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.

Phospholipid scramblase 3: a latent mediator connecting mitochondria and heavy metal apoptosis.

Lead and mercury are the ubiquitous heavy metals triggering toxicity and initiating apoptosis in cells. Though the toxic effects of heavy metals on various organs are known, there is a paucity of information on the mechanisms that instigate the current study. A plausible role of phospholipid scramblase 3 (PLSCR3) in Pb2+ and Hg2+ induced apoptosis was investigated with human embryonic kidney (HEK 293) cells. After 12 h of exposure, ~30-40% of the cells were in the early stage of apoptosis with increased reactive oxygen species (ROS), decreased mitochondrial membrane potential, and increased intracellular calcium levels. Also, ~20% of the cardiolipin localized within the inner mitochondrial membrane was translocated to the outer mitochondrial membrane along with the mobilization of truncated Bid (t-Bid) to the mitochondria and cytochrome c from the mitochondria. The endogenous expression levels of PLSCR3, caspase 8, and caspase 3 were upregulated in Pb2+ and Hg2+ induced apoptosis. The activation and upregulation of PLSCR3 mediate CL translocation playing a potential role in initiating the heavy metal-induced apoptosis. Therefore, PLSCR3 could be the linker between mitochondria and heavy metal apoptosis.

Investigation of a robust pretreatment technique based on ultrasound-assisted, cost-effective ionic liquid for enhancing saccharification and bioethanol production from wheat straw.

Application of cost-effective pretreatment of wheat straw is an important stage for massive bioethanol production. A new approach is aimed to enhance the pretreatment of wheat straw by using low-cost ionic liquid [TEA][HSO4] coupled with ultrasound irradiation. The pretreatment was conducted both at room temperature and at 130 °C with a high biomass loading rate of 20% and 20% wt water assisted by ultrasound at 100 W-24 kHz for 15 and 30 min. Wheat straw pretreated at 130 °C for 15 and 30 min had high delignification rates of 67.8% and 74.9%, respectively, and hemicellulose removal rates of 47.0% and 52.2%. Moreover, this pretreatment resulted in producing total reducing sugars of 24.5 and 32.1 mg/mL in enzymatic saccharification, respectively, which corresponds to saccharification yields of 67.7% and 79.8% with commercial cellulase enzyme CelluMax for 72 h. The ethanol generation rates of 38.9 and 42.0 g/L were attained for pretreated samples for 15 and 30 min, equivalent to the yields of 76.1% and 82.2% of the maximum theoretical yield following 48 h of fermentation. This demonstration provided a cheap and promising pretreatment technology in terms of efficiency and shortening the pretreatment time based on applying low-cost ionic liquid and efficient ultrasound pretreatment techniques, which facilitated the feasibility of this approach and could further develop the future of biorefinery.

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.

An overview of mammalian and microbial hormone-sensitive lipases (lipolytic family IV): biochemical properties and industrial applications.

In mammals, hormone-sensitive lipase (EC 3.1.1.79) is an intracellular lipase that significantly regulates lipid metabolism. Mammalian HSL is more active towards diacylglycerol but lacks a lid covering the active site. Dyslipidemia, hepatic steatosis, cancer, and cancer-associated cachexia are symptoms of HSL pathophysiology. Certain microbial proteins show a sequence homologous to the catalytic domain of mammalian HSL, hence called microbial HSL. They possess a funnel-shaped substrate-binding pocket and restricted length of acyl chain esters, thus known as esterases. These enzymes have broad substrate specificities and are capable of stereo, regio, and enantioselective, making them attractive biocatalysts in a wide range of industrial applications in the production of flavors, pharmaceuticals, biosensors, and fine chemicals. This review will provide insight into mammalian and microbial HSLs, their sources, structural features related to substrate specificity, thermal stability, and their applications.

Interaction of human phospholipid scramblase 1 with cholesterol via CRAC motif is essential for functional regulation and subcellular localization.

Human phospholipid scramblase 1 (hPLSCR1) possesses a putative cholesterol binding CRAC (cholesterol interaction/recognition amino acid consensus) motif at the C-terminal. The CRAC motif of hPLSCR1 interacts with cholesterol with an energy of interaction -64.39 KJ mol-1. Since palmitoylated hPLSCR1 localizes to the cholesterol-rich lipid rafts, the interaction between hPLSCR1 and raft cholesterol is highly likely. The present study investigated the hPLSCR1-cholesterol interaction in plasma membrane via putative CRAC motif. hPLSCR1 remains at cholesterol-rich lipid rafts as long as they interact. This interaction is inhibited by mutations in the CRAC motif or cholesterol depletion. Thus, CRAC mutants I300D hPLSCR1 and ΔCRAC hPLSCR1 diffused to the cytoplasm and nucleus. Cholesterol depletion by methyl-ß-cyclodextrin (MßCD) dose-dependently reduced cell viability in A549 cells. However, cholesterol depletion released 1.74 ± 0.12 times Ca2+ to the cytosol in A549 cells. Similarly, cholesterol depletion increased intracellular Ca2+ release by 1.81 ± 0.13 and 4.11 ± 0.19 times in RAJI cells expressing hPLSCR1 and ΔCRAC hPLSCR1, respectively. Moreover, the expression of hPLSCR1 and ΔCRAC hPLSCR1 increased apoptosis in RAJI cells by 21 ± 1.5% and 53.50 ± 4.40%, respectively. It was further increased to 43 ± 2.5% and 71.4 ± 1.4% upon cholesterol depletion. The current work links hPLSCR1 expression with cholesterol depletion, intracellular Ca2+ release, and induction of apoptosis.

Arjunetin as a promising drug candidate against SARS-CoV-2: molecular dynamics simulation studies.

Stem and bark of the tree Terminalia arjuna Wight & Arn. (Combretaceae) has been documented to exhibit therapeutic properties like cardiotonic, anticancer, antiviral, antibacterial, antifungal, hypercholesterolemia, hypolipidemic, and anti-coagulant. Our previous studies have shown that, ethanolic extract of T. arjuna bark exhibits radical scavenging anti-oxidant activity and also effectively inhibited catalase activity. In this study, oleanane triterpenoids type compounds viz., oleanolic acid, arjunolic acid, arjunolitin, arjunetin were isolated from ethanolic bark extract as bio-active compound and their structures were elucidated using 1H, 13C NMR, HR-ESIMS, IR. Of the various compounds, Arjunetin showed significant inhibition of catalase activity as compared to the other compounds. Based on the structural similarity between arjunetin and current antiviral drugs, we propose that arjunetin might exhibit antiviral activity. Molecular docking and molecular dynamics studies showed that arjunetin binds to the binds to key targets of SARS-CoV-2 namely, 3CLpro, PLpro, and RdRp) with a higher binding energy values (3CLpro, -8.4 kcal/mol; PLpro, -7.6 kcal/mol and RdRp, -8.1 kcal/mol) as compared with FDA approved protease inhibitor drugs to Lopinavir (3CLpro, -7.2 kcal/mole and PLpro -7.7 kcal/mole) and Remdesivir (RdRp -7.6 kcal/mole). To further investigate this, we performed 200-500 ns molecular dynamics simulation studies. The results transpired that the binding affinity of Arjunetin is higher than Remdesivir in the RNA binding cavity of RdRp. Based on structural similarity between arjunetin and Saikosaponin (a known antiviral agents) and based on our molecular docking and molecular dynamic simulation studies, we propose that arjunetin can be a promising drug candidate against Covid-19.Communicated by Ramaswamy H. Sarma.

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.

Regulation and functions of membrane lipids: Insights from Caenorhabditis elegans.

The Caenorhabditis elegans plasma membrane is composed of glycerophospholipids and sphingolipids with a small cholesterol. The C. elegans obtain the majority of the membrane lipids by modifying fatty acids present in the bacterial diet. The metabolic pathways of membrane lipid biosynthesis are well conserved across the animal kingdom. In C. elegans CDP-DAG and Kennedy pathway produce glycerophospholipids. Meanwhile, the sphingolipids are synthesized through a different pathway. They have evolved remarkably diverse mechanisms to maintain membrane lipid homeostasis. For instance, the lipid bilayer stress operates to accomplish homeostasis during any perturbance in the lipid composition. Meanwhile, the PAQR-2/IGLR-2 complex works with FLD-1 to balance unsaturated to saturated fatty acids to maintain membrane fluidity. The loss of membrane lipid homeostasis is observed in many human genetic and metabolic disorders. Since C. elegans conserved such genes and pathways, it can be used as a model organism.

Chlorpyrifos in environment and food: a critical review of detection methods and degradation pathways.

Chlorpyrifos (CP) is a class of organophosphorus (OP) pesticides, which find extensive applications as acaricide, insecticide and termiticide. The use of CP has been indicated in environmental contamination and disturbance in the biogeochemical cycles. CP has been reported to be neurotoxic and has a detrimental effect on immunological and psychological health. Therefore, it is necessary to design and develop effective degradation methods for the removal of CP from the environment. In the past few years, physicochemical (advanced oxidation process) and biological treatment approaches have been widely employed for the pesticide removal. However, the byproducts of this process are more toxic than the parent compound and along with an incomplete degradation of CP. This review focuses on the toxicity of CP, the sources of contamination, degradation pathways, physicochemical, biological, and nano-technology based methods employed for the degradation of CP. In addition, consolidated information on various detection methods and materials used for the detection have been provided in this review.

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.

Cholesterol interaction attenuates scramblase activity of SCRM-1 in the artificial membrane.

Phospholipid (PL) scramblases are single-pass transmembrane protein mediating bidirectional PL translocation. Previously in silico analysis of human PL scramblases, predicted the presence of an uncharacterized cholesterol-binding domain spanning partly in the transmembrane helix as well as in the adjacent extracellular coil. This domain was found to be universally conserved in diverse organisms like Caenorhabditis elegans. In this study, we investigated the saturable cholesterol-binding domain of SCRM-1 using fluorescence sterol binding assay, Stern-Volmer quenching, Förster resonance energy transfer, and CD spectroscopy. We observed high-affinity interaction between cholesterol and SCRM-1. Our results support a previous report, which showed that the cholesterol ordering effect reduced the scramblase activity of hPLSCR1. Considering the presence of a high-affinity binding sequence, we propose that the reduction in activity could partly be due to the cholesterol binding. To validate this, we generated a C-terminal helix (CTH) deletion construct (∆CTH SCRM-1) and a point mutation in the putative cholesterol-binding domain I273D SCRM-1. Deletion construct greatly reduced cholesterol affinity along with loss of scramblase activity. In contrast to this, I273D SCRM-1 retained scrambling activity in proteoliposomes containing ~30 mol% cholesterol but lost sterol binding ability. These results suggest that C-terminal helix is crucial for membrane insertion and in the lipid bilayer the scrambling activity of SCRM-1 is modulated through its interaction with cholesterol.

Simultaneous Optimization of Activity and Stability of Xylose Reductase from D. nepalensis NCYC 3413 Using Statistical Experimental Design.

BACKGROUND: Physical parameters like pH and temperature play a major role in the design of an industrial enzymatic process. Enzyme stability and activity are greatly influenced by these parameters; hence optimization and control of these parameters becomes a key point in determining the economic feasibility of the process. OBJECTIVE: This study was taken up with the objective to optimize physical parameters for maximum stability and activity of xylose reductase from D. nepalensis NCYC 3413 through separate and simultaneous optimization studies and comparison thereof. METHODS: Effects of pH and temperature on the activity and stability of xylose reductase from Debaryomyces nepalensis NCYC 3413 were investigated by enzyme assays and independent variables were optimised using surface response methodology. Enzyme activity and stability were optimised separately and concurrently to decipher the appropriate conditions. RESULTS: Optimized conditions of pH and temperature for xylose reductase activity were determined to be 7.1 and 27 °C respectively, with predicted responses of specific activity (72.3 U/mg) and half-life time (566 min). The experimental values (specific activity 50.2 U/mg, half-life time 818 min) were on par with predicted values indicating the significance of the model. CONCLUSION: Simultaneous optimization of xylose reductase activity and stability using statistical methods is effective as compared to optimisation of the parameters separately.

Polystyrene adsorbents: rapid and efficient surrogate for dialysis in membrane protein purification.

Membrane protein purification is a laborious, expensive, and protracted process involving detergents for its extraction. Purifying functionally active form of membrane protein in sufficient quantity is a major bottleneck in establishing its structure and understanding the functional mechanism. Although overexpression of the membrane proteins has been achieved by recombinant DNA technology, a majority of the protein remains insoluble as inclusion bodies, which is extracted by detergents. Detergent removal is essential for retaining protein structure, function, and subsequent purification techniques. In this study, we have proposed a new approach for detergent removal from the solubilized extract of a recombinant membrane protein: human phospholipid scramblase 3 (hPLSCR3). N-lauryl sarcosine (NLS) has been established as an effective detergent to extract the functionally active recombinant 6X-his- hPLSCR3 from the inclusion bodies. NLS removal before affinity-based purification is essential as the detergent interferes with the matrix binding. Detergent removal by adsorption onto hydrophobic polystyrene beads has been methodically studied and established that the current approach was 10 times faster than the conventional dialysis method. The study established the potency of polystyrene-based beads as a convenient, efficient, and alternate tool to dialysis in detergent removal without significantly altering the structure and function of the membrane protein.

Plant extract mediated synthesis enhanced the functional properties of silver ferrite nanoparticles over chemical mediated synthesis.

In this study, the antibacterial, antioxidant and cytotoxicity behaviour of silver ferrite nanoparticles (AgFeO2 NPs) synthesized through chemical and green routes were compared. Green synthesis (Bio) of AgFeO2 NPs were prepared by precipitation method using Amaranthus blitum leaves extract as a reducing agent. Chemical synthesis (Che) of AgFeO2 NPs was mediated by sodium borohydride as a reducing agent. [AgFeO2 (Bio)] NPs showed reduced size, better monodispersity and surface area compared to [AgFeO2 (Che)] NPs. The results showed that synthesized NPs have better antibacterial activity against E. coli than S. aureus. In addition, 250 µg of AgFeO2 (Bio) and (Che) NPs showed antioxidant efficiency of 98 and 86%. The results showed that [AgFeO2 (Bio)] NPs showed lower cytotoxicity [AgFeO2 (Che)] NPs against human human embryonic kidney (HEK 293) cells. These results suggest that [AgFeO2 (Bio)] NPs have improved physicochemical properties thereby they can be used as an effective biocatalytic material in biotechnology.

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.

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.

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.