Chemical analysis and anticancer activity of sesterterpenoid from an endophytic fungus Hypomontagnella monticulosa Zg15SU and its host Zingiber griffithii Baker.

Zingiber griffithii Baker is one of the native Zingiberaceous species in a tropical forest of North Sumatra, Indonesia. Zingiberaceous species have been intensively studied and reported as herbal ingredients in ethnomedicine and currently their endophytic fungal associates were studied for pharmacological importance. Fifteen endophytic fungi were isolated from Zingiber griffithii following morphological and molecular characterization. All isolates exhibited antibacterial properties to at least one of the tested pathogenic bacteria Staphylococcus aureus, Escherichia coli, Methicilin-resistant S. aureus (MRSA), and Enteropathogenic E. coli (EPEC). The isolate, identified as Hypomontagnella monticulosa strain Zg15SU (syn. Hypoxylon monticulosum Mont.) based on its rDNA/ITS sequence, displayed antibacterial activities to all tested pathogens. The EtOAc extract of the H. monticulosum Zg15SU showed the highest activity for gram-negative bacteria, the E. coli and EPEC, while the extract of Z. griffithii rhizome displayed activity only for E. coli. The gas chromatography-mass spectrometry analysis (GC-MS) indicated a major portion of similar compounds found in both the endophytic fungus and plant extract, revealing the compounds of oleic acid, cyclononasiloxane, octadecamethyl, and eicosanoic acid Furthermore, purification and structural elucidation on the EtOAc extract of both Z. griffithii rhizome and H. monticulos a Zg15SU yielded two bioactive compounds: a novel compound, griffithiiene, a terpenoid-alkaloid bearing the skeleton of a scalarane (1) and scalaradial (2) which were confirmed by 1H- (500 MHz) and 13C-NMR (125 MHz) spectroscopy. Importantly, the elucidated compounds showed a cytotoxicity activity against cancer cell lines, the Panc-1, NBT-T2, and HCT116 based on in vitro MTT proliferation assay. This is the first report of Z. griffithii harboring an endophytic fungus, H. monticulosa, which produced potential antibacterial and anticancer metabolites along with its host to be utilized for future prospects.

Isolation and enzyme bioprospection of bacteria associated to Bruguiera cylindrica, a mangrove plant of North Sumatra, Indonesia.

Mangrove-associated bacteria are of industrial interest due to their diverse and versatile enzyme properties. This study investigates the culturable bacteria from a wide range of habitat in a Bruguiera cylindrica mangrove ecosystem in North Sumatra. Screening of extracellular hydrolytic enzymes showed multiple potential traits in amylase, cellulase, chitinase, phosphatase, protease, and urease production by bacterial isolates. Molecular identification based on 16S rDNA region of a potential strain, Vibrio alginolyticus Jme3-20 is then reported as a newly proteolytic agent. The strain also showed a stable growth under salinity (NaCl) stress with considerable phosphate solubilization activities. Protease activity was enhanced by optimizing the 0.5 % (w/v) sucrose and soy peptone in the fermentation medium. SDS-PAGE and zymogram analysis showed the presence of a 35-kDa MW protease. Hence, our study revealed important insights into the bacterial diversity and activity in mangrove ecosystems, evidencing the importance of microbial exploration in this ecosystem.

Subcellular localization of glyoxylate cycle key enzymes involved in oxalate biosynthesis of wood-destroying basidiomycete Fomitopsis palustris grown on glucose.

This study investigated the subcellular localization of key enzymes of the glyoxylate cycle, i.e. isocitrate lyase (ICL; EC 4.1.3.1) and malate synthase (EC 2.3.3.9), that function constitutively in coordination with oxalate biosynthesis of glucose-grown Fomitopsis palustris. The ICL purified previously from F. palustris is termed FPICL1. Subcellular fractionation analysis of the cell homogenate by the sucrose density-gradient method showed that both key enzymes were present in peroxisomes, whereas acetyl-CoA synthase (EC 6.2.1.1) and oxalate-producing oxaloacetate acetylhydrolase (EC 3.7.1.1) were cytosolic. The peroxisomal localization of FPICL1 was further confirmed by electron microscopic and immunocytochemical analysis with anti-FPICL1 antibody. In addition, the peroxisomal target signal, composed of SKL at the C terminus of the cDNA encoding FPICL1, was found, which also suggests that FPICL1 is peroxisomal. Accordingly, it is postulated that transportation of succinate from peroxisomes to mitochondria, and vice versa, for the transportation of isocitrate or citrate, occurs in glucose-grown F. palustris for the constitutive metabolic coordination of the TCA and glyoxylate cycles with oxalate biosynthesis.

Purification and characterization of malate synthase from the glucose-grown wood-rotting basidiomycete Fomitopsis palustris.

Malate synthase (EC 4.1.3.2), the key enzyme of the glyoxylate cycle, was purified to a homogeneous protein from the wood-rotting basidiomycete Fomitopsis palustris grown on glucose. The purified enzyme, with a molecular mass of 520 kDa, was found to consist of eight 65-kDa subunits, and to have Km of 45 and 2.2 microM for glyoxylate and acetyl-CoA, respectively. The enzyme activity was competitively inhibited by oxalate (K1, 8.5 microM) and glycolate (Ki, 17 microM), and uncompetitively by coenzyme A (Ki, 100 microM). The potent inhibition of the activity by p-chloromercuribenzoate suggests that the enzyme has a sulfhydryl group at the active center. However, the enzyme was inhibited moderately by adenine nucleotides and weakly by some of the metabolic intermediates of glycolysis and tricarboxylic acid cycle. The enzyme was completely inactive in the absence of metal ions and was maximally activated by Mg2+ (Km, 0.4 microM), which also served to significantly prevent enzyme inactivation during storage.

Purification and characterization of isocitrate lyase from the wood-destroying basidiomycete Fomitopsis palustris grown on glucose.

Isocitrate lyase (EC 4.1.3.1), a key enzyme in the glyoxylate cycle, was purified 76-fold with 23% yield as an electrophoretically homogeneous protein from the wood-destroying basidiomycete Fomitopsis palustris grown on glucose. The native enzyme has a molecular mass of 186 kDa, consisting of three identical subunits of 60 kDa. The K(m) for DL-isocitrate was found to be 1.6 mM at the optimum pH (7.0). The enzyme required Mg(2+) (K(m) 92 microM) and sulfhydryl compounds for optimal activity. The enzyme activity was strongly inhibited by oxalate and itaconate with a K(i) of 37 and 68 microM, respectively. The inhibition by the glycolysis and tricarboxylic acid cycle intermediates and related compounds suggested that the isocitrate lyase was a regulatory enzyme playing a crucial role in the fungal growth.