Recombinant Enzyme Research Articles

Metabolism of Furanocoumarins by Three Recombinant CYP9A Proteins From the Polyphagous Cotton Bollworm Helicoverpa armigera.

Furanocoumarins are a class of chemical compounds with phototoxic properties. For herbivores, efficient detoxification of such defense compounds is the prerequisite to feed successfully on furanocoumarin-containing plants. The cotton bollworm Helicoverpa armigera is a very important polyphagous pest in agriculture, but how it copes with toxic furanocoumarins in some of its host plants is not well understood. Given that cytochrome P450s are well known for their capacity in xenobiotic metabolism, this study attempted to explore the potential roles of cytochrome P450s in furanocoumarin transformation in this pest. Our data showed that two linear structures (psoralen and xanthotoxin) could be metabolized by three recombinant CYP9A enzymes, but no detectable depletion was observed for the linear one with the 8-dimethylallyloxy substituent on the coumarin moiety (imperatorin) and the angular furanocoumarin (angelicin). Initial epoxidation of the double bond connecting C2' and C3' of the furano ring following by cleavage of the epoxidated furan ring, leading to the formation of more soluble, less reactive and nonphotosensitizing metabolites, was identified as a common mechanism of linear furanocoumarin metabolism using a quadrupole/time-of-flight (Q-TOF) mass spectrometry interfaced with a high performance liquid chromatography (HPLC) system. Our data demonstrated that multiple P450s were involved in the detoxification of linear furanocoumarins in the cotton bollworm. These findings contribute to a better understanding of the biochemical basis of adaptation to plant defense chemicals in this economically important pest.

Cloning, Expression, Purification and Enzymatic Characterization of Low-temperature Cholesterol Esterase from Marine Panthenia Agglutinosa

BACKGROUND: The low-temperature cholesterol esterase is primarily used in industries such as papermaking and healthcare. OBJECTIVE: To discover a microorganism with high cholesterol esterase activity and tolerance to low temperatures, leading to the promotion of the sustainable utilization of marine cold-adapted microbial resources and fostering industrial development. MATERIALS AND METHODS: This study isolated a strain producing low-temperature cholesterol esterase from marine samples in the China Bohai Sea. The strain was identified through 16S rDNA sequencing and named Panthenia agglutinosa Y03. The cholesterol esterase gene (PaChe) from P. agglutinosa Y03 was cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme PaChe was purified and characterized. The structure of PaChe was predicted using AlphaFold2, and molecular docking was performed with cholesterol linoleate as the ligand. RESULTS: The enzyme protein has a molecular weight of 56.35 KDa, a theoretical pI of 7.24, lacks a signal peptide, and exhibits structural features of the α/β hydrolase superfamily protein. The concentration of the purified PaChe is 0.5 mg/mL, with a specific activity of 42.7 U/mg. The optimal working temperature is 30 °C, and the enzyme retains activity at 4 °C , demonstrating weaker thermal stability. The optimal pH is 7, and the enzyme maintains over 70% activity at pH 9. Na +, Ca 2+ and Mg 2+ are the primary activators, while Ba 2+, Fe 2+, Mn 2+, Cu 2+ and chemical agents such as SDS as inhibitors, with Cu2+ exhibiting particularly significant inhibitory effects. CONCLUSION: This study establishes the theoretical groundwork for the development and utilization of a novel lowtemperature cholesterol esterase.

Cold-Active Lipase from the Ice Cave Psychrobacter SC65A.3 Strain, a Promising Biocatalyst for Silybin Acylation

Cold-active lipase from the psychrophilic bacterial strain Psychrobacter SC65A.3 isolated from Scarisoara Ice Cave (Romania) was cloned and characterized as an extremophilic biocatalyst for silybin acylation. Structural analyses highlighted conserved motifs confirming a functional lipase and the presence of primary structure elements for catalysis at low temperatures. The recombinant enzyme (PSL2) heterologously expressed in Escherichia coli was purified in one step by affinity chromatography with a yield of 12.08 ± 1.72 µg L−1 of culture and a specific activity of 20.1 ± 3.2 U mg−1 at 25 °C. Functional characterization of PSL2 showed a neutral (7.2) optimal pH and a high thermal stability up to 90 °C. Also, this lipase was stable in the presence of different organic solvents, with 60% residual activity when using 20% DMSO. Kinetic measurements indicated performant catalytic efficiency of PSL2 for different short and long chain fatty acids, with Km in the mM range. The catalytic activity of PSL2 was assessed for silybin acylation with various fatty acids and fatty acid methyl esters, demonstrating a 90% silybin conversion when methyl decanoate ester was used. This result clearly highlights the biocatalytic capability of this new cold-active lipase.

Obtaining and Characterizing Thermostable α-Amylases Secreted by Bacillus subtilis, Originating from Bacillus amyloliquefaciens and Bacillus subtilis

The production of recombinant enzymes, primarily used for obtaining pure and functional target molecules, holds significant importance in modern biotechnology. This study aimed to obtain and characterize recombinant, extracellularly expressed α-amylases (Amy3500 and Amy1974), derived from B. amyloliquefaciens MDC1974 and B. subtilis MDC3500, respectively, using the pBE-S shuttle vector. Both α-amylase genes were molecularly cloned into the E. coli/B. subtilis pBE-S shuttle vector, both with (Amy1974sig and Amy3500sig) and without their signal peptides (Amy1974 and Amy3500), along with a signal peptide originating from the plasmid, and tested in flask fermentations. For recombinant Amy3500, the amylase variants resulted in similar levels of volumetric activity (700–750 U/mL). In contrast, the expression of Amy1974 nearly doubled compared to Amy1974sig with double signal peptides, reaching 2000 U/mL. SDS-PAGE estimated the molecular weight of Amy1974 α-amylase to be 54.6 kDa, which is consistent with the theoretical molecular mass calculations. However, the estimated molecular weight of Amy3500 α-amylase was significantly lower upon exiting the producer cells. Ca2⁺ ions exhibit a modest activating effect on the activities of Amy1974 and Amy3500 amylases, likely due to their tight binding to the protein scaffold. Both enzymes exhibited broad activity peaks between 45 and 70 °C, with a maximum at 65 °C. The Amy1974 and Amy3500 α-amylases demonstrated broad pH optima and pH-dependent thermostability, with optimum pH values at 6.5 and 5.8, and thermal stability peaks at pH 7.6 and 5.9, respectively. Both α-amylases displayed high relative activity against various starches, including corn amylopectin and potato amylose, while showing comparatively lower activity towards α-, β-, and γ-cyclodextrins. The Amy1974 amylase is effective in converting starch into dextrins of varying lengths, while Amy3500 primarily converts starch into glucose. These characteristics make them promising candidate enzymes for industrial applications.

Molecular and Cellular Characterization of the Glutathione Transferases Involved in the Olfactory Metabolism of the Mammary Pheromone.

Odorant metabolizing enzymes, considered as critical olfactory perireceptor actors, control the odor molecules reaching the olfactory epithelium by biotransforming them. As an odorant, the mammary pheromone, i.e., 2-methylbut-2-enal (2MB2), emitted in the milk of lactating female rabbits triggers typical nipple searching-grasping behavior through orocephalic movements in newborn rabbits but not in weaned rabbits. We previously showed that 2MB2 perception is significantly modified when its glutathione transferase-dependent olfactory metabolism is affected in newborns. Here, enzymatic assays of the recombinant enzymes GSTA1, M1, and P1 revealed the activity of these enzymes toward the mammary pheromone. Histological experiments revealed strong expression of the GSTA class restricted to the Bowman glands and of GSTP1 in the nuclei of sustentacular cells. Moreover, some modulations of GSTs have been demonstrated, including a significant increase in GSTP1 expression (2-fold in mRNA, p value < 0.001; protein, p value: 0.031) after 45 min of mammary pheromone exposure at 10-2 g/mL and an increase in GSTA expression in weaned rabbits compared with newborn rabbits (3-fold in mRNA, p value: 0.011; protein, p value: 0.001). Our results provide new insights into the activity, cellular expression, and modulation of the mammary pheromone GST-metabolizing enzymes and clues about their olfactory function.

Detection and Quantification of Drug-Protein Adducts in Human Liver.

Covalent protein adducts formed by drugs or their reactive metabolites are risk factors for adverse reactions, and inactivation of cytochrome P450 (CYP) enzymes. Characterization of drug-protein adducts is limited due to lack of methods identifying and quantifying covalent adducts in complex matrices. This study presents a workflow that combines data-dependent and data-independent acquisition (DDA and DIA) based liquid chromatography with tandem mass spectrometry (LC-MS/MS) to detect very low abundance adducts resulting from CYP mediated drug metabolism in human liver microsomes (HLMs). HLMs were incubated with raloxifene as a model compound and adducts were detected in 78 proteins, including CYP3A and CYP2C family enzymes. Experiments with recombinant CYP3A and CYP2C enzymes confirmed adduct formation in all CYPs tested, including CYPs not subject to time-dependent inhibition by raloxifene. These data suggest adducts can be benign. DIA analysis showed variable adduct abundance in many peptides between livers, but no concomitant decrease of unadducted peptides. This study sets a new standard for adduct detection in complex samples, offering insights into the human adductome resulting from reactive metabolite exposure. The methodology presented will aid mechanistic studies to identify, quantify and differentiate between adducts that result in adverse drug reactions and those that are benign.

Establishment of recombinase polymerase amplification detection method for Dactylobotrys graminicola

Hulless barley sheath rot is a spike disease caused by Dactylobotrys graminicola. In recent years, it has generally occurred in hulless barley growing areas in China, resulting in reduced hulless barley yields. In this study, primers and probes were designed based on conserved genome sequences, and a method was established using recombinant enzyme polymerase amplification-lateral flow burette (RPA-LFD) technology for the rapid diagnosis of sheath rot in hulless barley. The method can be successfully established in five minutes at a constant temperature of 39℃, and the results are consistent with those of normal PCR analysis. The method demonstrated high sensitivity, with a detection limit of 10 fg/µL. Furthermore, the rapid method was able to successfully detect D. graminicola in hulless barley during field incubation, which highlighted the significant advantage of the method in practical applications. In conclusion, the RPA method established in this study exhibited several advantageous characteristics, including high efficiency, simplicity, rapidity and practicality, which provide a theoretical basis for the early detection and prevention of D. graminicola.

The vacuolar anti-Pseudomonal activity of neutrophil primary granule peptidyl-arginine deiminase enzymes.

The role of neutrophils in host defense involves several cell processes including phagocytosis, degranulation of antimicrobial proteins, and the release of neutrophil extracellular traps (NETs). In turn, dysregulated cell activity is associated with the pathogenesis of airway and rheumatic diseases, in which neutrophil-derived enzymes including peptidyl-arginine deiminases (PADs) play a role. Known physiological functions of PADs in neutrophils are limited to the activity of PAD isotype 4 in histone citrullination in NET formation. The aim of this study was to extend our knowledge on the role of PADs in neutrophils and, specifically, bacterial killing within the confines of the phagocytic vacuole. Human neutrophils were fractionated by sucrose gradient ultracentrifuge and PADs localized in subcellular compartments by Western blot analysis. Direct interaction of PADs with Pseudomonas aeruginosa (P. aeruginosa) was assessed by flow cytometry and Western blot overlay. The participation of neutrophil PAD2 and PAD4 in killing of P. aeruginosa was assessed by inclusion of PAD-specific inhibitors. In vitro, bactericidal activity of recombinant human PAD2 or PAD4 enzymes against P. aeruginosa was determined by enumeration of colony-forming units (CFU). Together with neutrophil elastase (NE), PAD2 and PAD4 were localized to primary granules and, following activation with particulate stimuli, were degranulated in to the phagocytic vacuole. In vitro, PAD2 and PAD4 bound P. aeruginosa (p = 0.04) and significantly reduced bacterial survival to 49.1 ± 17.0 (p < 0.0001) and 48.5 ± 13.9% (p < 0.0001), respectively. Higher antibacterial activity was observed at neutral pH levels with the maximum toxicity at pH 6.5 and pH 7.5, comparable to the effects of neutrophil bactericidal permeability increasing protein. In phagosomal killing assays, inclusion of the PAD2 inhibitor, AFM-30a, or PAD4 inhibitor, GSK484, significantly increased survival of P. aeruginosa (AFM-30a, p = 0.05; and GSK484, p = 0.0079). Results indicate that PAD2 and PAD4 possess antimicrobial activity and are directly involved in the neutrophil antimicrobial processes. This study supports further research into the development of PAD-based antimicrobials.

New Plasmid Constructs for Production of Yeast Based Oral Vaccines

In order to avoid conventional vaccines, there is an urgent need to look for new ways of vaccine generation that can cut down the cost, time, and the need for special requirements during storage and distribution. The development of oral peptide/protein-based vaccines could be an ideal alternative. Five expression plasmids are designed in this work for the production of Saccharomyces cerevisiae-based oral vaccines. Therefore, in this work genetic cassette with different organizations of the required genetic components for efficient gene expression is constructed in the pYES2 plasmid. The construction of five new plasmids for surface display of heterologous recombinant proteins in S. cerevisiae would enable easy insertion of the gene of interest into an optimized genetic cassette consisting of a strong host promoter GAL1, HA-tag for detection of recombinant enzymes, GAS1 gene with/without binding sequence coded for GAS1 cell wall protein and signal sequence for directing the protein into the secretory pathway. After the construction of the genetic cassettes, E gene coded for envelope protein of Chikungunya and Dengue virus is inserted in the genetic cassettes. Genetic models pYES2HAGAS1EgeneChikungunya, pYES2HAGAS1 Egene Dengue, pYES2GAS1 Egene ChikungunyaHA, and pYES2GAS1EGeneDengueHA are constructed with different genetic organizations to display envelope protein of Chikungunya and Dengue virus in the cell surface of S. cerevisiae. In the pYES2SignalEGeneDengueHA construction, envelope protein will be secreted by S. cerevisiae into the media. The constructed vectors are specialized to regulate the expression and surface display of heterologous recombinant proteins in Saccharomyces cerevisiae and will be suitable for large-scale oral vaccine production.

Selective Inhibition of Deamidated Triosephosphate Isomerase by Disulfiram, Curcumin, and Sodium Dichloroacetate: Synergistic Therapeutic Strategies for T-Cell Acute Lymphoblastic Leukemia in Jurkat Cells.

T-cell acute lymphoblastic leukemia (T-ALL) is a challenging childhood cancer to treat, with limited therapeutic options and high relapse rates. This study explores deamidated triosephosphate isomerase (dTPI) as a novel therapeutic target. We hypothesized that selectively inhibiting dTPI could reduce T-ALL cell viability without affecting normal T lymphocytes. Computational modeling and recombinant enzyme assays revealed that disulfiram (DS) and curcumin (CU) selectively bind and inhibit dTPI activity without affecting the non-deamidated enzyme. At the cellular level, treatment with DS and CU significantly reduced Jurkat T-ALL cell viability and endogenous TPI enzymatic activity, with no effect on normal T lymphocytes, whereas the combination of sodium dichloroacetate (DCA) with DS or CU showed synergistic effects. Furthermore, we demonstrated that dTPI was present and accumulated only in Jurkat cells, confirming our hypothesis. Finally, flow cytometry confirmed apoptosis in Jurkat cells after treatment with DS and CU or their combination with DCA. These findings strongly suggest that targeting dTPI represents a promising and selective target for T-ALL therapy.

Deciphering Pathways and Thermodynamics of Protein Assembly Using Native Mass Spectrometry.

Protein oligomerization regulates many critical physiological processes, and its dysregulation can contribute to dysfunction and diseases. Elucidating the assembly pathways and quantifying their underlying thermodynamic and kinetic parameters are crucial for a comprehensive understanding of biological processes and for advancing therapeutics targeting abnormal protein oligomerization. Established binding assays, with limited mass precision, often rely on simplified models for data interpretation. In contrast, high-resolution native mass spectrometry (nMS) can directly determine the stoichiometry of biomolecular complexes in vitro. However, quantification is hindered by the fact that the relative abundances of gas-phase ions generally do not reflect solution concentrations due to nonuniform response factors. Recently, slow mixing mode (SLOMO)-nMS, which can quantify the relative response factors of interacting species, has been demonstrated to reliably measure the affinity (Kd) of binary biomolecular complexes. Here, we introduce an extended form of SLOMO-nMS that enables simultaneous quantification of the thermodynamics in multistep association reactions. Application of this method to homo-oligomerization of concanavalin A and insulin confirmed the reliability of the assay and uncovered details about the assembly processes that had previously resisted elucidation. Results acquired using SLOMO-nMS implemented with charge detection shed new light on the binding of recombinant human angiotensin-converting enzyme 2 and the SARS-CoV-2 spike protein. Importantly, new assembly pathways were uncovered, and the affinities of these interactions, which regulate host cell infection, were quantified. Together, these findings highlight the tremendous potential of SLOMO-nMS to accelerate the characterization of protein assembly pathways and thermodynamics and, in so doing, enhance fundamental biological understanding and facilitate therapeutic development. https://orcid.org/0000-0002-3389-7112.

Production of Domain 9 from the cation-independent mannose-6-phosphate receptor fused with an Fc domain.

Lysosomal storage diseases (LSDs) are genetic disorders caused by mutations in lysosomal enzymes, lysosomal membrane proteins or genes related to intracellular transport that result in impaired lysosomal function. Currently, the primary treatment for several LSDs is enzyme replacement therapy (ERT), which involves intravenous administration of the deficient lysosomal enzymes to ameliorate symptoms. The efficacy of ERT largely depends on the mannose-6-phosphate (M6P) modification of the N-glycans associated with the enzyme, as M6P is a marker for the recognition and trafficking of lysosomal enzymes. In cells, N-glycan processing and M6P modification occur in the endoplasmic reticulum and Golgi apparatus. This is a complex process involving multiple enzymes. In the trans-Golgi network (TGN), M6P-modified enzymes are recognized by the cation-independent mannose-6-phosphate receptor (CIMPR) and transported to the lysosome to exert their activities. In this study, we used the 9th domain of CIMPR, which exhibits a high affinity for M6P binding, and fused it with the Fc domain of human immunoglobulin G1 (IgG1). The resulting fusion protein specifically binds to M6P-modified proteins. This provides a tool for the rapid detection and concentration of M6P-containing recombinant enzymes to assess the effectiveness of ERT. The advantages of this approach include its high specificity and sensitivity and may lead to the development of new treatments for LSDs.

A regio-specific 4’-O-methyltransferase from Epimedium pseudowushanense regiospecifically catalyzing 8-prenylkeampferol to icaritin

Epimedium is widely used in traditional Chinese medicine and contains rich bioactive compounds. These compounds often have a methyl group at their 4’-OH position catalyzed by methyltransferases. Therefore, studying methyltransferases in Epimedium plants is of great significance. In this study, a flavonol methyltransferase, EpOMT4, was isolated from Epimedium pseudowushanense B.L. Guo. The recombinant enzyme regiospecifically transferred a methyl group to the 4’-OH position of 8-prenylkaempferol forming icaritin. The study demonstrates that enzymatic methylation of flavonoids in Epimedium plants holds significant potential and could provide a promising alternative method for the biosynthetic production of bioactive methylated prenylflavonoids.

7963 Analysis of the inhibition of adrenal synthesis and peripheral metabolism of corticosteroids by oteseconazole and two preclinical tetrazole antifungals

Abstract Disclosure: M. Jäger: None. V. González-Ruiz: None. F.L. Joos: None. D.V. Winter: None. J. Boccard: None. T. Degenhardt: Employee; Self; Mycovia Inc. S. Brand: Employee; Self; Mycovia Inc.. S. Rudaz: None. G.R. Thompson: None. A. Odermatt: None. The triazole antifungals posaconazole and itraconazole can cause pseudohyperaldosteronism with hypertension and hypokalemia by blocking adrenal cortisol synthesis and its peripheral inactivation [1]. Mechanisms of pseudohyperaldosteronism include inhibition of adrenal cytochrome P450 enzymes CYP11B1 and CYP17A1, as well as peripherally expressed 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). To enhance specificity for fungal CYP51, tetrazole compounds have been developed [2]. Here, we used adrenal H295R cells and enzyme activity assays to assess the inhibition of steroidogenesis by the tetrazoles oteseconazole, VT-1129 and VT-1598. In addition, the three tetrazoles were tested for inhibition of 11β-HSD2. Steroidomic footprint analyses of H295R cell supernatants using untargeted LC-HRMS with steroid annotation indicated common features of the oteseconazole and VT-1129 patterns with that of the triazole itraconazole, and similarities between VT-1598 and the triazole isavuconazole. Targeted quantification of nine adrenal steroids by UHPLC-MS/MS in supernatants of treated H295R cells showed an overall inhibition of steroidogenesis by the tetrazoles and the triazoles itraconazole and isavuconazole. Experiments using recombinant human enzymes suggested that this effect is not caused by direct inhibition of steroidogenic enzymes because no or very weak inhibition could be observed. Furthermore, oteseconazole, VT-1129 and VT-1598 did not inhibit 11β-HSD2, suggesting that they unlikely cause pseudohyperaldosteronism. Further research needs to uncover the exact mechanism for the observed overall inhibition of steroidogenesis by the tetrazoles as well as itraconazole and isavuconazole, and whether concentrations reached in patients are high enough to cause adrenal insufficiency and hyperplasia.

Engineering synthetic and recombinant human lysosomal β-glucocerebrosidase for enzyme replacement therapy for Gaucher disease

Gaucher Disease (GD) is an autosomal recessive, lysosomal storage disease caused by pathogenic variants in the glucocerebrosidase gene, leading to the loss of β-glucocerebrosidase (GCase) enzymatic activity. Enzyme replacement therapy (ERT) with recombinant GCase is the standard of care in GD patients. Our study investigates the combined use of in silico molecular evolution, synthetic biology and gene therapy approaches to develop a new synthetic recombinant enzyme. We engineered four GCases containing missense mutations in the signal peptide (SP) from four selected mammalian species, and compared them with human GCase without missense mutations in the SP. We investigated transcriptional regulation with CMV and hEF1a promoters alongside a GFP control construct in 293-FT human cells. One hEF1a-driven mutant GCase shows a 5.2-fold higher level of transcription than control GCase. In addition, this mutant exhibits up to a sixfold higher activity compared with the mock-control, and the predicted tertiary structure of this mutant GCase aligns with human GCase. We also evaluated conserved and coevolved residues mapped to functionally important positions. Further studies are needed to assess its functionality in a GD animal model. Altogether, our findings provide in vitro evidence of the potential of this engineered enzyme for improved therapeutic effects for GD.Article highlights.Conservation and coevolution analysis of amino acid frequencies in GBA1 homologs of pathogenic variants associated with Gaucher disease, Parkinson’ disease risk or Dementia with Lewy bodies risk.Graphic

Enzymatic Production of Trehalose and Trehalulose by Immobilized Thermostable Trehalose Synthase.

Trehalose, a versatile disaccharide renowned for its unique physical and chemical properties, finds extensive application in the food, pharmaceutical, and cosmetic industries. While conventional extraction methods face challenges, enzymatic conversion offers a promising avenue for the industrial production of trehalose. This study delves into a novel synthetic approach utilizing a recombinant enzyme, merging the thermostable trehalose synthase domain from Thermus thermophiles with a cellulose binding domain. Immobilization of this enzyme on cellulose matrices enhances stability and facilitates product purification, opening avenues for efficient enzymatic synthesis. Notably, the engineered enzyme demonstrates additional activity, converting sucrose into trehalulose. This dual functionality, combined with immobilization strategies, holds immense potential for scalable and cost-effective production of trehalose and trehalulose, offering promising prospects in various industrial and biomedical applications.

Expression of thermostable MMLV reverse transcriptase in Escherichia coli by directed mutation

The functionality of Moloney murine leukemia virus reverse transcriptase (MMLV RT) will increase with the improvement of its solubility and thermal stability. Introduce directed mutation at specific positions of the MMLV RT sequence and codon optimization is needed to achieve these properties. The two RT coding sequences with (rRT-K) and without directed mutations (rRT-L) were versatility optimized and expressed to analyze the ribonuclease H (RNase H) inactivity and thermostable polymerase activity. For this purpose, the five-point mutations (438–442aa) and three-point mutations (530, 568, and 659 aa) were done at the RT connection domain and RNase H active site, respectively. High expression levels of rRT-L and rRT-K were obtained in E. coli BL21(DE3) and BL21(shuffle) strains, 0.5 mM IPTG concentration at 37 °C, and 8 hours’ post-induction condition. Then, recombinant enzymes were purified and verified by Ni-NTA resin and western blotting. Insilico analysis (IUpred 3.0) showed that the directed mutation in the RNase H domain caused the formation of disorder regions or instability in the RNase H domain of rRT-K compared to rRT-L. The modified RT-PCR and the RT-LAMP reactions proved the RNase H inactivity of rRT-K. In addition, increasing of thermostability of rRT-K compared to rRT-L and commercial RT was evaluated by the RT-PCR and RT-LAMP reactions. The results showed that rRT-K could successfully tolerate 60 ºC in the two methods. This study revealed that the directed mutations and the versatile sequence optimization can promise to produce thermostable commercial enzymes to decrease non-specific one-step RT-PCR and RT-LAMP products.

Investigating the antifungal potential of genetically modified hybrid chitinase enzymes derived from Bacillus subtilis and Serratia marcescens.

Chitinases are glycosyl hydrolase enzymes that break down chitin, an integral component of fungal cell walls. Bacteria such as Bacillus subtilis and Serratia marcescens produce chitinases with antifungal properties. In this study, we aimed to generate hybrid chitinase enzymes with enhanced antifungal activity by combining functional domains from native chitinases produced by B. subtilis and S. marcescens. Chitinase genes were cloned from both bacteria and fused together using overlap extension PCR. The hybrid constructs were expressed in E. coli and the recombinant enzymes purified. Gel electrophoresis and computational analysis confirmed the molecular weights and isoelectric points of the hybrid chitinases were intermediate between the parental enzymes. Antifungal assays demonstrated that the hybrid chitinases inhibited growth of the fungus Fusarium oxysporum significantly more than the native enzymes and also showed fungicidal activity against Candida albicans, Alternaria solani, and Rhizoctonia solani. The results indicate that hybrid bacterial chitinases are a promising approach to engineer novel antifungal proteins. This study provides insight into structure-function relationships of chitinases and strategies for generating biotherapeutics with enhanced bioactive properties. These hybrid chitinases result in a more potent and versatile antifungal agent.

Preclinical metabolism and metabolic drug-drug interaction profile of pedunculoside and rotundic acid.

Pedunculoside and rotundic acid, the most abundant components in plants of the genus Ilex L. (Aquifoliaceae), exhibit biological and pharmacological significance in the treatment of cardiovascular diseases. However, there have been few studies on their metabolism. This study performed a systematic metabolism study of pedunculoside and rotundic acid and evaluated their potential for herb-drug interaction. Pedunculoside or rotundic acid was incubated with human liver microsomes and recombinant human metabolic enzymes, and analyzed using LC-Q-TOF/MS and LC-MS/MS. Pedunculoside was found to be the most stable in human liver microsomes, whereas rotundic acid was easily metabolized. Eight pedunculoside metabolites and six rotundic acid metabolites were detected and tentatively identified through hydroxylation, glucuronidation, acetylation, and glucose conjugation. Hydroxylation of pedunculoside is mainly catalyzed by CYP3A4/5 and partly by CYP2C8. Hydroxylation of rotundic acid is almost exclusively catalyzed by CYP3A4/5, and its glucuronidation reaction is mediated by UGT1A4. Neither pedunculoside nor rotundic acid showed CYP inhibition (IC50 values > 50 μM) with the probe substrates of major CYP isoforms during incubation with human liver microsomes. This study is the first investigation into the invitro metabolism of pedunculoside and rotundic acid using human liver microsomes. It also aims to assess their potential as perpetrators of drug-drug interactions involving CYP enzymes. The comprehensive metabolism and drug interaction studies of pedunculoside and rotundic acid enable us to evaluate and manage potential risks with their use in pharmacotherapy.

Novel treatment for PXE: Recombinant ENPP1 enzyme therapy

Pseudoxanthoma elasticum (PXE) is a genetic multisystem ectopic calcification disorder caused by inactivating mutations in the ABCC6 gene encoding ABCC6, a hepatic efflux transporter. ABCC6-mediated ATP secretion by the liver is the main source of plasma inorganic pyrophosphate (PPi), a potent endogenous calcification inhibitor. The deficiency of plasma PPi underpins PXE. Recent studies demonstrated that INZ-701, a recombinant human ENPP1, the principal PPi-generating enzyme, restored plasma PPi levels and prevented ectopic calcification in the muzzle skin of an Abcc6-/- mouse model of PXE. This study examined the pharmacokinetics, pharmacodynamics, and potency of a new ENPP1-Fc isoform, BL-1118, in Abcc6-/- mice. When Abcc6-/- mice received a single subcutaneous injection of BL-1118 at 0.25, 0.5, or 1 mg/kg, they had dose-dependent elevations in plasma ENPP1 enzyme activity and PPi levels, with an enzyme half-life of approximately 100 hours. When Abcc6-/- mice were injected weekly from 5 to 15 weeks of age, BL-1118 dose-dependently increased steady-state plasma ENPP1 activity and PPi levels and significantly reduced ectopic calcification in the muzzle skin and kidneys. These results suggest that BL-1118 is a promising enzyme therapy for PXE, a disease currently lacking preventive or therapeutic options.