Reciprocal sharing of extracellular proteases and extracellular matrix molecules facilitates Bacillus subtilis biofilm formation.

Extracellular proteases are a class of public good that support growth of Bacillus subtilis when nutrients are in a polymeric form. Bacillus subtilis biofilm matrix molecules are another class of public good that are needed for biofilm formation and are prone to exploitation. In this study, we investigated the role of extracellular proteases in B. subtilis biofilm formation and explored interactions between different public good producer strains across various conditions. We confirmed that extracellular proteases support biofilm formation even when glutamic acid provides a freely available nitrogen source. Removal of AprE from the NCIB 3610 secretome adversely affects colony biofilm architecture, while sole induction of WprA activity into an otherwise extracellular protease-free strain is sufficient to promote wrinkle development within the colony biofilm. We found that changing the nutrient source used to support growth affected B. subtilis biofilm structure, hydrophobicity and architecture. We propose that the different phenotypes observed may be due to increased protease dependency for growth when a polymorphic protein presents the sole nitrogen source. We however cannot exclude that the phenotypic changes are due to alternative matrix molecules being made. Co-culture of biofilm matrix and extracellular protease mutants can rescue biofilm structure, yet reliance on extracellular proteases for growth influences population coexistence dynamics. Our findings highlight the intricate interplay between these two classes of public goods, providing insights into microbial social dynamics during biofilm formation across different ecological niches.

A TrmBL2-like transcription factor mediates the growth phase-dependent expression of halolysin SptA in a concentration-dependent manner in Natrinema gari J7-2.

To cope with a high-salinity environment, haloarchaea generally employ the twin-arginine translocation (Tat) pathway to transport secretory proteins across the cytoplasm membrane in a folded state, including Tat-dependent extracellular subtilases (halolysins) capable of autocatalytic activation. Some halolysins, such as SptA of Natrinema gari J7-2, are produced at late-log phase to prevent premature enzyme activation and proteolytic damage of cellular proteins in haloarchaea; however, the regulation mechanism for growth phase-dependent expression of halolysins remains largely unknown. In this study, a DNA-protein pull-down assay was performed to identify the proteins binding to the 5'-flanking sequence of sptA encoding halolysin SptA in strain J7-2, revealing a TrmBL2-like transcription factor (NgTrmBL2). The ΔtrmBL2 mutant of strain J7-2 showed a sharp decrease in the production of SptA, suggesting that NgTrmBL2 positively regulates sptA expression. The purified recombinant NgTrmBL2 mainly existed as a dimer although monomeric and higher-order oligomeric forms were detected by native-PAGE analysis. The results of electrophoretic mobility shift assays (EMSAs) showed that NgTrmBL2 binds to the 5'-flanking sequence of sptA in a non-specific and concentration-dependent manner and exhibits an increased DNA-binding affinity with the increase in KCl concentration. Moreover, we found that a distal cis-regulatory element embedded in the neighboring upstream gene negatively regulates trmBL2 expression and thus participates in the growth phase-dependent biosynthesis of halolysin SptA. IMPORTANCE: Extracellular proteases play important roles in nutrient metabolism, processing of functional proteins, and antagonism of haloarchaea, but no transcription factor involved in regulating the expression of haloaechaeal extracellular protease has been reported yet. Here we report that a TrmBL2-like transcription factor (NgTrmBL2) mediates the growth phase-dependent expression of an extracellular protease, halolysin SptA, of haloarchaeon Natrinema gari J7-2. In contrast to its hyperthermophilic archaeal homologs, which are generally considered to be global transcription repressors, NgTrmBL2 functions as a positive regulator for sptA expression. This study provides new clues about the transcriptional regulation mechanism of extracellular protease in haloarchaea and the functional diversity of archaeal TrmBL2.

Halocin H4 is activated through cleavage by halolysin HlyR4.

Halocins are antimicrobial peptides secreted by haloarchaea capable of inhibiting the growth of other haloarchaea or bacteria. Halocin H4 (HalH4) is secreted by the model halophilic archaeon Haloferax mediterranei ATCC 33500. Despite attempts to express halH4 heterologously in Escherichia coli and subsequent careful renaturation procedures commonly employed for haloarchaeal proteins, no active halocin was obtained. However, it was discovered that the antihaloarchaeal activity of this halocin could be activated through cleavage by halolysin R4 (HlyR4), a serine protease also secreted by Hfx. mediterranei ATCC 33500. Replacement of the cysteine at the number 115 amino acid with glycine and deletion of the internal trans-membrane region (15 aa) markedly abolished HalH4's antihaloarchaeal activity. Compared to the N-terminus, the C-terminal amino acid sequence was found to be more crucial for HalH4 to exert its antihaloarchaeal activity. Mass spectrometry analysis revealed that the biologically active antihaloarchaeal peptide produced after hydrolytic cleavage by HlyR4 was the C-terminus of HalH4, suggesting a potential mechanism of action involving pore formation within competitor species' cell membranes. Taken together, this study offers novel insights into the interplay between halocins and secreted proteases, as well as their contribution to antagonistic interaction within haloarchaea. IMPORTANCE: The antihaloarchaeal function of halocin H4 (HalH4) can be activated by extracellular proteases from haloarchaea, as demonstrated in this study. Notably, we report the first instance of halocin activation through proteolytic cleavage, highlighting its significance in the field. The C-terminus of HalH4 (CTH4) has been identified as the antihaloarchaeal peptide present in hydrolysates generated by HlyR4. The CTH4 exhibited inhibitory activity against a range of haloarchaeal species (Haloarchaeobius spp., Haloarcula spp., Haloferax spp., Halorubellus spp., and Halorubrum spp.), as well as selected bacterial species (Aliifodinibius spp. and Salicola spp.), indicating its broad-spectrum inhibitory potential across domains. The encoding gene of halocin HalH4, halH4, from the model halophilic archaeon Haloferax mediterranei ATCC 33500 can be expressed in Escherichia coli without codon optimization.

An alkaline protease from Bacillus cereus NJSZ-13 can act as a pathogenicity factor in infection of pinewood nematode.

Endophytic bacteria are an important biological control for nematodes. We isolated the nematicidal Bacillus cereus NJSZ-13 from healthy Pinus elliottii trunks. Bioassay experiments showed killing of all tested nematodes by proteins from the NJSZ-13 culture filtrate within 72 h. Degradation of the nematode cuticles was observed, suggesting the action of extracellular bacterial enzymes. The responsible protease was purified by ammonium sulfate precipitation, hydrophobic interaction chromatography, ion-exchange chromatography, and SDS-PAGE. The protease had a molecular weight of 28 kDa and optimal activity at 55 °C and pH 9, indicating an alkaline protease. The study suggests the potential for using this B. cereus NJSZ-13 strain protease to prevent pinewood nematode infection.

Novel Antioxidant Peptides Identified from Arthrospira platensis Hydrolysates Prepared by a Marine Bacterium Pseudoalteromonas sp. JS4-1 Extracellular Protease.

Crude enzymes produced by a marine bacterium Pseudoalteromonas sp. JS4-1 were used to hydrolyze phycobiliprotein. Enzymatic productions showed good performance on DPPH radical and hydroxyl radical scavenging activities (45.14 ± 0.43% and 65.11 ± 2.64%, respectively), especially small peptides with MWCO <3 kDa. Small peptides were fractioned to four fractions using size-exclusion chromatography and the second fraction (F2) had the highest activity in hydroxyl radical scavenging ability (62.61 ± 5.80%). The fraction F1 and F2 both exhibited good antioxidant activities in oxidative stress models in HUVECs and HaCaT cells. Among them, F2 could upregulate the activities of SOD and GSH-Px and reduce the lipid peroxidation degree to scavenge the ROS to protect Caenorhabditis elegans under adversity. Then, 25 peptides total were identified from F2 by LC-MS/MS, and the peptide with the new sequence of INSSDVQGKY as the most significant component was synthetized and the ORAC assay and cellular ROS scavenging assay both illustrated its excellent antioxidant property.

A novel halolysin without C-terminal extension from an extremely halophilic archaeon.

Halolysins are extracellular proteases secreted by halophilic archaea for nutritional purposes. They bear great application potentials in various industries. Yet the diversity of halolysins remains underexplored. In this study, a halolysin from the extremely halophilic archaeon Haladaptatus sp. DYF46 (HlyHap) was identified to be a novel type of halolysin without C-terminal extension (CTE). Addition of the CTE of a halolysin from Halococcus salifodinae to HlyHap did not significantly affect its extracellular proteolytic activity. Mature HlyHap was generated from recombinant HlyHap precursor by high-affinity column refolding. HlyHap displayed optimal activity at 0.25-0.50 M NaCl, 45 °C and pH 8.5-9.0. Interestingly, HlyHap preferred a low salinity and was stable in a broad range of salinity, albeit from an extremely halophilic archaeon. Ca2+ and Mg2+ significantly promoted HlyHap activity. HlyHap activity was stable with organic solvents and detergents. The Km and Vmax values of HlyHap against azocasein were 0.018 mM and 7,179 U/mg, and those against succinyl-Ala-Ala-Pro-Phe-pNA were 0.32 mM and 3×106 µmol/min/µg, respectively. The unusual traits of HlyHap, a novel type of halolysin without CTE, may endow it with strong potential for various industrial uses, such as biocatalysis in fluctuating salinities and aqueous-organic solvent. KEY POINTS: • This is the first report of a novel type of halolysin without C-terminal extension • HlyHap was obtained by heterologous expression and high-affinity column refolding • HlyHap exhibited good salinity tolerance.

LuxS modulates motility and secretion of extracellular protease in fish pathogen Vibrio harveyi.

Vibrio harveyi can cause infections and diseases in a variety of marine vertebrates and invertebrates, which are harmful to the aquaculture industry. The LuxS quorum-sensing system regulates the expression of virulence factors in a wide variety of pathogenic bacteria. In this study, an in-frame deletion of the luxS gene was constructed to reveal the role of LuxS in the physiology and virulence of V. harveyi. Statistical analysis showed no significant differences in the growth ability, biofilm formation, antibiotic susceptibility, virulence by intraperitoneal injection, and ability of V. harveyi to colonize the spleen and liver of the pearl gentian grouper between the wild-type (WT) and luxS mutant. However, deletion of luxS decreased the secretion of extracellular protease, while increasing swimming and swarming abilities. Simultaneously, a luxS-deleted mutant showed overproduction of lateral flagella, and an intact luxS complemented this defect. Since motility is flagella dependent, 16 V. harveyi flagella biogenesis related genes were selected for further analysis. Based on quantitative real-time reverse transcription-PCR (qRT-PCR), the expression levels of these genes, including the polar flagella genes flaB, flhA, flhF, flhB, flhF, fliS, and flrA and the lateral flagella genes flgA, flgB, fliE, fliF, lafA, lafK, and motY, were significantly upregulated in the ΔluxS: pMMB207 (ΔluxS+) strain as compared with the V. harveyi 345: pMMB207 (WT+) and C-ΔluxS strains during the early, mid-exponential, and stationary growth phases. Our results indicate that LuxS plays an important role in controlling motility, flagella biogenesis, and extracellular protease secretion in V. harveyi.

Study on spoilage potential and its molecular basis of Shewanella putrefaciens in response to cold conditions by Label-free quantitative proteomic analysis.

To elucidate how Shewanella putrefaciens survives and produces spoilage products in response to cold conditions, the metabolic and protease activity of S. putrefaciens DSM6067 cultured at three different temperatures (30 °C, 10 °C, and 4 °C) was studied by determining the bacterial growth, total volatile basic nitrogen (TVB-N), biogenic amines, extracellular protease activity, as well as the differential expressed proteins via Label-free quantitative proteomics analysis. The lag phase of the strain cultured at 10 °C and 4 °C was about 20 h and 120 h longer than at 30 °C, respectively. The TVB-N increased to 89.23 mg N/100 g within 28 h at 30 °C, and it needed at least 72 h and 224 h at 10 °C and 4 °C, respectively. Cold temperatures (10 °C and 4 °C) also inhibited the yield factors and the extracellular protease activity per cell at the lag phase. However, the protease activity per cell and the yield factors of the sample cultivated at 10 °C and 4 °C well recovered, especially at the mid and latter stages of the log phase. The further quantitative proteomic analysis displayed a complex biological network to tackle cold stress: cold stress responses, nutrient uptake, and energy conservation strategy. It was observed that the protease and peptidase were upregulated, so as to the degradation pathways of serine, arginine, and aspartate, which might lead to the accumulation of spoilage products. This study highlighted the spoilage potential of S. putrefaciens still should be concerned even at low temperatures.

Investment in secreted enzymes during nutrient-limited growth is utility dependent.

Pathogenic bacteria secrete toxins and degradative enzymes that facilitate their growth by liberating nutrients from the environment. To understand bacterial growth under nutrient-limited conditions, we studied resource allocation between cellular and secreted components by the pathogenic bacterium Pseudomonas aeruginosa during growth on a protein substrate that requires extracellular digestion by secreted proteases. We identified a quantitative relationship between the rate of increase of cellular biomass under nutrient-limiting growth conditions and the rate of increase in investment in secreted proteases. Production of secreted proteases is stimulated by secreted signals that convey information about the utility of secreted proteins during nutrient-limited growth. Growth modeling using this relationship recapitulated the observed kinetics of bacterial growth on a protein substrate. The proposed regulatory strategy suggests a rationale for quorum-sensing-dependent stimulation of the production of secreted enzymes whereby investment in secreted enzymes occurs in proportion to the utility they confer. Our model provides a framework that can be applied toward understanding bacterial growth in many environments where growth rate is limited by the availability of nutrients.

Comprehensive analysis of the phylogeny and extracellular proteases in genus Vibrio strain.

As one of the dominant bacteria in the ocean, Vibrio play important roles in maintaining the aquatic ecosystem. In this study, we studied the phylogenetic relationships of 32 Vibrio based on the 16S rRNA genes sequences and utilized substrate immersing zymography method to detect the trend of protease production and components of multiprotease system of Vibrio extracellular proteases. The result showed that different extracellular proteolytic profiles among various Vibrio strains demonstrated a large interspecific variation, and for strains from the same environments, the closer the evolutionary relationship of them, the more similar their zymograms were. In addition, these proteases displayed very different hydrolysis abilities to casein and gelatin. Moreover, the results of the inhibitor-substrate immersing zymography indicated that the proteases secreted by marine Vibrio mostly belonged to serine proteases or metalloproteases. These results implied that combined taxonomic information of the Vibrios with their extracellular protease zymograms maybe contributed to the study of the classification, phylogeny and pathogenic mechanism of Vibrio, and can serve as a theoretical basis for controlling the pathogenic Vibrio disease as well as exploiting proteases. More importantly, we can also eliminate many similar strains by this way, thus can greatly reduce the workload of the experiments for us.

Understanding the regulation of extracellular protease gene expression in fungi: a key step towards their biotechnological applications.

The secretion of proteases by certain species of yeast and filamentous fungi is of importance not only for their biological function and survival, but also for their biotechnological application to various processes in the food, beverage, and bioprocessing industries. A key step towards understanding the role that these organisms play in their environment, and how their protease-secreting ability may be optimally utilised through industrial applications, involves an evaluation of those factors which influence protease production. The objective of this review is to provide an overview of the findings from investigations directed at elucidating the regulatory mechanisms underlying extracellular protease secretion in yeast and filamentous fungi, and the environmental stimuli that elicit these responses. The influence of nitrogen-, carbon-, and sulphur-containing compounds, as well as proteins, temperature, and pH, on extracellular protease regulation, which is frequently exerted at the transcriptional level, is discussed in particular depth. Protease-secreting organisms of biotechnological interest are also presented in this context, in an effort to explore the areas of industrial significance that could possibly benefit from such knowledge. In this way, the establishment of a platform of existing knowledge regarding fungal protease regulation is attempted, with the particular goal of aiding in the practical application of these organisms to processes that require secretion of this enzyme.

Impact of curcumin liposomes with anti-quorum sensing properties against foodborne pathogens Aeromonas hydrophila and Serratia grimesii.

Quorum sensing (QS) is a signal sensing system by which bacteria monitor the density of their population, thus, targeting to QS system may be an alternative approach to control diverse cellular processes including the microbial contamination and the virulence of human and plant pathogens as bacteria have been evolving resistance to antibiotics. In this study, curcumin liposomes were prepared by film dispersion method to increase its bioavailability against QS systems of food-borne epathogenic bacteria, i.e. Aeromonas hydrophila and Serratia grimesii. And their physicochemical properties were measured including particle size, zeta-potential entrapment rate as well as drug-loading rate. Meanwhile, the effects of curcumin liposomes on QS phenomenon of food-borne pathogens including biofilm formation, extracellular protease, swimming motility et al. were determined. Results showed that the average diameter of curcumin liposomes was (207 ±â€¯8.2) nm and the entrapment rate and drug-loading rate were 82.71% and 23.33%. The zeta-potential of curcumin liposomes were (-37 ±â€¯1.56) mv. Curcumin liposomes showed uniform structures by scanning electron microscopy (SEM). The curcumin liposomes could significantly inhibit QS systems of the two pathogens and the biological availability of curcumin has been improved.

The extracellular proteases produced by Vibrio parahaemolyticus.

Vibrio parahaemolyticus, a Gram-negative bacterium, inhabits marine and estuarine environments and it is a major pathogen responsible globally for most cases of seafood-associated gastroenteritis in humans and acute hepatopancreatic necrosis syndrome in shrimps. There has been a dramatic worldwide increase in V. parahaemolyticus infections over the last two decades. The pathogenicity of V. parahaemolyticus has been linked to the expression of different kinds of virulence factors including extracellular proteases, such as metalloproteases and serine proteases. V. parahaemolyticus expresses the metalloproteases; PrtV, VppC, VPM and the serine proteases; VPP1/Protease A, VpSP37, PrtA. Extracellular proteases have been identified as potential virulence factors which directly digest many kinds of host proteins or indirectly are involved in the processing of other toxic protein factors. This review summarizes findings on the metalloproteases and serine proteases produced by V. parahaemolyticus and their roles in infections. Identifying the role of V. parahaemolyticus virulence-associated extracellular proteases deepens our understanding of diseases caused by this bacterium.

Lepidopteran species have a variety of defence strategies against bacterial infections.

The insect immune system has versatile ways of coping with microbial insults. Currently, innate immune priming has been described in several invertebrates, and the first insights into its mechanistic basis have been described. Here we studied infections with two different strains of Serratia marcescens bacteria in two different Lepidopteran hosts. The results reveal fundamental differences between the two hosts, a well-known model organism Galleria mellonella and a non-model species Arctia plantaginis. They differ in their strategies for resisting oral infections; priming their defences against a recurring sepsis; and upregulating immunity related genes as a response to the specific pathogen strains. The two bacterial strains (an environmental isolate and an entomopathogenic isolate) differ in their virulence, use of extracellular proteases, survival in the larval gut, and in the immune response they evoke in the hosts. This study explores the potential mechanistic explanations for both host and pathogen specific characters that significantly affect the outcome of Gram-negative bacterial infection in Lepidopteran larvae. The results highlight the need to pay greater attention to the differences between model and non-model hosts, and closely related pathogen strains, in immunological studies.

Construction of Aeromonas salmonicida subsp. achromogenes AsaP1-toxoid strains and study of their ability to induce immunity in Arctic char, Salvelinus alpinus L.

The metalloendopeptidase AsaP1 is one of the major extracellular virulence factors of A. salmonicida subsp. achromogenes, expressed as a 37-kDa pre-pro-peptide and processed to a 19-kDa active peptide. The aim of this study was to construct mutant strains secreting an AsaP1-toxoid instead of AsaP1-wt, to study virulence of these strains and to test the potency of the AsaP1-toxoid bacterin and the recombinant AsaP1-toxoids to induce protective immunity in Arctic char. Two A. salmonicida mutants were constructed that secrete either AsaP1E294A or AsaP1Y309F . The secreted AsaP1Y309F -toxoid had weak caseinolytic activity and was processed to the 19-kDa peptide, whereas the AsaP1E294A -toxoid was found as a 37-kDa pre-pro-peptide suggesting that AsaP1 is auto-catalytically processed. The LD50 of the AsaP1Y309F -toxoid mutant in Arctic char was significantly higher than that of the corresponding wt strain, and LD50 of the AsaP1E294A -toxoid mutant was comparable with that of an AsaP1-deficient strain. Bacterin based on AsaP1Y309F -toxoid mutant provided significant protection, comparable with that induced by a commercial polyvalent furunculosis vaccine. Detoxification of AsaP1 is very hard, expensive and time consuming. Therefore, an AsaP1-toxoid-secreting mutant is more suitable than the respective wt strain for production of fish bacterins aimed to protect against atypical furunculosis.

A hyperstable, low-salt adapted protease from halophilic archaeon with potential applications in salt-fermented foods.

Salt-tolerant proteases with remarkable stability are highly desirable biocatalysts in the salt-fermented food industry. In this study, the undigested autocleavage product of HlyA (halolysin A), a low-salt adapted halolysin from halophilic archaeon Halococcus salifodinae, was investigated. HlyA underwent autocleavage of its C-terminal extension (CTE) at temperatures over 40 °C or NaCl concentrations below 2 M to yield HlyAΔCTE. HlyAΔCTE demonstrated robust stability over a wide range of -20-60 °C, 0.5-4 M NaCl, and pH 6.0-10.0 for at least 72 h. Notably, HlyAΔCTE is the first reported halolysin with such exceptional stability. Compared with HlyA, HlyAΔCTE preferred high temperatures (50-75 °C), low salinities (0.5-2.5 M NaCl), and near-neutral (pH 6.5-8.0) conditions to achieve high activity, consistently with its production conditions. HlyAΔCTE displayed a higher Vmax value against azocasein than HlyA. During fish sauce fermentation, HlyAΔCTE significantly enhanced fish protein hydrolysis, indicating its potential as a robust biocatalyst in the salt-fermented food industry.

DegQ is an important policing link between quorum sensing and regulated adaptative traits in Bacillus subtilis.

Quorum sensing (QS) is a widespread bacterial communication system that controls important adaptive traits in a cell density-dependent manner. However, mechanisms by which QS-regulated traits are linked within the cell and mechanisms by which these links affect adaptation are not well understood. In this study, Bacillus subtilis was used as a model bacterium to investigate the link between the ComQXPA QS system, DegQ, surfactin and protease production in planktonic and biofilm cultures. The work tests two alternative hypotheses predicting that hypersensitivity of the QS signal-deficient mutant (comQ::kan) to exogenously added ComX, resulting in increased surfactin production, is linked to an additional genetic locus, or alternatively, to overexpression of the ComX receptor ComP. Results are in agreement with the first hypothesis and show that the P srfAA hypersensitivity of the comQ::kan mutant is linked to a 168 strain-specific mutation in the P degQ region. Hence, the markerless ΔcomQ mutant lacking this mutation is not overresponsive to ComX. Such hyper-responsiveness is specific for the P srfAA and not detected in another ComX-regulated promoter, the P aprE , which is under the positive control by DegQ. Our results suggest that DegQ by exerting differential effect on P srfAA and P aprE acts as a policing mechanism and the intracellular link, which guards the cell from an overinvestment into surfactin production. IMPORTANCE DegQ levels are known to regulate surfactin synthesis and extracellular protease production, and DegQ is under the control of the ComX-dependent QS. DegQ also serves as an important policing link between these QS-regulated processes, preventing overinvestment in these costly processes. This work highlights the importance of DegQ, which acts as the intracellular link between ComX production and the response by regulating extracellular degradative enzyme synthesis and surfactin production.

Expression, purification, and enzymatic characterization of an extracellular protease from Halococcus salifodinae.

Extracellular proteases from halophilic archaea displays increased enzymatic activities in hypersaline environment. In this study, an extracellular protease-coding gene, hly34, from the haloarchaeal strain Halococcus salifodinae PRR34, was obtained through homologous search. The protease activity produced by this strain at 20% NaCl, 42 °C, and pH 7.0 was 32.5 ± 0.5 (U·mL-1). The codon-optimized hly34 which is specific for Escherichia coli can be expressed in E. coli instead of native hly34. It exhibits proteolytic activity under a wide range of low- or high-salt concentrations, slightly acidic or alkaline conditions, and slightly higher temperatures. The Hly34 presented the highest proteolytic activity at 50 °C, pH 9.0, and 0-1 M NaCl. It was found that the Hly34 showed a higher enzyme activity under low-salt conditions. Hly34 has good stability at different NaCl concentrations (1-4 M) and pH (6.0-10.0), as well as good tolerance to some metal ions. However, at 60 °C, the stability is reduced. It has a good tolerance to some metal ions. The proteolytic activity was completely inhibited by phenylmethanesulfonyl fluoride, suggesting that the Hly34 is a serine protease. This study further deepens our understanding of haloarchaeal extracellular protease, most of which found in halophilic archaea are classified as serine proteases. These proteases exhibit a certain level of alkaline resistance and moderate heat resistance, and they may emerge with higher activity under low-salt conditions than high-salt conditions. The protease Hly34 is capable of degrading a number of proteins, including substrate proteins, such as azocasein, whey protein and casein. It has promising applications in industrial production.

Purification of Extracellular Protease from Staphylococcus simulans QB7and Its Ability in Generating Antioxidant and Anti-inflammatory Peptides from Meat Proteins.

Proteases, especially microbial proteases, are widely used in food processing. The purpose of this study was aimed to purify an extracellular protease produced by the strain Staphylococcus simulans QB7 and to evaluate its ability in hydrolyzing meat proteins and generating antioxidant and anti-inflammatory peptides. The optimal conditions for producing the enzyme were as follows: inoculum ratio, 10%; initial pH, 6.5; temperature, 32 °C; incubation time, 36 h; and rotation speed, 160 rpm. The protease had a molecular weight of approximately 47 kDa, possessing the optimal activity at 50 °C, pH 7.0, The protease was stable at pH 4.0-8.0 and 30-60 °C, and the activity was improved by Na+, Mg2+, Ca2+, and Zn2+ ions, whereas it was inhibited by Cu2+, Co2+, Fe3+, Ba2+, Fe2+, ß-M, and ethylene diamine tetraacetic acid disodium salt (EDTA). The protease could effectively hydrolyze meat proteins, and the generated hydrolysate could significantly inhibit tumor necrosis factor-alpha (TNFα)-induced oxidative stress, including superoxide and malondialdehyde levels and inflammation (vascular adhesion molecule-1 [VCAM-1] and cyclooxygenase 2 [COX2)) in human vascular EA.hy926 cells. The present findings support the ability of S. simulans QB7 protease in generating antioxidant and anti-inflammatory peptides during the fermentation of meat products.

Genomic Analysis of Two Representative Strains of Shewanella putrefaciens Isolated from Bigeye Tuna: Biofilm and Spoilage-Associated Behavior.

Shewanella putrefaciens can cause the spoilage of seafood and shorten its shelf life. In this study, both strains of S. putrefaciens (YZ08 and YZ-J) isolated from spoiled bigeye tuna were subjected to in-depth phenotypic and genotypic characterization to better understand their roles in seafood spoilage. The complete genome sequences of strains YZ08 and YZ-J were reported. Unique genes of the two S. putrefaciens strains were identified by pan-genomic analysis. In vitro experiments revealed that YZ08 and YZ-J could adapt to various environmental stresses, including cold-shock temperature, pH, NaCl, and nutrient stresses. YZ08 was better at adapting to NaCl stress, and its genome possessed more NaCl stress-related genes compared with the YZ-J strain. YZ-J was a higher biofilm and exopolysaccharide producer than YZ08 at 4 and 30 °C, while YZ08 showed greater motility and enhanced capacity for biogenic amine metabolism, trimethylamine metabolism, and sulfur metabolism compared with YZ-J at both temperatures. That YZ08 produced low biofilm and exopolysaccharide contents and displayed high motility may be associated with the presence of more a greater number of genes encoding chemotaxis-related proteins (cheX) and low expression of the bpfA operon. This study provided novel molecular targets for the development of new antiseptic antisepsis strategies.