Search Results (46)

Chitosan oligosaccharides (COSs) with defined degrees of polymerization (DP) exhibit distinct bioactivities with promising applications in food, pharmaceutical, and agricultural industries. However, the specific and sustainable production of COSs remains challenging due to the broad product distribution of wild-type chitosanases and the difficulties in enzyme recovery and reuse. In this study, we employed rational design to engineer a GH46 chitosanase (CsnB) from Bacillus sp. BY01 for chitobiose production. Through homology modeling and molecular docking analysis, 15 mutants were designed by targeting key residues structurally critical for substrate stabilization, product release, and active-site geometry in the substrate-binding subsites. The D78Y mutant exhibited exclusive specificity for chitobiose, demonstrating a specific activity of 102.4 U/mg and yielding chitobiose with a purity exceeding 98%, thereby surpassing the previously reported enzymes for chitobiose production. To address the challenges of enzyme stability, purification costs, and product separation, we developed a ReELP system by integrating elastin-like polypeptides (ELPs) with a ReverseCatcher/ReverseTag peptide pair. This system enabled one-step purification and co-immobilization of CsnB-D78Y directly from cell lysate onto biomimetic silica nanoparticles, achieving 96.8% immobilization efficiency and 90.7% activity recovery. The immobilized enzyme exhibited enhanced thermal and pH stability, retaining approximately 50% activity after 12 h at 40 °C compared to only 5.7% for the free enzyme. In reusability assays, the immobilized CsnB-D78Y maintained efficient chitobiose production over 5 consecutive cycles. This work provides a green and cost-effective strategy for the specific and sustainable production of chitobiose, offering new insights into enzyme engineering and immobilization for industrial COS production. Full article

The escalating crisis of antibiotic resistance, particularly concerning foodborne pathogens such as Staphylococcus aureus and its biofilm contamination, has emerged as a major global challenge to food safety and public health. Biofilm formation significantly enhances the pathogen’s resistance to environmental stresses and disinfectants, underscoring the urgent need for novel antimicrobial agents. In this study, we isolated Bacillus strain B673 from the saline–alkali environment of Xinjiang, conducted whole-genome sequencing, and applied antiSMASH analysis to identify ribosomally synthesized and post-translationally modified peptide (RiPP) gene clusters. By integrating an LSTM-Attention-BERT deep learning framework, we screened and predicted nine novel antimicrobial peptide sequences. Using a SUMO-tag fusion tandem strategy, we achieved efficient soluble expression in an E. coli system, and the purified products exhibited remarkable inhibitory activity against Staphylococcus aureus (MIC = 3.13 μg/mL), with inhibition zones larger than those of the positive control. Molecular docking and dynamic simulations demonstrated that the peptides can stably bind to MurE, a key enzyme in cell wall synthesis, with negative binding free energy, suggesting an antibacterial mechanism via MurE inhibition. This study provides promising candidate molecules for the development of anti-drug-resistant agents and establishes an integrated research framework for antimicrobial peptides, spanning gene mining, intelligent screening, efficient expression, and mechanistic elucidation. Full article

Small non-coding microRNAs (miRNAs) play crucial roles in the degradation of the messenger RNAs (mRNAs) that are involved in various biological processes post-transcriptionally and translationally. Many plants, especially Musa spp. (plantains and bananas), which are important perennial herbs of the family Musaceae, experience significant yield loss due to abiotic stressors, yet only a few miRNAs involved in this response have been identified. This study employed in silico analyses of transcriptome shotgun assembly (TSA) and expressed sequence tag (EST) sequences to identify Musa miRNAs and their target genes. Leaf and root tissues from three Musa genomic groups (AAA, AAB, and ABB) under drought stress were analyzed using quantitative real-time PCR (qRT-PCR) to validate the expression of miRNAs. A total of 17 potential conserved miRNAs from 11 families were identified, with the minimal folding free energies (-kcal/mol) of precursors ranging from −136.00 to −55.70, as observed through RNA folding analysis. Six miRNAs (miR530-5p, miR528-5p, miR482a, miR397a, miR160h, and miR399a) showed distinct tissue-specific expression patterns in the roots and leaves across the three groups. A total of 59 target regulatory transcription factors and enzymes involved in stress response, growth, and metabolism were predicted. Of these, 11 targets were validated for miR530-5p, miR528-5p, miR482a, and miR397a, using qRT-PCR. These four stress-responsive miRNAs exhibited an inverse expression relationship with their target genes across two different tissues in Musa groups. This research provides insights into miRNA-mediated drought stress responsiveness in Musa spp., potentially benefiting future studies on gene regulation under drought stress. Full article

Although drunken horse grass (Achnatherum inebrians) can be simultaneously infected by the foliar endophyte Epichloë gansuensis and colonized by Bacillus subtilis, it remains unclear whether Epichloë endophyte symbiosis influences B. subtilis colonization, as well as how their interaction affects nitrogen fixation and assimilation. The purpose of the present study was to investigate whether E. gansuensis endophyte infection facilitates the colonization of B. subtilis in the roots of host plants, with a focus on understanding the interaction effects of the E. gansuensis endophyte and B. subtilis on plant growth and nutrient absorption. In this study, we measured the colony growth rate of B. subtilis LZU7 when co-cultured with E. gansuensis strains. In addition to an in vitro test, we investigated the root colonization of Epichloë endophyte-infected plants (E+) and Epichloë endophyte-free plants (E−) with the GFP-tagged B. subtilis LZU7 in an inoculation test. Furthermore, we evaluated the interactions between E. gansuensis endophyte symbiosis and B. subtilis LZU7 colonization on the dry weight, nitrogen fixation, nitrogen converting-enzyme activity, and nutrients for E+ and E− plants by labeling with ¹⁵N₂. The results showed that the growth rates of B. subtilis LZU7 were altered and increased in a co-culture with the E. gansuensis endophyte. A significantly greater colonization of GFP-tagged B. subtilis LZU7 was detected in the roots of E+ plants compared with the roots of E− plants, suggesting that E. gansuensis endophyte symbiosis enhances the colonization of beneficial microorganisms. The combination of E. gansuensis endophyte symbiosis and B. subtilis LZU7 inoculation significantly altered the expression of the nitrogenase (nifH) gene, thereby promoting increased biological nitrogen fixation (BNF). The E. gansuensis endophyte infection and inoculation with B. subtilis LZU7 significantly increased δ15NAir in plants. Co-inoculation with the E. gansuensis endophyte and B. subtilis LZU7 significantly elevated NH₄⁺ accumulation in the roots, depleted the NH₄⁺ availability in the surrounding soil, and showed no measurable impact on the foliar NH₄⁺ content. The observed alterations in the NH₄⁺ content were linked to nitrogen-fixing microorganisms that promoted nitrogen fixation, thereby enhancing nitrogen uptake and contributing to greater biomass production in A. inebrians. Our findings highlighted the fact that a foliar symbiosis with the E. gansuensis endophyte enhances the recruitment of beneficial bacteria, and that the resulting interaction significantly impacts nitrogen fixation, assimilation, and allocation in host plants. Full article

The site-directed immobilization of enzymes has demonstrated significant potential in industrial applications due to its ability to minimize enzyme heterogeneity and maximize retained activity. However, existing approaches often require the introduction of unnatural amino acids or excessive specific ligase to achieve this goal. In this study, a self-catalyzed protein capture system (i.e., the SnoopCatcher/SnoopTag pair) was utilized for the directed immobilization of lipase on magnetic carriers. By tagging the Pseudomonas fluorescens lipase (PFL) with a SnoopTag at the C-terminal, the fused lipase PFL-SnoopTag (PSNT) readily conjugated with the SnoopCatcher partner via a spontaneously formed isopeptide bond between them. Novel magnetic particles functionalized by SnoopCatcher proteins were prepared using a co-precipitation method, achieving a loading capacity of around 0.8 mg/g carrier for the SnoopCatcher. This functionalized magnetic carrier enabled the site-directed immobilization of lipase PSNT at 81.4% efficiency, while the enzyme loading capacity reached 3.04 mg/g carriers. To further assess the practical performance of site-directed immobilized lipases, they were applied in biodiesel production and achieved a yield of 88.5%. Our results demonstrate a universal platform for the site-directed immobilization of enzymes with high performance, which offers significant advantages, e.g., single-step purification and catalyst-free immobilization of engineered enzymes, as well as easy recovery, highlighting its potential for industrial applications. Full article

Mucolipidosis type II (ML II) is a rare and fatal disease of acid hydrolase trafficking. It is caused by pathogenic variants in the GNPTAB gene, leading to the absence of GlcNAc-1-phosphotransferase activity, an enzyme that catalyzes the first step in the formation of the mannose 6-phosphate (M6P) tag, essential for the trafficking of most lysosomal hydrolases. Without M6P, these do not reach the lysosome, which accumulates undegraded substrates. The lack of samples and adequate disease models limits the investigation into the pathophysiological mechanisms of the disease and potential therapies. Here, we report the generation and characterization of an ML II induced pluripotent stem cell (iPSC) line carrying the most frequent ML II pathogenic variant [NM_024312.5(GNPTAB):c.3503_3504del (p.Leu1168fs)]. Skin fibroblasts were successfully reprogrammed into iPSCs that express pluripotency markers, maintain a normal karyotype, and can differentiate into the three germ layers. Furthermore, ML II iPSCs showed a phenotype comparable to that of the somatic cells that originated them in terms of key ML II hallmarks: lower enzymatic activity of M6P-dependent hydrolases inside the cells but higher in conditioned media, and no differences in an M6P-independent hydrolase and accumulation of free cholesterol. Thus, ML II iPSCs constitute a novel model for ML II disease, with the inherent iPSC potential to become a valuable model for future studies on the pathogenic mechanisms and testing potential therapeutic approaches. Full article

Background: Heme is an important cofactor and plays crucial roles in the correct folding of hemoproteins. The synthesis of heme can be enhanced by the plasmid-based expression of heme biosynthetic genes. However, plasmid-based expression is genetically unstable and requires the utilization of antibiotics to maintain high copy numbers of plasmids. Methods: The rate-limiting steps in heme biosynthesis were first analyzed based on previous studies and the accumulation of heme intermediates was achieved by adding heme precursor (5-aminolevulinic acid, ALA). Next, the intracellular accumulation of porphyrin was increased by deleting the porphyrin transporter TolC. Finally, the heme synthetic genes were modified by integrating the hemA and hemL genes into the cheW and yciQ locus, assembling the rate-limiting enzymes HemC and HemD with RIAD-RIDD tags, replacing the promoters of hemE/hemH genes with the constitutive promoter P_J23100, and deleting the heme degradation gene yfeX. Results: An enhanced heme supply HEME2 strain was obtained with a heme titer of 0.14 mg/L, which was 4.60-fold higher than that of the C41(DE3) strain. The HEME2 strain was applied to produce human hemoglobin and leghemoglobin. The titer and peroxidase activity of human hemoglobin were 1.29-fold and 42.4% higher in the HEME2-hHb strain than the values in the control strain C41-hHb. In addition, the peroxidase activity and heme content of leghemoglobin were increased by 39.2% and 53.4% in the HEME2-sHb strain compared to the values in the control strain C41-sHb. Conclusions: A plasmid-free Escherichia coli C41(DE3) strain capable of efficient and stable heme supply was constructed and can be used for the production of high-active hemoglobins. Full article

Enzymes play an essential role in many different industries; however, their operating conditions are limited due to the loss of enzyme activity in the presence of proteases and at temperatures significantly above physiological conditions. One way to improve the stability of these enzymes against high temperatures and proteases is to encapsulate them in protective shells or virus-like particles. This work presents a streamlined, three-step, cell-free protein synthesis (CFPS) procedure that enables rapid in vitro enzyme production, targeted encapsulation in protective virus-like particles (VLPs), and facile purification using a 6× His-tag fused to the VLP coat protein. This process is performed in under 12 h and overcomes several limitations of enzyme encapsulation, such as the control of packing density, speed, and complexity of the process. Here, we encapsulate the enzyme Candida antarctica lipase B in the VLP from the bacteriophage Qβ, while in the presence of a linking RNA aptamer. The encapsulated enzymes largely retained their activity in comparison to the free enzymes. Additionally, when subjected to 90 °C temperatures or 5 h incubation with proteases, the encapsulated enzymes maintained their activity, whereas the free enzymes lost their activity. In this work, we also demonstrate control over packing density by achieving packing densities of 4.7 and 6.5 enzymes per VLP based off the concentration of enzyme added to the encapsulation step. Full article

The synthesis of enantiomeric forms of D-amino acids can be achieved by a two-step “hydantoinase process” based on the sequential catalysis of substrates by specific enzymes, D-carbamoylase and D-hydantoinase. Here, we describe the structural features of D-carbamoylase from Pseudomonas, the encoded gene of which was chemically synthesized and cloned into Escherichia coli. A significant fraction of the overexpressed recombinant protein forms insoluble inclusion bodies, which are partially converted to a soluble state upon treatment with N-lauroylsarcosine or upon incubation of cells at 28 °C. Purified His-tagged protein exhibits the highest activity towards N-carbamoyl-D-alanine and N-carbamoyl-D-tryptophan. Comprehensive virtual analysis of the interactions of bulky carbamylated amino acids with D-carbamoylase provided valuable information. Molecular docking analysis revealed the location of the substrate binding site in the three-dimensional structure of D-carbamoylase. Molecular dynamics simulations showed that the binding pocket of the enzyme in complex with N-carbamoyl-D-tryptophan was stabilized within 100 nanoseconds. The free energy data showed that Arg176 and Asn173 formed hydrogen bonds between the enzyme and substrates. The studies of D-carbamoylases and the properties of our previously obtained D-hydantoinase suggest the possibility of developing a harmonized biotechnological process for the production of new drugs and peptide hormones. Full article

Ethyl (S)-4-chloro-3-hydroxybutyrate ((S)-CHBE) is an important chiral intermediate in the synthesis of the cholesterol-lowering drug atorvastatin. Studying the use of SpyTag/SpyCatcher and SnoopTag/SnoopCatcher systems for the asymmetric reduction reaction and directed coupling coenzyme regeneration is practical for efficiently synthesizing (S)-CHBE. In this study, Spy and Snoop systems were used to construct a double-enzyme directed fixation system of carbonyl reductase (BsCR) and glucose dehydrogenase (BsGDH) for converting 4-chloroacetoacetate (COBE) to (S)-CHBE and achieving coenzyme regeneration. We discussed the enzymatic properties of the immobilized enzyme and the optimal catalytic conditions and reusability of the double-enzyme immobilization system. Compared to the free enzyme, the immobilized enzyme showed an improved optimal pH and temperature, maintaining higher relative activity across a wider range. The double-enzyme immobilization system was applied to catalyze the asymmetric reduction reaction of COBE, and the yield of (S)-CHBE reached 60.1% at 30 °C and pH 8.0. In addition, the double-enzyme immobilization system possessed better operational stability than the free enzyme, and maintained about 50% of the initial yield after six cycles. In summary, we show a simple and effective strategy for self-assembling SpyCatcher/SnoopCatcher and SpyTag/SnoopTag fusion proteins, which inspires building more cascade systems at the interface. It provides a new method for facilitating the rapid construction of in vitro immobilized multi-enzyme complexes from crude cell lysate. Full article

The emergence and spread of drug-resistant Plasmodium falciparum parasites shed a serious concern on the worldwide control of malaria, the most important tropical disease in terms of mortality and morbidity. This situation has led us to consider the use of peptide-alkoxyamine derivatives as new antiplasmodial prodrugs that could potentially be efficient in the fight against resistant malaria parasites. Indeed, the peptide tag of the prodrug has been designed to be hydrolysed by parasite digestive proteases to afford highly labile alkoxyamines drugs, which spontaneously and instantaneously homolyse into two free radicals, one of which is expected to be active against P. falciparum. Since the parasite enzymes should trigger the production of the active drug in the parasite’s food vacuoles, our approach is summarized as “to dig its grave with its fork”. However, despite promising sub-micromolar IC₅₀ values in the classical chemosensitivity assay, more in-depth tests evidenced that the anti-parasite activity of these compounds could be due to their cytostatic activity rather than a truly anti-parasitic profile, demonstrating that the antiplasmodial activity cannot be based only on measuring antiproliferative activity. It is therefore imperative to distinguish, with appropriate tests, a genuinely parasiticidal activity from a cytostatic activity. Full article

Our previous research has indicated that bioactive protein hydrolysates derived from porcine by-products possess the potential to be utilized in the production of functional additives and food supplements. The objective of this investigation was to assess the antioxidant and antimicrobial characteristics and amino acid changes in hydrolysates of lyophilized meat of bovine livers and hearts. The relevant enzymes, papain and pepsin, were used to hydrolyze the meat by-products over periods of 3, 6, and 24 h. The antimicrobial properties of all enzymatically digested samples were assessed against Listeria monocytogenes, Bacillus cereus, Salmonella enterica subsp. enterica Serovar Typhimurium. Bacillus cereus, and Escherichia coli, S. aureus subsp. aureus. The assessment of antiradical activity involved the quantification of DPPH• and ABTS•+ absorbance in bovine by-product hydrolysates. The hydrolysates were subjected to amino acid analysis using AccQ Tag technology, which was performed by Waters Corporation in Milford, MA, USA. The bacteria L. monocytogenes had the highest antibacterial activity (inhibition zone) (20.00 ± 0.20 mm) and less against E. coli (10.00 ± 0.10 mm) of bovine heart hydrolysates and were prepared for 24 h with papain. The highest values of ABTS•+ (98.1 ± 0.30%) and of DPPH• scavenging activity (92.56 ± 0.56%) of cationic radicals were evaluated in the bovine liver hydrolysates after the effect of papain for 24 h. Longer hydrolysis time influenced the decrease in free hydrophobic amino acids (Ala, Val, Ile, Leu, Tyr, Phe, Pro, Met). The results confirmed the potential use of bovine liver and heart hydrolysates as functional or biologically active materials. Full article

Virus-specific antibodies are crucial for protective immunity against SARS-CoV-2. Assessing functional antibodies through conventional or pseudotyped virus neutralisation tests (pVNT) requires high biosafety levels. Alternatively, the virus-free surrogate virus neutralisation test (sVNT) quantifies antibodies interfering with spike binding to angiotensin-converting enzyme 2. We evaluated secreted nanoluciferase-tagged spike protein fragments as diagnostic antigens in the sVNT in a vaccination cohort. Initially, spike fragments were tested in a capture enzyme immunoassay (EIA), identifying the receptor binding domain (RBD) as the optimal diagnostic antigen. The sensitivity of the in-house sVNT applying the nanoluciferase-labelled RBD equalled or surpassed that of a commercial sVNT (cPass, GenScript Diagnostics) and an in-house pVNT four weeks after the first vaccination (98% vs. 94% and 72%, respectively), reaching 100% in all assays four weeks after the second and third vaccinations. When testing serum reactivity with Omicron BA.1 spike, the sVNT and pVNT displayed superior discrimination between wild-type- and variant-specific serum reactivity compared to a capture EIA. This was most pronounced after the first and second vaccinations, with the third vaccination resulting in robust, cross-reactive BA.1 construct detection. In conclusion, utilising nanoluciferase-labelled antigens permits the quantification of SARS-CoV-2-specific inhibitory antibodies. Designed as flexible modular systems, the assays can be readily adjusted for monitoring vaccine efficacy. Full article

Mdx mice with a spontaneous mutation in exon 23 of the Dmd gene represent the most common model to investigate the pathophysiology of Duchenne muscular dystrophy (DMD). The disease, caused by the lack of functional dystrophin, is characterized by irreversible impairment of muscle functions, with the diaphragm affected earlier and more severely than other skeletal muscles. We applied a label-free (LF) method and the more thorough tandem mass tag (TMT)-based method to analyze differentially expressed proteins in the diaphragm of 6-week-old mdx mice. The comparison of both methods revealed 88 commonly changed proteins. A more in-depth analysis of the TMT-based method showed 953 significantly changed proteins, with 867 increased and 86 decreased in dystrophic animals (q-value < 0.05, fold-change threshold: 1.5). Consequently, several dysregulated processes were demonstrated, including the immune response, fibrosis, translation, and programmed cell death. Interestingly, in the dystrophic diaphragm, we found a significant decrease in the expression of enzymes generating hydrogen sulfide (H₂S), suggesting that alterations in the metabolism of this gaseous mediator could modulate DMD progression, which could be a potential target for pharmacological intervention. Full article

Galactooligosaccharides obtained via β-galactosidase transgalactosylation have health-promoting properties and are widely recognized as effective prebiotics. Trehalose-based galactooligosaccharides could be introduced into food and pharmaceutical industries similarly to trehalose. In light of this, new technological approaches are needed. Recently, in vivo enzyme immobilizations for recombinant proteins have been introduced, and physiological aggregation into active inclusion bodies (aIBs) has emerged as one such method of in vivo immobilization. To prepare LacZ β-galactosidase in the form of aIBs, we used a short 10 amino acid aggregation-prone tag. These native protein particles were simply washed from the cell lysate and applied in trehalose galactosylation in a recycled batch mode. In this study, aIBs entrapped in alginate beads, encapsulated in alginate/cellulose sulfate/poly(methylene-co-guanidine) capsules and magnetized were compared with free aIBs. Alginate/cellulose sulfate/PMCG capsules showed more suitable properties and applicability for biotransformation of trehalose at its high concentration (25%, w/v) and elevated temperature (50 °C). Full article