A novel raw starch digesting thermostable α-amylase from Bacillus sp. I-3 and its use in the direct hydrolysis of raw potato starch

A new strain of Bacillus sp. I-3, isolated from natural soil samples, showed a high raw starch digesting activity towards potato starch. Upon optimization of various environmental and cultural conditions, the yield of α-amylase reached 642 U/mL. The kinetic characterization of partially purified enzyme exhibited the maximum activity at 70 °C, pH 7.0 and revealed a high thermostability in the presence of 10 mM CaCl₂·2H₂O where it could retain more than 90% residual activity at 70 °C after 3.5 h. At 80, 90 and 100 °C, the enzyme retained 80, 59 and 26% of its maximum activity after 2.5, 0.5 and 0.5 h, respectively. The enzyme preparation had a strong affinity towards raw potato starch granules and was almost completely adsorbed onto it. It also hydrolyzed raw potato starch at a concentration of 12.5% significantly in a short period of time of 12 h.     

Improved production of an enzyme that hydrolyses raw yam starch by Penicillium sp. S-22 using fed-batch fermentation

A newly isolated strain, Penicillium sp. S-22, was used to produce an enzyme that hydrolyses raw yam starch [raw yam starch digesting enzyme (RYSDE)]. The enzyme activity and overall enzyme productivity were respectively 16 U/ml and 0.19 U/ml h in the batch culture. The enzyme activity increased to 85 U/ml by feeding of partially hydrolyzed raw yam starch. When a mixture containing partially hydrolyzed raw yam starch and peptone was fed by a pH-stat strategy, the enzyme activity reached 366 U/ml, 23-fold of that obtained in the batch culture, and the overall productivity reached 3.4 U/ml h, which was 18-fold of that in the batch culture.     

Improvement of ethanol yield from raw corn flour by <Emphasis Type="Italic">Rhizopus</Emphasis> sp.

A novel raw-starch-digesting glucoamylase producer, Rhizopus sp. W-08, and Saccharomyces cerevisiae Z-06 were used in a fed batch process for simultaneous saccharification and fermentation of raw corn flour. Ethanol concentration of 21% (v/v) was obtained after 48 h. The conversion efficiency of raw corn flour to ethanol was 94.5% of the theoretical ethanol yield.     

Four novel yeasts from decaying organic matter: Blastobotrys robertii sp. nov., Candida cretensis sp. nov., Candida scorzettiae sp. nov. and Candida vadensis sp. nov

Four novel yeast species are described, two from decaying mushrooms, viz. Candida cretensis and Candida vadensis, and two from rotten wood, viz. Blastobotrys robertii and Candida scorzettiae. Accession numbers for the CBS and ARS Culture Collections, and GenBank accession numbers for the D1/D2 domains of the large subunit of ribosomal DNA are: B. robertii CBS 10106^T, NRRL Y-27775, DQ839395; C. cretensis CBS 9453^T, NRRL Y-27777, AY4998861 and DQ839393; C. scorzettiae CBS 10107^T, NRRL Y-27665, DQ839394; C. vadensis CBS 9454^T, NRRL Y-27778, AY498863 and DQ839396. The GenBank accession number for the ITS region of C. cretensis is AY498862 and that for C. vadensis is AY498864. C. cretensis was the only species of the four that displayed fermentative activity. All four type strains grew on n-hexadecane. C. scorzettiae is the only one of the new species that assimilates some phenolic compounds, viz. 3-hydroxy derivatives of benzoic, phenylacetic and cinnamic acids, but not the corresponding 4-hydroxy acids. This is indicative of an operative gentisate pathway.     

Sulfonolipids are localized in the outer membrane of the gliding bacterium Cytophaga johnsonae

Earlier work in our laboratory demonstrated that gliding bacteria of the Cytophaga-Flexibacter group contain, in their cell envelopes, large quantities of unusual sulfonolipids (N-fatty acyl 2-amino-3-hydroxyisoheptadecane-1-sulfonic acids). Recently, it has been shown that these lipids are necessary for the gliding motility of C. johnsonae. As one approach to determining the role of the lipids in motility, methods have now been developed for separating the inner (cytoplasmic) and outer membranes of a strain (ATCC 43786) of this Gram-negative bacterium. Sulfonolipid is at least five times as abundant in the outer membrane as in the inner. The inner membrane has properties similar to those found for other Gram-negative bacteria; it has a buoyant density of 1.14 g/ml and is highly enriched in cytochromes and succinate dehydrogenase. The outer membrane (1.18 g/ml) is enriched in bound carbohydrate and sulfonolipid, but contains little or no 2-keto-3-deoxyoctonate (such as is found in the enterobacteria). The localization of the sulfonolipids in the outer membrane permits focus on the possible roles these unusual substances may play in gliding motility.Abbreviations used IM inner membrane - OM outer membrane - KDO 2-keto-3-deoxyoctonate - EDTA ethylenediaminetetraacetic acid - SDH succinate dehydrogenase     

Inhibition of Cytophaga U67 gliding motility by inhibitors of polypeptide synthesis

Gliding motility of Cytophaga U67 and several other cytophagas was inhibited by a growth-permissive concentration of chloramphenicol (50 g/ml). Several other inhibitors of polypeptide synthesis also demonstrated this effect. Short-term exposure to several of these inhibitors resulted in reversible inhibition of gliding by growing cells. In wet mounts chloramphenicol-grown cells demonstrated non-translocational tumbling. Electrophoretic patterns of polypeptides released by ethylenediaminetetraacetate treatment of control and chloramphenicol-grown cells were distinct. Gliding of a spontaneous mutant was resistant to chloramphenicol at 50 g/ml; its motility was inhibited at the growth-permissive concentration of 400 g/ml.     

Direct production of L-lysine from raw corn starch by Corynebacterium glutamicum secreting Streptococcus bovis alpha-amylase using cspB promoter and signal sequence

Corynebacterium glutamicum is an important microorganism in the industrial production of amino acids. We engineered a strain of C. glutamicum that secretes α-amylase from Streptococcus bovis 148 (AmyA) for the efficient utilization of raw starch. Among the promoters and signal sequences tested, those of cspB from C. glutamicum possessed the highest expression level. The fusion gene was introduced into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. L-Lysine fermentation was conducted using C. glutamicum secreting AmyA in the growth medium containing 50 g/l of raw corn starch as the sole carbon source at various temperatures in the range 30 to 40°C. Efficient L-lysine production and raw starch degradation were achieved at 34 and 37°C, respectively. The α-amylase activity using raw corn starch was more than 2.5 times higher than that using glucose as the sole carbon source during L-lysine fermentation. AmyA expression under the control of cspB promoter was assumed to be induced when raw starch was used as the sole carbon source. These results indicate that efficient simultaneous saccharification and fermentation of raw corn starch to L-lysine were achieved by C. glutamicum secreting AmyA using the cspB promoter and signal sequence.     

Production of an amylase-degrading raw starch by Gibberella pulicaris

An endophytic fungus, Gibberella pulicaris, produced an amylase which degraded raw starches from cereals and other crops including raw potato, sago, tapioca, corn, wheat and rice starch. In each case, glucose was the main product. Among the raw starches used, raw potato starch gave the highest enzyme activity (85 units mg^–1 protein) and raw wheat starch the lowest (49 units mg^–1 protein). The highest amylase production (260 units mg^–1 protein) was achieved when the concentration of raw potato starch was increased to 60 g l^–1. Optimum hydrolysis was at 40°C and pH 5.5.     

Candida alocasiicola sp. nov., Candida hainanensis sp. nov., Candida heveicola sp. nov. and Candida musiphila sp. nov., novel anamorphic, ascomycetous yeast species isolated from plants

In a taxonomic study on the ascomycetous yeasts isolated from plant materials collected in tropical forests in Yunnan and Hainan Provinces, southern China, four strains isolated from tree sap (YJ2E(T)) and flowers (YF9E(T), YWZH3C(T) and YYF2A(T)) were revealed to represent four undescribed yeast species. Molecular phylogenetic analysis based on the large subunit (26S) rRNA gene D1/D2 domain sequences showed that strain YJ2E(T) was located in a clade together with Candida haemulonii and C. pseudohaemulonii. Strain YF9E(T) was most closely related to C. azyma and strain YWZH3C(T) to C. sorbophila and C. spandovensis. Strain YYF2A(T) was clustered in a clade containing small-spored Metschnikowia species and related anamorphic Candida species. The new strains differed from their closely related described species by more than 10% mismatches in the D1/D2 domain. No sexual states were observed for the four strains on various sporulation media. The new species are therefore assigned to the genus Candida and described as Candida alocasiicola sp. nov. (type strain, YF9E(T) = AS 2.3484(T) = CBS 10702(T)), Candida hainanensis sp. nov. (type strain, YYF2A(T) = AS 2.3478(T) = CBS 10696(T)), Candida heveicola sp. nov. (type strain, YJ2E(T) = AS 2.3483(T) = CBS 10701(T)) and Candida musiphila sp. nov. (type strain, YWZH3C(T) = AS 2.3479(T) = CBS 10697(T)).     

Microbial Metabolism of Quinoline by Comamonas sp.

An aerobic bacterial strain which can use quinoline as the sole carbon and energy source has been isolated from activated sludge and identified as Comamonas sp. The microbial metabolism of quinoline by this strain has been investigated. A pH 8 and a temperature of 30 °C were the optimum degradation conditions of quinoline. Five intermediates including 2-oxo-1,2-dihydroquinoline, 5-hydroxy-6-(2-carboxyethenyl)-1H-2-pyridone, 6-hydroxy-2-oxo-1,2-dihydroquinoline, 5,6-dihydroxy-2-oxo-1,2-dihydroquinoline, and 8-hydroxy-2-oxo-1,2-dihydroquinoline were found during quinoline biodegradation. The presence of these intermediates suggested that at least two pathways were involved for quinoline degradation by Comamonas sp. and a reasonable degradation route was proposed to account for the intermediates observed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Surface-induced synthesis of new sulfonolipids in the gliding bacterium Cytophaga johnsonae

Many simple gliding bacteria contain significant quantities of phosphate-free, sulfur-containing lipids (sulfonolipids; N-acylamino-3-hydroxyisoheptadecane-1-sulfonic acids, or N-acyl capnines) that recently were shown to function in the ability of Cytophaga johnsonae to migrate over solid surfaces. Reported here is the synthesis, by surface-grown Cytophaga johnsonae cells, of two additional sulfonolipids not present in cells grown in liquid media. These newly characterized sulfonolipids are more polar than the N-acylcapnines characteristic of liquid grown cells. Acid methanolysis of the sulfonolipids revealed that the aminosulfonate capnine was common to all, thus indicating that the chemical differences in the compounds resided in their N-fatty acyl groups, and not in the aminosulfonate moiety. Instead of the non-hydroxy and 3-hydroxy fatty acyl moieties present in sulfonolipids of liquid-grown cells, one new sulfonolipid contained a 2-hydroxy, branched C₁₅ fatty acid, while the other contained a 2,3-dihydroxy, isobranched C₁₆ fatty acid, as indicated by gas chromatographic and mass spectrometric analyses. Although the structure of sulfonolipids thus varies between surface- and liquid-grown cells, no difference was found between the total quantity of sulfonolipids present under either of these conditions. The surface-dependent synthesis of these more polar N-acyl-aminosulfonates ceased immediately when surface-grown populations were suspended in broth. The ability of Cytophaga johnsonae to synthesize these compounds in response to a solid surface may be significant in relation to the organism's ability to migrate over such surfaces; it is one of few instances where a physical interaction of the cell surface has been shown to influence the molecular composition of a prokaryote.Abbreviations LTY tryptone yeast extract medium - TLC thin layer chromatography - FAME fatty acid methyl ester - ECL equivalent chain length - T _r retention time - TMS trimethylsilyl - TFA trifluoroacetyl     

Lipoamino acids and sulfonoplipids in Cytophaga johnsonae Stanier strain C21 and six related species of Cytophaga

Cytophaga johnsonae Stanier strain C21 (C. johnsonae C21) contains phosphatidylethanolamine (PE), an unusual glycine-containing lipid (glycine lipid), and two kinds of unidentified lipid as major lipid components. One of the latter lipids was identified by chemical and physicochemical methods as iso-3-hydroxy fatty acid, -amide linked to ornithine and esterified to iso-nonhydroxy fatty acid (ornithine lipid). The other lipid was identified as a sulfonolipid by a tracer experiment using ³⁵S. PE, glycine lipid and sulfonolipid were found in all seven species of Cytophage examined, namely, C. huchinsonii, C. heparina, C. johnsonae C21, C. aquatilis, and three unidentified species of Cytophaga. However, ornithine lipid was found only in the latter five species. By contrast, a serine-containing lipid, which is a specific lipid component of Flavobacterium species, was not found in any species of Cytophaga examined. The possible use and significance of amino acid-containing lipids and sulfonolipids as chemosystematic markers of the Cytophaga species are discussed.     

Secondary carotenoids formation by the green alga Chlorococcum sp.

The effects of temperature, pH and oxygen level onsecondary carotenoids (SC) accumulation in Chlorococcum sp. were investigated. The optimaltemperature, pH and oxygen level for the yields ofsecondary carotenoids and astaxanthin were35 ^°C, pH 8 and 10% (v/v), respectively. Whilethe ratio (R) of products containing hydroxyl group(s) to those containing keto group (s) increased withthe increase of temperature and pH, R value decreasedwith the level of oxygen. These results indicate thathigher temperature and pH favor the introduction ofhydroxyl group to the corresponding substrates,however, under high oxygen level keto group wasrelatively easy to be added compared to the additionof hydroxyl.     

The role of kairomones in prey finding by Diomus sp. and Exochomus sp., two coccinellid predators of the cassava mealybug, Phenacoccus manihoti

We studied searching behaviour of Diomus sp., a coccinellid predator introduced into Africa as a natural enemy of the cassava mealybug, when searching on cassava leaves, and compared its behaviour with the searching behaviour of Exochomus sp., an African predator of mealybugs (MB's). Female adults of Diomus and Exochomus spent more time searching on cassava leaves previously infested with cassava MB than on clean cassava leaves, in response to substances produced by MB's (wax and/or honeydew) still present on these leaves as kairomones after removal of the MB's. Both species were also arrested by wax and/or honeydew of the citrus mealybug, Planococcus citri. When offered a choice between kairomones of both MB species only experienced Diomus (reared on cassava MB) showed a clear preference for kairomone of cassava MB. Separate influences of wax and honeydew were tested. Wax from cassava MB was an arrestment stimulus for both coccinellid species. Honeydew produced by cassava MB arrested Exochomus and inexperienced Diomus.

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Zusammenfassung Das Suchverhalten auf Cassaveblätter des coccinelliden Raübers Diomus, eines aus Süd Amerika importierten natürlichen Feindes der Cassaveschmierlaus wurde studiert und verglichen mit dem Suchverhalten eines afrikanischen Räubers von Schmierläusen, Exochomus sp.. Weiblichen Adulten von Diomus un Exochomus verwendeten mehr Zeit auf Cassaveblätter wenn diese Blätter vorher mit Cassaveschmierläuse infiziert waren als auf uninfizierte Blätter, und gebrauchten Substanzen (Wachs und/oder Honigtau) die von Schmierläuse produziert wurden und nach der Entfernung der Schmierläuse auf die Blätter zugeblieben waren als Kairomonen.Beide Arten wurden auch von Wachs und/oder Honigtau der Citrusschmierlaus, Planococcus citri, auf den Blätter arretiert. Wenn die Tiere wahlen könnten zwischen Kairomone der beiden Schmierlausarten wurden die Kairomonen der Cassaveschmierlaus nur von erfahrenen Diomus-Individuen, die auf Cassaveschmierlaüse aufgezogen wurden, bevorzugt.Die Einflüsse von Wachs und Honigtau wurden separat geprüft. Wachs von Exuvien der Cassaveschmierlaus ist ein Arretierungsstimulus für beide Räuberarten. Von Cassaveschmierläusen produzierten Honigtau arretierte Exochomus-und unerfahrene Diomus-Individuen.

     

Infection of Glaucocystis nostochinearum (Glaucophyta) by Lagenidium sp. (Oomycota) and its hyperparasite Pythiella sp. (Oomycota)

The glaucophyte Glaucocystis nostochinearum has to our knowledge been observed to be infected by a parasite for the first time. It was found in samples taken from the northernmost freshwater pond in Germany (on the island of Sylt). The fungal parasite was identified as the oomycete Lagenidium sp. which itself was parasitised by another oomycete, Pythiella sp.     

Production of a novel raw-starch-digesting glucoamylase by <Emphasis Type="Italic">Penicillium</Emphasis> sp. X-1 under solid state fermentation and its use in direct hydrolysis of raw starch

Penicillium sp. X−1, isolated from decayed raw corn, produced high level of raw-starch-digesting glucoamylase (RSDG) under solid state fermentation (SSF). Maximum enzyme yield of 306.2 U g⁻¹ dry mouldy bran (DMB) was obtained after 36 h of culture upon optimized production. The enzyme could hydrolyse both small and large granule starches but did not adsorb on raw starch. The enzyme exhibited maximum activity at 65°C and pH 6.5, which provided an opportunity of synergism with α-amylase. It significantly hydrolysed 15% (w/v) raw corn starch slurry in synergism with the commercial α-amylase and a degree of hydrolysis of 92.4% was obtained after 2 h of incubation.     

Investigation of lipase production by a new isolate of Aspergillus sp.

Fungi isolated from soil were screened for exogenous lipolytic activity. The highest lipase activity was found in a new soil isolate of Aspergillus sp. Some optimal cultural parameters influencing the growth and production of extracellular lipase from this Aspergillus sp. were investigated. The lipase yield was maximum on day 4 of incubation of the culture at pH 5.5 and 30 °C. When the medium was prepared using olive oil as carbon source and peptone as a nitrogen source, better lipase yields were obtained. Aeration enhanced growth and lipase production.     

Identification of three Wx proteins in wheat (Triticum aestivum L.)

Nullisomic analysis of waxy (Wx) protein of hexaploid wheat (Triticum aestivum L.) cv. “Chinese Spring” using two-dimensional polyacrylamide gel electrophoresis revealed that threeWx loci,Wx-A1, Wx-B1, andWx-D1, located on chromosome arms 7AS, 4AL, and 7DS, produce three distinct Wx subunit groups, subunit group-A (SGA), SGB, and SGD, respectively. SGA has a higher molecular weight and a more basic isoelectric point (pI) than the other two. SGB and SGD have the same molecular weight but a slightly different pI range. Owing to the detection of these three subunit groups, we were able to identify the expression of three waxy genes in wheat endosperm and to find two types of mutants among Japanese wheat cultivars, one lacking SGA and the others SGB. These results suggest the possibility of breeding a waxy wheat.     

Simultaneous production of hydrogen and bioflocculant by Enterobacter sp. BY-29

Hydrogen and a bioflocculant could be produced simultaneously by anaerobic culture of Enterobacter sp. BY-29. For production of hydrogen and the bioflocculant by cell culture of the bacterium in batch cultures, cultivation at 37 °C in a medium containing glucose as a carbon source and Polypepton as a nitrogen source was found to be suitable. In continuous production of hydrogen and the bioflocculant by cell culture or immobilized cells of the bacterium, the hydrogen production rate and hydrogen yield by the immobilized cells on porous glass beads in stirred and column reactors were higher than those by the cell culture in a stirred reactor. However, production of the bioflocculant by the cell culture was superior to that by the immobilized cells in continuous production.