 2 high-resolution crystal structures solved closely match the designed models. The generated protein sequences exhibit low similarity (minimum 23 %) to the original sequences, with significantly altered hydrophobic cores. We further redesigned the hydrophobic core of glutathione peroxidase 4, and 3 of the 5 designs showed improved enzyme activity. Although further testing is needed, the high experimental success rate in our testing demonstrates that GeoSeqBuilder is a powerful tool for designing novel sequences for predefined protein structures with atomic details. GeoSeqBuilder is available at https://github.com/PKUliujl/GeoSeqBuilder.

High-efficiency expression of alginate lyase in Pichia pastoris facilitated by Vitreoscilla hemoglobin

Vitreoscilla hemoglobin (VHb) can enhance the ability of recombinant strains to express heterologous proteins under low-oxygen conditions. However, its mechanism of action in the Pichia pastoris expression system remains unclear. In this study, three VHb construction strategies were designed to elucidate the mechanisms by which VHb promotes heterologous protein expression in P. pastoris. Notably, the co-expression pattern involving the sequential expression of the 102C300C gene followed by the Vgb gene significantly improved enzyme activity in the recombinant strain X33-102C300C-Vgb. The enzyme activity was 203.4 ± 0.57 U/mL at 180 h of fermentation in the 5-L system, which was 20.7 % higher than that of the starting strain X33-102C300C. Fluorescent labeling experiments revealed for the first time that a dual-transcription unit approach achieved superior VHb expression, indicating its potential for further development. Furthermore, transcriptomic and metabolomic analyses demonstrated that VHb enhanced the growth of recombinant yeast colonies by improving respiration-related metabolism under low-oxygen conditions. This, in turn, alleviated the repression of the expression alcohol oxidase (AOX) at high methanol concentrations, resulting in increased alginate lyase activity. This study provides a theoretical foundation for improving the target protein expression in recombinant P. pastoris during high-density fermentation.

Harnessing the potential of deep eutectic solvents in biocatalysis: design strategies using CO2 to formate reduction as a case study.

Deep eutectic solvents (DESs) have emerged as green solvents with versatile applications, demonstrating significant potential in biocatalysis. They often increase the solubility of poorly water-soluble substrates, serve as smart co-substrates, modulate enzyme stereoselectivity, and potentially improve enzyme activity and stability. Despite these advantages, screening for an optimal DES and determining the appropriate water content for a given biocatalytic reaction remains a complex and time-consuming process, posing a significant challenge. This paper discusses the rational design of DES tailored to a given biocatalytic system through a combination of experimental screening and computational tools, guided by performance targets defined by solvent properties and process constraints. The efficacy of this approach is demonstrated by the reduction of CO2 to formate catalyzed by NADH-dependent formate dehydrogenase (FDH). By systematically analyzing FDH activity and stability, NADH stability (both long-term and short-term stability after solvent saturation with CO2), and CO2 solubility in initially selected glycerol-based DESs, we were able to skillfully guide the DES screening process. Considering trade-offs between experimentally determined performance metrics of DESs, 20% solution of choline chloride:glycerol in phosphate buffer (ChCl:Gly80%B) was identified as the most promising solvent system for a given reaction. Using ChCl:Gly as a co-solvent resulted in an almost 15-fold increase in FDH half-life compared to the reference buffer and stabilized the coenzyme after the addition of CO2. Moreover, the 20% addition of ChCl:Gly to the buffer improved the volumetric productivity of FDH-catalyzed CO2 reduction in a batch system compared to the reference buffer. The exceptional stability of the enzyme in this co-solvent system shows great potential for application in continuous operation, which can significantly improve process productivity. Additionally, based on easily measurable physicochemical solvent properties and molecular descriptors derived from COSMO-RS, QSAR models were developed, which successfully predicted enzyme activity and stability, as well as coenzyme stability in selected solvent systems with DESs.

An Analysis of the Mechanism About CO2 Enrichment Promoting Carbohydrate Metabolism in Cucumber (Cucumis sativus L.) Leaves.

Elevated CO2 can affect the synthesis and distribution of photosynthetic assimilates. However, the carbohydrate metabolism molecular mechanism of cucumber leaves in response to CO2 enrichment is unclear. Therefore, it is of great significance to investigate the key functional regulatory genes in cucumber. In this study, the growth of cucumber leaves under different CO2 conditions was compared. The results showed that under CO2 enrichment, leaf area increased, the number of mesophyll cells increased, stomata enlarged, and more starch grains accumulated in the chloroplasts. Compared with the control, the starch and soluble sugar content of leaves were maximally increased by 194.1% and 55.94%, respectively; the activities of fructose-1,6-bisphosphatase (FBPase), ADPG pyrophosphorylase (AGPase), starch synthase (SSS), sucrose phosphate synthase (SPS), sucrose synthase (SS) and invertase (Inv) were maximally increased by 36.91%, 66.13%, 33.18%, 21.7%, 54.11%, and 46.01%, respectively. Through transcriptome analysis, a total of 1,582 differential expressed genes (DEGs) were identified, in which the starch and sucrose metabolism pathway was significantly enriched, and 23 genes of carbon metabolism were screened. Through metabolome analysis, a total of 22 differential accumulation metabolites (DAMs) were identified. Moreover, D-glucose and D(+)-glucose were significantly accumulated, showing upregulation 2.4-fold and 2.6-fold, respectively. Through combined analysis of transcriptome and metabolome, it was revealed that seven genes were highly related to D-glucose, and Csa6G153460 (AGPase), Csa5G612840 (β-glucosidase), and Csa4G420150 (4-α-glucanotransferase) were significantly correlated to the carbohydrate regulatory network. Furthermore, the mechanism of CO2 enrichment that promotes carbohydrate metabolism in leaves at the molecular level was revealed. This mechanism advances the development of the cell wall and leaf morphology by activating the expression of key genes and improving enzyme activity.

Production and Characterization of Mineral-Enriched Nadep Composts for Enhanced Soil Fertility

Aim: Production and characterization of NADEP composts using aquatic weeds as substrates and enriched with different minerals, were conducted to evaluate its feasibility for agricultural purpose. Study Design: The experiment was carried out using a completely randomized design comprising 12 treatments and three replications. Place and Duration of Study: The experiment was conducted at the model organic Farm of the Department of Soil Science and Agricultural Chemistry, College of Agriculture, Vellayani, Kerala Agricultural University, between December 2023 and January 2024. Methods: Aquatic weeds, Limnocharis flava and Eichhornia crassipes, were collected from Vellayani lake and mixed with banana pseudostem in different proportions for compost preparation. Once the compost reached maturity, it was enriched with minerals such as calcium apatite and epsom salt. The resultant compost was then analyzed for its physical, chemical, and biological properties. Results: The NADEP compost, made from a 1:1 mixture of L. flava and E. crassipes and enriched with calcium apatite and epsom salt, was found to be the most effective treatment. It showed higher levels of macro and micronutrients, an ideal C: N ratio, improved enzyme activity, and a significant microbial load. Conclusion: NADEP composting has emerged as a highly effective and environmentally sustainable approach to producing high-quality compost in a prompt and efficient way. This method transforms aquatic weeds, such as Limnocharis and Eichhornia crassipes, which pose a threat to aquatic ecosystems, into valuable compost, thereby promoting effective waste management and advancing sustainable agricultural practices.

Dietary early addition enzyme-treated soybean improves amino acid absorption and protein digestibility by promoting digestive enzyme activity in broilers

Early nutritional regulation has become a research hotspot. The present study was undertaken to assess dietary early addition enzyme-treated soybean meal (ESBM) on nutrient digestibility, amino acid absorption, and intestinal development of broilers. Four hundred and fifty 1-day-old broilers were divided into three groups with 10 replicates of 15 broilers and fed with a basic diet, 2.5 or 5% ESBM during 1–10 days. Then, all groups were fed the same basic diets until 42 d. The obtained results indicated that supplementation of ESBM in early stage resulted in heavier BW and a better feed conversion ratio during the experimental periods compared with the control group. Supplementing the broiler’s diet with 5% ESBM led to enhance digestibility of DM, CP and decrease abdominal fat yield (P < 0.05). Meanwhile, 5% ESBM in the early diet increased villus height (P < 0.05, d 21) and reduced the crypt depth in jejunum and ileum (P < 0.05, d 42). Dietary 5% ESBM supplementation improved enzyme activity and upregulated nutrient transporters expression in jejunum, increased plasma amino acid (AA) concentrations (P < 0.05, d 10). In conclusion, our data demonstrated that supplementation ESBM promote intestinal development, upregulate AAs and peptide transporters gene expression for physiological absorption of AAs, and improve protein digestibility caused by intestinal enzyme activity, thereby improving growth performance.

Site-directed mutagenesis enhances the activity of dextranase from Arthrobacter oxidans KQ11

Dextranase is an enzyme that specifically hydrolyzes the α-1, 6 glucoside bond. In order to improve the activity of dextranase from Arthrobacter oxidans KQ11, site-directed mutagenesis was used to modify the amino acids involved in the "tunnel-like binding site". A saturating mutation at position 507 was carried out on this basis. The mutant enzymes A356G, S357W, W507Y, and W507F with improved enzyme activities and catalytic efficiency were successfully obtained. Compared with wild type (WT), W507Y showed the specific activity increasing by 3.00 times, the kcat value increasing by 3.62 times, the Km value decreasing by 54%, and the catalytic efficiency (kcat/Km) increasing by 8.98 times. The three-dimensional structure analysis showed that the increase of the number of hydrogen bonds and the distance between "tunnel-like binding sites" were important factors affecting enzyme activity. Compared with WT, W507Y had a shortened distance from the residues on the other side of the "tunnel-like binding site", which made it easier to generate hydrogen binding forces. Accordingly, the substrate hydrolysis and product efflux were accelerated, which dramatically increased the enzyme activity and catalytic efficiency.

Streptomyces as a Novel Biotool for Azo Pigments Remediation in Contaminated Scenarios.

Azo pigments are widely used in the textile and leather industry, and they generate diverse contaminants (mainly in wastewater effluents) that affect biological systems, the rhizosphere community, and the natural activities of certain species. This review was performed according to the Systematic Reviews and Meta Analyses (PRISMA) methodology. In the last decade, the use of Streptomyces species as biological azo-degraders has increased, and these bacteria are mainly isolated from mangroves, dye-contaminated soil, and marine sediments. Azo pigments such as acid orange, indigo carmine, Congo red, and Evans blue are the most studied compounds for degradation, and Streptomyces produces extracellular enzymes such as peroxidase, laccase, and azo reductase. These enzymes cleave the molecule through asymmetric cleavage, followed by oxidative cleavage, desulfonation, deamination, and demethylation. Typically, some lignin-derived and phenolic compounds are used as mediators to improve enzyme activity. The degradation process generates diverse compounds, the majority of which are toxic to human cells and, in some cases, can improve the germination process in some horticulture plants. Future research should include analytical methods to detect all of the molecules that are generated in degradation processes to determine the involved reactions. Moreover, future studies should delve into consortium studies to improve degradation efficiency and observe the relationship between microorganisms to generate scale-up biotechnological applications in the wastewater treatment industry.

Melatonin Prolongs the Postharvest Storability of Strawberry Fruits through Reduced Oxidative Stress and Improved Enzyme Activity

Strawberry fruits are popular among consumers because of their unique flavor and reported health benefits. However, microbial growth and oxidative stress that occur in postharvest storage cause strawberry fruits to have a relatively short postharvest life, which reduces consumer acceptance. This study aimed to evaluate the effects of exogenous melatonin application on the enzymatic activity and postharvest quality of strawberry fruits that are stored at 4 ± 0.5 °C. A total of 288 fruits with four replicates for each of the three treatments (control, 200 μM melatonin, and 500 μM melatonin) were used. Several quality metrics were regularly assessed at 3-day intervals during the 18-day storage trials. The results suggested that the exogenous melatonin application significantly increased the activity of antioxidant enzymes at the physiological level, including catalase, ascorbate peroxidase, superoxide dismutase, and peroxidase which led to reductions in weight loss, the decay incidence, and the malondialdehyde level. The use of melatonin successfully delayed changes in the soluble solids concentration, ascorbic acid, titratable acidity, and fruit firmness. The results indicated that applying 500 μM melatonin to strawberries would be a useful strategy for increasing their shelf life.

Effective parallel evaluation of molecular design, expression and bioactivity of novel recombinant butyrylcholinesterase medical countermeasures

Current medical countermeasures (MCMs) for nerve agent poisoning have limited efficacy, and can cause serious adverse effects, prompting the requirement for new broad-spectrum therapeutics. Human plasma-derived butyrylcholinseterase (huBChE) is a promising novel bioscavenger MCM which has shown potential in animal studies, however, is economically prohibitive to manufacture at scale. This study addresses current challenges for the economical production of a bioactive and long-acting recombinant huBChE (rBChE) in mammalian cells by being the first to directly compare novel rBChE design strategies. These include co-expression of a proline rich attachment domain (PRAD) and fusion of BChE with a protein partner. Additionally, a pre-purification screening method developed in this study enables parallel comparison of the expression efficiency, activity and broad-spectrum binding to nerve agents for ten novel rBChE molecular designs. All designed rBChE demonstrated functionality to act as broad-spectrum MCMs to G, V and A series nerve agents. Expression using the ExpiCHO™ Max protocol provided greatest expression levels and activity for all constructs, with most rBChE expressing poorly in Expi293™. Fc- or hSA-fused rBChE significantly outperformed constructs designed to mimic huBChE, including PRAD-BChE, and proved an effective strategy to significantly improve enzyme activity and expression. Choice of protein partner, directionality and the addition of a linker also impacted fusion rBChE activity and expression. Overall, hSA fused rBChE provided greatest expression yield and activity, with BChE-hSA the best performing construct. The purified and characterised BChE-hSA demonstrated similar functionality to huBChE to be inhibited by GD, VX and A-234, supporting the findings of the pre-screening study and validating its capacity to assess and streamline the selection process for rBChE constructs in a cost-effective manner. Collectively, these outcomes contribute to risk mitigation in early-stage development, providing a systematic method to compare rBChE designs and a focus for future development.

Enhancing the Catalytic Activity of Geranylgeranyl Diphosphate Synthase through Ancestral Sequence Reconstruction and Semirational Design.

Geranylgeranyl diphosphate synthase (GGPPS) is the crucial bottleneck in carotenoid biosynthesis. However, low activity limits the broad application of GGPPS. In this study, OsGGPPS1 in rice was engineered based on ancestral sequence reconstruction (ASR) and semirational design to improve the catalytic performances of existing GGPPS. The better mutant of A22R/A26P with improved enzyme activity was generated based on ASR. Additionally, the improved enzyme activity of mutants as V162A/M218S/F227Y was designed using a semirational design. The combinatorial assembly of the d-OsGGPPS1 mutant (A22R/A26P/V162A/M218S/F227Y) exhibited higher conversion of IPP and each cosubstrate of DMAPP for 9.8-fold in GPP production, GPP for 6.4-fold in FPP production, and FPP for 1.4-fold in GGPP production relative to wild-type OsGGPPS1 at 25 °C, which showed higher conversion than wild-type OsGGPPS1 at temperatures as high as 50 °C. The successful design of OsGGPPS1 was representative of protein engineering, which will shed new light on GGPPS engineering and active plant pigment resource utilization.

Enzyme engineering in microbial biosynthesis of terpenoids: progress and perspectives

Terpenoids, known for their structural and functional diversity, are highly valued, especially in food, cosmetics, and cleaning products. Microbial biosynthesis has emerged as a sustainable and environmentally friendly approach for the production of terpenoids. However, the natural enzymes involved in the synthesis of terpenoids have problems such as low activity, poor specificity, and insufficient stability, which limit the biosynthesis efficiency. Enzyme engineering plays a pivotal role in the microbial synthesis of terpenoids. By modifying the structures and functions of key enzymes, researchers have significantly improved the catalytic activity, specificity, and stability of enzymes related to terpenoid synthesis, providing strong support for the sustainable production of terpenoids. This article reviews the strategies for the modification of key enzymes in microbial synthesis of terpenoids, including improving enzyme activity and stability, changing specificity, and promoting mass transfer through multi-enzyme collaboration. Additionally, this article looks forward to the challenges and development directions of enzyme engineering in the microbial synthesis of terpenoids.

Semi-rational engineering of ω-transaminase for enhanced enzymatic activity to 2-ketobutyrate

Transaminases (EC 2.6.1.X, TAs) are important biocatalysts in the synthesis of chiral amines, and have significant value in the field of medicine. However, TAs suffer from low enzyme activity and poor catalytic efficiency in the synthesis of chiral amines or non-natural amino acids, which hinders their industrial applications. In this study, a novel TA derived from Paracoccus pantotrophus (ppTA) that was investigated in our previous study was employed with a semi-rational design strategy to improve its enzyme activity to 2-ketobutyrate. By using homology modeling and molecular docking, four surrounding sites in the substrate-binding S pocket were selected as potential mutational sites. Through alanine scanning and saturation mutagenesis, the optimal mutant V153A with significantly improved enzyme activity was finally obtained, which was 578 % higher than that of the wild-type ppTA (WT). Furthermore, the mutant enzyme ppTA-V153A also exhibited slightly improved temperature and pH stability compared to WT. Subsequently, the mutant was used to convert 2-ketobutyrate for the preparation of L-2-aminobutyric acid (L-ABA). The mutant can tolerate 300 mM 2-ketobutyrate with a conversion rate of 74 %, which lays a solid foundation for the preparation of chiral amines.

Investigating process parameters to enhance (hemi)cellulolytic enzymes activity produced by Trichoderma reesei RUT-C30 using deoiled oil palm mesocarp fiber in solid-state fermentation

This study investigates the synthesis of (hemi)cellulolytic enzymes, including endoglucanase (CMCase), xylanase, and β-glucosidase, employing Trichoderma reesei RUT-C30 and deoiled oil palm mesocarp fiber (OPMF) through solid-state fermentation (SSF). The objective was to determine the optimal process conditions for achieving high enzyme activities through a one-factor-at-a-time approach. The study primarily focused on the impact of the solid-to-liquid ratio, incubation period, initial pH, and temperature on enzyme activity. The effects of OPMF pretreatment, particularly deoiling and fortification, were explored. This approach significantly improved enzyme activity levels compared to the initial conditions, with CMCase increasing by 111.6 %, xylanase by 665.2 %, and β-Glucosidase by 1678.1 %. Xylanase and β-glucosidase activities, peaking at 1346.75 and 9.89 IU per gram dry substrate (GDS), respectively, under optimized conditions (1:4 ratio, pH 7.5, 20 °C, 9-day incubation). With lower moisture levels, CMCase reached its maximum activity of 227.84 IU/GDS. The study highlights how important it is for agro-industrial byproducts to support environmentally sustainable practices in the palm oil industry. It also emphasizes how differently each enzyme reacts to changes in process parameters.

Effect of 3-year amendment measures on coastal saline–alkali soil conditions during the growing season

Seawater intrusion and fluctuations in the water table in coastal areas lead to seasonal variations in soil salinity and pH, which greatly limit the development of coastal protection forests. In a 3-year field study, the impact of five soil amendment measures were evaluated on soil conditions in coastal areas. Amendments included biochar, biochar with arbuscular mycorrhizal fungi (AMF), straw with AMF, straw alone, and AMF alone, compared to a control (CK) with no additive. Results indicated that combinations of straw, biochar, and AMF reduced soil pH across various layers and seasons, with electrical conductivity mainly decreasing in spring. During the summer, at the 0–20 cm soil depth, microbial biomass carbon notably increased due to these mixtures. Additionally, AMF alone and biochar with AMF significantly improved enzyme activities in the 0–40 cm layer in spring, while in fall, AMF alone notably increased nutrient availability in the same layer. Linear regression analysis revealed a negative correlation between electrical conductivity, microbial biomass carbon, enzyme activity, and nutrient availability with pH. The biochar–AMF mixture emerged as the most effective soil amendment, suggesting that using it in conjunction with seasonal management could optimize soil health and promote silviculture in coastal regions.

Sulfur/zinc co-doped biochar for stabilization remediation of mercury-contaminated soil: Performance, mechanism and ecological risk

In this study, a novel sulfur/zinc co-doped biochar (SZ-BC) stabilizer was successfully developed for the remediation of mercury-contaminated soil. Results from SEM, TEM, FTIR and XRD revealed that biochar (BC) was successfully modified by sulfur and zinc. In the batch adsorption experiments, the sulfur-zinc co-pyrolysis biochar displayed excellent Hg(II) adsorption performance, with the maximum adsorption capacity of SZ-BC (261.074 mg/g) being approximately 16.5 times that of BC (15.855 mg/g). Laboratory-scale static incubation, column leaching, and plant pot experiments were conducted using biochar-based materials. At an additional dosage of 5 % mass ratio, the SZ-BC exhibits the most effective stabilization of mercury in soil, leading to a significant reduction in leaching loss compared to the control group (CK) by 51.30 %. Following 4 weeks of incubation and 2 weeks of leaching with SZ-BC, the residual mercury in the soil increased by 27.84 %. As a result, potential ecological risk index of mercury decreased by 92 % compared to the CK group. In the pot experiment, SZ-BC significantly enhanced the growth of Chinese cabbage, with biomass and root dry weight reaching 3.20 and 2.80 times that of the CK group, respectively. Additionally, the Translocation Factor (TF) and Bioconcentration Factor (BF) were reduced by 44.86 % and 74.43 %, respectively, in the SZ-BC group compared to the CK group. Moreover, SZ-BC can effectively improve enzyme activities and increase microbial communities in mercury-contaminated soils. The mechanisms of adsorption and stabilization were elucidated through electrostatic adsorption, ion exchange, surface complexation, and precipitation. These findings provide a potentially effective material for stabilizing soils contaminated with mercury.

The Formation of D-Allulose 3-Epimerase Hybrid Nanoflowers and Co-Immobilization on Resins for Improved Enzyme Activity, Stability, and Processability.

As a low-calorie sugar, D-allulose is produced from D-fructose catalyzed by D-allulose 3-epimerase (DAE). Here, to improve the catalytic activity, stability, and processability of DAE, we reported a novel method by forming organic-inorganic hybrid nanoflowers (NF-DAEs) and co-immobilizing them on resins to form composites (Re-NF-DAEs). NF-DAEs were prepared by combining DAE with metal ions (Co2+, Cu2+, Zn2+, Ca2+, Ni2+, Fe2+, and Fe3+) in PBS buffer, and were analyzed by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, and X-ray diffraction. All of the NF-DAEs showed higher catalytic activities than free DAE, and the NF-DAE with Ni2+ (NF-DAE-Ni) reached the highest relative activity of 218%. The NF-DAEs improved the thermal stability of DAE, and the longest half-life reached 228 min for NF-DAE-Co compared with 105 min for the free DAE at 55 °C. To further improve the recycling performance of the NF-DAEs in practical applications, we combined resins and NF-DAEs to form Re-NF-DAEs. Resins and NF-DAEs co-effected the performance of the composites, and ReA (LXTE-606 neutral hydrophobic epoxy-based polypropylene macroreticular resins)-based composites (ReA-NF-DAEs) exhibited outstanding relative activities, thermal stabilities, storage stabilities, and processabilities. The ReA-NF-DAEs were able to be reused to catalyze the conversion from D-fructose to D-allulose, and kept more than 60% of their activities after eight cycles.

Design of NAMPTs with Superior Activity by Dual-Channel Protein Engineering Strategy.

The nicotinamide phosphoribosyltransferase (NAMPT)-catalyzed substitution reaction plays a pivotal role in the biosynthesis of nucleotide compounds. However, industrial applications are hindered by the low activity of NAMPTs. In this study, a novel dual-channel protein engineering strategy was developed to increase NAMPT activity by enhancing substrate accessibility. The best mutant (CpNAMPTY13G+Y15S+F76P) with a remarkable 5-fold increase in enzyme activity was obtained. By utilizing CpNAMPTY13G+Y15S+F76P as a biocatalyst, the accumulation of β-nicotinamide mononucleotide reached as high as 19.94 g L-1 within 3 h with an impressive substrate conversion rate of 99.8%. Further analysis revealed that the newly generated substrate channel, formed through crack propagation, facilitated substrate binding and enhanced byproduct tolerance. In addition, three NAMPTs from different sources were designed based on the dual-channel protein engineering strategy, and the corresponding dual-channel mutants with improved enzyme activity were obtained, which proved the effectiveness and practicability of the approach.