Anaerobic fungi were isolated from rumen digesta of sheep and cattle and were purified using a plate culture technique. The isolates were successfully cultured on a semi-defined medium which lacked rumen fluid, and on a defined medium.

The new cytotoxic agents rakicidins A and B were isolated from cultured broth of a Micromonospora sp. Spectroscopic and amino acid analysis has shown that rakicidin A is a new cyclic lipopeptide, consisting of 4-amino-penta-2,4-dienoic acid, 3-hydroxy-2,4,16-trimethylheptadecanoic acid, sarcosine, and 3-hydroxyasparagine. Rakicidin B differs by one methylene group in the lipid side chain. These compounds exhibited cytotoxicity against the Ml09 cell line. In our continuing search for newantitumor agents from microorganisms, an isolate of Micromonospora was selected for further evaluation. This research led to the discovery of two newcytotoxic lipopeptides, rakicidins A and B (Fig. 1). This paper describes the fermentation, biological evaluation, purification, physico-chemical characterization, and structure elucidation of these compounds. Fermentation Microorganism The producing organism was isolated from a soil sample collected at Andhra Pradesh, India and was identified as a Micromonospora sp., strain No. R385-2. Media and Culture Conditions Micromonospora strain No. R385-2 was grown on slants of modified Bennett's mediumwhich contained the following: 0.5% Japanese potato starch (Generichem DEC. 1995 Corp. Acadama Dextrin No. 3), 0.5% glucose, 0.1% fish meat extract (Mikuni Chem. Industry), 0.1% yeast extract (Difco), 0.2% N-Z Case (Sheffield Products), 0.2% NaCl, 0.1% CaCO3, 1.5% agar. The medium was sterilized at 121°C for 20 minutes. The culture was transferred from the slant into 100ml of GERmedium in a 500ml flask. The medium contained the following: 2.4% Japanese potato starch, 0.1% dextrose, 0.3% beef extract (BBL), 0.5% tryptone (Difco), 0.5% yeast extract (BBL), 0.2% CaCO3 and was adjusted to pH 7.6 before autoclaving. The mediumwas sterilized at 121°C for 20

Deletion mutants were constructed from pZEP12, which contained the intact Thermoanaerobacterium saccharolyticum endoxylanase gene (xynA). Deletion of 1.75 kb from the N-terminal end of xynA resulted in a mutant enzyme that retained activity but lost thermostability. Deletion of 1.05 kb from the C terminus did not alter thermostability or activity. The deduced amino acid sequence of T. saccharolyticum B6A-RI endoxylanase XynA was aligned with five other family F beta-glycanases by using the PILEUP program of the Genetics Computer Group package. This multiple alignment of amino acid sequences revealed six highly conserved motifs which included the consensus sequence consisting of a hydrophobic amino acid, Ser or Thr, Glu, a hydrophobic amino acid, Asp, and a hydrophobic amino acid in the catalytic domain. Endoxylanase was inhibited by EDAC [1-(3-dimethylamino propenyl)-3-ethylcarbodiimide hydrochloride], suggesting that Asp and/or Glu was involved in catalysis. Three aspartic acids, two glutamic acids, and one histidine were conserved in all six enzymes aligned. Hydrophobic cluster analysis revealed that two Asp and one Glu occur in the same hydrophobic clusters in T. saccharolyticum B6A-RI endoxylanase and two other enzymes belonging to family F beta-glycanases and suggests their involvement in a catalytic triad. These two Asp and one Glu in XynA from T. saccharolyticum were targeted for analysis by site-specific mutagenesis. Substitution of Asp-537 and Asp-602 by Asn and Glu-600 by Gln completely destroyed endoxylanase activity. These results suggest that these three amino acids form a catalytic triad that functions in a general acid catalysis mechanism.

The activities of cellulolytic and xylanolytic enzymes produced by an anaerobic fungus (R1) which resembled Neocallimastix sp. were investigated. Carboxymethylcellulase (CMCase), cellobiase, and filter paper (FPase) activities had pH optima of 6.0, 5.5, and 6.0, respectively. CMCase and cellobiase activities both had a temperature optimum of 50°C, whereas FPase had an optimum of 45°C. The pH and temperature optima for xylanase activity were pH 6.0 and 50°C, respectively. Growth of the fungus on wheat straw, wheat straw holocellulose, or cellulose resulted in substantial colonization, with at least 43 to 58% losses in substrate dry matter and accumulation of comparable amounts of formate. This end product was correlated to apparent loss of substrate dry weight and could be used as an indicator of fungal growth. Milling of wheat straw did not enhance the rate or extent of substrate degradation. Growth of the Rl isolate on the above substrates or xylan also resulted in accumulation of high levels of xylanase activity and lower cellulase activities. Of the cellulases, CMCase was the most active and was associated with either low or trace amounts of cellobiase and FPase activities. During growth on xylan, reducing sugars, including arabinose and xylose, rapidly accumulated in the medium. Xylose and other reducing sugars, but not arabinose, were subsequently used for growth. Reducing sugars also accumulated, but not as rapidly, when the fungus was grown on wheat straw, wheat straw holocellulose, or cellulose. Xylanase activities detected during growth of Rl on media containing glucose, xylose, or cellobiose suggested that enzyme production was constitutive. Xylanase activity was mainly cell associated in these cultures, but there was a considerable increase in activity during fungal autolysis.

Growth and succinate versus lactate production from glucose by Anaerobiospirillum succiniciproducens was regulated by the level of available carbon dioxide and culture pH. At pH 7.2, the generation time was almost doubled and extensive amounts of lactate were formed in comparison with growth at pH 6.2. The succinate yield and the yield of ATP per mole of glucose were significantly enhanced under excess-CO2-HCO3 growth conditions and suggest that there exists a threshold level of CO2 for enhanced succinate production in A. succiniciproducens. Glucose was metabolized via the Embden-Meyerhof-Parnas route, and phosphoenolpyruvate carboxykinase levels increased while lactate dehydrogenase and alcohol dehydrogenase levels decreased under excess-CO2-HC03growth conditions. Kinetic analysis of succinate and lactate formation in continuous culture indicated that the growth rate-linked production rate coefficient (K) cells was much higher for succinate (7.2 versus 1.0 g/g of cells per h) while the non-growth-rate-related formation rate coefficient (K') was higher for lactate (1.1 versus 0.3 g/g of cells per h). The data indicate that A. succiniciproducens, unlike other succinate-producing anaerobes which also form propionate, can grow rapidly and form high final yields of succinate at pH 6.2 and with excess CO2-HCO3 as a consequence of regulating electron sink metabolism.