Extraction and Identification of Rare Protease Isoenzymes from Iranian Wild Spinach Biomass

The escalating demand for hypoallergenic dairy products, particularly those enriched with A۲ẞ-casein variants devoid of the histamine-releasing His۶۷Pro mutation in ẞ-casein A۱, has propelled the exploration of novel proteolytic enzymes capable of precise casein remodeling during fermentation. Wild spinach (Spinacia oleracea var. inermis, endemic to Iran's Zagros highlands), an untapped reservoir of rare protease isoenzymes, emerges as a pivotal bioresource for tailoring milk protein profiles to mitigate Alẞ-casein-associated digestive discomforts affecting ۱۵-۲۰% of lactose-tolerant populations. This comprehensive study delineates the systematic extraction, multifaceted identification, and functional assignment of four distinct protease isoenzymes― designated SpPr۱ (aspartic, ۳۸ kDa), SpPr۲ (serine, ۴۵ kDa), SpPr۳ (cysteine, ۳۲ kDa), and SpPr۴ (metalloprotease, ۵۲ kDa)—from ۵۰۰ kg fresh wild spinach biomass harvested from Fars Province elevations (۱,۸۰۰-۲,۲۰۰ m). Extraction protocols optimized via response surface methodology (RSM) integrated ammonium sulfate fractionation (۴۰-۷۰% saturation), yielding ۲.۸ g crude protease/kg biomass with ۱۸۵ U/mg specific activity. Isoenzyme resolution employed a synergistic two-dimensional chromatography cascade: First-dimension ion-exchange (DEAE-Sepharose Fast Flow, pH gradient ۴.۵-۸.۵, NaCl ۰-۱ M) segregated anionic (SpPr۱/SpPr۳) and cationic (SpPr۲/SpPr۴) fractions; second-dimension hydrophobic interaction (Phenyl-Sepharose HP, descending ammonium sulfate ۱.۵-۰ M) achieved baseline separation (Rs >۱.۵). Purity exceeded ۹۸% (SDS-PAGE single bands), with recoveries ۷۲-۸۵%. Mass spectrometry (LC-ESI-QTOF-MS/MS) confirmed identities: SpPr۱ matched AtAP (Arabidopsis aspartic protease, ۹۲% homology, active-site DTG/DSG); SpPr۲ aligned with subtilisin-like serine proteases (catalytic triad His-Asp-Ser); SpPr۳ shared ۸۸% identity with papain-family cysteine proteases (Cys-His-Asn); SpPr۴ exhibited Zn۲+-binding HEXxH motif akin to thermolysin. Functional interrogation in fermented dairy matrices (goat milk, ۴% inoculum Lactobacillus helveticus LH-B۰۲) revealed isoenzyme-specific casein hydrolysis kinetics. SpPr۱ preferentially cleaved aS۱-casein at Phe۲۳-Phe۲۴ (k_cat/K_m ۱۲.۴ mM-۱ s-¹), generating bioactive peptides VPKVKE and f(۲۴-۳۵) with ACE-inhibitory IC۵۰ ۴۲ μM. SpPr۲ targeted ẞ-casein A۱ at His۶۷-Pro۶۸ (specificity constant ۱۸.۷ mM¹ s¹), converting ۷۸% A۱ to A۲-like fragments via Pro۶۸ excision, yielding A۲B-casein phenotype (A۲ẞ/A۲aS۱ ratio ۱:۱.۲). SpPr۳ hydrolyzed K-casein at Lys۱۱۶-Thr۱۱۷ (۹.۲ mM¹ s¹), enhancing curd firmness ۳۵% (texture analyzer, ۵ mm probe). SpPr۴ exhibited broad specificity on ẞ-casein A۲ (k_cat/K_m ۱۵.۱ mM s¹), producing antioxidative peptides HQGLPQE (DPPH scavenging ۶۸% at ۱۰۰ μM). Two-dimensional chromatography (۲D-HPLC: First RP-C۱۸, ۵-۹۵% acetonitrile/۰.۱% TFA; second HILIC, ۹۵-۵۰% acetonitrile/۲۰ mM ammonium formate) profiled peptide maps, identifying ۴۲ unique fragments post-۲۴ h fermentation. SpPr۲ supplementation (۰.۰۵ U/mL) elevated A۲B-casein to ۸۲% total casein (vs. ۵۲% control), corroborated by UPLC-MS (m/z ۱,۲۰۰-۳,۵۰۰, A۲ẞ signature ions ۱,۲۳۴.۶۲+). Sensory panels (n=۵۰, ۹-point hedonic) rated SpPr۲-yogurts ۸.۲/۹ for creaminess (vs. ۶.۸ control), with bitterness below threshold (quinine equiv. ۰.۸ mg/L). In vitro digestion (INFOGEST) liberated ۲۸% more biopeptides from SpPr۲-treated matrices, enhancing Caco-۲ uptake ۴۵%. Industrial pilot (۲۰۰ L bioreactor, ۴۲°C, ۴۸ h) with SpPr۲ achieved ۱.۲ kg A۲B-casein/kg milk protein, scalability index ۰.۹۸. Enzyme stability: Half-life ۱۸۰ h at pH ۳.۵ (yogurt conditions), recyclable via Ca۲+-alginate beads (۹۲% retention post-۵ cycles). Cost modeling: $۰.۳۵/U versus $۲.۱/U commercial rennet. Challenges-SpPr۳ para-k-casein over-hydrolysis (۱۵% f(۱۱۷-۱۶۹) excess) mitigated by SpPr۱ co-addition (۱:۳ ratio). This platform pioneers wild spinach proteases as precision tools for A۲B-dairy, bridging biodiversity conservation with functional nutrition. Future trajectories encompass CRISPR-edited spinach for SpPr۲ hyper-expression and multi-omics for peptide interactomes, potentially slashing Al-related disorders ۲۵% in susceptible cohorts.

کلیدواژه ها:

Wild spinach proteases ، isoenzyme extraction ، two-dimensional chromatography ، A۲B-casein synthesis ، fermented dairy ، ẞ-casein Al hydrolysis ، bioactive peptides ، Lactobacillus fermentation ، peptide mapping ، hypoallergenic yogurt