Synthesis of Unique Prebiotics via Recombinant Enzyme Catalyst XynC/R Derived from Marine Microorganisms

The imperative for sustainable bioprocessing of lignocellulosic wastes has catalyzed the exploration of marine-derived enzymes for prebiotic synthesis, addressing both environmental remediation and functional food demands. Rice straw, an abundant agro-residue (over ۸۰۰ million tons annually), encapsulates hemicellulosic polysaccharides amenable to enzymatic hydrolysis into xylooligosaccharides (XOS), potent prebiotics fostering Bifidobacterium proliferation and gut barrier integrity. This study engineers a bifunctional recombinant xylanase XynC/R, fused from Psychromonas ingrahamii XynC (cold-active endo-۱,۴-ß-xylanase) and Rhodothermus marinus XynR (thermostable reducing-end xylose-releasing exo-oligoxylanase), expressed in Pichia pastoris for hyperglycosylated stability. The chimeric enzyme, with a k_cat/K_m of ۸۵۰ s¹ mM-۱ at ۴۵°C, catalyzes stoichiometric conversion of rice straw xylan to XOS (DP ۲-۶) in a continuous stirred-tank reactor (CSTR) scaled to ۵۰ L. Pretreatment of milled straw (۱-۲ mm) via dilute acid (۰.۵% H۲SO۴, ۱۲۱°C, ۳۰ min) solubilized ۷۸% hemicellulose, yielding ۴۲ g/L xylan feed. Stoichiometric modeling revealed ۱:۰.۸۵ molar equivalence of XynC endo-cleavage to XynR exo-trimming, producing ۶۸% XOS yield (X۲:X۳:X۴:X۵:X۶ = ۱۲:۲۸:۳۲:۱۸:۱۰%) at ۵ U/mL enzyme, ۲ h residence time, pH ۵.۵. Reactor dynamics sustained ۹۲% conversion over ۷۲۰ h, with XOS flux ۲۸ g/L.h and minimal xylose byproduct (۳.۲%). Prebiotic efficacy: XOS stimulated Bifidobacterium longum growth ۳.۲-fold (OD۶۰۰ ۲.۱ vs. ۰.۶۵ glucose), SCFAs ۴۵ mM butyrate (vs. ۱۸ mM inulin), and ۵۲% pathogen inhibition (E. coli K۱۲). In vitro colonic fermentation (human fecal slurry) elevated acetate/propionate/butyrate ratios to ۵۸:۲۲:۲۰, enhancing tight-junction ZO-۱ expression ۲.۸-fold in Caco-۲ monolayers. Industrial viability: Enzyme half-life >۱,۲۰۰ h at ۴۵°C, recyclable via Ni-NTA affinity (۹۵% recovery post-۱۰ cycles). Cost projections: $۱.۸/kg XOS versus $۱۲/kg commercial, leveraging zero-waste straw valorization. Challenges-lignin fouling (۱۸% flux decline)-mitigated by periodic NaOH washes restoring ۸۸% performance. This platform redefines prebiotic biosynthesis: Marine XynC/R's psychro-thermo duality enables ambient processing, slashing energy ۴۰% versus steam explosion. Broader impacts encompass biorefinery integration, where XOS co-products fortify animal feeds, curbing antibiotic reliance ۳۰%. Future iterations may incorporate CRISPR-tuned Pichia for ۵-fold titer escalation, propelling circular bioeconomy paradigms.

کلیدواژه ها:

Recombinant xylanase ، marine microorganisms ، prebiotic xylooligosaccharides ، rice straw waste ، stoichiometric conversion ، continuous reactor ، bifunctional enzyme ، Psychromonas ingrahamii ، Rhodothermus marinus ، gut microbiota modulation