•A protease from a psychrotolerant yeast was characterized.•Protease production was dependent on temperature and medium composition.•Mass spectrometry analysis indicated that the protein belongs to ...the pepsin family.•We propose that the enzyme reported here could be Rodothorulapepsin.
Enzymes from cold-adapted microorganisms are of high interest to industries due to their high activity at low and mild temperatures, which makes them suitable for their use in several processes that either require a supply of exogenous energy or involve the use of heat labile products. In this work, the protease production by the strain Rhodotorula mucilaginosa CBMAI 1528, previously isolated from the Antarctic continent, was optimized, and the purified enzyme analyzed. It was found that protease production was dependent on culture medium composition and growth temperature, being 20 °C and a culture medium containing both glucose and casein peptone (20 and 10 g/L, respectively) the optimal growing conditions in batch as well as in bioreactor. Moreover, mass spectrometry analysis revealed that the enzyme under study has a 100 % sequence identity with the deduced amino acid sequence of a putative aspartic protease from Rhodotorula sp. JG-1b (protein ID: KWU42276.1). This result was confirmed by the decrease of 95 % proteolytic activity by pepstatin A, a specific inhibitor of aspartic proteases. We propose that the enzyme reported here could be Rodothorulapepsin, a protein characterized in 1972 that did not have an associated sequence to date and has been classified as an orphan enzyme.
The A.oryzae strains isolated from coffee were screened to evaluate their potential for production of acid aspartic protease. The strains with highest activity were then subjected to UV-mutagenesis ...and the lethality was determined by spore spread plate method. The mutant strain CPO 025 produced 1.3 × 104 U/ml, which is 1.53 fold higher than the parent with survival rate of 76 ± 2%. The Central Composite Rotatable Design(CCRD) based Response Surface Methodology(RSM) was employed to optimize the parameters for Aspartic protease production. The mutant strain subjected to solid-state fermentation (SSF) using wheat bran as substrate yielded 1820 U/mg specific activity with functional conditions of moisture (35%), inoculum (6.5 CFU/ml), fermentation duration (108 h), and temperature (28 °C). The partially purified proteases by DEAE-Sepharose had attributes with 6082 U/mg specific activity, optimum pH 3.6, temperature 35 °C and MW of 35 kDa. Enhanced activity by β-mercaptoethanol and inhibition by iodoacetic acid confirmed cysteine protease. Application of cysteine protease with cocoa beans upsurged flavour profile compared to the untreated beans. The elevated level of dimethylpyzarine and ketone3,5-Bis(tert-butyl)-4-hydroxy-propiophenone indicated efficient protein hydrolysis with desirable cocoa flavour. Thus cocoa organoleptic enhancement with flavourzyme can camouflage poorly fermented cocoa and economy of the industry.
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•Aspartic protease enzymes were extracted from the A. oryzae.•A. oryzae which was subjected to UV mutagenesis to increase the yield.•35%, moisture, 6.5 cfu/ml inoculum, 108 h time, 35 °C temperature were optimised for high yield.•Enhanced dimethyl pyzarine and ketone 3, 5-Bis (tert-butyl)-4-hydroxy-propiophenone represented desirable flavour.•Aspartic flavourzyme could be value addition in contributing to sustainable cocoa industry.
Tick and mite infestations pose significant challenges to animal health, agriculture, and public health worldwide. The search for effective and environmentally friendly acaricidal agents has led ...researchers to explore natural alternatives. In this study, we investigated the acaricidal potential of the Monotheca buxifolia plant extract against Rhipicephalus microplus ticks and Sarcoptes scabiei mites. Additionally, we employed a computational approach to identify phytochemicals from the extract that could serve as drug candidates against these ectoparasites. The contact bioassay results demonstrated that the M. buxifolia plant extract exhibited significant efficacy against R. microplus and S. scabiei, with higher concentrations outperforming the positive control acaricide permethrin in terms of mite mortality. Time exposure to the extract also showed a positive correlation with better lethal concentration (LC50 and LC90) values. Similarly, the adult immersion test revealed a notable inhibition of tick oviposition via the plant extract, especially at higher concentrations. The two-protein primary structure, secondary structure and stability were predicted using the Expasy’s ProtParam server, SOPMA and SUSUI server, respectively. Using Homology modeling, the 3D structure of the protein was obtained and validated through the ERRAT server, and active sites were determined through the CASTp server. The docking analysis revealed that Alpha-Amyrenyl acetate and alpha-Tocopherol exhibited the highest docking scores for S. scabiei and R. microplus aspartic protease proteins, respectively. These phytochemicals demonstrated strong binding interactions, suggesting their potential as acaricidal drug candidates. In conclusion, the M. buxifolia plant extract displayed significant acaricidal activity against R. microplus and S. scabiei. Moreover, the computational approach identified promising phytochemicals that could serve as potential drug candidates for controlling these ectoparasites.
Abstract
Programmed cell death (PCD) is essential for wood development in trees. However, the determination of crucial factors involved in xylem PCD of wood development is still lacking. Here, two ...Populus trichocarpa typical aspartic protease (AP) genes, AP17 and AP45, modulate xylem maturation, especially fibre PCD, during wood formation. AP17 and AP45 were dominantly expressed in the fibres of secondary xylem, as suggested by GUS expression in APpro::GUS transgenic plants. Cas9/gRNA-induced AP17 or AP45 mutants delayed secondary xylem fibre PCD, and ap17ap45 double mutants showed more serious defects. Conversely, AP17 overexpression caused premature PCD in secondary xylem fibres, indicating a positive modulation in wood fibre PCD. Loss of AP17 and AP45 did not alter wood fibre wall thickness, whereas the ap17ap45 mutants showed a low lignin content in wood. However, AP17 overexpression led to a significant decrease in wood fibre wall thickness and lignin content, revealing the involvement in secondary cell wall synthesis during wood formation. In addition, the ap17ap45 mutant and AP17 overexpression plants resulted in a significant increase in saccharification yield in wood. Overall, AP17 and AP45 are crucial modulators in xylem maturation during wood development, providing potential candidate genes for engineering lignocellulosic wood for biofuel utilization.
A vast ensemble of extracellular proteins influences the development and progression of cancer, shaped and reshaped by a complex network of extracellular proteases. These proteases, belonging to the ...distinct classes of metalloproteases, serine proteases, cysteine proteases, and aspartic proteases, play a critical role in cancer. They often become dysregulated in cancer, with increases in pathological protease activity frequently driven by the loss of normal latency controls, diminished regulation by endogenous protease inhibitors, and changes in localization. Dysregulated proteases accelerate tumor progression and metastasis by degrading protein barriers within the extracellular matrix (ECM), stimulating tumor growth, reactivating dormant tumor cells, facilitating tumor cell escape from immune surveillance, and shifting stromal cells toward cancer-promoting behaviors through the precise proteolysis of specific substrates to alter their functions. These crucial substrates include ECM proteins and proteoglycans, soluble proteins secreted by tumor and stromal cells, and extracellular domains of cell surface proteins, including membrane receptors and adhesion proteins. The complexity of the extracellular protease web presents a significant challenge to untangle. Nevertheless, technological strides in proteomics, chemical biology, and the development of new probes and reagents are enabling progress and advancing our understanding of the pivotal importance of extracellular proteolysis in cancer.
Plasmepsins IX (PMIX) and X (PMX) are essential aspartyl proteases for Plasmodium spp. egress, invasion, and development. WM4 and WM382 inhibit PMIX and PMX in Plasmodium falciparum and P. vivax. WM4 ...inhibits PMX, while WM382 is a dual inhibitor of PMIX and PMX. To understand their function, we identified protein substrates. Enzyme kinetic and structural analyses identified interactions responsible for drug specificity. PMIX and PMX have similar substrate specificity; however, there are distinct differences for peptide and protein substrates. Differences in WM4 and WM382 binding for PMIX and PMX map to variations in the S' region and engagement of the active site S3 pocket. Structures of PMX reveal interactions and mechanistic detail of drug binding important for development of clinical candidates against these targets.
Aspartic proteases use a pair of carboxylic acids to activate water molecules for nucleophilic attack. Here we report a nanoparticle catalyst with a similar catalytic motif capable of generating a ...hydroxide ion in its active site even under acidic reaction conditions. The synthetic enzyme accelerated the hydrolysis of para-nitrophenyl acetate (PNPA) by 91 000 times and could also hydrolyze nonactivated aryl esters at pH 7. The distance between the two acids and, in particular, the flexibility of the catalytic groups in the active site controlled the catalytic efficiency. The synthetic enzyme readily detected the addition of a single methyl on the acyl group of the substrate, as well as the substitution pattern on the phenyl ring.
Abstract
The carnivorous plants in the order Caryophyllales co-opted jasmonate signalling from plant defence to botanical carnivory. However, carnivorous plants have at least 11 independent origins, ...and here we ask whether jasmonate signalling has been co-opted repeatedly in different evolutionary lineages. We experimentally wounded and fed the carnivorous plants Sarracenia purpurea (order Ericales), Cephalotus follicularis (order Oxalidales), Drosophyllum lusitanicum (order Caryophyllales), and measured electrical signals, phytohormone tissue level, and digestive enzymes activity. Coronatine was added exogenously to confirm the role of jasmonates in the induction of digestive process. Immunodetection of aspartic protease and proteomic analysis of digestive fluid was also performed. We found that prey capture induced accumulation of endogenous jasmonates only in D. lusitanicum, in accordance with increased enzyme activity after insect prey or coronatine application. In C. follicularis, the enzyme activity was constitutive while in S. purpurea was regulated by multiple factors. Several classes of digestive enzymes were identified in the digestive fluid of D. lusitanicum. Although carnivorous plants from different evolutionary lineages use the same digestive enzymes, the mechanism of their regulation differs. All investigated genera use jasmonates for their ancient role, defence, but jasmonate signalling has been co-opted for botanical carnivory only in some of them.
Carnivorous plants use jasmonates for plant defence like ordinary plants, but jasmonates were co-opted for botanical carnivory only in some of them.
Plasmodium parasites that cause malaria produce plasmepsins (PMs), pepsin‐like aspartic proteases that are important antimalarial drug targets due to their role in host hemoglobin degradation. The ...enzymes are synthesized as inactive zymogens (pro‐PMs), and the mechanism of their conversion to the active, mature forms has not been clearly elucidated. Our structural investigations of vacuolar pro‐PMs with truncated prosegment (pro‐tPMs) reveal that the formation of the S‐shaped dimer is their innate property. Further structural studies, biochemical analysis, and molecular dynamics simulations indicate that disruption of the Tyr‐Asp loop (121p‐4), coordinated with the movement of the loop L1 (237–247) and helix H2 (101p–113p), is responsible for the extension of the pro‐mature region (harboring the cleavage site). Consequently, under acidic pH conditions, these structural changes result in the dissociation of the dimers to monomers and the protonation of the residues in the prosegment prompts its unfolding. Subsequently, we demonstrated that the active site of the monomeric pro‐tPMs with the unfolded prosegment is accessible for peptide substrate binding; in contrast, the active site is blocked in folded prosegment form of pro‐tPMs. Thus, we propose a novel mechanism of auto‐activation of vacuolar pro‐tPMs that under acidic conditions can form a catalytically competent active site. One monomer cleaves the prosegment of the other one through a trans‐activation process, resulting in formation of mature enzyme. As a result, once a mature enzyme is generated, it leads to the complete conversion of all the inactive pro‐tPMs to their mature form.
Database
Atomic coordinates and structure factors have been submitted in the Protein Data Bank (PDB) under the PDB IDs 6KUB, 6KUC, and 6KUD.
The activation of the truncated proplasmepsin zymogen (pro‐tPMs) in the acidic condition to its mature form entails the conversion of dimeric pro‐tPMs to it monomers. The protonation of the residues of the prosegment leads to its unfolding, resulting in a zymogen that is catalytically active, which initiates the initial cleavage of the prosegment of another zymogen, called trans‐activation. The mature enzyme formed converts all the zymogen to their active mature form.
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