S8 Family Research Articles

Structural adaptations for carboxypeptidase activity in putative S9 acylaminoacyl peptidase from Bacillus subtilis

Prolyl oligopeptidase, or S9 (MEROPS) family enzymes are crucial drug targets due to their association with various diseases, neurological disorders, cell growth, and survival. These implications render them an exceptionally fascinating field of research. Despite sharing similar structural features, they exhibit diverse enzyme activities, including endopeptidase, dipeptidyl peptidase, and acylaminoacyl peptidase. Additionally, a few members of the S9 family demonstrate carboxypeptidase activity. A recent study showed that the S9 peptidase of Bacillus subtilis (S9bs) possesses the conserved sequence feature necessary for carboxypeptidase activity despite being annotated as an acylaminoacyl peptidase in the UniProt database. However, the mechanism of action and identity of S9bs as carboxypeptidase remain unclear. Consequently, we focused our studies on thoroughly investigating S9bs for its carboxypeptidase activity. In the present study, we report biochemical and biophysical analyses of S9bs, confirming its identity as a carboxypeptidase. Further, structural analysis reveals the molecular basis of S9bs' carboxypeptidase activity, highlighting the crucial structural elements like the “cavity loop” and the “two-arginine” residues essential for this activity. Additionally, our studies confirmed that S9bs forms a stable tetrameric assembly and established its quaternary molecular arrangement, which reveals the presence of an oligomeric pore. Altogether, these structural features play a are crucial role in substrate selection for S9 carboxypeptidases. Overall, our findings reveal a distinct carboxypeptidase within the S9 family and significantly enhance our understanding of these enzymes. Moreover, this study sheds light on the mechanisms underlying carboxypeptidase activity, offering valuable insights that could contribute to therapeutic and drug design.

Characterization of the extracellular proteases from Bacillus inaquosorum strain E1-8 and its application in the preparation of hydrolysates from plant and animal proteins with antioxidant, antifreeze and anti-browning properties.

Bacillus inaquosorum strains is widely recognized for their plant-growth-promoting and biocontrol capabilities, yet their roles in protease production remain unclear. The present study aimed to comprehensively assess the protease-producing performance of B. inaquosorum strain E1-8, at the same time as exploring the novel application of agricultural Bacillus proteases in the preparation of protein hydrolysates for fresh-cut fruits preservation. First, genomic sequencing revealed the diversity of E1-8 proteases, indicating 15 putative extracellular proteases. Subsequently, the fermentation conditions for E1-8 protease production were optimized, with sweet potato powder and soybean meal identified as the most suitable carbon and nitrogen sources, respectively, resulting in a maximum protease activity of 321.48 U mL-1. Upon culturing the strain under these optimized conditions, only an S8 family serine protease and an M48 family metalloprotease were revealed by secretomic analysis and protease inhibitor assays. Additionally, the optimal protease conditions for generating protein hydrolysates from soy, pea, fish and porcine proteins were determined. The molecular weight of the hydrolysates primarily ranged from 2000 to 180 Da, with a total of 17 amino acids identified. The application of these hydrolysates demonstrated a 2,2-diphenyl-1-picrylhydrazyl (i.e. DPPH) scavenging activity ranging from 58.64% to 84.12%, significantly reducing of the melting peaks and the freezing points. Furthermore, the browning index of apple slices stored at 4 °C decreased by 14.81% to 22.15% on the second day, and similar effects were observed in fresh-cut banana stored at 4 °C for 7 days. The protein hydrolysates obtained exhibit remarkable antioxidant, antifreeze and anti-browning properties for fresh-cut fruits. © 2024 Society of Chemical Industry.

Thermostable Bacterial Collagenolytic Proteases: A Review.

Collagenolytic proteases are widely used in the food, medical, pharmaceutical, cosmetic, and textile industries. Mesophilic collagenases exhibit collagenolytic activity under physiological conditions, but have limitations in efficiently degrading collagen-rich wastes, such as collagen from fish scales, at high temperatures due to their poor thermostability. Bacterial collagenolytic proteases are members of various proteinase families, including the bacterial collagenolytic metalloproteinase M9 and the bacterial collagenolytic serine proteinase families S1, S8, and S53. Notably, the C-terminal domains of collagenolytic proteases, such as the pre-peptidase C-terminal domain, the polycystic kidney disease-like domain, the collagen-binding domain, the proprotein convertase domain, and the β-jelly roll domain, exhibit collagen-binding or -swelling activity. These activities can induce conformational changes in collagen or the enzyme active sites, thereby enhancing the collagen-degrading efficiency. In addition, thermostable bacterial collagenolytic proteases can function at high temperatures, which increases their degradation efficiency since heat-denatured collagen is more susceptible to proteolysis and minimizes the risk of microbial contamination. To date, only a few thermophile-derived collagenolytic proteases have been characterized. TSS, a thermostable and halotolerant subtilisin-like serine collagenolytic protease, exhibits high collagenolytic activity at 60°C. In this review, we present and summarize the current research on A) the classification and nomenclature of thermostable and mesophilic collagenolytic proteases derived from diverse microorganisms, and B) the functional roles of their C-terminal domains. Furthermore, we analyze the cleavage specificity of the thermostable collagenolytic proteases within each family and comprehensively discuss the thermostable collagenolytic protease TSS.

Protease SfpB plays an important role in cell membrane stability and immune system evasion in Streptococcus agalactiae

Bacteria possess the ability to develop diverse and ingenious strategies to outwit the host immune system, and proteases are one of the many weapons employed by bacteria. This study sought to identify S. agalactiae additional serine protease and determine its role in virulence. The S. agalactiae THN0901 genome features one S8 family serine peptidase B (SfpB), acting as a secreted and externally exposed entity. A S8 family serine peptidase mutant strain (ΔsfpB) and complement strain (CΔsfpB) were generated through homologous recombination. Compared to the wild-type strain THN0901, the absorption of EtBr dyes was significantly reduced (P < 0.01) in ΔsfpB, implying an altered cell membrane permeability. In addition, the ΔsfpB strain had a significantly lower survival rate in macrophages (P < 0.01) and a 61.85 % lower adhesion ability to the EPC cells (P < 0.01) compared to THN0901. In the in vivo colonization experiment using tilapia as a model, 210 fish were selected and injected with different bacterial strains at a concentration of 3 × 106 CFU/tail. At 6, 12, 24, 48, 72 and 96 h post-injection, three fish were randomly selected from each group and their brain, liver, spleen, and kidney tissues were isolated. Subsequently, it was demonstrated that the ΔsfpB strain exhibited a markedly diminished capacity for colonization in tilapia. Additionally, the cumulative mortality of ΔsfpB in fish after intraperitoneal injection was reduced by 19.92–23.85 %. In conclusion, the findings in this study have demonstrated that the SfpB plays a significant role in S. agalactiae cell membrane stability and immune evasion. The immune evasion is fundamental for the development and transmission of invasive diseases, the serine protease SfpB may be a promising candidate for the development of antimicrobial agents to reduce the transmission of S. agalactiae.

Bacillus velezensis strain NA16 shows high poultry feather-degrading efficiency, protease and amino acid production

Isolated Bacillus velezensis strain NA16, which produces proteases, amino acids and the transcription levels of different keratinolytic enzymes and disulfide reductase genes in whole gene sequencing, was evaluated during feather degradation. The result shows under optimum fermentation conditions, chicken feather fermentation showed total amino acid concentration of 7599 mg/L, degradation efficiency of 99.3% at 72 h, and protease activity of 1058 U/mL and keratinase activity of 288 U/mL at 48 h. Goose feather fermentation showed total amino acid concentration of 4918 mg/L (96 h), and degradation efficiency was 98.9% at 120 h. Chicken feather fermentation broth at 72 h showed high levels of 17 amino acids, particularly phenylalanine (1050 ± 1.90 mg/L), valine (960 ± 1.04 mg/L), and glutamic (950 ± 3.00 mg/L). Scanning electron microscopy and Fourier transform infrared analysis revealed the essential role of peptide bond cleavage in structural changes and degradation of feathers. Protein purification and zymographic analyses revealed a key role in feather degradation of the 39-kDa protein encoded by gene1031, identified as an S8 family serine peptidase. Whole genome sequencing of NA16 revealed 26 metalloproteinase genes and 22 serine protease genes. Among the proteins, S8 family serine peptidase (gene1031, gene1428) and S9 family peptidase (gene3132) were shown by transcription analysis to play major roles in chicken feather degradation. These findings revealed the transcription levels of different families of keratinolytic enzymes in the degradation of feather keratin by microorganisms, and suggested potential applications of NA16 in feather waste management and amino acid production.

Evolutionary genomics of three agricultural pest moths reveals rapid evolution of host adaptation and immune-related genes.

Understanding the genotype of pest species provides an important baseline for designing integrated pest management (IPM) strategies. Recently developed long-read sequence technologies make it possible to compare genomic features of nonmodel pest species to disclose the evolutionary path underlying the pest species profiles. Here we sequenced and assembled genomes for 3 agricultural pest gelechiid moths: Phthorimaea absoluta (tomato leafminer), Keiferia lycopersicella (tomato pinworm), and Scrobipalpa atriplicella (goosefoot groundling moth). We also compared genomes of tomato leafminer and tomato pinworm with published genomes of Phthorimaea operculella and Pectinophora gossypiella to investigate the gene family evolution related to the pest species profiles. We found that the 3 solanaceous feeding species, P. absoluta, K. lycopersicella, and P. operculella, are clustered together. Gene family evolution analyses with the 4 species show clear gene family expansions on host plant-associated genes for the 3 solanaceous feeding species. These genes are involved in host compound sensing (e.g., gustatory receptors), detoxification (e.g., ABC transporter C family, cytochrome P450, glucose-methanol-choline oxidoreductase, insect cuticle proteins, and UDP-glucuronosyl), and digestion (e.g., serine proteases and peptidase family S1). A gene ontology enrichment analysis of rapid evolving genes also suggests enriched functions in host sensing and immunity. Our results of family evolution analyses indicate that host plant adaptation and pathogen defense could be important drivers in species diversification among gelechiid moths.

Screening Dolosigranulum pigrum Strains for Secreted Factors that InhibitStaphylococcus aureus

Abstract Background Staphylococcus aureus is a leading cause of community-associated and hospital-acquired infections, and methicillin-resistant S. aureus (MRSA) is a significant global health concern. Approximately 30% of the human population is asymptomatically colonized by S. aureus in the upper respiratory tract, with this colonization typically preceding respiratory infections caused by S. aureus. Dolosigranulum pigrum is a putatively beneficial bacterium that is a frequent colonizer of the human upper respiratory tract. The presence and abundance of D. pigrum in the upper respiratory tract has been negatively associated with S. aureus colonization among infants and young children. Additionally, in cocultivation experiments, D. pigrum has been shown to inhibit S. aureus growth, although the mechanisms for this antistaphylococcal activity have not been identified. Methods We hypothesized that D. pigrum strains secrete a protein factor that inhibits growth of S. aureus. To evaluate this hypothesis, we screened D. pigrum strains for antistaphylococcal activity using a plate-based cocultivation assay. Briefly, 5 µL aliquots of D. pigrum strains in liquid brain heart infusion (BHI) medium (OD600=0.3) were spotted onto a BHI agar plate and incubated at 37°C and 5% CO2 for 48 hours. Next, 5 µL aliquots of S. aureus strain Newman in liquid BHI medium (OD600=0.1) were spotted onto this plate at varying distances from the D. pigrum inocula and incubated at 37°C and 5% CO2 for an additional 24 hours. Secretion of an anti-staphylococcal factor was assessed based on the presence of a visible zone of inhibition between the D. pigrum and S. aureus inocula. We excised agar fragments adjacent to 4 inhibitory and 4 non-inhibitory D. pigrum strains and submitted these samples for liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based proteomics. The proteomic data were searched against the TrEMBL D. pigrum database and a common contaminant/spiked protein database, and signal intensity values for identified peptides were quantified. Results Four of 43 (9%) D. pigrum strains screened in cocultivation assays consistently produced a zone of inhibition against S. aureus. We identified 531 unique proteins from proteomic analysis of agar fragments, including 28 D. pigrum proteins that were significantly more abundant in zones adjacent to inhibitory vs. non-inhibitory strains. The most differentially abundant protein was an S8 family serine protease (112 times more abundant in fragments adjacent to inhibitory strains). Using previously generated whole-genome sequencing data from these 43 D. pigrum strains, we found that the gene encoding this serine protease was present in 41 of 43 (95%) D. pigrum strains, including all 4 strains that were inhibitory in the cocultivation assay. The predicted active domain for the final protein was highly conserved across strains. Other secreted D. pigrum proteins that were more abundant in gel fragments adjacent to inhibitory strains included a peptidoglycan hydrolase and 7 uncharacterized D. pigrum proteins. Conclusion D. pigrum inhibits S. aureus growth in vitro through secretion of inhibitory factors in a strain-dependent manner. We identified an S8 family serine protease as a possible candidate mechanism for the anti-staphylococcal activity of D. pigrum. Future experiments will screen cell-free supernatants from inhibitory D. pigrum strains for S. aureus inhibition and evaluate the effect of protease inhibitor treatment on the antistaphylococcal activity of these supernatants.

Novel biological aqua crust enhances in situ metal(loid) bioremediation driven by phototrophic/diazotrophic biofilm

BackgroundUnderstanding the ecological and environmental functions of phototrophic biofilms in the biological crust is crucial for improving metal(loid) (e.g. Cd, As) bioremediation in mining ecosystems. In this study, in combination with metal(loid) monitoring and metagenomic analysis, we systematically evaluated the effect of biofilm in a novel biological aqua crust (biogenic aqua crust—BAC) on in situ metal(loid) bioremediation of a representative Pb/Zn tailing pond.ResultsWe observed strong accumulation of potentially bioavailable metal(loid)s and visible phototrophic biofilms in the BAC. Furthermore, dominating taxa Leptolyngbyaceae (10.2–10.4%, Cyanobacteria) and Cytophagales (12.3–22.1%, Bacteroidota) were enriched in biofilm. Along with predominant heterotrophs (e.g. Cytophagales sp.) as well as diazotrophs (e.g. Hyphomonadaceae sp.), autotrophs/diazotrophs (e.g. Leptolyngbyaceae sp.) in phototrophic biofilm enriched the genes encoding extracellular peptidase (e.g. family S9, S1), CAZymes (e.g. CBM50, GT2) and biofilm formation (e.g. OmpR, CRP and LuxS), thus enhancing the capacity of nutrient accumulation and metal(loid) bioremediation in BAC system.ConclusionsOur study demonstrated that a phototrophic/diazotrophic biofilm constitutes the structured communities containing specific autotrophs (e.g. Leptolyngbyaceae sp.) and heterotrophs (e.g. Cytophagales sp.), which effectively control metal(loid) and nutrient input using solar energy in aquatic environments. Elucidation of the mechanisms of biofilm formation coupled with metal(loid) immobilization in BAC expands the fundamental understanding of the geochemical fate of metal(loid)s, which may be harnessed to enhance in situ metal(loid) bioremediation in the aquatic ecosystem of the mining area.BQhiNsEmXgEaKyMcCjHWdrVideo

Biochemical and molecular characterization of novel keratinolytic protease from Bacillus licheniformis (KRLr1).

The keratin-degrading bacterium Bacillus licheniformis secretes a keratinase with potential industrial interest. Here, the Keratinase gene was intracellularly expressed in Escherichia coli BL21(DE3) using pET-21b (+) vector. Phylogenetic tree analysis showed that KRLr1 is closely related to Bacillus licheniformis keratinase that belongs to the serine peptidase/subtilisin-like S8 family. Recombinant keratinase appeared on the SDS-PAGE gel with a band of about 38 kDa and was confirmed by western blotting. Expressed KRLr1 was purified by Ni-NTA affinity chromatography with a yield of 85.96% and then refolded. It was found that this enzyme has optimum activity at pH 6 and 37°C. PMSF inhibited the KRLr1 activity and Ca2+ and Mg2+ increased the KRLr1 activity. Using keratin 1% as the substrate, the thermodynamic values were determined as Km 14.54 mM, kcat 912.7 × 10-3 (S-1), and kcat/Km 62.77 (M-1 S-1). Feather digestion by recombinant enzyme using HPLC method, showed that the amino acids cysteine, phenylalanine, tyrosine and lysine had the highest amount compared to other amino acids obtained from digestion. Molecular dynamics (MD) simulation of HADDOCK docking results exhibited that KRLr1 enzyme was able to interact strongly with chicken feather keratine 4 (FK4) compared to chicken feather keratine 12 (FK12). These properties make keratinase KRLr1 a potential candidate for various biotechnological applications.

Overexpression of a Novel Vacuolar Serine Protease-Encoding Gene (spt1) to Enhance Cellulase Production in Trichoderma Reesei

Trichoderma reesei is widely applied as the major industrial fungus for the production of cellulases used for the conversion of lignocellulosic biomass to biofuels and other biobased products. The protein secretion pathway is vital for cellulase secretion, but few reports are related to the role of the vacuole in cellulase production. Here, we identified a novel vacuolar serine protease gene spt1 and investigated the ability of T. reesei to secrete cellulases by disrupting, complementing and overexpressing the spt1 gene. Amino acid sequence analysis of the Spt1 protein showed that it belongs to the subtilisin S8 family and has the conserved catalytic triples (Asp, His, Ser) of the serine protease. The deletion of spt1 did not lead to a decrease in extracellular protease activity, and the observation of mycelia with the Spt1–eGFP fusion expression and the vacuolar membrane dye FM4-64 staining confirmed that Spt1 was an intracellular protease located in the vacuoles of T. reesei. However, the spt1 gene deletion significantly reduced spore production and cellulase secretion, while the spt1 complementation recovered these traits to those of the parental strain. When spt1 was overexpressed by using its native promoter and introducing multiple copies, the cellulase secretion was improved. Furthermore, a strong promoter, Pcdna1, was used to drive the spt1 overexpression, and it was found that the cellulase production was significantly enhanced. Specifically, the filter paper activity of the spt1 overexpression strain SOD-2 reached 1.36 U/mL, which was 1.72 times higher than that of the parental strain. These findings demonstrated that the spt1 gene can be a powerful target for increasing cellulase production in T. reesei, which suggests a possible important role of the vacuole in the cellulase secretion pathway and provides new clues for improving strains for efficient cellulase production.

Ochratoxin A degrading enzymes of Stenotrophomonassp. 043-1a.

Ochratoxin A is a secondary metabolite that acts as a mycotoxin and is produced by Aspergillus, Penicillium, and other fungal species. It is a threat to animal and human health due to nephrotoxic, carcinogenic, and genotoxic properties and its widespread incidence in agricultural products. To reduce this threat, biological remediation processes are of growing interest. The aerobic gram-negative bacterium Stenotrophomonassp. 043-1a, isolated from soil, was previously shown to degrade ochratoxin A into the non-toxic ochratoxin α and l-phenylalanine (Schatzmayr et al. 2002). However, the enzyme or enzymes catalyzing this reaction in this strain remained elusive. Here, we report the targeted purification of Stenotrophomonassp. 043-1a lysate via ammonium sulfate precipitation, size-exclusion chromatography, and hydrophobic interaction chromatography to identify the ochratoxin A degrading enzymes by subsequent peptide fragment fingerprinting. The metallo-dependent hydrolase Chr1_3858681_3267 and a member of the peptidase S9 family, Chr1_3858681_771, were shown to degrade ochratoxin A. This was, to our knowledge, the first report of an ochratoxin A degrading enzyme from the peptidase S9 family.

Genome-Wide Identification and Analysis of the Maize Serine Peptidase S8 Family Genes in Response to Drought at Seedling Stage.

Subtilisin-like proteases (subtilases) are found in almost all plant species and are involved in regulating various biotic and abiotic stresses. Although the literature on subtilases in different plant species is vast, the gene function of the serine peptidase S8 family and its maize subfamily is still unknown. Here, a bioinformatics analysis of this gene family was conducted by describing gene structure, conserved motifs, phylogenetic relationships, chromosomal distributions, gene duplications, and promoter cis-elements. In total, we identified 18 ZmSPS8 genes in maize, distributed on 7 chromosomes, and half of them were hydrophilic. Most of these proteins were located at the cell wall and had similar secondary and tertiary structures. Prediction of cis-regulatory elements in promoters illustrated that they were mainly associated with hormones and abiotic stress. Maize inbred lines B73, Zheng58, and Qi319 were used to analyze the spatial-temporal expression patterns of ZmSPS8 genes under drought treatment. Seedling drought results showed that Qi319 had the highest percent survival after 14 d of withholding irrigation, while B73 was the lowest. Leaf relative water content (LRWC) declined more rapidly in B73 and to lower values, and the nitrotetrazolium blue chloride (NBT) contents of leaves were higher in Qi319 than in the other inbreds. The qPCR results indicated that 6 serine peptidase S8 family genes were positively or negatively correlated with plant tolerance to drought stress. Our study provides a detailed analysis of the ZmSPS8s in the maize genome and finds a link between drought tolerance and the family gene expression, which was established by using different maize inbred lines.

Mechanistic Insight Into the Production of Collagen Oligopeptides by the S8 Family Protease A4095

Collagen oligopeptides have wide applications in foods, pharmaceuticals, cosmetics, and others due to their high bioactivities and bioavailability. The S8 family is the second-largest family of serine proteases. Several collagenolytic proteases from this family have been reported to have good potential in the preparation of collagen oligopeptides, however, the underlying mechanism remains unknown. A4095 was the most abundant S8 protease secreted by the protease-producing bacterium Anoxybacillus caldiproteolyticus 1A02591. Here, we characterized A4095 as an S8 collagenolytic protease and illustrated its structural basis to produce collagen oligopeptides. Protease A4095 preferentially hydrolyzed the Y-Gly peptide bonds in denatured bovine bone collagen, leading to high production (62.48% <1000 Da) of collagen oligopeptides. Structural and mutational analyses indicated that A4095 has a unique S1' substrate-binding pocket to preferentially bind Gly, which is the structural determinant for the high production of collagen oligopeptides. This study provides mechanistic insight into the advantage of the S8 collagenolytic proteases in preparing collagen oligopeptides.

Proteomic analysis of digestive tract peptidases and lipases from the invasive gastropod Pomacea canaliculata.

The invasive gastropod Pomacea canaliculata has received great attention in the last decades as a resultof its negative impact on crops agriculture, yet knowledge of their digestive physiology remains incomplete, particularly the enzymatic breakdown of macromolecules such as proteins and lipids. Discovery proteomics revealed aspartic peptidases, cysteine peptidases, serine peptidases, metallopeptidases and threonine peptidases, as well as acid and neutral lipases and phospholipases along the digestive tract of P.canaliculata. Peptides specific to peptidases (139) and lipases (14) were quantified by targeted mass spectrometry. Digestion begins in the mouth via diverse salivary peptidases (nine serine peptidases; seven cysteine peptidases, one aspartic peptidase and 22 metallopeptidases) and then continues in the oesophagus (crop) via three luminal metallopeptidases (Family M12) and six serine peptidases (Family S1). Downstream, the digestive gland provides a battery of enzymes composed of aspartic peptidase (one), cysteine peptidases (nine), serine peptidases (12) and metallopeptidases (24),including aminopeptidases, carboxypeptidases and dipeptidases). The coiled gut has M1 metallopeptidases that complete the digestion of small peptides. Lipid extracellular digestion is completed by triglyceride lipases. From an integrative physiological and anatomical perspective, P.canaliculata shows an unexpected abundance and diversity of peptidases, which participate mainly in extracellular digestion. Moreover, the previously unknown occurrence of luminal lipases from the digestive gland is reported for the first time. Salivary and digestive glands were the main tissues involved in the synthesis and secretion of these enzymes, but plausibly the few luminally exclusive peptidases are secreted by ventrolateral pouches or epithelial unicellular glands. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Comparative Proteomic Analysis of Bacillus subtilis and Aspergillus niger in Black Soldier Fly Co-Fermentation

Black soldier fly larvae have gained popularity as an organic waste bio-conversional tool and fodder protein replacement in recent decades. It can consume all kinds of animal feces, kitchen waste and agricultural waste with great efficiency and transform them into high-value insect protein, fatty acids, and amino acids, which makes the larva a good substitute for costly fish meal and bean pulp in animal diets. However, excess chitin in the larva skin limits its application as an animal feed additive, consequently, employing fermentation with zymocytes to remove the chitin is necessary. In this study, we raised black soldier fly larvae (BSFL) with different carbon sources, such as chicken feces, straws and glucose, and examined the growth condition; we applied Bacillus subtilis and Aspergillus niger to co-ferment BSFL paste to analyze its nutrition changes. Data revealed that among the four kinds of cultures, the body weight of the corn powder group increased most rapidly; the wood chip group was the most underweight; however, it increased faster than others before day 4, and contained the least fat. Label-free quantitative proteomic analysis revealed that the expression of multiple enzymes from B. subtilis and A. niger involved in polysaccharide hydrolysis, amino acid biosynthesis and fatty acid metabolism, such as peptidase of S8 family, maltogenic α-amylase, oligo-1,6-glucosidase and lysophospholipase like protein changed significantly compared to the control group. Production detection showed that free amino acids, acid-soluble proteins, and short-chain fatty acids increased after fermentation; 13 out of 17 amino acids were increased and total free amino acids were increased from 0.08 g/100 g to 0.3 g/100 g; organic acids increased by 4.81 to 17 fold through fermentation, respectively; the actual protein content declined from 3.03 g/100 g to 1.81 g/100 g, the peptide content increased from 1.3 g/100 g to 2.46 g/100 g, the chitin degradation rate was 40.3%, and fat decreased 30% (p &lt; 0.05). These findings might provide important information for future applications of black soldier fly larvae in different carbon waste recycling measures and material for animal feed/organic fertilizer after fermentation.

Phylogenetic survey of the subtilase family and a data-mining-based search for new subtilisins from Bacillaceae.

The subtilase family (S8), a member of the clan SB of serine proteases are ubiquitous in all kingdoms of life and fulfil different physiological functions. Subtilases are divided in several groups and especially subtilisins are of interest as they are used in various industrial sectors. Therefore, we searched for new subtilisin sequences of the family Bacillaceae using a data mining approach. The obtained 1,400 sequences were phylogenetically classified in the context of the subtilase family. This required an updated comprehensive overview of the different groups within this family. To fill this gap, we conducted a phylogenetic survey of the S8 family with characterised holotypes derived from the MEROPS database. The analysis revealed the presence of eight previously uncharacterised groups and 13 subgroups within the S8 family. The sequences that emerged from the data mining with the set filter parameters were mainly assigned to the subtilisin subgroups of true subtilisins, high-alkaline subtilisins, and phylogenetically intermediate subtilisins and represent an excellent source for new subtilisin candidates.

Protease from jute endophyte Micrococcus luteus MBL-Bac7 functions as a potential bating agent for the leather industry

Ever since the discovery of jute endophytes, testing their potential for commercial uses has been a matter of interest. Considering the same, jute endophyte Micrococcus luteus MBL-Bac7, capable of producing extracellular proteases, was selected for in vitro and in silico analysis to assess its role as a bating agent required in rawhide processing. The presence of extracellular protease was confirmed from the plate assay. As the enzyme is tested for commercial use, the effect of various metal ions and reaction conditions (pH, temperature) have been optimized. The protease activity appears to be retained even at 85°C. It also showed significant activity in a wide range of pH (pH 3.0-8.5). Metal ion Mn2+ increased the protease activity significantly, but Fe2+, Zn2+, and Co2+ ions showed the opposite effect. Molecular identification of the protease was done from the whole genome sequence data. Using PSORTb v.3.0.2, SecretomeP-1.0, TMHMM-2.0, and protein molecular weight software, the physicochemical properties of the protease were predicted. The isolated protease shared a strong evolutionary link with Micrococcus species' S8 family serine peptidase. Finally, in the bating of cowhide, effects similar to that of commercial agents were obtained during finger prick, lastometer, and tensile tests. The findings of this study corroborate the possibility of using this protease as a potential bating agent. However, further studies are necessary to reduce the production cost for higher yield and commercialization. J. Bangladesh Acad. Sci. 46(1); 31-43: June 2022

Post-Proline Cleaving Enzymes (PPCEs): Classification, Structure, Molecular Properties, and Applications.

Proteases or peptidases are hydrolases that catalyze the breakdown of polypeptide chains into smaller peptide subunits. Proteases exist in all life forms, including archaea, bacteria, protozoa, insects, animals, and plants due to their vital functions in cellular processing and regulation. There are several classes of proteases in the MEROPS database based on their catalytic mechanisms. This review focuses on post-proline cleaving enzymes (PPCEs) from different peptidase families, as well as prolyl endoprotease/oligopeptidase (PEP/POP) from the serine peptidase family. To date, most PPCEs studied are of microbial and animal origins. Recently, there have been reports of plant PPCEs. The most common PEP/POP are members of the S9 family that comprise two conserved domains. The substrate-limiting β-propeller domain prevents unwanted digestion, while the α/β hydrolase catalyzes the reaction at the carboxyl-terminal of proline residues. PPCEs display preferences towards the Pro-X bonds for hydrolysis. This level of selectivity is substantial and has benefited the brewing industry, therapeutics for celiac disease by targeting proline-rich substrates, drug targets for human diseases, and proteomics analysis. Protein engineering via mutagenesis has been performed to improve heat resistance, pepsin-resistant capability, specificity, and protein turnover of PPCEs for pharmacological applications. This review aims to synthesize recent structure–function studies of PPCEs from different families of peptidases to provide insights into the molecular mechanism of prolyl cleaving activity. Despite the non-exhaustive list of PPCEs, this is the first comprehensive review to cover the biochemical properties, biological functions, and biotechnological applications of PPCEs from the diverse taxa.

Neprosin belongs to a new family of glutamic peptidase based on in silico evidence

Neprosin was first discovered in the insectivorous tropical pitcher plants of Nepenthes species as a novel protease with prolyl endopeptidase (PEP) activity. Neprosin has two uncharacterized domains of neprosin activation peptide and neprosin. A previous study has shown neprosin activity in hydrolyzing proline-rich gliadin, a gluten component that triggers celiac disease. In this study, we performed in silico structure-function analysis to investigate the catalytic mechanism of neprosin. Neprosin sequences lack the catalytic triad and motifs of PEP family S9. Protein structures of neprosins from Nepenthes × ventrata (NvNpr) and N. rafflesiana (NrNpr1) were generated by ab initio methods and comparatively assessed to obtain high-quality models. Structural alignment of models to experimental structures in the Protein Data Bank (PDB) found a high structural similarity to glutamic peptidases. Further investigations reveal other resemblances to the glutamic peptidases with low optimum pH that activates the enzyme via autoproteolysis for maturation. Two highly conserved glutamic acid residues, which are stable according to the molecular dynamics simulation, can be found at the active site of the substrate cleft. Protein docking demonstrated that mature neprosins bind well with potent antigen αI-gliadin at the putative active site. Taken together, neprosins represent a new glutamic peptidase family, with a putative catalytic dyad of two glutamic acids. This study illustrates a hypothetical enzymatic mechanism of the neprosin family and demonstrates the useful application of an accurate ab initio protein structure prediction in the structure-function study of a novel protein family.

Purification and Characterization of a Fibrinolytic Enzyme from Marine Bacillus velezensis Z01 and Assessment of Its Therapeutic Efficacy In Vivo.

Fibrinolytic enzymes are the most effective agents for the treatment of thrombotic diseases. In the present study, we purified and characterized an extracellular fibrinolytic serine metalloprotease (named Velefibrinase) that is produced by marine Bacillus velezensis Z01 and assessed its thrombolysis in vivo. SDS-PAGE and MALDI-TOF-MS analyses showed that the molecular mass of Velefibrinase was 32.3 KDa and belonged to the peptidase S8 family. The optimal fibrinolytic activity conditions of Velefibrinase were 40 °C and pH 7.0. Moreover, Velefibrinase exhibited high substrate specificity to fibrin, and a higher ratio of fibrinolytic/caseinolytic (1.48) values, which indicated that Velefibrinase had excellent fibrinolytic properties. Based on the degradation pattern of fibrin and fibrinogen, Velefibrinase could be classified as α/β-fibrinogenase. In vitro, Velefibrinase demonstrated efficient thrombolytic ability, anti-platelet aggregation, and amelioration of blood coagulation (APTT, PT, TT, and FIB), which were superior to those of commercial anticoagulant urokinase. Velefibrinase showed no hemolysis for erythrocyte in vitro and no hemorrhagic activity in vivo. Finally, Velefibrinase effectively prevented mouse tail thrombosis in a dose-dependent (0.22–0.88 mg/kg) manner. These findings suggested that Velefibrinase has the potential to becoming a new thrombolytic agent.