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Computational enzyme design is a promising technique for producing novel enzymes for industrial and clinical needs. A key challenge that this technique faces is to consistently achieve the desired activity. Fundamental studies of natural enzymes revealed critical contributions from second-shell - and even more distant - residues to their remarkable efficiency. In particular, such residues organize the internal electrostatic field to promote the reaction. Engineering such fields computationally proved to be a promising strategy, which, however, has some limitations. Charged residues necessarily form specific patterns of local interactions that may be exploited for structural integrity. As a result, it is impossible to probe the electrostatic field alone by substituting amino acids. We hypothesize that an approach that isolates the influences of residues' charges from other influences could yield deeper insights. We use molecular modeling with AI-enhanced QM/MM reaction sampling to implement such an approach and apply it to a model serine protease subtilisin. We find that the negative charge 8 Å away from the catalytic site is crucial to achieving the enzyme's catalytic efficiency, contributing more than 2 kcal/mol to lowering the barrier. In contrast, a positive charge from the second-closest charged residue opposes the efficiency of the reaction by raising the barrier by 0.8 kcal/mol. This result invites discussion into the role of this residue and trade-offs that might have taken place in the evolution of such enzymes. Our approach is transferable and can help investigate the evolution of electrostatic preorganization in other enzymes. We believe that the study and engineering of electrostatic fields in enzymes is a promising direction to advance both fundamental and applied enzymology and lead to the design of new powerful biocatalysts.

Subtilisins are alkaline serine proteases secreted by various species of Bacillus and can produce peptides by autolysis. A peptide from subtilisin NAT was found to disrupt the membrane of Streptococcus pneumoniae and to be cytotoxic only against tumor cell lines was found from subtilisin NAT. However, there has been little research on peptides derived from subtilisin Carlsberg, another famous subtilisin variant. In this research, we found another unique short peptide from subtilisin Carlsberg, which is produced by the fermentation of Bacillus licheniformis. This peptide had a molecular mass of 3225 Da and was identified as the N-terminal 31-amino acid residues of subtilisin Carlsberg, which has not been reported before. The peptide, named SC-(1-31), contains several cationic (pI = 9.83) and hydrophobic amino acid residues. It killed both cancer (Caco-2 and HeLa) and normal cell lines (WI-38) in concentration-dependent manners. The peptide was identified as a cytotoxic peptide based on its comparable toxicity towards cancer and normal cell lines. Liposome disruption assay suggested that this peptide may kill cells by disrupting the cell membrane., Competing Interests: Declaration of competing interest The authors have no conflict of interest. The authors declare no competing financial interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Subtilisin-like proteins are serine proteases that use two types of catalytic triads: Ser-His-Asp and Ser-Glu-Asp. Here, we investigate the two known families of subtilisin-like proteins, the subtilases (Ser-His-Asp triad) and the serine-carboxyl proteinases (Ser-Glu-Asp triad), and describe the local structural arrangements (cores) that govern the catalytic residues in these proteins. We show the separation of the cores into conserved structural zones, which can be repeatedly found in different structures, and compare the structural cores in subtilisin-like proteins with those in trypsin-like serine proteases and alpha/beta-hydrolases.

This research intended to remove residual protein from chitin with proteases in deep eutectic solvents (DESs). The activities of some proteases in several DESs, including choline chloride/p-toluenesulfonic acid, betaine/glycerol (Bet/G), choline chloride/malic acid, choline chloride/lactic acid, and choline chloride/urea, which are capable of dissolving chitin, were tested, and only in Bet/G some proteases were found to be active, with subtilisin A, ficin, and bromelain showing higher activity than other proteases. However, the latter two proteases caused degradation of chitin molecules. Further investigation revealed that subtilisin A in Bet/G did not exhibit "pH memory", which is a universal characteristic displayed by enzymes dispersed in organic phases, and the catalytic characteristics of subtilisin A in Bet/G differed significantly from those in aqueous phase. The conditions for protein removal from chitin by subtilisin A in Bet/G were determined: Chitin dissolved in Bet/G with 0.5 % subtilisin A (442.0 U/mg, based on the mass of chitin) was hydrolyzed at 45 °C for 30 min. The residual protein content in chitin decreased from 5.75 % ± 0.10 % to 1.01 % ± 0.12 %, improving protein removal by 57.20 % compared with protein removal obtained by Bet/G alone. The crystallinity and deacetylation degrees of chitin remained unchanged after the treatment., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service, and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The aim of this study was to evaluate the effect of the enzymatic hydrolysis, performed using Alcalase and Protamex enzymes, on the technological functionalities and the antioxidant capacity of whey protein hydrolysates (WPHs) to identify the conditions allowing to obtain target functionality/ies. Samples were characterized for hydrolysis degree (DH), molecular weight distribution, structural properties, and food-related functionalities. Free sulfhydryl groups and surface hydrophobicity significantly decreased with the increase in DH, regardless of the used enzyme. The foaming and antioxidant properties of Alcalase WPHs were higher as compared to those of WPI, reaching the maximum value at DH = 18-20 %, while higher DH resulted in impaired functionality. Gelling properties were guaranteed when WPI was hydrolysed by Protamex at DH < 15 % while foaming and antioxidant abilities were fostered at 15 < DH < 21 %. These results were well correlated with MW distribution and were rationalized into a road map which represents a useful tool in the selection of proper hydrolysis conditions (time, DH, enzyme type) to obtain WPHs with tailored functionalities. Research outcomes highlighted the possibility to drive protein hydrolysis to optimize the desired functionality/ies., 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 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Subtilisins from microbial sources, especially from the Bacillaceae family, are of particular interest for biotechnological applications and serve the currently growing enzyme market as efficient and novel biocatalysts. Biotechnological applications include use in detergents, cosmetics, leather processing, wastewater treatment and pharmaceuticals. To identify a possible candidate for the enzyme market, here we cloned the gene of the subtilisin SPFA from Fictibacillus arsenicus DSM 15822 T (obtained through a data mining-based search) and expressed it in Bacillus subtilis DB104. After production and purification, the protease showed a molecular mass of 27.57 kDa and a pI of 5.8. SPFA displayed hydrolytic activity at a temperature optimum of 80 °C and a very broad pH optimum between 8.5 and 11.5, with high activity up to pH 12.5. SPFA displayed no NaCl dependence but a high NaCl tolerance, with decreasing activity up to concentrations of 5 m NaCl. The stability enhanced with increasing NaCl concentration. Based on its substrate preference for 10 synthetic peptide 4-nitroanilide substrates with three or four amino acids and its phylogenetic classification, SPFA can be assigned to the subgroup of true subtilisins. Moreover, SPFA exhibited high tolerance to 5% (w/v) SDS and 5% H 2 O 2 (v/v). The biochemical properties of SPFA, especially its tolerance of remarkably high pH, SDS and H 2 O 2 , suggest it has potential for biotechnological applications., (© 2023 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

This study investigated the multifunctional attributes such as, antibacterial, antioxidant and anticancer potential of recombinant subtilisin. A codon-optimized subtilisin gene was synthesized from Bacillus subtilis and was successfully transformed into E. coli DH5α cells which was further induced for high level expression in E. coli BL21 (DE3). An affinity purified ~40 kDa recombinant subtilisin was obtained that revealed to be highly alkali-thermostable based on the thermodynamic parameters. The kinetic parameters were deduced that indicated higher affinity of N-Suc-F-A-A-F-pNA substrate towards subtilisin. Recombinant subtilisin demonstrated strong antibacterial activity against several pathogens and showed minimum inhibitory concentration of 0.06 μg/mL against B. licheniformis and also revealed high stability under the influence of several biochemical factors. It also displayed antioxidant potential in a dose dependent manner and exhibited cell cytotoxicity against A549 and MCF-7 cancerous cell lines with IC 50 of 5 μM and 12 μM respectively. The identity of recombinant subtilisin was established by MALDI-TOF mass spectrum depicting desired mass peaks and N-terminal sequence as MRSK by MALDI-TOF-MS. The deduced N- terminal amino acid sequence by Edman degradation revealed high sequence similarity with subtilisins from Bacillus strains. The structural and functional analysis of recombinant antibacterial subtilisin was elucidated by Raman, circular dichroism and nuclear magnetic resonance spectroscopy and thermogravimetric analysis. The results contribute to the development of highly efficient subtilisin with enhanced catalytic properties making it a promising candidate for therapeutic applications in healthcare industries., Competing Interests: Declaration of competing interest All the authors confirm that they have no argument of interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Halophilic and halotolerant microorganisms represent a promising source of salt-tolerant enzymes suitable for various biotechnological applications where high salt concentrations would otherwise limit enzymatic activity. Considering the current growing enzyme market and the need for more efficient and new biocatalysts, the present study aimed at the characterization of a high-alkaline subtilisin from Alkalihalobacillus okhensis Kh10-101 T . The protease gene was cloned and expressed in Bacillus subtilis DB104. The recombinant protease SPAO with 269 amino acids belongs to the subfamily of high-alkaline subtilisins. The biochemical characteristics of purified SPAO were analyzed in comparison with subtilisin Carlsberg, Savinase, and BPN'. SPAO, a monomer with a molecular mass of 27.1 kDa, was active over a wide range of pH 6.0-12.0 and temperature 20-80 °C, optimally at pH 9.0-9.5 and 55 °C. The protease is highly oxidatively stable to hydrogen peroxide and retained 58% of residual activity when incubated at 10 °C with 5% (v/v) H 2 O 2 for 1 h while stimulated at 1% (v/v) H 2 O 2 . Furthermore, SPAO was very stable and active at NaCl concentrations up to 5.0 m. This study demonstrates the potential of SPAO for biotechnological applications in the future., (© 2022 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Enzymatic degradation of collagen is of great industrial and environmental significance; however, little is known about thermophile-derived collagenolytic proteases. Here, we report a novel collagenolytic protease (TSS) from thermophilic Brevibacillus sp. WF146. The TSS precursor comprises a signal peptide, an N-terminal propeptide, a subtilisin-like catalytic domain, a β-jelly roll (βJR) domain, and a prepeptidase C-terminal (PPC) domain. The maturation of TSS involves a stepwise autoprocessing of the N-terminal propeptide and the PPC domain, and the βJR rather than the PPC domain is necessary for correct folding of the enzyme. Purified mature TSS displayed optimal activity at 70°C and pH 9.0, a half-life of 1.5 h at 75°C, and an increased thermostability as the NaCl concentration increased up to 4 M. TSS possesses an increased number of surface acidic residues and ion pairs, as well as four Ca 2+ -binding sites, which contribute to its high thermostability and halotolerance. At high temperatures, TSS exhibited high activity toward insoluble type I collagen and azocoll but showed a low gelatinolytic activity, with a strong preference for Arg and Gly at the P1 and P1' positions, respectively. Both the βJR and PPC domains could bind but not swell collagen, and thus facilitate TSS-mediated collagenolysis via improving the accessibility of the enzyme to the substrate. Additionally, TSS has the ability to efficiently degrade fish scale collagen at high temperatures. IMPORTANCE Proteolytic degradation of collagen at high temperatures has the advantages of increasing degradation efficiency and minimizing the risk of microbial contamination. Reports on thermostable collagenolytic proteases are limited, and their maturation and catalytic mechanisms remain to be elucidated. Our results demonstrate that the thermophile-derived TSS matures in an autocatalytic manner and represents one of the most thermostable collagenolytic proteases reported so far. At elevated temperatures, TSS prefers hydrolyzing insoluble heat-denatured collagen rather than gelatin, providing new insight into the mechanism of collagen degradation by thermostable collagenolytic proteases. Moreover, TSS has the potential to be used in recycling collagen-rich wastes such as fish scales.

Increased protease activity has been linked to the pathogenesis of IBD. While most studies have been focusing on host proteases in gut inflammation, it remains unclear how to address the potential contribution of their bacterial counterparts. In the present study, we report a functional characterization of a newly identified serine protease, SP-1, from the human gut microbiota. The serine protease repertoire of gut Clostridium was first explored, and the specificity of SP-1 was analyzed using a combinatorial chemistry method. Combining in vitro analyses and a mouse model of colitis, we show that oral administration of recombinant bacteria secreting SP-1 (i) compromises the epithelial barrier, (ii) alters the microbial community, and (ii) exacerbates colitis. These findings suggest that gut microbial protease activity may constitute a valuable contributor to IBD and could, therefore, represent a promising target for the treatment of the disease.

Colloidal protein-protein interactions (PPIs) of attractive and repulsive nature modulate the solubility of proteins, their aggregation, precipitation and crystallization. Such interactions are very important for many biotechnological processes, but are complex and hard to control, therefore, difficult to be understood in terms of measurements alone. In diluted protein solutions, PPIs can be estimated from the osmotic second virial coefficient, B22, which has been calculated using different methods and levels of theory. The most popular approach is based on the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory and its extended versions, i.e. xDLVO. Despite much efforts, these models are not fully quantitative and must be fitted to experiments, which limits their predictive value. Here, we report an extended xDLVO-CG model, which extends existing models by a coarse-grained representation of proteins and the inclusion of an additional ion-protein dispersion interaction term. We demonstrate for four proteins, i.e. lysozyme (LYZ), subtilisin (Subs), bovine serum albumin (BSA) and immunoglobulin (IgG1), that semi-quantitative agreement with experimental values without the need to fit to experimental B22 values. While most likely not the final step in the nearly hundred years of research in PPIs, xDLVO-CG is a step towards predictive PPIs calculations that are transferable to different proteins.

Here we report heterologous expression, enzymatic characterization and structure homology modeling of a subtilisin-like alkaline serine protease (ASP) from Bacillus halodurans C-125. Encoding gene was successfully obtained by PCR and cloned into pMA0911 shuttle vector under the control of strong HpaII promoter and expressed extracellularly. ASP enzyme was successfully expressed in B. subtilis WB800 cell line lacking eight extracellular proteases and produced extracellularly in the culture medium. Km, Vmax and specific activity parameters of the recombinantly produced ASP were identified as 0.2899 mg/ml, 76.12 U/ml and 9500 U/mg, respectively. The purified enzyme revealed remarkable proteolytic activity at highly alkaline conditions with a pH optimum 12.0 and notable thermostability with temperature optimum at 60 °C. Furthermore, substrate-free enzyme revealed remarkable pH stability at pH 12.0 and maintained 93% of its initial activity when incubated at 37 °C for 24 h and 60% of its initial activity upon incubation at 60 °C for 1 h. Theoretically calculated molecular mass of ASP protein was confirmed through SDS-PAGE and western blot analysis (Mw: 28.3 kDa). The secondary and tertiary structures of ASP protein were also identified through homology modeling and further examined in detail. ASP harbors a typical S8/S53 peptidase domain comprising 17 β-sheets and 9 α-helixes within its secondary structure. The structure dynamics analysis of modeled 3D structure further revealed that transient inactivating propeptide chain is the most dynamic region of ASP enzyme with 8.52 Å 2 β-Factor value. Additional residue-dependent fluctuation plot analysis also confirmed the elevated structure dynamics patterning of ASP N-terminus which could be the potential prerequisite for the autonomous propeptide removal of alkaline serine peptidases. Yet the functional domain of ASP becomes quite stable after autonomous exclusion of its propeptide. Although the sequence homology between ASP and commercial detergent additive B. lentus protease (PDB ID:1GCI) was moderate (65.4% sequence similarity), their overlaid 3D structures revealed much higher similarity (98.14%) within 0.80 Å RMSD. In conclusions, with remarkable pH stability, notable thermostability and particularly high specific activity at extreme alkaline conditions, the unveiled ASP protein stands out as a novel protease candidate for various industrial sectors such as textile, detergent, leather, feed, waste, pharmaceutical and others.

The serine protease Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakarensis possesses three insertion loops (IS1-IS3) on its surface, as compared to its mesophilic counterparts. Although IS1 and IS2 are required for maturation of Tk-subtilisin at high temperatures, the role of IS3 remains unknown. Here, CD spectroscopy revealed that IS3 deletion arrested Tk-subtilisin folding at an intermediate state, in which the central nucleus was formed, but the subsequent folding propagation into terminal subdomains did not occur. Alanine substitution of the aspartate residue in IS3 disturbed the intraloop hydrogen-bonding network, as evidenced by crystallographic analysis, resulting in compromised folding at high temperatures. Taking into account the high conservation of IS3 across hyperthermophilic homologues, we propose that the presence of IS3 is important for folding of hyperthermophilic subtilisins in high-temperature environments., (© 2020 Federation of European Biochemical Societies.)

Aggregation of human wild-type transthyretin (hTTR), a homo-tetrameric plasma protein, leads to acquired senile systemic amyloidosis (SSA), recently recognised as a major cause of cardiomyopathies in 1-3% older adults. Fragmented hTTR is the standard composition of amyloid deposits in SSA, but the protease(s) responsible for amyloidogenic fragments generation in vivo is(are) still elusive. Here, we show that subtilisin secreted from Bacillus subtilis, a gut microbiota commensal bacterium, translocates across a simulated intestinal epithelium and cleaves hTTR both in solution and human plasma, generating the amyloidogenic fragment hTTR(59-127), which is also found in SSA amyloids in vivo. To the best of our knowledge, these findings highlight a novel pathogenic mechanism for SSA whereby increased permeability of the gut mucosa, as often occurs in elderly people, allows subtilisin (and perhaps other yet unidentified bacterial proteases) to reach the bloodstream and trigger generation of hTTR fragments, acting as seeding nuclei for preferential amyloid fibrils deposition in the heart.

Subtilisin-like serine peptidases (subtilases) play important roles in the life cycle of many organisms, including the protozoan parasites that are the causative agent of malaria, Plasmodium spp. As with other peptidases, subtilase proteolytic activity has to be tightly regulated in order to prevent potentially deleterious uncontrolled protein degradation. Maturation of most subtilases requires the presence of an N-terminal propeptide that facilitates folding of the catalytic domain. Following its proteolytic cleavage, the propeptide acts as a transient, tightly bound inhibitor until its eventual complete removal to generate active protease. Here we report the identification of a stand-alone malaria parasite propeptide-like protein, called SUB1-ProM, encoded by a conserved gene that lies in a highly syntenic locus adjacent to three of the four subtilisin-like genes in the Plasmodium genome. Template-based modelling and ab initio structure prediction showed that the SUB1-ProM core structure is most similar to the X-ray crystal structure of the propeptide of SUB1, an essential parasite subtilase that is discharged into the parasitophorous vacuole (PV) to trigger parasite release (egress) from infected host cells. Recombinant Plasmodium falciparum SUB1-ProM was found to be a fast-binding, potent inhibitor of P. falciparum SUB1, but not of the only other essential blood-stage parasite subtilase, SUB2, or of other proteases examined. Mass-spectrometry and immunofluorescence showed that SUB1-ProM is expressed in the PV of blood stage P. falciparum, where it may act as an endogenous inhibitor to regulate SUB1 activity in the parasite., (© 2020 The Author(s).)

Plasma levels of cholesterol, especially low-density lipoprotein cholesterol (LDL-C), are positively correlated with the risk of cardiovascular disease. Buildup of LDL in the intima promotes the formation of foam cells and consequently initiates atherosclerosis, one of the main underlying causes of cardiovascular disease. Hepatic LDL receptor (LDLR) is mainly responsible for the clearance of plasma LDL. Mutations in LDLR cause familiar hypercholesterolemia and increase the risk of premature coronary heart disease. Proprotein convertase subtilisin/kexin-type 9 (PCSK9) promotes LDLR degradation and thereby plays a critical role in the regulation of plasma cholesterol metabolism. PCSK9 can bind to LDLR and reroute the receptor to lysosomes for degradation, increasing both circulating LDL-C levels and the risk of cardiovascular disease. PCSK9 is mainly regulated by sterol response element binding protein 2 (SREBP2) at the transcriptional level. Furthermore, many proteins have been identified as interacting with PCSK9, regulating plasma cholesterol levels. Pharmacotherapeutic inhibition of PCSK9 dramatically reduces plasma levels of LDL cholesterol and significantly reduces cardiovascular events. In this article, we summarize the latest advances in PCSK9, mainly focusing on the structure, function, and regulation of the protein, the underlying molecular mechanisms, and its pharmacotherapeutic applications.

Rational design-guided improvement of protein thermostability typically requires identification of residues or regions contributing to instability and introduction of mutations into these residues or regions. One popular method, B-FIT, utilizes B-factors to identify unstable residues or regions and combines them with other strategies, such as directed evolution. Here, we performed structure-based engineering to improve the thermostability of the subtilisin E-S7 (SES7) peptidase. The B-value of each residue was redefined in a normalized B-factor calculation, which was implemented with a refined bioinformatics analysis strategy to identify the critical area (loop 158-162) related to flexibility and to screen for suitable thermostable motif sequences in the Protein Data Bank that can act as transplant loops. In total, we analyzed 445 structures and identified 29 thermostable motifs as candidates. Using these motifs as a starting point, we performed iterative homologous modeling to obtain a desirable chimera loop and introduced five different mutations into this loop to construct thermostable SES7 proteins. Differential scanning fluorimetry revealed increases of 7.3 °C in the melting temperature of an SES7 variant designated M5 compared with the WT. The X-ray crystallographic structure of this variant was resolved at 1.96 Å resolution. The crystal structure disclosed that M5 forms more hydrogen bonds than the WT protein, consistent with design and molecular dynamics simulation results. In summary, the modified B-FIT strategy reported here has yielded a subtilisin variant with improved thermostability and promising industrial applications, supporting the notion that this modified method is a powerful tool for protein engineering., (© 2019 Tang et al.)

The present study investigates the purification and biochemical characterization of a novel extracellular serine alkaline protease, subtilisin (called SAPN) from Melghiribacillus thermohalophilus Nari2A T . The highest yield of protease (395 IU/g) with white shrimp shell by-product (40 g/L) as a unique source of nutriments in the growth medium was achieved after 52 h at 55 °C. The monomeric enzyme of about 30 kDa was purified to homogeneity by ammonium sulfate fractionation, heat treatment, followed by sequential column chromatographies. The optimum pH and temperature values for subtilisin activity were pH 10 and 75 °C, respectively, and half lives of 9 and 5 h at 80 and 90 °C, respectively. The sequence of the 25 NH 2 -terminal residues pertaining of SAPN exhibited a high homology with those of Bacillus subtilisins. The inhibition by DFP and PMSF indicates that this enzyme belongs to the serine proteases family. SAPN was found to be effective in the deproteinization (DDP %) of blue swimming crab (Portunus segnis) and white shrimp (Metapenaeus monoceros) by-products, with a degree of 65 and 82%, respectively. The commercial and the two chitins obtained in this work showed a similar peak pattern in Fourier-Transform Infrared (FTIR) analysis, suggesting that SAPN is suitable for the bio-production of chitin from shell by-products.

Detoxification of gluten immunogenic epitopes is a promising strategy for the treatment of celiac disease. Our previous studies have shown that these epitopes can be degraded in vitro by subtilisin enzymes derived from Rothia mucilaginosa, a natural microbial colonizer of the oral cavity. The challenge is that the enzyme is not optimally active under acidic conditions as encountered in the stomach. We therefore aimed to protect and maintain subtilisin-A enzyme activity by exploring two pharmaceutical modification techniques: PEGylation and Polylactic glycolic acid (PLGA) microencapsulation. PEGylation of subtilisin-A (Sub-A) was performed by attaching methoxypolyethylene glycol (mPEG, 5 kDa). The PEGylation protected subtilisin-A from autolysis at neutral pH. The PEGylated Sub-A (Sub-A-mPEG) was further encapsulated by PLGA. The microencapsulated Sub-A-mPEG-PLGA showed significantly increased protection against acid exposure in vitro. In vivo, gluten immunogenic epitopes were decreased by 60% in the stomach of mice fed with chow containing Sub-A-mPEG-PLGA (0.2 mg Sub-A/g chow) (n = 9) compared to 31.9% in mice fed with chow containing unmodified Sub-A (n = 9). These results show that the developed pharmaceutical modification can protect Sub-A from auto-digestion as well as from acid inactivation, thus rendering the enzyme more effective for applications in vivo.

Membraneless organelles are aggregates of disordered proteins that form spontaneously to promote specific cellular functions in vivo. The possibility of synthesizing membraneless organelles out of cells will therefore enable fabrication of protein-based materials with functions inherent to biological matter. Since random copolymers contain various compositions and sequences of solvophobic and solvophilic groups, they are expected to function in nonbiological media similarly to a set of disordered proteins in membraneless organelles. Interestingly, the internal environment of these organelles has been noted to behave more like an organic solvent than like water. Therefore, an adsorbed layer of random copolymers that mimics the function of disordered proteins could, in principle, protect and enhance the proteins' enzymatic activity even in organic solvents, which are ideal when the products and/or the reactants have limited solubility in aqueous media. Here, we demonstrate via multiscale simulations that random copolymers efficiently incorporate proteins into different solvents with the potential to optimize their enzymatic activity. We investigate the key factors that govern the ability of random copolymers to encapsulate proteins, including the adsorption energy, copolymer average composition, and solvent selectivity. The adsorbed polymer chains have remarkably similar sequences, indicating that the proteins are able to select certain sequences that best reduce their exposure to the solvent. We also find that the protein surface coverage decreases when the fluctuation in the average distance between the protein adsorption sites increases. The results herein set the stage for computational design of random copolymers for stabilizing and delivering proteins across multiple media., Competing Interests: The authors declare no conflict of interest.

In the present work, subtilisin gene from Bacillus subtilis PTTC 1023 was synthesized, cloned into the vector pD441-NH and expressed in E. coli BL21 (DE3). Recombinant subtilisin was purified in a single-step procedure by affinity chromatography. The molecular mass of the purified protein was determined to be about 40kDa by SDS-PAGE. The optimum pH and temperature values of its proteolytic activity were 10.5 and 50°C, respectively and retained more than 70% and 89% of its activity in pH range of 7-12 and 30-60°C, respectively. Enzyme purity was estimated to be about 200- fold greater than that of the crude extract and subtilisin had a specific activity of 56.16U/mg, with a yield of about 87.9%. It was completely inhibited by phenylmethanesulfonyl fluoride, which strongly suggests its belonging to serine protease family. Interestingly, subtilisin protease displayed a significant compatibility with commercial detergents, and tolerance organic solvents, metallic ions and surfactants. The findings obtained demonstrated that protease of B. subtilis could potentially be used in future applications as an additive in detergent formulations., (Copyright © 2017. Published by Elsevier B.V.)

The functions of metal-containing proteins (metalloproteins) are determined by the reactivities of transition metal ions at their active sites. Because protein macromolecular structures have several molecular degrees of freedom, global structural flexibility may also regulate the properties of metalloproteins. However, the influence of this factor has not been fully delineated in mechanistic studies of metalloproteins. Accordingly, we have investigated the relationship between global protein flexibility and the characteristics of a transition metal ion in the protein core using thiol-subtilisin (tSTL) with a Cys-coordinated Cu 2+ ion as a model system. Although tSTL has two Ca 2+ -binding sites, the Ca 2+ -binding status hardly affects its secondary structure. Nevertheless, guanidinium-induced denaturation and amide H/D exchange indicated the increase in the structural flexibility of tSTL by the removal of bound Ca 2+ ions. Electron paramagnetic resonance and absorption spectral changes have revealed that the protein flexibility determines the characteristics of a Cu 2+ ion in tSTL. Therefore, global protein flexibility should be recognized as an important factor that regulates the properties of metalloproteins., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The recent advancement of peptide macrocycles as promising therapeutics creates a need for novel methodologies for their efficient synthesis and (large scale) production. Within this context, due to the favorable properties of biocatalysts, enzyme-mediated methodologies have gained great interest. Enzymes such as sortase A, butelase 1, peptiligase and omniligase-1 represent extremely powerful and valuable enzymatic tools for peptide ligation, since they can be applied to generate complex cyclic peptides with exquisite biological activity. Therefore, the use of enzymatic strategies will effectively supplement the scope of existing chemical methodologies and will accelerate the development of future cyclic peptide therapeutics. The advantages and disadvantages of the different enzymatic methodologies will be discussed in this review., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The use of enzymes in organic solvents has considerably widened their repertoire of applications. Such low water containing media also offer the possibility of carrying out enzymatic reactions at higher temperatures and enhancing reaction yields. The utility of such preparations is limited by the damage caused to the protein structure during freeze-drying. This work investigates the result of exposing the proteolytic enzyme subtilisin to high temperature in low water containing n-octane on its activity in aqueous and non-aqueous media. Exposing subtilisin at 90 °C for 5 h led to 18-fold improvement in its transesterification activity even at the normal assay temperature (37 °C) when compared with the untreated enzyme. The use of n-octane as the reaction medium was important as it helped to retain the three-dimensional architecture of the enzyme and should be considered while designing strategies for obtaining high activity preparations of other enzymes. Structural analysis using differential scanning fluorimetry showed that the enzyme lost its structure after heating in aqueous medium but retained it when heated in organic solvent. The simplicity and general applicability of the strategy should make it useful for obtaining highly active preparations of other enzymes as well., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The cyclic lipopeptide surfactin (SF) is one of the promising environmental friendly biosurfactants abundantly produced by microorganisms such as Bacillus subtilis. SF shows excellent surface properties at various pH, together with lower toxicity and higher biodegradability than commonly used petroleum-based surfactants. However, the effect of the dissociation degree of SF on self-assembly is still incompletely understood, even though two acidic amino acid residues (Asp and Glu) are known to influence eventual surface and biological functions. Here, we report changes in the secondary structure of SF induced by increased pH, and the effect on protease activity. We found that the β-sheet and β-turn formation of SF are significantly enhanced through increased dissociation of Asp and Glu as revealed by a titration experiment of SF solution to estimate apparent pK 1 and pK 2 values together with circular dichroism spectroscopy. We also studied the activity of the common detergent enzyme subtilisin in SF solution at above its pK 2 (pH 7.6) to understand the role of the dissociation degree in the interaction with the protein. The mixing of SF having a unique cyclic topological feature with subtilisin suppressed the decrease in protease activity observed in the presence of synthetic surfactants such as sodium dodecyl sulfate and polyoxyethylene alkyl ether. Thus, SF has great potential for use in laundry detergent formulations, to improve the stability and reliability of detergent enzymes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Enzymes are widely used in nonaqueous solvents to catalyze non-natural reactions. While experimental measurements showed that the solvent nature has a strong effect on the reaction kinetics, the molecular details of the catalytic mechanism in nonaqueous solvents have remained largely elusive. Here we study the transesterification reaction catalyzed by the paradigm subtilisin Carlsberg serine protease in an organic apolar solvent. The rate-limiting acylation step involves a proton transfer between active-site residues and the nucleophilic attack of the substrate to form a tetrahedral intermediate. We design the first coupled valence-bond state model that simultaneously describes both reactions in the enzymatic active site. We develop a new systematic procedure to parametrize this model on high-level ab initio QM/MM free energy calculations that account for the molecular details of the active site and for both substrate and protein conformational fluctuations. Our calculations show that the reaction energy barrier changes dramatically with the solvent and protein conformational fluctuations. We find that the mechanism of the tetrahedral intermediate formation during the acylation step is similar to that determined under aqueous conditions, and that the proton transfer and nucleophilic attack reactions occur concertedly. We identify the reaction coordinate to be mostly due to the rearrangement of some residual water molecules close to the active site.

The role of seemingly non-enzymatic proteins in complexes interconverting UDP-arabinopyranose and UDP-arabinofuranose (UDP-arabinosemutases; UAMs) in the plant cytosol remains unknown. To shed light on their function, crystallographic and functional studies of the seemingly non-enzymatic UAM2 protein from Oryza sativa (OsUAM2) were undertaken. Here, X-ray diffraction data are reported, as well as analysis of the oligomeric state in the crystal and in solution. OsUAM2 crystallizes readily but forms highly radiation-sensitive crystals with limited diffraction power, requiring careful low-dose vector data acquisition. Using size-exclusion chromatography, it is shown that the protein is monomeric in solution. Finally, limited proteolysis was employed to demonstrate DTT-enhanced proteolytic digestion, indicating the existence of at least one intramolecular disulfide bridge or, alternatively, a requirement for a structural metal ion.

Pyrolysin from the hyperthermophilic archaeon Pyrococcus furiosus is the prototype of the pyrolysin family of the subtilisin-like serine protease superfamily (subtilases). It contains four inserts (IS 147 , IS 29 , IS 27 , and IS 8 ) of unknown function in the catalytic domain. We performed domain deletions and showed that three inserts are either essential (IS 147 and IS 27 ) or important (IS 8 ) for efficient maturation of pyrolysin at high temperatures, whereas IS 29 is dispensable. The large insert IS 147 contains Ca3 and Ca4, two calcium-binding Dx[DN]xDG motifs that are conserved in many pyrolysin-like proteases. Mutagenesis revealed that the Ca3 site contributes to enzyme thermostability and the Ca4 site is necessary for pyrolysin to fold into a maturation-competent conformation. Mature insert-deletion variants were characterized and showed that IS 29 and IS 8 contribute to enzyme activity and stability, respectively. In the presence of NaCl, pyrolysin undergoes autocleavage at two sites: one within IS 29 and the other in IS 27 Disrupting the ion pairs in IS 27 and IS 8 induces autocleavage in the absence of salts. Interestingly, autocleavage products combine noncovalently to form an active, nicked enzyme that is resistant to SDS and urea denaturation. Additionally, a single mutation in IS 29 increases resistance to salt-induced autocleavage and further increases enzyme thermostability. Our results suggest that these extra structural elements play a crucial role in adapting pyrolysin to hyperthermal environments. IMPORTANCE Pyrolysin-like proteases belong to the subtilase superfamily and are characterized by large inserts and long C-terminal extensions; however, the role of the inserts in enzyme function is unclear. Our results demonstrate that four inserts in the catalytic domain of hyperthermostable pyrolysin contribute to the folding, maturation, stability, and activity of the enzyme at high temperatures. The modification of extra structural elements in pyrolysin-like proteases is a promising strategy for modulating global structure stability and enzymatic activity of this class of protease., (Copyright © 2017 American Society for Microbiology.)

Detailed studies comparing solid-supported l- or d-amino acid adhesion peptides based on the sequence KLHRIRA were performed. Stability towards proteases and levels of cellular adhesion to the otherwise inert surface of PEGA resin were compared by using fluorescently labelled peptides. A clear difference in the peptide stability towards cleavage by subtilisin, trypsin, or papain was observed. However, all of the on-bead peptides provided an optimal surface for cell adhesion and proliferation. In long-term experiments, these properties were still found to be similar on the resins modified either with l- or with d-amino acids and unaffected by the nature of their fluorescence labelling at either terminus. These results support that the more accessible l-amino acids can be utilized for cell adhesion experiments and confirm the nonspecific interaction mechanism of cell binding to these peptides on the bead surface. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd., (Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.)

Background: Purified from the supernatant of Bacillus subtilis QK02 culture broth, Subtilisin QK-2 is a type of effective thrombolytic reagent that has great exploitable potential. However, the unbearable flavor that occurs with fermentation and the complicated methods that are required to obtain pure products limit the application of this enzyme. Lactic acid bacteria (LAB)-based delivery vehicles are promising as cheap and safe options for medicinal compounds. The secretory expression and surface display using LAB may popularize Subtilisin QK-2 more easily and conveniently with minimal adverse effects., Results: Subtilisin QK-2 was expressed successfully in two forms using lactic acid bacteria. For the secretory expression in Lactococcus lactis, Subtilisin QK-2 was efficiently secreted into the culture using the promoter P nisA and signal peptide SPUsp. The expression levels were not different in L. lactis NZ9000 and NZ3900 without the effect of different selection markers. However, leaky expression was only detected in L. lactis NZ3900. The biological activity of this secreted Subtilisin QK-2 was enhanced by modulating the pH of medium to slightly alkaline during induction and by codon optimization of either the entire gene sequence (qk') or only the propeptide gene sequence (qkpro'). For surface display onto gram-positive enhancer matrix (GEM) particles, n LysM repeats from the C-terminal region of the major autolysin AcmA of L. lactis were fused to either the C-terminus (n = 1, 3, 5) or the N-terminus (n = 1) of the Subtilisin QK-2. These fusion proteins were secreted into the culture medium, and the QK-3LysM was able to bind to the surface of various LAB GEM particles without a loss of fibrinolytic activity. Furthermore, the binding capacity significantly increased with a higher concentration of QK-3LysM. Compared to the free-form Subtilisin QK-2, the QK-3LysM displayed on the surface of GEM particles was more stable in the simulated gastric juice., Conclusions: Combined with the safety and popularity of LAB, Subtilisin QK-2 may be easily applied worldwide to prevent and control thrombosis diseases.

In recent years, mass spectrometry-based phosphoproteomics has propelled our knowledge about the regulation of cellular pathways. Nevertheless, typically applied bottom-up strategies have several limitations. Trypsin, the preferentially used proteolytic enzyme shows impaired cleavage efficiency in the vicinity of phosphorylation sites. Moreover, depending on the frequency and distribution of tryptic cleavage sites (Arg/Lys), generated peptides can be either too short or too long for confident identification using standard LC-MS approaches. To overcome these limitations, we introduce an alternative and simple approach based on the usage of the nonspecific serine protease subtilisin, which enables a fast and reproducible digestion and provides access to "hidden" areas of the proteome. Thus, in a single LC-MS experiment >1800 phosphopeptides were confidently identified and localized from 125 μg of HeLa digest, compared to >2100 sites after tryptic digestion. While the overlap was less than 20 %, subtilisin allowed the identification of many phosphorylation sites that are theoretically not accessible via tryptic digestion, thus considerably increasing the coverage of the phosphoproteome.

It has been reported that pulse proteolysis may be used to investigate protein unfolding kinetics in cell lysate. However, the method has not become popular and we could not judge whether or not it is effective for protein folding study. In this work, we examined the folding and unfolding kinetics of a protein and its variants without purification by pulse proteolysis. The unfolding and refolding rates of the unpurified proteins were similar to those of the purified proteins determined by pulse proteolysis and circular dichroism. Furthermore, because we used a super-stable subtilisin as a protease, we could evaluate the kinetics at 50°C. The present work demonstrates the validity of pulse proteolysis for folding and unfolding studies of unpurified proteins.

Proteases are essential ingredients in modern laundry detergents. Over the past 30 years, subtilisin proteases employed in the laundry detergent industry have been engineered by directed evolution and rational design to tailor their properties towards industrial demands. This comprehensive review discusses recent success stories in subtilisin protease engineering. Advances in protease engineering for laundry detergents comprise simultaneous improvement of thermal resistance and activity at low temperatures, a rational strategy to modulate pH profiles, and a general hypothesis for how to increase promiscuous activity towards the production of peroxycarboxylic acids as mild bleaching agents. The three protease engineering campaigns presented provide in-depth analysis of protease properties and have identified principles that can be applied to improve or generate enzyme variants for industrial applications beyond laundry detergents., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Can one infer the amino acids of the enzymes that are responsible for the stability or the level of the catalytic activity by computationally experimenting on the inhibited enzyme in the enzyme-inhibitor complex? In this article, we answer this question positively both by designing molecular dynamics simulations and by devising coarse-grained methodologies on the subtilisin serine protease. Both methodologies are based on the cross-correlations of the fluctuations of the residues, obtained either by monitoring the trajectories from the simulation or by constructing the inverse Laplacian of the elastic network model, of the complex. A perturbation scanning is applied to the complex using these correlations. The results indicate that the two methods almost point out the same regions on the flexible of the enzyme. These regions are: (i) 50-61, (ii) 155-164 and (iii) 192-194, all of which are designated to be important by experimental studies in the literature.

Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

White nose syndrome (WNS) is a cutaneous fungal disease of bats. WNS is responsible for unprecedented mortalities in North American cave bat populations. There have been few descriptions of enzyme activities that may function in WNS host/pathogen interactions, while no study has isolated and described secreted proteases. To address the hypothesis that Pseudogymnoascus destructans secretes extracellular proteases that function in wing necrosis during WNS infection, the object of this study was to culture P. destructans on various media, then isolate and structurally identify those proteases accumulated stably in the culture medium. We found a single dominant protease activity on minimal nutrient broth enriched with protein substrates, which was strongly inhibited by phenylmethylsulfonyl fluoride. This P. destructans serine protease (PdSP1) was isolated by preparative isoelectric focusing and concanavalin A lectin affinity chromatography. PdSP1 showed a molecular weight 27,900 (estimated by SDS-PAGE), broad pH optimum 6-8, and temperature optimum 60°C. Structural characterization of PdSP1 by MALDI-TOF MS, Orbitrap MS/MS, and Edman amino-terminal peptide sequencing matched it directly to a hypothetical protein accession from the sequenced P. destructans genome that is further identified as a MEROPS family S8A subtilisin-like serine peptidase. Two additional isoforms, PdSP2 and PdSP3, were identified in the P. destructans genome with 90% and 53% homology, respectively. P. destructans S8A serine proteases showed closer sequence conservation to P. pannorum and plant pathogenic fungi than to human pathogenic dermatophytes. Peptide-specific polyclonal antibodies developed from the PdSP1 sequence detected the protein in western blots. These subtilisin-like serine proteases are candidates for further functional studies in WNS host-pathogen interaction.

Unusually stable proteins are a disadvantage for the metabolic turnover of proteins in cells. The CutA1 proteins from Pyrococcus horikoshii and from Oryza sativa (OsCutA1) have unusually high denaturation temperatures (Td) of nearly 150 and 100 °C, respectively, at pH 7.0. It seemed that the CutA1 protein from the human brain (HsCutA1) also has a remarkably high stability. Therefore, the thermodynamic stabilities of HsCutA1 and its protease susceptibility were examined. The Td was remarkably high, being over 95 °C at pH 7.0. The unfolding Gibbs energy (ΔG(0)H2O) was 174 kJ/mol at 37 °C from the denaturant denaturation. The thermodynamic analysis showed that the unfolding enthalpy and entropy values of HsCutA1 were considerably lower than those of OsCutA1 with a similar stability to HsCutA1, which should be related to flexibility of the unstructured properties in both N- and C-terminals of HsCutA1. HsCutA1 was almost completely digested after 1-day incubation at 37 °C by subtilisin, although OsCutA1 was hardly digested at the same conditions. These results indicate that easily available fragmentation of HsCutA1 with remarkably high thermodynamic stability at the body temperature should be important for its protein catabolism in the human cells., (© The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Estrogenic compounds include endogenous estrogens such as estradiol as well as soybean isoflavones, such as daidzein and its metabolite equol, which are known phytoestrogens that prevent osteoporosis in postmenopausal women. Indeed, mineralization of MC3T3-E1 cells, a murine osteoblastic cell line, was significantly decreased in medium containing fetal bovine serum treated with charcoal-dextran to deplete endogenous estrogens, but estradiol and these soybean isoflavones dose-dependently restored the differentiation of MC3T3-E1 cells; equol was tenfold more effective than daidzein. These differentiation-promoting effects were inhibited by the addition of fulvestrant, which is a selective downregulator of estrogen receptors. Analysis of the expression pattern of bone-related genes by reverse transcription PCR (RT-PCR)/quantitative real-time PCR (qRT-PCR), which focused on responsiveness to the estrogen stimuli, revealed that the transcription of PACE4, a subtilisin-like proprotein convertase, was tightly linked with the differentiation of MC3T3-E1 cells induced by estrogen stimuli. Moreover, treatment with RNAi of PACE4 in MC3T3-E1 cells resulted in a drastic decrease of mineralization in the presence of estrogen stimuli. These results strongly suggest that PACE4 participates in bone formation at least in osteoblast differentiation, and estrogen receptor-mediated stimuli induce osteoblast differentiation through the upregulation of PACE4 expression.

The characteristics of Bacillus pumilus subtilisin-like protease expression were investigated. Potential binding sites for the transcription factor DegU~P have been identified in the regulatory region of the enzyme gene, which have been optimized for interaction with the regulatory protein. The expression of the extracellular subtilisin-like protease modified gene has been examined. It was established that the modification of one of the sites has resulted in increased expression of the proteinase in 2 times. It is concluded that the optimization of the promoter led to increased expression of subtilisin-like protease.

Peculiarities of the expression, localization, and structure of the subtilisin-like protease from the microsporidium Paranosema locustae, a parasite of the migratory locust and other orthopteran species, are analyzed. Heterologous expression of the microsporidian ferment in the bacterium Escherichia coli allowed obtaining antibodies to the recombinant protein and to start its examination. In spite of the presence of the N-tail signal peptide in the ferment, potentially able to secret it into the cytoplasm of the infected cell, immunoblotting with obtained antibodies had demonstrated specific accumulation of the protease in the insoluble fraction of spore homogenate. At the same time, the ferment was absent in intracellular stages.of the parasite and also in the cytoplasm of infested host cells. Accumulation of mRNA, coding the studied protein in microsporidian spores was confirmed with the use of RT-PCR method. Heterologous expression of the protease in the methylotrophic yeast Pichiapastoris demonstrated the same result. The ferment of P. locustae was not secreted into a culture medium and was absent in the cytoplasm of yeast cells, accumulating in a dissoluble (membrane) fraction of the homogenate. On the whole, the obtained data testify to the fact that the subtilisin-like protease of P. locustae plays an important role in the physiology of spores rather than participates in host-parasite relations during intra-cellular development.

Retention of total activity of the subtilisin-like serine protease from Beauveria sp. MTCC 5184 (Bprot) in the vicinity of (1) 3 M GdnHCl for 12 h, (2) 50% methanol and dimethyl sulfoxide each for 24 h, and (3) proteolytic enzymes (trypsin, chymotrypsin, and proteinase K) for 48 h led to expect the enzyme to be a kinetically stable protein. Also, the structure of the protein was stable at pH 2.0. Biophysical characterization and conformational transitions were monitored using steady-state and time-resolved fluorescence, FTIR, and CD spectroscopy. Single tryptophan in the protein exists as two conformers, in hydrophobic and polar environment. The secondary structure of Bprot was stable in 3 M GdnHCl as seen in far-UV CD spectra. The active fraction of Bprot obtained from size-exclusion chromatography in the presence of GdnHCl (1.0-3.0 M) eluted at reduced retention time. The peak area of inactive or denatured protein with the same retention time as that of native protein increased with increasing concentration of denaturant (1.0-4.0 M GdnHCl). However, the kinetics of GdnHCl-induced unfolding as studied from intrinsic fluorescence revealed k unf of native protein to be 5.407 × 10(-5) s(-1) and a half-life of 3.56 h. The enzyme is thermodynamically stable in spite of being resistant to the denaturant, which could be due to the effect of GdnHCl imparting rigidity to the active fraction and simultaneously unfolding the partially unfolded protein that exists in equilibrium with the folded active protein. Thermal and pH denaturation of Bprot exhibited interesting structural transitions.

A microcrystalline suspension of Bacillus lentus subtilisin (Savinase) produced during industrial large-scale production was analysed by X-ray powder diffraction (XRPD) and X-ray single-crystal diffraction (MX). XRPD established that the bulk microcrystal sample representative of the entire production suspension corresponded to space group P212121, with unit-cell parameters a = 47.65, b = 62.43, c = 75.74 Å, equivalent to those for a known orthorhombic crystal form (PDB entry 1ndq). MX using synchrotron beamlines at the Diamond Light Source with beam dimensions of 20 × 20 µm was subsequently used to study the largest crystals present in the suspension, with diffraction data being collected from two single crystals (∼20 × 20 × 60 µm) to resolutions of 1.40 and 1.57 Å, respectively. Both structures also belonged to space group P2(1)2(1)2(1), but were quite distinct from the dominant form identified by XRPD, with unit-cell parameters a = 53.04, b = 57.55, c = 71.37 Å and a = 52.72, b = 57.13, c = 65.86 Å, respectively, and refined to R = 10.8% and Rfree = 15.5% and to R = 14.1% and Rfree = 18.0%, respectively. They are also different from any of the forms previously reported in the PDB. A controlled crystallization experiment with a highly purified Savinase sample allowed the growth of single crystals of the form identified by XRPD; their structure was solved and refined to a resolution of 1.17 Å with an R of 9.2% and an Rfree of 11.8%. Thus, there are at least three polymorphs present in the production suspension, albeit with the 1ndq-like microcrystals predominating. It is shown how the two techniques can provide invaluable and complementary information for such a production suspension and it is proposed that XRPD provides an excellent quality-control tool for such suspensions.

Activities of the enzymes, protease subtilisin and horse radish peroxidase (HRP) have been increased 50 and 40%, respectively, in the presence of the nonionic surfactant, alkyl polyglucoside, compared with the activities in buffer alone. This enzyme hyperactivity reaches a peak at 3.0 mm of surfactant. Investigation into the structure of surfactant aggregates indicates "giant" micelle superstructures at this range of surfactant concentration of 1.7 μm in diameter--dramatically decreasing to 60 and 70 nm at higher surfactant concentrations, while surface tension measurements indicate two critical micelle concentration inflection points at 0.2 and 5.0 mm, which suggests transitions in micelle structure with respect to concentration. Furthermore, electron spin resonance (ESR) indicates that the micelles in first critical micelle concentration regime are loosely packed relative to the second aggregate phase. We hypothesize that this loose packing results in diminished hydration shell repulsion between the micelles, leading to the large, micrometer-sized aggregates. We further hypothesize that it is the interaction with these loosely packed micelles that affects the flexibility of the HRP and protease enzyme structure. Time-resolved fluorescence anisotropy of subtilisin in Brij-30 indicates increasing flexibility of catalytic active site with surfactant concentration. This is correlated with an increase in enzymatic activity., (© 2013 The American Society of Photobiology.)

Biomimetic nanocrystalline calcium-deficient apatite compounds are particularly attractive for the setup of bioactive bone-repair scaffolds due to their high similarity to bone mineral in terms of chemical composition, structural and substructural features. As such, along with the increasingly appealing development of moderate temperature engineered routes for sample processing, they have widened the armamentarium of orthopedic and maxillofacial surgeons in the field of bone tissue engineering. This was made possible by exploiting the exceptional surface reactivity of biomimetic apatite nanocrystals, capable of easily exchanging ions or adsorbing (bio)molecules, thus leading to highly-versatile drug delivery systems. In this contribution we focus on the preparation of hybrid materials combining biomimetic nanocrystalline apatites and enzymes (lysozyme and subtilisin). This paper reports physico-chemical data as well as cytotoxicity evaluations towards Cal-72 osteoblast-like cells and finally antimicrobial assessments towards selected strains of interest in bone surgery. Biomimetic apatite/enzyme hybrids could be prepared in varying buffers. They were found to be non-cytotoxic toward osteoblastic cells and the enzymes retained their biological activity (e.g. bond cleavage or antibacterial properties) despite the immobilization and drying processes. Release properties were also examined. Beyond these illustrative examples, the concept of biomimetic apatites functionalized with enzymes is thus shown to be useable in practice, e.g. for antimicrobial purposes, thus widening possible therapeutic perspectives.

The abnormal prion protein (scrapie-associated prion protein, PrP(Sc)) is considered to be included in the group of infectious agents of transmissible spongiform encephalopathies. Since PrP(Sc) is highly resistant to normal sterilization procedures, the decontamination of PrP(Sc) is a significant public health issue. In the present study, a hyperthermostable protease, Tk-subtilisin, was used to degrade PrP(Sc). Although PrP(Sc) is known to be resistant toward proteolytic enzymes, Tk-subtilisin was able to degrade PrP(Sc) under extreme conditions. The level of PrP(Sc) in brain homogenates was found to decrease significantly in vitro following Tk-subtilisin treatment at 100 °C, whereas some protease-resistant fractions remain after proteinase K treatment. Rather small amounts of Tk-subtilisin (0.3 U) were required to degrade PrP(Sc) at 100 °C and pH 8.0. In addition, Tk-subtilisin was observed to degrade PrP(Sc) in the presence of sodium dodecyl sulfate or other industrial surfactants. Although several proteases degrading PrP(Sc) have been reported, practical decontamination procedures using enzymes are not available. This report aims to provide basic information for the practical use of a proteolytic enzyme for PrP(Sc) degradation.

Collagen is an insoluble protein that widely distributes in the extracellular matrix of marine animals. Collagen degradation is an important step in the marine nitrogen cycle. However, the mechanism of marine collagen degradation is still largely unknown. Here, a novel subtilisin-like collagenolytic protease, myroicolsin, which is secreted by the deep sea bacterium Myroides profundi D25, was purified and characterized, and its collagenolytic mechanism was studied. Myroicolsin displays low identity (<30%) to previously characterized subtilisin-like proteases, and it contains a novel domain structure. Protein truncation indicated that the Pro secretion system C-terminal sorting domain in the precursor protein is involved in the cleavage of the N-propeptide, and the linker is required for protein folding during myroicolsin maturation. The C-terminal β-jelly roll domain did not bind insoluble collagen fiber, suggesting that myroicolsin may degrade collagen without the assistance of a collagen-binding domain. Myroicolsin had broad specificity for various collagens, especially fish-insoluble collagen. The favored residue at the P1 site was basic arginine. Scanning electron microscopy and atomic force microscopy, together with biochemical analyses, confirmed that collagen fiber degradation by myroicolsin begins with the hydrolysis of proteoglycans and telopeptides in collagen fibers and fibrils. Myroicolsin showed strikingly different cleavage patterns between native and denatured collagens. A collagen degradation model of myroicolsin was proposed based on our results. Our study provides molecular insight into the collagen degradation mechanism and structural characterization of a subtilisin-like collagenolytic protease secreted by a deep sea bacterium, shedding light on the degradation mechanism of deep sea sedimentary organic nitrogen.

A number of proteases in the subtilisin family derived from environmental or pathogenic microorganisms have been reported to be collagenolytic serine proteases. However, their collagen degradation mechanisms remain unclear. Here, the degradation mechanism of type I collagen fibres by the S8 collagenolytic protease MCP-01, from Pseudoalteromonas sp. SM9913, was studied. Atomic force microscopy observation and biochemical analysis confirmed that MCP-01 progressively released single fibrils from collagen fibres and released collagen monomers from fibrils mainly by hydrolysing proteoglycans and telopeptides in the collagen fibres. Structural and mutational analyses indicated that an enlarged substrate-binding pocket, mainly composed of loops 7, 9 and 11, is necessary for collagen recognition and that the acidic and aromatic residues on these loops form a negatively charged, hydrophobic environment for collagen binding. MCP-01 displayed a non-strict preference for peptide bonds with Pro or basic residues at the P1 site and/or Gly at the P1' site in collagen. His211 is a key residue for the P1-basic-residue preference of MCP-01. Our study gives structural and mechanistic insights into collagen degradation of the S8 collagenolytic protease, which is helpful in developing therapeutics for diseases with S8 collagenolytic proteases as pathogenic factors and in studying environmental organic nitrogen degradation mechanisms., (© 2013 John Wiley & Sons Ltd.)

Tk-subtilisin (Gly70-Gly398) is a subtilisin homolog from Thermococcus kodakarensis. Active Tk-subtilisin is produced from its inactive precursor, Pro-Tk-subtilisin (Gly1-Gly398), by autoprocessing and degradation of the propeptide (Tk-propeptide, Gly1-Leu69). This activation process is extremely slow at moderate temperatures owing to high stability of Tk-propeptide. Tk-propeptide is stabilized by the hydrophobic core. To examine whether a single nonpolar-to-polar amino acid substitution at this core affects the activation rate of Pro-Tk-subtilisin, the Pro-Tk-subtilisin derivative with the Phe17 → His mutation (Pro-F17H), Tk-propeptide derivative with the same mutation (F17H-propeptide), and two active-site mutants of Pro-F17H (Pro-F17H/S324A and Pro-F17H/S324C) were constructed. The crystal structure of Pro-F17H/S324A was nearly identical to that of Pro-S324A, indicating that the mutation does not affect the structure of Pro-Tk-subtilisin. The refolding rate of Pro-F17H/S324A and autoprocessing rate of Pro-F17H/S324C were also nearly identical to those of their parent proteins (Pro-S324A and Pro-S324C). However, the activation rate of Pro-F17H greatly increased when compared with that of Pro-Tk-subtilisin, such that Pro-F17H is efficiently activated even at 40°C. The far-UV circular dichroism spectrum of F17H-propeptide did not exhibit a broad trough at 205-230 nm, which is observed in the spectrum of Tk-propeptide. F17H-propeptide is more susceptible to chymotryptic degradation than Tk-propeptide. These results suggest that F17H-propeptide is unfolded in an isolated form and is therefore rapidly degraded by Tk-subtilisin. Thus, destabilization of the hydrophobic core of Tk-propeptide by a nonpolar-to-polar amino acid substitution is an effective way to increase the activation rate of Pro-Tk-subtilisin., (© 2013 The Protein Society.)

Subtilisin-like proteases of malaria parasite Plasmodium falciparum (PfSUB1, 2 and 3) are expressed at late asexual blood stages. PfSUB1 and 2 are considered important drug targets due to their essentiality for parasite blood stages and role in merozoite egress and invasion of erythrocytes. We have earlier shown the in vitro serine protease activity of PfSUB3 and its localization at asexual blood stages. In this study, we attempted to identify the biological substrate(s) of PfSUB3 and found parasite profilin (PfPRF) as a substrate of the protease. Eukaryotic profilins are multifunctional proteins with primary role in regulation of actin filament assembly. PfPRF possesses biochemical features of eukaryotic profilins and its rodent ortholog is essential in blood stages. Profilin from related apicomplexan parasite Toxoplasma gondii (TgPRF) is known to be involved in parasite motility, host cell invasion, active egress from host cell, immune evasion and virulence in mice. In this study, mature PfSUB3 proteolysed recombinant PfPRF in a dose-dependent manner in in vitro assays. Recombinant PfPRF was assessed for its proinflammatory activity and found to induce high level of TNF-α and low but significant level of IL-12 from mouse bone marrow-derived dendritic cells. Proteolysis of PfPRF by PfSUB3 is suggestive of the probable role of the protease in the processes of motility, virulence and immune evasion., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Members of the kinesin-13 sub-family, including KIF2C, depolymerize microtubules. The positive charge-rich 'neck' region extending from the N-terminus of the catalytic head is considered to be important in the depolymerization activity. Chemically synthesized peptides, covering the basic region (A182-E200), induced a sigmoidal increase in the turbidity of a microtubule suspension. The increase was suppressed by salt addition or by reduction of basicity by amino acid substitutions. Electron microscopic observations revealed ring structures surrounding the microtubules at high peptide concentrations. Using the peptide A182-D218, we also detected free thin straight filaments, probably protofilaments disintegrated from microtubules. Therefore, the neck region, even without the catalytic head domain, may induce lateral disintegration of microtubules. With microtubules lacking anion-rich C-termini as a result of subtilisin treatment, addition of the peptide induced only a moderate increase in turbidity, and rings and protofilaments were rarely detected, while aggregations, also thought to be caused by lateral disintegration, were often observed in electron micrographs. Thus, the C-termini are not crucial for the action of the peptides in lateral disintegration but contribute to structural stabilization of the protofilaments. Previous structural studies indicated that the neck region of KIF2C is flexible, but our IR analysis suggests that the cation-rich region (K190-A204) forms β-structure in the presence of microtubules, which may be of significance with regard to the action of the neck region. Therefore, the neck region of KIF2C is sufficient to cause disintegration of microtubules into protofilaments, and this may contribute to the ability of KIF2C to cause depolymerization of microtubules., (© 2013 The Authors Journal compilation © 2013 FEBS.)