The sub-cellular localisation and regulatory properties of pyruvate carboxylase from Rhizopus arrhizus

Cell-free extracts of Rhizopus arrhizus contain exclusively cytosolic pyruvate carboxylase and NAD-glutamate dehydrogenase, a single mitochondrial isoenzyme of NADP-isocitrate dehydrogenase, and both mitochondrial and cytosolic isoenzymes of NADP-malate dehydrogenase (decarboxylating). Other enzymes examined have sub-cellular localisations similar to those characteristic of mammalian liver. Purified preparations of R. arrhizus pyruvate carboxylase are subject to partial regulatory inhibition by L-aspartate and 2-oxoadipate. L-Glutamate acts as a less effective analogue of L-aspartate while 2-oxoglutarate is ineffective. Competition studies indicate the presence of separate inhibitory sites for L-aspartate and 2-oxoadipate. Under routine assay conditions R. arrhizus pyruvate carboxylase shows significant activation by acyl derivatives of coenzyme A with long chain acyl CoA being more effective than acetyl-CoA. This activation is no longer observed in the presence of high concentrations of pyruvate, MgATP2- and HCO-3. The concentrations of L-aspartate and 2-oxoadipate required to give 50% inhibition ([I]0.5), and the maximal extents of inhibition, are increased by addition of acetyl-CoA. Acetyl-CoA increases the sigmoidal character of the relationship: initial rate/[L-aspartate], but decreases this parameter for the relationship: initial rate/[2-oxoadipate]. The studies indicate that R. arrhizus possesses an entirely cytosolic pathway for the conversion of glucose to fumaric acid and that both the organisation of pyruvate metabolism and the regulation of pyruvate carboxylase differ significantly in this organism as compared to that proposed previously for Aspergillus nidulans.     

基因组SNP揭示少根根霉种群结构

少根根霉在自然界分布广泛,是重要的食品发酵菌,又是著名的机会致病菌,在形态和生化上多样性丰富,包括原变种、德氏变种和东京变种等3个分类学变种,原型、德氏型、东京型和鲁氏型等4个形态型,以及乳酸组和富马苹果酸组等2个生化组。DNA分子多样性更为丰富。但这些多样性之间缺乏系统的关联研究。本研究选择能代表以上多样性的67个菌株,通过全基因组重测序提取ITS、IGS rDNA和SNP多态性位点进行分子系统发育和群体结构分析,结果将少根根霉分为4个主要的系统发育分支,分支1和2是姐妹群关系,共同构成德氏变种,另外两个分支分别对应东京变种和原变种。鲁氏形态型多系,源自原变种和德氏变种。群体结构分析表明在少根根霉物种内,德氏变种首先分歧出来,然后东京变种和原变种发生分化,最后3个变种分化出各自的亚群;这些亚群表明少根根霉物种正沿着8个相互杂交的分子群体进行演化。本研究首次利用基因组范围的信息支持所有的生态群、分类学变种、形态型、生化组和系统发育分支仍然属于同一个物种,物种分化尚未完成,同时实现了对少根根霉多个DNA分子群体的解析并推导出其演化规律。     

Biosorption of radionuclides by Rhizopus arrhizus

The sorption of various radionuclides viz. 233U, 239Pu, 241Am, 144Ce, 147Pm, 152+154Eu and 95Zr from aqueous nitrate medium has been studied using biomass Rhizopus arrhizus. The biosorption of 233U and 239Pu was found to be maximum at pH 6-7 whereas for other trivalent actinides and fission products viz. 241Am, 144Ce, 147Pm, 152+154Eu and tetravalent 95Zr, it was more effective at pH 2. This biomass is a promising sorbant for the treatment of radioactive effluents from nuclear industry. © Rapid Science Ltd. 1998     

Lipids of Rhizopus arrhizus Fischer

The lipids of Rhizopus arrhizus Fischer mycelia and sporangiospores were extracted, isolated, and separated by thin-layer, liquid, and gas chromatography. Structural confirmations of the compounds were made by a gas chromatographmass spectrometer combination. The n-heptane fraction contained squalene (1%) as a major hydrocarbon constituent. Other major lipid classes detected were free fatty acids, naturally occurring methyl esters of fatty acids, triglycerides, sterols, and polar lipids. The polar lipids (44.4%) were found in the highest concentrations, and the triglycerides (22.1%), sterols (16.7%), and free fatty acids (11.7%) were present in lesser concentrations. This is the first report of naturally occuring methyl esters of long-chain fatty acids being present in fungal mycelium. There appears to be a preference for incorporation of unsaturated acids into the complex lipids, with the exception of the triglycerides. The major saturated fatty acids in the mycelium were palmitic (C(16)) and arachidic (C(20)), whereas the major unsaturated acids were oleic (C(18:1)) and linoleic (C(18:2)), respectively.     

Optimization of fumaric acid production from potato flour by Rhizopus arrhizus

Summary Fumaric acid production by Rhizopus arrhizus from potato flour was studied at different initial substrate concentrations (S), C/N ratios and fermentation times (t f) in a composite design experiment. By using response surface methodology and canonical analysis, the experimental values of fumaric acid and mycelial biomass yields and productivity were fitted to the only statistically significant factors with mean percentage errors of 11, 26 and 13%, respectively. Owing to the limited statistical significance of the C/N ratio, it was possible to determine the values of S (25–50 g/l of glucose equivalent) and t f(44–100 h) associated with fumaric acid yields ranging from 60 to 75% and productivities varying from 6 to 8.4 g/l per day. Since such results were in quite good agreement with previous experiments carried out on other starchy materials, the above operating conditions might be used to minimize fumaric acid production costs as a function of the feed-stock used. Offsprint requests to: M. Moresi     

Uptake of Metal Ions by Rhizopus arrhizus Biomass

Rhizopus arrhizus biomass was found to absorb a variety of different metal cations and anions but did not absorb alkali metal ions. The amount of uptake of the cations was directly related to ionic radii of La³⁺, Mn²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Ba²⁺, Hg²⁺, Pb²⁺, UO₂²⁺, and Ag⁺. The uptake of all the cations is consistent with absorption of the metals by sites in the biomass containing phosphate, carboxylate, and other functional groups. The uptake of the molybdate and vanadate anions was strongly pH dependent, and it is proposed that the uptake mechanism involves electrostatic attraction to positively charged functional groups.     

The mechanism of uranium biosorption by Rhizopus arrhizus

Biosorption of elements is a little understood phenomenon exhibited by some types of even nonliving microbial biomass. A common fungus Rhizopus arrhizus has been reported to take up uranium from aqueous solutions to the extent of 180 mg U(6+)/g. The mechanism of uranium sequestering by this type of biomass was studied by using experimental techniques such as electron microscopy, x-ray energy dispersion analysis, IR spectroscopy, and supporting evidence was obtained for a biosorption mechanism consisting of at least three processes. Uranium coordination and adsorption in the cell-wall chitin structure occur simultaneously and rapidly whereas precipitation of uranylhydroxide within the chitin microcrystalline cell-wall structure takes place at a lower rate. Interference of Fe(2+) and Zn(2+) coions with uranium biosorption is indicated.     

The mechanism of thorium biosorption by Rhizopus arrhizus

Inactive cells of Rhizopus arrhizus have been documented to exhibit a high thorium biosorptive uptake (170 mg/g) from aqueous solutions. The mechanism of thorium sequestering by this biomass type was investigated following the same method as for the uranium biosorption mechanism. The thorium sequestering mechanism appeared somewhat different from that of uranium. Experimental evidence is presented which indicates that, at optimum biosorption pH (4), thorium coordinates with the nitrogen of the chitin cell wall network and, in addition, more thorium is absorbed by the external section of the fungal cell wall. At pH 2 the overall thorium uptake is reduced. The kinetic study of thorium biosorption revealed a very rapid rate of uptake. Unlike uranium at optimum solution pH, Fe(2+) and Zn(2+) did not interfere significantly with the thorium biosorptive uptake capacity of R. arrhizus.     

Physiological restriction of the L-lactic acid production by Rhizopus arrhizus

The influence of basic physiological factors on the quality of inocula and L(+)-lactic acid production by Rhizopus arrhizus CCM 81 09 were studied. The most effective preparation of the spores (5 × 10⁷ spores/ml) and subsequent good lactate production was achieved on the agar medium with soil extract and malt agar. The optimum initial amount of active spores for inoculation was 10³–10⁴ spores/ml. The preparation of inoculum required intensive stirring with lower aeration and pH maintained in the range from 4.8 to 6.0 by the addition of CaCO₃. The maximum yield of lactic acid production was achieved by using 5% (v/v) of 24-h-old inoculum. The intensity of lactic acid production in the inoculum was proportional to its production in the subsequent steps of fermentation and can be used as a fast control of the physiological state of the producers.     

Biochemical Aspects of Fumaric Acid Accumulation by Rhizopus arrhizus

The accumulation and excretion of fumaric acid, and to a lesser extent malic and succinic acids, by Rhizopus arrhizus occurs under aerobic conditions in a high-glucose medium containing a limiting amount of nitrogen and a neutralizing agent (CaCO₃). An overall four-carbon dicarboxylic acid molar yield of up to 145% (moles of acid produced per mole of glucose utilized) is obtained after incubation for 4 to 5 days. Evidence is presented that fumarate is synthesized from pyruvate via a carboxylation reaction yielding oxaloacetate, which is then converted to malate and further on to fumarate via the reductive reactions of the tricarboxylic acid cycle. The possible formation of fumarate from the normal (oxidative) operation of the tricarboxylic acid cycle was not excluded by the data. Yield, ¹³C nuclear magnetic resonance, and enzymatic activity studies were carried out in a strain of R. arrhizus which produces high levels of fumarate from glucose and carbonate. The observed high fumarate molar yield (greater than 100%) can therefore be explained in terms of the carboxylation of pyruvate and the operation of the reductive reactions of the tricarboxylic acid cycle under aerobic conditions.     

Influence of anions on metal adsorption by Rhizopus arrhizus biomass

The presence of anions in solution was found to inhibit the uptake of La(3+), Cd(2+), Pb(2+), UO(2+) (2), and Ag(+) by Rhizopus arrhizus biomass. The effects ranged from total inhibition of Cd(2+) and Pb(2+) uptake at equimolar concentrations of EDTA to no change in uptake of La(3+) or UO(2+) (2) at 12-fold molar excesses of Cl(-) or CO(2-) (3). No anion was found to enhance metal uptake levels, and the degree of inhibition generally followed the series: \documentclass{article}\pagestyle{empty}\begin{document}$${\rm EDTA } \ge \ge {\rm SO}_{;{;{;{\rm 4} } } };{{\rm 2} - } \ge {\rm Cl}; - \ge {\rm PO}_{;{;{;{\rm 4} } } };{{\rm 3} - } \ge {\rm glutamate} \ge {\rm CO}_{;{;{\rm 3} } };{{\rm 2} - } $$\end{document} The chemical equilibrium model REDEQL2 was adapted to treat metal uptake by R. arrhizus biomass and used to predict the effects of anions in solution. Comparisons with the experimental results are made and discussed in light of the assumptions underlying the model.     

Ultrastructural Details in Germinating Sporangiospores of Rhizopus stolonifer and Rhizopus arrhizus

Electron microscope examination of sporangiospore sections from Rhizopus stolonifer (Ehrenb. ex Fr.) Lind. and R. arrhizus Fischer revealed details on intracellular organization not previously reported. Aldehyde fixation followed by chromeosmium postfixation permitted clear depiction of ribosomes hitherto unrevealed in these cells. Mitochondria were diversiform. Spore wall structures in the two species were generally similar, but outer contours differed sufficiently to permit easy species identification in examination of sections. The spores of both species abounded in cytosomes, corresponding in size, shape, and heavy-metal "stain" affinities to spherosomes in cells of higher plants. The osmiophilic response of these spherosome-like inclusions was intensified by treatment of sections with thiocarbohydrazide solution and subsequent application of aqueous osmium tetroxide, which strengthens an assumption that they are lipid-rich. The margins of the spherosome-like inclusions in lead citrate-stained sections included dense particles, about 60 A across, whose crystalline-like arrangements suggested that protein as well as lipid was present. Frequent and close associations between the spherosome-like inclusions and various cell membranes suggested that such bodies participate in membrane elaboration during germination.     

Production enhancement of Rhizopus arrhizus lipase by feeding oleic acid

A strategy for Rhizopus arrhizus lipase production enhancement by feeding oleic acid was developed. The oleic acid was proved to have strong inducing effect on lipase production, but high concentration oleic acid could repress lipase production. The decrease rate of oleic acid concentration using peanut oil as initial carbon source was figured out according to the change of oleic acid concentration in the fermentation broth. Our feeding strategy designed based on the decrease rate of oleic acid could avoid the repression of lipase production that is caused by high concentration of oleic acid in the fermenting liquor, and this strategy worked as a new feeding method showing excellent performance. The maximum lipase activity was gained by feeding dilute oleic acid every 12 h starting at 60 h, which maintained the oleic acid concentration around 18 mg/L, and the lipase activity was 31% higher than that of no feeding.