Liquid fluidized bed starter culture ofTrichoderma reesei for cellulase production

A starter culture ofTrichoderma reesei (Rut-C30) prepared in a liquid fluidized bed reactor (LFBR) gave better growth and greater cellulase production in submerged fermentation than a conventional shake flask inoculum. The LFBR starter was prepared by first coatingT. reesei spores to 0.25 mm size corncob (1.0x10⁸g^-1) in a medium containing 1.0% corncob, 0.5 gL^-1 xylose and 0.1 gL^-1 lactose in a balanced salt solution, then fluidizing the particles in the LFBR for 36 h to allow germination of the spores, and covering the particles with an approx 30 μm thick biofilm. This biofilm that developed in constant adherence to the lignocellulosic carrier, apparently became well adapted to grow rapidly on insoluble cellulose substrates (Solca Floc), and had the enzymes of the cellulase complex induced for increased cellulase production. The LFBR starter used in a stirred tank reactor (STR) gave 15 gL^-1 biomass production and 6.5 IU mL^-1 overall cellulase activity with a volumetric productivity of 64 IU L^-1h^-1 in a 5 d fermentation, compared with a 7 d shake flask inoculum that gave 11 gL^-1 biomass and 3.2 IU mL^-1 cellulase activity, with a volumetric productivity of 31IU L^-1h^-1. The LFBR starter culture retained its viability in dry storage for 6–9 mo.     

Transformed steroids

A preparative method for 9a-hydroxylation of ⁵-3-hydroxysteroids using the fungi ofCircinella sp. 10Kh-1220 not capable of modifying theA ring has been developed. It is established that the yields of the main and the side products greatly depend on the transformation conditions, mycelium age, and the structure of the steroid substrate. Under the optimal transformation conditions novel 9-hydroxysubstituted derivatives of androstenolone, pregnenolone, 16-dehydro-16,17-epoxy-, and-16-methoxypregnenolone have been obtained in 36–80 % yields.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 4, pp. 737–743, April, 1994.     

A model explaining declining rate in hydrolysis of lignocellulose substrates with cellobiohydrolase I (cel7A) and endoglucanase I (cel7B) of Trichoderma reesei

It is commonly observed that the rate of enzymatic hydrolysis of solid cellulose substrates declines markedly with time. In this work the mechanism behind the rate reduction was investigated using two dominant cellulases of Trichoderma reesei: exoglucanase Cel7A (formerly known as CBHI) and endoglucanase Cel7B (formerly EGI). Hydrolysis of steam-pretreated spruce (SPS) was performed with Cel7A and Cel7B alone, and in reconstituted mixtures. Throughout the 48-h hydrolysis, soluble products, hydrolysis rates, and enzyme adsorption to the substrate were measured. The hydrolysis rate for both enzymes decreases rapidly with hydrolysis time. Both enzymes adsorbed rapidly to the substrate during hydrolysis. Cel7A and Cel7B cooperate synergistically, and synergism was approximately constant during the SPS hydrolysis. Thermal instability of the enzymes and product inhibition was not the main cause of reduced hydrolysis rates. Adding fresh substrate to substrate previously hydrolyzed for 24 h with Cel7A slightly increased the hydrolysis of SPS; however, the rate increased even more by adding fresh Cel7A. This suggests that enzymes become inactivated while adsorbed to the substrate and that unproductive binding is the main cause of hydrolysis rate reduction. The strongest increase in hydrolysis rate was achieved by adding Cel7B. An improved model is proposed that extends the standard endo-exo synergy model and explains the rapid decrease in hydrolysis rate. It appears that the processive action of Cel7A becomes hindered by obstacles in the lignocellulose substrate. Obstacles created by disordered cellulose chains can be removed by the endo activity of Cel7B, which explains some of the observed synergism between Cel7A and Cel7B. The improved model is supported by adsorption studies during hydrolysis.     

Treatment of dairy wastewater using a selected bacterial isolate, Alcaligenes sp. MMRR7

Physicochemical and biologic analysis of dairy wastewater showed that the effluent had a high organic load (chemical oxygen demand [COD]: 5095 mg/L), an acidic pH (6.4), and a high probability of coliforms (most probable number [MPN]>1100). The various bacterial strains isolated and purified were identified as Sporolactobacillus sp., Citrobacter sp., Pseudomonas sp., Alcaligenes sp., Bacillus sp., Staphylococcus sp., and Proteus sp., as per the Bergey’s manual of systematic bacteriology. Among the five selected bacterial strains, the strain designated as MMRR₇ and identified as Alcaligenes sp. was found to give a maximum reduction in COD (62%) in 5 d of incubation. Chemical coagulation using alum at a concentration of 0.5 g/100 mL was found to be effective in the primary treatment of the effluent. Studies on free-cell treatment of the coagulated effluent with the selected bacterial strain Alcaligenes sp. MMRR₇ gave a maximum COD reduction of 91% in 120 h. This study clearly indicates the possibility of using Alcaligenes sp. MMRR₇ for the effective treatment of dairy wastewater.     

Effects of fatty acids on growth and poly-3-hydroxybutyrate production in bacteria

The effects of saturated and unsaturated fatty acids (lauric acid, palmitic acid, steric acid, oleic acid, linoleic acid, soybean oil) on Sphaerotilus natans, 0B17 (Pseudomonas sp.), and recombinant Escherichia coli DH5(/pUC19/CAB were studied. Oleic acid enhances Poly-3-hydroxybutyrate (PHB) production in these three bacterial strains, suggesting that the single double bond of the acid activates the polyhydroxylkanoate accumulation enzymatic reaction. Under the effect of lauric acid and linoleic acid, the growth of S. natans and 0B17 were totally inhibited. However, the enhanced PHB accumulation in recombinant E. coli was observed.     

Production ofL-glutamate oxidase andin situ monitoring of oxygen uptake in solid state fermentation ofstreptomyces sp. Nl

Effects of water content and carbon and nitrogen sources on the production ofL-glutamate oxidase (GOD) by solid state fermentation (SSF) ofStreptomyces sp. N₁ were investigated in a 250-mL shake flask. The results show that in the solid medium containing wheat bran 98% (w/w), KCl 0.2% (w/w), and MgCl₂ 0.2% (w/w), addition of 2.0-mL water per gram solid medium and 0.4% (w/w) (NH₄)₂SO₄ was the best for GOD production. In this work, we also developed a simple technique forin situ measuring oxygen uptake rate (OUR) and carbon dioxide evolution rate (CER) in SSF in a shake flask based on the principle of Warburg manometer. The method was successfully applied to determine OUR and CER values in SSF ofStreptomyces sp. N₁. The results indicate that the largest OUR value was detected about one or two days ahead of the highest GOD activity reached depending on the fermentation conditions, and the OUR may be used as anin situ indicator of GOD production in the SSF process.     

Antiplasmodial cembradiene diterpenoids from a Southwestern Caribbean gorgonian octocoral of the genus Eunicea

Five new representatives of the cembrane class of marine natural products have been isolated as minor metabolites from an undescribed species of Eunicea, a sea whip collected near the Colombian Southwestern Caribbean Sea. The structure of the crystalline metabolite 1 was solved by single-crystal X-ray diffraction analysis. Structures could then be proposed for cembradienes 2-5 by comprehensive spectral analyses involving 2D NMR, IR, UV, and high-resolution mass spectrometry, as well as chemical interconversion studies. Compound 1 exhibited weak cytotoxicity in the NCI 3-cell line panel human cancer screening program, whereas compounds 1, 2 and 4 displayed significant antiplasmodial activity against Plasmodium falciparum.     

Xylanase production by Trichoderma reesei rut C-30 on rice straw

Xylanase production of Trichoderma reesei Rut C-30 was examined at different initial pH values (4.8, 5.9, and 7.0) on rice straw in shake flasks, and in a fermentor, for the best pH condition. Enzyme performance was tested on ammonia-treated dwarf elephant grass. The maximum xylanase activities, 92 and 122 IU/mL, were obtained at pH 4.8 in the shake flasks and fermentor, respectively, in which good growth of the fungus was observed during the first 24 h and consumption of proteins dissolved from the rice straw caused the pH to rise later to values between 6.4 and 6.7 (optimal for xylanase production). The xylanases from T. reesei were as effective as Multifect XL, a commercial enzyme preparation, in hydrolyzing ammonia-treated elephant grass.     

Novel biotransformation of pentacyclic triterpenoid acids by Nocardia sp. NRRL 5646

Six pentacyclic triterpene acids, ursolic acid, oleanolic acid, betulinic acid, 23-hydroxybetulinic acid, glycyrrhetinic acid, and senegenin, were metabolized by the microbe Nocardia sp. NRRL 5646 to selectively furnish their corresponding 28-methyl esters. Notably, ursolic acid (1) was converted to oleanolic acid methyl ester (4) via two intermediates, oleanolic acid (2), and ursolic acid methyl ester (3), which are formed by participation of ‘retro-biosynthetic’ methyl migration from C-19 to C-20. Senegenin (11) was selectively converted to a nortriterpene methyl ester, senegenic acid methyl ester (12), via an unprecedented C-C bond cleavage. The stereochemical assignments of compounds 11 and 12 were made unambiguously for the first time using 2D NMR spectroscopy.     

Palladium‐Catalyzed Direct Cross‐Coupling of Carboranyllithium with (Hetero)Aryl Halides

A palladium‐catalyzed direct C‐arylation reaction of readily available cage carboranyllithium reagents with aryl halides has been developed for the first time. This method is applicable to a wide range of aryl halide substrates including aryl iodides, aryl bromides, and heteroaromatic halides.