Search

Background: A hydrolytic enzyme called protease from microbial sources has expansive applications in leather as well as tannery industries and thus leaves no choice but to discover a potential candidate for efficient yield of protease with eccentric characteristics., Methods and Results: In the current study, using skim milk agar medium, three bacterial strains (Pro SS14, Pro NTL1, Pro SM) were identified for protease production out of thirty tannery waste samples. The bacterial isolates were identified through morphological, biochemical and molecular basis and the highest protease producer (134 U/mL) having the paramount protein content (8.75%) with the greatest biomass (absorbance 1.7 at 600 nm) was further bioinformatically identified as Bacillus subtilis Pro NTL1. Moreover, the cultural conditions-pH, temperature, incubation period and salinity were tested to get the optimized condition for the three isolates where Bacillus subtilis Pro NTL1 gave best growth at pH 9 demonstrating alkaline protease producing capability, at 45 °C for 72 h proving its thermotolerance characteristics and at 3% NaCl concentration declaring its halotolerance property. Furthermore, following Kirby-Bauer agar disc diffusion technique, the antimicrobial susceptibility pattern was investigated showing Bacillus subtilis Pro NTL1 was resistant to ampicillin, penicillin, sulfafurazole and sensitive to amoxicillin, gentamycin, cefotaxime, kanamycin, imipenem, tetracycline. Additionally, Bacillus subtilis Pro NTL1 showed antagonistic activity against three pathogens named Bacillus cereus, Bacillus megaterium and Escherichia coli., Conclusions: Thus, these findings suggest that Bacillus subtilis Pro NTL1 is a newly isolated strain with unique features which might help to contribute in several biotechnological purposes with significant industrial benefits., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Enzymes are integral to numerous industrial processes, with a growing global demand for various enzyme types. Protease enzymes, in particular, have proven to be cost-effective, stable, and compatible alternatives to traditional chemical processes in both industrial and environmental applications. In this study, an alkaline protease-producing strain of Aeromonas spp. was isolated from a wastewater treatment plant in Iran. The protease production was confirmed by culturing the strain on casein agar medium. The bacterium was identified through morphological, biochemical, and 16 S rRNA sequencing analyses. The optimal culture medium for bacterial growth and enzyme production was obtained using peptone, salt, yeast extract, galactose, and CaCl₂ at an initial pH of 8. Maximum protease production was achieved after 20 h of incubation at 40 °C. To partially purify the enzyme, the supernatant of the bacterial culture medium was first centrifuged, and the enzyme was precipitated using ammonium sulfate, followed by dialysis. Zymography revealed the production of one type of protease during bacterial growth. The partially purified protease exhibited optimal activity at pH 8.5 and maximum stability at pH 9. The optimum temperature for maximum enzyme activity was observed at 50 °C, with 100% residual activity retained for 1 h at 0 °C. The effect of metal ions on enzyme activity was assessed, revealing that KCl induced the most significant effects (p < 0.0001) on enzyme activity. Chemical amino acid modifiers and inhibitors, such as EDTA, DEPSI, and IAA, did not exhibit significant inhibition. In contrast, PMSF and HNBB significantly (p < 0.0001) reduced enzyme activity, suggesting that the enzyme could be classified as a serine protease. The protease also demonstrated high stability in the presence of 2% SDS, showing no signs inactivation. The alkaline pH optimum, thermal stability, and resistance to SDS exhibited by the protease produced by the Aeromonas strain are particularly promising characteristics that warrant further investigation. Based on preliminary tests and the enzyme's characteristics, this protease can be recommended for various applications, pending further studies., (© 2024. The Author(s).)

Rhomboid proteases are universally conserved and facilitate the proteolysis of peptide bonds within or adjacent to cell membranes. While eukaryotic rhomboid proteases have been demonstrated to harbor unique cellular roles, prokaryotic members have been far less characterized. For the first time, we demonstrate that Vibrio cholerae expresses two active rhomboid proteases that cleave a shared substrate at distinct sites, resulting in differential localization of the processed protein. The rhomboid protease rhombosortase (RssP) was previously found to process a novel C-terminal domain called GlyGly-CTERM, as demonstrated by its effect on the extracellular serine protease VesB during its transport through the V. cholerae cell envelope. Here, we characterize the substrate specificity of RssP and GlpG, the universally conserved bacterial rhomboid proteases. We show that RssP has distinct cleavage specificity from GlpG, and specific residues within the GlyGly-CTERM of VesB target it to RssP over GlpG, allowing for efficient proteolysis. RssP cleaves VesB within its transmembrane domain, whereas GlpG cleaves outside the membrane in a disordered loop that precedes the GlyGly-CTERM. Cleavage of VesB by RssP initially targets VesB to the bacterial cell surface and, subsequently, to outer membrane vesicles, while GlpG cleavage results in secreted, fully soluble VesB. Collectively, this work builds on the molecular understanding of rhomboid proteolysis and provides the basis for additional rhomboid substrate recognition while also demonstrating a unique role of RssP in the maturation of proteins containing a GlyGly-CTERM., Importance: Despite a great deal of insight into the eukaryotic homologs, bacterial rhomboid proteases have been relatively understudied. Our research aims to understand the function of two rhomboid proteases in Vibrio cholerae . This work is significant because it will help us better understand the catalytic mechanism of rhomboid proteases as a whole and assign a specific role to a unique subfamily whose function is to process a subset of effector molecules secreted by V. cholerae and other pathogenic bacteria., Competing Interests: The authors declare no conflict of interest.

Enzymatic resolution of ethyl tetrahydrofuroate to produce (S)-2-ethyl tetrahydrofuroate and (R)-2-tetrahydrofuroic acid is a green biomanufacturing strategy. However, enzymatic activity and selectivity are still limiting factors of their industrial applications and development. In previous study, we incidentally found that a Bacillus licheniformis alkaline protease (BLAP), not a lipase, could specifically resolve ethyl tetrahydrofuroate to produce (S)-2-ethyl tetrahydrofuroate and (R)-2-tetrahydrofuroic acid. In this study, the point-saturation-mutation libraries based on the seven amino acid sites (L105, I113, P114, L115, V309, Y310, and M326) were constructed and screened using the molecular docking technology. It was found that activity of the mutant BLAP Y310E reached 182.78 U/mL with high stereoselectivity, 3.14 times higher than that of the wild-type BLAP. Further simulated mutation analysis showed that the Y310E mutation increased the distance from the substrate ligand to the binding pocket from 2.3 Å to 4.5 Å, reducing steric hindrance to the active center. Under the optimal conditions and after 3.5 h of reaction catalyzed by BLAP Y310E , 200 mM ethyl tetrahydrofuroate was converted to (S)-2-ethyl tetrahydrofuroate and (R)-2-tetrahydrofuroic acid with the ee values of 99.9 % and 68.63 %, respectively. The enantiomeric ratio of BLAP Y310E was 105.5, which was 30.23 times higher than that of BLAP. This study advances the comprehension of protease activity and selectivity mechanisms in resolving ester substances and lays a robust foundation for the industrial production of the optically pure (S)-2-ethyl tetrahydrofuroate and (R)-2-tetrahydrofuroic acid via biological enzymatic methods., Competing Interests: Competing interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The Clostridia produce and secrete Large Clostridial Glucosylating Toxins (LCGTs) responsible for disease symptoms, but the secretion mechanism is largely unknown. Recently, a holin-like protein was shown to be essential for toxin secretion. Holins, typically bacteriophage-specific proteins, are part of the holin-endo(lysin) system that releases phage progeny. To determine if the clostridia also use a lysin, we investigated two conserved putative lysins, M7404&#95;01910 and M7404&#95;02200, in the release of the LCGTs TcdA and TcdB from a Clostridioides difficile ribotype 027 strain, M7404. Sequence analysis and structural modelling indicates that both proteins are related to N-acetylmuramoyl-l-alanine amidases, similar to CD27L, a lysin from the C. difficile phage ΦCD27. Disruption of these genes reveal that only M7404&#95;02200 contributes to toxin secretion and does so in a non-lytic fashion. Peptidoglycan hydrolysis assays show that recombinant M7404&#95;02200 is an active peptidoglycan amidase, confirming its role in TcdA and TcdB secretion in C. difficile M7404., (© 2024. The Author(s).)

Peptidoglycan DL-endopeptidases locally cleave the peptide stem of peptidoglycan in the bacterial cell wall. This process facilitates bacterial growth and division by loosening the rigid peptidoglycan layer. IseA binds to the active site of multiple DL-endopeptidases and inhibits excessive peptidoglycan degradation that leads to cell lysis. To better understand how IseA inhibits DL-endopeptidase activity, we determined the crystal structure of the peptidoglycan DL-endopeptidase CwlO/IseA complex and compared it with that of the peptidoglycan DL-endopeptidase LytE/IseA complex. Structural analyses showed significant differences between the hydrophobic pocket-binding residues of the DL-endopeptidases (F361 of CwlO and W237 of LytE). Additionally, binding assays showed that the F361 mutation of CwlO to the bulkier hydrophobic residue, tryptophan, increased its binding affinity for IseA, whereas mutation to alanine reduced the affinity. These analyses revealed that the hydrophobic pocket-binding residue of DL-endopeptidases determines IseA-binding affinity and is required for substrate-mimetic inhibition by IseA., (© 2024 Federation of European Biochemical Societies.)

Background: The present study was conducted to investigate the effects of dietary novel alkaline protease from Bacillus licheniformis on the growth performance, meat quality, antioxidant status and intestinal morphology of broilers. In total, 4000 broilers were randomly assigned into five groups and treated with normal control, normal control + 100 mg kg -1 protease, normal control + 200 mg kg -1 protease, normal control + 300 mg kg -1 protease and normal control + 400 mg kg -1 protease., Results: Supplementing protease impacted final body weight (linear, P = 0.003; quadratic, P = 0.006) and decreased feed conversion rate (linear, P = 0.036) in broilers. Moreover, dietary protease significantly increased breast muscle rate (linear, P = 0.005; quadratic, P = 0.021) and decreased drip loss (linear, P < 0.001; quadratic, P < 0.001). In addition, dietary protease notably increased protein digestibility (linear, P = 0.001; quadratic, P = 0.006) and trypsin activity (linear, P = 0.002; quadratic, P = 0.009) in jejunum. Light microscopy revealed that the jejunum villi in the 300 mg kg -1 and 400 mg kg -1 groups exhibited greater height and a denser arrangement compared to those in the control group. The addition of protease decreased malondialdehyde content (linear, P < 0.001; quadratic, P < 0.001) and increased total antioxidant capacity (linear, P = 0.001; quadratic, P < 0.001) in pectoral muscles., Conclusion: The results of the present study suggest that dietary novel alkaline protease from B. licheniformis improved growth performance by affecting trypsin activity, protein digestibility, antioxidant capacity and intestinal health. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Microbial proteases have proven their efficiency in various industrial applications; however, their application in accelerating the wound healing process has been inconsistent in previous studies. In this study, heterologous expression was used to obtain an over-yielding of the serine alkaline protease. The serine protease-encoding gene aprE was isolated from Bacillus safensis lab 418 and expressed in E. coli BL21 (DE3) using the pET28a (+) expression vector. The gene sequence was assigned the accession number OP610065 in the NCBI GenBank. The open reading frame of the recombinant protease (aprEsaf) was 383 amino acids, with a molecular weight of 35 kDa. The yield of aprEsaf increased to 300 U/mL compared with the native serine protease (SAFWD), with a maximum yield of 77.43 U/mL after optimization conditions. aprEsaf was immobilized on modified amine-functionalized films (MAFs). By comparing the biochemical characteristics of immobilized and free recombinant enzymes, the former exhibited distinctive biochemical characteristics: improved thermostability, alkaline stability over a wider pH range, and efficient reusability. The immobilized serine protease was effectively utilized to expedite wound healing. In conclusion, our study demonstrates the suitability of the immobilized recombinant serine protease for wound healing, suggesting that it is a viable alternative therapeutic agent for wound management., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Improving the ability of bacteria to secrete protein is essential for large-scale production of food enzymes. However, due to the lack of effective tracking technology for target proteins, the optimization of the secretory system is facing many problems. In this study, we utilized the split-GFP system to achieve self-assembly into mature GFP in Bacillus amyloliquefaciens and successfully tracked the alkaline protease AprE. The split-GFP system was employed to assess the signal peptidases, a crucial component in the secretory system, and signal peptidase sipA was identified as playing a role in the secretion of AprE. Deletion of sipA resulted in a higher accumulation of the precursor protein of AprE compared to other signal peptidase deletion strains. To explore the mechanism of signal peptidase on signal peptide, molecular docking and calculation of free energy were performed. The action strength of the signal peptidase is determined by its binding affinity with the tripeptides at the C-terminal of the signal peptide. The functions of signal peptides YdbK and NucB rely on sipA, and overexpression of sipA by integrating it into genome of B. amyloliquefaciens increased the activity of extracellular AprE by 19.9 %. These findings provide insights into enhancing the secretion efficiency of chassis strains., Competing Interests: Declaration of competing interest There are no conflicts of interest to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Alkaline protease AprE, produced by Bacillus licheniformis 2709 is an important edible hydrolase, which has potential applications in nutrient acquisition and medicine. The expression of AprE is finely regulated by a complex transcriptional regulation system. However, there is little study on transcriptional regulation mechanism of AprE biosynthesis in Bacillus licheniformis, which limits system engineering and further enhancement of AprE. Here, the severely depressed expression of aprE in degU and degS deletion mutants illustrated that the regulator DegU and its phosphorylation played a crucial part in AprE biosynthesis. Further electrophoretic mobility shift assay (EMSA) in vitro indicated that phosphorylated DegU can directly bind to the regulatory region though the DNase I foot-printing experiments failed to observe protected region. The plasmid-mediated overexpression of degU32 (Hy) obviously improved the yield of AprE by 41.6 % compared with the control strain, which demonstrated the importance of phosphorylation state of DegU on the transcription of aprE in vivo. In this study, the putative binding sequence of aprE (5'-TAAAT……AAAAT…….AACAT…TAAAA-3') located upstream -91 to -87 bp, -101 to -97 bp, -195 to -191 bp, -215 to -211 bp of the transcription start site (TSS) in B. licheniformis was computationally identified based on the DNA-binding sites of DegU in Bacillus subtilis. Overall, we systematically investigated the influence of the interplay between phosphorylated DegU and its cognate DNA sequence on expression of aprE, which not only contributes to the further AprE high-production in a genetically modified host in the future, but also significantly increases our understanding of the aprE transcription mechanism., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Pandemic Pseudomonas aeruginosa clone C strains encode two inner-membrane associated ATP-dependent FtsH proteases. PaftsH1 is located on the core genome and supports cell growth and intrinsic antibiotic resistance, whereas PaftsH2, a xenolog acquired through horizontal gene transfer from a distantly related species, is unable to functionally replace PaftsH1. We show that purified PaFtsH2 degrades fewer substrates than PaFtsH1. Replacing the 31-amino acid-extended linker region of PaFtsH2 spanning from the C-terminal end of the transmembrane helix-2 to the first seven highly divergent residues of the cytosolic AAA+ ATPase module with the corresponding region of PaFtsH1 improves hybrid-enzyme substrate processing in vitro and enables PaFtsH2 to substitute for PaFtsH1 in vivo. Electron microscopy indicates that the identity of this linker sequence influences FtsH flexibility. We find membrane-cytoplasmic (MC) linker regions of PaFtsH1 characteristically glycine-rich compared to those from FtsH2. Consequently, introducing three glycines into the membrane-proximal end of PaFtsH2's MC linker is sufficient to elevate its activity in vitro and in vivo. Our findings establish that the efficiency of substrate processing by the two PaFtsH isoforms depends on MC linker identity and suggest that greater linker flexibility and/or length allows FtsH to degrade a wider spectrum of substrates. As PaFtsH2 homologs occur across bacterial phyla, we hypothesize that FtsH2 is a latent enzyme but may recognize specific substrates or is activated in specific contexts or biological niches. The identity of such linkers might thus play a more determinative role in the functionality of and physiological impact by FtsH proteases than previously thought., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Methicillin-resistant Staphylococcus aureus (MRSA) causes life-threatening human infections. Bacteriophage-encoded endolysins degrade the cell walls of Gram-positive bacteria by selectively hydrolyzing the peptidoglycan layer and thus are promising candidates to combat bacterial infections. PlyGRCS, the S. aureus-specific bacteriophage endolysin, contains a catalytic CHAP domain and a cell-wall binding SH3&#95;5 domain connected by a linker. Here, we show the crystal structure of full-length PlyGRCS refined to 2.1 Å resolution. In addition, a serendipitous finding revealed that PlyGRCS binds to cold-shock protein C (CspC) by interacting with its CHAP and SH3&#95;5 domains. CspC is an RNA chaperone that plays regulatory roles by conferring bacterial adaptability to various stress conditions. PlyGRCS has substantial lytic activity against S. aureus and showed only minimal change in its lytic activity in the presence of CspC. Whereas the PlyGRCS-CspC complex greatly reduced CspC-nucleic acid binding, the aforesaid complex may downregulate the CspC function during bacterial infection. Overall, the crystal structure and biochemical results of PlyGRCS provide a molecular basis for the bacteriolytic activity of PlyGRCS against S. aureus., (© 2023 The Protein Society.)

Salmonella enterica serovar Typhimurium is a common cause of gastroenteritis in humans and occasionally causes systemic infection. Salmonella's ability to survive and replicate within macrophages is an important characteristic during systemic infection. The outer membrane protease PgtE of S. enterica is a member of the Omptin family of outer membrane aspartate proteases which has well-characterized proteolytic activities in-vitro against a wide range of physiologically relevant substrates. However, no study has been done so far that draws a direct correlation between these in-vitro observations and the biology of the pathogen in-vivo. The main goals of this study were to characterize the pathogenesis-associated functions of pgtE and study its role in the intracellular survival and in-vivo virulence of Salmonella Typhimurium. Our study elucidated a possible role of Salmonella Typhimurium pgtE in combating host antimicrobial peptide- bactericidal/ permeability increasing protein (BPI) to survive in human macrophages. The pgtE-deficient strain of Salmonella showed attenuated proliferation and enhanced colocalization with BPI in U937 and Thp1 cells. In the presence of polymixin B, the attenuated in-vitro survival of STM ΔpgtE suggested a role of PgtE against the antimicrobial peptides. In addition, our study revealed that compared to the wild type Salmonella, the pgtE mutant is replication-deficient in C57BL/6 mice. Further, we showed that PgtE interacts directly with several antimicrobial peptides (AMPs) in the host gut. This gives the pathogen a survival advantage and helps to mount a successful infection in the host., Competing Interests: Declaration of Competing Interest The authors are unaware of any conflicting interests and declare no conflict of interest., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

In prokaryotes, CRISPR-Cas systems provide adaptive immune responses against foreign genetic elements through RNA-guided nuclease activity. Recently, additional genes with non-nuclease functions have been found in genetic association with CRISPR systems, suggesting that there may be other RNA-guided non-nucleolytic enzymes. One such gene from Desulfonema ishimotonii encodes the TPR-CHAT protease Csx29, which is associated with the CRISPR effector Cas7-11. Here, we demonstrate that this CRISPR-associated protease (CASP) exhibits programmable RNA-activated endopeptidase activity against a sigma factor inhibitor to regulate a transcriptional response. Cryo-electron microscopy of an active and substrate-bound CASP complex reveals an allosteric activation mechanism that reorganizes Csx29 catalytic residues upon target RNA binding. This work reveals an RNA-guided function in nature that can be leveraged for RNA-sensing applications in vitro and in human cells.

The ring-forming AAA+ hexamer ClpC1 associates with the peptidase ClpP1P2 to form a central ATP-driven protease in Mycobacterium tuberculosis (Mtb). ClpC1 is essential for Mtb viability and has been identified as the target of antibacterial peptides like CyclomarinA (CymA) that exhibit strong toxicity toward Mtb. The mechanistic actions of these drugs are poorly understood. Here, we dissected how ClpC1 activity is controlled and how this control is deregulated by CymA. We show that ClpC1 exists in diverse activity states correlating with its assembly. The basal activity of ClpC1 is low, as it predominantly exists in an inactive nonhexameric resting state. We show that CymA stimulates ClpC1 activity by promoting formation of supercomplexes composed of multiple ClpC1 hexameric rings, enhancing ClpC1-ClpP1P2 degradation activity toward various substrates. Both the ClpC1 resting state and the CymA-induced alternative assembly state rely on interactions between the ClpC1 coiled-coil middle domains (MDs). Accordingly, we found that mutation of the conserved aromatic F444 residue located at the MD tip blocks MD interactions and prevents assembly into higher order complexes, thereby leading to constitutive ClpC1 hexamer formation. We demonstrate that this assembly state exhibits the highest ATPase and proteolytic activities, yet its heterologous expression in Escherichia coli is toxic, indicating that the formation of such a state must be tightly controlled. Taken together, these findings define the basis of control of ClpC1 activity and show how ClpC1 overactivation by an antibacterial drug generates toxicity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The bacterial HslVU complex consists of two different proteins, i.e., the HslV protease and the HslU ATPase. The functional HslVU enzyme complex forms only when the HslU c-terminal helix is inserted into the cavity located between two adjacent HslV monomers in order to allosterically activate the HslV protease. Based on its essential role in maintaining microbial proteostasis as well its absence from human beings, it is considered a promising therapeutic target for designing antibacterial agents. The goal of the present study was to find out potential drug candidates that could over-activate the HslV protease and produce aberrant proteolysis in pathogenic bacteria. Derivatives of 3-substituted coumarin have been identified as potential HslV protease activators based on their highest docking scores, ideal interaction patterns, and significant in-vitro HslV activation potential. Their ED 50 values were in the sub-micromolar range, i.e., 0.4-0.48µM. The conformational stability of the contacts between the HslV dimer and the active compounds was further confirmed by molecular dynamics studies. Correspondingly, the ADMET characteristics of these lead molecules considerably demonstrated their significant non-toxic drug-like abilities. This research not only identified small non-peptidic HslV protease activators but also improved the understanding of the mode of action of 3-substituted coumarin derivatives as antibacterials.

Intramembrane proteases (IPs) function in numerous signaling pathways that impact health, but elucidating the regulation of membrane-embedded proteases is challenging. We examined inhibition of intramembrane metalloprotease SpoIVFB by proteins BofA and SpoIVFA. We found that SpoIVFB inhibition requires BofA residues in and near a predicted transmembrane segment (TMS). This segment of BofA occupies the SpoIVFB active site cleft based on cross-linking experiments. SpoIVFB inhibition also requires SpoIVFA. The inhibitory proteins block access of the substrate N-terminal region to the membrane-embedded SpoIVFB active site, based on additional cross-linking experiments; however, the inhibitory proteins did not prevent interaction between the substrate C-terminal region and the SpoIVFB soluble domain. We built a structural model of SpoIVFB in complex with BofA and parts of SpoIVFA and substrate, using partial homology and constraints from cross-linking and co-evolutionary analyses. The model predicts that conserved BofA residues interact to stabilize a TMS and a membrane-embedded C-terminal region. The model also predicts that SpoIVFA bridges the BofA C-terminal region and SpoIVFB, forming a membrane-embedded inhibition complex. Our results reveal a novel mechanism of IP inhibition with clear implications for relief from inhibition in vivo and design of inhibitors as potential therapeutics., Competing Interests: SO, LH, MF, LK No competing interests declared, (© 2022, Olenic et al.)

A novel ANAP (Aspergillus niger from alkaline protease) catalyzed one pot three component approach in the synthesis of new thiazolidinedione festooned quinoline analogues via Knoevenagel condensation and N-alkylation have been reported. The catalytic effect of enzyme was monitored and optimized by adjusting various parameters including catalyst concentration, choice of solvent and temperature. The isolated alkaline protease exhibits favorable features for the reaction response such as the shorter reaction time, simple work-up procedure, clean reaction profiles and excellent product yields through reusability of the catalyst upto five cycles. In silico molecular docking simulations were carried out to find out the effective binding affinity of the synthesized quinoline analogues 4(a-i) towards PPARγ protein (Id-2XKW). In vitro α-amylase and α-glucosidase assays were performed for hypoglycemic activity evaluation. In vivo hypoglycemic studies carried out on streptozotocin (SZT) induced diabetic male albino rats have shown that compounds 4e and 4f significantly reduced blood glucose levels with percentage reduction of 43.7 ± 0.91 and 45.6 ± 0.28 at a concentration of 50 mg/kg body wt. The results obtained from molecular docking simulations and in vitro enzyme assays are in consistent with in-vivo studies which clearly demonstrated that out of the synthesized quinoline analogues, compounds 4e and 4f possess promising hypoglycemic activity which was on par to that of standards pioglitazone and rosiglitazone respectively., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The peptidoglycan cell wall is a predominant structure of bacteria, determining cell shape and supporting survival in diverse conditions. Peptidoglycan is dynamic and requires regulated synthesis of new material, remodeling, and turnover - or autolysis - of old material. Despite exploitation of peptidoglycan synthesis as an antibiotic target, we lack a fundamental understanding of how peptidoglycan synthesis and autolysis intersect to maintain the cell wall. Here, we uncover a critical physiological role for a widely misunderstood class of autolytic enzymes, lytic transglycosylases (LTGs). We demonstrate that LTG activity is essential to survival by contributing to periplasmic processes upstream and independent of peptidoglycan recycling. Defects accumulate in Vibrio cholerae LTG mutants due to generally inadequate LTG activity, rather than absence of specific enzymes, and essential LTG activities are likely independent of protein-protein interactions, as heterologous expression of a non-native LTG rescues growth of a conditional LTG-null mutant. Lastly, we demonstrate that soluble, uncrosslinked, endopeptidase-dependent peptidoglycan chains, also detected in the wild-type, are enriched in LTG mutants, and that LTG mutants are hypersusceptible to the production of diverse periplasmic polymers. Collectively, our results suggest that LTGs prevent toxic crowding of the periplasm with synthesis-derived peptidoglycan polymers and, contrary to prevailing models, that this autolytic function can be temporally separate from peptidoglycan synthesis., Competing Interests: AW, LA, KR, AA, GW, Mv, FC, TD No competing interests declared, (© 2022, Weaver et al.)

In this study, a high protease-producing strain was screened by spread plate method and identified by molecular biology and morphological identification. It was identified as Bacillus sp. LCB14. A neutral protease gene was cloned and heterologous expressed by B. subtilis SCK6. Then, the recombinant protease was used to dehair the goat skins. The fermentation conditions of neutral protease production by B. subtilis SCK6 were optimized. The single factor experiments, Plackett-Burma experiment, and response surface method were conducted to determine fermentation medium and culture conditions. The optimized medium contained corn meal 49 g/L, soluble starch 28 g/L, soybean meal 17 g/L, corn steep liquor powder 8 g/L, yeast extract 10 g/L, Na 2 HPO 4 2.3 g/L, KH 2 PO 4 1.9 g/L, MgSO 4 0.5 g/L, MnCl 2 0.1 g/L and ZnSO 4 0.05 g/L. The optimized culture conditions were 35 °C and pH 7.0. Under the optimum conditions, the recombinant strain reached 33467.28 U/mL after 72 hr ferment. Moreover, by fed batch in 30 L fermenters, neutral protease production reached 39,440.78 U/mL and shortened fermentation time from 72 hr to 46 hr. Finally, the crude enzyme was utilized to replace sodium sulfide for dehairing of goatskins. The enzymatic dehaired pelts were white, smooth, and soft; the grain side of enzymatic dehaired pelts were clear; there was no obvious damage to the grain side of enzymatic dehaired pelts by visual observation and tactile test. Furthermore, there were no hair roots, hair follicles and other glands in enzymatic dehaired belts, and the collagen fibers of enzymatic dehaired belt were dispersed well by histological analysis.

Background: Peptidoglycan is an essential component of the cell wall in all bacteria. In particular, the cell walls of Gram-positive bacteria are composed mostly of a thick layer of peptidoglycan. Its accessibility has important implications for their sensing in whole bacterial detection methodologies. Indeed, there is an urgent demand for rapid tests which can identify whole bacteria, e.g., directly at the point of care., Objective: The aim of this work is to explore the suitability of RipA, a key cell division protein of M. tuberculosis, for whole cell biosensing of Gram-positive bacteria., Methods: We here conducted Molecular Dynamics (MD) studies aimed at the understanding of the structural and dynamic features of active RipA and at the design of a suitable bioreceptor. Based on these studies, we engineered a RipA variant for covalent oriented immobilisation on golden surfaces and are able to bind peptidoglycan, albeit without degrading it. Surface Plasmon Resonance (SPR) was employed to check the ability of functionalized golden chips to recognize whole bacteria., Results: MD analyses elucidated the structural details of the active form of RipA and suggested that this enzyme, once inactivated, presents a rigid and well-exposed peptidoglycan recognition cleft. We engineered RipA for proper oriented immobilisation on golden chips for SPR studies. Results show that once chemically coupled to a golden chip, the developed RipA-based bioreceptor is able to detect B. subtilis, used as a model in a concentration-dependent mode., Conclusion: Results highlight the potential of the engineered molecule to be integrated in the development of early warning biosensors for Gram-positive contamination in clinical diagnosis or food-borne infections., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Mycobacterium tuberculosis, the causative agent of tuberculosis remains a global health concern, further compounded by the high rates of HIV-TB co-infection and emergence of multi- and extensive drug resistant TB, all of which have hampered efforts to eradicate this disease. As a result, novel anti-tubercular interventions are urgently required, with the peptidoglycan component of the M. tuberculosis cell wall emerging as an attractive drug target. Peptidoglycan M23 endopeptidases can function as active cell wall hydrolases or degenerate activators of hydrolases in a variety of bacteria, contributing to important processes such as bacterial growth, division and virulence. Herein, we investigate the function of the Rv0950-encoded putative M23 endopeptidase in M. tuberculosis. In silico analysis revealed that this protein is conserved in mycobacteria, with a zinc-binding catalytic site predictive of hydrolytic activity. Transcript analysis indicated that expression of Rv0950c was elevated during lag and log phases of growth and reduced in stationary phase. Deletion of Rv0950c yielded no defects in growth, colony morphology, antibiotic susceptibility or intracellular survival but caused a reduction in cell length. Staining with a monopeptide-derived fluorescent D-amino acid, which spatially reports on sites of active PG biosynthesis or repair, revealed an overall reduction in uptake of the probe in ΔRv0950c. When stained with a dipeptide probe in the presence of cell wall damaging agents, the ΔRv0950c mutant displayed reduced sidewall labelling. As bacterial peptidoglycan metabolism is important for survival and pathogenesis, the role of Rv0950c and other putative M23 endopeptidases in M. tuberculosis should be explored further., Competing Interests: The authors have declared that no competing interests exist.

We have developed a new binary epitope-presenting CVP platform based on bamboo mosaic virus (BaMV) by using the sortase A (SrtA)-mediated ligation technology. The reconstructed BaMV genome harbors two modifications: 1) a coat protein (CP) with N-terminal extension of the tobacco etch virus (TEV) protease recognition site plus 4 extra glycine (G) residues as the SrtA acceptor; and 2) a TEV protease coding region replacing that of the triple-gene-block proteins. Inoculation of such construct, pKB5G, on Nicotiana benthamiana resulted in the efficient production of filamentous CVPs ready for SrtA-mediated ligation with desired proteins. The second part of the binary platform includes an expression vector for the bacterial production of donor proteins. We demonstrated the applicability of the platform by using the recombinant envelope protein domain III (rEDIII) of Japanese encephalitis virus (JEV) as the antigen. Up to 40% of the BaMV CP subunits in each CVP were loaded with rEDIII proteins in 1 min. The rEDIII-presenting BaMV CVPs (BJLPET5G) could be purified using affinity chromatography. Immunization assays confirmed that BJLPET5G could induce the production of neutralizing antibodies against JEV infections. The binary platform could be adapted as a useful alternative for the development and mass production of vaccine candidates., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yang, Hu, Wong, Liang, Ko, He, Lin, Lin and Hsu.)

Objectives: Proteases have gained great attention due to their enormous applications in food, tannery, detergent, photography and many other industries. Proteases rank third position in the production of enzymes. This paper targets to isolate a bacterium with high alkaline protease activity and optimization of its production conditions using Response Surface Methodology (RSM)., Results: A bacterium isolated from soil contaminated with detergent exhibited clearance zone on skim milk agar medium with a protease activity of 22 U/ml. The bacterial strain was identified as Bacillus cereus KM05 and optimization of its production conditions were performed using statistical methods. Further optimization with Box Behnken design resulted in an increase in protease activity by 1.5-fold (28.6 U/ml). The protease enzyme was thermotolerant up to 70 °C with stability towards alkaline pH (pH 9). The enzyme was not affected by most of the metal ions and solvents. Moreover, the protease was also compatible with six commercial detergents tested. Densitometric analysis of the destained fabric materials following the detergent-enzyme treatment, revealed a stain removal efficiency of 97%., Conclusion: The alkaline protease enzyme obtained was stable at different conditions with stain removal efficacy. Hence, the present alkaline protease could be used for detergent formulations., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

The bacterial cell wall is composed primarily of peptidoglycan (PG), a poly-aminosugar that is essential to sustain cell shape, growth, and structural integrity. PG is synthesized by class A/B penicillin-binding proteins (a/bPBPs) and shape, elongation, division, and sporulation (SEDS) proteins like RodA (as part of the Rod system cell elongation machinery) and degraded by "autolytic" enzymes to accommodate growth processes. It is thought that autolysins (particularly endopeptidases [EPs]) are required for PG synthesis and incorporation by creating gaps that are patched and paved by PG synthases, but the exact relationship between autolysins and PG synthesis remains incompletely understood. Here, we have probed the consequences of EP depletion for PG synthesis in the diarrheal pathogen Vibrio cholerae We found that EP depletion resulted in severe morphological and division defects, but these cells continued to increase in mass and aberrantly incorporated new cell wall material. Mass increase proceeded in the presence of Rod system inhibitors, but cells lysed upon inhibition of aPBPs, suggesting that aPBPs are required for structural integrity under these conditions. The Rod system, although not essential for the observed mass increase, remained functional even after prolonged EP depletion. Last, heterologous expression of an EP from Neisseria gonorrhoeae fully complemented growth and morphology of an EP-insufficient V. cholerae , highlighting the possibility that the PG synthases may not necessarily function via direct interaction with EPs. Overall, our findings suggest that during EP insufficiency in V. cholerae , aPBPs become essential for structural integrity while the Rod system is unable to promote proper cell expansion. IMPORTANCE Synthesis and turnover of the bacterial cell wall must be tightly coordinated to avoid structural integrity failure and cell death. Details of this coordination are poorly understood, particularly if and how cell wall turnover enzymes are required for the activity of the different cell wall synthesis machines, the aPBPs and the Rod system. Our results suggest that in Vibrio cholerae , one class of turnover enzymes, the endopeptidases, are necessary for proper cell elongation and division. aPBPs become essential for maintaining structural integrity during EP insufficiency, while the Rod system remains active but contributes little to cell expansion under these conditions. Our results suggest that aPBPs are more versatile than the Rod system in their ability to recognize cell wall gaps formed by autolysins other than the major endopeptidases, adding to our understanding of the coordination between autolysins and cell wall synthases. A detailed understanding of autolysin biology may promote the development of antibiotics that target these essential turnover processes., (Copyright © 2021 Murphy et al.)

Extracellular traps released by neutrophils (NETs) are essential for the clearance of Pseudomonas aeruginosa . Alkaline protease (AprA) secreted by P. aeruginosa negatively correlates with clinical improvement. Moreover, anti-AprA in patients with cystic fibrosis (CF) can help identify patients with aggressive forms of chronic infection. However, the mechanism underlying the clinical outcomes remains unclear. We demonstrated that aprA deficiency in P. aeruginosa decreased the bacterial burden and reduced lung infection. AprA degraded NET components in vitro and in vivo but did not affect NET formation. Importantly, antibodies induced by AprA acted as an agonist and directly enhanced the degrading activities of AprA. Moreover, antisera from patients with P. aeruginosa infection exhibited antibody-dependent enhancement (ADE) similar to that of the antibodies we prepared. Our further investigations showed that the interaction between AprA and the specific antibodies might make the enzyme active sites better exposed, and subsequently enhance the recognition of substrates and accelerate the degradation. Our findings revealed that AprA secreted by P. aeruginosa may aggravate infection by destroying formed NETs, an effect that was further enhanced by its antibodies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jing, Liu, Liu, Feng, Cao, Guan, Xuan, Gao, Wang, Yang, Ma, Lan, Feng, Shen, Wang, Yu and Yang.)

Proteolytic enzymes hold a pivotal position in numerous industrial processes where hydrolysis of protein molecules is required under precise conditions. The emerging trend of biotechnological applications in recent years has witnessed a renewed interest in alkaline serine proteases extending their utility in detergent, leather, textile, food and pharmaceutical industries. A variety of microorganisms have been reported to produce alkaline serine proteases on a large scale, however, extensive research to find an alkaline serine protease with desirable characteristics such as significant catalytic efficiency, expanded stability and broad substrate specificity is still ongoing. Although submerged fermentation dominates the commercial enzyme production, recent reports have emphasized on solid state fermentation technology which can reduce major cost associated with the enzyme production. In the present review, recent research on alkaline serine proteases along with their novel properties and production using solid state fermentation have been discussed.

Background: Proteases are important for hydrolysis of proteins to generate peptides with many bioactivities. Thus, the development of novel proteases with high activities is meaningful to discover bioactive peptides. Because natural isolation from animal, plant and microbial sources is impractical to produce large quantities of proteases, gene cloning and expression of target protease are preferred., Results: In this study, an alkaline serine protease gene (GsProS8) from Geobacillus stearothermophilus was successfully cloned and expressed in Bacillus subtilis. The recombinant GsProS8 was produced with high protease activity of 3807 U/mL after high cell density fermentation. GsProS8 was then purified through ammonium sulfate precipitation and a two-step chromatographic method to obtain the homogeneous protease. The molecular mass of GsProS8 was estimated to be 27.2 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and 28.3 kDa by gel filtration. The optimal activity of GsProS8 was found to be pH 8.5 and 50 °C, respectively. The protease exhibited a broad substrate specificity and different kinetic parameters to casein and whey protein. Furthermore, the hydrolysis of whey protein using GsProS8 resulted in a large amount of peptides with high angiotensin-I-converting enzyme (ACE) inhibitory activity (IC 50 of 0.129 mg/mL)., Conclusions: GsProS8 could be a potential candidate for industrial applications, especially the preparation of antihypertensive peptides.

The Yersinia genus comprises pathogens that can adapt to an environmental life cycle stage as well as to mammals. Yersinia enterocolitica strain W22703 exhibits both insecticidal and nematocidal activity conferred by the tripartite toxin complex (Tc) that is encoded on the 19-kb pathogenicity island Tc-PAI Ye All tc genes follow a strict temperature regulation in that they are silenced at 37°C but activated at lower temperatures. Four highly conserved phage-related genes, located within the Tc-PAI Ye , were recently demonstrated to encode a biologically functional holin-endolysin gene cassette that lyses its own host W22703 at 37°C. Conditions transcriptionally activating the cassette are not yet known. In contrast to Escherichia coli , the overproduction of holin and endolysin did not result in cell lysis of strain W22703 at 15°C. When the holin-endolysin genes were overexpressed at 15°C in four Y. enterocolitica biovars and in four other Yersinia spp., a heterogenous pattern of phenotypes was observed, ranging from lysis resistance of a biovar 1A strain to the complete growth arrest of a Y. kristensenii strain. To decipher the molecular mechanism underlying this temperature-dependent lysis, we constructed a Lon protease-negative mutant of W22703 in which the overexpression of the lysis cassette leads to cell death at 15°C. Overexpressed endolysin exhibited a high proteolytic susceptibility in strain W22703 but remained stable in the W22703 Δ lon strain or in Y. pseudotuberculosis Although artificial overexpression was applied here, the data indicate that Lon protease plays a role in the control of the temperature-dependent lysis in Y. enterocolitica W22703. IMPORTANCE The investigation of the mechanisms that help pathogens survive in the environment is a prerequisite to understanding their evolution and their virulence capacities. In members of the genus Yersinia , many factors involved in virulence, metabolism, motility, or biofilm formation follow a strict temperature-dependent regulation. While the molecular mechanisms underlying the activation of determinants at body temperature have been analyzed in detail, the molecular basis of low-temperature-dependent phenotypes is largely unknown. Here, we demonstrate that a novel phage-related lysis cassette, which is part of the insecticidal and nematocidal pathogenicity island of Y. enterocolitica , does not lyse its own host following overexpression at 15°C and that the Lon protease is involved in this phenotype., (Copyright © 2021 American Society for Microbiology.)

Bacterial biofilms are composed of aggregates of cells encased within a matrix of extracellular polymeric substances (EPS). One key EPS component is extracellular DNA (eDNA), which acts as a 'glue', facilitating cell-cell and cell-substratum interactions. We have previously demonstrated that eDNA is produced in Pseudomonas aeruginosa biofilms via explosive cell lysis. This phenomenon involves a subset of the bacterial population explosively lysing, due to peptidoglycan degradation by the endolysin Lys. Here we demonstrate that in P. aeruginosa three holins, AlpB, CidA and Hol, are involved in Lys-mediated eDNA release within both submerged (hydrated) and interstitial (actively expanding) biofilms, albeit to different extents, depending upon the type of biofilm and the stage of biofilm development. We also demonstrate that eDNA release events determine the sites at which cells begin to cluster to initiate microcolony formation during the early stages of submerged biofilm development. Furthermore, our results show that sustained release of eDNA is required for cell cluster consolidation and subsequent microcolony development in submerged biofilms. Overall, this study adds to our understanding of how eDNA release is controlled temporally and spatially within P. aeruginosa biofilms.

Protein-fragment complementation assays are used ubiquitously for probing protein-protein interactions. Most commonly, the reporter protein is split in two parts, which are then fused to the proteins of interest and can reassemble and provide a readout if the proteins of interest interact with each other. The currently known split fluorescent proteins either can be used only in aerobic conditions and assemble irreversibly, or require addition of exogenous chromophores, which complicates the design of experiments. In recent years, light-oxygen-voltage (LOV) domains of several photoreceptor proteins have been developed into flavin-based fluorescent proteins (FbFPs) that, under some circumstances, can outperform commonly used fluorescent proteins such as GFP. Here, we show that CagFbFP, a small thermostable FbFP based on a LOV domain-containing protein from Chloroflexus aggregans , can serve as a split fluorescent reporter. We use the available genetic and structural information to identify three loops between the conserved secondary structure elements, Aβ-Bβ, Eα-Fα, and Hβ-Iβ, that tolerate insertion of flexible poly-Gly/Ser segments and eventually splitting. We demonstrate that the designed split pairs, when fused to interacting proteins, are fluorescent in vivo in E. coli and human cells and have low background fluorescence. Our results enable probing protein-protein interactions in anaerobic conditions without using exogenous fluorophores and provide a basis for further development of LOV and PAS (Per-Arnt-Sim) domain-based fluorescent reporters and optogenetic tools.

In this study, serine alkaline protease from halotolerant alkaliphilic Salipaludibacillus agaradhaerens strain AK-R was purified and immobilized onto double mesoporous core-shell silica (DMCSS) nanospheres. Covalent immobilization of AK-R protease onto activated DMCSS-NH 2 nanospheres was more efficient than physical adsorption and was applied in further studies. DMCSS-NH 2 nanospheres showed high loading capacity of 103.8 μg protein/mg nanospheres. Relative to free AK-R protease, the immobilized enzyme exhibited shifts in the optimal temperature and pH from 60 to 65 °C and pH 10.0 to 10.5, respectively. While the soluble enzyme retained 47.2% and 9.1% of its activity after treatment for 1 h at 50 and 60 °C, the immobilized protease maintained 87.7% and 48.3%, respectively. After treatment for 2 h at pH 5 and 13, the immobilized protease maintained 73.6% and 53.4% of its activity, whereas the soluble enzyme retained 32.9% and 1.4%, respectively. Furthermore, the immobilized AK-R protease showed significant improvement of enzyme stability in high concentration of NaCl, organic solvents, surfactants, and commercial detergents. In addition, the immobilized protease exhibited a very good operational stability, retaining 79.8% of its activity after ten cycles. The results clearly suggest that the developed immobilized protease system is a promising nanobiocatalyst for various protease applications., Competing Interests: Declaration of competing interest The authors have no potential conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Traditional therapy for malignant neoplasms involving surgical procedures, radiotherapy and chemotherapy aims to kill neoplastic cells, but also affects normal cells. Therefore, exogenous proteases are the target of studies in cancer therapy, as they have been shown to be effective in suppressing tumors and reducing metastases. Pluronic F127 (F127) is a copolymer of amphiphilic blocks that has shown significant potential for drug administration, as it is capable of incorporating hydrophobic drugs and self-assembling in micrometers of nanometric size. This study investigated the effects of immobilization of the alkaline protease PR4A3 with pluronic F127 micelles on the enzyme-induced cytotoxicity. Protease immobilization was demonstrated through UV-visible and circular dichroism (CD) spectroscopies, as the enzyme interacts with the polymeric micelle of Pluronic F127 without changing its secondary structure. In addition, the immobilized form of the enzyme showed greater bioavailability after passing through the simulated gastrointestinal transit. Cell viability was assessed using the tetrazoic methylthiazole (MTT) assay. The results open perspectives for new research and development for PR4A3 in the treatment of colorectal carcinoma., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The Streptococcus suis serotype 2 (SS2) is a significant zoonotic pathogen that is responsible for various swine diseases, even causing cytokine storms of Streptococcal toxic shock-like syndromes amongst human. Cell wall anchoring proteins with a C-terminal LPxTG are considered to play vital roles during SS2 infection; however, their exporting mechanism across cytoplasmic membranes has remained vague. This study found that YSIRK-G/S was involved in the exportation of LPxTG-anchoring virulence factors MRP and SspA in virulent SS2 strain ZY05719. The whole-genome analysis indicated that diverse LPxTG proteins fused with an N-terminal YSIRK-G/S motif are encoded in strain ZY05719. Two novel LPxTG proteins SspB and YzpA were verified to be exported via a putative transport system that was dependent on the YSIRK-G/S directed translocation, and portrayed vital functions during the infection of SS2 strain ZY05719. Instead of exhibiting an inactivation of C5a peptidase in SspB, another LPxTG protein with an N-terminal YSIRK-G/S motif from Streptococcus agalactiae was depicted to cleave the C5a component of the host complement. The consequent domain-architecture retrieval determined more than 10,000 SspB/YzpA like proteins that are extensively distributed in the Gram-positive bacteria, and most of them harbor diverse glycosyl hydrolase or peptidase domains within their middle regions, thus presenting their capability to interact with host cells. The said findings provide compelling evidence that LPxTG proteins with an N-terminal YSIRK-G/S motif are polymorphic effectors secreted by Gram-positive bacteria, which can be further proposed to define as cell wall anchoring effectors in a new subset.

Organisms specialized to thrive in cold environments (so-called psychrophiles) produce enzymes with the remarkable ability to catalyze chemical reactions at low temperature. Cold activity relies on adaptive changes in the proteins' sequence and structural organization that result in high conformational flexibility. As a consequence of flexibility, several such enzymes are inherently heat sensitive. Cold-active enzymes are of interest for application in a number of bioprocesses, where cold activity coupled with easy thermal inactivation can be of advantage. We describe the biochemical and functional properties of two glycosyl hydrolases (named LYS177 and LYS188) of family 19 (GH19), identified in the genome of an Antarctic marine Pseudomonas . Molecular evolutionary analysis placed them in a group of characterized GH19 endolysins active on lysozyme substrates, such as peptidoglycan. Enzyme activity peaks at about 25-35 °C and 40% residual activity is retained at 5 °C. LYS177 and LYS188 are thermolabile, with Tm of 52 and 45 °C and half-lives of 48 and 12 h at 37 °C, respectively. Bioinformatics analyses suggest that low heat stability may be associated to temperature-driven increases in local flexibility occurring mainly in a specific region of the polypeptide that is predicted to contain hot spots for aggregation.

FtsEX is a bacterial ABC transporter that regulates the activity of periplasmic peptidoglycan amidases via its interaction with the murein hydrolase activator, EnvC. In Escherichia coli , FtsEX is required to separate daughter cells after cell division and for viability in low-osmolarity media. Both the ATPase activity of FtsEX and its periplasmic interaction with EnvC are required for amidase activation, but the process itself is poorly understood. Here we present the 2.1 Å structure of the FtsX periplasmic domain in complex with its periplasmic partner, EnvC. The EnvC-FtsX periplasmic domain complex has a 1-to-2 stoichiometry with two distinct FtsX-binding sites located within an antiparallel coiled coil domain of EnvC. Residues involved in amidase activation map to a previously identified groove in the EnvC LytM domain that is here found to be occluded by a "restraining arm" suggesting a self-inhibition mechanism. Mutational analysis, combined with bacterial two-hybrid screens and in vivo functional assays, verifies the FtsEX residues required for EnvC binding and experimentally test a proposed mechanism for amidase activation. We also define a predicted link between FtsEX and integrity of the outer membrane. Both the ATPase activity of FtsEX and its periplasmic interaction with EnvC are required for resistance to membrane-attacking antibiotics and detergents to which E. coli would usually be considered intrinsically resistant. These structural and functional data provide compelling mechanistic insight into FtsEX-mediated regulation of EnvC and its downstream control of periplasmic peptidoglycan amidases., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

With the modern world focusing on environmental friendly products, more and more chemical processes are being replaced by enzymatic methods. Alkaline proteases (APases) place more than 50% of the total world enzyme production. Marine microorganisms are capable of producing an extensive spectrum of APases which have important ecological roles and promising industrial applications. Marine microbial APases can meet the required market demand for various industrial processes due to their strong specificity, mild reaction conditions, environmental friendliness and easy inactivation or control in comparison with chemical catalysts. In this review, a bird's-eye view on recent research works in the field of APase production from marine microorganisms as well as their potential industrial applications. The effect of various physical and chemical parameters on marine microbial APase is discussed. Isolation, purification, optimum pH and temperature of marine microbial APases are also reported. We anticipate that this review will provide an outline of potential industrial application of marine microbial APases and open new avenues to help the academicians, researchers and industrialists., (Copyright © 2020. Published by Elsevier B.V.)

The expression of enzymes in Bacillus licheniformis, such as the valuable extracellular alkaline protease AprE, is highly regulated by a complex transcriptional regulation mechanism. Here, we found that the transcript abundance of aprE varies >343-fold in response to the supply of nutrients or to environmental challenges. To identify the underlying regulatory mechanism, the core promoter of aprE and several important upstream regulatory regions outside the promoter were firstly confirmed by 5'-RACE and mutagenesis experiments. The specific proteins that bind to the identified sequences were subsequently captured by DNA pull-down experiments, which yielded the transcriptional factors (TFs) Spo0A, CggR, FruR, YhcZ, as well as fragments of functionally unassigned proteins. Further electrophoretic mobility shift assay (EMSA) and DNase I foot-printing experiments indicated that Spo0A can directly bind to the region from -92 to -118 nucleotides upstream of the transcription start site, and the deletion of this specific region drastically decreased the production of AprE. Taken together, these results indicated that the expression of aprE was mainly regulated by the interplay between Spo0A and its cognate DNA sequence, which was successfully applied to overproduce AprE in a genetically modified host harboring three aprE expression cassettes. The DNA binding proteins may serve to increase the efficiency of transcription by creating an additional binding site for RNA polymerase. The discovery of this mechanism significantly increases our understanding of the aprE transcription mechanism, which is of great importance for AprE overproduction., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The emergence of B. cereus as an opportunistic food-borne pathogen has intensified the need to distinguish strains of public health concern. The heterogeneity of the diseases associated with B. cereus infections emphasizes the versatility of these bacteria strains to colonize their host. Nevertheless, the molecular basis of these differences remains unclear. Several toxins are involved in virulence, particularly in gastrointestinal disorders, but there are currently no biological markers able to differentiate pathogenic from harmless strains. We have previously shown that CwpFM is a cell wall peptidase involved in B. cereus virulence. Here, we report a sequence/structure/function characterization of 39 CwpFM sequences, chosen from a collection of B. cereus with diverse virulence phenotypes, from harmless to highly pathogenic strains. CwpFM is homology-modeled in silico as an exported papain-like endopeptidase, with an N-terminal end composed of three successive bacterial Src Homology 3 domains (SH3b 1-3 ) likely to control protein-protein interactions in signaling pathways, and a C-terminal end that contains a catalytic NLPC&#95;P60 domain primed to form a competent active site. We confirmed in vitro that CwpFM is an endopeptidase with a moderate peptidoglycan hydrolase activity. Remarkably, CwpFMs from pathogenic strains harbor a specific stretch of twenty residues intrinsically disordered, inserted between the SH3b 3 and the catalytic NLPC&#95;P60 domain. This strongly suggests this linker as a marker of differentiation between B. cereus strains. We believe that our findings improve our understanding of the pathogenicity of B. cereus while advancing both clinical diagnosis and food safety.

ISP-SW5 is an intracellular alkaline serine protease gene from Bacillus velezensis SW5 that was heterologously expressed in Escherichia coli BL21 (DE3). Sequence analysis indicated that the ISP-SW5 gene has 960 bp open reading frame and encodes a protein of 319 amino acid residues. Three-dimensional structure of ISP-SW5 with the fibrinolytic activity from Bacillus velezensis was predicted by in silico analysis. Gly219 was the most likely active site for the fibrinolytic activity of ISP-SW5. The recombinant enzyme ISP-SW5 was purified by Ni-NTA Superflow Column. SDS-PAGE showed that this enzyme had a molecular mass of 34 kDa. The result of native-PAGE and N-terminal sequencing showed that the N-terminal propeptide of ISP-SW5 was cleaved during the maturation of protease. The optimum pH and temperature were 8.0 and 40 °C, respectively. Enzyme activity was markedly inhibited by PMSF and EDTA but enhanced by 5 mM Ca 2+ and 2 mM Zn 2+ by up to 143% and 115%, respectively. Additionally, ISP-SW5 retained 93%, 78%, and 49% relative enzyme activity after incubation with 0.5 M, 1 M and 2 M NaCl, respectively, at 4 °C for 12 h. The enzyme activity determined by casein as substrate was 1261 U/mg. ISP-SW5 could degrade fibrin at an activity of 3428 U/mg, and its properties reflect its potential application in developing a novel biological catalyst for efficient fibrin hydrolysis in medical treatment.

Understanding the relationship between the metallocofactor and its protein environment is the key to uncovering the mechanism of metalloenzymes. PqqE, a radical S- adenosylmethionine enzyme in pyrroloquinoline quinone (PQQ) biosynthesis, contains three iron-sulfur cluster binding sites. Two auxiliary iron-sulfur cluster binding sites, designated as AuxI and AuxII, use distinctive ligands compared to other proteins in the family while their functions remain unclear. Here, we investigate the electronic properties of these iron-sulfur clusters and compare the catalytic efficiency of wild-type (WT) Methylorubrum extorquens AM1 PqqE to a range of mutated constructs. Using native mass spectrometry, protein film electrochemistry, and electron paramagnetic resonance spectroscopy, we confirm the previously proposed incorporation of a mixture of [2Fe-2S] and [4Fe-4S] clusters at the AuxI site and are able to assign redox potentials to each of the three iron-sulfur clusters. Significantly, a conservative mutation at AuxI, C268H, shown to selectively incorporate a [4Fe-4S] cluster, catalyzes an enhancement of uncoupled S- adenosylmethionine cleavage relative to WT, together with the elimination of detectable peptide cross-linked product. While a [4Fe-4S] cluster can be tolerated at the AuxI site, the aggregate findings suggest a functional [2Fe-2S] configuration within the AuxI site. PqqE variants with nondestructive ligand replacements at AuxII also show that the reduction potential at this site can be manipulated by changing the electronegativity of the unique aspartate ligand. A number of novel mechanistic features are proposed based on the kinetic and spectroscopic data. Additionally, bioinformatic analyses suggest that the unique ligand environment of PqqE may be relevant to its role in PQQ biosynthesis within an oxygen-dependent biosynthetic pathway.

The Dispase autolysis-inducing protein (DAIP) from Streptomyces mobaraensis attracts M4 metalloproteases, which results in inhibition and autolysis of bacillolysin (BL) and thermolysin (TL). The present study shows that aureolysin (AL) from Staphylococcus aureus and pseudolysin (LasB) from Pseudomonas aeruginosa are likewise impaired by DAIP. Complete inhibition occurred when DAIP significantly exceeded the amount of the target protease. At low DAIP concentrations, AL and BL performed autolysis, while LasB and TL degradation required reductants or detergents that break intramolecular disulfide bonds or change the protein structure. Site directed mutagenesis of DAIP and removal of an exposed protein loop either influenced binding or inhibition of AL and TL but had no effect on LasB and BL. The Y170A and Δ239-248 variants had completely lost affinity for TL and AL. The exchange of Asn-275 also impaired the interaction of DAIP with AL. In contrast, DAIP Phe-297 substitution abolished inhibition and autolysis of both target proteases but still allowed complex formation. Our results give rise to the conclusion that other, yet unknown DAIP amino acids inactivate LasB and BL. Obviously, various bacteria in the same habitat caused Streptomyces mobaraensis to continuously optimize DAIP in inactivating the tackling metalloproteases., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Bacterial dormancy can take many forms, including formation of Bacillus endospores, Streptomyces exospores, and metabolically latent Mycobacterium cells. In the actinobacteria, including the streptomycetes and mycobacteria, the rapid resuscitation from a dormant state requires the activities of a family of cell-wall lytic enzymes called resuscitation-promoting factors (Rpfs). Whether Rpf activity promotes resuscitation by generating peptidoglycan fragments (muropeptides) that function as signaling molecules for spore germination or by simply remodeling the dormant cell wall has been the subject of much debate. Here, to address this question, we used mutagenesis and peptidoglycan binding and cleavage assays to first gain broader insight into the biochemical function of diverse Rpf enzymes. We show that their LysM and LytM domains enhance Rpf enzyme activity; their LytM domain and, in some cases their LysM domain, also promoted peptidoglycan binding. We further demonstrate that the Rpfs function as endo-acting lytic transglycosylases, cleaving within the peptidoglycan backbone. We also found that unlike in other systems, Rpf activity in the streptomycetes is not correlated with peptidoglycan-responsive Ser/Thr kinases for cell signaling, and the germination of rpf mutant strains could not be stimulated by the addition of known germinants. Collectively, these results suggest that in Streptomyces , Rpfs have a structural rather than signaling function during spore germination, and that in the actinobacteria, any signaling function associated with spore resuscitation requires the activity of additional yet to be identified enzymes., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Sexton et al.)

Recent recurrent outbreaks of bacterial resistance to antibiotics have shown the critical need to identify new lytic agents to combat them. The species Lysobacter capsici VKM B-2533 T possesses a potent antimicrobial action against a number of bacteria, fungi and yeasts. Its activity can be due to the impact of bacteriolytic enzymes, antibiotics and peptides. This work isolated four homogeneous bacteriolytic enzymes and a mixture of two proteins, which also had a bacteriolytic activity. The isolates included proteins identical to L. enzymogenes α- and β-lytic proteases and lysine-specific protease. The proteases of 26 kDa and 29 kDa and a protein identified as N-acetylglycosaminidase had not been isolated in Lysobacter earlier. The isolated β-lytic protease digested live methicillin-resistant staphylococcal cells with high efficiency (minimal inhibitory concentration, 2.85 μg/mL). This property makes the enzyme deserving special attention. A recombinant β-lytic protease was produced. The antimicrobial potential of the bacterium was contributed to by outer membrane vesicles (OMVs). L. capsici cells were found to form a group of OMVs responsible for antifungal activity. The data are indicative of a significant antimicrobial potential of this bacterium that requires thorough research.

In this study, three active alkaline proteases (AprEs) (BaApr1, BaApr2, and BaApr9) from Bacillus altitudinis W3 were obtained through bioinformatics analysis and verification. Multiple sequence alignment showed low identity of 64.60% and suggested that the three AprEs belonged to the S8A subfamily of serine proteases. They showed maximal activity with pH of 9.5 at 55 °C, 8.5 at 50 °C, and 10.5 at 45 °C, respectively. They were stable at alkaline condition and below 50 °C. In the presence of Ca 2+ , the optimal temperatures and thermostability of them were significantly improved. They were activated by Ca 2+ and Mg 2+ but inhibited by ethylenediaminetetraacetic acid (EDTA) and phenylmethanesulfonyl fluoride (PMSF). Surfactants had little effect on them, but most organic solvents had some inhibitory effect except for n-hexane. They were effective in hydrolyzing natural proteins such as casein and NON-fat powdered milk. BaApr1 exhibited the highest catalytic efficiency towards casein and showed an excellent effect on the desensitization of milk proteins. The present study reveals some useful characteristics of the three AprEs, and indicates that AprEs have potential application values in the desensitization process of milk proteins., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The article reviews the discovery, properties and functional activities of new bacterial enzymes, proteases grimelysin (ECP 32) of Serratia grimesii and protealysin of Serratia proteamaculans , characterized by both a highly specific "actinase" activity and their ability to stimulate bacterial invasion. Grimelysin cleaves the only polypeptide bond Gly42-Val43 in actin. This bond is not cleaved by any other proteases and leads to a reversible loss of actin polymerization. Similar properties were characteristic for another bacterial protease, protealysin. These properties made grimelysin and protealysin a unique tool to study the functional properties of actin. Furthermore, bacteria Serratia grimesii and Serratia proteamaculans , producing grimelysin and protealysin, invade eukaryotic cells, and the recombinant Escherichia coli expressing the grimelysin or protealysins gene become invasive. Participation of the cellular c-Src and RhoA/ROCK signaling pathways in the invasion of eukaryotic cells by S. grimesii was shown, and involvement of E-cadherin in the invasion has been suggested. Moreover, membrane vesicles produced by S. grimesii were found to contain grimelysin, penetrate into eukaryotic cells and increase the invasion of bacteria into eukaryotic cells. These data indicate that the protease is a virulence factor, and actin can be a target for the protease upon its translocation into the host cell.

Most bacteria surround themselves with a cell wall, a strong meshwork consisting primarily of the polymerized aminosugar peptidoglycan (PG). PG is essential for structural maintenance of bacterial cells, and thus for viability. PG is also constantly synthesized and turned over; the latter process is mediated by PG cleavage enzymes, for example, the endopeptidases (EPs). EPs themselves are essential for growth but also promote lethal cell wall degradation after exposure to antibiotics that inhibit PG synthases (e.g., β-lactams). Thus, EPs are attractive targets for novel antibiotics and their adjuvants. However, we have a poor understanding of how these enzymes are regulated in vivo, depriving us of novel pathways for the development of such antibiotics. Here, we have solved crystal structures of the LysM/M23 family peptidase ShyA, the primary EP of the cholera pathogen Vibrio cholerae Our data suggest that ShyA assumes two drastically different conformations: a more open form that allows for substrate binding and a closed form, which we predicted to be catalytically inactive. Mutations expected to promote the open conformation caused enhanced activity in vitro and in vivo, and these results were recapitulated in EPs from the divergent pathogens Neisseria gonorrheae and Escherichia coli Our results suggest that LysM/M23 EPs are regulated via release of the inhibitory Domain 1 from the M23 active site, likely through conformational rearrangement in vivo., Competing Interests: The authors declare no competing interest.

Chitin is the second-most abundant bioresource and widely used in the food, agricultural, biomedicine, and other industries. However, under the mutual restriction of extraction cost and environmental protection, it is relatively difficult to prepare chitin from natural sources by pure separation. The aim of this study is to extract chitin from fresh crab shell waste by decalcification (DC) and deproteinization (DP) using glutamic acid and alkaline protease. The optimum technological conditions for DC and DP were as follows: (1) 5% (w/v) glutamic acid solution was used as decalcifying agent, the ratio of material to liquid was 1:10 (m/v), and the ash content in chitin was 0.83 ± 0.027% after decalcification at 75° C for 12 h. (2) Using alkaline protease as enzymatic hydrolyzer, 1500 U of alkaline protease was added per gram of crab shell. Under the conditions of material-liquid ratio of 1:10 (m/v) and pH value of hydrolysate of 9.0, N content in chitin was 6.63 ± 0.10% after 6 h of enzymatic hydrolysis at 55° C. And the extraction rate of chitin was 92.25 ± 0.51%. As a decalcifying agent, glutamic acid could be recycled with a recovery rate of 77.42 ± 2.16%. Calcium carbonate in crab shell was converted into calcium hydrogen phosphate by calcium glutamate, and protein into amino acids and polypeptides, which could be used as feed additives. The "glutamic acid-enzymolysis" for extracting chitin from crab shell is a relatively closed process, which has the advantages of mild reaction, greatly reducing the discharge of three wastes and high comprehensive utilization rate of raw materials.

Background: Aiming at the characteristics of nonlinear, multi-parameter, strong coupling and difficulty in direct on-line measurement of key biological parameters of marine low-temperature protease fermentation process, a soft-sensing modeling method based on artificial bee colony (ABC) and multiple least squares support vector machine (MLSSVM) inversion for marine protease fermentation process is proposed., Methods: Firstly, based on the material balance and the characteristics of the fermentation process, the dynamic "grey box" model of the fed-batch fermentation process of marine protease is established. The inverse model is constructed by analyzing the inverse system existence and introducing the characteristic information of the fermentation process. Then, the inverse model is identified off-line using MLSSVM. Meanwhile, in order to reduce the model error, the ABC algorithm is used to correct the inverse model. Finally, the corrected inverse model is connected in series to the marine alkaline protease MP fermentation process to form a composite pseudo-linear system, thus, real-time on-line prediction of key biological parameters in fermentation process can be realized., Results: Taking the alkaline protease MP fermentation process as an example, the simulation results demonstrate that the soft-sensing modeling method can solve the real-time prediction problem of key biological parameters in the fermentation process on-line, and has higher accuracy and generalization ability than the traditional soft-sensing method of support vector machine., Conclusions: The research provides a new method for soft-sensing modeling of key biological parameters in fermentation process, which can be extended to soft-sensing modeling of general nonlinear systems.

Alkaline proteases having activity and stability at alkaline pH possess a large variety of applications in many industries. Growing renewed interest urges the need to find a single alkaline protease with promising properties to be used in different industrial processes. Herein, alkaline proteases produced through fermentation of cheap and easily available organic municipal solid wastes by Bacillus subtilis AKAL7 and Exiguobacterium indicum AKAL11 were purified to investigate their kinetic and thermodynamic parameters, detergent compatibility, dehairing and feather-degrading capability. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the purified protease from B. subtilis and E. indicum had molecular mass of ∼45 and 75 kDa, respectively. The protease from B. subtilis and E. indicum showed highest activity at 55 and 50 °C having low K m 1.17 and 0.567 mg/mL and high V max 416.67 and 333.33 µmole/min, respectively. The activation energy and temperature quotient of protease from B. subtilis and E. indicum were 26.52 and 65.75 kJ/mole, and 1.0004 and 1.0003 at 20-55 and 20-50 °C, respectively. Thermodynamics analysis revealed the formation of more ordered enzyme-substrate complexes along with spontenity of enzyme reaction. The protease from E. indicum exhibited better compatibility at higher concentration of detergents compared to that from B. subtilis. However, both proteases could retain more than 80% of the activity in the presence of 0.1% commercial laundry detergents. The purified protease from the both sources could degrade almost 90% of barbs and 40% of dry weight of the native feather and that from E. indicum could dehair cow skin. Results reported herein suggest that the alkaline protease from B. subtilis AKAL7 and E. indicum AKAL11 has biotechnological implications in detergent, leather and poultry feather processing industries.