The effects of ovomucoid (OVM), a by-product of egg white, and its hydrolysates on adipocyte differentiation and lipid accumulation were investigated. The OVM hydrolyzed using Alcalase® and pepsin was named AH and PH, respectively. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis revealed significant changes in molecular weight of both hydrolysates, with AH showing a higher degree of hydrolysis. AH exhibited a more pronounced inhibitory effect on fat accumulation than PH. In in vitro experiments, AH and PH suppressed lipid accumulation during 3T3-L1 adipocyte differentiation, with AH inhibiting lipid accumulation most effectively. Oil red O staining and triglyceride measurements revealed lipid reduction in AH-treated cells, indicating that AH plays a major role in preventing lipid accumulation in adipocytes. In addition, AH inhibited the expression of lipid transcription factors (CCAAT/enhancer-binding protein alpha (C/EBP-α), peroxisome proliferator-activated receptor gamma (PPAR-γ), and sterol regulatory element-binding proteins (SREBP-1c)), adipogenesis-related factors (fatty acid synthase (FAS) and ACC1), insulin-related factors (insulin receptor substrate (IRS2) and protein kinase B (AKT2)), and lipolysis-related factors (glycerol-3-phosphate acyltransferase (GPAT), CD36, and lipoprotein lipase (LPL)) in a concentration-dependent manner. Specifically, the effect of AH was most pronounced in the early stages of adipocyte differentiation, where it activated AMPK early to associate energy homeostasis and downregulate genes important for cell cycle and lipid formation. This study suggests that OVM hydrolysates prepared using Alcalase® may contribute to the development of new strategies for the obesity treatment market.

Spermidine, a naturally occurring polyamine compound, has garnered significant attention due to its versatile physiological functions, including induction of cellular autophagy, antioxidant activity, and maintenance of mitochondrial homeostasis. In this study, we established a novel spermidine biosynthesis system in E. coli BL21 (DE3) by heterologously introducing the carboxyaminopropylagmatine (CAPA) pathway derived from cyanobacterium. To enhance precursor supply, we overexpressed key enzymes in the L-aspartate β-semialdehyde and agmatine branch metabolic pathways within the precursor metabolic module, while simultaneously knocking out competing metabolic pathways to redirect metabolic flux toward spermidine biosynthesis. To address the challenge of intracellular spermidine accumulation and its associated cytotoxicity, the high-efficiency spermidine efflux pump protein AmvA from Acinetobacter baumannii was heterologously expressed in E. coli BL21 (DE3). This engineering strategy enabled efficient extrusion of spermidine from the cells, alleviating the toxic effects of high intracellular spermidine concentrations on the host strain. Through these metabolic and efflux pump engineering modifications, the engineered strain SPD06-P5-P6 was constructed. Following 48 h of shake flask fermentation, SPD06-P5-P6 achieved a spermidine titer of 163.11 mg/L, which further increased to 1164.22 mg/L after 96 h of scale-up cultivation in a 5 L bioreactor.

Commercial production of 2-keto-L-gulonic acid (2-KGA), a crucial precursor in the synthesis of vitamin C, is carried out using the two-step fermentation process. This process uses a dual-strain system in the second step (conversion of L-sorbose to 2-KGA), necessitating complex process control. In this study, we report development of a recombinant Escherichia coli strain capable of producing 2-KGA from L-sorbose. L-Sorbose dehydrogenase (sdh) and L-sorbosone dehydrogenase (sndh) genes from Gluconobacter oxydans were expressed in E. coli as a synthetic operon in three different configurations under the control of the strong inducible T7 promoter. High-density whole-cell biotransformation was carried out to produce 2-KGA from L-sorbose, resulting in a yield of 0.69 g/g when the two genes were arranged in the non-native sdh-sndh configuration. Our design eliminates the need for multi-organism co-culture, intricate redox balancing, or nutrient-enriched media, representing a robust and scalable alternative to conventional production. This work demonstrates the feasibility of employing E. coli as a chassis strain for vitamin C precursor biosynthesis and offers a modular operon framework adaptable to other dehydrogenase-driven bioconversions. This approach enables efficient, scalable production of the vitamin C precursor in a genetically tractable host.

The site-specific methylation of (-)-epigallocatechin-3-O-gallate (EGCG), especially 3`` hydroxyl group in D-ring, significantly enhances its stability and bioavailability in human body. Therefore, the methylation is the way to make EGCG more orally active and potent nutraceutical agents. In this study, for the effective synthesis of the methylated EGCG, S-adenosyl-L-methionine dependent O-methyltransferase (OMT) was studied to synthesize the methylated EGCG. OMT from Bacillus licheniformis and B. megaterium were known to have methylation activity for EGCG. Because OMT from B. licheniformis (Bl_OMT) showed higher activity and regioselectivity between two OMTs, rational design was tried using Bl_OMT. F163W mutant of Bl_OMT showed 2-fold increased initial velocity for the methylation of EGCG than wild type Bl_OMT. This engineering result was further utilized as a basis for the identification of highly active OMT from sequence database. 100 homolog sequences of OMT of B. licheniformis and 100 homolog sequences of OMT of B. megaterium, were collected using BLAST. Multiple alignment of 202 sequences was used to generate subgroups. Four representative sequences from each subgroup were further studied. As a result, OMTs from Thermolongibacillus altinseunsis and B. subtilis, which were from homolog group of Bl_OMT, showed higher activity than Bl_OMT while showing the same high regioselectivity. OMTs from T. altinseunsis and B. subtilis showed k/K of 17.4 Ms and 11.3 Ms, respectively, while Bl_OMT showed 8.7 Ms. Therefore, we could find that phenylalanine residue of the active site of OMT is very important to make strong binding of hydrophobic moiety of substrate and mutation to tryptophan is able to give higher binding strength.

Several commercially important vinyl phenolic compounds can be produced by enzymatic decarboxylation of phenolic acids such as ferulic and p-coumaric acids which can be extracted from agro-industrial waste. Phenolic acid decarboxylase is an enzyme that acts in the decarboxylation of these acids to 4-vinylguaiacol and 4-vinylphenol, respectively. In this study, the gene encoding phenolic acid decarboxylase from Klebsiella pneumoniae TD 4.7 was isolated and identified as a 504 bp fragment, encoding a polypeptide of 167 amino acid residues. A 98 % predicted amino acid sequence identity between ferulic acid decarboxylase from other bacteria of the same genus was determined. The gene was successfully expressed in Escherichia coli BL21 (DE3), and the recombinant enzyme was purified as active in absence of cofactor. The protein had a mass of 22-kDa protein, with greater activity at pH 5.5 and 40 °C. The decarboxylase activity was inhibited by Hg , Zn, Cu, and Cd ions and increased by 20 % in the presence of Co. The K and V values for the recombinant enzyme were estimated at 2.95 mM and 102.10 µmol min mg, respectively. The enzyme's structure was modelled using the structural prediction programs AlphaFold Multimer and SWISS-MODEL, with an RMSD of just 0.7 Å, demonstrating the absence of cysteine and disulfide bonds in the homodimer, with the presence of a high number of lysine residues. The amino acids involved in the catalytic site were Tyr27, Glu134, and Asn23. The Enzyme activity on substrates ferulic and p-coumaric acids extracted from sugarcane bagasse, resulted in 4-vinylguaiacol and 4-vinylphenol, respectively, with conversion yields of 43 % for ferulic acid and 55 % for p-coumaric acid. These data are important in terms of obtaining an enzyme that decarboxylates ferulic and p-coumaric acids obtained from sugarcane bagasse hydrolyzed with similar efficiency, in a single step and without the need for a cofactor, making it an excellent option for bioprocesses using lignocellulosic biomass derivatives.

This study aims to diversify the structure of Soyasapogenol B (1) and generate novel metabolites through microbial-catalyzed biotransformation by two highly efficient microbial strains, Streptomyces griseus ATCC 13273 and Penicillium griseofulvum CICC 40293. Consequently, ten (2-11) unique bioactive metabolites were isolated. Their structures were determined using 1D/2D NMR and HR-ESI-MS data, revealing multiple tailoring reactions, including oxidation, C-C double bond rearrangement, hydroxylation, and dehydrogenation. This highlights the enzymatic ability of these strains to catalyze specific and diverse regioselective modifications on the Soyasapogenol B scaffold. Therefore, this study demonstrates that microbial-catalyzed biotransformation offers a promising approach to increase the chemical diversity of Soyasapogenol B (1), providing a sustainable alternative to chemical synthesis.

Vibrio parahaemolyticus is a major seafood-associated foodborne pathogen whose lipopolysaccharide (LPS) plays an important role in virulence and antimicrobial resistance. The LPS of V. parahaemolyticus contains a single 3-deoxy-D-manno-octulosonic acid (Kdo) sugar with phosphorylation. Previously, we have characterized the gene VP_RS01035 is responsible for the addition of Kdo; in this study, we characterized another gene VP_RS00960 which is responsible for the Kdo phosphorylation of V. parahaemolyticus LPS. To investigate its function, we first constructed an LPS-deficient Escherichia coli WH600 strain using CRISPR/Cas9. Heterologous expression of VP_RS01035 alone or in combination with VP_RS00960 yielded recombinant strains WH600/pB1-1 and WH600/pB2-12, respectively. Analysis of total lipids from the recombinant strains by thin-layer chromatography and high-performance liquid chromatography-tandem mass spectrometry demonstrated that the VP_RS00960 gene encodes a Kdo kinase responsible for phosphorylating the 4-OH site of the Kdo sugar in V. parahaemolyticus. To further validate its role, we deleted VP_RS00960 gene in V. parahaemolyticus, resulting in mutant ΔRS00960. Notably, ΔRS00960 failed to produce polysaccharide-linked lipid A, although free lipid A synthesis remained unaffected. Furthermore, defective long-chain LPS assembly compromised outer membrane integrity, increasing permeability and hydrophobicity while reducing biofilm formation. Consequently, ΔRS00960 exhibited heightened susceptibility to membrane-targeting antibiotics, such as erythromycin and novobiocin. Macrophage infection assays using RAW264.7 cells revealed that VP_RS00960 deletion attenuated bacterial pathogenicity. These findings enhance the understanding of the pathogenicity and drug resistance of V. parahaemolyticus, and provide novel insights and strategies for addressing antibiotic resistance and food safety challenges posed by V. parahaemolyticus.

Recombinant protein production in prokaryotic systems remains a major topic in biotechnology because of their rapid growth, cost-effectiveness, and ease of genetic manipulation. However, the production of functionally active proteins still faces significant challenges due to folding failures, insolubility, and the lack of the capability of most prokaryotes for complex post-translational processing. This review dwells into both traditional and emerging strategies for optimizing recombinant protein expression in various prokaryotic systems. It also highlights recent advances in genetic engineering and synthetic biology for expanding the toolkit available for protein production, which include refined expression vectors, engineered hosts with improved folding capabilities, and high-throughput screening platforms. Additionally, it provides a thorough discussion of how to optimize heterologous expression using fusion tag approaches, codon bias elimination, promoter and ribosome binding site (RBS) engineering, and chaperone-assisted folding. This review explores diverse prokaryotic expression systems that offer unique advantages for heterologous expression that extend far beyond the limitations of traditional hosts. Additionally, this review also emphasizes the need for the selection of the right expression system and optimizing conditions to fulfill the increasing demands for recombinant protein production in various fields.

The ability of Nocardiopsis dassonvillei NCIM 5124 to synthesize polyhydroxybutyrate depolymerase (PHBD) was recently reported. In this investigation, in vitro codon optimized gene synthesis, overexpression, and biochemical characterization of this enzyme along with molecular docking studies are presented. The sequence of the PHBD was inserted in pET-28a(+) along with the PelB_Signal and His tag to generate the recombinant vector pET-Nd-pelB_PHBD. The transformed Escherichia coli BL21(DE3) could produce active PHBD. This enzyme was purified using Ni-NTA affinity chromatography, producing a product with a molecular weight of roughly 50 kDa. The optimum temperature and pH of the recombinant enzyme were 35°C and 8.0, respectively. Triton X100 and Tween 20 inhibited the enzyme activity by 90 %, indicating the role of hydrophobic residues in the active site of the enzyme, as also noted during docking studies. On the basis of the Michaelis-Menten equation, apparent K and V of recombinant PHBD were found to be 1.782 mg/mL and 4.79 U/mL/min, respectively. Molecular docking studies indicated that the hydrophobic amino acids Cys 39, Ala 40, Cys 77, Phe 158, Met 161, Val 201, Ala 272, and Tyr 273 present in the catalytic site were providing the necessary hydrophobic environment for binding of the ligand. The purified enzyme could also degrade films of PHB and poly (3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate (PHBVH). As far as we are aware, this is the first report on the overexpression of PHBD from Nocardiopsis sp.

To enhance the esterification and stability of lipase in organic solvents, the bio-imprinted Aspergillus niger lipase combined with cross-linked aggregate immobilization was investigated. The bio-imprinted lipase cross-linked aggregates were applied to the catalytic esterification for the synthesis of Vitamin E succinate in N, N-dimethylformamide (DMF) solution. Lauric acid, serving as a succinic acid analogue, was selected as the bio-imprinting molecule, 0.10 g lauric acid was added to 36 mL of 2.10 mg/mL lipase solution, imprinting 40 mins at pH 8.0, the immobilization yield achieved 91.5 % with cross-linked aggregates by glutaraldehyde. The catalytic activity of the bio-imprinted lipase cross-linked aggregates was significantly enhanced, achieving an esterification yield of 87.4 ± 0.43 % for Vitamin E succinate. Moreover, the bio-imprinted lipase cross-linked aggregates maintained their catalytic activity over five consecutive reaction cycles in DMF. Fluorescence spectroscopy analysis revealed that bio-imprinting promoted the exposure of the lipase active sites, which corresponded with the observed increase in esterification activity. In addition, the mechanism of the substrate analogue-imprinted lipase was characterized. This study provides a theoretical foundation for improving the catalytic esterification performance of lipase as well as a process basis for the enzymatic synthesis of Vitamin E succinate.

Forty-five fungal strains from decomposing wood, representing eight genera, were isolated. Among them, 27 cellulolytic strains were identified. The genera Trichoderma, Penicillium, and Phanerochaete (6, 2, and 4 isolates, respectively) demonstrated the highest cellulase production. The isolates TW-25, TW-28, and TW-33 exhibited superior enzyme activities, CMCase (5.4-6.5 U/mL), FPase (3.2-3.8 U/mL), pNPCase (2.6-3.0 U/mL), and pNPGase (3.5-4.0 U/mL) and were selected for further study. Internal transcribed spacer (ITS) region sequencing, combined with phenotypic characteristics, identified these strains as Trichoderma sp. TW-25, Trichoderma sp. TW-28, and Penicillium sp. TW-33. Maximum protoplast release was observed in Trichoderma sp. TW-25 (3.6 × 10⁶ protoplasts/mL), followed by Trichoderma sp. TW-28 (3.0 × 10⁶ protoplasts/mL) and Penicillium sp. TW-33 (2.8 × 10⁶ protoplasts/mL). Fusion frequencies were 2.8 × 10⁻³ for TW-25 × TW-28, 2.0 × 10⁻³ for TW-25 × TW-33, and 1.8 × 10⁻³ for TW-28 × TW-33. A total of 13 colonies obtained from TW-25 × TW-28, and 18 from intergeneric fusions (10 from TW-25 × TW-33 and 8 from TW-28 × TW-33). The cellulase activity of the fusants TWF1/1, TWF1/6, and TWF2/5 was the same as TW-25 and the fusants TWF1/3 and TWF3/2 the same as TW-28 while none of the fusants had the cellulase activity of TW-33. Fusants differed from their parental strains in their DNA content (3.25-3.65 µg/mg dry weight) and showed high cellulase activities in general. Among them, TWF1/10 demonstrated the highest enzymatic activity, producing CMCase, FPase, pNPCase, and pNPGase (10.5, 6.5, 5.8, and 7.5 U/mL), respectively, followed by TWF1/13, TWF2/8, TWF2/10, and TWF3/8. DNA banding patterns of TWF1/10, TWF1/13, TWF2/8, TWF2/10, and TWF3/8, analyzed using four RAPD and three ISSR primers, differed from their parental strains, except for ISSR-3 with fusants TWF1/10 and TWF1/13. These variations underscore the effectiveness of interspecific and intergeneric protoplast fusion. The supernatant of the hybrid strain TWF1/10 was concentrated and purified via ultrafiltration, and SDS-PAGE and zymogram assays confirmed its cellulase activity using CMC as the substrate.

Zymomonas mobilis has an extremely high specific glucose uptake rate and produces ethanol at high yields and productivities. For synthesizing products beyond ethanol in Z. mobilis, reducing ethanol yields is a major challenge. Previous efforts have sought to decrease PDC activity to minimize ethanol formation. Here, we sought to modulate ADH (encoded by adhA and adhB genes) activity to redirect flux away from ethanol. We found that deletion of adhB combined with co-expression of a heterologous NAD(P) regenerating enzyme (L-LDH) diverted more than 50 % of carbon flux away from ethanol formation. The sequential deletion of adhA in the adhB-deleted strain led to ∼90 % reduction in ADH activity. During batch growth, the strain showed ∼90 % reduction in ethanol titres (27 mM) compared to WT (209 mM) and significant flux redirection toward L-LA formation (169 mM). The results demonstrate that modulation of ADH activity by deletion of adhA and adhB genes is an effective strategy for rediverting flux toward products beyond ethanol in Z. mobilis.

Environmental microplastic leads to bioaccumulation in humans, animals, and plants with potential toxicity. Polyethylene terephthalate (PET) degrading enzymes (PETases) present an opportunity to depolymerize PET in key intervention points, such as wastewater treatment. While PETase has been extensively studied since its discovery and modified for enhanced performance (especially thermostability), knowledge on immobilization for reusability remains limited. This study investigated the effect of linker peptides between functional, active, stable, and tolerant (FAST) PETase and a silica binding protein for immobilization onto silica and the reusability of the enzyme. Linker peptides and a silica binding protein were assembled onto FAST-PETase and expressed in Escherichia coli. The activity of the constructs was tested on PET before and after binding to silica. In the free system, repeating (GGGGS) flexible linker achieved the same activity as FAST-PETase parent enzyme after 48 h of degradation. Once immobilized to silica, repeating (GGGGS) flexible linker preserved 50 % of enzymatic activity, compared to free FAST-PETase, and 80 % compared to its free form. Silica-immobilized enzyme constructs all retain at least 15 % of relative activity compared to the first cycle of use after 5 reuse cycles. Integration of linker peptides between the enzyme and the silica binding peptide had a significant effect on the overall catalytic activity of FAST-PETase and advances our understanding of immobilized PETase for potential recovery and reuse in applications such as wastewater treatment. SYNOPSIS: Minimal research exists on the immobilization of polyethylene terephthalate degrading enzymes for reuse in environmental systems. This study reports the ability of silica immobilized enzyme, with aid of linker peptides, to minimize microplastic contamination from wastewater treatment plants.

Adipic acid is an important six-carbon dicarboxylic acid with numerous industrial applications in polymers (nylon) and the food industry. Traditional manufacturing of adipic acid relies on petroleum feedstocks and involves energy-intensive chemical processes with negative environmental impacts. Consequently, alternative synthesis methods are being developed, including the hydrogenation of biobased muconic acid to adipic acid via chemical catalysis or enzymatic reduction with 2-enoate reductases. This study revealed that purified full-length 2-enoate reductase ERBC from Heyndrickxia (Bacillus) coagulans can reduce the three muconic acid isomers (cis,cis, cis,trans, trans,trans) using NADH as a reductant. Titration of the purified ERBC with different chemical reductants showed that its redox cofactors (FMN, FAD, and [4Fe-4S]) can also be reduced by dithionite and Ti(III)-citrate. However, only dithionite and NADH supported the biohydrogenation of trans-cinnamic acid and cis,cis-muconic acid. The individually expressed and purified large domain of ERBC also catalyzed muconic acid reduction with these reductants, but exhibited lower activity and produced only 2-hexenedioic acid as the product. Efficient conversion of muconic acid to adipic acid was demonstrated using lyophilized E. coli cells expressing full-length ERBC as the catalyst, with dithionite acting as both a reductant and an oxygen scavenger. The use of lyophilized recombinant Escherichia coli cells with dithionite for ERBC-mediated biohydrogenation of muconic acid eliminates the need for protein purification and costly natural cofactors (NAD(P)H), as well as enhances ERBC tolerance to high substrate concentrations and creates anaerobic conditions for ERBC activity. This approach shows promise for biobased adipic acid production and other applications of 2-enoate reductases.

Proteases have diverse industrial applications including keratinase, which degrade keratin-rich substrates, such as hair, nails, feathers, and skin. This study aimed to express a protease of Bacillus velezensis LPL061 (WP_003155195.1) in Escherichia coli BL21(DE3) and to evaluate its keratinolytic as a new source of keratinase. Two gene constructs were designed, one containing the propeptide domain (kerfull) and one without it (kerhalf). Expression was induced with 0.05 mM IPTG at 20°C for 20 h. KerFull was partially purified using sequential ultrafiltration. SDS-PAGE analysis showed that KerHalf as a 32.2 kDa protein, while KerFull appeared as a 38 kDa fusion and a 28.5 kDa mature form. QTOF-MS confirmed the amino acid sequence. Only KerFull exhibited proteolytic and keratinolytic, highlighting the importance of the I9 domain for proper folding and enzyme activation. The partially purified mature KerFull protein (P50 fraction) retained activity despite low yield. KerFull showed a broad pH stability (6-11) with optimum at pH 9 and active over a wide temperature range (37-70℃), with an optimum at 60℃. Protein remained stable at 20-40℃ and pH 8. Specific activity reached 16.67 U/mg in the crude and 34.14 U/mg of P50 fraction. When combined with DTT, KerFull effectively degraded chicken feather barbules within 4 h at 37 °C. These findings suggest that one of the proteases from B. velezensis LPL061 (WP_003155195.1) is a robust keratinase candidate, offering broad operational pH and temperature, and efficient keratin degradation even at low concentrations, making it a promising candidate for industrial keratinase applications.

Lignin is a major component of plant cell walls; however, its heterogeneous and complex structure has hindered its efficient utilization. Recently, strategies that combine chemical pretreatment with microbial conversion to produce valuable chemicals have attracted attention. To develop an ideal microbial platform for this purpose, it is essential to elucidate the complete bacterial catabolic system for lignin-derived aromatic compounds. Here, we identified an inner membrane transporter gene involved in the uptake of a metabolite of dehydrodiconiferyl alcohol (DCA), a lignin-derived β-5 dimer, in Sphingobium lignivorans SYK-6. SLG_12820 (phcK) was found to encode a major facilitator superfamily transporter belonging to the endosomal spinster family and is regulated by PhcR, a transcriptional regulator of DCA catabolism genes. Through mutant analysis and a specific uptake assay based on PhcR effector recognition, we demonstrated that PhcK functions as an inner membrane transporter that specifically imports the DCA metabolite, 3-(2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-7-methoxy-2,3-dihydrobenzofuran-5-yl)acrylic acid.

Carbohydrate-binding modules (CBMs) enhance enzymatic degradation of polysaccharides by improving substrate binding. CBM54, a family of carbohydrate-binding modules, is found in surface-displayed multimodular glycoside hydrolases (MGHs) of gram-positive bacteria. These modules bind insoluble polysaccharides, including chitosan, chitin, xylan, cellulose, and fungal cell wall β-glucans. In MGHs, the CBM54 module is invariably positioned downstream of three S-layer homology modules (SLHs), which anchor the enzyme to the bacterial cell surface. The SLHs-CBM54 tandem promotes bacterial adherence to substrates, concentrating hydrolytic enzymes at the interface and facilitating efficient uptake of soluble degradation products. CBM54 contains a cleavage site that divides it into two structurally distinct parts, which remain associated via hydrogen bonding. The processing of CBM54 promotes bacterial detachment from the substrate and their migration toward new nutrient sources. Beyond MGHs, the SLHs-CBM54 tandem occurs in many other bacterial proteins with uncharacterized functions. SLH modules may self-assemble into 2D arrays on synthetic supports, enabling their use as matrices for protein nucleation and crystal growth. The SLHs-CBM54 tandem fusion with functional modules offers an innovative approach to surface modification, with broad biomedical and biotechnological applications. Due to its versatile architecture, this system holds promise for antigen display, vaccine development, and enhanced polysaccharide degradation processes.

Nitrotryptophan and its derivatives are valuable building blocks for synthesizing bioactive compounds and functional materials. This study reports the development of an efficient and novel bio-catalytic bioreactor in Escherichia coli capable of the direct aromatic nitration of tryptophan, enabling the synthesis of nitrotryptophan isomers. The biosynthetic pathway incorporates a self-sufficient P450 enzyme (TB14, consisting of TxtE-linker14-BM3R) from Streptomyces for the direct insertion of a nitro group into the indole ring of L-tryptophan. This process is supported by a nitric oxide synthetase (BsNOS) from Bacillus subtilis or its chemical alternative, sodium nitroprusside (SNP), to produce nitric oxide (NO) from L-arginine, which facilitates the direct nitration. As both TB14 and BsNOS require the reductant NADPH for their respective biochemical reactions, a glucose dehydrogenase (GDH) from Bacillus subtilis was included in the experimental design to ensure NADPH regeneration within the system.The initial engineered strain produced 133.2 mg/L of nitrotryptophan in TB medium. Through systematic optimization, including pathway balancing, fermentation condition enhancement, and elimination of competing metabolic pathways, the final titer was successfully increased to 209.9 mg/L within 48 h. This work establishes a robust platform for the microbial production of valuable nitroaromatic compounds and provides key insights for future biocatalytic nitration strategies.

This study presents a novel fluorescence-based assay for quantifying Glyoxalase II (Glo II) enzymatic activity, using N-(9-Acridinyl)maleimide (NAM) as a fluorescent probe. The assay is designed to measure glutathione (GSH) production resulting from the hydrolysis of S-D-lactoylglutathione by Glo II, providing a sensitive and reliable method for assessing enzyme activity across various biological samples. The protocol involves incubating Glo II samples at 37 °C, then adding NAM, which reacts with thiol groups to form a fluorescent adduct. The fluorescence intensity is measured at excitation and emission wavelengths of 360 nm and 432 nm, respectively, allowing for precise quantification of Glo II activity. The NAM-Glo II method demonstrates exceptional sensitivity and specificity, with limits of detection (LOD) and quantification (LOQ) of 0.01 U/L and 0.033 U/L, respectively. This high sensitivity is crucial for accurately measuring Glo II activity in diverse bacterial strains, where enzyme levels may vary. Comparative studies with established methods reveal that the NAM-Glo II assay consistently yields results comparable to, and in some cases superior to, those obtained using UV-based techniques. Notably, the method effectively minimizes interference from common biomolecules, such as amino acids and carbohydrates, which can confound traditional assays. The NAM-Glo II method is a reliable, sensitive tool for quantifying Glo II activity, crucial for neurological and microbial studies. It enables accurate enzyme measurement, reveals higher activity in E. coli, aids bacterial metabolism research, and supports insights into detoxification, resistance, and targeted antimicrobial therapies.

Trichomonas vaginalis is the etiologic agent of trichomoniasis, the non-viral sexually transmitted infection most prevalent in world. It is important to investigate biochemical aspects of the parasite that contribute to our understanding of the biology and applications in the treatment and diagnosis of the infection. The nucleoside triphosphate diphosphohydrolase (NTPDase) is an enzyme that hydrolyses extracellular adenine and guanine nucleotides, forming nucleosides adenosine and guanosine. This is important for parasite survival through the purine salvage pathway, since adenosine is the precursor for the entire purine nucleotides pool in T. vaginalis. Herein we expressed TvNTPDase4 in the bacterial system Escherichia coli. Our data demonstrate that the enzyme is active, being able to hydrolyze ATP, ADP and AMP at a concentration of 10 μg of purified protein/reaction. The inhibitors gadolinium and adenosine 5'-[α,β-methylene]diphosphate (AMPCP) inhibited the hydrolysis of rTvNTPDase4. The inhibition of ATPase/ADPase activity was more effective with gadolinium, while the inhibition of AMPase activity was more effective with AMPCP. The enzyme rTvNTPDase4 was not cytotoxic to HMVII cells. In molecular dynamics, we observed that the ability of TvNTPDase4 to hydrolyze ATP, ADP, and AMP substrates occurs through direct interactions with the apyrase-conserved regions (ACR), especially ACR1 and ACR4. In this work, we did not find any candidate sequence for ecto-5'-nucleotidase (E-5'-N) in T. vaginalis, which leads us to believe that the parasite does not have this enzyme in its proteomic repertoire. Finally, we report that rTvNTPDase4 expressed and purified from a bacterial is active and has potential for biotechnological applications.

Thermoplastic polyurethane (TPU) with complex physical crosslinking is difficult to degrade under natural environmental conditions. Current degradation methods, particularly for aromatic TPU, suffer from poor degradation efficiency. This study investigated the degradation performance of lignin peroxidase (LiP) on aromatic TPU through protein engineering and multi-enzyme system construction. Results indicated that wild-type LiP (WT-LiP), expressed from the recombinant Pichia pastoris GS115, exhibited a certain degradation effect on aromatic TPU. By using molecular docking techniques to identify key mutation sites, three LiP mutants (F46W, H47W, and H175R) were successfully constructed. Under optimal conditions (30°C, pH 2.5, 1 mM H₂O₂, and 5 U/mg enzyme), the F46W mutant achieved a molecular weight degradation rate of 11.97 % after 3 days of degradation, which is 2.2 times higher than that of the WT-LiP with a weight loss of 2.22 %, and the degradation efficiency in 28 days was 26.59 %. Furthermore, the constructed multi-enzyme systems (LiP-manganese peroxidase-laccase and LiP-carboxylesterase) substantially improved the degradation efficiency of TPU. Specifically, the LiP-carboxylesterase system demonstrated superior performance, achieving molecular weight degradation rates of 29.20 % and weight loss of 5.07 % after 3 days of treatment. This study provides a green enzymatic approach for efficient aromatic TPU plastics degradation and offers more sustainable solutions for plastic waste management.