KM Values Research Articles

Mandelonitrile lyase MDL2-mediated regulation of seed amygdalin and oil accumulation of Prunus Sibirica

BackgroundThe Prunus sibirica seeds with rich oils has great utilization, but contain amygdalin that can be hydrolyzed to release toxic HCN. Thus, how to effectively reduce seed amygdalin content of P. sibirica is an interesting question. Mandelonitrile is known as one key intermediate of amygdalin metabolism, but which mandelonitrile lyase (MDL) family member essential for its dissociation destined to low amygdalin accumulation in P. sibirica seeds still remains enigmatic. An integration of our recent 454 RNA-seq data, amygdalin and mandelonitrile content detection, qRT-PCR analysis and function determination is described as a critical attempt to determine key MDL and to highlight its function in governing mandelonitrile catabolism with low amygdalin accumulation in Prunus sibirica seeds for better developing edible oil and biodiesel in China.ResultsTo identify key MDL and to unravel its function in governing seed mandelonitrile catabolism with low amygdalin accumulation in P. sibirica. Global identification of mandelonitrile catabolism-associated MDLs, integrated with the across-accessions/developing stages association of accumulative amount of amygdalin and mandelonitrile with transcriptional level of MDLs was performed on P. sibirica seeds of 5 accessions to determine crucial MDL2 for seed mandelonitrile catabolism of P. sibirica. MDL2 gene was cloned from the seeds of P. sibirica, and yeast eukaryotic expression revealed an ability of MDL2 to specifically catalyze the dissociation of mandelonitrile with the ideal values of Km (0.22 mM) and Vmax (178.57 U/mg). A combination of overexpression and mutation was conducted in Arabidopsis. Overexpression of PsMDL2 decreased seed mandelonitrile content with an increase of oil accumulation, upregulated transcript of mandelonitrile metabolic enzymes and oil synthesis enzymes (involving FA biosynthesis and TAG assembly), but exhibited an opposite situation in mdl2 mutant, revealing a role of PsMDL2-mediated regulation in seed amygdalin and oil biosynthesis. The PsMDL2 gene has shown as key molecular target for bioengineering high seed oil production with low amygdalin in oilseed plants.ConclusionsThis work presents the first integrated assay of genome-wide identification of mandelonitrile catabolism-related MDLs and the comparative association of transcriptional level of MDLs with accumulative amount of amygdalin and mandelonitrile in the seeds across different germplasms and developmental periods of P. sibirica to determine MDL2 for mandelonitrile dissociation, and an effective combination of PsMDL2 expression and mutation, oil and mandelonitrile content detection and qRT-PCR assay was performed to unravel a mechanism of PsMDL2 for controlling amygdalin and oil production in P. sibirica seeds. These findings could offer new bioengineering strategy for high oil production with low amygdalin in oil plants.

Importance of Michaelis Constants for Cancer Cell Redox Balance and Lactate Secretion-Revisiting the Warburg Effect.

Cancer cells metabolize a large fraction of glucose to lactate, even under a sufficient oxygen supply. This phenomenon-the "Warburg Effect"-is often regarded as not yet understood. Cancer cells change gene expression to increase the uptake and utilization of glucose for biosynthesis pathways and glycolysis, but they do not adequately up-regulate the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). Thereby, an increased glycolytic flux causes an increased production of cytosolic NADH. However, since the corresponding gene expression changes are not neatly fine-tuned in the cancer cells, cytosolic NAD+ must often be regenerated by loading excess electrons onto pyruvate and secreting the resulting lactate, even under sufficient oxygen supply. Interestingly, the Michaelis constants (KM values) of the enzymes at the pyruvate junction are sufficient to explain the priorities for pyruvate utilization in cancer cells: 1. mitochondrial OXPHOS for efficient ATP production, 2. electrons that exceed OXPHOS capacity need to be disposed of and secreted as lactate, and 3. biosynthesis reactions for cancer cell growth. In other words, a number of cytosolic electrons need to take the "emergency exit" from the cell by lactate secretion to maintain the cytosolic redox balance.

Characterization of a glycosyltransferase from Paris polyphylla for application in biosynthesis of rare ophiopogonins and ginsenosides

Saponins are bioactive components of many medicinal plants, possessing complicated chemical structures and extensive pharmacological activities, but the production of high-value saponins remains challenging. In this study, a 6′-O-glucosyltransferase PpUGT7 (PpUGT91AH7) was functionally characterized from Paris polyphylla Smith var. yunnanensis (Franch.) Hand. -Mazz., which can transfer a glucosyl group to the C-6′ position of diosgenin-3-O-rhamnosyl-(1 → 2)-glucoside (1), pennogenin-3-O-rhamnosyl-(1 → 2)-glucoside (2), and diosgenin-3-O-glucoside (5). The KM and Kcat values of PpUGT7 towards the substrate 2 were 8.4 μM and 2 × 10−3 s−1, respectively. Through molecular docking and site-directed mutagenesis, eight residues were identified to interact with the sugar acceptor 2 and be crucial for enzyme activity. Moreover, four rare ophiopogonins and ginsenosides were obtained by combinatorial biosynthesis, including an undescribed compound ruscogenin-3-O-glucosyl-(1 → 6)-glucoside (10). Firstly, two monoglycosides 9 and 11 were generated using a known sterol 3-O-β-glucosyltransferase PpUGT80A40 with ruscogenin (7) and 20(S)-protopanaxadiol (8) as substrates, which were further glycosylated to the corresponding diglycosides 10 and 12 under the catalysis of PpUGT7. In addition, compounds 7–11 were found to show inhibitory effects on the secretion of TNF-α and IL-6 in macrophages RAW264.7. The findings provide valuable insights into the enzymatic glycosylation processes in the biosynthesis of bioactive saponins in P. polyphylla var. yunnanensis, and also serve as a reference for utilizing UDP-glycosyltransferases to construct high-value or rare saponins for development of new therapeutic agents.

Biochemical characterization of purified phytase produced from Aspergillus awamori AFE1 associated with the gastrointestinal tract of longhorn beetle (Cerambycidae latreille)

ABSTRACT The need for industrially and biotechnologically significant enzymes, such as phytase, is expanding daily as a result of the increased use of these enzymes in a variety of operations, including the manufacture of food, animal feed, and poultry feed. This study sought to characterize purified phytase from A. awamori AFE1 isolated from longhorn beetle for its prospect in industrial applications. Ammonium sulfate precipitation, ion-exchange chromatography, and gel-filtration chromatography were used to purify the crude enzyme obtained from submerged fermentation using phytase-producing media, and its physicochemical characteristics were examined. The homogenous 46.8-kDa phytase showed an 8.1-fold purification and 40.7% recovery. At 70 C and pH 7, the optimum phytase activity was noted. At acidic pH 4–6 and alkaline pH 8–10, it likewise demonstrated relative activity of 88–95% and 67–88%, respectively. It showed 67–70% residual activity between 30 and 70 C after 40 min, and 68–94% residual activity between pH 2 and 12 after 2 h. The presence of Hg+, Mg2+, and Al3+ significantly decreased the enzymatic activity, whereas Ca2+ and Cu2+ enhanced it. Ascorbic acid increased the activity of the purified enzyme, whereas ethylenediaminetetraacetic acid (EDTA) and mercaptoethanol inhibited it. The calculated values for Km and Vmax were 55.4 mM and1.99 μmol/min/mL respectively. A. awamori phytase, which was isolated from a new source, showed unique and remarkable qualities that may find use in industrial operations such as feed pelleting and food processing.

Catalytic mechanism and kinetics of malate dehydrogenase.

Malate dehydrogenase (MDH) is a ubiquitous and central enzyme in cellular metabolism, found in all kingdoms of life, where it plays vital roles in the cytoplasm and various organelles. It catalyzes the reversible NAD+-dependent reduction of L-malate to oxaloacetate. This review describes the reaction mechanism for MDH and the effects of mutations in and around the active site on catalytic activity and substrate specificity, with a particular focus on the loop that encloses the active site after the substrates have bound. While MDH exhibits selectivity for its preferred substrates, mutations can alter the specificity of MDH for each cosubstrate. The kinetic characteristics and similarities of a variety of MDH isozymes are summarized, and they illustrate that the KM values are consistent with the relative concentrations of the substrates in cells. As a result of its existence in different cellular environments, MDH properties vary, making it an attractive model enzyme for studying enzyme activity and structure under different conditions.

Purification and Characterization of a Small Thermostable Protease from Streptomyces sp. CNXK100.

Proteases derived from Streptomyces demonstrate numerous commendable properties, rendering it extensively applicable in biotechnology and various industrial sectors. This study focused on the purification and characterization of the thermostable protease obtained from Streptomyces sp. CNXK100. The purified protease exhibited an estimated molecular weight of 27 kDa, with optimal activity at 75°C and pH 8.0. Notably, the enzyme remained active even without any metal ions and fully active in the presence of Na+, K+, Mg2+, and Cu2+metal ions. The kinetic parameters were determined with a KM value of 3.13 mg/ml and a Vmax value of 3.28 × 106 U/mg. Furthermore, the protease has demonstrated notable stability when subjected to a treatment temperature of up to 65°C for 60 minutes, and across a broad pH range extending from 5.0 to 10.0. This protease also demonstrated resilience against a spectrum of harsh conditions, including exposure to organic solvents, surfactants, bleaching agents, and proteolytic enzymes. Additionally, the enzyme maintained its activity following treatment with commercial detergents, accomplishing complete thrombus lysis at a concentration of 2.50 mg/ml within 4 hours. Remarkably, the protease exhibited stability in terms of activity and protein concentration for 70 days at 4°C. These findings underscore the potential industrial applications of the thermostable protease from Streptomyces sp. CNXK100.

The critical role of residues Phe120 and Val161 of (2 R,3 R)‑2,3‑butanediol dehydrogenase from Neisseria gonorrhoeae as probed by molecular docking and site-directed mutagenesis.

NAD+-dependent (2 R,3 R)‑2,3‑butanediol dehydrogenase (BDH) from Neisseria gonorrhoeae (NgBDH) is a representative member of the medium-chain dehydrogenase/reductase (MDR) superfamily. To date, little information is available on the substrate binding sites and catalytic residues of BDHs from this superfamily. In this work, according to molecular docking studies, we found that conserved residues Phe120 and Val161 form strong hydrophobic interactions with both (2 R,3 R)‑2,3‑butanediol (RR-BD) and meso-2,3‑butanediol (meso-BD) and that mutations of these residues to alanine or threonine impair substrate binding. To further evaluate the roles of these two residues, Phe120 and Val161 were mutated to alanine or threonine. Kinetic analysis revealed that, relative to those of wild type, the apparent KM values of the Phe120Ala mutant for RR-BD and meso-BD increased 36- and 369-fold, respectively; the catalytic efficiencies of this mutant with RR-BD and meso-BD decreased approximately 586- and 3528-fold, respectively; and the apparent KM values of the Val161Ala mutant for RR-BD and meso-BD increased 4- and 37-fold, respectively, the catalytic efficiencies of this mutant with RR-BD and meso-BD decreased approximately 3- and 28-fold, respectively. Additionally, the Val161Thr mutant slightly decreased catalytic efficiencies (twofold with RR-BD; 7.3-fold with meso-BD) due to an increase in KM (sixfold for RR-BD; 24-fold for meso-BD) and a slight increase (2.8-fold with RR-BD; 3.3-fold with meso-BD) in kcat. These findings validate the critical roles of Phe120 and Val161 of NgBDH in substrate binding and catalysis. Overall, the current study provides a better understanding of the substrate binding and catalysis of BDHs within the MDR superfamily.

Statistical optimized production of Phytase from Hanseniaspora guilliermondii S1 and studies on purification, homology modelling and growth promotion effect

This investigation was performed to obtain a promising phytase enzyme producing yeast. In this regard, the PSM was used to isolate the phytase-producing Hanseniaspora guilliermondii S1 (MG663578) from sugarcane juice. The SSF optimum conditions for phytase generation were optimized using (OVAT) one-variable-at-a-time strategy using both Box-Behnken design and shake flask method (g/100 ml: 0.05 yeast extract, 0.15 Peptone, 0.05 malt extract 0.50 dextrose, pH 5.8 and 28ᵒC). The protein model developed was shown to be adequate for phytase production (91% accuracy), with the greatest phytase productivity in shake flask with substrate jack fruit seed powder being 395 ± 0.43 U/ml compared to 365U/ml for the BBD projected value. Crude Phytase was partially purified with a protein recovery of 43%, revealing a molecular weight of 120 kDa. It had an enzyme kinetic value of Km 3.3 mM and a Vmax of 19.1 mol/min. The 3D structure of PhyS1 amino acid sequences (PhyS1. B99990002) was simulated using Modeler 9.23, and the validated result revealed that 86.7% were in the favored region by Ramachandran plot. The SAVES server verified the 3D PDB file as satisfactory, and the model (in.pdb format) was uploaded in the PMDB database with the accession number ID: PM0082974. At the lab level, Hanseniaspora guilliermondii S1 (MG663578) producing phytase exhibited successful plant growth promotion activity in Ragi - CO 19 (Eleusine coracana L.) and Rice -Navarai - IR 64 (Oryza sativa L.). As a result, a phytase-based formulation for sustainable agriculture must be developed and tested on a large scale in diverse geographical areas of agricultural lands to determine its effect and potential on plant development.

A Simple and Sensitive Approach for Real‐Time Sensing of Enzymatically Catalyzed Hydrogelation

AbstractDespite the critical role of hydrogels in material science and biotechnology, current methods for analyzing their formation lack real‐time monitoring and require complex sample preparation and instrumentation. In this work, an innovative methodology is introduced for the real‐time analysis of enzymatically catalyzed hydrogelation. Electrochemical impedance spectroscopy (EIS) coupled with interdigitated electrodes (IDEs) to sense and transduce the gelation reaction of model precursor carboxymethyl cellulose‐tyramine (CMC‐TA) conjugates that undergoes enzymatic cross‐linking by horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). Real‐time monitoring involves single‐frequency analyses at 3 × 105 Hz, where the measured impedance consists solely of a resistive component, and the admittance equates to solution conductance. The gelation trajectories for all tested enzymatically cross‐linked hydrogel component combinations are determined by substituting the conductance data in the modified Michaelis–Menten kinetic model. Specifically, for CMC‐TA cross‐linked by HRP, the authors calculate apparent KM and kcat values of 82.1 µM and 95.5 s−1, respectively. These findings are further validated through rheological characterization, including oscillatory shear measurements and microrheology. Overall, this research paves the way for a streamlined, accurate, and cost‐effective approach to controllable enzymatically initiated hydrogel synthesis, enhancing their successful application in various fields ranging from material science to biotechnology.

Characterization of the extended substrate spectrum of the class A β-lactamase CESS-1 from Stenotrophomonas sp. and structure-based investigation into its substrate preference

Stenotrophomonas spp. intrinsically resistant to many β-lactam antibiotics are found throughout the environment. CESS-1 identified in Stenotrophomonas sp. KCTC 12332 is an uncharacterized class A β-lactamase. The goal of this study was to reveal biochemical and structural characteristics of CESS-1. The hydrolytic activities of CESS-1 towards penicillins (penicillin G and ampicillin), cephalosporins (cephalexin, cefaclor, and cefotaxime), and carbapenems (imipenem and meropenem) was spectrophotometrically monitored. Structural information on E166Q mutants of CESS-1 acylated by cefaclor, cephalexin, or ampicillin were determined by X-ray crystallography. CESS-1 displayed hydrolytic activities toward penicillins and cephalosporins, with negligible activity toward carbapenems. Although cefaclor, cephalexin, and ampicillin have similar structures with identical R1 side chains, the catalytic parameters of CESS-1 toward them were distinct. The kcat values for cefaclor, cephalexin, and ampicillin were 1249.6 s-1, 204.3 s-1, and 69.8 s-1, respectively, with the accompanying KM values of 287.6 μM, 236.7 μM, and 28.8 μM, respectively. CESS-1 was able to discriminate between cefaclor and cephalexin with a single structural difference at C3 position: -Cl (cefaclor) and -CH3 (cephalexin). Structural comparisons among three E166Q mutants of CESS-1 acylated by cefaclor, cephalexin, or ampicillin, revealed that cooperative positional changes in the R1 side chain of substrates and their interaction with the β5-β6 loop affect the distance between Asn170 and the deacylating water at the acyl-enzyme intermediate state. This is directly associated with the differential hydrolytic activities of CESS-1 toward the three structurally similar β-lactam antibiotics.

Facile Synthesis of Copper Telluride Nanoparticles as a Robust Nanoenzymatic Catalyst for Intrinsic Peroxidase‐Like Activity

AbstractCopper telluride nanoparticles are classified as the family of metal chalcogenides and were synthesized using a simple solvothermal method using a new source of telluride, ditelluride diphenyl. The characterized techniques revealed that the atomic composition of copper tellurides is Cu2Te and particles are spherical. This study is greatly examined by different designed experiments for the proof of the intrinsic peroxidase‐like activity of copper telluride nanoparticles and its effectiveness in a variety of similar structures. Cu2Te nanoparticles can convert peroxidase substrates (TMB and OPD) to colored products in the presence of H2O2. In addition, the as‐prepared nanoparticles have the potential to oxidize DPPH and ABTS substrates. Michaelis–Menten constant KM value for TMB and H2O2 was found to be 12 mM and 0.60 mM for H2O2 and the Vmax value of the Cu2Te was found to be TMB and H2O2 was 2.50×10−7 and 0.82×10−8 MS−1. These data are found to be almost comparable with natural peroxidase HRP and other artificial enzymes reported in the literature. The natural peroxidase‐like activities of Cu2Te nanoparticles originated from the catalytic decomposition of H2O2 into ⋅OH radical, which was confirmed from the ESR spin‐trapping technique and using a fluorescence probe, terephthalic acid.

Purification, characterization of glutathion reductase enzyme from sheep splen tissue and investıgatıon of the effects of some antibiotics on enzyme activity

In this study, glutathione reductase (EC 1.8.1.7; GR, Glutathione: NADP+ oxidoreductase), which is the key enzyme of antioxidant metabolism, was purified from sheep spleen using ammonium sulfate precipitation and 2', 5'-ADP Sepharose-4B affinity chromatography. As a result of purification, the GR enzyme was purified, with 20.03 EU/mg.protein of specific activity, 1564.8 times a yield of 40.61%. The purity of the enzyme was checked by SDS-PAGE. In the characterization studies, optimum pH, optimum ionic strength, stable pH, optimum temperature and subunit molecular mass of the enzyme were determined. In addition, KM and Vmax values were found to determine the enzyme's affinity for GSSG and NADPH substrates. It was determined as KM constant 0.0061 mM and Vmax value 0.259 EU/mL, for NADPH, KM constant was determined as 0.351 mM and Vmax value was determined as 0.604 EU/mL for GSSG. In addition, the effects of ampicillin, streptomycin sulfate, gentamicin, cefoperazone sodium and precort-lyo on enzyme activity were investigated. It was determined that these drugs showed an inhibitory effect on GR enzyme activity purified from sheep spleen tissue. The IC50 values for precort-lyo, ampicillin, streptomycin sulfate, cefoperazone sodium, and gentamicin were 1.27 mM, 3.22 mM, 7.95 mM, 16.97 mM, and 17.20 Mm, respectively; and Ki constants were calculated as 0.466±0.387 mM (competitive), 1.057±0.110 mM (non-competitive), 3.386±1.305 mM (competitive), 4.910±0.960 mM (competitive), and 20.770±8.169 mM (non-competitive), respectively.

Engineering diaryl alcohol dehydrogenase KpADH reveals importance of retaining hydration shell in organic solvent tolerance.

Alcohol dehydrogenases (ADHs) are synthetically important biocatalysts for the asymmetric synthesis of chiral alcohols. The catalytic performance of ADHs in the presence of organic solvents is often important since most prochiral ketones are highly hydrophobic. Here, the organic solvent tolerance of KpADH from Kluyveromyces polyspora was semi-rationally evolved. Using tolerant variants obtained, meticulous experiments and computational studies were conducted to explore properties including stability, activity and kinetics in the presence of various organic solvents. Compared with WT, variant V231D exhibited 1.9-fold improvement in ethanol tolerance, while S237G showed a 6-fold increase in catalytic efficiency, a higher , as well as 15% higher tolerance in 7.5% (v/v) ethanol. Based on 3 × 100 ns MD simulations, the increased tolerance of V231D and S237G against ethanol may be ascribed to their enhanced ability in retaining water molecules and repelling ethanol molecules. Moreover, 6.3-fold decreased KM value of V231D toward hydrophilic ketone substrate confirmed its capability of retaining hydration shell. Our results suggest that retaining hydration shell surrounding KpADH is critical for its tolerance to organic solvents, as well as catalytic performance. This study provides useful guidance for engineering organic solvent tolerance of KpADH and other ADHs.

Critical Roles of Acidic Residues in Loop Regions of the Structural Surface for the Salt Tolerance of a GH39 β-d-Xylosidase.

Xylan is the main component of hemicellulose. Complete hydrolysis of xylan requires synergistically acting xylanases, such as β-d-xylosidases. Salt-tolerant β-d-xylosidases have significant application benefits, but few reports have explored the critical amino acids affecting the salt tolerance of xylosidases. Herein, the site-directed mutation was used to demonstrate that negative electrostatic potentials generated by 19 acidic residues in the loop regions of the structural surface positively correlated with the improved salt tolerance of GH39 β-d-xylosidase JB13GH39P28. These mutants showed reduced negative potentials on structural surfaces as well as a 13-43% decrease in stability in 3.0-30.0% (w/v) NaCl. Six key residue sites, D201, D259, D297, D377, D395, and D474, were confirmed to influence both the stability and activity of GH39 β-d-xylosidase. The activity of the GH39 β-d-xylosidase was found promoting by SO42- and inhibiting by NO3-. Values of Km and Kcat/Km decreased aggravatedly in 30.0% (w/v) NaCl when mutation operated on residues E179 and D182 in the loop regions of the catalytic domain. Taken together, mutation on acidic residues in loop regions from catalytic and noncatalytic domains may cause the deformation of catalytic pocket and aggregation of protein particles then decrease the stability, binding affinity, and catalytic efficiency of the β-d-xylosidase.

Cloning and characterization of luciferase from an Asian firefly Pygoluciola qingyu and its comparison with other beetle luciferases.

The bioluminescence system of luminescent beetles has extensive applications in biological imaging, protein labeling and drug screening. To explore wild luciferases with excellent catalytic activity and thermal stability, we cloned the luciferase of Pygoluciola qingyu, one species living in areas of high temperature and with strong bioluminescence, by combining transcriptomic sequencing and reverse transcription polymerase chain reaction (RT-PCR). The total length of luciferase gene is 1638bp and the luciferase consists 544 amino acids. The recombinant P. qingyu luciferase was produced in vitro and its characteristics were compared with those of eight luciferases from China firefly species and two commercial luciferases. Compared with these luciferases, the P. qingyu luciferase shows the highest luminescence activity at room temperature (about 25-28℃) with similar KM value for D-luciferin and ATP to the Photinus pyralis luciferase. The P. qingyu luciferase activity was highest at 35℃ and can keep high activity at 30-40℃, which suggests the potential of P. qingyu luciferase for in vivo and cell application. Our results provide new insights into P. qingyu luciferase and give a new resource for the application of luciferases.

Cloning and expression heterologous alanine dehydrogenase genes: Investigation of reductive amination potential of L-alanine dehydrogenases for green synthesis of alanine derivatives

Unnatural amino acids (UAAs) offer significant promise in a wide range of applications, including drug discovery, the custom design of peptides and proteins, and their utility and use as markers for monitoring molecular interactions in biological research. The synthesis of UAAs presents a formidable challenge and can be classified into two primary categories: enzymatic and chemical synthesis. Notably, the enzymatic route, specifically asymmetric synthesis, emerges as a an attractive method for procuring enantiopure UAAs with high efficiency, owing to its streamlined and concise reaction mechanism.The current study investigated the reductive amination activity mechanisms of alanine dehydrogenase (L-AlaDH), sourced from a combination of newly and previously characterized microorganisms. Our principal aim was to evaluate the catalytic efficiency of these L-AlaDH enzymes concerning a range of specific ketoacids and pyruvate to ascertain their capability for facilitating the production of both natural and unnatural amino acids. After the characterization processes, mutation points for TtAlaDH were determined and as a result of the mutations, mutants that could use ketocaproate and ketovalerate more effectively than the wild type were obtained.Among the enzymes studied, MetAlaDH exhibited the highest specific activity against pyruvate, 173 U/mg, and a KM value of 1.3 mM. VlAlaDH displayed the most favourable catalytic efficiency with a rate constant of 170 s−1mM−1. On the other hand, AfAlaDH demonstrated the highest catalytic efficiency against α-ketobutyrate (34.0 s−1mM−1) and α-ketovalerate (2.7 s−1mM−1). Of the enzymes investigated in the study, TtAlaDH exhibited the highest effectiveness among bacterial enzymes in catalyzing ketocaproate with a measured catalytic efficiency of about 0.6 s−1mM−1 and a KM value of approximately 0.3 mM. These findings provide valuable insights into the substrate specificity and catalytic performance of L-AlaDHs, enhancing our understanding of their potential applications in various biocatalytic processes.

Plant root associated chitinases: structures and functions.

Chitinases degrade chitin, a linear homopolymer of β-1,4-linked N-acetyl-D-glucosamine (GlcNAc) residues found in the cell walls of fungi and the exoskeletons of arthropods. They are secreted by the roots into the rhizosphere, a complex and dynamic environment where intense nutrient exchange occurs between plants and microbes. Here we modeled, expressed, purified, and characterized Zea mays and Oryza sativa root chitinases, and the chitinase of a symbiotic bacterium, Chitinophaga oryzae 1303 for their activities with chitin, di-, tri-, and tetra-saccharides and Aspergillus niger, with the goal of determining their role(s) in the rhizosphere and better understanding the molecular mechanisms underlying plant-microbe interactions. We show that Zea mays basic endochitinase (ZmChi19A) and Oryza sativa chitinase (OsChi19A) are from the GH19 chitinase family. The Chitinophaga oryzae 1303 chitinase (CspCh18A) belongs to the GH18 family. The three enzymes have similar apparent K M values of (20-40 µM) for the substrate 4-MU-GlcNAc3. They vary in their pH and temperature optima with OsChi19A activity optimal between pH 5-7 and 30-40°C while ZmChi19A and CspCh18A activities were optimal at pH 7-9 and 50-60°C. Modeling and site-directed mutation of ZmChi19A identified the catalytic cleft and the active residues E147 and E169 strategically positioned at ~8.6Å from each other in the folded protein. Cleavage of 4-MU-GlcNAc3 was unaffected by the absence of the CBD but diminished in the absence of the flexible C-terminal domain. However, unlike for the soluble substrate, the CBD and the newly identified flexible C-terminal domain were vital for inhibiting Aspergillus niger growth. The results are consistent with the involvement of the plant chitinases in defense against pathogens like fungi that have chitin exoskeletons. In summary, we have characterized the functional features and structural domains necessary for the activity of two plant root chitinases that are believed to be involved in plant defense and a bacterial chitinase that, along with the plant chitinases, may participate in nutrient recycling in the rhizosphere.

Dephosphorization Kinetics of Hot Metal at Low Slag Basicity and Low Temperature under the Different Fe2O3 Addition Amounts

In this article, the influence of the Fe2O3 addition amount on the dephosphorization kinetics of hot metal at the low slag basicity (CaO/SiO2) of about 1.6 and low temperature of 1623 K is investigated with laboratorial experiments and the kinetic model. Si and P contents in the hot metal during the whole dephosphorization process, and the endpoint FeO content in the slag, show good agreements between the calculated results with the kinetic model and experimental results. The peak value of dephosphorization rate linearly increases from 8.35 × 10−5 to 4.52 × 10−4 mass% s−1 when the weight ratio of Fe2O3 is increased from 5 to 10/100 g hot metal. With the increase in Fe2O3 addition amount, the mass transfer coefficient in the hot metal phase (km) and that in the slag phase (ks) and dephosphorization rate constant (k) all increase. The value of km is found to be 4.34 times that of ks. During the dephosphorization process, the rate‐controlling step converts from the mass transfer in slag phase to the mass transfer in hot metal phase in less than 10 min. With the increase in Fe2O3 addition amount, the value of interfacial oxygen activity of increases from 0.0102 to 0.0201.

The mechanism of ceftazidime and cefiderocol hydrolysis by D179Y variants of KPC carbapenemases is similar and involves the formation of a long-lived covalent intermediate.

Klebsiella pneumoniae carbapenemase (KPC) variants have been described that confer resistance to both ceftazidime-avibactam and cefiderocol. Of these, KPC-33 and KPC-31 are D179Y-containing variants derived from KPC-2 and KPC-3, respectively. To better understand this atypical phenotype, the catalytic mechanism of ceftazidime and cefiderocol hydrolysis by KPC-33 and KPC-31 as well as the ancestral KPC-2 and KPC-3 enzymes was studied. Steady-state kinetics showed that the D179Y substitution in either KPC-2 or KPC-3 is associated with a large decrease in both kcat and KM such that kcat/KM values were largely unchanged for both ceftazidime and cefiderocol substrates. A decrease in both kcat and KM is consistent with a decreased and rate-limiting deacylation step. We explored this hypothesis by performing pre-steady-state kinetics and showed that the acylation step is rate-limiting for KPC-2 and KPC-3 for both ceftazidime and cefiderocol hydrolysis. In contrast, we observed a burst of acyl-enzyme formation followed by a slow steady-state rate for the D179Y variants of KPC-2 and KPC-3 with either ceftazidime or cefiderocol, indicating that deacylation of the covalent intermediate is the rate-limiting step for catalysis. Finally, we show that the low KM value for ceftazidime or cefiderocol hydrolysis of the D179Y variants is not an indication of tight binding affinity for the substrates but rather is a reflection of the deacylation reaction becoming rate-limiting. Thus, the hydrolysis mechanism of ceftazidime and cefiderocol by the D179Y variants is very similar and involves the formation of a long-lived covalent intermediate that is associated with resistance to the drugs.

Exploration of inhibitor effect of Gly-Pro (GP), Arg-Gly-Asp-Ser (RGDS) and Ser-Asp-Gly-Arg-Gly (SDGRG) bioactive peptides on angiotensin-converting enzyme activity purified from human serum.

Bioactive peptides (BPs) are a natural and important alternative to synthetic angiotensin-converting enzyme (ACE) inhibitors used in the treatment of hypertension. In this study, ACE was 3575-fold purified from human serum with the affinity chromatography process in one step. The molecular weight and purity of ACE were identified using the SDS-PAGE process and seen in two bands at around 60 kDa and 70 kDa on the gel. Vmax and KM values from the Lineweaver-Burk graphic were determined as 96.15 (µmol/min) mL-1 and 0.2 mM, respectively. The effects of Gly-Pro (GP), Arg-Gly-Asp-Ser (RGDS) and Ser-Asp-Gly-Arg-Gly (SDGRG) BPs on purified ACE were researched. Also, lisinopril was used as a reference inhibitor. GP, RGDS and SDGRG on purified ACE demonstrated an inhibitory effect. IC50 values for these peptides were found as 184.71, 107.16 and 32.54 µM, respectively. Ki values and type of inhibitory for GP, RGDS and SDGRG by the Lineweaver-Burk chart were found. The type of inhibitory for these peptides was calculated as reversible-competitive inhibitory. Ki values for GP, RGDS and SDGRG were calculated to be 260.02, 63.44 and 11.42 µM, respectively. Also, the SDGRG indicated a higher inhibition effect on ACE activity than the GP and RGDS. The IC50 value of lisinopril was designated as 0.35 nM. The inhibition type of lisinopril was designated as reversible noncompetitive inhibition from the Lineweaver-Burk chart and the Ki value was 0.15 nM. Herein, it was concluded that GP, RGDS and SDGRG have ACE inhibitor potential.Communicated by Ramaswamy H. Sarma.