Specific Activity Of Enzyme Research Articles

Mitochondrial proteome analysis reveals that an augmented cytochrome c oxidase assembly and activity potentiates respiratory capacity in sarcoma

Mitochondrial oxidative phosphorylation (OXPHOS) is an obligatory process in sarcoma. Despite that, the metabolic programming of sarcoma mitochondria is still unknown. To obtain a comprehensive metabolic insight of mitochondria, we developed a mouse fibrosarcoma model by injecting 3-methylcholanthrene and compared mitochondrial proteomes between sarcoma and its contralateral normal muscle using mass spectrometry. Our study identified ∼449 proteins listed in the SwissProt databases, and all the data sets are available via ProteomeXchange with the identifier PXD044903. In sarcoma, 49 mitochondrial proteins were found differentially expressed, including 36 proteins up-regulated and 13 proteins down-regulated, with the significance of p-value <0.05 and the log2[fold change] > 1 and < −1 as compared to normal muscle. Our data revealed that various anaplerotic reactions actively replenish the TCA cycle in sarcoma. The comparative expression profile and Western blotting analysis of OXPHOS subunits showed that complex-IV subunits, MT-CO3 and COX6A1, were significantly up-regulated in sarcoma vs. normal muscle. Further, biochemical and physiological assays confirmed enhanced complex-IV specific enzymatic and supercomplex activities with a concomitant increase of oxygen consumption rate in sarcoma mitochondria compared to normal muscle. Validation with human post-operative sarcoma tissues also confirms an increased MT-CO3 expression compared to normal tissue counterparts. Thus, our data comprehensively analyses the mitochondrial proteome and identifies augmented complex-IV assembly and activity in sarcoma.

Insight into the role of droplets in quinoa starch-based Pickering high internal phase emulsion for enzymatic hydrolysis reaction

Pickering high internal phase emulsion has emerged as potential platforms for biocatalytic reactions. Herein, native quinoa starch was used as the emulsifier to form a stable Pickering HIPE with an internal phase up to 80 %. The catalytic performance of lipase loaded in HIPE was evaluated for the enzymatic hydrolysis of hexyl hexanoate. Compared with the typical Pickering emulsion, biphasic, and monophasic systems, the Pickering HIPE system displayed higher conversion (83.54 % at 1440 min) and specific enzyme activity (1.41 U/mg), thus emphasizing the crucial role of microdroplets and large interface area created by both the starch particles and high internal phase volume. Subsequently, HIPEs with different starch concentrations were fabricated to further understand the catalysis behavior happening at compartmentalized microdroplets and interfaces. The microstructural, interfacial, and rheological properties of the HIPEs were systematically determined to explain the correlation between emulsion properties and catalytic efficiency. High starch concentration reduced the droplet size and increased the interfacial area, thus shortening the mass transfer distance and enlarging the reaction area. Meanwhile, the increased surface coverage and viscosity led to poor accessibility and limited mass movement. This trade-off made the maximum conversion be obtained at 2 wt% concentration. The study provides novel information about the role of emulsion droplets in the catalytic performance of lipase in starch-based HIPE.

Extraction and Partial Purification of Glucoamylase from Rhizopus oryzae by Solid State Fermentation using Agro-Industrial Residues

Background: Filamentous fungi are used to produce many industrial enzymes because these fungi have a great ability to produce such enzymes, especially when using solid substrates that are usually low in cost. The widespread use of this enzyme has made it very important to work on improving its production to achieve the maximum possible benefit.Methods: Five fungal isolates were obtained from soil and decaying fruits and vegetables. All isolates were identified as Rhizopus spp. and cultured on Potato Dextrose Agar (PDA) plates escorted by starch 1%. The appearance of clearance zones around the fungal colonies represents the ability of fungi to produce Glucoamylase. Different solid substrates, nitrogen sources, and temperatures were used to improve enzyme production.Results: Wheat bran gave the highest enzyme production with specific activity 6.25 U/mg, and yeast extract was the potent inducer for enzyme production with specific activity 9.28 U/mg. The optimal temperature for enzyme production was 30°C with specific activity10.27U/mg. Maximum specific activity 18.40U/mg was recorded at 55% saturation with ammonium sulfate precipitation. The result showed increased specific activity 24.84U/mg with dialysis. Partial purification of the enzyme revealed that pH5 was optimum for enzyme-specific activity 9.2U/ml; higher enzyme-specific activity was found with MnSO4 (11.83U/ml) as the best enzyme inducer.Conclusion: This investigation attempted to examine R. oryzae as an effective maker of GA. Innovative work into the nearby creation of GA for industrial use utilizing local resources has demonstrated financial effectiveness. It requires improvement into consistency as needed by worldwide associations dealing with industrial enzymes. Keywords: Glucoamylase; Production; Partial purification; Rhizopus oryzae; Solid state fermentation

Isolation and characterization of polygalacturonase producing thermophilic Aspergillus niger isolated from decayed tomato fruits

This study aimed to isolate a fungal strain capable of producing acidophilic and thermostable polygalacturonase. In this study, the fungal isolate was isolated from decaying tomatoes. Based on the colony characteristics, microscopic and morphological observations, the isolated fungal pathogen has been identified as Aspergillus niger. The isolated fungus was used in solid-state fermentation to produce an acidic polygalacturonase enzyme. The enzyme was then purified using ammonium sulphate precipitation and column chromatography, and its activity was assayed by measuring the releasing sugar group from citrus pectin using a 3, 5-dinitrosalicylic acid (DNSA) reagent assay. The crude extract obtained from solid-state fermentation had an activity of 94.6 U/mL. Ammonium sulphate precipitation increased the enzyme's specific activity from 6.89 U/mg to 12.42 U/mg. Sephadex G-200 was used to purify the enzyme 3.58 times, and its specific activity was determined to be 24.66 U/mg. The Sephacryl S-100 column achieved a final fold purification of 9.93 times and a specific activity of 68.41 U/mg. The purified enzyme performed best when polygalacturonic acid was used as a substrate. The enzyme's optimum temperature and pH were 55°C and 5, respectively. CaCl2 was found to be the best chelating ion for the enzyme. This enzyme is recommended for use in a variety of industrial applications as the enzyme was found to be stable at acidic pH and high temperature.

Synthesis and application of mesoporous MIL-88A for enhanced lipase immobilization and high value oil conversion

MIL-88A, a metal–organic framework (MOF), has significant promise for lipase immobilization due to its robust acid and water tolerance coupled with cost-effectiveness. Nonetheless, enhancing the dimensional compatibility of carrier with lipase and substrate is imperative for its broader application. In this study, we innovatively synthesized mesoporous MIL-88A (Meso-MIL-88A) with a fairly wide pore distribution around 10 nm using a soft-template method followed by a citrate dissociation strategy. The adsorption isotherm of Meso-MIL-88A conformed to the Langmuir model, exhibiting a notable increase in the adsorption capacity of lipase ET 2.0 compared to common MIL-88A (C-MIL-88A), attributed to the augmented external specific surface area (59 to 19 m2/g). Moreover, the immobilized lipase on Meso-MIL-88A showcased superior specific activity and enzyme activity recovery in contrast to C-MIL-88A, owing to enhanced structural preservation of lipase protein and reduced mass transfer resistance. Greater stability towards pH and temperature and better dynamic performance of immobilized lipase compared to free lipases exhibited the great advantages of Meso-MIL-88A as immobilized carrier. Notably, in the synthesis of phosphatidyl DHA (docosahexaenoic acid), immobilized CalB (Candida antarctica lipase B) on Meso-MIL-88A exhibited a 41.89 % incorporation of DHA into PC (phosphatidylcholine) within 48 h and an 82.60 % recovery after five batches of use. SEM observations confirmed the sustained integrity of the immobilized lipase structure post-recycling, underscoring the good stability of Meso-MIL-88A in the catalysis system. This study unveils the substantial potential of Meso-MIL-88A in lipase immobilization and offers a facile approach to tailoring the pore size of MOFs for practical application system.

Proteomic analysis of Rana sylvatica reveals differentially expressed proteins in liver in response to anoxia, dehydration or freezing stress

Ectothermic animals that live in seasonally cold regions must adapt to seasonal variation and specific environmental conditions. During the winter, some amphibians hibernate on land and encounter limited environmental water, deficient oxygen, and extremely low temperatures that can cause the whole body freezing. These stresses trigger physiological and biochemical adaptations in amphibians that allow them to survive. Rana sylvatica, commonly known as the wood frog, shows excellent freeze tolerance. They can slow their metabolic activity to a near halt and endure freezing of 65–70% of their total body water as extracellular ice during hibernation, returning to normal when the temperatures rise again. To investigate the molecular adaptations of freeze-tolerant wood frogs, a comprehensive proteomic analysis was performed on frog liver tissue after anoxia, dehydration, or freezing exposures using a label-free LC–MS/MS proteomic approach. Quantitative proteomic analysis revealed that 87, 118, and 86 proteins were significantly upregulated in dehydrated, anoxic, and frozen groups, suggesting potential protective functions. The presence of three upregulated enzymes, glutathione S-transferase (GST), aldolase (ALDOA), and sorbitol dehydrogenase (SORD), was also validated. For all enzymes, the specific enzymatic activity was significantly higher in the livers of frozen and anoxic groups than in the controls. This study reveals that GST, ALDOA, and SORD might participate in the freeze tolerance mechanism by contributing to regulating cellular detoxification and energy metabolism.

Phenol content and β-glucosidase activity during the ripening period of olive fruits (Erkence cv.) from different locations

In the present study, Erkence olive fruits were examined during two harvest periods and in two orchards close to the sea at low altitude (A) and far from the sea at higher altitude (B). Olives from the sea-facing northeast (NE) and land-facing southwest (SW) orientations of the trees were analyzed in terms of maturity index, total phenol content, phenolic compounds, β-glucosidase enzyme activity, total protein content and specific enzyme activity. Climate data were collected from the trees and from the meteorology station. The maturity index, β-glucosidase enzyme activity, total protein content and specific enzyme activity of the olives increased during the ripening period. On the other hand, the total phenol content, oleuropein and other phenolic compound contents in the olives decreased. The altitude and location of the fruits on the tree affected the ripening process. Lower altitude and sea-facing side of the tree resulted in an increased enzyme activity, thus aiding in the ripening process.

Non-destructive detection of specific enzyme activities in tomato plants using visible-near infrared spectroscopy

Non-destructive detection of specific enzyme activities in tomato plants using visible-near infrared spectroscopy

Intermolecular FRET Pairs as An Approach to Visualize Specific Enzyme Activity in Model Biomembranes and Living Cells

Herein, we propose an analytical approach based on intermolecular fluorescent resonant energy transfer (FRET) pairs for the visualization of specific enzyme activity in model biomembranes and in living cells. Cell visualizations with fluorescent confocal laser microscopy usually rely on fluorescent probes, such as Fluorescein isothiocyanate (FITC), Alexa488, Tetramethylrhodamine isothiocyanate (TRITC) and many others. However, for more specific tasks, such as the detection of certain enzymatic activity inside the living cell, the toolbox is quite limited. In the case of enzyme-hydrolases for example, the choice is limited to organic molecules comprising a fluorescent dye (typically, 4-methylumbelliferone (MUmb) or 7-amino-4-methylcoumarin (AMC) derivatives) and a fluorescence quencher, bound via an enzyme-sensitive linker—so that when the linker is degraded, the fluorescent signal increases. Unfortunately, both MUmb and AMC are quenched and have a relatively low quantum yield in cells, and their excitation and emission ranges overlap with that of intracellular fluorophores, often producing a strong background noise. R6G, on the other hand, has excellent quantum yield apart from intracellular fluorophores, but there are no efficient quenchers that could be chemically linked to R6G. Herein, we show that R6G is able to form intermolecular FRET pairs with MUmb or AMC, with the latter serving as fluorescence donors. This yields a combination of R6G’s excellent fluorescence properties with a possibility to use an enzyme-sensitive linker in MUTMAC or AMC derivatives. This phenomenon was initially discovered in a model system, reversed micelles, where the donor, the acceptor, and the enzyme are forced to be in close proximity to each other, so that proximity could serve as an explanation for the intermolecular FRET effect. Surprisingly enough, the phenomenon has been reproduced in living cells. Moreover, we were able to create working intermolecular donor–acceptor FRET pairs for several different enzymes, including chymotrypsin, phosphatase, and asparaginase. This appears counterintuitive, as besides the overlap of the emission spectra of the donor and the absorption spectra of the acceptor, there are other criteria for the FRET effect, including the convergence of two fluorophores at a distance of about 1–10 nm, and the orientation of their dipoles at a certain angle, which is difficult to imagine in a bulk system like a living cell. We hypothesize that FRET-enabling donor–acceptor interaction may be taking place at the inner surface of the lipid bilayer, to which both donor and acceptor molecules would likely have an affinity. This hypothesis would require a more detailed investigation. Therefore, we have shown that the method suggested has good potential in the visualization of enzyme functioning inside living cells, which is often a challenging task. Shifting of the fluorescence signal to the long-wavelength region would increase the signal selectivity, making it easily distinguishable from autofluorescence.

Influence of Some Heterocyclic, Cyclic, and Nitrogen-Containing Compounds on Oxidative Deamination of Polyamines in a Cell-Free Test System.

We studied the effects of some nitrogen-containing, heterocyclic, and cyclic compounds on the rate of oxidative deamination of polyamines and putrescine in tissues with a high proliferation rate. For this purpose, the specific activities of the main enzymes of polyamine oxidative degradation - spermine oxidase (SMO), polyamine oxidase (PAO), and diamine oxidase (DAO) were determined using a cell-free test system from regenerating rat liver. The compounds methyl 2-(5-formylfuran-2-yl)benzoate and 2,7-bis-[2-(diethylamino)ethoxy]-9H-fluoren-9-one (and in the form of dihydrochloride) showed mainly activating effect on oxidative degradation of putrescine, spermidine, and spermine, which indirectly indicates their antiproliferative effect. Nitrogen-free compounds inhibited this process, thus exhibiting potentially carcinogenic properties. Correlations were calculated for activity of DAO, PAO, and SMO with 5 topological indices: Wiener (W), Rouvray (R), Balaban (J) in the Trinaistich modification, detour (Ip), and electropy (Ie). The highest dependence was noted for DAO and the Balaban index (R=-0.55), for PAO and the detour index (R=0.78), and for SMO and the electropy index (R=0.53). The remaining dependencies showed insignificant correlation strength.

Partial purification and pectinase activity of lactic acid bacteria and pectinolytic bacteria consortium

Abstract Pectinase is an enzyme that can degrade pectin and often applied to the food industry. Microbes predominantly produce pectinases. This study aims to obtain the partial pectinase enzyme activity of bacteria from Liberica coffee fermentation. Lactic acid bacteria (LAB) isolates and pectinolytic bacteria were tested qualitatively to determine their pectinolytic activity. The highest pectinolytic activity will be used as a consortium. Optimal growth of lactic acid bacteria consortium and pectinolytic bacteria consortium incubation results were centrifuged to obtain enzyme extracts. The pectinase enzyme extract was partially purified by ammonium sulfate precipitation and dialysis. Based on the analysis, the qualitative index of pectinolytic lactic acid bacteria with isolate code BAL 1, BAL 2, and BAL 3 was 3,62%, 5,17%, and 7,27%, respectively. In pectinolytic bacteria, the qualitative index of pectinolytic with isolate codes IF, 2C, and 3S was 14.56%, 6.26%, 6.74%. Precipitation 70% ammonium sulfate has the highest enzyme-specific activity.

Effect of pasteurization time and temperature on the physico-biochemical properties of soursop (Annona muricata L.) juice

This study aimed to determine the effect of pasteurization time and temperature on soursop juice characteristics. Soursop (Anonna muricata L.), a climacteric fruit, is a high source of natural antioxidants and contains polyphenol oxidase (PPO) enzyme, which causes browning and can reduce the phenol content in fruit which can reduce antioxidant activity. The pasteurization process was carried out to inactivate PPO in soursop juice production. Soursop juice was prepared from soursop fruit pulp pasteurized at temperatures (56, 62, 68, 74, and 80 oC) for various pasteurization times (5, 10, and 15 minutes). Time and temperature of pasteurization had a significant effect (ρ &lt; 0.05) on physical characteristics (Total Soluble Solid/TSS, viscosity, pH, and colour), polyphenol oxidase enzyme activity, antioxidant activity, and microbial population. Pasteurization at 62 ˚C for 5 minutes can maintain antioxidant activity with the greatest total phenol of 85.45 ± 3.5 mg GAE/100 g, total flavonoid of 71.38 ± 6.9 mg QE/100 g, and antioxidant activity of 76.91 ± 0, 33%. Pasteurization at 80 ˚C for 15 minutes can reduce the total number of bacteria from 350 ± 24 CFU/mL to 54 ± 22 CFU/mL, which has a TSS value of 13.73 ˚brix, viscosity of 928.67 mPa, pH of 4.40, a total colour change of 0.556, and has the lowest PPO enzyme specific activity of 0.0005 U/mg with enzyme inactivation of 93%.

Effect of ultrasound on the activity and structure of polyphenol oxidase purified from mango (Mangifera indica cv. "Xiaotainong")

This study separated, purified, and identified mango polyphenol oxidase (PPO) in Xiaotainong mango, and the inactivation effect and possible inactivation mechanism of mango PPO by ultrasound (US) was investigated. The purified mango PPO, identified as Alfonso mango chloroplast PPO (NCBI number: XP:044462061.1), exhibited specific enzyme activity of 178,192 U/mg, with a yield of 1.2%, which was 9.85-fold higher than crude enzyme solution. Bioinformatics analysis of XP_044462061.1 indicated that the PPO consisted of a stable hydrophilic protein with a molecular weight of 66.77 kDa, an amino acid number of 593, and a structural domain similar to the TYROSINASE_1 (PS00497, tyrosinase) CuA and TYROSINASE_2 (PS00498; tyrosinase) CuB binding regions. The result showed that increased US treatment intensity and time better facilitated PPO inactivation, with a higher US intensity performing better than extended treatment time. US changed and disrupted the secondary and endogenous hydrophobic structures of the mango PPO, altered the exogenous hydrophobicity, and caused PPO molecule aggregation, ultimately leading to PPO inactivation. This article provided a theoretical basis for the application of US technology for processing mango products.

First Clarification of the Mechanism of Action of the Apple Glycosyltransferase MdUGT91AJ2 Involved in the Detoxification Metabolism of the Triketone Herbicide Sulcotrione.

Sulcotrione is a member of triketone herbicides, a class of HPPD (4-hydroxyphenylpyruvate dioxygenase) inhibitors with broad-spectrum herbicidal activity. Modifications of glycosylation mediated by glycosyltransferases (GT) are involved in plant detoxification. In this study, we analyzed chip data published online and found that eight glycosyltransferases from group A of the apple glycosyltransferase family 1 may be involved in the metabolic mechanism of detoxification of triketone herbicides. To verify this prediction, we induced apple seedlings with six types of triketone herbicides, and then detected the expression levels of eight glycosyltransferase genes through real-time PCR. We found that triketone herbicides induced up-regulation of eight glycosyltransferase genes to varying degrees, with MdUGT91AJ2 being the most significantly up-regulated by sulcotrione-induced glycosyltransferase gene expression. Then, through in vitro enzymatic reactions and HPLC identification of glycoside substrates, it was found that the glycosyltransferase MdUGT91AJ2 had the highest specific enzyme activity against the triketone herbicide sulcotrione. Furthermore, the in vivo mechanism of the glycosyltransferase MdUGT91AJ2 in the detoxification metabolism of sulcotrione was further validated by overexpressing the strain in the plant. HPLC analysis showed that the content of sulcotrione glycosides in the overexpressing strain of MdUGT91AJ2 was significantly higher than that in the wild type. This result indicated that the apple glycosyltransferase MdUGT91AJ2 can still glycosylate and modify sulfotrione in plants, and participate in its detoxification metabolism. In summary, this study identified for the first time a novel apple glycosyltransferase MdUGT91AJ2 and elucidated its mechanism of action in the detoxification and metabolism of the triketone herbicide sulfotriene.

Molecular insight into the potential functional role of pseudoenzyme GFOD1 via interaction with NKIRAS2.

The glucose-fructose oxidoreductase/inositol dehydrogenase/rhizopine catabolism protein (Gfo/Idh/MocA) family includes a variety of oxidoreductases with a wide range of substrates that utilize NAD or NADP as redox cofactor. Human contains two members of this family, namely glucose-fructose oxidoreductase domain-containing protein 1 and 2 (GFOD1 and GFOD2). While GFOD1 exhibits low tissue specificity, it is notably expressed in the brain, potentially linked to psychiatric disorders and severe diseases. Nevertheless, the specific function, cofactor preference, and enzymatic activity of GFOD1 remain largely unknown. In this work, we find that GFOD1 does not bind to either NAD or NADP. Crystal structure analysis unveils that GFOD1 exists as a typical homodimer resembling other family members, but lacks essential residues required for cofactor binding, suggesting that it may function as a pseudoenzyme. Exploration of GFOD1-interacting partners in proteomic database identifies NF-κB inhibitor-interacting Ras-like 2 (NKIRAS2) as one potential candidate. Co-immunoprecipitation (co-IP) analysis indicates that GFOD1 interacts with both GTP- and GDP-bound forms of NKIRAS2. The predicted structural model of the GFOD1-NKIRAS2 complex is validated in cells using point mutants and shows that GFOD1 selectively recognizes the interswitch region of NKIRAS2. These findings reveal the distinct structural properties of GFOD1 and shed light on its potential functional role in cellular processes.

Establishing an Artificial Pathway for the Biosynthesis of Octopamine and Synephrine.

In this study, we designed an artificial pathway composed of tyramine β-hydroxylase (TBH) and phenylethanolamine N-methyltransferase (PNMT) for the biosynthesis of both octopamine and synephrine. As most TBH and PNMT originate from eukaryotic animals and plants, the heterologous expression and identification of functional TBH and PNMT are critical for establishing the pathway in mode microorganisms like Escherichia coli. Here, three TBHs were evaluated, and only TBH from Drosophila melanogaster was successfully expressed in the soluble form in E. coli. Its expression was promoted by evaluating the effects of different expression strategies. The specific enzyme activity of TBH was optimized up to 229.50 U·g-1, and the first step in the biosynthetic pathway was successfully established and converted tyramine to synthesize 0.10 g/L of octopamine. Furthermore, the second step to produce synephrine from octopamine was developed by screening PNMT, enhancing enzyme activity, and optimizing reaction conditions, with a maximum synephrine production of 2.02 g/L. Finally, based on the optimization of the reaction conditions for each individual reaction, the one-pot cascade reaction for synthesizing synephrine from tyramine was constructed by combining the TBH and PNMT. The synthetic synephrine reached 30.05 mg/L with tyramine as substrate in the two-step enzyme cascade system. With further optimization and amplification, the titers of octopamine and synephrine were increased to 0.45 and 0.20 g/L, respectively, with tyramine as substrate. This work was the first achievement of the biosynthesis of octopamine and synephrine to date.

Titanium dioxide nanoparticles induce energy perturbation by stimulating glycolytic metabolic profile in rats

The safety of titanium dioxide nanoparticles (TiO2-NPs) remains uncertain due to a scarcity of data regarding its absorption, distribution, elimination, and potential adverse effects following oral exposure. As emerging evidence suggests perturbations in energy metabolism play a pivotal role in the toxicity induced by various toxicants, this investigation aimed to assess the effect of TiO2-NPs on the activities of specific glycolytic enzymes in rats. In this study, seventy-two (72) male Wistar rats (200 ± 20g) were divided into 12 groups, each consisting of 6 animals. The rats were orally exposed to TiO2-NPs (8-12nm) at doses of 50, 150, and 250 mg/kg body weight (BW) for durations of 4, 8, and 12 weeks. The control groups received distilled water. The results showed that, except for the 150 mg/kg BW dose of 12 weeks, TiO2-NPs exposure led to an up-regulation in hexokinase activity in lymphocytes, plasma, erythrocytes, and the liver. Hepatic and lymphocyte aldolase activities were also up-regulated, except at 8 and 12 weeks for the 50 and 150 mg/kg BW doses, where slight decreases were observed. Plasma aldolase increased, except at 12 weeks for 150 mg/kg BW dose, while erythrocyte aldolase increased only during the 4 and 8-week exposure but decreased throughout the 12-week exposure. Notably, compared to erythrocyte lactate dehydrogenase activity, which decreased, a consistent pattern observed in lymphocytes, plasma, and hepatic enzymes was a down-regulation at 8 and 12 weeks for the 50 and 150 mg/kg BW doses of TiO2-NPs. In other instances, and time intervals, lactate dehydrogenase was up-regulated. The data from this study underscores that exposure to TiO2-NPs can disrupt the glycolytic pathway of energy metabolism, particularly during the initial 4 weeks of exposure. These perturbations are characterized by increased glycolytic enzymes activity in the lymphocytes, plasma, and the liver.

Enhanced Protease Production from Diabetic Ulcer-Derived E. coli

This study explored a local Escherichia coli isolate from diabetic foot ulcer patients for protease enzyme production. Qualitative screening on skim milk agar showed a 6 mm hydrolysis halo, while quantitative screening with casein revealed a specific activity of 144.55 U/mg protein. Purification via ion exchange chromatography on DEAE-cellulose increased the specific activity to 767.5 U/mg, with a 5.3-fold enhancement and a 29.5% yield. The findings suggest E. coli as a viable source for protease production and demonstrate the efficiency of ion-exchange chromatography for enzyme purification, with potential significant industrial applications. Highlight: E. coli sourced from diabetic ulcers shows promise for protease production. Skim milk agar and casein used for qualitative and quantitative screening. Ion exchange chromatography significantly enhances enzyme specific activity. Keywoard: Escherichia coli, protease production, diabetic foot ulcer, enzyme purification, ion exchange chromatography

Functional characterization of two reductase KshBs in Mycobacterium fortuitum and their applications to C9 non-hydroxylated steroid production

3-ketosteroid-9α-hydroxylase (KSH), a two-component monooxygenase consisting of a Rieske oxygenase KshA and a ferredoxin reductase KshB, is a crucial enzyme involved in C-9 hydroxylation and is indispensable in the microbial catabolism of sterols. However, the in vivo function of KshB remains unclear. In this study, two reductase subunits, KshB1 and KshB2 from Mycobacterium fortuitum ATCC 35855, were first characterized and then engineered in the strain MFΔkstD, which produces 9-OHAD (9α-hydroxy-4-androstene-3,17-dione), to construct the strains producing AD (4-androstene-3,17-dione). The transcriptional level and specific enzyme activity of KshB1 under phytosterols induction was significantly higher compared to KshB2. After knocking out kshB1, AD peaked at 1.77 g/L at 48 h, subsequently being fully converted to 9-OHAD. Furthermore, we developed the stable AD-producer MFΔkstDΔkshB by null mutation both kshB1 and kshB2, although no AD was detected by single knocking out kshB2. Through gene complementation and the bioconversion of phytosterols, KshB1 emerged as the primary reductase in the KSH system, with KshB2 serving a complementary role. The stable AD-producer MFΔkstDΔkshB could produce 5.18 g/L, 6.91 g/L, and 9.03 g/L AD from the transformation of 10 g/L, 15 g/L, and 20 g/L phytosterols, respectively. In conclusion, these findings highlight a new strategy for the metabolic engineering of Mycobacterium fortuitum, involving the inactivation of the reductase KshB to facilitate the production of C9 non-hydroxylated steroidal intermediates.

Bi-directionalized promoter systems allow methanol-free production of hard-to-express peroxygenases with Komagataella Phaffii

BackgroundHeme-incorporating peroxygenases are responsible for electron transport in a multitude of organisms. Yet their application in biocatalysis is hindered due to their challenging recombinant production. Previous studies suggest Komagataella phaffi to be a suitable production host for heme-containing enzymes. In addition, co-expression of helper proteins has been shown to aid protein folding in yeast. In order to facilitate recombinant protein expression for an unspecific peroxygenase (AnoUPO), we aimed to apply a bi-directionalized expression strategy with Komagataella phaffii.ResultsIn initial screenings, co-expression of protein disulfide isomerase was found to aid the correct folding of the expressed unspecific peroxygenase in K. phaffi. A multitude of different bi-directionalized promoter combinations was screened. The clone with the most promising promoter combination was scaled up to bioreactor cultivations and compared to a mono-directional construct (expressing only the peroxygenase). The strains were screened for the target enzyme productivity in a dynamic matter, investigating both derepression and mixed feeding (methanol-glycerol) for induction. Set-points from bioreactor screenings, resulting in the highest peroxygenase productivity, for derepressed and methanol-based induction were chosen to conduct dedicated peroxygenase production runs and were analyzed with RT-qPCR. Results demonstrated that methanol-free cultivation is superior over mixed feeding in regard to cell-specific enzyme productivity. RT-qPCR analysis confirmed that mixed feeding resulted in high stress for the host cells, impeding high productivity. Moreover, the bi-directionalized construct resulted in a much higher specific enzymatic activity over the mono-directional expression system.ConclusionsIn this study, we demonstrate a methanol-free bioreactor production strategy for an unspecific peroxygenase, yet not shown in literature. Hence, bi-directionalized assisted protein expression in K. phaffii, cultivated under derepressed conditions, is indicated to be an effective production strategy for heme-containing oxidoreductases. This very production strategy might be opening up further opportunities for biocatalysis.