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Purpose: The purpose of this study is to enable the efficient production of the industrial enzyme subtilisin, a serine protease with extensive applications in various industries such as detergents, food processing, textiles, and pharmaceuticals. By leveraging recombinant DNA technology to produce subtilisin locally, the study aims to decrease reliance on foreign imports and enhance the sustainability of enzyme supply chains. This initiative seeks not only to meet the growing demand for subtilisin but also to promote economic independence, reduce costs, and foster innovation within the local biotechnology sector. Material and Methods: Following bioinformatic calculations and processes for pET28 and subtilisin, the enzyme was produced. Results: The results confirm successful cloning and expression of the subtilisin gene in the pET28b vector, creating the recombinant construct pET28b-subt. Agarose gel electrophoresis verified the transformation, showing distinct bands for the pET28b backbone and subtilisin insert. The recombinant subtilisin was purified from lysed E. coli using Ni-NTA affinity chromatography, with SDS-PAGE analysis revealing a molecular mass of 41,646.82 Da (Figure 5, Figure 6). These findings demonstrate successful production of subtilisin, though further optimization is needed for industrial applications. Conclusion: Production has been carried out and the sds-page result supports this. After this, it was seen that it was necessary to focus on activity studies. [ABSTRACT FROM AUTHOR]

Some types of collagens, including transmembrane MACIT collagens and C. elegans cuticle collagens, are N-terminally cleaved at a dibasic site that resembles the consensus for furin or other proprotein convertases of the subtilisin/kexin (PCSK) family. Such cleavage may release transmembrane collagens from the plasma membrane and affect extracellular matrix assembly or structure. However, the functional consequences of such cleavage are unclear and evidence for the role of specific PCSKs is lacking. Here, we used endogenous collagen fusions to fluorescent proteins to visualize the secretion and assembly of the first collagen-based cuticle in C. elegans and then tested the role of the PCSK BLI-4 in these processes. Unexpectedly, we found that cuticle collagens SQT-3 and DPY-17 are secreted into the extraembryonic space several hours before cuticle matrix assembly. Furthermore, this early secretion depends on BLI-4/PCSK; in bli-4 and cleavage-site mutants, SQT-3 and DPY-17 are not efficiently secreted and instead form large intracellular puncta. Their later assembly into cuticle matrix is reduced but not entirely blocked. These data reveal a role for collagen N-terminal processing in intracellular trafficking and the control of matrix assembly in vivo. Our observations also prompt a revision of the classic model for C. elegans cuticle matrix assembly and the pre-cuticle-to-cuticle transition, suggesting that cuticle layer assembly proceeds via a series of regulated steps and not simply by sequential secretion and deposition.

The food enzyme subtilisin (EC 3.4.21.62) is produced with the non‐genetically modified Bacillus paralicheniformis strain AP‐01 by Nagase (Europa) GmbH. It was considered free from viable cells of the production organism. The food enzyme is intended to be used in five food manufacturing processes. Since residual amounts of food enzyme‐total organic solids (TOS) are removed in one process, dietary exposure was calculated only for the remaining four food manufacturing processes. It was estimated to be up to 0.875 mg TOS/kg body weight per day in European populations. The production strain of the food enzyme has the capacity to produce bacitracin and thus failed to meet the requirements of the Qualified Presumption of Safety approach. Bacitracin was detected in the industrial fermentation medium but not in the food enzyme itself. However, the limit of detection of the analytical method used for bacitracin was not sufficient to exclude the possible presence of bacitracin at a level representing a risk for the development of antimicrobial resistant bacteria. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and twenty‐eight matches with respiratory allergens, one match with a contact allergen and two matches with food allergens (melon and pomegranate) were found. The Panel considered that the risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to melon or pomegranate, cannot be excluded, but would not exceed the risk of consuming melon or pomegranate. Based on the data provided, the Panel could not exclude the presence of bacitracin, a medically important antimicrobial, and consequently the safety of this food enzyme could not be established. [ABSTRACT FROM AUTHOR]

The food enzyme with two declared activities, bacillolysin (EC 3.4.24.28) and subtilisin (EC 3.4.21.62), is produced with the non‐genetically modified Bacillus amyloliquefaciens strain AR‐383 by AB Enzymes GmbH. The food enzyme is intended to be used in nine food manufacturing processes. Since residual amounts of total organic solids (TOS) are removed in the production of distilled alcohol, dietary exposure was calculated only for the remaining eight food manufacturing processes. Exposure was estimated to be up to 1.958 mg TOS/kg body weight per day in European populations. As the production strain qualifies for the qualified presumption of safety approach to safety assessment and no issues of concern arising from the production process of the food enzyme were identified, the Panel considered that no toxicological studies other than the assessment of allergenicity were necessary. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made, and 30 matches were found, including one food allergen (melon). The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure to this food enzyme cannot be excluded, but for individuals sensitised to melon, this would not exceed the risk of consuming melon. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use. [ABSTRACT FROM AUTHOR]

The food enzyme subtilisin (EC 3.4.21.62) is produced with the genetically modified Bacillus licheniformis strain NZYM‐CB by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme is considered free from viable cells of the production organism and its DNA. It is intended to be used in six food manufacturing processes. The dietary exposure to the food enzyme‐TOS was estimated to be up to 0.722 mg TOS/kg body weight (bw) per day in European populations. The production strain of the food enzyme fulfils the requirements for the qualified presumption of safety approach to safety assessment. As no other concerns arising from the manufacturing process were identified, the Panel considered that toxicological tests were not required for the assessment of this food enzyme. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and 20 matches were found, including two food allergens (melon and pomegranate). The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, particularly in individuals sensitised to melon and pomegranate, but would not exceed the risk from consumption of melon or pomegranate. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use. [ABSTRACT FROM AUTHOR]

Background: Tinea of vellus hair is a dermatophyte infection with or without localised infection of adjacent skin. The epidemiology and pathogenesis of this disorder have insufficiently been established. Objectives: To gain insight into the epidemiology and analysis dissection of patterns of vellus hair infection. Methods: A comprehensive examination of our series of cases and existing literature, clinical information was performed. Microsporum species were assayed for levels of keratinase and SUB1‐3 level after different keratin‐induced cultures. Results: The infection affects both children and adults, with a male‐to‐female ratio of 1:1.3. The most afflicted areas are the face, neck and upper limbs. Both typical (ringworm‐like) and atypical (tinea incognito) tinea lesions can be observed. The disorder is caused primarily by the zoophilic Microsporum canis (40.39%). Topical antifungal drugs alone are ineffective; mycological cure is achieved in combination with systemic antifungal therapy for 4–8 weeks. In vitro, M. canis exhibited significantly elevated levels of secreted protease and SUB1 and SUB3 transcripts in cat hair cultures. Conversely, the findings of anthropophilic dermatophytes M. audouinii and M. ferrugineum demonstrated the opposite trend, suggesting that there are associations between keratinase activity and expression of SUB1 and SUB3 induced by different keratin substrates among M. canis complex members. Conclusions: The epidemiological characteristics of tinea of vellus hair help us to understand its transmission patterns and develop effective prevention strategies. There are associations between keratinase activity and expression of SUB1‐3 induced by different substrates, which may explain the host shift and adaptation from pet animals to particular micro‐habitats on the human host. [ABSTRACT FROM AUTHOR]

Abstract The food enzyme subtilisin (EC 3.4.21.62) is produced with the non‐genetically modified Bacillus paralicheniformis strain AP‐01 by Nagase (Europa) GmbH. It was considered free from viable cells of the production organism. The food enzyme is intended to be used in five food manufacturing processes. Since residual amounts of food enzyme‐total organic solids (TOS) are removed in one process, dietary exposure was calculated only for the remaining four food manufacturing processes. It was estimated to be up to 0.875 mg TOS/kg body weight per day in European populations. The production strain of the food enzyme has the capacity to produce bacitracin and thus failed to meet the requirements of the Qualified Presumption of Safety approach. Bacitracin was detected in the industrial fermentation medium but not in the food enzyme itself. However, the limit of detection of the analytical method used for bacitracin was not sufficient to exclude the possible presence of bacitracin at a level representing a risk for the development of antimicrobial resistant bacteria. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and twenty‐eight matches with respiratory allergens, one match with a contact allergen and two matches with food allergens (melon and pomegranate) were found. The Panel considered that the risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to melon or pomegranate, cannot be excluded, but would not exceed the risk of consuming melon or pomegranate. Based on the data provided, the Panel could not exclude the presence of bacitracin, a medically important antimicrobial, and consequently the safety of this food enzyme could not be established.

Abstract The food enzyme with two declared activities, bacillolysin (EC 3.4.24.28) and subtilisin (EC 3.4.21.62), is produced with the non‐genetically modified Bacillus amyloliquefaciens strain AR‐383 by AB Enzymes GmbH. The food enzyme is intended to be used in nine food manufacturing processes. Since residual amounts of total organic solids (TOS) are removed in the production of distilled alcohol, dietary exposure was calculated only for the remaining eight food manufacturing processes. Exposure was estimated to be up to 1.958 mg TOS/kg body weight per day in European populations. As the production strain qualifies for the qualified presumption of safety approach to safety assessment and no issues of concern arising from the production process of the food enzyme were identified, the Panel considered that no toxicological studies other than the assessment of allergenicity were necessary. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made, and 30 matches were found, including one food allergen (melon). The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure to this food enzyme cannot be excluded, but for individuals sensitised to melon, this would not exceed the risk of consuming melon. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

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 15822T (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% H2O2 (v/v). The biochemical properties of SPFA, especially its tolerance of remarkably high pH, SDS and H2O2, suggest it has potential for biotechnological applications.

The food enzyme subtilisin (EC 3.4.21.62) is produced with the non‐genetically modified Bacillus licheniformis strain NZYM‐CX by Novozymes A/S. The production strain met the requirements for the qualified presumption of safety (QPS) approach. The food enzyme is intended to be used in eight food manufacturing processes: processing of cereals and other grains for the production of brewed products; processing of dairy products for the production of modified milk proteins and flavouring preparations; processing of plant‐ and fungal‐derived products for the production of plant‐based analogues of milk and milk products, protein hydrolysates and edible oils from algae; processing of meat and fish products for the production of protein hydrolysates; processing of yeast and yeast products. Since residual amounts of total organic solids (TOS) are removed in the production of edible oils from algae, dietary exposure was calculated only for the remaining seven food manufacturing processes. Exposure was estimated to be up to 2.393 mg TOS/kg body weight (bw) per day in European populations. As the production strain qualified for the QPS approach and no issues of concern arose from the production process of the food enzyme, the Panel considered that toxicological studies were unnecessary. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was performed, and a total of 20 matches were found, 17 to respiratory allergens, two to food allergens (found in muskmelon and pomegranate) and one to a contact allergen. The Panel considered that the risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, especially in individuals sensitised to muskmelon or pomegranate, but would not exceed the risk of consuming these foods. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use. [ABSTRACT FROM AUTHOR]

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 15822T (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% H2O2 (v/v). The biochemical properties of SPFA, especially its tolerance of remarkably high pH, SDS and H2O2, suggest it has potential for biotechnological applications. [ABSTRACT FROM AUTHOR]

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 15822T (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% H2O2 (v/v). The biochemical properties of SPFA, especially its tolerance of remarkably high pH, SDS and H2O2, suggest it has potential for biotechnological applications. [ABSTRACT FROM AUTHOR]

Abstract The food enzyme subtilisin (EC 3.4.21.62) is produced with the genetically modified Bacillus licheniformis strain NZYM‐CB by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme is considered free from viable cells of the production organism and its DNA. It is intended to be used in six food manufacturing processes. The dietary exposure to the food enzyme‐TOS was estimated to be up to 0.722 mg TOS/kg body weight (bw) per day in European populations. The production strain of the food enzyme fulfils the requirements for the qualified presumption of safety approach to safety assessment. As no other concerns arising from the manufacturing process were identified, the Panel considered that toxicological tests were not required for the assessment of this food enzyme. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and 20 matches were found, including two food allergens (melon and pomegranate). The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, particularly in individuals sensitised to melon and pomegranate, but would not exceed the risk from consumption of melon or pomegranate. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Abstract The food enzyme subtilisin (EC 3.4.21.62) is produced with the non‐genetically modified Bacillus licheniformis strain NZYM‐CX by Novozymes A/S. The production strain met the requirements for the qualified presumption of safety (QPS) approach. The food enzyme is intended to be used in eight food manufacturing processes: processing of cereals and other grains for the production of brewed products; processing of dairy products for the production of modified milk proteins and flavouring preparations; processing of plant‐ and fungal‐derived products for the production of plant‐based analogues of milk and milk products, protein hydrolysates and edible oils from algae; processing of meat and fish products for the production of protein hydrolysates; processing of yeast and yeast products. Since residual amounts of total organic solids (TOS) are removed in the production of edible oils from algae, dietary exposure was calculated only for the remaining seven food manufacturing processes. Exposure was estimated to be up to 2.393 mg TOS/kg body weight (bw) per day in European populations. As the production strain qualified for the QPS approach and no issues of concern arose from the production process of the food enzyme, the Panel considered that toxicological studies were unnecessary. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was performed, and a total of 20 matches were found, 17 to respiratory allergens, two to food allergens (found in muskmelon and pomegranate) and one to a contact allergen. The Panel considered that the risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, especially in individuals sensitised to muskmelon or pomegranate, but would not exceed the risk of consuming these foods. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

The food enzyme subtilisin (serine endopeptidase, EC 3.4.21.62) is produced with the non‐genetically modified Bacillus paralicheniformis strain DP‐Dzx96 by Genencor International B.V. The food enzyme was considered free from viable cells of the production organism. The food enzyme is intended to be used in five food manufacturing processes: production of protein hydrolysates from plants and fungi, production of protein hydrolysates from meat and fish proteins, production of cooked rice, production of modified meat and fish products, and yeast processing. The production strain of the food enzyme contains known antimicrobial resistance genes. Bacitracin, a medically important antimicrobial, was detected in the food enzyme. The presence of bacitracin represents a risk for the development of antimicrobial resistant bacteria. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and three matches with respiratory and two matches with food allergens were found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to muskmelon or pomegranate, cannot be excluded, but would not exceed the risk of consuming these foods. Due to the presence of bacitracin, the Panel concluded that the food enzyme subtilisin produced with the non‐genetically modified Bacillus paralicheniformis strain DP‐Dzx96 cannot be considered safe. [ABSTRACT FROM AUTHOR]

The constant selection and propagation of multi‐resistant Plasmodium sp. parasites require the identification of new antimalarial candidates involved in as‐yet untargeted metabolic pathways. Subtilisin‐like protease 1 (SUB1) belongs to a new generation of drug targets because it plays a crucial role during egress of the parasite from infected host cells at different stages of its life cycle. SUB1 is characterized by an unusual pro‐region that tightly interacts with its cognate catalytic domain, thus precluding 3D structural analysis of enzyme–inhibitor complexes. In the present study, to overcome this limitation, stringent ionic conditions and controlled proteolysis of recombinant full‐length P. vivax SUB1 were used to obtain crystals of an active and stable catalytic domain (PvS1Cat) without a pro‐region. High‐resolution 3D structures of PvS1Cat, alone and in complex with an α‐ketoamide substrate‐derived inhibitor (MAM‐117), showed that, as expected, the catalytic serine of SUB1 formed a covalent bond with the α‐keto group of the inhibitor. A network of hydrogen bonds and hydrophobic interactions stabilized the complex, including at the P1′ and P2′ positions of the inhibitor, although P′ residues are usually less important in defining the substrate specificity of subtilisins. Moreover, when associated with a substrate‐derived peptidomimetic inhibitor, the catalytic groove of SUB1 underwent significant structural changes, particularly in its S4 pocket. These findings pave the way for future strategies for the design of optimized SUB1‐specific inhibitors that may define a novel class of antimalarial candidates. [ABSTRACT FROM AUTHOR]

Abstract The food enzyme subtilisin (serine endopeptidase, EC 3.4.21.62) is produced with the non‐genetically modified Bacillus paralicheniformis strain DP‐Dzx96 by Genencor International B.V. The food enzyme was considered free from viable cells of the production organism. The food enzyme is intended to be used in five food manufacturing processes: production of protein hydrolysates from plants and fungi, production of protein hydrolysates from meat and fish proteins, production of cooked rice, production of modified meat and fish products, and yeast processing. The production strain of the food enzyme contains known antimicrobial resistance genes. Bacitracin, a medically important antimicrobial, was detected in the food enzyme. The presence of bacitracin represents a risk for the development of antimicrobial resistant bacteria. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and three matches with respiratory and two matches with food allergens were found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to muskmelon or pomegranate, cannot be excluded, but would not exceed the risk of consuming these foods. Due to the presence of bacitracin, the Panel concluded that the food enzyme subtilisin produced with the non‐genetically modified Bacillus paralicheniformis strain DP‐Dzx96 cannot be considered safe.

The aim of the present study was the characterisation of three true subtilisins and one phylogenetically intermediate subtilisin from halotolerant and halophilic microorganisms. Considering the currently growing enzyme market for efficient and novel biocatalysts, data mining is a promising source for novel, as yet uncharacterised enzymes, especially from halophilic or halotolerant Bacillaceae, which offer great potential to meet industrial needs. Both halophilic bacteria Pontibacillus marinus DSM 16465T and Alkalibacillus haloalkaliphilus DSM 5271T and both halotolerant bacteria Metabacillus indicus DSM 16189 and Litchfieldia alkalitelluris DSM 16976T served as a source for the four new subtilisins SPPM, SPAH, SPMI and SPLA. The protease genes were cloned and expressed in Bacillus subtilis DB104. Purification to apparent homogeneity was achieved by ethanol precipitation, desalting and ion-exchange chromatography. Enzyme activity could be observed between pH 5.0–12.0 with an optimum for SPPM, SPMI and SPLA around pH 9.0 and for SPAH at pH 10.0. The optimal temperature for SPMI and SPLA was 70 °C and for SPPM and SPAH 55 °C and 50 °C, respectively. All proteases showed high stability towards 5% (w/v) SDS and were active even at NaCl concentrations of 5 M. The four proteases demonstrate potential for future biotechnological applications. Key points: • Halophilic and halotolerant Bacillaceae are a valuable source of new subtilisins. • Four new subtilisins were biochemically characterised in detail. • The four proteases show potential for future biotechnological applications. [ABSTRACT FROM AUTHOR]

The food enzyme subtilisin (serine endopeptidase, EC 3.4.21.62) is produced with the non‐genetically modified microorganism Bacillus paralicheniformis strain LMG S‐30155 by ENMEX SA de CV, now part of Kerry Food Ingredients (Cork) Ltd. The food enzyme is intended to be used in oil production, hydrolysis of vegetable/microbial/animal proteins, yeast processing and production of flavouring preparations. The production strain of the food enzyme contains known antimicrobial resistance genes and genes involved in bacitracin biosynthesis. Consequently, it does not fulfil the requirements for the QPS approach to safety assessment. Bacitracin was detected in the food enzyme and the ■■■■■ The presence of bacitracin, a medically important antimicrobial, in the food enzyme represents a risk for the development of resistance in bacteria. Due to the presence of bacitracin, the Panel concluded that the food enzyme subtilisin produced with the non‐genetically modified Bacillus paralicheniformis strain LMG S‐30155 cannot be considered safe. [ABSTRACT FROM AUTHOR]

The bacterial protease inhibitor domains known as Streptomyces subtilisin inhibitors (SSI) are rarely found in fungi. Genome analysis of a fungal pathogen, Choanephora cucurbitarum KUS-F28377, revealed 11 SSI-like domains that are horizontally transferred and sequentially diverged during evolution. We investigated the molecular function of fungal SSI-like domains of C. cucurbitarum, designated "choanepins." Among the proteins tested, only choanepin9 showed inhibitory activity against subtilisin as the target protease, accounting for 47% of the inhibitory activity of bacterial SSI. However, the binding affinity (expressed as the dissociation constant [Kd ]) of choanepin9 measured via microscale thermophoresis was 21 nM, whereas that for bacterial SSI is 34 nM. The trend of binding and inhibitory activity suggests that the two inhibitors exhibit different inhibitory mechanisms for subtilisin protease. Interestingly, choanepin9 was identified as a monomer in studies in vitro, whereas bacterial SSI is a homodimer. Based on these observations, we constructed a monomeric bacterial SSI protein with decreased binding affinity to abrogate its inhibitory activity. By altering the reactive sites of choanepin9 deduced from the P1 and P4 sites of bacterial SSI, we reestablished that these residues in choanepins are also crucial for modulating inhibitory activity. These findings suggest that the fungal SSI evolved to target specific cognate proteases by altering the residues involved in inhibitory reactivity (reactive sites) and binding affinity (structural integrity). The function of fungal SSI proteins identified in this study provides not only a clue to fungal pathogenesis via protease inhibition but also a template for the design of novel serine protease inhibitors.IMPORTANCE Until recently, Streptomyces subtilisin inhibitors (SSI) were reported and characterized only in bacteria. We found SSI-like domains in a plant-pathogenic fungus, Choanephora cucurbitarum KUS-F28377, which contains 11 sequentially diverged SSI-like domains. None of these fungal SSI-like domains were functionally characterized before. The active form of fungal SSI-like protein is a monomer, in contrast to the homodimeric bacterial SSI. We constructed a synthetic monomer of bacterial SSI to demonstrate the modulation of its activity based on structural integrity and not reactive sites. Our results suggest the duplication and divergence of SSI-like domains of C. cucurbitarum within the genome to inhibit various cognate proteases during evolution by modulating both binding and reactivity. The molecular functional characterization of fungal SSI-like domains will be useful in understanding their biological role and future biotechnological applications.

Dermatophytes are responsible for the majority of human and animal cutaneous mycoses. Proteases secreted by dermatophytes offer potential virulence factors. Multiple proteases have been discovered as possible virulence factors. In Iran, however, no human nor animal isolates of Trichophyton mentagrophytes have been examined for the presence of the subtilisin (SUB) gene family. Therefore, we investigated the existence of SUB1-7 genes in T. mentagrophytes isolated from dermatophytosis lesions in humans and animals. Ten and eight molecularly verified T. mentagrophytes isolates obtained from human and bovine dermatophytosis, respectively, were examined for the presence of SUB genes. In two multiplex PCR panels, all T. mentagrophytes strains were examined. The initial multiplex PCR primers detected the presence of SUB1, SUB4, SUB5, and SUB6. The second multiplex PCR panel includes SUB2, SUB3, and SUB7 specific primers. DNA sequencing of the ribosomal internal transcribed spacer (ITS) region has been used to positively identify all T. mentagrophytes isolates (rDNA). All T. mentagrophytes strains tested positive for SUB1, SUB2, SUB4, SUB6, and SUB7. While SUB5 was not found in any of the isolates, SUB3 was present in 90% of human and 100% of animal dermatophytosis samples. The presence of dermatophyte virulence factors, or SUB genes, is indicative of the existence of many dermatophyte species that share a common ancestor. However, the fact that our clinical isolates lacked the SUB5 gene suggests that not all SUB genes contribute to pathogenesis and infection. [ABSTRACT FROM AUTHOR]

Abstract The food enzyme subtilisin (serine endopeptidase, EC 3.4.21.62) is produced with the non‐genetically modified microorganism Bacillus paralicheniformis strain LMG S‐30155 by ENMEX SA de CV, now part of Kerry Food Ingredients (Cork) Ltd. The food enzyme is intended to be used in oil production, hydrolysis of vegetable/microbial/animal proteins, yeast processing and production of flavouring preparations. The production strain of the food enzyme contains known antimicrobial resistance genes and genes involved in bacitracin biosynthesis. Consequently, it does not fulfil the requirements for the QPS approach to safety assessment. Bacitracin was detected in the food enzyme and the ■■■■■ The presence of bacitracin, a medically important antimicrobial, in the food enzyme represents a risk for the development of resistance in bacteria. Due to the presence of bacitracin, the Panel concluded that the food enzyme subtilisin produced with the non‐genetically modified Bacillus paralicheniformis strain LMG S‐30155 cannot be considered safe.

Nattokinase (NK) is a protease derived from food used mainly in the Japanese diet that has several properties. The main activity is related to improving fibrinolytic activities. Other activities have been demonstrated in the regulation of blood pressure by the action toward angiotensin proteases and in the antiplatelet activities. NK can be given orally and reaches its maximal concentration after 12 hours. In addition, an antithrombotic activity based on various NK activities has been proposed. First, increased fibrinolytic activity increases thrombus dissolution and/or the formation of atherosclerotic plaques; second, its enhanced antiplatelet action adds to clot dissolution. All activities have been studied in animals and humans in vitro and in vivo. Relevant adverse effects of NK therapy have not been described, however clinical experience is restricted to case series and volunteers and is not based on clinical studies, thus clinical trials are required to confirm.

Subtilisin proteases, found in all organisms, are enzymes important in the post-translational steps of protein processing. In Leishmania major and L. donovani, this enzyme has been described as essential to their survival; however, few compounds that target subtilisin have been investigated for their potential as an antileishmanial drug. In this study, we first show, by electron microscopy and flow cytometry, that subtilisin has broad localization throughout the cytoplasm and membrane of the parasite in the promastigote form with foci in the flagellar pocket. Through in silico analysis, the similarity between subtilisin of different Leishmania species and that of humans were determined, and based on molecular docking, we evaluated the interaction capacity of a serine protease inhibitor against both life cycle forms of Leishmania. The selected inhibitor, known as PF-429242, has already been used against the dengue virus, arenaviruses, and the hepatitis C virus. Moreover, it proved to have antilipogenic activity in a mouse model and caused hypolipidemia in human cells in vitro. Here, PF-429242 significantly inhibited the growth of L. amazonensis promastigotes of four different strains (IC50 values = 3.07 ± 0.20; 0.83 ± 0.12; 2.02 ± 0.27 and 5.83 ± 1.2 µM against LTB0016, PH8, Josefa and LV78 strains) whilst having low toxicity in the host macrophages (CC50 = 170.30 µM). We detected by flow cytometry that there is a greater expression of subtilisin in the amastigote form; however, PF-429242 had a low effect against this intracellular form with an IC50 of >100 µM for intracellular amastigotes, as well as against axenic amastigotes (94.12 ± 2.8 µM for the LV78 strain). In conclusion, even though PF-429242 does not affect the intracellular forms, this drug will serve as a tool to explore pharmacological and potentially leishmanicidal targets.

Proteases, catalysing protein hydrolysis, and arylsulfatases, catalysing sulfate-ester hydrolysis, are key microbial enzymes for N and S mineralization in soil. However, knowledge gaps remain regarding the effect of crop successions and seasonal and interannual meteorological variations on microbial communities responsible for those activities. Here, we compared the effect of six cropping sequences on the abundance and activity of microbial communities involved in proteolysis and sulfate-ester hydrolysis in northern France over four years, with two sampling dates per year. Crop sequences impacted soil microbial communities involved in proteolysis but not those involved in sulfate-ester hydrolysis. Oilseed rape following wheat presented a higher abundance of fungal 18S rDNA, culturable bacteria and alkaline metalloprotease genes and higher protease activity than other crop sequences (wheat following oilseed rape or pea, barley following wheat and pea following barley). Net N and S mineralization was not impacted by the cropping sequence. However, interannual variability of microbial parameters was large, and largely overcame the effect of crop sequences. Precipitation variability between years was the likely cause of this effect. In conclusion, the interaction between current crop, previous crops and yearly meteorology can strongly impact the soil microbial communities in agroecosystems. [ABSTRACT FROM AUTHOR]

Due to its thermostability and high pH compatibility, subtilisin is most known for its role as an additive for detergents in which it is categorized as a serine protease according to MEROPS database. Subtilisin is typically isolated from various bacterial species of the Bacillus genus such as Bacillus subtilis, B. amyloliquefaciens, B. licheniformis, and various other organisms. It is composed of 268–275 amino acid residues and is initially secreted in the precursor form, preprosubtilisin, which is composed of 29‐residues signal peptide, 77‐residues propeptide, and 275‐residues active subtilisin. Subtilisin is known for the presence of high and low affinity calcium binding sites in its structure. Native subtilisin has general properties of thermostability, tolerance to neutral to high pH, broad specificity, and calcium‐dependent stability, which contribute to the versatility of subtilisin applicability. Through protein engineering and immobilization technologies, many variants of subtilisin have been generated, which increase the applicability of subtilisin in various industries including detergent, food processing and packaging, synthesis of inhibitory peptides, therapeutic, and waste management applications. [ABSTRACT FROM AUTHOR]

Pine processionary caterpillar nests are made from raw silk. Fibroin protein is the main component of silk which, in the case of pine processionary caterpillar, has some unusual properties such as a higher resistance to chemical hydrolysis. Isolation of microorganisms naturally present in silk nests led to identification of Bacillus licheniformis and Pseudomonas aeruginosa strains that in a defined minimal medium were able to carry out extensive silk biodegradation. A LasB elastase-like protein from P. aeruginosa was shown to be involved in silk biodegradation. A recombinant form of this protein expressed in Escherichia coli and purified by affinity chromatography was able to efficiently degrade silk in an in vitro assay. However, silk biodegradation by B. licheniformis strain was mediated by a SubC subtilisin-like protease. Homologous expression of a subtilisin Carlsberg encoding gene (subC) allowed faster degradation compared to the biodegradation kinetics of a wildtype B. licheniformis strain. This work led to the identification of new enzymes involved in biodegradation of silk materials, a finding which could lead to possible applications for controlling this pest and perhaps have importance from sanitary and biotechnological points of view. [ABSTRACT FROM AUTHOR]

Microbial Vpr-like proteases are extracellular multidomain subtilases with diverse functions and can form oligomers, but their maturation and oligomerization mechanisms remain to be elucidated. Here, we report a novel Vpr-like protease (BTV) from thermophilic bacterium Brevibacillus sp. WF146. The BTV precursor comprises a signal peptide, an N-terminal propeptide, a subtilisin-like catalytic domain with an inserted protease-associated (PA) domain, two tandem fibronectin type III domains (Fn1 and Fn2), and a C-terminal propeptide. The BTV proform (pro-BTV) could be autoprocessed into the mature form (mBTV) via two intermediates lacking the N- or C-terminal propeptide, respectively, and the C-terminal propeptide delays the autocatalytic maturation of the enzyme. By comparison, pro-BTV is more efficiently processed into mBTV by protease TSS from strain WF146. Purified mBTV is a Ca21-dependent thermostable protease, showing optimal activity at 60°C and retaining more than 60% of activity after incubation at 60°C for 8 h. The PA domain is important for enzyme stability and contributes to the substrate specificity of BTV by restricting the access of protein substrates to the active site. The proform and mature form of BTV exist as a monomer and a homodimer, respectively, and the dimerization is mediated by the Fn1 and Fn2 domains. The N-terminal propeptide of BTV not only acts as intramolecular chaperone and enzymatic inhibitor but also inhibits the homodimerization of the enzyme. The removal of the N-terminal propeptide leads to a structural adjustment of the enzyme and thus promotes enzyme dimerization. [ABSTRACT FROM AUTHOR]

Abstract Using parameters of optimal conditions from laboratory experiments often results in the loss of significant time and resources when trying to scale up the process. In this study, the comparison of results of laboratory and semi-industrial experiments of enzymatic hydrolysis of soy protein isolate is considered. The kinetics of peptides accumulation was investigated by colorimetric method in both microtube (volume reaction is 0.7 ml, 7.14 mg/ml of substrate, incubation in solid state thermostat) and industrial homogenizer (volume reaction is 4,000 ml, 100 mg/ml of substrate, rotor–stator type mixer). The enzyme preparation Protosubtilin G3x (main component is subtilisin) was used as an analogue of the Alcalase preparation, which is already widely used in the food industry. It was found that the pH and the number of proteolytic units in the reaction mixture of both scales had slightly different results of the kinetics, while the temperature showed significantly one. The laboratory scale of the reaction had a wide range of optimal temperature (40–60 $$^{\circ }$$ ∘ C, 30 $$^{\circ }$$ ∘ C showed slowest rate of kinetics reaction), whereas the semi-industrial scale had 50 $$^{\circ }$$ ∘ C of optimal temperature (30, 40, 60 $$^{\circ }$$ ∘ C had the same kinetics). It also was found that maintaining the pH value of the reaction mixture was not mandatory. The obtained results indicate the need to refine the process conditions using semi-industrial experiments before attracting industrial-scale resources. In the case of selection of conditions for the hydrolysis of soy protein isolate in production, it is necessary first of all to take into account the reaction temperature as the most irreproducible parameter when scaling. Graphical Abstract

The recent identification of catalytically active peptidylglycine α-amidating monooxygenase (PAM) in Chlamydomonas reinhardtii, a unicellular green alga, suggested the presence of a PAM-like gene and peptidergic signaling in the last eukaryotic common ancestor (LECA). We identified prototypical neuropeptide precursors and essential peptide processing enzymes (subtilisin-like prohormone convertases and carboxypeptidase B-like enzymes) in the C.reinhardtii genome. Reasoning that sexual reproduction by C. reinhardtii requires extensive communication between cells, we used mass spectrometry to identify proteins recovered from the soluble secretome of mating gametes, and searched for evidence that the putative peptidergic processing enzymes were functional. After fractionation by SDS-PAGE, signal peptide-containing proteins that remained intact, and those that had been subjected to cleavage, were identified. The C. reinhardtii mating secretome contained multiple matrix metalloproteinases, cysteine endopeptidases, and serine carboxypeptidases, along with one subtilisin-like proteinase. Published transcriptomic studies support a role for these proteases in sexual reproduction. Multiple extracellular matrix proteins (ECM) were identified in the secretome. Several pherophorins, ECM glycoproteins homologous to the Volvox sex-inducing pheromone, were present; most contained typical peptide processing sites, and many had been cleaved, generating stable N- or C-terminal fragments. Our data suggest that subtilisin endoproteases and matrix metalloproteinases similar to those important in vertebrate peptidergic and growth factor signaling play an important role in stage transitions during the life cycle of C.reinhardtii.

Nattokinase (NK) is a protease derived from food used mainly in the Japanese diet that has several properties. The main activity is related to improving fibrinolytic activities. Other activities have been demonstrated in the regulation of blood pressure by the action toward angiotensin proteases and in the antiplatelet activities. NK can be given orally and reaches its maximal concentration after 12 hours. In addition, an antithrombotic activity based on various NK activities has been proposed. First, increased fibrinolytic activity increases thrombus dissolution and/or the formation of atherosclerotic plaques; second, its enhanced antiplatelet action adds to clot dissolution. All activities have been studied in animals and humans in vitro and in vivo. Relevant adverse effects of NK therapy have not been described, however clinical experience is restricted to case series and volunteers and is not based on clinical studies, thus clinical trials are required to confirm. [ABSTRACT FROM AUTHOR]

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

Colour is considered as a foremost parameter for physical appearance and personal identification. The distinct coloration of skin, hair and eyes of humas are categorized based on the natural pigment melanin which is the final product of melanogenesis. Melanin establishes a primary protection to the skin by inducing photo protection. But the rapid mechanism of absorbing free radicals from the cytoplasm is defensive against UV light, thereby its enormous production and accretion leads to perturbing skin ailments. During the process of melanogenesis the core enzyme tyrosinase is employed and moderated by a main transcription factor called microphthalmia associated transcription factor (MITF), which is enhanced by both tyrosinase-related 1 and 2 proteins (TRP-1/TRP-2). Amending this physiological process is considered to be the foremost mechanism in improving skin fairness. Conversely, there are numerous cosmetics commercially available to depigment skin colour, the alarm of adverse effects and reoccurrence of hyperpigmentation prevails as a hitch. Synthetic skin products cause remedy with adverse effects and hence there is a high demand for novel skin colouring agent. This work lays a pedal stone in promoting the appliance of a naturally derived protein subtilisin secluded from soil isolates of Bacillus sp. in cosmetic industry as skin whitening aspect. The extraction of subtilisin was detected by biochemical and HPLC assays coordinated with anti-melanogenesis activity by in vitro studies. Further, synchronization of molecular docking studies hoisted the protein as an effective ligand targeting TRP1 and TRP2 with energy minimization of -675569 KJ/Mol and -36957KJ/Mol. [ABSTRACT FROM AUTHOR]

Measuring the activity of proteases is essential for investigating both the physiological functions and commercial applications of these enzymes. In contrast to the numerous protease assays that are based on chromogenic or fluorogenic peptide substrates, there is a lack of approaches to monitor degradation of proteins in real time. Here we report a protease assay where SYPRO Orange is employed as a fluorogenic probe to follow proteolysis. The functionality of the assay was demonstrated with the two subtilases of varying thermostability, using four different protein substrates. The assay is compatible with a real-time PCR instrument which allows continuous fluorescence measurements in low-volume samples even at high temperatures. This makes the assay especially suitable for high-throughput characterization of thermostable proteases., 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 Authors. Published by Elsevier Inc. All rights reserved.)

Background: Beyond therapeutic implications, PCSK9 (proprotein convertase subtilisin/kexin 9) has emerged as a promising cardiovascular biomarker. The exact role of PCSK9 in the setting of acute ST-elevation myocardial infarction (STEMI) is incompletely understood. We aimed to investigate the association of PCSK9 with ischemia-reperfusion injury, visualized by cardiac magnetic resonance imaging, in patients with STEMI revascularized by primary percutaneous coronary intervention (PCI)., Methods: In this prespecified substudy from the prospective MARINA-STEMI (NCT04113356) registry, we included 205 patients with STEMI. PCSK9 concentrations were measured from venous blood samples by an immunoassay 24 and 48 hours after PCI. The primary end point was defined as presence of intramyocardial hemorrhage according to cardiac magnetic resonance T2* mapping. Secondary imaging end points were the presence of microvascular obstruction (MVO) and infarct size. The clinical end point was the occurrence of major adverse cardiac events., Results: We observed a significant increase in PCSK9 levels from 24 to 48 hours (268-304 ng/mL; P <0.001) after PCI. PCSK9 24 hours after PCI did not show any relation to intramyocardial hemorrhage, MVO, and infarct size (all P >0.05). PCSK9 concentrations 48 hours post-STEMI were higher in patients with intramyocardial hemorrhage (333 versus 287 ng/mL; P =0.004), MVO (320 versus 292 ng/mL; P =0.020), and large infarct size (323 versus 296 ng/mL; P =0.013). Furthermore, patients with increased PCSK9 levels >361 ng/mL at 48 hours were more likely to experience major adverse cardiac events (15% versus 8%; P =0.002) during a median follow-up of 12 months., Conclusions: In patients with STEMI, a significant increase in PCSK9 was observed from 24 to 48 hours after PCI. While PCSK9 levels after 24 hours were not related to myocardial or microvascular injury, PCSK9 after 48 hours was significantly associated with intramyocardial hemorrhage, MVO, and infarct size as well as worse subsequent clinical outcomes., Registration: URL: https://www.clinicaltrials.gov; Unique identifier; NCT04113356., Competing Interests: None.

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

The efficacy and safety of a novel Subtilisin protease from a Bacillus sp. produced in Bacillus licheniformis was investigated in broiler chickens, and a range of toxicological tests, respectively. The B. licheniformis production strain culture supernatant was not found cytotoxic in a Vero cell assay. Subtilisin was non-mutagenic and non-clastogenic in in-vitro tests, and did not exhibit irritating potential to the eye or skin in ex-vivo/in-vitro models. Oral administration of Subtilisin to rats did not cause any adverse effects in a 13-week sub-chronic toxicity study. In addition, a 35-day dose response broiler performance trial conducted with Subtilisin (30,000 and 60,000 NFP/kg diet), showed a significant linear improvement in both body weight gain and feed conversion ratio up to 35 days of protease supplementation.In conclusion, there are no safety concerns using this novel Subtilisin as a feed additive, and the protease is efficient in improving broiler growth performance, making it a good candidate for use as a feed additive.

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.

In this review, we discuss the progress in the study and modif ication of subtilisin proteases. Despite longstanding applications of microbial proteases and a large number of research papers, the search for new protease genes, the construction of producer strains, and the development of methods for their practical application are still relevant and important, judging by the number of citations of the research articles on proteases and their microbial producers. This enzyme class represents the largest share of the industrial production of proteins worldwide. This situation can explain the high level of interest in these enzymes and points to the high importance of designing domestic technologies for their manufacture. The review covers subtilisin classif ication, the history of their discovery, and subsequent research on the optimization of their properties. An overview of the classes of subtilisin proteases and related enzymes is provided too. There is a discussion about the problems with the search for (and selection of) subtilases from natural strains of various microorganisms, approaches to (and specifics of) their modif ication, as well as the relevant genetic engineering techniques. Details are provided on the methods for expression optimization of industrial subtilases of various strains: the details of the most important parameters of cultivation, i. e., composition of the media, culture duration, and the inf luence of temperature and pH. Also presented are the results of the latest studies on cultivation techniques: submerged and solid-state fermentation. From the literature data reviewed, we can conclude that native enzymes (i. e., those obtained from natural sources) currently hardly have any practical applications because of the decisive advantages of the enzymes modified by genetic engineering and having better properties: e. g., thermal stability, general resistance to detergents and specif ic resistance to various oxidants, high activity in various temperature ranges, independence from metal ions, and stability in the absence of calcium. The vast majority of subtilisin proteases are expressed in producer strains belonging to different species of the genus Bacillus. Meanwhile, there is an effort to adapt the expression of these enzymes to other microbes, in particular species of the yeast Pichia pastoris.

In response to high-salt conditions, haloarchaea export most secretory proteins through the Tat pathway in folded states; however, it is unclear why some haloarchaeal proteins are still routed to the Sec pathway. SptE is an extracellular subtilase of Natrinema sp. strain J7-2. Here, we found that SptE precursor comprises a Sec signal peptide, an N-terminal propeptide, a catalytic domain, and a long C-terminal extension (CTE) containing seven domains (C1 to C7). SptE is produced extracellularly as a mature form (M180) in strain J7-2 and a proform (Î"S) in the Î"sptA mutant strain, indicating that halolysin SptA mediates the conversion of the secreted proform into M180. The proper folding of Î"S is more efficient in the presence of NaCl than KCl. Î"S requires SptA for cleavage of the N-terminal propeptide and C-terminal C6 and C7 domains to generate M180, accompanied by the appearance of autoprocessing product M120 lacking C5. At lower salinities or elevated temperatures, M180 and M120 could be autoprocessed into M90, which comprises the catalytic and C1 domains and has a higher activity than M180. When produced in Haloferax volcanii, SptE could be secreted as a properly folded proform, but its variant (TSptE) with a Tat signal peptide does not fold properly and suffers from severe proteolysis extracellularly; meanwhile, TSptE is more inclined to aggregate intracellularly than SptE. Systematic domain deletion analysis reveals that the long CTE is an important determinant for secretion of SptE via the Sec rather than Tat pathway to prevent enzyme aggregation before secretion. [ABSTRACT FROM AUTHOR]

Using parameters of optimal conditions from laboratory experiments often results in the loss of significant time and resources when trying to scale up the process. In this study, the comparison of results of laboratory and semi-industrial experiments of enzymatic hydrolysis of soy protein isolate is considered. The kinetics of peptides accumulation was investigated by colorimetric method in both microtube (volume reaction is 0.7 ml, 7.14 mg/ml of substrate, incubation in solid state thermostat) and industrial homogenizer (volume reaction is 4,000 ml, 100 mg/ml of substrate, rotor–stator type mixer). The enzyme preparation Protosubtilin G3x (main component is subtilisin) was used as an analogue of the Alcalase preparation, which is already widely used in the food industry. It was found that the pH and the number of proteolytic units in the reaction mixture of both scales had slightly different results of the kinetics, while the temperature showed significantly one. The laboratory scale of the reaction had a wide range of optimal temperature (40–60 ∘ C, 30 ∘ C showed slowest rate of kinetics reaction), whereas the semi-industrial scale had 50 ∘ C of optimal temperature (30, 40, 60 ∘ C had the same kinetics). It also was found that maintaining the pH value of the reaction mixture was not mandatory. The obtained results indicate the need to refine the process conditions using semi-industrial experiments before attracting industrial-scale resources. In the case of selection of conditions for the hydrolysis of soy protein isolate in production, it is necessary first of all to take into account the reaction temperature as the most irreproducible parameter when scaling. [ABSTRACT FROM AUTHOR]

Thraustochytrids have attracted attention due to the high contents of useful lipids and growth rate. Genus Schizochytrium is commonly used for docosahexaenoic acid (DHA) production, while a strain, which produces a high amount of squalene, has been reported in the genus Aurantiochytrium. These organisms are heterotrophic, and Schizochytrium degrades the extracellular macromolecules, e.g., proteins and polysaccharides, as the nutrients. However, the extracellular lytic enzymes are not well-studied yet. Here, we investigated the induction of extracellular proteases of Schizochytrium aggregatum ATCC 28209. A casein-hydrolytic activity was induced in the nitrogen-limited conditions, and that was also detected by zymography after fractionation by non-heat denatured SDS-PAGE. The proteinous band corresponding to the protease activity was analyzed by MALDI-TOF mass spectrometry after digestion with trypsin. The molecular mass data of the protein fragments were compared to the protein database of S. aggregatum ATCC 28209 in the Joint Genome Institute, and we found that the molecular masses of the six peptides were matched with the prediction from the sequence of a protein, ID 63992, which was annotated as a peptidase S8 family protein. Interestingly, we found that a paralogous protein, ID 99856, was encoded by a gene flanking at the downstream of the gene for ID 63992, and the expression of both genes was similarly induced under the nitrogen-limited conditions. These findings may provide us a key to disclose the induction mechanisms of the extracellular lytic enzymes and the function of the proteolytic enzyme for the nutrition acquisition in thraustochytrids. [ABSTRACT FROM AUTHOR]

Background and objectives: Industrial wastewater is worldwide health concern. Microorganisms present in the environment have an important role in the biodegradation of lipids, fats and proteins from wastewater. In this regard, microbial lipases and proteases are interesting research targets because of high stability, broad substrate specificity, high yields and availability. In this study, we analyze sequences encoding lipase of Pseudomonas putida and subtilisin of Bacillus subtilis for generation of a new recombinant protein for degradation of environmental contaminations caused by lipids and proteins. Methods: In this study, sequences of the genes encoding lipase and subtilisin were obtained from GenBank. To predict the 3D structure of the protein, modeling was carried out. The prediction of secondary structure, tertiary structure and solvent accessibility was carried using bioinformatics tools including I-TASSER, GoR4 and ExPasy. Results: The lipase-subtilisin fusion protein was well-characterized by bioinformatical studies with appropriate spatial and secondary structures. The protein had appropriate hydrophilicity, biological half-life and thermal and acidic stability. The codon optimization was performed appropriately. Conclusion: Overall, the bioinformatical analysis of the designed protein showed that the recombinant lipase-subtilisin protein has a stable structure both in vitro and in vivo, a negative normalized B-factor and lipolytic and proteolytic activities, which makes it suitable for treatment of lipid and protein contaminations.

The growth and development of organisms depend on nutrient availability. Dermatophytes must sense nutrient levels and adapt to the host environment to colonize human and animal keratinized tissues. Owing to the clinical importance of the Trichophyton genus, this study compared the expression profile of genes involved in metabolism, cell cycle control, and proteases in two Trichophyton species, Trichophyton rubrum, and Trichophyton interdigitale, in response to nutrients and environmental pH. In addition, we evaluated the activity of enzymes in the tricarboxylic acid, glyoxylate, and methylcitrate cycles. Moreover, the effects of interruption of the transcription factor pacC on T. interdigitale in the same conditions as for the wild-type strain were determined. Our analyses revealed specific responses in each species to the nutritional and pH variation. An improved adaptation of T. interdigitale to keratin was observed, compared with that of T. rubrum. T. rubrum growth in buffered keratin media indicated pH 8.0 as an optimal pH condition for metabolic activity, which differed from that for T. interdigitale. Tricarboxylic acid components in T. rubrum showed increased enzymatic activity and transcript accumulation. In T. interdigitale, a higher activity of enzymes in glyoxylate and methylcitrate cycles was observed, with no direct correlation to the transcriptional profile. T. interdigitale fungal metabolism suggests the requirement of anaplerotic pathways in the late cultivation period. The identified differences between T. rubrum and T. interdigitale may represent determinants for adaptation to the host and the incidence of infection with each species.

: Keratinase enzymes are important virulence factors that play critical roles at the invasive stage of dermatophyte infection. In the present study, we evaluate the presence of genes encoding subtilisins (SUB) in four dermatophyte species includes that isolated from human dermatophytosis. Clinical specimens obtained from patients suspected of dermatophytosis were subjected to direct examination and culture. PCR technique using panfungal primers targeting the ITS region of the rDNA gene was used to confirm the identity of dermatophyte isolates. Finally, specific primers were used to determine the presence of SUB gene families in clinical isolates were evaluated. The prevalence of dermatophytosis in suspected patients was 13.7%. Tinea corporis (37.1%) was the predominant type of dermatophytosis. Totally, the difference reflected the low distribution of SUB1 (50%) and SUB5 (54.2%). In T. mentagrophytes, all isolates have SUB2, SUB3, SUB4, SUB6 and SUB7. As well as 100% of E. floccosum strains have SUB2 and SUB7. The most frequent SUB gene in M. canis isolates were SUB3 (75%). In T. rubrum SUB1 was not detected in any of isolates but SUB2, SUB5 and SUB7 were more prominent. In conclusion, the possessionof SUB genes by different dermatophytes species armed the fungi with virulence factors needed to invade keratinized tissue and cause dermatophytosis. However, the low profile of some SUB genes in our clinical isolates indicates that not all SUB genes are involved in the pathogenesis of dermatophytosis. The characterization of proteins coded by SUB genes remains crucial to providing useful data that may disclose the contributory role of SUB genes in dermatophytes pathogenesis. [ABSTRACT FROM AUTHOR]

Proteases, catalysing protein hydrolysis, and arylsulfatases, catalysing sulfate-ester hydrolysis, are key microbial enzymes for N and S mineralization in soil. However, knowledge gaps remain regarding the effect of crop successions and seasonal and interannual meteorological variations on microbial communities responsible for those activities. Here, we compared the effect of six cropping sequences on the abundance and activity of microbial communities involved in proteolysis and sulfate-ester hydrolysis in northern France over four years, with two sampling dates per year. Crop sequences impacted soil microbial communities involved in proteolysis but not those involved in sulfate-ester hydrolysis. Oilseed rape following wheat presented a higher abundance of fungal 18S rDNA, culturable bacteria and alkaline metalloprotease genes and higher protease activity than other crop sequences (wheat following oilseed rape or pea, barley following wheat and pea following barley). Net N and S mineralization was not impacted by the cropping sequence. However, interannual variability of microbial parameters was large, and largely overcame the effect of crop sequences. Precipitation variability between years was the likely cause of this effect. In conclusion, the interaction between current crop, previous crops and yearly meteorology can strongly impact the soil microbial communities in agroecosystems.

The ribosomally synthesized and post-translationally modified peptide mersacidin is a class II lanthipeptide with good activity against Gram-positive bacteria. The intramolecular lanthionine rings, that give mersacidin its stability and antimicrobial activity, are specific structures with potential applications in synthetic biology. To add the mersacidin modification enzymes to the synthetic biology toolbox, a heterologous expression system for mersacidin in Escherichia coli has recently been developed. While this system was able to produce fully modified mersacidin precursor peptide that could be activated by Bacillus amyloliquefaciens supernatant and showed that mersacidin was activated in an additional proteolytic step after transportation out of the cell, it lacked a mechanism for clean and straightforward leader processing. Here, the protease responsible for activating mersacidin was identified and heterologously produced in E. coli , improving the previously reported heterologous expression system. By screening multiple proteases, the stringency of proteolytic activity directly next to a very small lanthionine ring is demonstrated, and the full two-step proteolytic activation of mersacidin was elucidated. Additionally, the effect of partial leader processing on diffusion and antimicrobial activity is assessed, shedding light on the function of two-step leader processing. [ABSTRACT FROM AUTHOR]