Maximum Enzyme Activity Research Articles

A novel alkali and thermotolerant protease from Aeromonas spp. retrieved from wastewater

Enzymes are integral to numerous industrial processes, with a growing global demand for various enzyme types. Protease enzymes, in particular, have proven to be cost-effective, stable, and compatible alternatives to traditional chemical processes in both industrial and environmental applications. In this study, an alkaline protease-producing strain of Aeromonas spp. was isolated from a wastewater treatment plant in Iran. The protease production was confirmed by culturing the strain on casein agar medium. The bacterium was identified through morphological, biochemical, and 16 S rRNA sequencing analyses. The optimal culture medium for bacterial growth and enzyme production was obtained using peptone, salt, yeast extract, galactose, and CaCl₂ at an initial pH of 8. Maximum protease production was achieved after 20 h of incubation at 40 °C. To partially purify the enzyme, the supernatant of the bacterial culture medium was first centrifuged, and the enzyme was precipitated using ammonium sulfate, followed by dialysis. Zymography revealed the production of one type of protease during bacterial growth. The partially purified protease exhibited optimal activity at pH 8.5 and maximum stability at pH 9. The optimum temperature for maximum enzyme activity was observed at 50 °C, with 100% residual activity retained for 1 h at 0 °C. The effect of metal ions on enzyme activity was assessed, revealing that KCl induced the most significant effects (p < 0.0001) on enzyme activity. Chemical amino acid modifiers and inhibitors, such as EDTA, DEPSI, and IAA, did not exhibit significant inhibition. In contrast, PMSF and HNBB significantly (p < 0.0001) reduced enzyme activity, suggesting that the enzyme could be classified as a serine protease. The protease also demonstrated high stability in the presence of 2% SDS, showing no signs inactivation. The alkaline pH optimum, thermal stability, and resistance to SDS exhibited by the protease produced by the Aeromonas strain are particularly promising characteristics that warrant further investigation. Based on preliminary tests and the enzyme’s characteristics, this protease can be recommended for various applications, pending further studies.

Bimetallic FeCo metal–organic framework based cascade reaction system: Enhanced peroxidase activity for antibacterial performance

Metal-organic frameworks (MOFs), as a peroxidase (POD), can catalyze the conversion of H2O2 to reactive oxygen species (ROS) for antibacterial application. To achieve strong antibacterial activity, it is necessary to improve the enzyme-like activity of MOFs. In this study, FexCoMOF was synthesized by incorporating Co(II) to improve the electron transfer of Fe(III)/Fe(II), which enhanced its redox capacity as a nanozyme and further promoted the generation of hydroxyl radical (⋅OH), exhibiting higher POD activity. Doping with different amounts of Co(II) altered the particle size, specific surface area, and surface defects of FexCoMOF, thus exhibiting differential enzyme-like activities. Additionally, a glucose-responsive enzyme cascade reaction system based on Fe4CoMOF/glucose oxidase (GOx) was established. In the presence of glucose, the in situ-generated substrate H2O2 was in contact with the catalytic site and the generated gluconic acid enabled Fe4CoMOF to maintain maximum enzyme activity under physiological pH conditions, avoiding damage caused by the use of exogenous high concentrations of H2O2. In the in vitro antibacterial experiment, the minimum inhibitory concentration (MIC) of Fe4CoMOF/GOx was 10 μg mL−1 for Escherichia coli (E. coli) and 5 μg mL−1 for Staphylococcus aureus (S. aureus) (∼108 CFU mL−1). The increase in enzyme activity resulted in a considerable reduction in the dose of the antibacterial agent, which was conducive to improving bio-safety. The in vivo experiment in mice demonstrated that the glucose-responsive Fe4CoMOF-based cascade reaction system had excellent antibacterial properties and remarkably promoted wound healing. The antibacterial agent based on bimetallic MOF developed in this work provides a new idea for cascade catalytic antibacterial therapy and will have remarkable application prospects in biomaterials and nanomedicine.

Engineering Bacterial Chitinases for Industrial Application: From Protein Engineering to Bacterial Strains Mutation! A Comprehensive Review of Physical, Molecular, and Computational Approaches.

Bacterial chitinases are integral in breaking down chitin, the natural polymer in crustacean and insect exoskeletons. Their increasing utilization across various sectors such as agriculture, waste management, biotechnology, food processing, and pharmaceutical industries highlights their significance as biocatalysts. The current review investigates various scientific strategies to maximize the efficiency and production of bacterial chitinases for industrial use. Our goal is to optimize the heterologous production process using physical, molecular, and computational tools. Physical methods focus on isolating, purifying, and characterizing chitinases from various sources to ensure optimal conditions for maximum enzyme activity. Molecular techniques involve gene cloning, site-directed mutation, and CRISPR-Cas9 gene editing as an approach for creating chitinases with improved catalytic activity, substrate specificity, and stability. Computational approaches use molecular modeling, docking, and simulation techniques to accurately predict enzyme-substrate interactions and enhance chitinase variants' design. Integrating multidisciplinary strategies enables the development of highly efficient chitinases tailored for specific industrial applications. This review summarizes current knowledge and advances in chitinase engineering to serve as an indispensable guideline for researchers and industrialists seeking to optimize chitinase production for various uses.

Screening and Optimization of Pathogenic Strains of Pseudomonas sp. for Cholinesterase Enzyme Activity

The identification and optimization of pathogenic strains of Pseudomonas sp. with significant Cholinesterase enzyme activity has promising implications for environmental and medical applications. This study aimed to screen various Pseudomonas strains to identify those exhibiting high cholinesterase activity and to optimize the conditions for maximal enzyme production. Multiple Pseudomonas strains were isolated and characterized from diverse clinical samples, employing selective media and biochemical assays to determine cholinesterase activity. The strains with maximum enzyme activity were further subjected to optimization experiments, including variations in growth conditions such as Temperature, pH and Nutrient availability. The findings revealed sample no.39 among the collected 50 samples had significant enzyme activity of 0.19151 IU is obtained when choline chloride was 0.3999%, Ammonium Chloride was 0.01M, pH was 7.0, Temperature was 27°C and Time of incubation was 24hrs. These results not only advance our understanding of Pseudomonas sp. enzyme capabilities but also offer potential applications in bioremediation, biosensor development and pharmaceutical industries. Future research will focus on the molecular characterization of the cholinesterase enzyme and its applications in various industrial processes.

Producción de esterasa por Fusarium culmorum crecido en presencia de diferentes concentraciones di(2-etilhexil) ftalato y de ion calcio en fermentación sumergida

Phthalates are plastic additives used as plasticizers in the manufacture of flexible plastic materials. These compounds, such as di(2-ethylhexyl) phthalate (DEHP) are toxic substances to the environment and human health, and have been detected as environmental pollutants. In this study, the activity of esterase produced by Fusarium culmorum grown in media supplemented with different concentrations of DEHP (1.5, 3 and 4 g/L) and calcium ion (0.1 and 0.5 g/L) in submerged fermentation was characterized by biochemical tests and polyacrylamide gel electrophoresis. The greatest esterase activity was observed in cultures grown in media added with 3 g of DEHP/L and 0.5 g of calcium ion/L, followed by those cultures grown in media containing 1.5 g of DEHP/L. Whereas cultures grown on 4 g of DEHP/L had the lowest esterase activity. In general, cultures added with 0.5 g of calcium ion/L had the maximum enzymatic activity and the highest enzymatic productivity in the media tested. High concentration of DEHP were used as the sole source of carbon and energy by F. culmorum. It is suggested that the calcium ion favored the esterase production during the DEHP biodegradation. However, further studies are needed to understand the relationship between calcium ion, fungal growth, and enzyme production to provide the appropriate concentration of this ion in the culture medium.

Metabolic Adaptations in Phosphine-Resistant Tribolium castaneum Driven by Mitochondrial Enzyme Variability and Gene Expression.

Phosphine fumigation is essential for controlling storage pests like Tribolium castaneum, but its frequent application has resulted in resistance, primarily due to mutations in the Dihydrolipoamide dehydrogenase (DLD) gene associated with the rph2 allele. This study demonstrates that the Patiala population exhibits homozygous resistance variations across populations, contrasting with the susceptibility observed in laboratory cultures. Our assessment of mitochondrial DLD and Cytochrome c oxidase (COX) activities showed significantly elevated levels in the Patiala population, with increases of approximately sevenfold for DLD and 6.92-fold for COX, indicating mitochondrial adaptations for enhanced energy production. Kinetic analyses of DLD in the resistant Patiala population showed a higher Vmax (0.005 mmol/min) and a significantly increased Km (16.66 mM), indicating variations in maximal enzyme activity and substrate affinity. Furthermore, resistant T. castaneum populations displayed substantial upregulation of DLD and COX gene expression, with DLD expression increasing by 10.53-fold and COX expression peaking at 102.57-fold in Patiala. Pearson correlation analysis indicated strong positive correlations (r > 0.8) between enzymatic activity and gene expression for both DLD and COX, suggesting a coordinated role in resistance mechanisms. The PCA biplot illustrated distribution patterns of enzymatic activity and gene expression among field-resistant populations, highlighting the association between increased resistance and elevated enzymatic activity and gene expression levels. Therefore, the upregulation of DLD and COX activities in resistant populations underscores their critical roles in counteracting phosphine, reflecting metabolic reprogramming for improved energy production under stress.

Carbon dot unravels accumulation of triterpenoid in Evolvulus alsinoides hairy roots culture by stimulating growth, redox reactions and ANN machine learning model prediction of metabolic stress response

Evolvulus alsinoides, a therapeutically valuable shrub can provide consistent supply of secondary metabolites (SM) with pharmaceutical significance. Nonetheless, because of its short life cycle, fresh plant material for research and medicinal diagnostics is severely scarce throughout the year. The effects of exogenous carbon quantum dot (CD) application on metabolic profiles, machine learning (ML) prediction of metabolic stress response, and SM yields in hairy root cultures of E. alsinoides were investigated and quantified. The range of the particle size distribution of the CDs was between 3 and 7 nm. The CDs EPR signal and spin trapping experiments demonstrated the formation of O2-•spin-adducts at (g = 2.0023). Carbon dot treatment increased the levels of hydrogen peroxide and malondialdehyde concentrations as well as increased antioxidant enzyme activity. CD treatments (6 μg mL-1) significantly enhanced the accumulation of squalene and stigmasterol (7 and 5-fold respectively). The multilayer perceptron (MLP) algorithm demonstrated remarkable prediction accuracy (MSE value = 1.99E-03 and R2 = 0.99939) in both the training and testing sets for modelling. Based on the prediction, the maximum oxidative stress index and enzymatic activities were highest in the medium supplemented with 10 μg mL-1 CDs. The outcome of this study indicated that, for the first time, using CD could serve as a novel elicitor for the production of valuable SM. MLP may also be used as a forward-thinking tool to optimize and predict SM with high pharmaceutical significance. This study would be a touchstone for understanding the use of ML and luminescent nanomaterials in the production and commercialization of important SM.

Improved levan production by novel Calidifontibacillus erzurumensis LEV207 using one variable at a time approach.

Levan, an exopolysaccharide, has applications in the medical and food sectors. Under natural conditions, levan is produced at a low concentration by microorganisms. This current study focuses on optimizing levan production by a one-variable-at-a-time approach from a novel marine bacterium, Calidifontibacillus erzurumensis LEV207. The growth and levansucrase production by C. erzurumensis LEV207 were studied to optimize the media components and conditions. Sucrose and yeast extract proved to be a good levansucrase inducer. The pH of 6.0 and temperature of 30°C, along with MgSO4 as salt, increased the enzyme activity. Sucrose and yeast extract at the concentration of 50% and 1%, respectively, produced the maximum enzyme activity of 427.4µmol/mL/min after 48h of incubation. Sevag reagent was used for the purification of levan. The optimization process increased the levan (EPSLEV207) production sevenfold with a final yield of 65.58g/L. To confirm the structure and nature of EPSLEV207, NMR and XRD were performed, and the functional groups present in EPSLEV207 were determined using FTIR. Chemical hydrolysis of levan confirmed fructose as the monomer, making EPSLEV207 a homopolysaccharide. EPSLEV207 exhibited 49% and 22% radical scavenging activity against DPPH and ABTS radicals, respectively, and promoted the growth of probiotic strain. EPSLEV207 showed an 88% water solubility index and 78% water retention capacity. The optimization process has increased the yield of levan, highlighting the potential of C. erzurumensis LEV207 to be a viable option for industrial applications.

Temperature influence on the stability of the precursor cluster of the thermolysin crystal

We used the molecular dynamics method to assess the stability of the precursor-cluster (hexamer) of thermolysin crystal over a wide range of temperatures (10–90°C). The simulation results showed that as the temperature increases, the stability of the hexamer, in general, decreases, however, the hexamer does not dissociate at any of the considered temperatures. At a temperature of 60°C, an increase in the stability of the hexamer was observed. This value is close to the temperature of maximum enzymatic activity of thermolysin (70°C). Based on the analysis of the results, it was assumed that the crystallization of thermolysin could be carried out at 60°C.

Isolation of a novel Bacillus strain with industrial potential of producing alkaline chitosanase.

A novel Bacillus strain, designated as HZ20-1, was discovered. This strain can produce naturally alkaline chitosanase without induction. Genomic analysis revealed that this chitosanase belongs to glycoside hydrolase family 8. When colloidal chitosan is used as a substrate, the maximum enzyme activity of 2.39±0.03U/mL is observed at a pH range of 8.5-9. This alkaline enzyme holds advantages over common acidic enzymes in various fields such as medicine, the environment, and daily chemical products, and thus has great market value. Moreover, as the chitosanase is a constitutive enzyme, there is no need for substrate induction. This can simplify fermentation equipment and reduce production cost. Additionally, in a preliminary study, we found that this strain can also degrade chitin and chitosan derivatives. After analysis, it was discovered that it has genes in glycoside hydrolase families 18 and 23. By controlling the enzymatic hydrolysis time, it is possible to produce products with different molecular weights, including N-acetylglucosamine, glucosamine, chito-oligosaccharides, and chitin oligosaccharides. Consequently, Bacillus sp. HZ20-1 is considered to have great potential for future industrial production of enzymes and oligosaccharides.

Bioethanol production from groundnut shell using microorganisms derived from sugarcane root soil

Bioethanol is a form of renewable energy that can be produced from fuel or energy crops. Ethanol is produced by the fermentation of sugars present in agricultural feed stocks and crop residues. This study investigates the use of agro wastes like groundnut shells for ethanol production. Initially, the groundnut shells were washed, dried and grinded into powder. Then it was subjected to ethanol production using yeast. After 20 days of incubation the ethanol was estimated using potassium dichromate method. The maximum ethanol yield (1.55%) was obtained when 1% yeast was used. To increase the efficiency of ethanol production, the cellulolytic bacteria was isolated from the cow dung dumped soil site. The 10 bacterial isolates were screened for cellulase enzyme production. Among them one bacterium showed maximum decolourization of congored which was subjected for enzyme production in nutrient broth. The organism showed maximum enzyme activity of 558.12 U/ml. The isolated cellulolytic bacteria were identified as Bacillus anthracis using 16s rDNA sequencing. Ethanol production from the groundnut shells was again carried out with various concentration of crude cellulase enzyme isolated from the bacteria. The estimation result showed 3.8% of ethanol as maximum. Then the ethanol was condensed using a rotary evaporator and when again estimated, showed 7.3% of ethanol. Finally, the presence of ethanol was confirmed by iodoform test. Thus, groundnut shells can efficiently be used for the production of ethanol, which can be used as a high potential fuel source for transportation in the future.

Preparation of micron-sized benzamidine-modified magnetic agarose beads for trypsin purification from fish viscera

The effective method for trypsin purification should be established because trypsin has important economic value. In this work, a novel and simple strategy was proposed for fabricating micron-sized magnetic Fe3O4@agarose-benzamidine beads (MABB) with benzamidine as a ligand, which can efficiently and selectively capture trypsin. The micro-sized MABB, with clear spherical core-shell structure and average particle size of 6.6 μm, showed excellent suspension ability and magnetic responsiveness in aqueous solution. The adsorption capacity and selectivity of MABB towards target trypsin were significantly better than those of non-target lysozyme. According to the Langmuir equation, the maximum adsorption capacity of MABB for trypsin was 1946 mg g−1 at 25 °C, and the adsorption should be a physical sorption process. Furthermore, the initial adsorption rate and half equilibrium time of MABB toward trypsin were 787.4 mg g−1 min−1 and 0.71 min, respectively. To prove the practicability, MABB-based magnetic solid-phase extraction (MSPE) was proposed, and the related parameters were optimized in detail to improve the purification efficiency. With Tris-HCl buffer (50 mM, 10 mM CaCl2, pH 8.0) as extraction buffer, Tris-HCl buffer (50 mM, 100 mM CaCl2, pH 8.0) as rinsing buffer, acidic eluent (0.01 M HCl, 0.5 M NaCl, pH 2.0) as eluent buffer and alkaline buffer (1 M Tris-HCl buffer, pH 10.0) as neutralization solution, the MABB-based MSPE was successfully used for trypsin purification from the viscera of grass carp (Ctenopharyngodon idella). The molecular weight of purified trypsin was determined as approximate 23 kDa through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified trypsin was highly active from 30 °C to 60 °C, with an optimum temperature of 50 °C, and was tolerant to pH variation, exhibiting 85 % of maximum enzyme activity from pH 7.0 to 10.0. The results demonstrated that the proposed MABB-based MSPE could effectively purify trypsin and ensure the biological activity of purified trypsin. Therefore, we believe that the novel MABB could be applicable for efficient purification of trypsin from complex biological systems.

In silico and experimental characterization of a new polyextremophilic subtilisin-like protease from Microbacterium metallidurans and its application as a laundry detergent additive.

Considering the current growing interest in new and improved enzymes for use in a variety of applications, the present study aimed to characterize a novel detergent-stable serine alkaline protease from the extremophilic actinobacterium Microbacterium metallidurans TL13 (MmSP) using a combined in silico and experimental approach. The MmSP showed a close phylogenetic relationship with high molecular weight S8 peptidases of Microbacterium species. Moreover, its physical and chemical parameters computed using Expasy's ProtParam tool revealed that MmSP is hydrophilic, halophilic and thermo-alkali stable. 3D structure modelling and functional prediction of TL13 serine protease resulted in the detection of five characteristic domains: [catalytic subtilase domain, fibronectin (Fn) type-III domain, peptidase inhibitor I9, protease-associated (PA) domain and bacterial Ig-like domain (group 3)], as well as the three amino acid residues [aspartate (D182), histidine (H272) and serine (S604)] in the catalytic subtilase domain. The extremophilic strain TL13 was tested for protease production using agricultural wastes/by-products as carbon substrates. Maximum enzyme activity (390U/gds) was obtained at 8th day fermentation on potato peel medium. Extracellular extract was concentrated and partially purified using ammonium sulfate precipitation methodology (1.58 folds purification fold). The optimal pH, temperature and salinity of MmSP were 9, 60°C and 1M NaCl, respectively. The MmSP protease showed broad pH stability, thermal stability, salt tolerance and detergent compatibility. In order to achieve the maximum stain removal efficacy by the TL 13 serine protease, the operation conditions were optimized using a Box-Behnken Design (BBD) with four variables, namely, time (15-75min), temperature (30-60°C), MmSP enzyme concentration (5-10U/mL) and pH (7-11). The maximum stain removal yield (95 ± 4%) obtained under the optimal enzymatic operation conditions (treatment with 7.5U/mL of MmSP during 30min at 32°C and pH9) was in good agreement with the value predicted by the regression model (98 ± %), which prove the validity of the fitted model. In conclusion, MmSP appears to be a good candidate for industrial applications, particularly in laundry detergent formulations, due to its high hydrophilicity, alkali-halo-stability, detergent compatibility and stain removal efficiency.

A kinetic modelling approach to explore mechanism of Cr(VI) detoxification by a novel strain Pseudochrobactrum saccharolyticum NBRI-CRB 13 using response surface methodology.

A novel Pseudochrobactrum saccharolyticum strain NBRI-CRB 13, isolated from tannery sludge, was studied to grow up to 500 mgL-1 of Cr(VI) and showed Cr(VI) detoxification by reducing > 90% of Cr(VI) at different concentrations 25, 50 and 100 mgL-1. Kinetic studies showed that first-order models were fitted (R2 = 0.998) to the time-dependent Cr(VI) reduction with degradation rate constant (k) (1.03-0.429h-1). Cr(VI) detoxification was primarily related to the extracellular fraction of microbial cells, which showed a maximum extracellular reductase enzyme activity led to 94.6% reduction of Cr(VI). Moreover, the strain showed maximum extracellular polymeric substances (EPS) production at 100 mgL-1 Cr(VI), which is presumably the reason for Cr(VI) removal as EPS serves as the metal binding site for Cr(VI) ions. Further, an optimization study using Box-Behnken design was conducted considering parameters viz., pH, temperature, and initial concentration of Cr(VI). The maximum percent reduction of Cr(VI) was obtained at pH 6.5, temperature 30°C with 62.5 mgL-1Cr(VI) concentration. Further, the Cr(VI) reduction and adsorption ability of strain P. saccharolyticum NBRI-CRB13 were confirmed by SEM-EDS, FTIR, and XRD analyses. FTIR analysis confirmed the presence of functional groups (-OH, -COOH, -PO4) on bacterial cell walls, which were more likely to interact with positively charged chromium ions. The study elucidated the reduction of Cr(VI) by the novel bacterium within 24h using the response surface methodology approach and advocated its application in real-time situations.

Microbial degradation of paddy (Oryza sativa) straw by submerged state fermentation

This study elucidates the effective lignocellulose degrading potential of microbial consortium by using paddy straw as the sole source under submerged fermentation conditions. Straw degradation was enhanced by bioaugmentation procedures where consortia of isolated and standard cultures resulted in maximum enzyme activities of endoglucanase (1 U/ml), exoglucanase (1.31 U/ml), β-glucosidase (2.42 U/ml), xylanase (12.63 U/ml) and manganese peroxidase (5.43 U/ml) after 28 days of incubation along with laccase (4.36 U/ml) and lignin peroxidase (6.71 U/ml) after 21 days of incubation. By the action of consortium, maximum reduction of cellulose, hemicellulose and lignin was found to be 35.31, 22.50 and 44.72%, respectively after 28 days of incubation. A significant increase in percentage loss in dry matter of paddy straw was observed till 4 weeks of fermentation. Field emission scanning electron microscope (FE-SEM) micrographs revealed sufficient loosening of cellulose and hemicellulose showing successful degradation of straw tissue. Overall, the above stated findings emphasize on potential of lignocellulosic microorganisms to be used as an alternative to traditional stubble burning practice. Although, these extracellular enzymes are well equipped in breaking down lignocelluloses to their monomeric or oligomeric subunits, but they may not be exploited at commercial level due to very slow rate of reaction. The efficacy of these cultures for straw degradation in field condition further needs elaborative experimental trials as environmental parameters are quite variable in the field.

Optimization of Lignocellulosic Enzyme production by Microbial Strains: Study of Kinetics and Environmental Influences

In the context of valorizing lignocellulosic biomass for biofuel production a mixture of forest residues (FW) and aromatic-medicinal plant wastes (AMPW) was considered in this study to assess its ability to support the production of cellulolytic enzymes by Aspergillus niger, Trichoderma longibrachiatum and Zymomonas mobilis. The residue mixture promoted growth and cellulase production by the microbial strains after 6 days of incubation. Enzymatic activity was determined using filter paper activity (FPase) and endoglucanase carboxymethylcellulase (CMase) assays. Optimization of enzymatic activity was investigated at pH levels (2, 4, 6, 8), various temperatures (22°C, 30°C, 37°C, 45°C) and different inoculum rates (1.5 × 107, 2 × 107 and 2.5 × 107 spores per gram of substrate). Maximum enzymatic activity was observed in Aspergillus niger with FPase and CMCase yields of 23.12 ± 0.1 and 29.78 ± 0.15 U/gds respectively. The highest enzymatic activity was recorded at temperatures of 30°C and 37°C, pH levels of 4 and 6 and with an inoculum rate of 2.5 × 107. The selection of the strain and the optimization of operational conditions are crucial for efficient enzymatic production in the context of biorefinery.

Isolation and Identification of Xylanase Producing Bacteria from Soil Samples of Kerala

Microorganisms play a very vital role in the biological recycling of plant bio-matter. Xylan is the second most plentiful polysaccharide and is found as an interface between lignin and other polysaccharide in the secondary cell wall. When these polysaccharides are hydrolyzed by a suitable strain of bacteria it will become an end to many environmental issues and had many useful applications such as in paper and pulp industry, food and feed industry, bread quality improvement, agro waste treatment, seed germination, generation of bio-fuels etc. To isolate a suitable strain of bacteria which possess good xylanase activity several samples of soils were collected from various regions of Kerala and were undergone a three step enrichment process. In the primary screening 85 bacterial strains were obtained. 22 isolates produces clear zone during plate assay using beech wood xylan. The isolates which possess more than 0.5cm clear zone were undergone final screening process and the strain PSS 1 showed the maximum enzyme activity of 112.38 U/ml.

Stoichiometry and architecture of the human pyruvate dehydrogenase complex.

The pyruvate dehydrogenase complex (PDHc) is a key megaenzyme linking glycolysis with the citric acid cycle. In mammalian PDHc, dihydrolipoamide acetyltransferase (E2) and the dihydrolipoamide dehydrogenase-binding protein (E3BP) form a 60-subunit core that associates with the peripheral subunits pyruvate dehydrogenase (E1) and dihydrolipoamide dehydrogenase (E3). The structure and stoichiometry of the fully assembled, mammalian PDHc or its core remained elusive. Here, we demonstrate that the human PDHc core is formed by 48 E2 copies that bind 48 E1 heterotetramers and 12 E3BP copies that bind 12 E3 homodimers. Cryo-electron microscopy, together with native and cross-linking mass spectrometry, confirmed a core model in which 8 E2 homotrimers and 12 E2-E2-E3BP heterotrimers assemble into a pseudoicosahedral particle such that the 12 E3BP molecules form six E3BP-E3BP intertrimer interfaces distributed tetrahedrally within the 60-subunit core. The even distribution of E3 subunits in the peripheral shell of PDHc guarantees maximum enzymatic activity of the megaenzyme.

Magnetic ligand fishing protocol combined with HPLC-FT-ICR-MS for screening potential α-Glucosidase inhibitors from UCG and in silico analysis

Uremic Clearance Granule (UCG) is a traditional Chinese medicines (TCMs) that has been recognized as potentially effective treatment for diabetic nephropathy (DN). However, the chemical composition and the hypoglycemic ingredients of UCG remain unclear. In this experiment, a magnetic ligand fishing protocol combined with HPLC coupled Fourier transform ion cyclotron resonance mass spectrometry (HPLC-FT-ICR-MS) technology was developed to screen and identify α-Glucosidase inhibitors from UCG. First, constituents of UCG was identified by HPLC-FT-ICR-MS. Next, α-Glucosidase coated Fe3O4@SiO2@NH2 (Fe3O4@SiO2@NH2@α-Glucosidase) nano composites was synthesized. The essential parameters (concentration of Glutaraldehyde (GA), ratio of Fe3O4@SiO2@NH2 to enzyme, crosslinking time and immobilization time) were optimized to obtain maximum enzyme activity and the performance of immobilized enzyme was compared with that of free enzyme. The α-Glucosidase inhibitory activities were then evaluated using autodock and the 4-Nitrophenyl-α-D-glucopyranoside (pNPG) method. As a result, 142 compounds were identified were identified by comparing the retention time, molecular ions and fragmentation behaviors with the reference compounds or the in-house database. 20 possible α-Glucosidase activity inhibitory components were identified using HPLC-FT-ICR-MS. Molecular docking and inhibition studies showed that 4 compuunds including Limonexic acid, Sanggenol A, Glabrone, Matrine were more likely to stronger α-Glucosidase inhibitory activities than acarbose. In conclusion, the proposed approach, which combined highly specific screening with HPLC-FT-ICR-MS, provided a powerful platform for discovering bioactive components from multi-component and multi-target traditional Chinese medicines (TCMs).

Biofabrication of TiO2 nanoparticles via Aspergillus niger DS22 supernatant: bioreactor optimization and multi-activity profiling

AbstractThe study presents a safe and eco-friendly green synthesis of titanium dioxide nanoparticles (TiO2 NPs) using Aspergillus niger DS22 (ON076463.1) cell-free filtrate, focusing on optimizing factors affecting nitrate reductase enzyme production within the framework TiO2 NP biosynthesis. Maximum enzyme activity was accomplished by growing A. niger DS22 in a modified MYGP medium at pH 6, 0.5% peptone, 0.15% yeast extract, 0.25% KNO3, 2% glucose, and 200 rpm for 4 days at 30 °C. Statistical optimization takes place, where a central composite design was employed for testing the reaction variables. The individual and interactive effects of process variables lead to optimal biosynthesis conditions with 10−4 M (K2TiF6) concentration, for 96 h, 28 °C, pH 9, and Ti+4 salt solution:filtrate ratio (10%, v/v). Kinetic conversion rates in 1-L shake flask and 10-L stirred tank bioreactor were calculated and compared. Current findings revealed that the yield coefficient of biomass dry weight (Yx/s) and the yield coefficient of TiO2 NP dry weight (Y pn/s) in the bioreactor exceed those of the shake flask (0.85 g/L and 0.51 g/L; 0.04 g/L and 0.11 g/L, respectively). TiO2 NPs showed anticancer activities with high biocompatibility (at 1000 µg/mL) against MCF-7 and HepG-2 cell lines, with 97.35% and 97.71% cytotoxicity, respectively. TiO2 NPs had a moderate antioxidant activity of 57.8% recorded by DPPH assay. Moreover, TiO2 NPs had anticoagulant activities and decolorization efficiency for methyl orange dye. The current study paves the way for maximizing TiO2 NP production, which can be used in industrial and medical sectors. Graphical Abstract