Enzymatic Modification Research Articles

3D Carbon-Based Conductive Network Printed for Glucose Sensors on Curved and Flexible Substrates.

The rising prevalence of diabetes has led to an increased focus on real-time glucose monitoring. Wearable glucose sensor patches allow noninvasive, real-time monitoring, reducing patient discomfort compared to invasive sensors. However, most existing glucose sensor patches rely on complex and contaminating metal vapor deposition technologies, which pose limitations in practical production. In this study, we propose a novel approach for preparing graphite/multiwall carbon nanotubes (MWCNT)/reduced graphene oxide (rGO) using a high-viscosity ink, which can be easily obtained through simple mechanical stirring. To create intricate patterns and enable printing on curved substrates, we employed a 3D printer equipped with an infrared laser ranging system. The ink served as a working electrode, and we developed a three-electrode system patch with a concentric circle structure. Subsequently, the working electrode underwent enzymatic modification with glucose dehydrogenase with flavin adenine dinucleotide (GDH-FAD) using a polymer embedding method. The resulting wearable glucose sensor exhibited a sensitivity of 2.42 μA mM-1 and a linear detection range of 1-12 mM. In addition, the glucose sensor has excellent anti-interference capability and demonstrates good repeatability in simulated real human wear scenarios, which meets the requirements for accurate human detection. These findings provide valuable insights into the development of human health monitoring technologies.

Enzymatic Modifications, as Biomarker for Heavy Metal Toxicity in Catla catla a Fresh Water Fish

Over the last couple of decades, pollution of the environment has grown to be a significant problem, attracting the curiosity of countless experts from developed as well as underdeveloped countries. Sewage dumping into waterways has a detrimental effect on the aquatic ecosystem and biota since it is the principal sink. As a result of their ability to bioaccumulate and intensify as they ascend food chains, toxic metals are not completely eliminated from the environment. For instance, heavy metals have a tendency to accumulate in the muscular tissues of aquatic species; hence, tissues with elevated levels of toxic metals may be detrimental to the well-being of humans and other animals. Fish have been used as indicator organisms to track contamination in aquatic environments. The fish known as catla catla, an Indian Major carp, is edible and extremely susceptible to even the slightest amount of stress. Fish biomarkers are now often employed to assess the internal and exterior health conditions brought on by toxins.
 The purpose of our investigation was to clarify how biomarkers may be used to track and assess the levels of toxic heavy metals in freshwater Catla catla fish.
 Results: SOD can be used as a bioindicator for heavy metal pollution, according to the current study's findings. These investigations ultimately aim to quantify pollution concentrations that can cause aquatic environments to undergo permanent biological alterations. Though still mild, the amount of toxicity was approaching to danger. To preserve a healthy and pristine environment, public and government-sponsored initiatives can be undertaken to restrict those activities that discharge contaminants into the ecosystem in an unscientific way.
 Conclusion: The study comes to the conclusion that in order to evaluate and track the biological state of the aquatic environment, a multiparameter assessment is required.

PARP-2 mediates cardiomyocyte aging and damage induced by doxorubicin through SIRT1 Inhibition.

Doxorubicin (DOX) is an anthracycline antibiotic used as an antitumor treatment. However, its clinical application is limited due to severe side effects such as cardiotoxicity. In recent years, numerous studies have demonstrated that cellular aging has become a therapeutic target for DOX-induced cardiomyopathy. However, the underlying mechanism and specific molecular targets of DOX-induced cardiomyocyte aging remain unclear. Poly (ADP-ribose) polymerase (PARP) is a family of protein post-translational modification enzymes in eukaryotic cells, including 18 members. PARP-1, the most well-studied member of this family, has become a potential molecular target for the prevention and treatment of various cardiovascular diseases, such as DOX cardiomyopathy and heart failure. PARP-1 and PARP-2 share 69% homology in the catalytic regions. However, they do not entirely overlap in function. The role of PARP-2 in cardiovascular diseases, especially in DOX-induced cardiomyocyte aging, is less studied. In this study, we found for the first time that down-regulation of PARP-2 can inhibit DOX-induced cellular aging in cardiomyocytes. On the contrary, overexpression of PARP-2 can aggravate DOX-induced cardiomyocyte aging and injury. Further research showed that PARP-2 inhibited the expression and activity of SIRT1, which in turn was involved in the development of DOX-induced cardiomyocyte aging and injury. Our findings provide a preliminary experimental basis for establishing PARP-2 as a new target for preventing and treating DOX cardiomyopathy and related drug development.

Omniligase-1-Mediated Phage-Peptide Library Modification and Insulin Engineering.

Chemical and enzymatic modifications of peptide-displayed libraries have been successfully employed to expand the phage display library. However, the requirement of specific epitopes and scaffolds has limited the scope of protein engineering using phage display. In this study, we present a novel approach utilizing omniligase-1-mediated selective and specific ligation on the phage pIII protein, offering a high conversion rate and compatibility with commercially available phage libraries. We applied this method to perform high-throughput engineering of insulin analogues with randomized B chain C-terminal regions. Insulin analogues with different B chain C-terminal segments were selected and exhibited biological activity equivalent to that of human insulin. Molecular dynamics studies of insulin analogues revealed a novel interaction between the insulin B27 residue and insulin receptor L1 domain. In summary, our findings highlight the potential of omniligase-1-mediated phage display in the development and screening of disulfide-rich peptides and proteins. This approach holds promise for the creation of novel insulin analogues with enhanced therapeutic properties and exhibits potential for the development of other therapeutic compounds.

Proteome-Wide Analysis of Lysine 2-Hydroxyisobutyrylation in Aspergillus fumigatus

Aspergillus fumigatus is the significant causative agent in cases of invasive aspergillosis, leading to a high mortality rate in immunocompromised patients. A comprehensive understanding of its growth patterns and metabolic processes within the host is a critical prerequisite for the development of effective antifungal strategies. Lysine 2-hydroxyisobutyrylation (Khib) is a highly conserved protein posttranslational modifications (PTM) found in various organisms. In this study, we investigate the biological impact of Khib in A. fumigatus. Using a combination of antibody enrichment with the conventional LC–MS/MS method, the pattern of Khib-modification in proteins and their respective sites were analyzed in a wild type strain of A. fumigatus. Our findings revealed 3494 Khib-modified proteins with a total of 18,091 modified sites in this strain. Functional enrichment analysis indicated that these Khib-modified proteins participate in a diverse range of cellular functions, spanning various subcellular locations such as ribosome biosynthesis, protein synthesis and nucleocytoplasmic transport. Notably, when compared with other reported eukaryotes, A. fumigatus exhibited consistently higher numbers of Khib-modified proteins, suggesting the potential significance of this modification in this organism. An interesting observation is the prevalence of Khib modifications in most enzymes involved in the ergosterol synthesis pathway. The insights gathered from this study provide new avenue for studying PTM-associated mechanisms in fungal growth and offer potential implication for antifungal drug development.

Isolation and Cs+ resistance mechanism of Escherichia coli strain ZX-1.

This research aims to elucidate the physiological mechanisms behind the accidental acquisition of high-concentration cesium ions (Cs+) tolerance of Escherichia coli and apply this understanding to develop bioremediation technologies. Bacterial Cs+ resistance has attracted attention, but its physiological mechanism remains largely unknown and poorly understood. In a prior study, we identified the Cs+/H+ antiporter TS_CshA in Microbacterium sp. TS-1, resistant to high Cs+ concentrations, exhibits a low Cs+ affinity with a Km value of 370 mM at pH 8.5. To enhance bioremediation efficacy, we conducted random mutagenesis of TS_cshA using Error-Prone PCR, aiming for higher-affinity mutants. The mutations were inserted downstream of the PBAD promoter in the pBAD24 vector, creating a mutant library. This was then transformed into E. coli-competent cells. As a result, we obtained a Cs+-resistant strain, ZX-1, capable of thriving in 400 mM CsCl-a concentration too high for ordinary E. coli. Unlike the parent strain Mach1™, which struggled in 300 mM CsCl, ZX-1 showed robust growth even in 700 mM CsCl. After 700 mM CsCl treatment, the 70S ribosome of Mach1™ collapsed, whereas ZX-1 and its derivative ΔZX-1/pBR322ΔAp remained stable. This means that the ribosomes of ZX-1 are more stable to high Cs+. The inverted membrane vesicles from strain ZX-1 showed an apparent Km value of 28.7 mM (pH 8.5) for Cs+/H+ antiport activity, indicating an approximately 12.9-fold increase in Cs+ affinity. Remarkably, the entire plasmid isolated from ZX-1, including the TS_cshA region, was mutation-free. Subsequent whole-genome analysis of ZX-1 identified multiple SNPs on the chromosome that differed from those in the parent strain. No mutations in transporter-related genes were identified in ZX-1. However, three mutations emerged as significant: genes encoding the ribosomal bS6 modification enzyme RimK, the phage lysis regulatory protein LysB, and the flagellar base component protein FlgG. These mutations are hypothesized to affect post-translational modifications, influencing the Km value of TS_CshA and accessory protein expression. This study unveils a novel Cs+ resistance mechanism in ZX-1, enhancing our understanding of Cs+ resistance and paving the way for developing technology to recover radioactive Cs+ from water using TS_CshA-expressing inverted membrane vesicles.

Bidirectional Regulation of Intracellular Enzyme Activity Using Light-Driven Nano-Inhibitors.

Photochemical regulation provides precise control over enzyme activities with high spatiotemporal resolution. A promising approach involves anchoring "photoswitches" at enzyme active sites to modulate substrate recognition. However, current methods often require genetic mutations and irreversible enzyme modifications for the site-specific anchoring of "photoswitches", potentially compromising the enzyme activities. Herein, we present a pioneering reversible nano-inhibitor based on molecular imprinting technique for bidirectional regulation of intracellular enzyme activity. The nano-inhibitor employs a molecularly imprinted polymer nanoparticle as its body and azobenzene-modified inhibitors ("photoswitches") as the arms. By using a target enzyme as the molecular template, the nano-inhibitor acquires oriented binding sites on its surface, resulting in a high affinity for the target enzyme and non-covalently firm anchoring of the azobenzene-modified inhibitor to the enzyme active site. Harnessing the reversible isomerization of azobenzene units upon exposure to ultraviolet and visible light, the nano-inhibitor achieves bidirectional enzyme activity regulation by precisely docking and undocking inhibitor at the active site. Notably, this innovative approach enables the facile in situ regulation of intracellular endogenous enzymes, such as carbonic anhydrase. Our results represent a practical and versatile tool for precise enzyme activity regulation in complex intracellular environments.

Bidirectional Regulation of Intracellular Enzyme Activity Using Light‐Driven Nano‐Inhibitors

AbstractPhotochemical regulation provides precise control over enzyme activities with high spatiotemporal resolution. A promising approach involves anchoring “photoswitches” at enzyme active sites to modulate substrate recognition. However, current methods often require genetic mutations and irreversible enzyme modifications for the site‐specific anchoring of “photoswitches”, potentially compromising the enzyme activities. Herein, we present a pioneering reversible nano‐inhibitor based on molecular imprinting technique for bidirectional regulation of intracellular enzyme activity. The nano‐inhibitor employs a molecularly imprinted polymer nanoparticle as its body and azobenzene‐modified inhibitors (“photoswitches”) as the arms. By using a target enzyme as the molecular template, the nano‐inhibitor acquires oriented binding sites on its surface, resulting in a high affinity for the target enzyme and non‐covalently firm anchoring of the azobenzene‐modified inhibitor to the enzyme active site. Harnessing the reversible isomerization of azobenzene units upon exposure to ultraviolet and visible light, the nano‐inhibitor achieves bidirectional enzyme activity regulation by precisely docking and undocking inhibitor at the active site. Notably, this innovative approach enables the facile in situ regulation of intracellular endogenous enzymes, such as carbonic anhydrase. Our results represent a practical and versatile tool for precise enzyme activity regulation in complex intracellular environments.

MicroRNA-93-5p regulates odontogenic differentiation and dentin formation via KDM6B

BackgroundEpigenetic factors influence the odontogenic differentiation of dental pulp stem cells and play indispensable roles during tooth development. Some microRNAs can epigenetically regulate other epigenetic factors like DNA methyltransferases and histone modification enzymes, functioning as epigenetic-microRNAs. In our previous study, microarray analysis suggested microRNA-93-5p (miR-93-5p) was differentially expressed during the bell stage in human tooth germ. Prediction tools indicated that miR-93-5p may target lysine-specific demethylase 6B (KDM6B). Therefore, we explored the role of miR-93-5p as an epi-miRNA in tooth development and further investigated the underlying mechanisms of miR-93-5p in regulating odontogenic differentiation and dentin formation.MethodsThe expression pattern of miR-93-5p and KDM6B of dental pulp stem cells (DPSCs) was examined during tooth development and odontogenic differentiation. Dual luciferase reporter and ChIP-qPCR assay were used to validate the target and downstream regulatory genes of miR-93-5p in human DPSCs (hDPSCs). Histological analyses and qPCR assays were conducted for investigating the effects of miR-93-5p mimic and inhibitor on odontogenic differentiation of hDPSCs. A pulpotomy rat model was further established, microCT and histological analyses were performed to explore the effects of KDM6B-overexpression and miR-93-5p inhibition on the formation of tertiary dentin.ResultsThe expression level of miR-93-5p decreased as odontoblast differentiated, in parallel with elevated expression of histone demethylase KDM6B. In hDPSCs, miR-93-5p overexpression inhibited the odontogenic differentiation and vice versa. MiR-93-5p targeted 3′ untranslated region (UTR) of KDM6B, thereby inhibiting its protein translation. Furthermore, KDM6B bound the promoter region of BMP2 to demethylate H3K27me3 marks and thus upregulated BMP2 transcription. In the rat pulpotomy model, KDM6B-overexpression or miR-93-5p inhibition suppressed H3K27me3 level in DPSCs and consequently promoted the formation of tertiary dentin.ConclusionsMiR-93-5p targets epigenetic regulator KDM6B and regulates H3K27me3 marks on BMP2 promoters, thus modulating the odontogenic differentiation of DPSCs and dentin formation.

Hematopoietic stem cell quiescence and DNA replication dynamics maintained by the resilient β-catenin/Hoxa9/Prmt1 axis

AbstractMaintenance of quiescence and DNA replication dynamics are 2 paradoxical requirements for the distinct states of dormant and active hematopoietic stem cells (HSCs), which are required to preserve the stem cell reservoir and replenish the blood cell system in response to hematopoietic stress, respectively. Here, we show that key self-renewal factors, β-catenin or Hoxa9, largely dispensable for HSC integrity, in fact, have dual functions in maintaining quiescence and enabling efficient DNA replication fork dynamics to preserve the functionality of hematopoietic stem and progenitor cells (HSPCs). Although β-catenin or Hoxa9 single knockout (KO) exhibited mostly normal hematopoiesis, their coinactivation led to severe hematopoietic defects stemmed from aberrant cell cycle, DNA replication, and damage in HSPCs. Mechanistically, β-catenin and Hoxa9 function in a compensatory manner to sustain key transcriptional programs that converge on the pivotal downstream target and epigenetic modifying enzyme, Prmt1, which protects the quiescent state and ensures an adequate supply of DNA replication and repair factors to maintain robust replication fork dynamics. Inactivation of Prmt1 phenocopied both cellular and molecular phenotypes of β-catenin/Hoxa9 combined KO, which at the same time could also be partially rescued by Prmt1 expression. The discovery of the highly resilient β-catenin/Hoxa9/Prmt1 axis in protecting both quiescence and DNA replication dynamics essential for HSCs at different key states provides not only novel mechanistic insights into their intricate regulation but also a potential tractable target for therapeutic intervention.

Enzymatic Synthesis of Arachidonic Acid-Rich Phosphatidylcholine from Arachidonic Acid Ethyl Esters

Arachidonic acid (ARA) is an essential fatty acid with numerous biological activities that benefits human health. However, ARA-rich phosphatidylcholine (PtdCho), which has a higher bioavailability than ARA-rich triacylglycerols, is scarce in the natural source. In this study, we developed an enzymatic modification approach for the synthesis of ARA-rich PtdCho from ARA-rich ethyl esters (EE). The maximum incorporation of ARA into PtdCho (24.02%) was achieved from the optimized conditions, including ARA-rich EE/PtdCho mass ratio of 2:1, hexane, lipase Novozym 435 as a biocatalyst (15% of enzyme load) and reaction time of 24 h. The 31P nuclear magnetic resonance (NMR) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) results revealed that the PtdCho content decreased to 17.53% and the ARA-containing PtdCho species was primarily identified as PtdCho (18:2/20:4). Taken together, this investigation offers a new reference for the efficient production of ARA-rich PtdCho via enzymatic modification and paves a theoretical groundwork of industrial production practice.

Production of hybrid macrolide antibiotics by exploiting the specific substrate recognition characteristics of multifunctional cytochrome P450 enzyme MycG.

Macrolide antibiotics are biosynthesized via enzymatic modifications, including glycosylation, methylation, and oxidation, after the core macro-lactone ring is generated by a polyketide synthase system. This study explored the diversification of macrolides by combining biosynthetic enzymes and reports an approach to produce unnatural hybrid macrolide antibiotics. The cytochrome (CYP) P450 monooxygenase MycG exhibits bifunctional activity, catalyzing late-stage hydroxylation at C-14 followed by epoxidation at C-12/13 during mycinamicin biosynthesis. The mycinose sugar of mycinamicin serves as a key molecular recognition element for binding to MycG. Thus, we subjected the hybrid macrolide antibiotic 23-O-mycinosyl-20-deoxo-20-dihydro-12,13-deepoxyrosamicin (IZI) to MycG, and confirmed that MycG catalyzed hydroxylation at C-22 and epoxidation at C-12/13 in IZI. In addition, the introduction of mycinose biosynthesis-related genes and mycG into rosamicin-producing Micromonospora rosaria enabled the fermentative production of 22-hydroxylated and 12,13-epoxidized forms of IZI. Interestingly, MycG catalyzed the sequential oxidation of hydroxylation and epoxidation in mycinamicin biosynthesis, but only single reactions in IZI. These findings highlight the potential for expanding the application of the multifunctional P450 monooxygenase MycG for the production of unnatural compounds.

Overexpression of BRG1 improves early development of porcine somatic cell nuclear transfer embryos

The epigenetic modification levels of donor cells directly affect the developmental potential of somatic cell nuclear transfer (SCNT) embryos. BRG1, as an epigenetic modifying enzyme, has not yet been studied in donor cells and SCNT embryos. In this study, BRG1 was overexpressed in porcine fetal fibroblasts (PFFs), its effect on chromatin openness and gene transcription was examined, subsequently, the development potential of porcine SCNT embryos was investigated. The results showed that compared with the control group, the percentage of G1 phase cells was significantly increased (32.3 % ± 0.87 vs 25.7 % ± 0.81, P < 0.05) in the experimental group. The qRT-PCR results showed that the expression of H3K9me3-related genes was significantly decreased (P < 0.05), HAT1 was significantly increased (P < 0.05). Assay of Transposase Accessible Chromatin sequencing (ATAC-seq) results revealed that SMARCA4、NANOG、SOX2、MAP2K6 and HIF1A loci had more open chromatin peaks in the experimental group. The RNA-seq results showed that the upregulated genes were mainly enriched in PI3K/AKT and WNT signaling pathways, and the downregulated genes were largely focused on disease development. Interestingly, the developmental rate of porcine SCNT embryos was improved (27.33 % ± 1.40 vs 17.83 % ± 2.02, P < 0.05), the expression of zygotic gene activation-related genes in 4-cell embryos, and embryonic development-related genes in blastocysts was significantly upregulated in the experimental group (P < 0.05). These results suggest that overexpression of BRG1 in donor cells is benefit for the developmental potential of porcine SCNT embryos.

Idiosyncratic genome evolution of the thermophilic cyanobacterium Synechococcus at the limits of phototrophy.

Thermophilic microorganisms are expected to have smaller cells and genomes compared with mesophiles, a higher proportion of horizontally acquired genes, and distinct nucleotide and amino acid composition signatures. Here, we took an integrative approach to investigate these apparent correlates of thermophily for Synechococcus A/B cyanobacteria, which include the most heat-tolerant phototrophs on the planet. Phylogenomics confirmed a unique origin of different thermotolerance ecotypes, with low levels of continued gene flow between ecologically divergent but overlapping populations, which has shaped the distribution of phenotypic traits along these geothermal gradients. More thermotolerant strains do have smaller genomes, but genome reduction is associated with a decrease in community richness and metabolic diversity, rather than with cell size. Horizontal gene transfer played only a limited role during Synechococcus evolution, but, the most thermotolerant strains have acquired a Thermus tRNA modification enzyme that may stabilize translation at high temperatures. Although nucleotide base composition was not associated with thermotolerance, we found a general replacement of aspartate with glutamate, as well as a dramatic remodeling of amino acid composition at the highest temperatures that substantially differed from previous predictions. We conclude that Synechococcus A/B genome diversification largely does not conform to the standard view of temperature adaptation. In addition, carbon fixation was more thermolabile than photosynthetic oxygen evolution for the most thermotolerant strains compared with less tolerant lineages. This suggests that increased flow of reducing power generated during the light reactions to an electron sink(s) beyond carbon dioxide has emerged during temperature adaptation of these bacteria.

Enhancement of functional properties, digestive properties, and in vitro digestion product physiological activity of extruded corn gluten meal by enzymatic modification.

Enzymatic modification is an effective means of improving the functional properties, digestive properties, and in vitro digestion product physiological activity of proteins, thus significantly expanding protein uses in various food applications. In this study, the addition of chymotrypsin (CT) at pH 9.0 and 11.0 was found to significantly improve the functional properties (solubility, foaming properties, water holding capacity, oil holding capacity, etc.) and digestive properties of extruded corn gluten meal (ECGM). Similar changes were observed when treating ECGM with glutaminase, protein glutaminase, and papain. These changes were likely due to the increase in number of carboxyl groups and the multiple effects of change in protein net charge and conformation caused by enzymatic deamidation. Of note, ECGM deamidated by CT showed the highest degree of deamidation, solubility, and gastrointestinal digestibility at pH 11.0, up to 44.92%, 43.75%, and 82.22%, respectively. In addition, CT-ECGM digestion product exhibited strong antioxidant activity and potential to promote alcohol metabolism in both a static digestion model and dynamic digestion model, even comparable to commercial corn peptides (CCP), while being inexpensive and of low bitterness compared to CCP. Meanwhile, the physiological activity enhanced as the molecular weight of digestion product decreased with the digested component having strongest activity. This study may promote the application of ECGM as a food component in the food industry or even as a substitute for CCP. © 2023 Society of Chemical Industry.

An UMAMIT-GTR transporter cascade controls glucosinolate seed loading in Arabidopsis.

Many plant species translocate maternally synthesized specialized metabolites to the seed to protect the developing embryo and later the germinating seedling before it initiates its own de novo synthesis. While the transport route into the seed is well established for primary metabolites, no model exists for any class of specialized metabolites that move from maternal source tissue(s) to embryo. Glucosinolate seed loading in Arabidopsis depends on plasma membrane localized exporters (USUALLY MULTIPLE AMINO ACIDS MOVE IN AND OUT TRANSPORTERs, UMAMITs) and importers (GLUCOSINOLATE TRANSPORTERs, GTRs), but the critical barriers in the seed loading process remain unknown. Here we dissect the transport route of glucosinolates from their source in the reproductive organ to the embryo by re-introducing the transporters at specific apoplastic barriers in their respective mutant backgrounds. We find that UMAMIT exporters and GTR importers form a transporter cascade that is both essential and sufficient for moving glucosinolates across at least four plasma membrane barriers along the route. We propose a model in which UMAMITs export glucosinolates out of the biosynthetic cells to the apoplast, from where GTRs import them into the phloem stream, which moves them to the unloading zone in the chalazal seed coat. From here, the UMAMITs export them out of maternal tissue and ultimately, the GTRs import them into the embryo symplasm, where the seed-specific glucosinolate profile is established by enzymatic modifications. Moreover, we propose that methylsulfinylalkyl glucosinolates are the predominant mobile form in seed loading. Elucidation of the seed loading process of glucosinolates identifies barrier-specific targets for transport engineering strategies to eliminate or over-accumulate a specialized metabolite in seeds with minimal interruption of other cellular processes.

Dissemination and Molecular Characterization of 16s Rna Methylase and Aminoglycoside Modifying Enzymes- Producing P. Aeruginosa Isolated from Burn Specimens in Najaf City

Background: Pseudomonas aeruginosa is a significant species of bacteria connected with nosocomial infections involving, pneumonia, infections of urinary tract, and damage of the surgical site. Aim of the Study: The aim of this thesis is to investigate the occurrence of genes encoding clinically important 16S rRNA methylases and AMEs enzymes in aminoglycosides resistance P.aeruginosa isolates recovered from Najaf hospitals and the characteristics of these isolates. Methods: a total of 68 specimens were isolated from patients at burn center in Najaf City from the whole study period. The 24 isolates were screened for the sensitivity versus 3 various antibiotics disks of the one class by using Kirby-Bauer disk diffusion method. Results: The resistance rate of 24 isolates of XDR Pseudomonas aeruginosa against aminoglycoside groups: gentamicin, amikacin, and tobramycin were 91.6 %, 83.3 %, and 66.6 %, respectively. However, the resistance profiles of the isolates show that 22 (91.6 %) were resistant to 1 or more antibiotics of aminoglycoside. Present study show that only 3 (12.5%) of XDR isolates harbored ant(4')-IIb gene isolates and none of the XDR isolates investigated were positive for rmtA and aac(6')-IIb gene. Conclusions: This survey has showed for the initial time three isolates harbors the ant(4')-IIb aminoglycoside resistance genes in Iraq.

Enzymatic Modification of Starch: A Review

Starch is the most abundant naturally occurring carbohydrate reserve in plants and is found in cereals, roots, tubers, legumes and some immature fruits like bananas or mangos. Starch is usually employed as a food additive, such as a thickening, stabilizer, or texture enhancer to improve some of the products quality characteristics, pharmaceutical and among other. The application of native starch is often restricted owing to its constricted solubility, weak functional attributes and limited tolerance to a wide array of processing conditions. Its low resistance to shear, high retrogradation, and poor freeze-thaw stability, limit the use of starch in industrial applications. These natural shortcomings can be overcome by different methods of modification. In recent decades, enzymatic modifications have been adopted, partly replacing the chemical and physical methods for the preparation of modified starch, as enzymes are safer and healthier than chemical method for both the environment and food consumers. Several enzymes viz., alfa-amylase, beta-amylase, glucose isomerase, pullulanase, xylanase, among others are use in modification of starch. The enzymatic modification of starch molecules directly affected properties of the modified starch especially in freeze-thaw stability of gels and retardation of retrogradation during storage. Combined enzymatic modification resulted in a marked increase in resistant starch and enzyme modified starch can be well utilized as a fat replacer. It is environment-friendly method and can provide desired functional characteristics.

Rational Design and Site‐Directed Mutagenesis Promote the Heterologous Production of the Antitumor Natural Product Dehydroabietic Acid

AbstractCytochrome P450s participate in the biosynthesis of natural active ingredients and catalyze the formation of hydroxyl, carboxyl, epoxy and other groups from C−H bonds. CYP728B70 from Tripterygium wilfordii oxidizes the miltiradiene‐C18 methyl group to produce dehydroabietic acid, a diterpenoid intermediate with anticancer, antibacterial and anti‐inflammatory activities. To elevate the carboxylation product output, we excavated serveral homologous CYP728Bs and an N‐terminal truncated CYP728B70 transcript from T. wilfordii genome, of which CYP728B79 possessed a weak carboxylation effect towards miltiradiene through heterologous expression in Saccharomyces cerevisiae. We employed RoseTTAFold method and molecular dynamic simulations to predict its protein structure and docking pose of CYP728B70, and obtained several mutants with increased yield using Funclib and site‐directed mutagenesis methods. Our study confirmed the influence of peripheral residues on protein activity, improved the catalytic activity of CYP728B70, and would provide ideas for related enzyme modification.

Влияние способов получения резистентного крахмала на жизнедеятельность лактобактерий

This study investigated the influence of different methods for obtaining resistant starch on the growth of lactobacilli. Resistant starch was obtained by modifying starch suspension using enzymes, low, and high temperatures. Enzymatic modification was carried out using amylolytic enzyme preparations – Disticime BA-T and Disticime AG at a temperature of 50°C–80°C–60°C for 3 h; at a temperature of –18°C for 12 h; in a liquid nitrogen environment at –196°C for 5 min; as well as at a high temperature of 120°C for 15; 30; 45, and 60 min. The stability of resistant starch was assessed by the amount of reducing substances obtained after repeated treatment of modified starch suspensions with enzyme preparations after the introduction of modified starch suspensions into media with low and high pH values. The effectiveness of each method of obtaining resistant starch was assessed by the growth of lactobacilli. It was established that the most stable resistant starch is obtained after autoclaving the starch suspension at 120°C for 15 minutes. In the same sample, the greatest growth of microorganisms was observed – 2 times higher than in the sample where starch modification was carried out with enzyme preparations. These results are important to consider when incorporating resistant starch into the formulations of functional food.