Purification and biochemical characterization of novel α-amylase and cellulase from Bacillus sp. PM06.

Bacillus sp. PM06, previously isolated from sugarcane waste pressmud, could produce dual enzymes α-amylase and cellulase. The isolate's crude enzymes were purified homogeneously using ammonium sulfate precipitation followed by High Quaternary amine anion exchange chromatography. Purified enzymes revealed the molecular weights of α-amylase and cellulase as 55 and 52 kDa, with a purification fold of 15.4 and 11.5, respectively. The specific activity of purified α-amylase and cellulase were 740.7 and 555.6 U/mg, respectively. It demonstrated a wide range of activity from pH 5.0 to 8.5, with an optimum pH of 5.5 and 6.4 for α-amylase and cellulase. The optimum temperature was 50 °C for α-amylase and 60 °C for cellulase. The kinetic parameters of purified α-amylase were 741.5 ± 3.75 µmol/min/mg, 1.154 ± 0.1 mM, and 589 ± 3.5/(s mM), using starch as a substrate. Whereas cellulase showed 556.3 ± 1.3 µmol/min/mg, 1.78 ± 0.1 mM, and 270.9 ± 3.8/(s mM) of Vmax, Km, Kcat/Km, respectively, using carboxymethyl cellulose (CMC) as substrate. Among the various substrates tested, α-amylase had a higher specificity for amylose and CMC for cellulase. Different inhibitors and activators were also examined. Ca2+ Mg2+, Co2+, and Mn2+ boosted α-amylase and cellulase activities. Cu2+ and Ni2+ both inhibited the enzyme activities. Enzymatic saccharification of wheat bran yielded 253.61 ± 1.7 and 147.5 ± 1.0 mg/g of reducing sugar within 12 and 24 h of incubation when treated with purified α-amylase and cellulase. A more significant amount of 397.7 ± 1.9 mg/g reducing sugars was released from wheat bran due to the synergetic effect of two enzymes. According to scanning electron micrograph analysis, wheat bran was effectively broken down by both enzymes.

Advances in the applications of Bacteriophages and phage products against food-contaminating bacteria.

Food-contaminating bacteria pose a threat to food safety and the economy by causing foodborne illnesses and spoilage. Bacteriophages, a group of viruses that infect only bacteria, have the potential to control bacteria throughout the "farm-to-fork continuum". Phage application offers several advantages, including targeted action against specific bacterial strains and minimal impact on the natural microflora of food. This review covers multiple aspects of bacteriophages applications in the food industry, including their use as biocontrol and biopreservation agents to fight over 20 different genera of food-contaminating bacteria, reduce cross-contamination and the risk of foodborne diseases, and also to prolong shelf life and preserve freshness. The review also highlights the benefits of using bacteriophages in bioprocesses to selectively inhibit undesirable bacteria, such as substrate competitors and toxin producers, which is particularly valuable in complex microbial bioprocesses where physical or chemical methods become inadequate. Furthermore, the review briefly discusses other uses of bacteriophages in the food industry, such as sanitizing food processing environments and detecting specific bacteria in food products. The review also explores strategies to enhance the effectiveness of phages, such as employing multi-phage cocktails, encapsulated phages, phage products, and synergistic hurdle approaches by combining them with antimicrobials.

Role of key enzymes in the production of docosahexaenoic acid (DHA) by Thraustochytrium sp. T01.

Docosahexaenoic acid (DHA) is an essential dietary supplement that is highly coveted due to its benefits for human health. Extensive research has been conducted for the sustainable commercial production of DHA by various strains in thraustochytrid family due to the accumulation of higher lipid content in the cells. The current study is focused on improving DHA production by investigating various key enzymes like glucose-6-phosphate dehydrogenase (G6PDH), malic enzyme (ME), and ATP-citrate lyase (ACL) involved in DHA production using Thraustochytrium sp. T01. The growth of this strain was compared in batch and fed-batch mode. The fed-batch yielded better Dry cell weight (40 g L-1), lipid (27.75 g L-1 or 693 mg g-1 of DCW), and DHA contents (11.10 g L-1 or 277 mg g-1 of DCW). G6PDH activity increased 4-fold during the glucose fed-batch, but ME and ACL did not increase significantly. Furthermore, a study was conducted to determine the effects of organic acids (pyruvate and malate) on key enzyme activities. The addition of pyruvate increased the lipid content by 1.35-fold, and ACL activity by 10-fold as compared with control (without added organic acids). Malate addition into the culture media increased DHA content 1.4-fold, and ME activity increased 14-fold compared with control.

Biophysical characterization of the DNA binding motif of human phospholipid scramblase 1.

Human phospholipid scramblase 1 (hPLSCR1) is a 37 kDa multi-compartmental protein, which was initially identified as a Ca2+-dependent phospholipid translocator upon localizing to the plasma membrane. However, under certain physiological conditions, hPLSCR1 is localized to the nucleus where it interacts with the IP3R1 promoter (IP3R1P) and regulates its expression. In this study, the DNA binding properties of hPLSCR1 and ∆100-hPLSCR1 (N-terminal 100 amino acids deleted from hPLSCR1) were investigated by using a synthetic IP3R1P oligonucleotide and nonspecific scrambled-sequence oligonucleotides. Our results revealed that hPLSCR1 and ∆100-hPLSCR1 were bound to IP3R1P oligos in a 1:1 stoichiometry. In addition, ∆160-hPLSCR1 could not bind to IP3R1P oligonucleotide suggesting that the proposed DNA binding motif is the actual DNA binding motif. Specific binding of hPLSCR1 and ∆100-hPLSCR1 to IP3R1P oligos was demonstrated by fluorescence anisotropy assay. ITC analysis revealed that hPLSCR1 binds to IP3R1P oligos with Kd = 42.91 ± 0.23 nM. Binding of IP3R1P oligos induces ß-sheet formation in hPLSCR1 and increases the thermal stability of hPLSCR1 and ∆100-hPLSCR1. Binding of IP3R1P oligos to hPLSCR1 altered the B-form of the DNA, which was not observed with ∆100-hPLSCR1. Results from this study suggest that (i) ∆100-hPLSCR1 possesses a minimal DNA binding region and (ii) structural alterations of IP3R1P oligo by hPLSCR1 require proline-rich N-terminal region.

α-Amylase and cellulase production by novel halotolerant Bacillus sp.PM06 isolated from sugarcane pressmud.

A novel Bacillus sp.PM06 isolated from sugarcane waste pressmud was tested for extracellular α-amylase and cellulase enzyme production. The effect of different substrates, nitrogen sources, pH, and temperature on growth and extracellular enzyme production was examined. Bacillus sp.PM06 was able to grow with starch and carboxymethyl cellulose (CMC) as a sole source of carbon and ammonium chloride was found to be the best nitrogen source. Maximum enzyme production was obtained at 48 H for both α-amylase and cellulase. The optimal condition for measuring enzyme activity was found to be pH 5.5 at 50 °C for α-amylase and pH 6.4 at 60 °C for cellulase respectively. It was found that Bacillus sp.PM06 exhibited halotolerance up to 2 M Sodium chloride (NaCl) and Potassium chloride (KCl). The isolate could produce α-amylase in the presence of 2 M NaCl and 1 M KCl. However, the strain produced cellulase even in the presence of 2 M NaCl and KCl. Concomitant production of both enzymes was observed when the medium was supplemented with starch and CMC. A maximum of 31 ± 1.15 U/mL of amylase and 15 ± 1.5 U/mL of cellulase was produced in 48 H. The enzyme was partially purified by Ammonium sulphate (NH4 )2 SO4 precipitation with 2.2 and 2.3-fold purification.

Are cysteine residues of human phospholipid scramblase 1 essential for Pb2+ and Hg2+ binding-induced scrambling of phospholipids?

Lead and mercury being common environmental pollutants are often associated with erythrocytes, where phosphatidylserine (PS) exposure-mediated procoagulant activation is induced. Human phospholipid scramblase 1 (hPLSCR1) identified in the erythrocyte membrane is a type II transmembrane protein involved in Ca2+-dependent bidirectional scrambling of phospholipids (PL) during blood coagulation, cell activation, and apoptosis. The prominent role of hPLSCR1 in Pb2+ and Hg2+ poisoning was demonstrated by a biochemical assay, where recombinant hPLSCR1 induced PL scrambling across bilayer with a higher binding affinity (Kd) towards Hg2+ (4.1 µM) and Pb2+ (5.8 µM) than Ca2+ (25.6 mM). The increased affinity could be the outcome of heavy metals interacting at auxiliary sites other than the calcium-binding motif of hPLSCR1. Similar to other metal-binding proteins, cysteine-based metal-binding motifs could be the potential additional binding sites in hPLSCR1. To explore the hypothesis, the cysteines were chemically modified, which significantly reduced only the Hg2+- and Pb2+-induced scrambling activity leaving Ca2+-induced activity unaltered. Recombinant constructs with deletion of prominent cysteine residues and point mutation in the calcium-binding motif including Δ100-hPLSCR1, Δ160-hPLSCR1, and D275A-hPLSCR1 were generated, purified, and assayed for scramblase activity. The cysteine-deleted constructs of hPLSCR1 showed reduced binding affinity (Kd) for Hg2+ and Pb2+ without altering the Ca2+-binding affinity whereas the point mutant had completely lost its affinity for Ca2+ and reduced affinities for Hg2+ and Pb2+. The results accentuated the significance of cysteine residues as additional binding sites for heavy metal ions in hPLSCR1.

Heavy-Metals-Mediated Phospholipids Scrambling by Human Phospholipid Scramblase 3: A Probable Role in Mitochondrial Apoptosis.

Human phospholipid scramblases are a family of four homologous transmembrane proteins (hPLSCR1-4) mediating phospholipids (PLs) translocation in plasma membrane upon Ca2+ activation. hPLSCR3, the only homologue localized to mitochondria, plays a vital role in mitochondrial structure, function, maintenance, and apoptosis. Upon Ca2+ activation, hPLSCR3 mediates PL translocation at the mitochondrial membrane enhancing t-bid-induced cytochrome c release and apoptosis. Mitochondria are important target organelles for heavy-metals-induced apoptotic signaling cascade and are the central executioner of apoptosis to trigger. Pb2+ and Hg2+ toxicity mediates apoptosis by increased reactive oxygen species (ROS) and cytochrome c release from mitochondria. To discover the role of hPLSCR3 in heavy metal toxicity, hPLSCR3 was overexpressed as a recombinant protein in Escherichia coli Rosetta (DE3) and purified by affinity chromatography. The biochemical assay using synthetic proteoliposomes demonstrated that hPLSCR3 translocated aminophospholipids in the presence of micromolar concentrations of Pb2+ and Hg2+. A point mutation in the Ca2+-binding motif (F258V) led to a ∼60% loss in the functional activity and decreased binding affinities for Pb2+ and Hg2+ implying that the divalent heavy metal ions bind to the Ca2+-binding motif. This was further affirmed by the characteristic spectra observed with stains-all dye. The conformational changes upon heavy metal binding were monitored by circular dichroism, intrinsic tryptophan fluorescence, and light-scattering studies. Our results revealed that Pb2+ and Hg2+ bind to hPLSCR3 with higher affinity than Ca2+ thus mediating scramblase activity. To summarize, this is the first biochemical evidence for heavy metals binding to the mitochondrial membrane protein leading to bidirectional translocation of PLs specifically toward phosphatidylethanolamine.

Molecular cloning and biochemical characterization of the phospholipid scramblase SCRM-1 from Caenorhabditis elegans.

In this study, the SCRM-1 gene from Caenorhabditis elegans was cloned and overexpressed in E. coli to study the biochemical properties of scramblase. This is the first report showing that this scramblase from C. elegans possesses a Ca2+-dependent and head group-independent scramblase activity. The SCRM-1 of C.elegans possesses functional domains including a single EF-hand-like Ca2+ binding domain, as human scramblases do. A point mutation in the EF-hand-like Ca2+ binding motif results in loss of scramblase activity. Other biochemical assays like carbocyanine staining, Tb3+ luminescence, Tryptophan fluorescence, and CD spectroscopy strongly proved the role of the EF-hand motif for functional activity. The increase in protein size in solution upon incubating with Ca2+ shows ligand-dependent oligomerization and conformational changes.

Counteraction of osmolytes on pH-induced unfolding of xylose reductase from Debaryomyces nepalensis NCYC 3413.

The stability of Debaryomyces nepalensis NCYC 3413 xylose reductase, a homodimeric enzyme recombinantly expressed and purified from E. coli Rosetta cells, was studied at different pH ranging from 5.0 to 10.0. Deactivation kinetics at different pH were studied by analyzing residual activity of the recombinant enzyme over time at 40 °C whereas conformational changes and stability dependence were investigated by using circular dichroism and differential scanning calorimetry. Four osmolytes viz. glycerol, sucrose, trehalose and sorbitol were explored for their effect on the deactivation and melting temperatures of the enzyme under neutral and extreme pH conditions. The enzyme was found to be catalytically and structurally stable at pH 7.0 with half-life of 250 min and a melting temperature of 50 °C. It was found that alteration in both secondary and tertiary structures caused enzyme deactivation in acidic pH while increased deactivation rates at alkaline pH was attributed to the variation of tertiary structure over time. Estimated thermodynamic parameters also showed that the enzyme stability was highest at neutral pH with ΔH of 348 kcal/mole and ΔG40 of 9.53 kcal/mole. All four osmolytes were effective in enhancing enzyme stability by several folds at extreme pH with sorbitol being the most efficient, which increased enzyme half-life by 11-fold at pH 10.0 and 8-fold at pH 5.0.

Biochemical characterization of an esterase from Clostridium acetobutylicum with novel GYSMG pentapeptide motif at the catalytic domain.

Gene CA_C0816 codes for a serine hydrolase protein from Clostridium acetobutylicum (ATCC 824) a member of hormone-sensitive lipase of lipolytic family IV. This gene was overexpressed in E. coli strain BL21and purified using Ni2+-NTA affinity chromatography. Size exclusion chromatography revealed that the protein is a dimer in solution. Optimum pH and temperature for recombinant Clostridium acetobutylicum esterase (Ca-Est) were found to be 7.0 and 60 °C, respectively. This enzyme exhibited high preference for p-nitrophenyl butyrate. KM and kcat/KM of the enzyme were 24.90 µM and 25.13 s-1 µM-1, respectively. Sequence analysis of Ca-Est predicts the presence of catalytic amino acids Ser 89, His 224, and Glu 196, presence of novel GYSMG conserved sequence (instead of GDSAG and GTSAG motif), and undescribed variation of HGSG motif. Site-directed mutagenesis confirmed that Ser 89 and His 224 play a major role in catalysis. This study reports that Ca-Est is hormone-sensitive lipase with novel GYSMG pentapeptide motif at a catalytic domain.

Screening of new yeast Pichia manchurica for arabitol production.

Arabitol has several applications in food and pharmaceutical industries as a natural sweetener, dental caries inhibitor, and texturing agent. Newly isolated yeast strains from seawater, sugarcane plantation soil samples, and Zygosaccharomyces rouxii 2635 from MTCC were tested for arabitol production. The yield of arabitol was found to be higher in seawater isolate (24.6 g L-1 ) compared to two soil isolates (22.5 g L-1 ) and Z. rouxii (19.4 g L-1 ). Based on ITS 26S rDNA sequence analysis, the seawater isolate was identified as Pichia manchurica. In the present study, the effect of different substrates, trace elements, nitrogen sources, pH, and temperature on arabitol production was examined. Three different carbon sources viz. glucose, arabinose, and galactose were studied. Glucose was determined to be the best substrate for arabitol production (27.6 g L-1 ) followed by arabinose (13.7 g L-1 ) and galactose (7.7 g L-1 ). Maximum production of arabitol was observed at pH 6.0 (34.7 g L-1 ). In addition, arabitol production was high (35.7 g L-1 ) at temperature of 30 °C. Among the different concentrations of ammonium sulfate tested (3, 4.5, 6, 7.5, and 9 g L-1 ) concentration of 6 g L-1 resulted in higher arabitol Individual metal ions had no effect on arabitol production by this strain as compared to control. Results obtained in this study identify ways for improved arabitol production with natural isolates using microbial processes.

The role of human phospholipid scramblases in apoptosis: An overview.

Human phospholipid scramblases (hPLSCRs) are a family of four homologous single pass transmembrane proteins (hPLSCR1-4) initially identified as the proteins responsible for Ca2+ mediated bidirectional phospholipid translocation in plasma membrane. Though in-vitro assays had provided evidence, the role of hPLSCRs in phospholipid translocation is still debated. Recent reports revealed a new class of proteins, TMEM16 and Xkr8 to exhibit scramblase activity challenging the function of hPLSCRs. Apart from phospholipid scrambling, numerous reports have emphasized the multifunctional roles of hPLSCRs in key cellular processes including tumorigenesis, antiviral defense, protein and DNA interactions, transcriptional regulation and apoptosis. In this review, the role of hPLSCRs in mediating cell death through phosphatidylserine exposure, interaction with death receptors, cardiolipin exposure, heavy metal and radiation induced apoptosis and pathological apoptosis followed by their involvement in cancer cells are discussed. This review aims to connect the multifunctional characteristics of hPLSCRs to their decisive involvement in apoptotic pathways.

In vitro reconstitution and biochemical characterization of human phospholipid scramblase 3: phospholipid specificity and metal ion binding studies.

Human phospholipid scramblase 3 (hPLSCR3) is a single pass transmembrane protein that plays a vital role in fat metabolism, mitochondrial function, structure, maintenance and apoptosis. The mechanism of action of scramblases remains still unknown, and the role of scramblases in phospholipid translocation is heavily debated. hPLSCR3 is the only member of scramblase family localized to mitochondria and is involved in cardiolipin translocation at the mitochondrial membrane. Direct biochemical evidence of phospholipid translocation by hPLSCR3 is yet to be reported. Functional assay in synthetic proteoliposomes upon Ca2+ and Mg2+ revealed that, apart from cardiolipin, recombinant hPLSCR3 translocates aminophospholipids such as NBD-PE and NBD-PS but not neutral phospholipids. Point mutation in hPLSCR3 (F258V) resulted in decreased Ca2+ binding affinity. Functional assay with F258V-hPLSCR3 led to ~50% loss in scramblase activity in the presence of Ca2+ and Mg2+. Metal ion-induced conformational changes were monitored by intrinsic tryptophan fluorescence, circular dichroism, surface hydrophobicity changes and aggregation studies. Our results revealed that Ca2+ and Mg2+ bind to hPLSCR3 and trigger conformational changes mediated by aggregation. In summary, we suggest that the metal ion-induced conformational change and the aggregation of the protein are essential for the phospholipid translocation by hPLSCR3.

Rapid method for an enhanced recovery of biologically active human phospholipid scramblase1 from inclusion bodies.

Human phospholipid scramblase 1 (hPLSCR1) is an ATP independent, Ca2+ dependent transmembrane protein mediating bidirectional translocation of phospholipids across the lipid bilayer but the mechanism of scrambling is unknown. Determination of the hPLSCR1 structure would help understand the mechanism and its multi-functional property. Recombinant hPLSCR1 forms inclusion bodies (IBs), when over-expressed in E. coli BL21 (DE3) and recovery of active hPLSCR1 from IBs, were time-consuming and resulted in low protein yield. This study aims to optimize and enhance the expression and purification of active recombinant hPLSCR1 by various strategies. Additives including stabilizers and detergents were added during cell lysis to improve the recovery of soluble hPLSCR1. Five E. coli strains, BL21 (DE3), C43 (DE3), Rosetta, BL21-CodonPlus-RP, and BL21 (DE3) pLysS were screened for maximum yield of soluble protein but localized in IBs. To recover hPLSCR1 from IBs, different additives were added of which, 0.3% N-lauroyl sarcosine (NLS) recovered ∼50% of bioactive hPLSCR1 from IBs. E. coli C43 (DE3) gave higher yields of purified protein (7.76 mg/g cell) followed by E. coli BL21 (DE3) pLysS (5.87 mg/g cell). This report describes a rapid and efficient method for solubilizing membrane proteins from inclusion bodies with a higher recovery.

Identification and characterization of oxidoreductase component (NdmD) of methylxanthine oxygenase system in Pseudomonas sp. NCIM 5235.

Pseudomonas sp. NCIM 5235 is a caffeine-degrading bacterial strain that metabolizes caffeine by sequential demethylation using methylxanthine demethylases. These enzymes belong to the class of two-component Rieske oxygenases and require an oxidoreductase, NdmD, for efficient catalysis. NdmD in Pseudomonas sp. has a unique domain fusion in its N-terminal that is not observed in any other Rieske oxygenase reductases reported so far. In this report, a ~ 1.7 kb ndmD gene from the gDNA of Pseudomonas sp. has been isolated and has been cloned in a pET28a expression vector. Soluble NdmD was over-expressed in Escherichia coli BL21 cells and purified by Ni2+ NTA chromatography. Monomeric molecular mass of the protein was found to be ~ 65 kDa and optimal activity was observed at 35 °C and pH 8.0. It showed broad substrate specificity with highest Kcat/km of 490.8 ± 17.7 towards cytochrome c. To determine the role of N-terminal Rieske domain in its reductase activity, two deletion constructs Δ114NdmD and Δ250NdmD were made. Cytochrome c reductase (ccr) activity of the NdmD constructs and demethylase activity of NdmA in the presence of NdmD constructs showed that there is no significant difference in the catalytic activity of NdmD upon deletion of its N-terminal Rieske domain. However, there might be some functional and evolutionary significance for the fusion of Rieske domain to NdmD and we hypothesize that this domain fusion is an intermediate phase of evolution towards the development of a more efficient enzyme system for xenobiotic degradation.

Comparison of the elution characteristics of individual forms of lovastatin in both isocratic and gradient modes and HPLC-PDA method development for pure and fermentation-derived lovastatin.

The elution characteristics of lovastatin were studied by varying the composition of mobile phase in both isocratic and gradient elution modes to comprehend the role of organic modifier and acidifier on the overall analysis time and retention time of individual forms of lovastatin. Acetonitrile has influenced on the overall analysis time, whereas the acidifier determines the retention time of hydroxy acid form of lovastatin and the retention time gap between the individual forms. A combination of acetonitrile and 0.1% trifluoroacetic acid (TFA) (60:40, v/v) in isocratic elution mode eluted both hydroxy acid and lactone forms of lovastatin at 4.5 and 5.4 min, respectively. This appears to be a better approach for the separation of pharmaceutical and clinical lovastatin samples. At isocratic elution mode, a mixture of acetonitrile and either 0.05% TFA or 0.1% H3PO4 of 60:40 (v/v) has eluted both hydroxy acid and lactone forms of lovastatin at 10 ± 0.5 and 17 ± 0.5 min, respectively. This is suitable for the fermentation-derived samples or for the complex mixtures of structural analogs. The fermentation broth (pH not adjusted) extracted with ethyl acetate at a ratio of 1:1 (v/v) at 60°C for 30 min was the optimal extraction condition for lovastatin.

Two c-Myc binding sites are crucial in upregulating the expression of human phospholipid scramblase 1 gene.

Human phospholipid scramblase 1 (hPLSCR1) is a type II endofacial membrane protein which mediates bi-directional transport of phospholipids across the plasma membrane. hPLSCR1, a multifunctional protein with variety of roles in apoptosis, tumor progression, cell signaling and anti-viral defense. The expression of such a multifunctional protein should be under tight regulation. Apart from a single report showing snail mediated down regulation of hPLSCR1, the molecular mechanisms regulating the expression of scramblases are not well elucidated. In this study we identified c-Myc as a transcriptional regulator of hPLSCR1. Transcription factor prediction tool ConSite predicted three binding sites for c-Myc. Reporter gene assays and western blot analysis revealed c-Myc mediated up regulation of hPLSCR1 expression. Deletion construct -790 lacking one c-Myc binding site showed a 27% decrease in promoter activity while deletion construct -469 lacking two c-Myc binding sites showed a 62% decrease in promoter activity. Site directed mutagenesis revealed the importance of c-Myc binding sites from -751 to -756 and -548 to -553 on the promoter of hPLSCR1in transcriptionally regulating the expression of hPLSCR1. The results were further confirmed by shRNA mediated knock down of endogenous c-Myc and in vivo interactions by ChIP assay.

Identification and characterization of the novel nuclease activity of human phospholipid scramblase 1.

BACKGROUND: Human phospholipid scramblase 1 (hPLSCR1) was initially identified as a Ca(2+) dependent phospholipid translocator involved in disrupting membrane asymmetry. Recent reports revealed that hPLSCR1 acts as a multifunctional signaling molecule rather than functioning as scramblase. hPLSCR1 is overexpressed in a variety of tumor cells and is known to interact with a number of protein molecules implying diverse functions. RESULTS: In this study, the nuclease activity of recombinant hPLSCR1 and its biochemical properties have been determined. Point mutations were generated to identify the critical region responsible for the nuclease activity. Recombinant hPLSCR1 exhibits Mg(2+) dependent nuclease activity with an optimum pH and temperature of 8.5 and 37 °C respectively. Experiments with amino acid modifying reagents revealed that histidine, cysteine and arginine residues were crucial for its function. hPLSCR1 has five histidine residues and point mutations of histidine residues to alanine in hPLSCR1 resulted in 60 % loss in nuclease activity. Thus histidine residues could play a critical role in the nuclease activity of hPLSCR1. CONCLUSIONS: This is the first report on the novel nuclease activity of the multi-functional hPLSCR1. hPLSCR1 shows a metal dependent nuclease activity which could play a role in key cellular processes that needs to be further investigated.

Characterization and biological activities of cyclic (1 → 3, 1 → 6)-ß-glucans from Bradyrhizobium japonicum.

OBJECTIVE: To isolate cyclic (1 â†’ 3, 1 â†’ 6)-ß-glucan from Bradyrhizobium japonicum MTCC120, to characterize its structure and to study its biological activities. RESULTS: The degree of polymerization of cyclic (1 â†’ 3, 1 â†’ 6)-ß-glucan varied between 10 and 13 and with substituents acetyl, succinyl and phosphocholine. The cyclic glucans showed bimodal particle size distribution, with hydrodynamic diameters of 1.92 and 231 nm corresponding to monomeric and aggregated cyclic glucans, respectively. SEM and TEM images showed that the glucans formed aggregates of nanorods. The glucans were biocompatible, exhibited good antioxidant activity and had the abilities to bind to Aniline Blue dye to form a fluorescence complex which was concentration dependent. CONCLUSION: The glucans isolated are cyclic and have good antioxidant activities, hence have potential application in food and pharmaceutical industries. Their dye binding ability could be exploited in medical imaging to reduce the cytotoxicity of the dyes.

N-terminal proline-rich domain is required for scrambling activity of human phospholipid scramblases.

Human phospholipid scramblase 1 (hPLSCR1), a type II integral class membrane protein, is known to mediate bidirectional scrambling of phospholipids in a Ca(2+)-dependent manner. hPLSCR2, a homolog of hPLSCR1 that lacks N-terminal proline-rich domain (PRD), did not show scramblase activity. We attribute this absence of scramblase activity of hPLSCR2 to the lack of N-terminal PRD. Hence to investigate the above hypothesis, we added the PRD of hPLSCR1 to hPLSCR2 (PRD-hPLSCR2) and checked whether scramblase activity was restored. Functional assays showed that the addition of PRD to hPLSCR2 restored scrambling activity, and deletion of PRD in hPLSCR1 (ΔPRD-hPLSCR1) resulted in a lack of activity. These results suggest that PRD is crucial for the function of the protein. The effects of the PRD deletion in hPLSCR1 and the addition of PRD to hPLSCR2 were characterized using various spectroscopic techniques. Our results clearly showed that hPLSCR1 and PRD-hPLSCR2 showed Ca(2+)-dependent aggregation and scrambling activity, whereas hPLSCR2 and ΔPRD-hPLSCR1 did not show aggregation and activity. Thus we conclude that scramblases exhibit Ca(2+)-dependent scrambling activity by aggregation of protein. Our results provide a possible mechanism for phospholipid scrambling mediated by PLSCRs and the importance of PRD in its function and cellular localization.