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Journal of Agricultural and Food... May 2024In the development of biomaterials with specific structural domains associated with various cellular activities, the limited integrin specificity of commonly used...
In the development of biomaterials with specific structural domains associated with various cellular activities, the limited integrin specificity of commonly used adhesion sequences, such as the RGD tripeptide, has resulted in an inability to precisely control cellular responses. To overcome this limitation, we conducted multiple replications of the integrin αβ-specific ligand, the collagen hexapeptide Gly-Phe-Pro-Gly-Glu-Arg (GFPGER) in . This enabled the development of recombinant collagen with high biological activity, which was subsequently expressed, isolated, and purified for structural and functional analysis. The proteins carrying the multiple replications GFPGER sequence demonstrated significant bioactivity in cells, leading to enhanced cell adhesion, osteoblast differentiation, and mineralization when compared to control groups. Importantly, these effects were mediated by integrin αβ. The new collagen constructed in this study is expected to be a biomaterial for regulating specific cell functions and fates.
PubMed: 38801678
DOI: 10.1021/acs.jafc.4c00582 -
Brazilian Journal of Microbiology :... May 2024Pediocin PA-1, an antimicrobial peptide derived from Pediococcus acidilactici PAC1.0, has a potential application as a food preservative thanks to its strong inhibitory...
OBJECTIVE
Pediocin PA-1, an antimicrobial peptide derived from Pediococcus acidilactici PAC1.0, has a potential application as a food preservative thanks to its strong inhibitory activity against the foodborne pathogen L. monocytogenes. This study aimed to produce Pediocin PA-1 from the yeast P. pastoris and evaluate its characteristics.
METHODS
Gene encoding Pediocin PA-1 was integrated into P. pastoris X33 genome to establish the strain X33::ped, which could produce and secrete this peptide into culture medium. The antimicrobial activity of Pediocin PA-1 was examined using agar diffusion assay. The stability of pediocin PA-1 was determined based on its remaining antibacterial activity after exposure to proteases and extreme pH and temperatures. The potential use of this bacteriocin in food preservation was demonstrated using the L. monocytogenes infected pork bologna. The anticancer activity of Pediocin PA-1 was also investigated on some cancer cells using MTT assay.
RESULTS
We established the yeast P. pastoris X33::ped capable of producing pediocin PA-1 with antimicrobial activity against L. monocytogenes and some other harmful bacteria. Pediocin PA-1 was stable at 100˚C and resistant against pH 1-12 for 1 h, but susceptible to trypsin, α-chymotrypsin, and proteinase K. This peptide could reduce the number of L. monocytogenes in pork bologna by 3.59 log CFU/g after 7 days of storage at 4˚C. Finally, Pediocin PA-1 (25 µg/ml) inhibited the proliferation of A549 and Hela cancer cells.
CONCLUSION
We succeeded in producing active Pediocin PA-1 from P. pastoris and demonstrated its potential use in food preservation and pharmaceutical industry.
PubMed: 38801640
DOI: 10.1007/s42770-024-01388-w -
Biotechnology Journal May 2024Human carboxypeptidase B1 (hCPB1) is vital for recombinant insulin production, holding substantial value in the pharmaceutical industry. Current challenges include...
High-activity recombinant human carboxypeptidase B expression in Pichia pastoris through rational protein engineering and enhancing secretion from the Golgi apparatus to the plasma membrane.
Human carboxypeptidase B1 (hCPB1) is vital for recombinant insulin production, holding substantial value in the pharmaceutical industry. Current challenges include limited hCPB1 enzyme activity. In this study, recombinant hCPB1 efficient expression in Pichia pastoris was achieved. To enhance hCPB1 secretion, we conducted signal peptides screening and deleted the Vps10 sortilin domain, reducing vacuolar mis-sorting. Overexpression of Sec4p increased the fusion of secretory vesicles with the plasma membrane and improved hCPB1 secretion by 20%. Rational protein engineering generated twenty-two single-mutation mutants and identified the A178L mutation resulted in a 30% increase in hCPB1 specific activity. However, all combinational mutations that increased specific activities decreased protein expression levels. Therefore, computer-aided global protein design with PROSS was employed for the aim of improving specific activities and preserving good protein expression. Among the six designed mutants, hCPB1-P6 showed a remarkable 114% increase in the catalytic rate constant (k), a 137% decrease in the Michaelis constant (K), and a 490% increase in catalytic efficiency. Most mutations occurred on the surface of hCPB1-P6, with eight sites mutated to proline. In a 5 L fermenter, hCPB1-P6 was produced by the secretion-enhanced P. pastoris chassis to 199.6 ± 20 mg L with a specific activity of 96 ± 0.32 U mg, resulting in a total enzyme activity of 19137 ± 1131 U L, demonstrating significant potential for industrial applications.
Topics: Humans; Recombinant Proteins; Protein Engineering; Carboxypeptidase B; Cell Membrane; Golgi Apparatus; Saccharomycetales; Mutation; Pichia; Protein Sorting Signals; Protein Transport
PubMed: 38797728
DOI: 10.1002/biot.202400098 -
Critical Reviews in Biotechnology May 2024Owing to the unmet demand, the pharmaceutical industry is investigating an alternative host to mammalian cells to produce antibodies for a variety of therapeutic and... (Review)
Review
Owing to the unmet demand, the pharmaceutical industry is investigating an alternative host to mammalian cells to produce antibodies for a variety of therapeutic and research applications. Regardless of some disadvantages, and are the preferred microbial hosts for antibody production. Despite the fact that the production of full-length antibodies has been successfully demonstrated in , which has mostly been used to produce antibody fragments, such as: antigen-binding fragments (Fab), single-chain fragment variable (scFv), and nanobodies. In contrast, , a eukaryotic microbial host, is mostly used to produce glycosylated full-length antibodies, though hypermannosylated glycan is a major challenge. Advanced strategies, such as the introduction of human-like glycosylation in endotoxin-edited . and cell-free system-based glycosylation, are making progress in creating human-like glycosylation profiles of antibodies in these microbes. This review begins by explaining the structural and functional requirements of antibodies and continues by describing and analyzing the potential of and as hosts for providing a favorable environment to create a fully functional antibody. In addition, authors compare these microbes on certain features and predict their future in antibody production. Briefly, this review analyzes, compares, and highlights and as potential hosts for antibody production.
PubMed: 38797692
DOI: 10.1080/07388551.2024.2342969 -
Journal of Photochemistry and... Jul 2024In this study, for the first time, red LED light radiation was applied to the fermentation process of table olives using the Negrinha de Freixo variety. Photostimulation...
In this study, for the first time, red LED light radiation was applied to the fermentation process of table olives using the Negrinha de Freixo variety. Photostimulation using LED light emission (630 ± 10 nm) is proposed to shorten and speed up this stage and reduce time to market. Several physical-chemical characteristics and microorganisms (total microbial count of mesophilic aerobic, molds, yeasts, and lactic acid bacteria) and their sequence during fermentation were monitored. The fermentation occurred for 122 days, with two irradiation periods for red LED light. The nutritional composition and sensory analysis were performed at the end of the process. Fermentation under red LED light increased the viable yeast and lactic acid bacteria (LAB) cell counts and decreased the total phenolics in olives. Even though significant differences were observed in some color parameters, the hue values were of the same order of magnitude and similar for both samples. Furthermore, the red LED light did not play a relevant change in the texture profile, preventing the softening of the fruit pulp. Similarly, LED light did not modify the existing type of microflora but increased species abundance, resulting in desirable properties and activities. The species identified were yeasts - Candida boidinii, Pichia membranifaciens, and Saccharomyces cerevisiae, and bacteria - Lactobacillus plantarum and Leuconostoc mesenteroides, being the fermentative process dominated by S. cerevisiae and L. plantarum. At the end of fermentation (122 days), the irradiated olives showed less bitterness and acidity, higher hardness, and lower negative sensory attributes than non-irradiated. Thus, the results of this study indicate that red LED light application can be an innovative technology for table olives production.
Topics: Olea; Fermentation; Light; Yeasts; Phenols; Fruit; Food Microbiology
PubMed: 38795655
DOI: 10.1016/j.jphotobiol.2024.112945 -
Sensors (Basel, Switzerland) May 2024This paper proposes a novel soft sensor modeling approach, MIC-TCA-INGO-LSSVM, to address the decline in performance of soft sensor models during the fermentation...
This paper proposes a novel soft sensor modeling approach, MIC-TCA-INGO-LSSVM, to address the decline in performance of soft sensor models during the fermentation process of , caused by changes in working conditions. Initially, the transfer component analysis (TCA) method is utilized to minimize the differences in data distribution across various working conditions. Subsequently, a least squares support vector machine (LSSVM) model is constructed using the dataset adapted by TCA, and strategies for improving the northern goshawk optimization (INGO) algorithm are proposed to optimize the parameters of the LSSVM model. Finally, to further enhance the model's generalization ability and prediction accuracy, considering the transfer of knowledge from multiple-source working conditions, a sub-model weighted ensemble scheme is proposed based on the maximum information coefficient (MIC) algorithm. The proposed soft sensor model is employed to predict cell and product concentrations during the fermentation process of . Simulation results indicate that the of the INGO-LSSVM model in predicting cell and product concentrations is reduced by 47.3% and 42.1%, respectively, compared to the NGO-LSSVM model. Additionally, TCA significantly enhances the model's adaptability when working conditions change. Moreover, the soft sensor model based on TCA and the MIC-weighted ensemble method achieves a reduction of 41.6% and 31.3% in the for predicting cell and product concentrations, respectively, compared to the single-source condition transfer model TCA-INGO-LSSVM. These results demonstrate the high reliability and predictive performance of the proposed soft sensor method under varying working conditions.
Topics: Fermentation; Algorithms; Support Vector Machine; Least-Squares Analysis; Pichia; Saccharomycetales
PubMed: 38793872
DOI: 10.3390/s24103017 -
Microorganisms May 2024Palm kernel cake (PKC), a byproduct of palm oil extraction, serves an important role in Ecuador's animal feed industry. The emergence of yellow-orange fungal growth in...
Palm kernel cake (PKC), a byproduct of palm oil extraction, serves an important role in Ecuador's animal feed industry. The emergence of yellow-orange fungal growth in PKC on some cattle farms in Ecuador sparked concerns within the cattle industry regarding a potential mycotoxin-producing fungus on this substrate. Due to the limited availability of analytical chemistry techniques in Ecuador for mycotoxin detection, we chose to isolate and identify the fungus to determine its association with mycotoxin-producing genera. Through molecular identification via ITS region sequencing, we identified the yellow-orange fungus as the yeast . Furthermore, we isolated two other fungi-the yeast , and the fungus Molecular identification confirmed that all three species are not classified as mycotoxin-producing fungi but in contrast, the literature indicates that all three have demonstrated antifungal activity against and species, genera associated with mycotoxin production. This suggests their potential use in biocontrol to counter the colonization of harmful fungi. We discuss preventive measures against the fungal invasion of PKC and emphasize the importance of promptly identifying fungi on this substrate. Rapid recognition of mycotoxin-producing and pathogenic genera holds the promise of mitigating cattle intoxication and the dissemination of mycotoxins throughout the food chain.
PubMed: 38792767
DOI: 10.3390/microorganisms12050937 -
Microorganisms May 2024Because data on the fungal gut community structure of African children are scarce, we aimed to describe it by reanalysing rRNA ITS1 and ITS2 metabarcoding data from a...
Because data on the fungal gut community structure of African children are scarce, we aimed to describe it by reanalysing rRNA ITS1 and ITS2 metabarcoding data from a study designed to assess the influence of microbiota in malaria susceptibility in Malian children from the Dogon country. More specifically, we aimed to establish the core gut mycobiome and compare the gut fungal community structure of breastfed children, aged 0-2 years, with other age groups. Briefly, DNA was extracted from 296 children's stool samples. Both rRNA ITS1 and ITS2 genomic barcodes were amplified and subjected to Illumina MiSeq sequencing. The ITS2 barcode generated 1,975,320 reads and 532 operational taxonomic units (OTUs), while the ITS1 barcode generated 647,816 reads and 532 OTUs. The alpha diversity was significantly higher by using the ITS1 compared to the ITS2 barcode ( < 0.05); but, regardless of the ITS barcode, we found no significant difference between breastfed children, aged 0-2 years, compared to the other age groups. The core gut mycobiome of the Malian children included , , , , and section , which were present in at least 50% of the 296 children. Further studies in other African countries are warranted to reach a global view of African children's core gut mycobiome.
PubMed: 38792756
DOI: 10.3390/microorganisms12050926 -
International Journal of Molecular... May 2024Mycoses are one of the major causes of morbidity/mortality among immunocompromised individuals. Considering the importance of these infections, the World Health...
Mycoses are one of the major causes of morbidity/mortality among immunocompromised individuals. Considering the importance of these infections, the World Health Organization (WHO) defined a priority list of fungi for health in 2022 that include as belonging to the critical priority group and () to the medium priority group. The existence of few available antifungal drugs, their high toxicity, the acquired fungal resistance, and the appearance of new species with a broader spectrum of resistance, points out the need for searching for new antifungals, preferably with new and multiple mechanisms of action. The cyclam salt H[H(PhCH)Cyclam]Cl was previously tested against several fungi and revealed an interesting activity, with minimal inhibitory concentration (MIC) values of 8 µg/mL for and of 128 µg/mL for . The main objective of the present work was to deeply understand the mechanisms involved in its antifungal activity. The effects of the cyclam salt on yeast metabolic viability (resazurin reduction assay), yeast mitochondrial function (JC-1 probe), production of reactive oxygen species (DCFH-DA probe) and on intracellular ATP levels (luciferin/luciferase assay) were evaluated. H[H(PhCH)Cyclam]Cl induced a significant decrease in the metabolic activity of both and , an increase in Reactive Oxygen Species (ROS) production, and an impaired mitochondrial function. The latter was observed by the depolarization of the mitochondrial membrane and decrease in ATP intracellular levels, mechanisms that seems to be involved in the antifungal activity of H[H(PhCH)Cyclam]Cl. The interference of the cyclam salt with human cells revealed a CC value against HEK-293 embryonic kidney cells of 1.1 μg/mL and a HC value against human red blood cells of 0.8 μg/mL.
Topics: Antifungal Agents; Candida albicans; Humans; Reactive Oxygen Species; Candida; Microbial Sensitivity Tests; Mitochondria; Membrane Potential, Mitochondrial; Pichia
PubMed: 38791254
DOI: 10.3390/ijms25105209 -
Journal of Biotechnology Jul 2024To reduce food spoilage and deterioration caused by microbial contamination, antimicrobial peptides (AMPs) have gradually gained attention as a biological preservative....
To reduce food spoilage and deterioration caused by microbial contamination, antimicrobial peptides (AMPs) have gradually gained attention as a biological preservative. Odorranain-C1 is an α-helical cationic antimicrobial peptide extracted from the skin of frogs with broad-spectrum antimicrobial activity. In this study, we achieved the expression of Odorranain-C1 in Pichia pastoris (P. pastoris) (also known as Komagataella phaffii) by employing DNA recombination technology. The recombinant Odorranain-C1 showed broad-spectrum antibacterial activity and displayed a minimum inhibitory concentration within the range of 8-12 μg.mL. Meanwhile, Odorranain-C1 exhibited superior stability and lower hemolytic activity. Mechanistically, Odorranain-C1 disrupted the bacterial membrane's integrity, ultimately causing membrane rupture and subsequent cell death. In tilapia fillets preservation, Odorranain-C1 inhibited the total colony growth and pH variations, while also reducing the production of total volatile basic nitrogen (TVB-N) and thiobarbituric acid (TBA). In conclusion, these studies demonstrated the efficient recombinant expression of Odorranain-C1 in P. pastoris, highlighting its promising utilization in food preservation.
Topics: Animals; Saccharomycetales; Food Preservation; Microbial Sensitivity Tests; Antimicrobial Cationic Peptides; Recombinant Proteins; Antimicrobial Peptides; Anti-Bacterial Agents; Hemolysis; Pichia; Amphibian Proteins; Anura
PubMed: 38789049
DOI: 10.1016/j.jbiotec.2024.05.009