Production and Characteristics of a Bioflocculant by Klebsiella pneumoniae YZ-6 Isolated from Human Saliva

The production and characterization of a bioflocculant, MBF-6, by Klebsiella pneumoniae was investigated. Optimum culture conditions for bioflocculant production were an initial medium pH of 7, an incubation temperature of 30 °C, and an inoculum size of 1 % (v/v) of cell density 1.0?×?10⁸ cfu/mL. The carbon, nitrogen, and cation sources for optimum bioflocculant production were glucose, peptone, and ZnCl₂. The bioflocculant mainly consisted of protein (3.4 %) and sugar (95.1 %). Fourier transform infrared (FTIR) spectrum revealed the presence of carboxyl and hydroxyl groups while the thermogravimetric analysis (TGA) showed a degradation temperature (T _d) of 81.4 °C. MBF-6 had a good flocculating rate in kaolin suspension without cation addition and was stable over a wide range of pH and temperature. Investigation on the flocculation efficacy of the characterized MBF-6 for wastewater treatment of dairy, woolen, brewery, and sugar industries suggested it to be effective.     

Adding Value to the Oil Cake as a Waste from Oil Processing Industry: Production of Lipase and Protease by <Emphasis Type="Italic">Candida utilis</Emphasis> in Solid State Fermentation

Olive oil cake is a by-product from the olive oil processing industry and can be used for the lipase and protease production by Candida utilis in solid state fermentation. Different carbon and nitrogen sources were evaluated, and the results showed that the supplementation of the substrate with maltose and starch as carbon sources and yeast extract as a nitrogen source significantly increased the lipase production. The best results were obtained with maltose, whereas rather low lipase and protease activities were found with glucose and oleic acid. Response surface methodology and a five-level–three-factor central composite rotatable design were used to evaluate the effects of the initial moisture content, inoculum size and fermentation time on both lipase and protease activity levels. A lipase activity value of ≈25 U g^-1 and a protease activity value of 110 U g^-1 were obtained under the optimized fermentation conditions. An alkaline treatment of the substrate appeared to be efficient, leading to increases of 39% and 133% in the lipase and protease production, respectively. The results showed that the olive cake could be a good source for enzyme production by solid state fermentation.     

Psychrotolerant Mesorhizobium sp. Isolated from Temperate and Cold Desert Regions Solubilizes Potassium and Produces Multiple Plant Growth Promoting Metabolites

Soil potassium (K) supplement depends intensively on the application of chemical fertilizers, which have substantial harmful environmental effects. However, some bacteria can act as inoculants by converting unavailable and insoluble K forms into plant-accessible forms. Such bacteria are an eco-friendly approach for enhancing plant K absorption and consequently reducing utilization of chemical fertilization. Therefore, the present research was undertaken to isolate, screen, and characterize the K solubilizing bacteria (KSB) from the rhizosphere soils of northern India. Overall, 110 strains were isolated, but only 13 isolates showed significant K solubilizing ability by forming a halo zone on solid media. They were further screened for K solubilizing activity at 0 °C, 1 °C, 3 °C, 5 °C, 7 °C, 15 °C, and 20 °C for 5, 10, and 20 days. All the bacterial isolates showed mineral K solubilization activity at these different temperatures. However, the content of K solubilization increased with the upsurge in temperature and period of incubation. The isolate KSB (Grz) showed the highest K solubilization index of 462.28% after 48 h of incubation at 20 °C. The maximum of 23.38 µg K/mL broth was solubilized by the isolate KSB (Grz) at 20 °C after 20 days of incubation. Based on morphological, biochemical, and molecular characterization (through the 16S rDNA approach), the isolate KSB (Grz) was identified as Mesorhizobium sp. The majority of the strains produced HCN and ammonia. The maximum indole acetic acid (IAA) (31.54 µM/mL) and cellulase (390 µM/mL) were produced by the isolate KSB (Grz). In contrast, the highest protease (525.12 µM/mL) and chitinase (5.20 µM/mL) activities were shown by standard strain Bacillus mucilaginosus and KSB (Gmr) isolate, respectively.     

Characterization of Thermostable Serine Alkaline Protease from an Alkaliphilic Strain Bacillus pumilus MCAS8 and Its Applications

This study describes the characterization and optimization of medium components for an extracellular detergent, surfactant, organic solvent and thermostable serine alkaline protease produced by alkaliphilic Bacillus pumilus MCAS8 strain isolated from Pulicat lake sediments, Tamil Nadu, India. The strain yielded maximum protease (2,214?U/ml) under optimized conditions: carbon source, citric acid??1.5?% (w/w); inducer, soyabean meal??2?% (w/w); pH?11.0; shaking condition 37?°C for 48?h. The enzyme had pH and temperature optima of 9.0 and 60?°C, respectively. The enzyme displayed the molecular mass of 36?kDa in sodium dodecyl sulphate?Cpolyacrylamide gel electrophoresis study and exhibited activity at a wide range of pH (6.0?C11.0) and thermostability (20?C70?°C). More than 70?% residual activity was observed when the enzyme was incubated with dithiothreitol, ethylenediaminetetraacetic acid, ethylene glycol tetraacetic acid and H₂O₂ for 30?min. The protease activity was also enhanced by divalent cations such as Ba²⁺, Ca²⁺ and Mg²⁺ and was strongly inhibited by Fe²⁺, Zn²⁺, Sr²⁺, Hg²⁺ and urea. The enzyme retained more than 50?% of its initial activity after pre-incubation for 1?h in the presence of 5?% (v/v) organic solvents such as dimethyl sulphoxide and acetone. The protease could hydrolyse various native proteinaceous substrates (1?%?w/v) such as bovine serum albumin, casein, skim milk, gelatine, azocasein and haemoglobin. Wash performance analysis of enzyme revealed that it could effectively remove blood stains from the cotton fabric, thus making it suitable to use as an effective detergent additive. The protease enzyme also exhibited promising result in the dehairing of goat skin. The potency of the eco-friendly enzyme without using any chemicals against washing and dehairing showed that the enzyme could be used for various industrial applications.     

Production of Alkaline Protease by Bacillus altitudinis GVC11 using Castor Husk in Solid-State Fermentation

Castor (Ricinus communis L.) is an important oil seed crop having its main cultivated area in India, China, and Brazil in dry land farming. Castor husk is generated as waste in castor oil production. Use of castor husk waste as substrate is studied for alkaline protease production by Bacillus altitudinis GVC11 in solid-state fermentation. Various parameters like moisture content, incubation period, particle size, effect of carbon and nitrogen sources are studied and optimized for enzyme production. Highest enzyme production of 419,293 units per gram husk is obtained. Cost of enzyme production can be reduced by using castor husk as substrate.     

Fluorescence detection of Escherichia coli on mannose modified ZnTe quantum dots

Rapid detection and identification of Escherichia coli(E.coli) is essential to prevent its quickly spread.In this study,a novel fluorescence probe based on ZnTe quantum dots(QDs) modified by mannose(MAN)had been prepared for the determination of E.coli.The results showed that the obtained QDs showed excellent selectivity toward E.coli,and presented a good linearity in range of 1.0×10~5～1.0×10~8 CFU/mL.The optimum fluorescence intensity for detecting E. coli was found to be at pH 7.0 with a temperature of25℃ and incubation time of 20 min.Under these optimum conditions,the detection limit of E.coli was4.6×10~4 CFU/mL.The quenching was discussed to be a static quenching procedure,which was proved by the quenching efficiency of QDs decreased with the temperature increasing.     

Biosynthesis of Benzoylformic Acid from Benzoyl Cyanide with a New Bacterial Isolate of Brevibacterium sp. CCZU12-1

Brevibacterium sp. CCZU12-1 with high nitrilase activity could effectively hydrolyze benzoyl cyanide into benzoylformic acid. After the culture optimization, the preferred carbon sources, nitrogen sources, and inducer were glucose (10 g/L), a composite of peptone (10 g/L) plus yeast extract (2.5 g/L), and ε-caprolactam (2.0 mM), respectively. After the reaction optimization, the optimum reaction temperature, reaction pH, organic cosolvent, and metal ion were 30 °C, 7.0, ethanol (2 %, v/v), and Ca²⁺ (0.1 mM), respectively. At biotransformation of 120-mM benzoyl cyanide for 24 h, the yield of benzoylformic acid reached 91.8 %. Moreover, the microbial nitrilase from Brevibacterium sp. CCZU12-1 could hydrolyze various nitriles, and it significantly exhibited high nitrilase activity against benzoyl cyanide, 3-cyanopyridine, and α-cyclohexyl-mandelonitrile.     

Influence of media nutrients on synthesis of lignin peroxidase from Aspergillus sp

The effect of carbon and nitrogen sources, lignocellulosic substrates, and metal ions on lignin peroxidase (LiP) activity of Aspergillus sp., which was isolated from a mangrove area, was studied. Glucose (1%) was found to be the best carbon source. Among the various lignocellulosic substrates used, coir pith at 3% concentration increased LiP activity twofold on the second day of incubation. Peptone and KNO₃ completely inhibited the enzyme synthesis while (NH₄)₂SO₄ at 12.5 mM produced maximum activity. Since seawater contained all the requisite metal ions, any added ions had a negative effect on activity. Cu²⁺ had the most inhibiting effect while K⁺ the least. When all the optimized conditions were provided, in nitrogen- and carbon-sufficient medium, a maximum LiP activity of 345 U/mL was obtained on the second day of incubation.     

Green Synthesis of Silver Nanoparticles by Extracellular Extracts from Aspergillus japonicus PJ01

The present study focuses on the biological synthesis, characterization, and antibacterial activities of silver nanoparticles (AgNPs) using extracellular extracts of Aspergillus japonicus PJ01.The optimal conditions of the synthesis process were: 10 mL of extracellular extracts, 1 mL of AgNO₃ (0.8 mol/L), 4 mL of NaOH solution (1.5 mol/L), 30 °C, and a reaction time of 1 min. The characterizations of AgNPs were tested by UV-visible spectrophotometry, zeta potential, scanning electron microscope (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and thermogravimetric (TG) analyses. Fourier transform infrared spectroscopy (FTIR) analysis showed that Ag⁺ was reduced by the extracellular extracts, which consisted chiefly of soluble proteins and reducing sugars. In this work, AgNO₃ concentration played an important role in the physicochemical properties and antibacterial properties of AgNPs. Under the AgNO₃ concentration of 0.2 and 0.8 mol/L, the diameters of AgNPs were 3.8 ± 1.1 and 9.1 ± 2.9 nm, respectively. In addition, smaller-sized AgNPs showed higher antimicrobial properties, and the minimum inhibitory concentration (MIC) values against both E. coli and S. aureus were 0.32 mg/mL.     

Phytochemical Constituents,Antioxidant Activity,and Toxicity Assessment of the Aerial Part Extracts from the Infraspecific Taxa of Matthiola fruticulosa (Brassicaceae) Endemic to Sicily

In a project designed to investigate the specific and infraspecific taxa of Matthiola endemic to Sicily (Italy) as new potential sources of bioactive compounds in this work, the infraspecific taxa of Matthiola fruticulosa were studied, namely, subsp. fruticulosa and subsp. coronopifolia. HPLC–PDA/ESI–MS and SPME–GC/MS analyses of hydroalcoholic extracts obtained from the aerial parts of the two subspecies led to the detection of 51 phenolics and 61 volatile components, highlighting a quite different qualitative–quantitative profile. The antioxidant properties of the extracts were explored through in vitro methods: 1,1-diphenyl-2-picrylhydrazyl (DPPH), reducing power and Fe²⁺ chelating activity assays. The results of the antioxidant tests showed that the extracts possess a different antioxidant ability: particularly, the extract of M. fruticulosa subsp. fruticulosa exhibited higher radical scavenging activity than that of subsp. coronopifolia (IC₅₀ = 1.25 ± 0.02 mg/mL and 2.86 ± 0.05 mg/mL), which in turn displayed better chelating properties (IC₅₀ = 1.49 ± 0.01 mg/mL and 0.63 ± 0.01 mg/mL). Lastly, Artemia salina lethality bioassay was performed for toxicity assessment. The results of the bioassay showed lack of toxicity against brine shrimp larvae for both extracts. The data presented indicate the infraspecific taxa of M. fruticulosa as new and safe sources of antioxidant compounds.     

Mechanochemical synthesis,structure, and properties of solid solutions of alkaline earth metal fluorides: Ma1−xMbxF2 (M: Ca,Sr, Ba)

The capability of mechanochemical synthesis for the formation of solid solutions of alkaline earth metal fluorides M^a_1−xM^b_xF₂ (M: Ca, Sr, Ba) was tested by fluorination of metal acetates and metal hydroxides with ammonium fluoride directly at milling. Evidence was found for a mutual substitution of cations on their lattice positions in Ca_1−xSr_xF₂ and Ba_1−xSr_xF₂ samples. For the Ba/Ca-system this synthesis route is only partially successful. X-ray diffraction and ¹⁹F MAS NMR spectroscopy were used to characterize all samples concerning their crystal structure and local fluorine coordination. Calculations of ¹⁹F chemical shifts with the superposition model along with probability calculations for the intensity of the individual ¹⁹F lines, performed in dependence on the molar composition of the samples, perfectly agree with the experimental findings. The fluoride ion conductivity of as-prepared samples, determined by temperature dependent DC conductivity measurements, is significantly higher than those of crystalline binary fluorides. Moreover, a higher F⁻ ion conductivity is observed for samples with higher mixing grade in the Ca/Sr-and the Ba/Sr-systems.     

Ferulated Pectins and Ferulated Arabinoxylans Mixed Gel for Saccharomyces boulardii Entrapment in Electrosprayed Microbeads

Ferulated polysaccharides such as pectin and arabinoxylan form covalent gels which are attractive for drug delivery or cell immobilization. Saccharomyces boulardii is a probiotic yeast known for providing humans with health benefits; however, its application is limited by viability loss under environmental stress. In this study, ferulated pectin from sugar beet solid waste (SBWP) and ferulated arabinoxylan from maize bioethanol waste (AX) were used to form a covalent mixed gel, which was in turn used to entrap S. boulardii (2.08 × 10⁸ cells/mL) in microbeads using electrospray. SBWP presented a low degree of esterification (30%), which allowed gelation through Ca²⁺, making it possible to reduce microbead aggregation and coalescence by curing the particles in a 2% CaCl₂ cross-linking solution. SBWP/AX and SBWP/AX+ S. boulardii microbeads presented a diameter of 214 and 344 µm, respectively, and a covalent cross-linking content (dimers di-FA and trimer tri-FA of ferulic acid) of 1.15 mg/g polysaccharide. The 8-5′, 8-O-4′and 5-5′di-FA isomers proportions were 79%, 18%, and 3%, respectively. Confocal laser scanning microscopy images of propidium iodide-stained yeasts confirmed cell viability before and after microbeads preparation by electrospray. SBWP/AX capability to entrap S. boulardii would represent an alternative for probiotic immobilization in tailored biomaterials and an opportunity for sustainable waste upcycling to value-added products.     

Antimicrobial Constituents from Machaerium Pers.: Inhibitory Activities and Synergism of Machaeriols and Machaeridiols against Methicillin-Resistant Staphylococcus aureus,Vancomycin-Resistant Enterococcus faecium,and Permeabilized Gram-Negative Pathogens

Two new epimeric bibenzylated monoterpenes machaerifurogerol (1a) and 5-epi-machaerifurogerol (1b), and four known isoflavonoids (+)-vestitol (2), 7-O-methylvestitol (3), (+)-medicarpin (4), and 3,8-dihydroxy-9-methoxypterocarpan (5) were isolated from Machaerium Pers. This plant was previously assigned as Machaerium multiflorum Spruce, from which machaeriols A-D (6–9) and machaeridiols A-C (10–12) were reported, and all were then re-isolated, except the minor compound 9, for a comprehensive antimicrobial activity evaluation. Structures of the isolated compounds were determined by full NMR and mass spectroscopic data. Among the isolated compounds, the mixture 10 + 11 was the most active with an MIC value of 1.25 μg/mL against methicillin-resistant Staphylococcus aureus (MRSA) strains BAA 1696, −1708, −1717, −33591, and vancomycin-resistant Enterococcus faecium (VRE 700221) and E. faecalis (VRE 51299) and vancomycin-sensitive E. faecalis (VSE 29212). Compounds 6–8 and 10–12 were found to be more potent against MRSA 1708, and 6, 11, and 12 against VRE 700221, than the drug control ciprofloxacin and vancomycin. A combination study using an in vitro Checkerboard method was carried out for machaeriols (7 or 8) and machaeridiols (11 or 12), which exhibited a strong synergistic activity of 12 + 8 (MIC 0.156 and 0.625 µg/mL), with >32- and >8-fold reduction of MIC’s, compared to 12, against MRSA 1708 and −1717, respectively. In the presence of sub-inhibitory concentrations on polymyxin B nonapeptide (PMBN), compounds 10 + 11, 11, 12, and 8 showed activity in the range of 0.5–8 µg/mL for two strains of Acinetobacter baumannii, 2–16 µg/mL against Pseudomonas aeruginosa PAO1, and 2 µg/mL against Escherichia coli NCTC 12923, but were inactive (MIC > 64 µg/mL) against the two isolates of Klebsiella pneumoniae.     

A New Synthetic Methodology in the Preparation of Bimetallic Chalcogenide Clusters via Cluster-to-Cluster Transformations

A decanuclear silver chalcogenide cluster, [Ag₁₀(Se){Se₂P(OⁱPr)₂}₈] (2) was isolated from a hydride-encapsulated silver diisopropyl diselenophosphates, [Ag₇(H){Se₂P(OⁱPr)₂}₆], under thermal condition. The time-dependent NMR spectroscopy showed that 2 was generated at the first three hours and the hydrido silver cluster was completely consumed after thirty-six hours. This method illustrated as cluster-to-cluster transformations can be applied to prepare selenide-centered decanuclear bimetallic clusters, [Cu_xAg_10-x(Se){Se₂P(OⁱPr)₂}₈] (x = 0–7, 3), via heating [Cu_xAg_7−x(H){Se₂P(OⁱPr)₂}₆] (x = 1–6) at 60 °C. Compositions of 3 were accurately confirmed by the ESI mass spectrometry. While the crystal 2 revealed two un-identical [Ag₁₀(Se){Se₂P(OⁱPr)₂}₈] structures in the asymmetric unit, a co-crystal of [Cu₃Ag₇(Se){Se₂P(OⁱPr)₂}₈]_0.6[Cu₄Ag₆(Se){Se₂P(OⁱPr)₂}₈]_0.4 ([3a]_0.6[3b]_0.4) was eventually characterized by single-crystal X-ray diffraction. Even though compositions of 2, [3a]_0.6[3b]_0.4 and the previous published [Ag₁₀(Se){Se₂P(OEt)₂}₈] (1) are quite similar (10 metals, 1 Se²⁻, 8 ligands), their metal core arrangements are completely different. These results show that different synthetic methods by using different starting reagents can affect the structure of the resulting products, leading to polymorphism.     

Characterization of a Novel Organic Solvent Tolerant Protease from a Moderately Halophilic Bacterium and Its Behavior in Ionic Liquids

An extracellular protease was purified from a novel moderately halophilic bacterium Salinivibrio sp. strain MS-7 by the combination of an acetone precipitation (40–80 %) step and a DEAE-cellulose anion exchange column chromatography. Kinetic parameters of the enzyme exhibited V _max and K _m of 130 U/mg and 1.14 mg/ml, respectively, using casein as a substrate. The biochemical properties of the enzyme revealed that the 21-kDa protease had a temperature and pH optimum of 50 °C and 8.0, respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride, Pefabloc SC, chymostatin, and also EDTA, indicating that it belongs to the class of serine metalloproteases. Interestingly, Ba²⁺ and Ca²⁺ (2 mM) strongly enhanced the enzyme activity, while Fe²⁺ and Mg²⁺ activated moderately and Zn²⁺, Ni²⁺, and Hg²⁺ decreased the enzyme activity. The effect of organic solvents with different logP on the purified protease revealed complete stability in toluene, ethyl acetate, chloroform, and n-hexane at 10 and 50 % (v/v) and moderate stability even in 50 % of DMSO and ethanol. The behavior of the MS-7 protease in three imidazolium-based ionic liquids exhibited suitable activity in these green solvent systems, especially in 1-hexyl-3-methylimidazolium hexafluorophosphate ([C₆MIM][PF₆]). Comparison of the purified protease with other previously reported proteases suggests that strain MS-7 secrets a novel organic solvent-tolerant protease with outstanding activity in organic solvents and imidazolium-based ionic liquids, which could be applied in low water synthetic section of industrial biotechnology.     

Monoterpene Indole Alkaloids with Cav3.1 T-Type Calcium Channel Inhibitory Activity from Catharanthus roseus

Catharanthus roseus is a well-known traditional herbal medicine for the treatment of cancer, hypertension, scald, and sore in China. Phytochemical investigation on the twigs and leaves of this species led to the isolation of two new monoterpene indole alkaloids, catharanosines A (1) and B (2), and six known analogues (3–8). Structures of 1 and 2 were established by ¹H-, ¹³C- and 2D-NMR, and HREIMS data. The absolute configuration of 1 was confirmed by single-crystal X-ray diffraction analysis. Compound 2 represented an unprecedented aspidosperma-type alkaloid with a 2-piperidinyl moiety at C-10. Compounds 6–8 exhibited remarkable Ca_v3.1 low voltage-gated calcium channel (LVGCC) inhibitory activity with IC₅₀ values of 11.83 ± 1.02, 14.3 ± 1.20, and 14.54 ± 0.99 μM, respectively.     

Optimization of Fermentation Process of Pomegranate Peel and Schisandra Chinensis and the Biological Activities of Fermentation Broth: Antioxidant Activity and Protective Effect Against H2O2-induced Oxidative Damage in HaCaT Cells

In this study, the lactobacillus fermentation process of pomegranate (Punica granatum L.) peel and Schisandra chinensis (Turcz.) Baill (PP&SC) was optimized by using the response surface method (RSM) coupled with a Box-Behnken design. The optimum fermentation condition with the maximal yield of ellagic acid (99.49 ± 0.47 mg/g) was as follows: 1:1 (w:w) ratio of pomegranate peel to Schisandra chinensis, 1% (v:v) of strains with a 1:1 (v:v) ratio of Lactobacillus Plantarum to Streptococcus Thermophilus, a 37 °C fermentation temperature, 33 h of fermentation time, 1:20 (g:mL) of a solid–liquid ratio and 3 g/100 mL of a glucose dosage. Under these conditions, the achieved fermentation broth (FB) showed stronger free radical scavenging abilities than the water extract (WE) against the ABTS⁺, DPPH, OH⁻ and O₂⁻ radicals. The cytotoxicity and the protective effect of FB on the intracellular ROS level in HaCaT cells were further detected by the Cell Counting Kit-8 (CCK-8) assay. The results showed that FB had no significant cytotoxicity toward HaCaT cells when its content was no more than 8 mg/mL. The FB with a concentration of 8 mg/mL had a good protective effect against oxidative damage, which can effectively reduce the ROS level to 125.94% ± 13.46% (p < 0.001) compared with 294.49% ± 11.54% of the control group in H₂O₂-damaged HaCaT cells. The outstanding antioxidant ability and protective effect against H₂O₂-induced oxidative damage in HaCaT cells promote the potential for the FB of PP&SC as a functional raw material of cosmetics.     

Two dimensional inorganic electride-promoted electron transfer efficiency in transfer hydrogenation of alkynes and alkenes

A simple and highly efficient transfer hydrogenation of alkynes and alkenes by using a two-dimensional electride, dicalcium nitride ([Ca₂N]⁺·e^–), as an electron transfer agent is disclosed. Excellent yields in the transformation are attributed to the remarkable electron transfer efficiency in the electride-mediated reactions. It is clarified that an effective discharge of electrons from the [Ca₂N]⁺·e^– electride in alcoholic solvents is achieved by the decomposition of the electride via alcoholysis and the generation of ammonia and Ca(OⁱPr)₂. We found that the choice of solvent was crucial for enhancing the electron transfer efficiency, and a maximum efficiency of 80% was achieved by using a DMF mixed isopropanol co-solvent system. This is the highest value reported to date among single electron transfer agents in the reduction of C–C multiple bonds. The observed reactivity and efficiency establish that electrides with a high density of anionic electrons can readily participate in the reduction of organic functional groups.     

Purification and Biochemical Characterization of a New Protease Inhibitor from Conyza dioscoridis with Antimicrobial,Antifungal and Cytotoxic Effects

The main objective of the current study was the extraction, purification, and biochemical characterization of a protein protease inhibitor from Conyza dioscoridis. Antimicrobial potential and cytotoxic effects were also examined. The protease inhibitor was extracted in 0.1 M phosphate buffer (pH 6–7). Then, the protease inhibitor, named PDInhibitor, was purified using ammonium sulfate precipitation followed by filtration through a Sephadex G-50 column and had an apparent molecular weight of 25 kDa. The N-terminal sequence of PDInhibitor showed a high level of identity with those of the Kunitz family. PDInhibitor was found to be active at pH values ranging from 5.0 to 11.0, with maximal activity at pH 9.0. It was also fully active at 50 °C and maintained 90% of its stability at over 55 °C. The thermostability of the PDInhibitor was clearly enhanced by CaCl₂ and sorbitol, whereas the presence of Ca²⁺ and Zn²⁺ ions, Sodium taurodeoxycholate (NaTDC), Sodium dodecyl sulfate (SDS), Dithiothreitol (DTT), and β-ME dramatically improved the inhibitory activity. A remarkable affinity of the protease inhibitor with available important therapeutic proteases (elastase and trypsin) was observed. PDInhibitor also acted as a potent inhibitor of commercial proteases from Aspergillus oryzae and of Proteinase K. The inhibitor displayed potent antimicrobial activity against gram+ and gram- bacteria and against fungal strains. Interestingly, PDInhibitor affected several human cancer cell lines, namely HCT-116, MDA-MB-231, and Lovo. Thus, it can be considered a potentially powerful therapeutic agent.     

The Antiproliferative and Apoptosis-Inducing Effects of the Red Macroalgae Gelidium latifolium Extract against Melanoma Cells

The red macroalga Gelidium latifolium is widely distributed in the coastal areas of Indonesia. However, current knowledge on its potential biological activities is still limited. In this study, we investigated the potential bioactive compounds in Gelidium latifolium ethanol extract (GLE), and its cytotoxic effects against the murine B16-F10 melanoma cell line. GLE shows high total phenolic content (107.06 ± 17.42 mg GAE/g) and total flavonoid content (151.77 ± 3.45 mg QE/g), which potentially contribute to its potential antioxidant activity (DPPH = 650.42 ± 2.01 µg/mL; ABTS = 557.01 ± 1.94 µg/mL). ESI-HR-TOF-MS analysis revealed large absorption in the [M-H]^- of 327.2339 m/z, corresponding to the monoisotopic molecular mass of brassicolene. The presence of this compound potentially contributes to GLE’s cytotoxic activity (IC₅₀ = 84.29 ± 1.93 µg/mL). Furthermore, GLE significantly increased the number of apoptotic cells (66.83 ± 3.06%) compared to controls (18.83 ± 3.76%). Apoptosis was also confirmed by changes in the expression levels of apoptosis-related genes (i.e., p53, Bax, Bak, and Bcl2). Downregulated expression of Bcl2 indicates an intrinsic apoptotic pathway. Current results suggest that components of Gelidium latifolium should be further investigated as possible sources of novel antitumor drugs.