Preparation of Optically Pure cis-2,4-Dimethyl-l-cyclohexanones,the Key Intermediates in Cycloheximide Synthesis,Using Microbial Resolution

Asymmetric hydrolysis of acetate (10) of (±)-t-2,t-4-dimethyl-r-l-cyclohexanol with Bacillus subtilis var. niger gave (?)-(lS,2S,4S)-2,4-dimethyl-l-cyclohexanol (6a) and (+)-(1R,2R,4R)-acetate (10b) with high optical purities. Optically pure (?) and (+)-alcohols (6a and 6b) were prepared via corresponding 3,5-dinitrobenzoates. Oxidation of alcohols (6a and 6b) with chromic acid gave optically pure (?)-(2S,4S) and (+)-(2R,4R)-2,4-dimethyl-l-cyclohexanones (2a and 2b), respectively.     

Effect of deglycosylation on the properties of β-fructofuranosidaseP-1 fromAureobasidium sp. ATCC 20524

Summary Most of the carbohydrate moiety of -fructofuranosidaseP-1 fromAureobasidium sp. ATCC 20524 was removed by endo--N-acetylglucosaminidase F. A subunit of 94000 Da was observed in SDS-PAGE after deglycosylation. TheK _m value for sucrose was not changed by deglycosylation but the stability at pH 4–5 and 50°C was decreased. The deglycosylated enzyme was more sensitive to proteases such as pronase E and subtilisin than the native enzyme. It is considered that the carbohydrate moiety of -fructofuranosidaseP-1 contributes to the stability of the enzyme but is not essential in its catalytic function.     

Biopolymer flocculant produced by an Enterobacter sp.

A new biopolymer flocculant was produced by Enterobacter sp. BY-29. Flocculating activity increased in the presence of Al , Fe or Fe . The flocculant had flocculating activity not only in inorganic suspensions of kaolin and active carbon but also in organic suspensions of cellulose and yeast. The flocculant was an acidic polysaccharide consisting of glucose, galactose, xylose and galacturonic acid, and its MW was about 2.5 ¥ 10 6 Da.     

Flocculation properties of xanthan produced by Xanthomonas campestris

Summary Xanthan had a flocculating activity in a kaolin suspension and high flocculating activity was obtained in the suspension (pH 7.0) adding Al³⁺, Fe³⁺ or Fe²⁺. Xanthan had high flocculating activity not only in other inorganic suspensions such as active carbon and acid clay but also in organic suspensions of cellulose and yeast. From these flocculation properties, xanthan is anticipated to be utilized in wide areas as a new biodegradable, harmless biopolymer flocculant.     

Quantitative evaluation of predation by spiders on the green rice leafhopper,Nephotettix cincticeps Uhler,by a sight-count method

A sight-count method for evaluation of predation by spiders on the green rice leafhopper, Nephotettix cincticeps was proposed and its applicability was tested under natural conditions. The number (n) of leafhoppers preyed on by spiders per rice hill per day was estimated by the formula: (1) where F is the frequency of predation observed per hill:P is given by dividing the time spent feeding on prey by 24 hours; and C refers to the total amount of feeding activity expressed in terms of the activity during the standard time interval. The total number (N) of prey attacked during the specified period can be given as follows: (2) With this method, the role of paddy-inhabiting spiders, Lycosa pseudoannulata, Oedothorax insecticeps, Tetragnatha spp, and Enoplognatha japonica, as predator of N. cincticeps was evaluated with reference to life tables of the prey. The advantages and limitation of the sight-count method were discussed as compared with other methods so far proposed.     

H production by immobilized cells of Clostridium butyricum on porous glass beads

Clostridium butyricum immobilized on porous glass beads in a column reactor evolved H 2 at 715 and 1,150 ml/l.h, with H 2 yields of 2.3 and 1.9 mol H 2 /mol glucose, at retention times of 2.0 and 1.0 h, respectively, with a medium containing 0.5 g glucose/l in continuous cultures without pH control.     

Production of (R)-3-amino-3-phenylpropionic acid and (S)-3-amino-3-phenylpropionic acid from (R,S)-N-acetyl-3-amino-3-phenylpropionic acid using microorganisms having enantiomer-specific amidohydrolyzing activity

(R)-3-Amino-3-phenylpropionic acid ((R)-beta-Phe) and (S)-3-amino-3-phenylpropionic acid ((S)-beta-Phe) are key compounds on account of their use as intermediates in synthesizing pharmaceuticals. Enantiomerically pure non-natural amino acids are generally prepared by enzymatic resolution of the racemic N-acetyl form, but despite the intense efforts this method could not be used for preparing enantiomerically pure beta-Phe, because the effective enzyme had not been found. Therefore, screening for microorganisms capable of amidohydrolyzing (R,S)-N-acetyl-3-amino-3-phenylpropionic acid ((R,S)-N-Ac-beta-Phe) in an enantiomer-specific manner was performed. A microorganism having (R)-enantiomer-specific amidohydrolyzing activity and another having both (R)-enantiomer- and (S)-enantiomer-specific amidohydrolyzing activities were obtained from soil samples. Using 16S rDNA analysis, the former organism was identified as Variovorax sp., and the latter as Burkholderia sp. Using these organisms, enantiomerically pure (R)-beta-Phe (>99.5% ee) and (S)-beta-Phe (>99.5% ee) with a high molar conversion yield (67%-96%) were obtained from the racemic substrate.     

Systematic analysis of the relation of electron transport and ATP synthesis to the photodamage and repair of photosystem II in Synechocystis

The photosynthetic machinery and, in particular, the photosystem II (PSII) complex are susceptible to strong light, and the effects of strong light are referred to as photodamage or photoinhibition. In living organisms, photodamaged PSII is rapidly repaired and, as a result, the extent of photoinhibition represents a balance between rates of photodamage and the repair of PSII. In this study, we examined the roles of electron transport and ATP synthesis in these two processes by monitoring them separately and systematically in the cyanobacterium Synechocystis sp. PCC 6803. We found that the rate of photodamage, which was proportional to light intensity, was unaffected by inhibition of the electron transport in PSII, by acceleration of electron transport in PSI, and by inhibition of ATP synthesis. By contrast, the rate of repair was reduced upon inhibition of the synthesis of ATP either via PSI or PSII. Northern blotting and radiolabeling analysis with [(35)S]Met revealed that synthesis of the D1 protein was enhanced by the synthesis of ATP. Our observations suggest that ATP synthesis might regulate the repair of PSII, in particular, at the level of translation of the psbA genes for the precursor to the D1 protein, whereas neither electron transport nor the synthesis of ATP affects the extent of photodamage.     

MbIDGF, a novel member of the imaginal disc growth factor family in Mamestra brassicae, stimulates cell proliferation in two lepidopteran cell lines without insulin

Imaginal disc growth factor (IDGF) is a soluble polypeptide growth factor that was first identified from the conditioned medium of Drosophilia imaginal disc C1.8+ cells. Working with insulin, IDGF stimulated the growth of cultured imaginal disk cells, which suggested that IDGF might function as a cofactor of Drosophila insulin or insulin like peptide. Here we report a new member of the IDGF family, named MbIDGF, from the cabbage armyworm, Mamestra brassicae. Using a cloned cDNA of MbIDGF, recombinant MbIDGF protein was expressed in baculovirus-infected Sf9 cells and purified. Without insulin, the recombinant MbIDGF protein stimulated cell growth of SES-MaBr-4 and NIAS-MaBr-93 cell lines that were derived from the fat bodies and hemocytes of M. brassicae, in a dose-dependent manner. The saturation of growth stimulation by MbIDGF was attained for the two types of cells at 80 ng/ml (0.8 nM) and 300 ng/ml (6 nM), respectively. The results suggest that MbIDGF may stimulate the growth of lepidopteran cells by a new mechanism without associating with the insulin pathway.     

Ultrafiltration-based assay for heparanase activity

Heparanase, a mammalian endoglycosidase that specifically cleaves heparan sulfate (HS), has been found in many tissues. Platelet, liver, and placenta have been abundant sources for the study of the enzyme. Notably, certain malignant cells also have been found to produce large amounts of the enzyme, the levels of which often correlate with their invasive and metastatic properties. To study roles of heparanase in various biological situations, a reliable method measuring the enzyme activity is indispensable. In the past, measurement of heparanase enzyme activity was done either by the detection of the degradation of fluorescent or radiolabeled HS chains by gel filtration procedures or by the use of radiolabeled substrate conjugated to solid matrices for the easy separation of degraded HS chains. A newly developed procedure, presented in this article, measures degradation of radiolabeled HS chains in the aqueous buffer by detecting their degradation products using an ultrafiltration device, the Centricon 30. This procedure has several advantages over previous assay procedures that involved tedious processing such as gel filtration chromatography of each sample or the preparation of substrate HS proteoglycans conjugated to a solid matrix. The simplicity of the new procedure allows a short setup time and a rapid processing of a large number of samples. Furthermore, the enzymatic reaction during the aqueous phase allows kinetic analyses in standard conditions.