Yazar "Kayili, Haci Mehmet" seçeneğine göre listele

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    Exploring the Activity of a Novel N-Glycosidase (EndoBI-2): Recombinant Production to Release Bioactive Glycans
    (Mdpi, 2025) Duman, Hatice; Avci, Izzet; Salih, Bekir; Kayili, Haci Mehmet; Bechelany, Mikhael; Karav, Sercan
    The gut microbiome evolves in response to host development, health state, lifestyle, nutrition, and microbial interactions. The survival of gut microbiota depends on its ability to utilize its host-indigestible complex oligosaccharides. Certain gut microbes produce glycosidases that cleave N-glycoproteins to release N-glycans that are then used as a carbon source. However, commercial glycosidases are inefficient and, thus, require improved deglycosylation strategies to study their functions and scale up their production. Therefore, the main objective of this study was to recombinantly produce and characterize the novel endo-beta-N-acetylglucosaminidase 2 (EndoBI-2) from Bifidobacterium longum subsp. infantis (B. infantis) and to evaluate its enzymatic performance for controlled N-glycan release. Furthermore, the optimum reaction conditions for EndoBI-2 were investigated on model glycoprotein RNAse B using model glycoprotein. The released N-glycans were profiled by hydrophilic interaction liquid chromatography-fluorescence detection-quadrupole time-of-flight tandem mass spectrometry (HILIC-FLD-QTOF-MS/MS). We demonstrated that EndoBI-2 possesses a strong temperature tolerance and efficiently cleaves N-glycans under mild reaction conditions, exhibiting high activity at pH 5. These findings highlight EndoBI-2 as a robust and efficient biocatalyst for the production of bioactive N-glycans from diverse N-glycoproteins, with potential applications in glycobiotechnology.
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    Immobilization of a Bifidobacterial Endo-ss-N-Acetylglucosaminidase to Generate Bioactive Compounds for Food Industry
    (Frontiers Media Sa, 2022) Pekdemir, Burcu; Duman, Hatice; Arslan, Aysenur; Kaplan, Merve; Karyelioglu, Melda; ozer, Tolgahan; Kayili, Haci Mehmet
    Conjugated N-glycans are considered next-generation bioactive prebiotic compounds due to their selective stimulation of beneficial microbes. These compounds are glycosidically attached to proteins through N-acetylglucosamines via specific asparagine residue (AsN-X-Ser/Thr). Certain bacteria such as Bifidobacterium longum subspecies infantis (B. infantis) have been shown to be capable of utilizing conjugated N-glycans, owing to their specialized genomic abilities. B. infantis possess a unique enzyme, Endo-ss-N-acetylglucosaminidase (EndoBI-1), which cleaves all types of conjugated N-glycans from glycoproteins. In this study, recombinantly cloned EndoBI-1 enzyme activity was investigated using various immobilization methods: 1) adsorption, 2) entrapment-based alginate immobilization, 3) SulfoLink-, and 4) AminoLink-based covalent bonding immobilization techniques were compared to develop the optimum application of EndoBI-1 to food processes. The yield of enzyme immobilization and the activity of each immobilized enzyme by different approaches were investigated. The N-glycans released from lactoperoxidase (LPO) using different immobilized enzyme forms were characterized using MALDI-TOF mass spectrometry (MS). As expected, regardless of the techniques, the enzyme activity decreased after the immobilization methods. The enzyme activity of adsorption and entrapment-based alginate immobilization was found to be 71.55% +/- 0.6 and 20.32% +/- 3.18, respectively, whereas the activity of AminoLink- and SulfoLink-based covalent bonding immobilization was found to be 58.05 +/- 1.98 and 47.49% +/- 0.30 compared to the free form of the enzyme, respectively. However, extended incubation time recovery achieved activity similar to that of the free form. More importantly, each immobilization method resulted in the same glycan profile containing 11 different N-glycan structures from a model glycoprotein LPO based on MALDI-TOF MS analysis. The glycan data analysis suggests that immobilization of EndoBI-1 is not affecting the enzyme specificity, which enables full glycan release without a limitation. Hence, different immobilization methods investigated in this study can be chosen for effective enzyme immobilization to obtain bioactive glycans. These findings highlight that further optimization of these methods can be a promising approach for future processing scale-up and commercialization of EndoBI-1 and similar enzymes.
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    Proteomic and N-glycomic comparison of synthetic and bovine whey proteins and their effect on human gut microbiomes in vitro
    (Amer Soc Microbiology, 2025) Bolino, Matthew; Duman, Hatice; Avci, Izzet; Kayili, Haci Mehmet; Petereit, Juli; Zundel, Chandler; Salih, Bekir
    Advances in food production systems and customer acceptance have led to the commercial launch of dietary proteins produced via modern biotechnological approaches as alternatives to traditional agricultural sources. At the same time, a deeper understanding of how dietary components interact with the gut microbiome has highlighted the importance of understanding the nuances underpinning diet-microbiome interactions. Novel food proteins with distinct post-translational modifications resulting from their respective production systems have not been characterized, nor how they may differ from their traditionally produced counterparts. Here, we have characterized the protein composition and N-glycome of a yeast-synthesized and commercially available whey protein ingredient and compared this novel ingredient to whey protein isolate powder derived from bovine milk. Despite strong similarities in protein composition, we found that the N-glycome significantly differs between the two protein sources, reflecting the biosynthetic machinery of the production systems. Furthermore, the diversity of proteins found in yeast-synthesized whey protein were lower relative to bovine whey protein, despite both being predominantly beta-lactoglobulin. Finally, to understand whether these differences in N-glycome profiles may affect the human gut microbiome, we compared these proteins in an in vitro fecal fermentation model. The two whey protein sources generated significant differences among three representative gut microbiomes in vitro, most likely due to differences in N-glycan composition and degradation by these representative microbial communities. This work highlights the need to understand how differences in novel biotechnological systems affect the bioactivity of synthesized proteins and how these differences impact the human gut microbiome.IMPORTANCERecent advances in food technology have led to the production of animal-free products from yeast that are traditionally derived from animals, such as milk proteins. These new processes raise important questions about the use of synthetic proteins as a replacement for traditionally sourced protein, especially in the context of the gut microbiome. Importantly, yeast produce N-glycans comprised primarily of mannose, while animals synthesize structurally and compositionally complex N-glycan structures. Given these differences, we characterized a new, yeast-derived whey protein ingredient and compared it to bovine whey protein. We found that yeast-derived whey protein differs in its impact on human gut microbiomes because of differences in N-glycan structures, despite similarity in protein composition. These findings raise important questions as to whether these differences in synthetic proteins lead to significant changes to the gut microbiome in vivo, and whether this may impact the utility of these novel ingredients.