Effect of benzoic acid and its hydroxy-derivatives on the carbohydrate-metabolism of starved and of sucrose-fed etiolated barley leaves

Summary In etiolated barley leaves benzoic acid and its hydroxy-derivatives lowered the rate of carbon dioxide output, o-hydroxybenzoic acid (OHBA) being most effective, while p-hydroxybenzoic acid (PHBA) was least. Sucrose furthered the inhibitory effects of these acids when they were administered in 0.01 M concentration, but seemed only to alleviate the effect of PHBA when the latter was present in 0.001 M concentration.High concentrations of BA or its hydroxy-derivatives caused the excretion of hexoses, more prominently in the presence than in the absence of sucrose; OHBA being the exception, where sucrose stopped hexose-excretion. In the presence of lower concentrations BA retarded, while OHBA accelerated sucrose-uptake.BA and its hydroxy-derivatives depleted the tissue of its sucrosecontent and lowered the polysaccharide value, whether the leaves were starved or sugar-fed.     

Acid-stable α-Amylase of Black Aspergilli

The amino acid compositions of the acid-stable α-amylase and the acid-unstable α-amylase obtained from Aspergillus niger were determined by automatic column chromatography. The amino acid composition of the acid-unstable α-amylase was very similar to that of the α-amylase of Aspergillus oryzae. The amino acid composition of the acid-stable α-amylase was also similar in most part, but differed from that of the acid-unstable α-amylase in the following features, (a) The lysine content was lower, (b) Although the totals of carboxyl and amide were almost equal, there were considerably more free carboxyl residues, (c) The serine content was higher, (d) The proline content was lower. These facts may be related to the lower isoelectric point (pH 3.44) of the acid-stable α-amylase.

Amino-terminal amino acid analysis demonstrated one mole of amino-terminal leucine or isoleucine per mole of the acid-stable α-amylase and one mole of amino-terminal alanine per mole of the acid-unstable α-amylase.     

Effect of changed cultivation conditions on the morphology ofAspergillus niger and on citric acid biosynthesis in laboratory cultivation

Cultivation ofAspergillus niger 97A in baffled flasks on a shaker brings about metabolic changes demonstrated in increased citric acid biosynthesis, morphological changes and alterations in the mycelial cell wall.     

<Emphasis Type="Italic">Aspergillus niger</Emphasis> lipase: gene cloning,over-expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> and in vitro refolding

From the N-terminal amino acid sequence of the lipase from Aspergillus niger F044, a potential homologous gene A84689 to the lipanl (the gene encoding the lipase from Aspergillus niger F044) was identified. A pair of primers were designed according to the nucleotide sequence of A84689, and the lipanl was cloned by PCR. Nucleotide sequencing revealed that the lipanl has an ORF of 1,044 bp, containing three introns. The deduced amino acid sequence corresponds to 297 amino acid residues. The cloned cDNA fragment encoding the mature lipase from Aspergillus niger F044 was over-expressed in Escherichia coli BL21(De3) and the recombinant protein was refolded in vitro by dilution followed by DEAE Sepharose Fast Flow chromatography.     

Aerobiological,biochemical and immunological studies on some of the dominant <Emphasis Type="Italic">Aspergillus</Emphasis> species of South Assam (India)

Two years atmospheric survey of air-borne Aspergillus was carried out in the environmental conditions of South Assam. The survey revealed a total of 16 different species of Aspergillus with marked seasonal and annual variations. Aspergillus fumigatus was found to be the dominant atmospheric fungal species followed by Aspergillus flavus, Aspergillus niger, etc. Among the sample extracts tested, highest quantity of soluble protein was recorded in Aspergillus fumigatus (95.0 mg/g) whereas highest quantity of soluble carbohydrate (40.8 mg/g) and free amino acid (135.0 mg/g) was recorded in the sample extract of Aspergillus niger per gram of dry weight, respectively. The highest numbers of protein polypeptide bands were detected in the sample extract of Aspergillus fumigatus followed by Aspergillus flavus and lowest in Aspergillus niger. The maximum numbers of immunoglobulin E binding protein fractions were found in Aspergillus fumigatus, followed by Aspergillus flavus, Aspergillus clavatus, etc.     

Physiological characterisation of <Emphasis Type="Italic">acuB</Emphasis> deletion in <Emphasis Type="Italic">Aspergillus niger</Emphasis>

The acuB gene of Aspergillus niger is an ortholog of facB in Aspergillus nidulans. Under carbon-repression conditions, facB is repressed, thereby preventing acetate metabolism when the repressing carbon source is present. Even though facB is reported to be repressed directly by CreA, it is believed that a basal level of FacB activity exists under glucose-repressive conditions. In the present study, the effect of deletion of acuB on the physiology of A. niger was assessed. Differences in organic acid and acetate production, enzyme activities and extracellular amino and non-amino organic acid production were determined under glucose-repressing and -derepressing conditions. Furthermore, consumption of alternative carbon sources (e.g. xylose, citrate, lactate and succinate) was investigated. It was shown that AcuB has pleiotropic effects on the physiology of A. niger. The results indicate that metabolic pathways that are not directly involved in acetate metabolism are influenced by acuB deletion. Clear differences in organic acid consumption and production were detected between the ∆acuB and reference strain. However, the hypothesis that AcuB is responsible for basal AcuA activity necessary for activation of acetate metabolic pathways, even during growth on glucose, could not be confirmed. The experiments demonstrated that also when acuB was deleted, no acetate was formed. Therefore, AcuB cannot be the only activator of AcuA, and another control mechanism has to be available for activating AcuA.     

Phytic acid content reduction in canola meal by various microorganisms in a solid state fermentation process

Solid state fermentation of canola meal has been carried out for the reduction of its phytic acid content using the following microorganisms: Rhizopus oligosporus NRRL 2990, Aspergillus niger NRC 5765 and NRC 401 121, Aspergillus ficuum NRRL 3135 and a wild Saccharomyces cerevisiae strain. The results showed that all these microorganisms can be used for the reduction of the phytic acid content in the tested material. A. ficuum which completely hydrolyzed the phytic acid in 48 hours was the most efficient. Buffered systems, aeration and an increase in inoculum concentration caused faster and higher reduction of phytic acid content in canola meal.     

Physico-Chemical Properties of Linseed Oil as Affected by Various Seed-Borne Fungi

The effect of six seed-borne fungi on linseed oil was studied. Alternaria alternate, Aspergillus flavus, Aspergillus repens, Fusarium culmorum and Torula allii reduced oil content while Cladosporum herbarum slightly increased it. All fungi increased the free fatty acid content and saponification number linseed oil. Iodine number was decreased except in Cladosporium herbarum and Fusarium culmorum. Aspergillus flavus was the most destructive parasite in altering quantity and quality of linseed oil.     

Cellular localization of acid carboxypeptidase inAspergillus oryzae

Fresh mycelia ofAspergillus oryzae were shown to have strong acid carboxypeptidase (EC 3.4.12.-) activity. The cellular localization of acid carboxypeptidase was investigated using the broken mycelia and the protoplasts of the fungusAspergillus oryzae IAM 2640. In the broken mycelia, about 40% of the total activity was found in the “cell wall” fraction (2,000×g), with most of the remainder in the soluble fraction (100,000×g supernatant). During the formation of protoplasts, most of the acid carboxypeptidase in the mycelia was solubilized and released. The specific activity of acid carboxypeptidase in the lysed protoplasts was about 10-fold lower than that found in the broken mycelial preparation. These data indicate that most of the acid carboxypeptidase is probably located in the cell surface, which includes the cell wall, the periplasm, and the cell membrane.     

Isolation and Sequencing of a New Glucoamylase Gene from an Aspergillus niger Aggregate Strain (DSM 823) Molecularly Classified as Aspergillus tubingensis

Based on morphological characteristics the taxa included in the Aspergillus aggregate can hardly be differentiated. For that reason the phylogeny of this genus was revised several times as different criteria, from morphological to later molecular, were used. We found, comparing nucleotide sequences of the ITS-region, that the strain Aspergillus niger (DSM 823) which is claimed to be identical to the strains ATCC 10577, IMI 027809, NCTC 7193 and NRRL 2322 can be molecularly classified as Aspergillus tubingensis, exhibiting 100% identity with the A. tubingensis CBS strains 643.92 and 127.49. We amplified, cloned and sequenced a new glucoamylase gene (glaA) from this strain of A. tubingensis (A. niger DSM 823) using primers derived from A. niger glucoamylase G1. The amplified cDNA fragment of 2013 bp contained an open reading frame encoding 648 amino acid residues. The calculated molecular mass of the glucoamylase, deduced from the amino acid sequence, was 68 kDa. The nucleotide sequence of glaA showed 99% similarity with glucoamylases from Aspergillus kawachii and Aspergillus shirousami, whereas the similarity with the glucoamylase G1 from A. niger was 92% An erratum to this article is available at .     

Aspergillus oryzae laeA Regulates Kojic Acid Synthesis Genes

Kojic acid synthesis genes regulation was investigated in Aspergillus oryzae. Our results indicate that kojic acid production was lost in the laeA disruption strain, but was recovered in the LaeA complement strain. Real-time PCR also confirmed that expression of kojic acid biosynthesis genes decreased in the laeA disruption strain, indicating that these genes are under the control of LaeA.     

Based on biochemical and physiological behavior,where isAspergillus egyptiacus better placed?

Physiological and biochemical properties were tested in 45 isolates ofAspergillus egyptiacus (16 isolates),Emericella nidulans (16) andAspergillus versicolor (13). The three fungal species exhibited common and similar features. The big similarity betweenA. egyptiacus andE. nidulans was greater than betweenA. egyptiacus andA. versicolor. It included the inability to produce base either from sodium citrate or lactic acid media, growth at 45 °C (thermophilicity), and production of very similar pigmentations onAspergillus flavus andparasiticus agar.A. egyptiacus is therefore better placed in theAspergillus nidulans-Emericella assemblage.     

Characterization of a chimeric enzyme comprising feruloyl esterase and family 42 carbohydrate-binding module

We engineered a chimeric enzyme (AwFaeA-CBM42) comprising of type-A feruloyl esterase from Aspergillus awamori (AwFaeA) and family 42 carbohydrate-binding module (AkCBM42) from glycoside hydrolase family 54 α-l-arabinofuranosidase of Aspergillus kawachii. The chimeric enzyme was successfully produced in Pichia pastoris and accumulated in the culture broth. The purified chimeric enzyme had an apparent relative molecular mass (M _r) of 53,000. The chimeric enzyme binds to arabinoxylan; this indicates that the AkCBM42 in AwFaeA-CBM42 binds to arabinofuranose side chain moiety of arabinoxylan. The thermostability of the chimeric enzyme was greater than that of AwFaeA. No significant difference of the specific activity toward methyl ferulate was observed between the AwFaeA and chimeric enzyme, but the release of ferulic acid from insoluble arabinoxylan by the chimeric enzyme was approximately 4-fold higher than that achieved by AwFaeA alone. In addition, the chimeric enzyme and xylanase acted synergistically for the degradation of arabinoxylan. In conclusion, the findings of our study demonstrated that the components of the AwFaeA-CBM42 chimeric enzyme act synergistically to bring about the degradation of complex substrates and that the family 42 carbohydrate-binding module has potential for application in the degradation of polysaccharides.     

Continuous production of citric acid from dairy wastewater using immobilized<Emphasis Type="Italic">Aspergillus niger</Emphasis> ATCC 9142

The continuous production of citric acid from dairy wastewater was investigated using calcium-alginate immobilizedAspergillus niger ATCC 9142. The citric acid productivity and yield were strongly affected by the culture conditions. The optimal pH, temperature, and dilution rate were 3.0, 30°C, and 0.025 h⁻¹, respectively. Under optimal culture conditions, the maximum productivity, concentration, and yield of citric acid produced by the calcium-alginate immobilizedAspergillus niger were 160 mg L⁻¹ h⁻¹, 4.5 g/L, and 70.3% respectively. The culture was continuously perfored for 20 days without any apparent loss in citric acid productivity. Conversely, under the same conditions with a batch shake-flask culture, the maximum productivity, citric acid concentration, and yield were only 63.3 mg L⁻¹ h⁻¹, 4.7 g/L and 51.4%, respectively. Therefore, the results suggest that the bioreactor used in this study could be potentially used for continuous citric acid production from dairy wastewater by applying calcium-alginate immobilizedAspergillus niger.     

Galactose inhibition of citric acid production from glucose by Aspergillus niger

Summary Previous work in this laboratory has demonstrated that although Aspergillus niger can readily utilize galactose, no citric acid is produced from this carbon source (Hossain et al. 1984). Experiments were now conducted where galactose was added at various concentrations to synthetic growth medium containing glucose as carbon source, so that the effect of galactose on citric acid production from glucose could be observed. The results showed that the presence of galactose or a product of galactose metabolism caused inhibition of citric acid production, and also reduced the rate of glucose utilization. Enzyme analyses using mycelial cell-free extracts indicated that galactose interfered with the glucose-repression of the key enzyme 2-oxoglutarate dehydrogenase.     

Aspergillus oryzae NRRL 35191 from coffee, a non-toxigenic endophyte with the ability to synthesize kojic acid

Aspergillus oryzae NRRL 35191 was isolated as an endophyte from coffee leaves and found to produce kojic acid (KA) in culture. When inoculated into cacao seedlings (Theobroma cacao), A. oryzae grew endophytically and synthesized KA in planta. Cacao seedlings inoculated with A. oryzae produced higher levels of caffeine than non-inoculated ones. Aspergillus oryzae may be a useful endophyte to introduce to cacao since it grows non-pathogenically and induces the caffeine defense response that may make the plant more tolerant to insects and pathogens.     

Studies on the Proteolytic Enzymes of Black Aspergilli

The production of acid protease, as well as that of other enzymes, involves many variable factors, such as rate of aeration and agitation, temperature control during the operation, components of the media and type of antiform agents. The proper control of such factors can be achieved through effective utilization of a well-equipped pilot plant, designed specifically for the acid protease research and development. The fermentation unit described ranges in capacity from 500 ml shake-flask to 20-liter jar fermentor. Detailed studies on the various conditions for the production of acid protease by Aspergillus usamii, Aspergillus saitoi and other related species are described. It was found that high rates of agitation and aeration were required to obtain acid protease in maximum yield.     

Isolation and characterization of a proteinaceous antifungal compound from Lactobacillus plantarum YML007 and its application as a food preservative

Korean kimchi is known for its myriad of lactic acid bacteria (LAB) with diverse bioactive compounds. This study was undertaken to isolate an efficient antifungal LAB strain among the isolated kimchi LABs. One thousand and four hundred LABs isolated from different kimchi samples were initially screened against Aspergillus niger. The strain exhibiting the highest antifungal activity was identified as Lactobacillus plantarum YML007 by 16S rRNA sequencing and biochemical assays using API 50 CHL kit. Lact. plantarum YML007 was further screened against Aspergillus oryzae, Aspergillus flavus, Fusarium oxysporum and other pathogenic bacteria. The morphological changes during the inhibition were assessed by scanning electron microscopy. Preliminary studies on the antifungal compound demonstrated its proteinaceous nature with a molecular weight of 1256·617 Da, analysed by matrix‐assisted laser desorption ionization‐time‐of‐flight mass spectrometry (MALDI‐TOF). The biopreservative activity of Lact. plantarum YML007 was evaluated using dried soybeans. Spores of A. niger were observed in the negative control after 15 days of incubation. However, fungal growth was not observed in the soybeans treated with fivefold concentrated cell‐free supernatant of Lact. plantarum YML007. The broad activity of Lact. plantarum YML007 against various food spoilage moulds and bacteria suggests its scope as a food preservative.

Significance and Impact of the Study

After screening 1400 kimchi bacterial isolates, strain Lactobacillus plantarum YML007 was selected with strong antifungal activity against various foodborne pathogens. From the preliminary studies, it was found that the bioactive compound is a low molecular weight novel protein of 1256·617 Da. Biopreservative potential of Lact. plantarum YML007 was demonstrated on soybean grains, and the results point out YML007 as a potent biopreservative having broad antimicrobial activity against various foodborne pathogens.