Heterologous Expression and Catalytic Properties of Codon-Optimized Small-Sized Bromelain from MD2 Pineapple.

Bromelain is a unique enzyme-based bioactive complex containing a mixture of cysteine proteases specifically found in the stems and fruits of pineapple (Ananas comosus) with a wide range of applications. MD2 pineapple harbors a gene encoding a small bromelain cysteine protease with the size of about 19 kDa, which might possess unique properties compared to the other cysteine protease bromelain. This study aims to determine the expressibility and catalytic properties of small-sized (19 kDa) bromelain from MD2 pineapple (MD2-SBro). Accordingly, the gene encoding MD2-SBro was firstly optimized in its codon profile, synthesized, and inserted into the pGS-21a vector. The insolubly expressed MD2-SBro was then resolubilized and refolded using urea treatment, followed by purification by glutathione S-transferase (GST) affinity chromatography, yielding 14 mg of pure MD2-SBro from 1 L of culture. The specific activity and catalytic efficiency (kcat/Km) of MD2-SBro were 3.56 ± 0.08 U mg-1 and 4.75 ± 0.23 × 10-3 µM-1 s-1, respectively, where optimally active at 50 °C and pH 8.0, and modulated by divalent ions. The MD2-SBro also exhibited the ability to scavenge the 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) with an IC50 of 0.022 mg mL-1. Altogether, this study provides the production feasibility of active and functional MD2-Bro as a bioactive compound.

Catalytic Properties of Caseinolytic Protease Subunit of Plasmodium knowlesi and Its Inhibition by a Member of δ-Lactone, Hyptolide.

The caseinolytic protease (Clp) system plays an essential role in the protein homeostasis of the malaria parasite, particularly at the stage of apicoplast development. The inhibition of this protein is known to have a lethal effect on the parasite and is therefore considered an interesting avenue for antimalaria drugs discovery. The catalytic activity of the Clp system is modulated by its proteolytic subunit (ClpP), which belongs to the serine protease family member and is therefore extensively studied for further inhibitors development. Among many inhibitors, the group of ß-lactone is known to be a specific inhibitor for ClpP. Nevertheless, other groups of lactones have never been studied. This study aims to characterize the catalytic properties of ClpP of Plasmodium knowlesi (Pk-ClpP) and the inhibition properties of a δ-lactone hyptolide against this protein. Accordingly, a codon-optimized synthetic gene encoding Pk-ClpP was expressed in Escherichia coli BL21(DE3) and purified under a single step of Ni2+-affinity chromatography, yielding a 2.20 mg from 1 L culture. Meanwhile, size-exclusion chromatography indicated that Pk-ClpP migrated primarily as homoheptameric with a size of 205 kDa. The specific activity of pure Pk-ClpP was 0.73 U µg-1, with a catalytic efficiency kcat/KM of 0.05 µM-1 s-1, with optimum temperature and pH of 50 °C and 7.0-7.5, respectively. Interestingly, hyptolide, a member of δ-lactone, was shown to inhibit Pk-ClpP with an IC50 value of 17.36 ± 1.44 nM. Structural homology modelling, secondary structure prediction, and far-UV CD spectra revealed that helical structures dominate this protein. In addition, the structural homology modeling showed that this protein forms a barrel-shaped homoheptamer. Docking simulation revealed that the inhibition was found to be a competitive inhibition in which hyptolide was able to dock into the catalytic site and block the substrate. The competitiveness of hyptolide is due to the higher binding affinity of this molecule than the substrate.

The workability of Escherichia coli BL21 (DE3) and Pseudomonas putida KT2440 expression platforms with autodisplayed cellulases: a comparison.

This article comparatively reports the workability of Escherichia coli BL21(DE3) and Pseudomonas putida KT2440 cell factories for the expression of three model autodisplayed cellulases (i.e., endoglucanase, BsCel5A; exoglucanase, CelK; ß-glucosidase, BglA). The differentiation of the recombinant cells was restricted to their cell growth and enzyme expression/activity attributes. Comparatively, the recombinant E. coli showed higher cell growth rates but lower enzyme activities than the recombinant P. putida. However, the endo-, exoglucanase, and ß-glucosidase on the surfaces of both cell factories showed activity over a broad range of pH (4-10) and temperature (30-100 °C). The pH and temperature optima were pH 6, 60 °C (BsCel5A); pH 6, 60-70 °C (CelK); and pH 6, 50 °C (BglA). Overall, the P. putida cell factory with autodisplayed enzymes demonstrated higher bioactivity and remarkable biochemical characteristics and thus was chosen for the saccharification of filter paper. A volumetric blend of the three cellulases with P. putida as the host yielded a ratio of 1:1:1.5 of endoglucanase, exoglucanase, and ß-glucosidase, respectively, as the optimum blend composition for filter paper degradation. At an optical density (578 nm) of 50, the blend generated a maximum sugar yield of about 0.7 mg/ml (~ 0.08 U/g) from Whatman filter paper (Ø 6 mm, ~ 2.5 mg) within 24 h.

Adaptive Strategies of Bacillus thuringiensis Isolated from Acid Mine Drainage Site in Sabah, Malaysia.

The adaptive process in bacteria is driven by specific genetic elements which regulate phenotypic characteristics such as tolerance to high metal ion concentrations and the secretion of protective biofilms. Extreme environments such as those associated with heavy metal pollution and extremes of acidity offer opportunities to study the adaptive mechanisms of microorganisms. This study focused on the genome analysis of Bacillus thuringiensis (Bt MCMY1), a gram positive rod shaped bacterium isolated from an acid mine drainage site in Sabah, Malaysia by using a combination of Single Molecule Real Time DNA Sequencing, Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR). The genome size of Bt MCMY1 was determined to be 5,458,152 bases which was encoded on a single chromosome. Analysis of the genome revealed genes associated with resistance to Copper, Mercury, Arsenic, Cobalt, Zinc, Cadmium and Aluminum. Evidence from SEM and FTIR indicated that the bacterial colonies form distinct films which bear the signature of polyhydroxyalkanoates (PHA) and this finding was supported by the genome data indicating the presence of a genetic pathway associated with the biosynthesis of PHAs. This is the first report of a Bacillus sp. isolated from an acid mine drainage site in Sabah, Malaysia and the genome sequence will provide insights into the manner in which B. thuringiensis adapts to acid mine drainage.

Complete genome sequence data of an Antarctic bacterium Arthrobacter sp. EM1 from the freshwater lake of the King George Island.

Arthrobacter sp. EM1 is a cold-adapted bacterium isolated from the Antarctic region, which was known to exhibit mannan-degrading activity. Accordingly, this strain not only promises a cell factory for mannan-degrading enzymes, widely used in industry but also serves as a model organism to decipher its cold adaptation mechanism. Accordingly, whole genome sequencing of the EM1 strain was performed via Single Molecule Real Time sequencing under the PacBio platform, followed by genome HGAP de novo assembly and genome annotation through Rapid Annotation System Technology (RAST) server. The chromosome of this strain is 3,885,750 bp in size with a GC content of 65.8. The annotation predicted a total of 3607 protein-coding genes and 65 RNA genes, which were classified under 398 subsystems. The subsystem with the highest number of genes is carbohydrate metabolism (397 genes), which includes two genes encoding mannan-degrading enzymes (endoglucanase and α-mannosidase). This confirmed that the EM1 strain is able to produce cold-adapted mannan degrading enzymes. The complete genome sequence data have been submitted to the National Center for Biotechnology Information (NCBI) and have been deposited at GenBank (Bioproject ID Accession Number: PRJNA963062; Biosample ID Accession Number: SAMN34434776; GenBank: CP124836.1; https://www.ncbi.nlm.nih.gov/nuccore/CP124836).

Whole genome sequence data of an Antarctic bacterium, Arthrobacter sp. ES1 from the Schirmacher Oasis, East Antarctica.

Arthrobacter is a coryneform bacterium in the family of Micrococcaceae. Arthrobacter species isolated from hostile environments are capable of producing interesting bioactive compounds, some of which may be a new class of antibiotics. Here, we present the complete genome sequence of Arthrobacter sp. ES1 isolated from Schirmacher Oasis in East Antarctica. Genomic DNA sequencing was performed using the Illumina MiSeq sequencer. Arthrobacter sp. ES1 has a genome size of 3,964,927 bp and a GC content of 65.73%. The raw genome sequences have been deposited in the NCBI Sequence Read Archive database under the accession number, SRR20664316.

Recent Advances in Overexpression of Functional Recombinant Lipases.

Heterologous functional expression of the recombinant lipases is typically a bottleneck due to the expression in the insoluble fraction as inclusion bodies (IBs) which are in inactive form. Due to the importance of lipases in various industrial applications, many investigations have been conducted to discover suitable approaches to obtain functional lipase or increase the expressed yield in the soluble fraction. The utilization of the appropriate prokaryotic and eukaryotic expression systems, along with the suitable vectors, promoters, and tags, has been recognized as a practical approach. One of the most powerful strategies to produce bioactive lipases is using the molecular chaperones co-expressed along with the target protein's genes into the expression host to produce the lipase in soluble fraction as a bioactive form. The refolding of expressed lipase from IBs (inactive) is another practical strategy which is usually carried out through chemical and physical methods. Based on recent investigations, the current review simultaneously highlights strategies to express the bioactive lipases and recover the bioactive lipases from the IBs in insoluble form.

Antimicrobial Activity of Propolis Extract and Their Application as a Natural Preservative in Livestock Products: A Meta-Analysis.

This study aimed to evaluate the effectiveness of propolis extract as a natural preservative for livestock products in term of chemical and microbiological characteristics by meta-analysis. The stages carried out in this study were identification, selection, checking suitability, and the resulting selected articles were used in the meta-analysis. The selection results obtained a total of 22 selected journal articles consisting of 9 articles for analysis of the antimicrobial activity of propolis extract and 13 articles for analysis of the chemical and mirobiological characteristics of livestock products. The articles were obtained from electronic databases, namely Science Direct and Google Scholar. The model used in this study is the random-effect model involving two groups, control and experimental. Heterogeneity and effect size values were carried out in this study using Hedge's obtained through openMEE software. Forest plot tests and data validation on publication bias was obtained using Kendall's test throught JASP 0.14.1 software. The results showed that there is a significant relationship between propolis extract with the results of the antimicrobial activity (p<0.05). In addition, the results of the application of propolis extract on the livestock products for the test microbes and the value of thiobarbituric acid reactive substances (TBARs) showed significant results (p<0.05). Conclusion based on the random-effect model on the effectiveness of antimicrobial activity of propolis extract and their apllication as a natural preservative of the chemical and microbiological characteristics of livestock products is valid by Kendall's test (p>0.05). Propolis in this case effectively used as natural preservatives in livestock products.

Characterization of Bioactive Sialyl Oligosaccharides Separated from Colostrum of Indonesia Dairy Goat.

The bioactive functions of oligosaccharides from human milk have been reported by many studies. Many of oligosaccharides isolated from colostrum and/or milk of dairy animals have been reported to have similar chemical structures with those in human colostrum and/or milk. It has been proved by several studies that the oligosaccharides with similar chemical structure shared common bioactivities. Among domesticated dairy animals, bovine/cattle, caprine/goat, and ovine/sheep are the most commonly used species to isolate oligosaccharides from their colostrum and/or milk. Several studies on the oligosaccharides from goat colostrum and milk have revealed similar properties to that of human milk and possess the highest content of sialyl oligosaccharides (SOS) as compared to other ruminants. Indonesia ranks first in Association of Southeast Asian Nations (ASEAN) for goat milk production. Therefore, goat milk is the second most consumed milk in the country. The most reared dairy goat breed in Indonesia is Etawah Grade. However, oligosaccharides from Indonesia dairy animals including goat, have not been characterized. This is the first study to characterize oligosaccharides from Indonesia dairy animals. The present study was aimed to isolate and characterize oligosaccharides, specifically SOS from the colostrum of Etawah Grade goats by using proton/1H-nuclear magnetic resonance. The SOS successfully characterized in this study were: Neu5Ac(α2-3)Gal(ß1-4)Glc (3'-N-acetylneuraminyllactose), Neu5Ac(α2-6)Gal(ß1-4)Glc (6'-N-acetylneuraminyllactose), Neu5Gc(α2-3)Gal(ß1-4)Glc (3'-N-glycolylneuraminyllactose), Neu5Gc(α2-6)Gal(ß1-4)Glc (6'-N-glycolylneuraminyllactose), Neu5Ac(α2-6)Gal(ß1-4) GlcNAc (6'-N-acetylneuraminyllactosamine) and Neu5Gc(α2-6)Gal(ß1-4)GlcNAc (6'-N-glycolylneuraminyllactosamine). This finding shows that Etawah Grade, as a local dairy goat breed in Indonesia, is having significant potential to be natural source of oligosaccharides that can be utilized in the future food and pharmaceutical industries.

Novel Peptide Sequences with ACE-Inhibitory and Antioxidant Activities Derived from the Heads and Bones of Hybrid Groupers (Epinephelus lanceolatus × Epinephelus fuscoguttatus).

The heads and bones of hybrid groupers are potential precursors for angiotensin-converting enzyme (ACE)-inhibitory and antioxidant peptides. The aim of this study was to isolate the dual-action peptides from the Alcalase-treated head and bone hydrolysate of hybrid groupers followed by identification of the novel peptides. The stability of these peptides against stimulated in vitro gastrointestinal digestion (SGID) was also determined. Fraction HB-IV (less than 1 kDa) obtained from ultrafiltration showed the strongest ACE-inhibition ability (IC50: 0.28 mg/mL), which was comparable to the potency of the commercial supplement, PeptACE (IC50: 0.22 mg/mL). This fraction also demonstrated the highest hydroxyl radical scavenging and metal-chelating activities. However, further fractionation of HB-IV by a series of chromatography resulted in peptide fractions of reduced ACE-inhibitory and antioxidant activities. The hydroxyl radical scavenging and reduction potential of HB-IV were enhanced, whereas ACE-inhibitory and metal-chelating activities were reduced following SGID. A total of 145 peptide sequences were identified from HB-IV, of which 137 peptides were novel to the BIOPEP database. The results suggested that the bioactive peptides isolated from the heads and bones of hybrid groupers could be used as functional foods/ingredients with potential ACE-inhibitory and antioxidant effects.

FK506-Binding protein 22 from a psychrophilic bacterium, a cold shock-inducible peptidyl prolyl isomerase with the ability to assist in protein folding.

Adaptation of microorganisms to low temperatures remains to be fully elucidated. It has been previously reported that peptidyl prolyl cis-trans isomerases (PPIases) are involved in cold adaptation of various microorganisms whether they are hyperthermophiles, mesophiles or phsycrophiles. The rate of cis-trans isomerization at low temperatures is much slower than that at higher temperatures and may cause problems in protein folding. However, the mechanisms by which PPIases are involved in cold adaptation remain unclear. Here we used FK506-binding protein 22, a cold shock protein from the psychrophilic bacterium Shewanella sp. SIB1 (SIB1 FKBP22) as a model protein to decipher the involvement of PPIases in cold adaptation. SIB1 FKBP22 is homodimer that assumes a V-shaped structure based on a tertiary model. Each monomer consists of an N-domain responsible for dimerization and a C-catalytic domain. SIB1 FKBP22 is a typical cold-adapted enzyme as indicated by the increase of catalytic efficiency at low temperatures, the downward shift in optimal temperature of activity and the reduction in the conformational stability. SIB1 FKBP22 is considered as foldase and chaperone based on its ability to catalyze refolding of a cis-proline containing protein and bind to a folding intermediate protein, respectively. The foldase and chaperone activites of SIB1 FKBP22 are thought to be important for cold adaptation of Shewanella sp. SIB1. These activities are also employed by other PPIases for being involved in cold adaptation of various microorganisms. Despite other biological roles of PPIases, we proposed that foldase and chaperone activities of PPIases are the main requirement for overcoming the cold-stress problem in microorganisms due to folding of proteins.

Structure-Function Characteristics of SARS-CoV-2 Proteases and Their Potential Inhibitors from Microbial Sources.

The COVID-19 pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is considered the greatest challenge to the global health community of the century as it continues to expand. This has prompted immediate urgency to discover promising drug targets for the treatment of COVID-19. The SARS-CoV-2 viral proteases, 3-chymotrypsin-like protease (3CLpro) and papain-like cysteine protease (PLpro), have become the promising target to study due to their essential functions in spreading the virus by RNA transcription, translation, protein synthesis, processing and modification, virus replication, and infection of the host. As such, understanding of the structure and function of these two proteases is unavoidable as platforms for the development of inhibitors targeting this protein which further arrest the infection and spread of the virus. While the abundance of reports on the screening of natural compounds such as SARS-CoV-2 proteases inhibitors are available, the microorganisms-based compounds (peptides and non-peptides) remain less studied. Indeed, microorganisms-based compounds are also one of the potent antiviral candidates against COVID-19. Microbes, especially bacteria and fungi, are other resources to produce new drugs as well as nucleosides, nucleotides, and nucleic acids. Thus, we have compiled various reported literature in detail on the structures, functions of the SARS-CoV-2 proteases, and potential inhibitors from microbial sources as assistance to other researchers working with COVID-19. The compounds are also compared to HIV protease inhibitors which suggested the microorganisms-based compounds are advantageous as SARS-CoV2 proteases inhibitors. The information should serve as a platform for further development of COVID-19 drug design strategies.

Soluble Expression and Catalytic Properties of Codon-Optimized Recombinant Bromelain from MD2 Pineapple in Escherichia coli.

Bromelain, a member of cysteine proteases, is found abundantly in pineapple (Ananas comosus), and it has a myriad of versatile applications. However, attempts to produce recombinant bromelain for commercialization purposes are challenging due to its expressibility and solubility. This study aims to express recombinant fruit bromelain from MD2 pineapple (MD2Bro; accession no: OAY85858.1) in soluble and active forms using Escherichia coli host cell. The gene encoding MD2Bro was codon-optimized, synthesized, and subsequently ligated into pET-32b( +) for further transformation into Escherichia coli BL21-CodonPlus(DE3). Under this strategy, the expressed MD2Bro was in a fusion form with thioredoxin (Trx) tag at its N-terminal (Trx-MD2Bro). The result showed that Trx-MD2Bro was successfully expressed in fully soluble form. The protein was successfully purified using single-step Ni2+-NTA chromatography and confirmed to be in proper folds based on the circular dichroism spectroscopy analysis. The purified Trx-MD2Bro was confirmed to be catalytically active against N-carbobenzoxyglycine p-nitrophenyl ester (N-CBZ-Gly-pNP) with a specific activity of 6.13 ± 0.01 U mg-1 and inhibited by a cysteine protease inhibitor, E-64 (IC50 of 74.38 ± 1.65 nM). Furthermore, the catalytic efficiency (kcat/KM) Trx-MD2Bro was calculated to be at 5.64 ± 0.02 × 10-2 µM-1 s-1 while the optimum temperature and pH were at 50 °C and pH 6.0, respectively. Furthermore, the catalytic activity of Trx-MD2Bro was also affected by ethylenediaminetetraacetic acid (EDTA) or metal ions. Altogether it is proposed that the combination of codon optimization and the use of an appropriate vector are important in the production of a soluble and actively stable recombinant bromelain.

FKBP22 from the psychrophilic bacterium Shewanella sp. SIB1 selectively binds to the reduced state of insulin to prevent its aggregation.

FKBP22 of a psychrophilic bacterium, Shewanella sp. SIB1 (SIB1 FKBP22), is a member of peptidyl-prolyl cis-trans isomerase (PPIase) and consists of N- and C-domains responsible for chaperone-like and PPIase catalytic activities, respectively. The chaperone-like activity of SIB1 FKBP22 was previously evidenced by its ability to prevent dithiothreitol (DTT)-induced insulin aggregation. Nevertheless, the mechanism by which this protein inhibits the aggregation remains unclear. To address this, the binding affinity of SIB1 FKBP22 to the native or reduced states of insulin was examined using surface plasmon resonance (SPR). The native and reduced states refer to insulin in the absence or DTT presence, respectively. The SPR sensorgram showed that SIB1 FKBP22 binds specifically to the reduced state of insulin, with a KD value of 37.31 ± 3.20 µM. This binding was facilitated by the N-domain, as indicated by the comparable KD values of the N-domain and SIB1 FKBP22. Meanwhile, the reduced state of insulin was found to have no affinity towards the C-domain. The KD value of SIB1 FKBP22 was slightly decreased by NaCl but was not severely affected by FK506, a specific FKBP inhibitor. Similarly, the prevention of DTT-induced aggregation by SIB1 FKBP22 was also modulated by the N-domain and was not affected by FK506. Further, the reduced and native states of insulin had no effect on the catalytic efficiency (kcat/KM) of SIB1 FKBP22 towards a peptide substrate. Nevertheless, the reduced state of insulin slightly reduced the catalytic efficiency towards refolding RNase T1, at up to 1.5-fold lower than in the absence of insulin. These results suggested that the binding event was mainly facilitated by hydrophobic interaction and was independent from its PPIase activity. Altogether, a possible mechanism by which SIB1 FKBP22 prevents DTT-induced insulin aggregation was proposed.

Kinetic and Thermodynamic Study of Plantaricin IIA-1A5, a Bacteriocin Produced by Indonesian Probiotic Lactobacillus plantarum IIA-1A5.

BACKGROUND: Plantaricin IIA-1A5 is a bacteriocin produced by Lactobacillus plantarum IIA-1A5, a locally isolated probiotic from Indonesia. Plantaricin IIA-1A5 exhibits antibacterial activity against wide spectrum of pathogenic bacteria, thus promising to be applied in various food products. Nevertheless, thermal stability of this bacteriocin remains to be fully investigated. OBJECTIVE: This study aims to determine thermal stability of plantaricin IIA-1A5 through kinetic and thermodynamic parameters. METHOD: To address, plantaricin IIA-1A5 was purified from Lactobacillus plantarum IIA-1A5, which was growth under whey media, using ammonium sulfate precipitation followed by ionexchange chromatography. Purified plantaricin IIA-IA5 was then subjected to analysis of its bacteriocin activity. The thermal inactivation of bacteriocin from L. plantarum IIA-1A5 was calculated by incubating the bacteriocin at different temperatures ranging from 60-80 °C for 30 to 90 min, which was then used to calculate its kinetic and thermodynamic parameters. RESULTS: The result showed the inactivation rates (k-value) were ranging from 0.008 to 0.013 min-1. Heat resistance of plantaricin IIA-1A5 (D-value) at constant heating temperature of 60, 65, 70, 75, and 80 °C were 311.6, 305.9, 294.5, 198.9, and 180.2 min, which indicated a faster inactivation at higher temperatures. D-value sensitivity for temperature changes (z-value) was calculated to be 75.76 °C. Further, thermodynamic analysis suggested that plantaricin IIA-1A5 is thermostable, with activation energy (Ea) of 29.02 kJ mol-1. CONCLUSION: This result showed that plantaricin IIA-1A5 is considerably more heat-stable than plantaricin members and promises to be applied in food industries where heat treatments are applied. Furthermore, a possible mechanism by which plantaricin IIA-1A5 maintains its stability was also discussed by referring to its thermodynamic parameters.

Inhibition of Human Cervical Cancer Hela Cell Line by Meat-Derived Lactic Acid Bacteria of Lactobacillus plantarum IIA-1A5 and Lactobacillus acidophilus IIA-2B4.

<b>Background and Objective:</b> Two Indonesian lactic acid bacteria of<i> L. plantarum </i>I IA-1A5 and <i>L. acidophilus </i>IIA-2B4 were previously isolated from beef with some functional probiotic properties. Nevertheless, the possibility of these strains to have anticancer activity remains unknown. Current study aimed to evaluate the inhibitory properties of intra-and extracellular protein extracts of these two strains against cervical cancer HeLa cells. <b>Materials and Methods:</b> The intracellular and extracellular proteins extract from <i>L. plantarum </i>IIA-1A5 and <i>L. acidophilus </i>IIA-2B4 were collected and designated as IP-LP, IP-LA, EP-LP and EP-LA, respectively. The effect of these extracts on the viability and morphology of HeLa cells were observed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and confocal microscopy, respectively. <b>Results:</b> Both IP-LP and IP-LA inhibited HeLa cells in a concentration-dependent manner, with IC₅₀ values of 352.62 and 120.97 µg mL¹, respectively. Meanwhile, the inhibition activity was also observed for EP-LP and EP-LA, <i>albeit</i> very low. The inhibition effect was also confirmed by morphological analysis under confocal electron microscopy which showed the changes in the cell shapes and numbers. <b>Conclusion:</b> Altogether, for the first time this study proposed that the probiotic isolated from Indonesian beef are promising to inhibit cancer cell lines.

Technical data on the inhibition properties of some medicinal plant extracts towards caseinolytic protease proteolytic subunit of Plasmodium knowlesi.

Proteolytic subunit of the caseinolytic protease system of Plasmodium knowlesi (Pk-ClpP; EC 3.4.21.92) is considered a viable target for antimalarial drug development to eradicate P. knowlesi malaria infection in Malaysia and Southeast Asian region. Inhibition of this system leads to a disruption in the protein homeostasis molecular machinery and therefore be lethal for the parasite. While plants are considered excellent sources of bioactive compounds exhibiting inhibition activity towards Pk-ClpP, many local medicinal plants remain unexplored. This article expands the data collected from the inhibition properties of the methanolic extract of Asystasia gangetica (Chinese Violet), Alstonia scholaris (Pulai Tree), Piper retrofractum (Javanese Long Pepper) and Smallanthus sonchifolius (Yacon) towards Pk-ClpP. These plants are widely found in Malaysia and Indonesia and have been traditionally used in various medical treatments. The present dataset showed that the extracts contained phenolic and flavonoid compounds in various concentrations, whereby S. sonchifolius was found to have the lowest content of phenolic and flavonoid contents, while A. gangetica and A. scholaris were statistically comparable, yet higher than P. retrofactum and S. sonchifolus. Further inhibition data assay towards Pk-ClpP revealed that A. gangetica, A. scholaris and P. retrofactum demonstrated remarkable inhibition activity with IC50 values of 39.06 ± 1.98, 48.92 ± 1.52, and 87.63 ± 3.55, respectively. However, the inhibition activity of these extracts was significantly lower than a serine protease inhibitor of phenylmethylsulfonyl fluoridenone (PMSF). Meanwhile, S. sonchifolus did not exhibit significant inhibition activity towards Pk-ClpP. In addition, Pk-ClpP was not inhibited by a cysteine protease inhibitor of E64.

Inhibition and Substrate Specificity Properties of FKBP22 from a Psychrotrophic Bacterium, Shewanella sp. SIB1.

SIB1 FKBP22 is a peptidyl prolyl cis-trans isomerase (PPIase) member from a psychrotrophic bacterium, Shewanella sp. SIB1, consisting of N- and C-domains responsible for dimerization and catalytic PPIase activity, respectively. This protein was assumed to be involved in cold adaptation of SIB1 cells through its dual activity of PPIase activity and chaperone like-function. Nevertheless, the catalytic inhibition by FK506 and its substrate specificity remain unknown. Besides, ability of SIB1 FKBP22 to inhibit phosphatase activity of calcinuerin is also interesting to be studied since it may reflect wider cellular functions of SIB1 FKBP22. In this study, we found that wild type (WT) SIB1 FKBP22 bound to FK506 with IC50 of 77.55 nM. This value is comparable to that of monomeric mutants (NNC-FKBP22, C-domain+ and V37R/L41R mutants), yet significantly higher than that of active site mutant (R142A). In addition, WT SIB1 FKBP22 and monomeric variants were found to prefer hydrophobic residues preceding proline. Meanwhile, R142A mutant has wider preferences on bulkier hydrophobic residues due to increasing hydrophobicity and binding pocket space. Surprisingly, in the absence of FK506, SIB1 FKBP22 and its variants inhibited, with the exception of N-domain, calcineurin phosphatase activity, albeit low. The inhibition of SIB1 FKBP22 by FK506 is dramatically increased in the presence of FK506. Altogether, we proposed that local structure at substrate binding pocket of C-domain plays crucial role for the binding of FK506 and peptide substrate preferences. In addition, C-domain is essential for inhibition, while dimerization state is important for optimum inhibition through efficient binding to calcineurin.

Expression and characterization of functional domains of FK506-binding protein 35 from Plasmodium knowlesi.

The FK506-binding protein of Plasmodium knowlesi (Pk-FKBP35) is considerably a viable antimalarial drug target, which belongs to the peptidyl-prolyl cis-trans isomerase (PPIase) protein family member. Structurally, this protein consists of an N-terminal FK506-binding domain (FKBD) and a C-terminal tetratricopeptide repeat domain (TPRD). This study aims to decipher functional properties of these domains as a platform for development of novel antimalarial drugs. Accordingly, full-length Pk-FKBP35 as well as its isolated domains, Pk-FKBD and Pk-TPRD were overexpressed, purified, and characterized. The results showed that catalytic PPIase activity was confined to the full-length Pk-FKBP35 and Pk-FKBD, suggesting that the catalytic activity is structurally regulated by the FKBD. Meanwhile, oligomerization analysis revealed that Pk-TPRD is essential for dimerization. Asp55, Arg60, Trp77 and Phe117 in the Pk-FKBD were considerably important for catalysis as underlined by significant reduction of PPIase activity upon mutations at these residues. Further, inhibition activity of Pk-FKBP35 towards calcineurin phosphatase activity revealed that the presence of FKBD is essential for the inhibitory property, while TPRD may be important for efficient binding to calcineurin. We then discussed possible roles of FKBP35 in Plasmodium cells and proposed mechanisms by which the immunosuppressive drug, FK506, interacts with the protein.

Elucidating endotoxin-biomolecule interactions with FRET: extending the frontiers of their supramolecular complexation.

Endotoxin has been one of the topical chemical contaminants of major concern to researchers, especially in the field of bioprocessing. This major concern of researchers stems from the fact that the presence of Gram-negative bacterial endotoxin in intracellular products is unavoidable and requires complex downstream purification steps. For instance, endotoxin interacts with recombinant proteins, peptides, antibodies and aptamers and these interactions have formed the foundation for most biosensors for endotoxin detection. It has become imperative for researchers to engineer reliable means/techniques to detect, separate and remove endotoxin, without compromising the quality and quantity of the end-product. However, the underlying mechanism involved during endotoxin-biomolecule interaction is still a gray area. The use of quantitative molecular microscopy that provides high resolution of biomolecules is highly promising, hence, may lead to the development of improved endotoxin detection strategies in biomolecule preparation. Förster resonance energy transfer (FRET) spectroscopy is one of the emerging most powerful tools compatible with most super-resolution techniques for the analysis of molecular interactions. However, the scope of FRET has not been well-exploited in the analysis of endotoxin-biomolecule interaction. This article reviews endotoxin, its pathophysiological consequences and the interaction with biomolecules. Herein, we outline the common potential ways of using FRET to extend the current understanding of endotoxin-biomolecule interaction with the inference that a detailed understanding of the interaction is a prerequisite for the design of strategies for endotoxin identification and removal from protein milieus.