Production of proline and protease with different organic wastes in bacteria (Production proline and protease with organic wastes) / Produção de prolina e protease com diferentes resíduos orgânicos em bactérias (produção de prolina e protease com resíduos orgânicos)

In this study, we investigated the proline and protease production of different bacteria in several organic waste materials. Our aim was to produce proline and protease economically in waste that is abundantly available while reducing its environmental impact. 5 ml of different organic waste materials (OWW: Olive waste water; N.B: Nutrient Broth; EW: Eggshell; PBS: PBS buffer; PLW: Peach leaf wastes; TCW: Turkish coffee wastes; TWW: Tea waste water; WCW: Waste cheese whey; WFO: Waste frying oil) were placed in 10 ml grow tubes, inoculated and incubated for 24 h. Phosphate-buffered saline and 10% solutions of different organic wastes were added. These cultures were subsequently incubated at 37°C for 24 h. Cells were harvested at 24 h for L-proline assay. 1 ml of culture was transferred by pipette into an Eppendorf tube and centrifuged at 14,000 rpm for 20 min at room temperature. Cellular debris was removed by centrifuge and the supernatant was used for proline activity assays. Protease activity was determined using a modified method with casein as the substrate. We found that proline and protease can easily be produced economically using Turkish coffee wastes (TCW), Waste cheese whey (WCW) and Olive waste water (OWW) organic waste. We believe that this study will result in similar research leading to the economical use of these waste materials thus reducing their impact on the environment.

Neste estudo, investigamos a produção de prolina e protease de diferentes bactérias em diversos resíduos orgânicos. Nosso objetivo era produzir prolina e protease economicamente em resíduos que estão disponíveis em abundância, reduzindo seu impacto ambiental. Cinco ml de diferentes materiais de resíduos orgânicos (OWW: resíduos de azeitona; NB: caldo nutriente; EW: casca de ovo; PBS: tampão PBS; PLW: resíduos de folhas de pêssego; TCW: resíduos de café turco; TWW: resíduos de chá; WCW: resíduos de queijo soro de leite; WFO: óleo de fritura residual) foram colocados em tubos de cultivo de 10 ml, inoculados e incubados por 24 horas. Adicionaram-se solução salina tamponada com fosfato e soluções a 10% de diferentes resíduos orgânicos. Essas culturas foram subsequentemente incubadas a 37° C durante 24 h. As células foram colhidas às 24 h para o ensaio de L-prolina. Um ml de cultura foi transferido por pipeta para um tubo Eppendorf e centrifugado a 14.000 rpm, por 20 min, em temperatura ambiente. Os detritos celulares foram removidos por centrifugação e o sobrenadante foi usado para ensaios de atividade de prolina. A atividade da protease foi determinada usando um método modificado com caseína como substrato. Descobrimos que a prolina e a protease podem ser facilmente produzidas economicamente, usando resíduos de café turco (TCW), resíduos de soro de queijo (WCW) e resíduos orgânicos de água de oliva (OWW). Acreditamos que este estudo resultará em pesquisas semelhantes, levando ao uso econômico desses materiais residuais, reduzindo, assim, seu impacto no meio ambiente.

Protease Produced by Endophytic Fungi: A Systematic Review.

The purpose of this systematic review was to identify the available literature of production, purification, and characterization of proteases by endophytic fungi. There are few complete studies that entirely exhibit the production, characterization, and purification of proteases from endophytic fungi. This study followed the PRISMA, and the search was conducted on five databases: PubMed, PMC, Science Direct, Scopus Articles, and Web of Science up until 18 May 2021, with no time or language restrictions. The methodology of the selected studies was evaluated using GRADE. Protease production, optimization, purification, and characterization were the main evaluated outcomes. Of the 5540 initially gathered studies, 15 met the inclusion criteria after a two-step selection process. Only two studies optimized the protease production using statistical design and two reported enzyme purification and characterization. The genus Penicillium and Aspergillus were the most cited among the eleven different genera of endophytic fungi evaluated in the selected articles. Six studies proved the ability of some endophytic fungi to produce fibrinolytic proteases, demonstrating that endophytic fungi can be exploited for the further production of agents used in thrombolytic therapy. However, further characterization and physicochemical studies are required to evaluate the real potential of endophytic fungi as sources of industrial enzymes.

Proteases Production and Chitin Preparation from the Liquid Fermentation of Chitinous Fishery By-Products by Paenibacillus elgii.

Chitinous fishery by-products have great application in the production of various bioactive compounds. In this study, Paenibacillus elgii TKU051, a protease-producing bacterial strain, was isolated using a medium containing 1% squid pens powder (SPP) as the sole carbon/nitrogen (C/N) source. P. elgii TKU051 was found to produce at least four proteases with molecular weights of 100 kDa, 57 kDa, 43 kDa, and 34 kDa (determined by the gelatin zymography method). A P. elgii TkU051 crude enzyme cocktail was optimally active at pH 6-7 and 60 °C. The 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and α-glucosidase inhibitory activity of the hydrolysates obtained from the hydrolysis of shrimp shell powder, shrimp head powder, shrimp meat powder, fish head powder and soya bean powder catalyzed by the P. elgii TkU051 crude enzyme cocktail were also evaluated. P. elgii TKU051 exhibited a high deproteinization capacity (over 94%) on different kinds of shrimp waste (shrimp heads and shells; fresh and cooked shrimp waste; shrimp waste dried by oven and lyophilizer), and the Fourier-transform infrared spectroscopy profile of the chitin obtained from the deproteinization process displayed the characteristic of chitin. Finally, the obtained chitin exhibited an effect comparable to commercial chitin in terms of adsorption against Congo Red (90.48% and 90.91%, respectively). Thus, P. elgii TKU051 showed potential in the reclamation of chitinous fishery by-products for proteases production and chitin extraction.

Induction of amylase and protease as antibiofilm agents by starch, casein, and yeast extract in Arthrobacter sp. CW01.

BACKGROUND: In unfavourable environment, such as nutrient limitation, some bacteria encased themselves into a three dimensional polymer matrix called biofilm. The majority of microbial infections in human are biofilm related, including chronic lung, wound, and ear infections. The matrix of biofilm which consists of extracellular polymeric substances (EPS) causes bacterial colonization on medical implanted device in patients, such as catheter and lead to patient's death. Biofilm infections are harder to treat due to increasing antibiotic resistance compared to planktonic microbial cells and escalating the antibiotic concentration may result into in vivo toxicity for the patients. Special compounds which are non-microbicidal that could inhibit or destroy biofilm formation are called antibiofilm compounds, for example enzymes, anti-quorum sensing, and anti-adhesins. Arthrobacter sp. CW01 produced antibiofilm compound known as amylase. This time our preliminary study proved that the antibiofilm compound was not only amylase, but also protease. Therefore, this research aimed to optimize the production of antibiofilm agents using amylase and protease inducing media. The five types of production media used in this research were brain heart infusion (BHI) (Oxoid), BHI with starch (BHIS), casein with starch (CS), yeast extract with starch (YS), and casein-yeast extract with starch (CYS). Biofilm eradication and inhibition activities were assayed against Pseudomonas aeruginosa (ATCC 27,853) and Staphylococcus aureus (ATCC 25,923). RESULTS: The results showed that different production media influenced the antibiofilm activity. Addition of starch, casein and yeast extract increased the production of amylase and protease significantly. Higher amylase activity would gradually increase the antibiofilm activity until it reached the certain optimum point. It was shown that crude extracts which contained amylase only (BHI, BHIS and YS) had the optimum eradication activity against P. aeruginosa and S. aureus biofilm around 60-70 %. Meanwhile, CS and CYS crude extracts which contained both amylase and protease increased the biofilm eradication activity against both pathogens, which were around 70-90 %. CONCLUSIONS: It was concluded that the combination of amylase and protease was more effective as antibiofilm agents against P. aeruginosa and S. aureus rather than amylase only.

The COP9 signalosome subunit 3 is necessary for early embryo survival by way of a stable protein deposit in mouse oocytes.

Investigations of genes required in early mammalian development are complicated by protein deposits of maternal products, which continue to operate after the gene locus has been disrupted. This leads to delayed phenotypic manifestations and underestimation of the number of genes known to be needed during the embryonic phase of cellular totipotency. Here we expose a critical role of the gene Cops3 by showing that it protects genome integrity during the 2-cell stage of mouse development, in contrast to the previous functional assignment at postimplantation. This new role is mediated by a substantial deposit of protein (94th percentile of the proteome), divided between an exceptionally stable cortical rim, which is prevalent in oocytes, and an ancillary deposit in the embryonic nuclei. Since protein abundance and stability defeat prospects of DNA- or RNA-based gene inactivation in oocytes, we harnessed a classical method next to an emerging method for protein inactivation: antigen masking (for functional inhibition) versus TRIM21-mediated proteasomal degradation, also known as 'Trim away' (for physical removal). Both resulted in 2-cell embryo lethality, unlike the embryos receiving anti-green fluorescent protein. Comparisons between COPS3 protein-targeted and non-targeted embryos revealed large-scale transcriptome differences, which were most evident for genes associated with biological functions critical for RNA metabolism and for the preservation of genome integrity. The gene expression abnormalities associated with COPS3 inactivation were confirmed in situ by the occurrence of DNA endoreduplication and DNA strand breaks in 2-cell embryos. These results recruit Cops3 to the small family of genes that are necessary for early embryo survival. Overall, assigning genes with roles in embryogenesis may be less safe than assumed, if the protein products of these genes accumulate in oocytes: the inactivation of a gene at the protein level can expose an earlier phenotype than that identified by genetic techniques such as conventional gene silencing.

PDIA6 promotes pancreatic cancer progression and immune escape through CSN5-mediated deubiquitination of ß-catenin and PD-L1.

Protein Disulfide Isomerase Family A Member 6 (PDIA6) is an endoplasmic reticulum protein that is capable of catalyzing protein folding and disulfide bond formation. Abnormally elevated expression of PDIA6 has been reported to predict poor outcomes in various cancers. Herein, gain-of- and loss-of-function experiments were performed to investigate how PDIA6 participated in the carcinogenesis of pancreatic cancer (PC). By analyzing the protein expression of PDIA6 in 28 paired PC and para carcinoma specimens, we first found that PDIA6 expression was higher in PC samples. Both the overall survival and disease-free survival rates of PC patients with higher PDIA6 expression were poorer than those with lower PDIA6 (n = 178). Furthermore, knockdown of PDIA6 impaired the malignancies of PC cells - suppressed cell proliferation, invasion, migration, cisplatin resistance, and xenografted tumor growth. PDIA6-silenced PC cells were more sensitive to cytotoxic natural killer (NK) cells. Overexpression of PDIA6 had opposite effects on PC cells. Interestingly, COP9 signalosome subunit 5 (CSN5), a regulator of E3 ubiquitin ligases known to promote deubiquitination of its downstream targets, was demonstrated to interact with PDIA6, and its expression was increased in PC cells overexpressing PDIA6. Additionally, PDIA6 overexpression promoted deubiquitination of ß-catenin and PD-L1 and subsequently upregulated their expression in PC cells. These alterations were partly reversed by CSN5 shRNA. Collectively, the above results demonstrate that PDIA6 contributes to PC progression, which may be associated with CSN5-regulated deubiquitination of ß-catenin and PD-L1. Our findings suggest PDIA6 as a potential target for the treatment of PC.

Application of Co-Culture Technology to Enhance Protease Production by Two Halophilic Bacteria, Marinirhabdus sp. and Marinobacter hydrocarbonoclasticus.

Although axenic microbial cultures form the basis of many large successful industrial biotechnologies, the production of single commercial microbial strains for use in large environmental biotechnologies such as wastewater treatment has proved less successful. This study aimed to evaluate the potential of the co-culture of two halophilic bacteria, Marinirhabdus sp. and Marinobacter hydrocarbonoclasticus for enhanced protease activity. The co-culture was significantly more productive than monoculture (1.6-2.0 times more growth), with Marinobacter hydrocarbonoclasticus being predominant (64%). In terms of protease activity, enhanced total activity (1.8-2.4 times) was observed in the co-culture. Importantly, protease activity in the co-culture was found to remain active over a much broader range of environmental conditions (temperature 25 °C to 60 °C, pH 4-12, and 10-30% salinity, respectively). This study confirms that the co-culturing of halophilic bacteria represents an economical approach as it resulted in both increased biomass and protease production, the latter which showed activity over arange of environmental conditions.

Coproduction of Enzymes and Beta-Glucan by Aspergillus oryzae Using Solid-State Fermentation of Brown Rice.

The effect of medium composition on enzyme and ß-glucan production by Aspergillus oryzae KCCM 12698 was investigated. Brown rice, rice bran, nitrogen, and ascorbic acid are key components of the synthetic medium used in liquid-state fermentation. To determine the optimal concentrations of these components for enzyme and ß-glucan production, we conducted one factor at a time experiments, which showed that the optimal concentrations were 30 g/l brown rice, 30 g/l rice bran, 10 g/l soytone, and 3 g/l ascorbic acid. Pretreatment of brown rice for 60 min prior to inoculation enhanced fungal biomass, while increasing the production of enzymes and ß-glucan using solidstate fermentation. Maximum fungal biomass of 0.76 mg/g, amylase (26,551.03 U/g), protease (1,340.50 U/g), and ß-glucan at 9.34% (w/w) were obtained during fermentation. Therefore, solidstate fermentation of brown rice is a process that could enhance yield and overall production of enzymes and ß-glucan for use in various applications.

Structural-genetic insight and optimization of protease production from a novel strain of Aeromonas veronii CMF, a gut isolate of Chrysomya megacephala.

Structural-genetic characterization of protease producing genes and enzymes from microbial sources are seldom appreciated despite having its substantial utilization in protein engineering or genetic manipulation for biotechnological applications. Aeromonas veronii CMF, a mesophilic bacterium isolated from the gut of Chrysomya megacephala, was found to exhibited significant level of protease activity. For the revelation of genetic potential in relation to protease production, whole genome of this organism was sequenced and analysed while structure-function of different protease enzyme was predicated using various in silico analysis. The 4.5 mb CMF genome was found to encompass various types of protease and mostly they are neutral in nature. Enzyme production was highest in an optimum pH and temperature of 6.0 (32.09 ± 1.015 U/ml) and 35ºC (41.65 ± 1.152 U/ml), respectively. Other culture parameters for optimum production of protease were determined to be inoculum size (1%), incubation period (72 h), shaking condition (125 rpm), carbon and nitrogen source [2% lactose (92.21 ± 3.16 U/ml) and 0.5% urea (163.62 ± 4.31 U/ml), respectively] and effect of surfactants [0.02 mg/ml Tween 80 (174.72 ± 4.48 U/ml)]. Furthermore, A. veronii CMF exhibited significant enzyme production like serine protease (15.22 ± 0.563 U/ml), aspartate protease (33.16 ± 0.762 U/ml) and collagenase (17.26 ± 0.626 U/ml). Genomic information and results of physio-biochemical assays indicate its cost-effective potential use in different enzyme-industry.

Effect of Agitation and Aeration on Keratinase Production in Bioreactors Using Bioprocess Engineering Aspects.

Streptomyces sp. 2M21 was evaluated for keratinase production in bioreactors using chicken feathers. Firstly, optimization of bioengineering parameters (agitation and aeration rates) using Response Surface Methodology was carried out in 2 L bioreactors. Optimized conditions identified by the modified quadratic model were verified as 150 rpm and 1 vvm experimentally corresponding to 351 U/ml of keratinase activity. Moreover, scaling up sequentially to 20 L bioreactors was implemented using constant impeller tip speed and constant mass transfer coefficient as key scale-up parameters. The keratinase activity in 5, 10 and 20 L bioreactors showed similar results with the one of shake flasks (412 U/ml) and 2 L bioreactors (351 U/ml)with respect to the keratinase activity values of 336, 385 and 344 U/ml, respectively. The results suggest keratinase production by evaluating chicken feathers in commercial level. Furthermore, this study has potential to contribute industrial scale production of keratinase by Streptomyces sp. 2M21 using the proposed bioreactor conditions.

Screening for optimal protease producing Bacillus licheniformis strains with polymer-based controlled-release fed-batch microtiter plates.

BACKGROUND: Substrate-limited fed-batch conditions have the favorable effect of preventing overflow metabolism, catabolite repression, oxygen limitation or inhibition caused by elevated substrate or osmotic concentrations. Due to these favorable effects, fed-batch mode is predominantly used in industrial production processes. In contrast, screening processes are usually performed in microtiter plates operated in batch mode. This leads to a different physiological state of the production organism in early screening and can misguide the selection of potential production strains. To close the gap between screening and production conditions, new techniques to enable fed-batch mode in microtiter plates have been described. One of these systems is the ready-to-use and disposable polymer-based controlled-release fed-batch microtiter plate (fed-batch MTP). In this work, the fed-batch MTP was applied to establish a glucose-limited fed-batch screening procedure for industrially relevant protease producing Bacillus licheniformis strains. RESULTS: To achieve equal initial growth conditions for different clones with the fed-batch MTP, a two-step batch preculture procedure was developed. Based on this preculture procedure, the standard deviation of the protease activity of glucose-limited fed-batch main culture cultivations in the fed-batch MTP was ± 10%. The determination of the number of replicates revealed that a minimum of 6 parallel cultivations were necessary to identify clones with a statistically significant increased or decreased protease activity. The developed glucose-limited fed-batch screening procedure was applied to 13 industrially-relevant clones from two B. licheniformis strain lineages. It was found that 12 out of 13 clones (92%) were classified similarly as in a lab-scale fed-batch fermenter process operated under glucose-limited conditions. When the microtiter plate screening process was performed in batch mode, only 5 out of 13 clones (38%) were classified similarly as in the lab-scale fed-batch fermenter process. CONCLUSION: The glucose-limited fed-batch screening process outperformed the usual batch screening process in terms of the predictability of the clone performance under glucose-limited fed-batch fermenter conditions. These results highlight that the implementation of glucose-limited fed-batch conditions already in microtiter plate scale is crucial to increase the precision of identifying improved protease producing B. licheniformis strains. Hence, the fed-batch MTP represents an efficient high-throughput screening tool that aims at closing the gap between screening and production conditions.

Limiting protease production plays a key role in the pathogenesis of the divergent clinical isolates of Staphylococcus aureus LAC and UAMS-1.

Using the USA300, methicillin-resistant Staphylococcus aureus strain LAC, we previously examined the impact of regulatory mutations implicated in biofilm formation on protease production and virulence in a murine sepsis model. Here we examined the impact of these mutations in the USA200, methicillin-sensitive strain UAMS-1. Mutation of agr, mgrA, rot, sarA and sigB attenuated the virulence of UAMS-1. A common characteristic of codY, rot, sigB, and sarA mutants was increased protease production, with mutation of rot having the least impact followed by mutation of codY, sigB and sarA, respectively. Protein A was undetectable in conditioned medium from all four mutants, while extracellular nuclease was only present in the proteolytically cleaved NucA form. The abundance of high molecular weight proteins was reduced in all four mutants. Biofilm formation was reduced in codY, sarA and sigB mutants, but not in the rot mutant. Eliminating protease production partially reversed these phenotypes and enhanced biofilm formation. This was also true in LAC codY, rot, sarA and sigB mutants. Eliminating protease production enhanced the virulence of LAC and UAMS-1 sarA, sigB and rot mutants in a murine sepsis model but did not significantly impact the virulence of the codY mutant in either strain. Nevertheless, these results demonstrate that repressing protease production plays an important role in defining critical phenotypes in diverse clinical isolates of S. aureus and that Rot, SigB and SarA play critical roles in this regard.

Expression and characterization of a novel organic solvent tolerant protease from Bacillus sphaericus DS11.

Organic solvent-tolerant proteases have many applications in the synthesis of peptides. In this study, we have developed a low-cost and convenient method to produce highly concentrated organic solvent-tolerant protease. Organic solvent tolerant protease (OSP) gene from Bacillus sphaericus DS11 was cloned and expressed in Bacillus subtilis WB800. The optimum pH of the recombinant protease was 9.0. The optimum temperature of the recombinant protease was 40 °C. The recombinant protease was purified by ethanol with the yield of (87.33%). The yield of OSP enriched by ethanol was higher than that of by Ni-chelating affinity chromatography, which indicated that precipitation of the recombinant OSP with ethanol is a relatively low-cost and fast method for organic solvent -tolerant protease preparation. These results showed that this enzyme could be very useful in different industrial applications.

Proteomic analysis and optimized production of Alkalihalobacillus patagoniensis PAT 05T extracellular proteases.

Extracellular proteolytic extracts from the haloalkalitolerant strain Alkalihalobacillus patagoniensis PAT 05T have proved highly efficient to reduce wool felting, as part of an ecofriendly treatment suitable for organic wool. In the present study, we identified the extracellular proteases produced by PAT 05T and we optimized its growth conditions for protease production through statistical methods. A total of 191 proteins were identified in PAT 05T culture supernatants through mass spectrometry analysis. Three of the 6 detected extracellular proteases belonged to the serine-endopeptidase family S8 (EC 3.4.21); two of them showed 86.3 and 67.9% identity with an alkaline protease from Bacillus alcalophilus and another one showed 50.4% identity with Bacillopeptidase F. The other 3 proteases exhibited 55.3, 49.4 and 61.1% identity with D-alanyl-D-alanine carboxypeptidase DacF, D-alanyl-D-alanine carboxypeptidase DacC and endopeptidase LytE, respectively. Using a Fractional Factorial Design followed by a Central Composite Design optimization, a twofold increase in protease production was reached. NaCl concentration was the most influential factor on protease production. The usefulness of PAT 05T extracellular proteolytic extracts to reduce wool felting was possible associated with the activity of the serine-endopeptidases closely related to highly alkaline keratinolytic proteases. The other identified proteases could cooperate, improving protein hydrolysis. This study provided valuable information for the exploitation of PAT 05T proteases which have potential for the valorization of organic wool as well as for other industrial applications.

Isolation and Characterization of Serratiopeptidase Producing Bacteria from Mulberry Phyllosphere.

Serratiopeptidase (EC 3.4.24.40), a proteolytic enzyme, is one of the most promising enzymes being used in biopharmaceutical industry. Mulberry phyllosphere, being an unexplored niche for exploration of protease production, was chosen for the present study. Protease producing bacteria were isolated from the tissues of mulberry plant as well as its rhizospheric soil. Two protease producing bacteria belonging to Serratia genus were found to be potential serratiopeptidase producers. Among them, the endophyte, i.e., Serratia marcescens MES-4 presented 95 Units/mL activity, while the soil isolate i.e., Serratia marcescens MRS-11 presented 156 Units/mL activity.

The nucleoid-associated protein IHF acts as a 'transcriptional domainin' protein coordinating the bacterial virulence traits with global transcription.

Bacterial pathogenic growth requires a swift coordination of pathogenicity function with various kinds of environmental stress encountered in the course of host infection. Among the factors critical for bacterial adaptation are changes of DNA topology and binding effects of nucleoid-associated proteins transducing the environmental signals to the chromosome and coordinating the global transcriptional response to stress. In this study, we use the model phytopathogen Dickeya dadantii to analyse the organisation of transcription by the nucleoid-associated heterodimeric protein IHF. We inactivated the IHFα subunit of IHF thus precluding the IHFαß heterodimer formation and determined both phenotypic effects of ihfA mutation on D. dadantii virulence and the transcriptional response under various conditions of growth. We show that ihfA mutation reorganises the genomic expression by modulating the distribution of chromosomal DNA supercoils at different length scales, thus affecting many virulence genes involved in both symptomatic and asymptomatic phases of infection, including those required for pectin catabolism. Altogether, we propose that IHF heterodimer is a 'transcriptional domainin' protein, the lack of which impairs the spatiotemporal organisation of transcriptional stress-response domains harbouring various virulence traits, thus abrogating the pathogenicity of D. dadantii.

Cloning, expression and characterization of P1 and NIa proteases from banana bract mosaic virus (BBrMV).

Banana bract mosaic virus (BBrMV) causes the banana bract mosaic disease in banana. It belongs to the genus Potyvirus within the family Potyviridae. To the best of our knowledge apart from BBrMV coat protein gene, there are no reports on cloning, expression and characterization of any other genes from BBrMV. In this study, the BBrMV P1 and NIa protease genes were amplified from BBrMV infected banana plant cultivar Nendran and were cloned into the protein expression vector pET28b. Recombinant plasmids were transferred to BL21-CodonPlus (DE3)-RP cells and the IPTG (Isopropyl ß-d-1-thiogalactopyranoside) induced BBrMV P1 and NIa proteins with molecular weights of 42 and 32 KDa respectively were purified on Ni-NTA resin column under denaturing conditions using 8 M urea. BBrMV P1 and NIa purified proteins were detected by Western blot using anti-histidine antibody. The activity of both P1 and NIa proteases in native form was analyzed through in-gel zymographic assay. The activities of both the proteases were strongly inhibited by PMSF, suggesting that both the proteases are the serine type proteases. Interestingly both the proteases showed a temperature optimum of 50 °C while the pH optimum was 8. Both proteases lost their activity when incubated at 70 °C for 1 h. This is the first report of expression, purification and characterization of BBrMV P1 and NIa proteases.

Production, properties and some applications of protease from alkaliphilic Bacillus sp. EBTA6.

Extracellular protease production by a novel strain, Bacillus sp. EBTA6, has been optimized by using central composite design of response surface methodology and properties and industrial applications of crude enzyme have been investigated. Three independent variables (temperature, pH and yeast extract concentration) chosen in the experimental design were significant terms and reduced cubic model fit with the design at p < 0.0001 level. The recommended temperature, pH and yeast extract concentration were 30 °C, 8, and 15 g/L, respectively. Crude enzyme displayed activity over a wide pH and temperature ranges having the optimum at 50-60 °C and pH 8. It was quite stable at high pH values and at 50 °C. Amongst the metal ions (Mg+, Cu2+, Ca2+, Zn2+, K2+, and Sn2+), Ca2+ enhanced the activity and the others either decreased or did not change it. The enzyme activity was reduced by phenyl-methyl-sulfonyl fluoride (PMSF), and ethylene diamine tetra acetic acid (EDTA). The results revealed that the protease was serine alkaline type. Tween 20 and Tween 80 did not inhibit the enzyme, however, sodium dodecyl sulfate (SDS), reduced it by 39%. It completely removed blood stain in 20 min and coagulated milk in the presence of CaCl2.

SarA plays a predominant role in controlling the production of extracellular proteases in the diverse clinical isolates of Staphylococcus aureus LAC and UAMS-1.

Using DNA affinity chromatography we demonstrate that the S. aureus regulatory proteins MgrA, Rot, SarA, and SarS bind DNA baits derived from the promoter regions associated with the genes encoding aureolysin, ScpAB, SspABC, and SplA-F. Three of four baits also bound SarR and SarZ, the exception in both cases being the ScpAB-associated bait. Using the USA300, methicillin-resistant strain LAC and the USA200, methicillin-sensitive strain UAMS-1, we generated mutations in the genes encoding each of these proteins alone and in combination with sarA and examined the impact on protease production, the accumulation of high molecular weight proteins, and biofilm formation. These studies confirmed that multiple regulatory loci are involved in limiting protease production to a degree that impacts all of these phenotypes, but also demonstrate that sarA plays a predominant role in this regard. Using sarA mutants unable to produce individual proteases alone and in combination with each other, we also demonstrate that the increased production of aureolysin and ScpA is particularly important in defining the biofilm-deficient phenotype of LAC and UAMS-1 sarA mutants, while aureolysin alone plays a key role in defining the reduced accumulation of alpha toxin and overall cytotoxicity as assessed using both osteoblasts and osteoclasts.

Zinc deficiency induces abnormal development of the myocardium by promoting SENP5 overexpression.

Gestational zinc deficiency is a cause of congenital heart disease in the fetus, and sentrin/small ubiquitin-like modifier (SUMO)-specific proteases (SENPs) as deSUMOylation enzymes play a crucial role in the development of cardiac structures. However, current studies of the regulation and function of SENP in zinc-deficient status during heart development remain limited. In this study, SUMO1 modification was found to gradually decrease during heart development, and the level of SENP5 exhibited a similar trend to SUMO1 conjugation. In addition, zinc deficiency resulted in cardiac dysplasia, increased cell apoptosis, decreased cell viability, and differentiation inhibition of hiPSC-CMs. In order to investigate the function of SENP5 in zinc deficiency, hiPSC-CMs were transfected with SENP5 small interfering RNA. The negative effects of zinc lacking conditions were reversed with depletion of SENP5. It was confirmed that zinc deficiency induced abnormal differentiation of hiPSCs and increased apoptosis of hiPSC-CMs by promoting SENP5 overexpression, which led to cardiac dysplasia. Thus, it was concluded that SENP5 regulates the SUMO1 deconjugation during heart development and zinc deficiency may reduce conjugated SUMO by promoting SENP5 overexpression, which induces abnormal development of the myocardium.