A Novel Artificial Hemoglobin Carrier Based on Heulandite-Calcium Mesoporous Aluminosilicate Particles.

Tetraethyl-orthosilicate (TEOS)-based nanoparticles are most extensively used as a silica-based hemoglobin carrier system. However, TEOS-based nanoparticles induce adverse effects on the hemoglobin structure. Therefore, a heulandite-calcium-based carrier was investigated as a novel silica-based hemoglobin carrier system. The heulandite-calcium mesoporous aluminosilicate particles (MSPs) were fabricated by a patented tribo-mechanical activation process, according to the manufacturer, and its structure was assessed by X-ray diffraction analysis. Upon hemoglobin encapsulation, alternation in the secondary and tertiary structure was observed. The hemoglobin-particle interactions do not cause heme degradation or decreased activity. Once encapsulated inside the particle pores, the hemoglobin shows increased thermal stability, and higher loading capacity per gram of particles (by a factor of >1.4) when compared to TEOS-based nanoparticles. Futhermore, we introduced a PEGlyted lipid bilayer which significantly decreases the premature hemoglobin release and increases the colloidal stability. The newly developed hemoglobin carrier shows no cytotoxicity to human umbilical vein endothelial cells (HUVEC).

Diverse Mechanisms of Antimicrobial Activities of Lactoferrins, Lactoferricins, and Other Lactoferrin-Derived Peptides.

Lactoferrins are an iron-binding glycoprotein that have important protective roles in the mammalian body through their numerous functions, which include antimicrobial, antitumor, anti-inflammatory, immunomodulatory, and antioxidant activities. Among these, their antimicrobial activity has been the most studied, although the mechanism behind antimicrobial activities remains to be elucidated. Thirty years ago, the first lactoferrin-derived peptide was isolated and showed higher antimicrobial activity than the native lactoferrin lactoferricin. Since then, numerous studies have investigated the antimicrobial potencies of lactoferrins, lactoferricins, and other lactoferrin-derived peptides to better understand their antimicrobial activities at the molecular level. This review defines the current antibacterial, antiviral, antifungal, and antiparasitic activities of lactoferrins, lactoferricins, and lactoferrin-derived peptides. The primary focus is on their different mechanisms of activity against bacteria, viruses, fungi, and parasites. The role of their structure, amino-acid composition, conformation, charge, hydrophobicity, and other factors that affect their mechanisms of antimicrobial activity are also reviewed.

Basic Methods for Preparation of Liposomes and Studying Their Interactions with Different Compounds, with the Emphasis on Polyphenols.

Studying the interactions between lipid membranes and various bioactive molecules (e.g., polyphenols) is important for determining the effects they can have on the functionality of lipid bilayers. This knowledge allows us to use the chosen compounds as potential inhibitors of bacterial and cancer cells, for elimination of viruses, or simply for keeping our healthy cells in good condition. As studying those effect can be exceedingly difficult on living cells, model lipid membranes, such as liposomes, can be used instead. Liposomal bilayer systems represent the most basic platform for studying those interactions, as they are simple, quite easy to prepare and relatively stable. They are especially useful for investigating the effects of bioactive compounds on the structure and kinetics of simple lipid membranes. In this review, we have described the most basic methods available for preparation of liposomes, as well as the essential techniques for studying the effects of bioactive compounds on those liposomes. Additionally, we have provided details for an easy laboratory implementation of some of the described methods, which should prove useful especially to those relatively new on this research field.

Polysaccharide Hydrogels for the Protection of Dairy-Related Microorganisms in Adverse Environmental Conditions.

Adverse environmental conditions are severely limiting the use of microorganisms in food systems, such as probiotic delivery, where low pH causes a rapid decrease in the survival of ingested bacteria, and mixed-culture fermentation, where stepwise changes and/or metabolites of individual microbial groups can hinder overall growth and production. In our study, model probiotic lactic acid bacteria (L. plantarum ATCC 8014, L. rhamnosus GG) and yeasts native to dairy mixed cultures (K. marxianus ZIM 1868) were entrapped in an optimized (cell, alginate and hardening solution concentration, electrostatic working parameters) Ca-alginate system. Encapsulated cultures were examined for short-term survival in the absence of nutrients (lactic acid bacteria) and long-term performance in acidified conditions (yeasts). In particular, the use of encapsulated yeasts in these conditions has not been previously examined. Electrostatic manufacturing allowed for the preparation of well-defined alginate microbeads (180-260 µm diameter), high cell-entrapment (95%) and viability (90%), and uniform distribution of the encapsulated cells throughout the hydrogel matrix. The entrapped L. plantarum maintained improved viabilities during 180 min at pH 2.0 (19% higher when compared to the free culture), whereas, L. rhamnosus appeared to be less robust. The encapsulated K. marxianus exhibited double product yields in lactose- and lactic acid-modified MRS growth media (compared to an unfavorable growth environment for freely suspended cells). Even within a conventional encapsulation system, the pH responsive features of alginate provided superior protection and production of encapsulated yeasts, allowing several applications in lacto-fermented or acidified growth environments, further options for process optimization, and novel carrier design strategies based on inhibitor charge expulsion.

Polymers and protein-associated vesicles for the microencapsulation of anthocyanins from grape skins used for food applications.

BACKGROUND: Anthocyanins were extracted from grape skins by a combination of ethanolic-ultrasonic assisted methods and were then encapsulated by freeze-drying in soy phosphatidylcholine vesicles with the addition of different polymers, such as pectin, acacia gum, and whey protein isolate. The goal of this research was to microencapsulate anthocyanin compounds extracted from grape skins, to characterize the stability and behavior of the vesicles and then to use them to obtain a new light formulated mayonnaise. RESULTS: The particle size ranged from 900 nm in the control condition to 250 nm in vesicles loaded with whey proteins. The powders showed higher encapsulation efficiency for all variants, ranging from 81 to 96%. Vibrational spectroscopy revealed better inclusion of anthocyanins in polysaccharide-based coacervates, whereas in protein-based coacervates a possible interaction of anthocyanins with amine groups was observed. The vesicles were tested for in vitro release, and the results confirmed the gradual release of the anthocyanins in both stages of digestion, with a residual content of about 50% in the vesicles. The powders displayed high stability during storage in the dark at 4 °C. The panelists appreciated the new light formulated mayonnaises enriched with 10% dried vesicles compared with the control sample, in particular samples with acacia gum. CONCLUSION: The study revealed that polymer-loaded vesicles presented stability in simulated gastrointestinal fluids and have proved successful in obtaining new light enriched mayonnaises. © 2020 Society of Chemical Industry.

Insights into the Maturation of Pernisine, a Subtilisin-Like Protease from the Hyperthermophilic Archaeon Aeropyrum pernix.

Pernisine is a subtilisin-like protease that was originally identified in the hyperthermophilic archaeon Aeropyrum pernix, which lives in extreme marine environments. Pernisine shows exceptional stability and activity due to the high-temperature conditions experienced by A. pernix Pernisine is of interest for industrial purposes, as it is one of the few proteases that has demonstrated prion-degrading activity. Like other extracellular subtilisins, pernisine is synthesized in its inactive pro-form (pro-pernisine), which needs to undergo maturation to become proteolytically active. The maturation processes of mesophilic subtilisins have been investigated in detail; however, less is known about the maturation of their thermophilic homologs, such as pernisine. Here, we show that the structure of pro-pernisine is disordered in the absence of Ca2+ ions. In contrast to the mesophilic subtilisins, pro-pernisine requires Ca2+ ions to adopt the conformation suitable for its subsequent maturation. In addition to several Ca2+-binding sites that have been conserved from the thermostable Tk-subtilisin, pernisine has an additional insertion sequence with a Ca2+-binding motif. We demonstrate the importance of this insertion for efficient folding and stabilization of pernisine during its maturation. Moreover, analysis of the pernisine propeptide explains the high-temperature requirement for pro-pernisine maturation. Of note, the propeptide inhibits the pernisine catalytic domain more potently at high temperatures. After dissociation, the propeptide is destabilized at high temperatures only, which leads to its degradation and finally to pernisine activation. Our data provide new insights into and understanding of the thermostable subtilisin autoactivation mechanism.IMPORTANCE Enzymes from thermophilic organisms are of particular importance for use in industrial applications, due to their exceptional stability and activity. Pernisine, from the hyperthermophilic archaeon Aeropyrum pernix, is a proteolytic enzyme that can degrade infective prion proteins and thus has a potential use for disinfection of prion-contaminated surfaces. Like other subtilisin-like proteases, pernisine needs to mature through an autocatalytic process to become an active protease. In the present study, we address the maturation of pernisine and show that the process is regulated specifically at high temperatures by the propeptide. Furthermore, we demonstrate the importance of a unique Ca2+-binding insertion for stabilization of mature pernisine. Our results provide a novel understanding of thermostable subtilisin autoactivation, which might advance the development of these enzymes for commercial use.

Periplasmic production of pernisine in Escherichia coli and determinants for its high thermostability.

Pernisine is a subtilisin-like serine proteinase secreted by the hyperthermophilic archaeon Aeropyrum pernix. The significant properties of this proteinase are remarkable stability and ability to degrade the infectious prion proteins. Here we show the production of pernisine in the periplasm of Escherichia coli. This strategy prevented the aggregation of pernisine in the cytoplasm and increased the purity of the isolated pernisine. The thermostability of this recombinant pernisine was significantly increased compared with previous studies. In addition, several truncated pernisine variants were constructed and expressed in E. coli to identify the minimally active domain. The catalytic domain of pernisine consists of the αẞα structurally similar core flanked by the N-terminal and C-terminal outer regions. The deletion of the C-terminal α helix did not affect the pernisine activity at 90 °C. However, the complete deletion of the C-terminal outer region resulted in loss of proteolytic activity. The pernisine variant, in which the N-terminal outer region was deleted, had a reduced activity at 90 °C. These results underline the importance of the Ca2+ binding sites predicted in these outer regions for stability and activity of pernisine. KEY POINTS: • Aggregation of produced pernisine was prevented by translocation into periplasm. • Thermostability of mature pernisine was increased. • The outer regions of the catalytic core are required for pernisine thermostability.

Chiroptical Sensing: A Conceptual Introduction.

Chiroptical responses have been an essential tool over the last decades for chemical structural elucidation due to their exceptional sensitivity to geometry and intermolecular interactions. In recent times, there has been an increasing interest in the search for more efficient sensing by the rational design of tailored chiroptical systems. In this review article, advances made in chiroptical systems towards their implementation in sensing applications are summarized. Strategies to generate chiroptical responses are illustrated. Theoretical approaches to assist in the design of these systems are discussed. The development of efficient chiroptical reporters in different states of matter, essential for the implementation in sensing devises, is reviewed. In the last part, remarkable examples of chiroptical sensing applications are highlighted.

Enhanced Yield of Bioactivities from Onion (Allium cepa L.) Skin and Their Antioxidant and Anti-α-Amylase Activities.

There is increasing concern for reduction of the ecological impacts of industrial waste caused by fruits and vegetables. To reduce costs of onion waste disposal while obtaining value-added products, onion skin can be used to extract quercetin, a natural flavonoid with antioxidant, anti-inflammatory and anti-cancer effects. The aim was to optimize quercetin extraction from brown onion (Allium cepa L.) skin through investigation of the effects of different parameters on quercetin yield. Operational parameters for conventional maceration extraction and for ultrasound-assisted extraction were compared: solvent type, mass-to-liquid ratio, extraction time and temperature. Antioxidant capacity was determined using DPPH· radical scavenging assays and quercetin yield using HPLC/DAD. Anti-α-amylase activity of onion skin extracts was investigated using α-amylase inhibition assays. Optimal extraction conditions of quercetin from onion skin were obtained with maceration extraction, 50% ethanol, 1:100 mass-to-liquid ratio, 25 °C, for 15 min. Under these conditions, the antioxidant capacity (expressed as quercetin equivalents) was 18.7 mg/g and the mass fraction of quercetin was 7.96 mg/g. The onion skin extracts showed a dose-dependent relationship between dry extract concentration and α-amylase inhibition, which confirms that this onion skin extract can be considered as an anti-diabetes agent.

Extracellular production of the engineered thermostable protease pernisine from Aeropyrum pernix K1 in Streptomyces rimosus.

BACKGROUND: The thermostable serine protease pernisine originates from the hyperthermophilic Archaeaon Aeropyrum pernix and has valuable industrial applications. Due to its properties, A. pernix cannot be cultivated in standard industrial fermentation facilities. Furthermore, pernisine is a demanding target for heterologous expression in mesophilic heterologous hosts due to the relatively complex processing step involved in its activation. RESULTS: We achieved production of active extracellular pernisine in a Streptomyces rimosus host through heterologous expression of the codon-optimised gene by applying step-by-step protein engineering approaches. To ensure secretion of fully active enzyme, the srT signal sequence from the S. rimosus protease was fused to pernisine. To promote correct processing and folding of pernisine, the srT functional cleavage site motif was fused directly to the core pernisine sequence, this way omitting the proregion. Comparative biochemical analysis of the wild-type and recombinant pernisine confirmed that the enzyme produced by S. rimosus retained all of the desired properties of native pernisine. Importantly, the recombinant pernisine also degraded cellular and infectious bovine prion proteins, which is one of the particular applications of this protease. CONCLUSION: Functional pernisine that retains all of the advantageous properties of the native enzyme from the thermophilic host was successfully produced in a S. rimosus heterologous host. Importantly, we achieved extracellular production of active pernisine, which significantly simplifies further downstream procedures and also omits the need for any pre-processing step for its activation. We demonstrate that S. rimosus can be used as an attractive host for industrial production of recombinant proteins that originate from thermophilic organisms.

White Hop Shoot Production in Slovenia: Total Phenolic, Microelement and Pesticide Residue Content in Five Commercial Cultivars.

Harvesting of white hop shoots might be justified if they can be shown to be beneficial to human health. The aim of the present study is to determine the effects of hop cultivars and year of production on total phenolics, antioxidant potential, microelements and pesticide residues. Biomass per plant was highly variable across the cultivars (3.1-7.1 g dry mass per plant) and depended on hop cultivar and year (2009-2011). Total phenolics as chlorogenic acid equivalents (CAE) on dry mass basis varied from 0.60 to 1.80 mg/g, and showed significant effects across hop cultivar and year. The radical scavenging activities of the samples collected in years 2010-2012 ranged from 11 to 19 µg CAE. Ferric reducing activity was <0.01, with significantly different effects across hop cultivars (pC≤0.05) and year (py≤0.05) observed only in 2012. Traces of microelements and potentially active compounds from the use of pesticides in white hop shoots of Humulus lupulus 'Dana' were analysed. The content of zinc in the hop shoots on dry mass basis was very low (4 mg/kg), and it was below the limit of detection in the soil. The content of copper in the hop shoots was also very low (2.3 mg/kg), while in the soil it was below the critical emission (100 vs 300 mg/kg, respectively). All 182 active ingredients from the residues of the previously used pesticides were below the limits of detection. It can be concluded that these white hop shoots are better antioxidants than hop cones and hop leaves, and that they do not contain any pesticide residues.

An Integrated Characterization of Jujube (Ziziphus jujuba Mill.) Grown in the North Adriatic Region.

Jujube (Ziziphus jujuba Mill.) has favourable horticultural properties including adaptation to arid conditions, abiotic and biotic stresses, as well as positive impact on human health. The present study describes the characterization of genetic diversity of the germplasm of jujube from the Istrian peninsula, the determination of important chemical compounds, antioxidative properties in relation to antibacterial and antifungal activities of jujube fruit extracts, and the determination of nutritional properties of jujube fruit. The results of the genetic analysis showed that most of the samples from the Istrian peninsula belong to two recently introduced varieties, 'Li' and 'Lang', and the most widespread local variety 'Navadna zizola'. The local variety has smaller fruit than the 'Li' and 'Lang' varieties, with thick and fleshy mesocarp. Chemical analysis indicated that fruits of the local variety contained a valuable source of dietary fibre ((9.7±0.6) g/100 g) and were rich in minerals such as (in g/100 g dry mass): potassium (829±51), calcium (177±11) and phosphorus (129±19). Aqueous extracts showed slight antibacterial activity, while ethanol extracts had higher mass fractions of phenolic compounds (expressed as gallic acid equivalents (GAE), 5.8-8.7 mg/g) than the aqueous extracts, but did not show antimicrobial activity. Compounds other than phenolic compounds in jujube fruit may be more biologically active. Based on the results of these analyses, the local Istrian jujube variety is a promising candidate for cultivation potential.

Antioxidative Activity of Methanolic and Water Extracts from the Hyperthermophilic Archaeon Aeropyrum pernix K1.

The hyperthermophilic archaeon Aeropyrum pernix has adapted to optimal growth under high temperatures in saline environments and under oxidizing conditions. In the present study, we focused on the antioxidative activity of proteins from A. pernix K1. Following high temperature methanol and water extractions of the protein from the biomass of A. pernix K1, the total sulphydryl groups and radical scavenging activities were investigated. The total protein in the methanolic extract was 36% lower and showed 10% fewer sulphydryl groups than that from the water extract. However, the radical scavenging activity of the water extract was four-fold greater than for the methanolic extract. The proteins of both of these extracts were separated by two-dimensional electrophoresis, and selected proteins were identified using mass spectrometry. The majority of these identified proteins were intracellular proteins, such as those involved in oxidative stress responses and osmotic stress responses, and proteins with hydrolase and dehydrogenase activities. These proteins are also common to most organisms, and included putative uncharacterized proteins.

Analytical techniques for the study of polyphenol-protein interactions.

This mini review focuses on advances in biophysical techniques to study polyphenol interactions with proteins. Polyphenols have many beneficial pharmacological properties, as a result of which they have been the subject of intensive studies. The most conventional techniques described here can be divided into three groups: (i) methods used for screening (in-situ methods); (ii) methods used to gain insight into the mechanisms of polyphenol-protein interactions; and (iii) methods used to study protein aggregation and precipitation. All of these methods used to study polyphenol-protein interactions are based on modifications to the physicochemical properties of the polyphenols or proteins after binding/complex formation in solution. To date, numerous review articles have been published in the field of polyphenols. This review will give a brief insight in computational methods and biosensors and cell-based methods, spectroscopic methods including fluorescence emission, UV-vis adsorption, circular dichroism, Fourier transform infrared and mass spectrometry, nuclear magnetic resonance, X-ray diffraction, and light scattering techniques including small-angle X-ray scattering and small-angle neutron scattering, and calorimetric techniques (isothermal titration calorimetry and differential scanning calorimetry), microscopy, the techniques which have been successfully used for polyphenol-protein interactions. At the end the new methods based on single molecule detection with high potential to study polyphenol-protein interactions will be presented. The advantages and disadvantages of each technique will be discussed as well as the thermodynamic, kinetic or structural parameters, which can be obtained. The other relevant biophysical experimental techniques that have proven to be valuable, such electrochemical methods, hydrodynamic techniques and chromatographic techniques will not be described here.

Bilberry: Chemical Profiling, in Vitro and in Vivo Antioxidant Activity and Nephroprotective Effect against Gentamicin Toxicity in Rats.

We assessed possible protective effect of bilberry diet in rat model of nephrotoxicity. In vivo and in vitro antioxidant activity and chemical profiling of this functional food was performed. With aid of HPLC-DAD and spectrophotometric method, 15 individual anthocyanins were quantified alongside total tannin, phenylpropanoid, and anthocyanin content. The study was conducted on four groups of rats: control, treated with only gentamicin, treated with only bilberry, and treated with both gentamicin and bilberry. Kidney function was evaluated by tracking urea and creatinine. Morphology of renal tissue and its changes were recorded pathohistologically and quantified morphometrically. Bilberry (100 mg/kg daily) showed strong nephroprotective effect against gentamicin toxicity in rats (as shown through MDA, AOPP, and catalase levels). In conclusion, the demonstrated protective activity of bilberry extract matched well with the assessed in vivo and in vitro antioxidant activity as well as with its polyphenolic content, particularly with high anthocyanin levels. Copyright © 2016 John Wiley & Sons, Ltd.

A Kinetic Approach in the Evaluation of Radical-Scavenging Efficiency of Sinapic Acid and Its Derivatives.

A kinetic approach was used to determine the radical scavenging activities of sinapic acid and its derivatives: sinapine, 4-vinylsyringol, syringic acid, syringaldehyde, and ethyl, propyl and butyl sinapate. The responses were expressed as rates of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH˙) scavenging (RS), superoxide radical (O2˙-) scavenging (RFF), and ß-carotene bleaching in the emulsion system (RB). For RS and RB, the esters of sinapic acid showed the highest responses while, for RFF, this was seen for syringic acid. The effectiveness of the selected compounds for scavenging these free radicals was also determined at a fixed endpoint. The early response parameters were demonstrated to be good discriminators in assessing differences for antioxidants with comparable fixed endpoint activity. The primary feature that ranks the kinetic data and the endpoint determinations is interpreted in terms of the mechanisms of the reactions involved in each of the assays conducted.

Encapsulation of (-)-epigallocatechin gallate into liposomes and into alginate or chitosan microparticles reinforced with liposomes.

BACKGROUND: (-)-Epigallocatechin gallate (EGCG) was encapsulated into liposomes that were further incorporated into alginate and chitosan microparticles. The stability of free and encapsulated EGCG in all three systems was evaluated at different pH values and in fruit nectar. Furthermore, the interactions between EGCG and the compounds of the microparticles were studied using Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). RESULTS: All three encapsulation systems showed high encapsulation efficiency (>97%) and sustained release; in 14 days, no more than 15% of EGCG was released. The encapsulation systems successfully protected EGCG against degradation at alkaline pH. For non-encapsulated EGCG, >70% was degraded after 14 days, while there was no significant degradation of encapsulated EGCG in these three systems. In fruit nectar, >30% of non-encapsulated EGCG was degraded in 14 days, while only 6% of EGCG encapsulated into liposomes or chitosan microparticles reinforced with liposomes was degraded at that time. The DSC and FTIR analyses showed that the main interactions occurred between the liposomes and the EGCG. CONCLUSION: This study demonstrates that liposomes as well as alginate and chitosan microparticles reinforced with liposomes have the potential to enhance EGCG stability in food products during storage. © 2016 Society of Chemical Industry.

The effect of tyrosine residues on α-synuclein fibrillation.

Aggregation of the intrinsically disordered protein α-synuclein into ordered amyloid fibrils is implicated in the pathogenesis of Parkinson's disease. To unravel the role of Tyr residues in α-synuclein fibrillation, we prepared recombinant N-terminal (Y39A) and C-terminal (Y(125,133,136)A) mutants of α-synuclein and examined their fibrillation propensities by thioflavin T and 1-anilinonaphthalene-8-sulfonate (ANS) fluorescent probes, SDS-PAGE and atomic force microscopy. We demonstrate that in contrast to wild-type α-synuclein, both mutants show large, but comparable delays in the fibrillation process and exhibit enhanced hydrophobicity during fibril-like assembly. Both Tyr mutants form fibril-like structures after prolonged incubation periods, which are morphologically distinct from those of the wild-type protein. Our results suggest that the N-terminal and C-terminal Tyr residues of α-synuclein are important primarily for the initiation of the fibrillation process.

Aspects of quercetin stability and its liposomal enhancement in yellow onion skin extracts.

Quercetin is a flavonoid that occurs in many types of fruit and vegetables and is stable for no longer than 4.5 h in the investigated pH range (6.0-8.0), even at 4 °C in the dark. At higher temperatures, the degradation/oxidation process is much faster. Simple but effective proliposomal encapsulation was used to protect the quercetin from environmental conditions such as pH. With this approach, 65 to 90% of pure quercetin and quercetin-rich onion extract was kept after >60 days under conditions that favoured its oxidation (pH 7.4). In addition, the encapsulated quercetin decreases the lipid peroxidation induced by pulsed UV light by >50%. At a mass ratio of 1:100 quercetin to lipids (w/w), the liposomes remained intact in solutions for six months. Quercetin in lipid bilayers simultaneously protects the unsaturated lipids from peroxidation.

High selectivity of the hyperthermophilic subtilase propeptide domain toward inhibition of its cognate protease.

Microbial extracellular subtilases are highly active proteolytic enzymes commonly used in commercial applications. These subtilases are synthesized in their inactive proform, which matures into the active protease under the control of the propeptide domain. In mesophilic bacterial prosubtilases, the propeptide functions as both an obligatory chaperone and an inhibitor of the subtilase catalytic domain. In contrast, the propeptides of hyperthermophilic archaeal prosubtilases act mainly as tight inhibitors and are not essential for subtilase folding. It is unclear whether this stronger inhibitory activity of hyperthermophilic propeptides results in their higher selectivity toward their cognate subtilases, in contrast to promiscuous mesophilic propeptides. Here, we showed that the propeptide of pernisine, a hyperthermostable archaeal subtilase, strongly interacts with and inhibits pernisine, but not the homologous subtilisin Carlsberg and proteinase K. Instead, the pernisine propeptide was readily degraded by subtilisin Carlsberg and proteinase K. In addition, the catalytic domain of unprocessed propernisine was also susceptible to degradation but became proteolytically stable after autoprocessing of propernisine into the inactive, noncovalent complex propeptide:pernisine. This allowed efficient transactivation of the autoprocessed complex propeptide:pernisine through degradation of pernisine propeptide by subtilisin Carlsberg and proteinase K at mesophilic temperature. Moreover, we demonstrated that active pernisine molecules are inhibited by the propeptide that is released after pernisine-catalyzed degradation of the unprocessed propernisine catalytic domain. This highlights the high inhibitory potency of the hyperthermophilic propeptide toward its cognate subtilase and its importance in regulating subtilase maturation, to prevent the degradation of the unprocessed subtilase precursors by the prematurely activated molecules. IMPORTANCE Many microorganisms secrete proteases into their environment to degrade protein substrates for their growth. The important group of these extracellular enzymes are subtilases, which are also widely used in practical applications. These subtilases are inhibited by their propeptide domain, which is degraded during the prosubtilase maturation process. Here, we showed that the propeptide of pernisine, a prion-degrading subtilase from the hyperthermophilic archaeon, strongly inhibits pernisine with extraordinarily high binding affinity. This interaction proved to be highly selective, as pernisine propeptide was rapidly degraded by mesophilic pernisine homologs. This in turn allowed rapid transactivation of propernisine by mesophilic subtilases at lower temperatures, which might simplify the procedures for preparation of active pernisine for commercial use. The results reported in this study suggest that the hyperthermophilic subtilase propeptide evolved to function as tight and selective regulator of maturation of the associated prosubtilase to prevent its premature activation under high temperatures.