Effect of manganese deficiency on plasma-membrane lipid composition and glucose uptake in Aspergillus niger

Abstract Plasma membranes were isolated by means of the concanavalin A technique from protoplasts of manganese deficient (< 10⁻⁸ M Mn²⁺) and sufficient (10⁻⁵ M Mn²⁺) grown mycelium. The membranes differed with respect to their quantitative contents of fatty acids, sterols and phospholipids. These changes did not influence the glucose transport system, as shown by kinetic investigations using intact mycelia.     

Effect of ammonium-N on malic enzyme and lipid production in Rhodotorula glutinis grown on monosodium glutamate wastewater

Rhodotorula glutinis, an oil producing strain, can utilize monosodium glutamate (MSG) wastewater as a raw material for lipid production. The effects of ammonium-N in the MSG wastewater (ammonium 15,000–25,000?mg/L, COD 30,000–50,000?mg/L) on cell growth, lipid accumulation and malic enzyme activity of R. glutinis have been studied. Four initial ammonium sulfate concentrations in the medium were set, which were 20, 60, 100, and 140?g/L. With an increase in the ammonium sulfate concentration, the uptake of ammonia nitrogen and lipid accumulation increased while the biomass decreased at 72?h. The maximum value of ammonia nitrogen consumption reached 5.77?g/L for an initial ammonium sulfate concentration of 140?g/L at 72?h. In addition, 60?g/L ammonium sulfate concentration may be an appropriate concentration for R. glutinis cultivation. The activity of the malic enzyme was measured and the results showed that there was a linear relationship between the intracellular lipid content and the total malic enzyme activity.     

Efficient synthesis of triazole moiety-containing nucleotide analogs and their inhibitory effects on a malic enzyme

Eleven triazole moiety-containing nucleotide analogs were synthesized starting form tetra-O-acetylribose in 55-63% total yields. The synthesis involved two key steps, the lipase-mediated selective deacylation of 1-azido-2,3,5-tri-O-acetyl-β-d-ribofuranoside and the Huisgen 1,3-dipolar cycloaddition between terminal alkynes and the 1-azido ribofuranoside derivative. These analogs showed inhibitory effects against a recombinant Escherichia coli NAD-dependent malic enzyme.     

Impact of overexpressing NADH kinase on glucoamylase production in Aspergillus niger

Glucoamylase has a wide range of applications in the production of glucose, antibiotics, amino acids, and other fermentation industries. Fungal glucoamylase, in particular, has attracted much attention because of its wide application in different industries, among which Aspergillus niger is the most popular strain producing glucoamylase. The low availability of NADPH was found to be one of the limiting factors for the overproduction of glucoamylase. In this study, 3 NADH kinases (AN03, AN14, and AN17) and malic enzyme (maeA) were overexpressed in aconidial A. niger by CRISPR/Cas9 technology, significantly increasing the size of the NADPH pool, resulting in the activity of glucoamylase was improved by about 70%, 50%, 90%, and 70%, respectively; the total secreted protein was increased by about 25%, 22%, 52%, and 26%, respectively. Furthermore, the combination of the mitochondrial NADH kinase (AN17) and the malic enzyme (maeA) increased glucoamylase activity by a further 19%. This study provided an effective strategy for enhancing glucoamylase production of A. niger.     

The physiological role of malic enzyme in grape ripening

The high specificity of malic enzyme (ME; EC 1.1.1.40) from grape berries (Vitis vinifera L.) for the naturally occurring l-enantiomer of malic acid, its very selective C₄-decarboxylation, and certain allosteric properties, reported previously, favour the conjecture of a regulatory function of ME in fruit malic acid degradation. On the other hand, high ME activity was detected even during the acid-accumulating phase of berry development. Also, the in vitro reversibility of the reaction supports the possibility of malate formation under conditions facilitating carboxylation of pyruvate, notably high CO₂/HCO ₃ ^- and NADPH/NADP ratios. However, a very limited incorporation of ¹⁴C into malate and the uniform labeling pattern of the dicarboxylic acid after administration of [U-¹⁴C] alanine to grape berries before and after the onset of ripening, indicate that the reverse reaction does not contribute essentially to grape malate synthesis. A regulatory mechanism mediating malic acid remetabolization on the basis of cosubstrate availability, comparable to the control of the hexose monophosphate shunt, is discussed.Abbreviation ME Malic enzyme (l-malate: NADP oxidoreductase)     

Isolation and biochemical characterization of grape malic enzyme

Malic enzyme (ME=L-malate: NADP oxidoreductase; E.C. 1.1.1.40) was extracted by Triton X-100-induced resolubilization of enzyme proteins which denaturize spontaneously upon homogenization of grape berry material. The purification procedure included fractionating with (NH₄)₂SO₄, preparative IEF, and Sephadex G-100 chromatography. ME was identified by TLC of the radioactive product after supplementing the assay mixture with [¹⁴C]malate. Cofactor dependence, pH-optimum and affinities for substrates and cosubstrates were determined. Enzymic pI was found to be 5.8, the Hill coefficients range from 1 to 3. In malate decarboxylating direction at pH 7.4, grape ME displayed positive cooperativity toward the substrate, the curve approaching normal Michaelis-Menten-kinetics at pH 7.0. Substituting Mn²⁺ for Mg²⁺ not only increased maximal turnover rates, but also enzymic affinity for malate. These features were considered indicative of the regulatory properties of the enzyme. Their relevance for grape malate metabolism and fruit ripening is discussed.Abbreviations EDTA ethylenediaminetetraacetic acid - IFF isoelectric focusing - MDH malate dehydrogenase - ME malic enzyme - OAA oxaloacetic acid - PAG polyacrylamide gel - TCA trichloroacetic acid - TLC thin layer chromatography     

Effect of some metabolic inhibitors on citric acid production by Aspergillus niger

Stationary cultures of Aspergillus niger grown on a synthetic medium have been used to study the effect of some metabolic inhibitors on citric acid production. Addition of 0.05 to 1 mM sodium malonate or 0.01 to 0.1 mM potassium ferricyanide, iodoacetate, sodium azide, sodium arsenate or sodium fluoride stimulated citric acid production (3.6 to 45%), but not total titratable acids. Addition of higher concentrations (0.2 to 10 mM) of later inhibitors caused a marked inhibition of fungal growth and citric acid production. The implications of these preliminary findings are discussed.     

Terminal differentiation in the avian uropygial gland. Accumulation of fatty acid synthase and malic enzyme in non-dividing cells

Summary The secretory tissue of the uropygial gland is of the holocrine type, containing both dividing progenitor cells and lipid-filled differentiated cells. In this study, we examined the relationship between cell division and differentiation. The location of dividing cells was determined by autoradiography of tissue sections from ducklings injected intra-abdominally with ³H-thymidine. Only cells on the basal lamina of the tubules contained labeled nuclei. Dividing cells were distributed uniformly over the length of the tubules. Over the next five days, most of the labeled cells migrated to the lumen of the tubules and disappeared. Cells containing the lipogenic enzymes, fatty acid synthase and malic enzyme, were localized either immunocytochemically using affinity-purified antibodies or cytochemically using a specific assay for malic enzyme activity. Fatty acid synthase and malic enzyme were undetectable in dividing basal cells but present at high levels in differentiating and differentiated cells. Thus, basal cells lying along the basal lamina of the tubules were replacing lipid-laden cells that were continually sloughed into the lumens of the tubules. The signals for differentiation and enzyme accumulation appear to be linked to one another and to cessation of cell division.     

碳源对固定化细胞生产柠檬酸的影响

柠檬酸是利用微生物代谢生产的一种极为重要的有机酸．广泛应用于食品、饮料、化工、冶金、印染等各个领域。在国外,近10年来,利用固定化细胞生产柠檬酸已获得较广泛的研究^〔1－6〕,国内也有学者指出,柠檬酸发酵的趋向是利用固定化细胞进行连续化生产⑺。而国内这方面的研究报道很少〔8,9〕。我们利用海藻酸钙凝胶包埋固定化黑曲霉细胞生产柠檬酸．探讨了碳源种类及其浓度对固定化细胞生产柠檬酸的影响。现将结果报道如下。     

Identification of malic enzyme mutants depending on 1,2,3-triazole moiety-containing nicotinamide adenine dinucleotide analogs

An activity screening between 1,2,3-triazole moiety-containing nicotinamide adenine dinucleotide (NAD) analogs and malic enzyme (ME) mutants identified some mutants capable of taking NAD analogs as the cofactor. One particular pair, ME-L310K/L404S and the analog B-8 had good catalytic efficiency and cofactor specificity. The new system gained about 1200-fold cofactor specificity shift from NAD toward B-8 in terms of oxidative decarboxylation of l-malate. Our results provided insightful information for the development of orthogonal redox system that is of particular important to precisely control engineered metabolic pathways.     

Cultivation of Aspergillus niger in a pilot plant external-loop air-lift bioreactor

Abstract: Changes of the main hydrodynamic and oxygen transfer parameters during Aspergillus niger cultivation in an external-loop air-lift bioreactor of 200 dm³ operating capacity were investigated. The final average concentrations of biomass and citric acid obtained in batch fermentations were about 17 g 1^-1 and 90 g 1^-1, respectively. Significant influence of the increasing biomass concentration on the rheological properties of the broth and operating parameters was found. Volumetric oxygen transfer coefficient. k ^L a , was found to be dependent on the apparent viscosity of the broth with an exponent of -0.984.     

Two forms of NADP-dependent malic enzyme in expanding maize leaves

Etiolated maize leaves (Zea mays L.) contain a major isozyme of NADP-dependent malic enzyme (L-malate dehydrogenase, decarboxylating, EC 1.1.1.40) having an isoelectric point of 5.28±0.03, a K_m (L-malate) 0.3–0.6 mM at pH 7.45; a broad pH optimum around pH 6.9 under the conditions of assay; a molecular weight of 280,000 (sometimes accompanied by a minor component of 150,000); and an NAD-dependent activity about 1/50 the NADP-dependent activity. This isozyme, resembling the NADP-malic enzyme of vertebrates, is labeled type 1. The dominant isozyme of young green leaves (type 2) has, however, a pI 4.90±0.03, a K_m (L-malate) 0.10–0.15 mM, a pH optimum of 8, and a molecular weight of 280,000. It is also more stable and exhibits an appreciable NAD-dependent activity (1/5–1/7 the NADP activity). Both isozymes show linear kinetics, dependence on Mn or Mg ions, similar K_m (NADP⁺), and the typical increase of K_m for L-malate with increasing pH values. Type 1 isozyme of maize is assumed to be cytosolic. Type 2 corresponds in each property to the chloroplast enzyme of bundle-sheath cells. It is present at a low level in etiolated leaves and develops to a high specific activity (up to 100 nmol min^-1 mg protein^-1 by 150 h illumination) during photosynthetic differentiation, replacing the type 1 form.Abbreviation MES 2 (N-morpholino)ethane sulfonic acid Work supported by grants from the Consiglio Nazionale delle Ricerche for years 1975 and 1976     

The identification and properties of a naturally occurring inhibitor of malic enzyme

The inhibitor of malic enzyme present in potato tubers has been identified as oxalic acid. Oxalic acid proves to be a particularly potent inhibitor with a K_I = 50 μM. A kinetic analysis indicates that inhibition is not due to chelation of Mg²⁺ and suggests that oxalate binds tightly to malic enzyme after NADPH has been bound.     

High malic enzyme activity in tumor cells and its cross-reaction with antipigeon liver malic enzyme serum

Summary Rabbit antibodies against pigeon liver malic enzyme (EC 1.1.1.40) were prepared. The antiserum gave single precipitation line with crude pigeon liver extract. Cross reaction was observed with partially purified malic enzyme or crude extract from chicken liver. Positive cross reaction was also observed with the concentrated cytosolic fraction of two human carcinoma cell lines which were demonstrated to contain high malic enzyme activity. All other proteins examined did not react with the antibodies. When purified pigeon liver malic enzyme was mixed with the antiserumin vitro, a time-dependent inactivation of the enzyme activity was observed. Protection of the enzyme activity against antiserum inactivation was afforded by NADP⁺ orL-malate. Metal Mn²⁺ gave little protection.     

Cloning and sequence analysis of the gene encoding Lactococcus lactis malolactic enzyme: Relationships with malic enzymes

Abstract Malolactic enzyme is the key enzyme in the degradation of L-malic acid by lactic acid bacteria. Using degenerated primers designed from the first 20 N-terminal amino acid sequence of lactococcal malolactic enzyme, a 60-bp DNA fragment containing part of the mleS gene was amplified from Lactococcus lactis in a polymerase chain reaction. This specific probe was used to isolate two contiguous fragments covering the gene as a whole. The 1.9-kb region sequenced contains an open reading frame of 1623 bp, coding a putative protein of 540 amino acids. The deduced amino acid sequence reveals that lactococcal putative protein (Mlep) is highly homologous to the malic enzyme of other organisms. Expression of the mleS gene in Escherichia coli results in malolactic activity.     

The 53-kDa proteolytic product of precursor starch-hydrolyzing enzyme of Aspergillus niger has Taka-amylase-like activity

The 53-kDa amylase secreted by Aspergillus niger due to proteolytic processing of the precursor starch-hydrolyzing enzyme was resistant to acarbose, a potent α-glucosidase inhibitor. The enzyme production was induced when A. niger was grown in starch medium containing the inhibitor. Antibodies against the precursor enzyme cross-reacted with the 54-kDa Taka-amylase protein of A. oryzae. It resembled Taka-amylase in most of its properties and also hydrolyzed starch to maltose of α-anomeric configuration. However, it did not degrade maltotriose formed during the reaction and was not inhibited by zinc ions.     

黑曲霉纤维素酶的纯化及酶学性质研究

黑曲霉(Aspergillusniger)固态发酵后粗酶液经硫酸铵盐析,2次SephadexG-200柱层析后可提纯8倍左右.CMC酶最适作用温度为60℃,最适作用pH为3.5,30℃～70℃区间酶活力较稳定,在pH3.0～5.0范围内,50℃保温30min能保持80%的酶活力.CMC酶的Km、Vmax值分别为7.69%CMCg/ml、0.33mg/ml·     

甲醛低温消毒对黑曲霉柠檬酸发酵影响的初步研究

在柠檬酸发酵中,加入较低浓度的甲醛后８５℃灭菌,可以灭活培养基中的杂菌,确保发酵正常进行,加甲醛灭菌后不仅不影响柠檬酸的产量,而且可能还有利于菌丝生长,在大生产中加甲醛低温灭菌还减少蒸汽用量,有明显经济意义。     

Correlation between manganese-deficiency,loss of respiratory chain complex I activity and citric acid production in Aspergillus niger

The correlation between manganese deficiency, loss of mitochondrial respiratory chain NADH: ubiquinone oxidoreductase (complex I) activity and citric acid overproduction in the Aspergillus niger strain B 60 was analysed. With increasing manganese-supplementation of the production medium the loss of complex I activity and the production of citric acid was reduced. Addition of manganese during growth stopped further loss of complex I activity and further increase of citric acid production. A possible causality between complex I deficiency and citric acid overproduction is discussed.     

Functional expression in Saccharomyces cerevisiae of the Lactococcus lactis mleS gene encoding the malolactic enzyme

Abstract Malolactic fermentation, a crucial step in winemaking, results mostly in degradation by lactic acid bacteria of L-malic acid into L-lactic acid. This direct decarboxylation is catalysed by the malolactic enzyme. Recently we, and others, have cloned the mleS gene of Lactococcus lactis encoding malolactic enzyme. Heterologous expression of mleS in Saccha-romyces cerevisiae was tested to perform simultaneously alcoholic and malolactic fermentations by yeast. mleS gene was cloned in a yeast multicopy vector under a strong promoter. Malolactic activity was present in crude extracts of recombinant yeasts. Malic acid degradation was tested during alcoholic fermentation in synthetic media and must. Yeasts expressing the mleS gene actually produced L-lactate from L-malate; nevertheless malate degradation was far from complete.