Supporting role of lignin in immobilization of yeast on sugarcane bagasse for continuous pectinase production.

BACKGROUND: Lignocellulosic wastes are pretreated prior to their utilization in fermentation processes. Such pretreatment also alters the topological features of the substrates, and therefore the suitability of pretreated waste as immobilization matrix for microbial cells needs investigation. RESULTS: In this study, the effect of chemical pretreatment of sugarcane bagasse (SB) for its subsequent utilization as a matrix to immobilize a pectinolytic yeast, Geotrichum candidum AA15, was evaluated using cell retention, concentration of immobilized cells, immobilization efficiency, scanning electron microscopy and Fourier transform infrared spectroscopy of the substrate and pectinase titers obtained after recycling. The results revealed that untreated SB is more efficient for immobilization with higher values of cell retention and pectinase productivity (99.78%) retained for up to six production cycles. It was deduced that removal of lignin by pretreatment negatively influenced the ability of SB to support cell adhesion, as lignin acts as a sealing agent that provides strength to the substrate. CONCLUSIONS: The strategy of utilizing SB as immobilization matrix was found effective at the laboratory scale as it improved pectinase production and may be investigated further for large-scale and cost-effective production. © 2020 Society of Chemical Industry.

Inhibitory effects of glutaraldehyde on Geotrichum citri-aurantii and its possible mechanism.

AIMS: This study investigated the inhibitory effect of glutaraldehyde (GA) on sour rot in citrus fruit and the underlying antifungal mechanism on mycelial growth of the causative pathogen Geotrichum citri-aurantii. METHODS AND RESULTS: Glutaraldehyde exhibited a strong inhibitory effect on G. citri-aurantii, with a minimum inhibitory and fungicidal concentration (MFC) of 1·00 µl ml-1 . In addition, in vivo application of GA (1 × MFC and 5 × MFC) reduced the disease incidence of sour rot in citrus fruit by 60 and 80% respectively. Scanning electron microscopy results revealed that the morphology of G. citri-aurantii mycelia was greatly altered by GA treatment. Propidium iodide and Calcofluor White Staining revealed that the membrane permeability, rather than the cell wall integrity, of G. citri-aurantii mycelia was severely disrupted after the addition of GA. Massive accumulation of malonaldehyde and reactive oxygen species as well as an increase in lipoxygenase activity were observed. CONCLUSION: These results indicate that GA may inhibit the mycelia growth of G. citri-aurantii through a membrane damage mechanism induced by membrane peroxidation. SIGNIFICANCE AND IMPACT OF THE STUDY: Glutaraldehyde is expected to be a novel fungicide for controlling sour rot in citrus fruit.

Date Palm Trees Root-Derived Endophytes as Fungal Cell Factories for Diverse Bioactive Metabolites.

Endophytic fungi of healthy and brittle leaf diseased (BLD) date palm trees (Phoenix dactylifera L.) represent a promising source of bioactive compounds with biomedical, industrial, and pharmaceutical applications. The fungal endophytes Penicillium citrinum isolate TDPEF34, and Geotrichum candidum isolate TDPEF20 from healthy and BLD date palm trees, respectively, proved very effective in confrontation assays against three pathogenic bacteria, including two Gram-positive bacteria Bacillus thuringiensis (Bt) and Enterococcus faecalis (Ef), and one Gram-negative bacterium Salmonella enterica (St). They also inhibited the growth of three fungi Trichoderma sp. (Ti), Fusarium sporotrichioides (Fs), Trichoderma sp. (Ts). Additionally, their volatile organic compounds (VOCs) were shown to be in part responsible for the inhibition of Ti and Ts and could account for the full inhibition of Fs. Therefore, we have explored their potential as fungal cell factories for bioactive metabolites production. Four extracts of each endophyte were prepared using different solvent polarities, ethanol (EtOH), ethyl acetate (EtOAc), hexane (Hex), and methanol (MetOH). Both endophyte species showed varying degrees of inhibition of the bacterial and fungal pathogens according to the solvent used. These results suggest a good relationship between fungal bioactivities and their produced secondary metabolites. Targeting the discovery of potential anti-diabetic, anti-hemolysis, anti-inflammatory, anti-obesity, and cytotoxic activities, endophytic extracts showed promising results. The EtOAc extract of G. candidum displayed IC50 value comparable to the positive control diclofenac sodium in the anti-inflammatory assays. Antioxidant activity was evaluated using α,α-diphenyl-ß-picrylhydrazyl (DPPH), ß-carotene bleaching, reducing power (RP), and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonique) (ABTS) radical scavenging assays. The findings revealed strong anti-oxidant power with an IC50 of 177.55 µg/mL for G. candidum EtOAc extract using DPPH assay, probably due to high polyphenol and flavonoid content in both fungal extracts. Finally, LC-HRMS (Liquid Chromatography­High Resolution Mass Spectrometry) and GC-MS (Gas Chromatography­Mass Spectrometry) analysis of G. candidum and P. citrinum extracts revealed an impressive arsenal of compounds with previously reported biological activities, partly explaining the obtained results. Finally, LC-HRMS analysis indicated the presence of new fungal metabolites that have never been reported, which represent good candidates to follow for the discovery of new bioactive molecules.

Biodiversity in targeted metabolomics analysis of filamentous fungal pathogens by 1H NMR-based studies.

The taxonomical classification among fungi kingdom in the last decades was evolved. In this work the targeted metabolomics study based on 1H NMR spectroscopy combined with chemometrics tools was reported to be useful for differentiation of three model of fungal strains, which represent various genus of Ascomycota (Aspergillus pallidofulvus, Fusarium oxysporum, Geotrichum candidum) were selected in order to perform metabolomics studies. Each tested species, revealed specific metabolic profile of primary endo-metabolites. The species of A. pallidofulvus is represented by the highest concentration of glycerol, glucitol and Unk5. While, F. oxysporum species is characterised by increased level of propylene glycol, ethanol, 4-aminobutyrate, succinate, xylose, Unk1 and Unk4. In G. candidum, 3-methyl-2-oxovalerate, glutamate, pyruvate, glutamine and citrate were elevated. Additionally, a detailed analysis of metabolic changes among A. pallidofulvus, F. oxysporum and G. candidum showed that A. pallidofulvus seems to be the most pathogenic fungi. The obtained results demonstrated that targeted metabolomics analysis could be utilized in the future as a supporting taxonomical tool for currently methods.

Optimization of the production and characterization of lipase from Candida rugosa and Geotrichum candidum in soybean molasses by submerged fermentation.

This present work describes the production and biochemical characterization of lipase by Candida rugosa and Geotrichum candidum in a culture supplemented with soybean molasses. After optimizing the fermentation times for both microorganisms, the effects of changing the soybean molasses concentration, the fermentative medium pH and the fermentation temperature were evaluated using the Central Composite Planning. When soybean molasses was used at a concentration of 200 g/L at 27 ± 1 °C and pH 3.5, the lipolytic activity measured in the broth was 12.3 U/mL after 12 h for C. rugosa and 11.48 U/mL after 24 h for G. candidum. The molecular masses were 38.3 kDa to G. candidum lipase and 59.7 kDa to C. rugosa lipase, determined by SDS-PAGE. The lipase from both microorganisms exhibited maximal hydrolytic activity at a temperature of 40 °C and were inhibited at pH 10.0. Using different concentration of p-nitrophenylbutyrate (p-NPB), the kinetic parameters were calculated, as follows: the Km of lipase from G. candidum was 465.44 µM and the Vmax 0.384 µmol/min; the Km and Vmax of lipase from C. rugosa were 129.21 µM and 0.034 µmol/min, respectively. Lipases activity were increased by metallic ions Mg(2+) and Na(+) and inhibited by metallic ion Cu(3+).

Isolation and evaluation of probiotics from traditional Chinese foods for aflatoxin B1 detoxification: Geotrichum candidum XG1 (yeast) and mechanistic insights.

Identifying aflatoxin-detoxifying probiotics remains a significant challenge in mitigating the risks associated with aflatoxin contamination in crops. Biological detoxification is a popular technique that reduces mycotoxin hazards and garners consumer acceptance. Through multiple rounds of screening and validation tests, Geotrichum candidum XG1 demonstrated the ability to degrade aflatoxin B1 (AFB1) by 99-100%, exceeding the capabilities of mere adsorption mechanisms. Notably, the degradation efficiency was demonstrably influenced by the presence of copper and iron ions in the liquid medium, suggesting a potential role for proteases in the degradation process. Subsequent validation experiments with red pepper revealed an 83% reduction in AFB1 levels following fermentation with G. candidum XG1. Furthermore, mass spectrometry analysis confirmed the disruption of the AFB1 furan ring structure, leading to a subsequent reduction in its toxicity. Collectively, these findings establish G. candidum XG1 as a promising candidate for effective aflatoxin degradation, with potential applications within the food industry.

Structure of Acidothermus cellulolyticus family 74 glycoside hydrolase at 1.82 Å resolution.

Here, a 1.82 Šresolution X-ray structure of a glycoside hydrolase family 74 (GH74) enzyme from Acidothermus cellulolyticus is reported. The resulting structure was refined to an R factor of 0.150 and an Rfree of 0.196. Structural analysis shows that five related structures have been reported with a secondary-structure similarity of between 75 and 89%. The five similar structures were all either Clostridium thermocellum or Geotrichum sp. M128 GH74 xyloglucanases. Structural analysis indicates that the A. cellulolyticus GH74 enzyme is an endoxyloglucanase, as it lacks a characteristic loop that blocks one end of the active site in exoxyloglucanases. Superimposition with the C. thermocellum GH74 shows that Asp451 and Asp38 are the catalytic residues.

Total synthesis and antibacterial screening of ( ± )-7-butyl-6,8-dihydroxy-3-pentyl-3,4-dihydroisochromen-1-one.

A new total synthesis of ( ± )-7-butyl-6,8-dihydroxy-3-pentyl-3,4-dihydroisochromen-1-one, isolated as R-enantiomer from Geotrichum sp., has been described. Reaction of 4-butyl-3,5-dimethoxyhomophthalic anhydride with hexanoyl chloride in the presence of 1,1,3,3-tetramethyl guanidine and triethyl amine afforded 7-butyl-6,8-dimethoxy-3-pentylisochromen-1-one, which was converted into corresponding 3,4-dihydroisochromen-1-one by successive ring opening and reduction. Final demethylation to furnish the natural product was achieved using anhydrous aluminum chloride in ethanethiol. The target compound and the intermediates were subjected to antibacterial evaluation against 10 bacterial strains using levofloxacin as standard.

Decolorization of oxygen-delignified bleaching effluent and biobleaching of oxygen-delignified kraft pulp by non-white-rot fungus Geotrichum candidum Dec 1.

Decolorization of oxygen-delignified bleaching effluent (abbreviated as OBE) and biobleaching of oxygen-delignified kraft pulp (OKP) were conducted using a non-white rot fungus Geotrichum candidum Dec 1 (abbreviated as Dec 1) which has ability to decolorize various synthetic dyes and molasses. Dec 1 decolorized up to 77% of OBE for 6 days. In addition, Dec 1 increased the brightness of OKP from 47.8% to 51.2% and decreased the kappa value of OKP from 12.4 to 10.4 points during a 6-day incubation period at a 25% of pulp-concentration. At 2% pulp-concentration, the brightness of OKP increased by 13% and the kappa value of OKP decreased by 4 points only for a 3-day incubation period. When the decolorized OBE was used for bleaching of OKP, the brightness of OKP increased to 62.7% under the shaking culture to a 2% pulp-concentration using culture fluid of decolorized OBE. It was revealed that Dec 1 is a potential to apply for decolorization of wastewater and biobleaching of pulp in paper-mills.

The use of FAME analyses to discriminate between different strains of Geotrichum klebahnii with different viabilities.

A considerable decline in viability of spray dried cells of Geotrichum klebahnii was observed and was attributed to an undefined alteration of the used strain. As common techniques were not able to distinguish the altered from the still viable strains, we used the fatty acid methyl ester (FAME) analysis. On the basis of FAME data we were able to discriminate the three strains under investigation. Especially the ratios of cis/trans fatty acid ratios and of saturated/unsaturated fatty acid were significantly reduced in the less viable strain, pointing to an increased stress level in this strain. These findings clearly show the applicability of the FAME analysis to detect strain alterations and that this method is therefore a suitable, fast and feasible tool for quality assurance.

Screening of Oxygenated Aromatic Compounds for Potential Antifungal Activity against Geotrichum citri-aurantii through Structure-Activity Relationship Analysis.

Sour rot caused by Geotrichum citri-aurantii (G. citri-aurantii) is responsible for huge economic losses during citrus fruit storage. However, the availability of chemical fungicides for controlling this disease is rather limited. In the present study, the antifungal activities of 25 oxygenated aromatic compounds against the mycelial growth of G. citri-aurantii were determined, and their corresponding structure-activity relationships were illustrated. Salicylaldehyde (pMIC = 2.689) possessed the strongest inhibitory effect on G. citri-aurantii growth, followed by thymol (pMIC = 2.478) and o-phthalaldehyde (pMIC = 2.429). Molecular electrostatic potential and molecular orbital analysis showed that the antifungal efficiency of test compounds was determined by the number and location of hydroxyl and aldehyde groups and the length of the ester chain. All compounds were selected for quantitative structure-antifungal activity relationship (QSAR) analysis. A three-dimensional-QSAR model of G. citri-aurantii inhibitors was established and demonstrated good predictive capability [comparative molecular field analysis, q2 = 0.532, optimum number of components (ONC) =10, R2 = 0.996, F = 560.325, standard error of estimation (SEE) = 0.034, and two descriptors; comparative similarity index analysis, q2 = 0.675, ONC = 6, R2 = 0.989, F = 263.354, SEE = 0.054, and five descriptors]. QSAR analysis showed that substitution at position 1 with hydrophilic and electron-withdrawing groups produced a hydrogen donor and thus improved the antifungal activity. In contrast, substitution at positions 4 or 5 with hydrophilic and electron-donating groups decreased its antifungal activity. These findings can provide theoretical guidance for preparing effective antifungal drugs for controlling sour rot in citrus.

Differences in hydrolytic abilities of two crude lipases from Geotrichum candidum 4013.

The fungus Geotrichum candidum 4013 produces two types of lipases (extracellular and cell-bound). Both enzymes were tested for their hydrolytic ability to p-nitrophenyl esters and compounds having a structure similar to the original substrate (triacylglycerols). Higher lipolytic activity of extracellular lipase was observed when triacylglycerols of medium- (C12) and long- (C18) chain fatty acids were used as substrates. Cell-bound lipase preferentially hydrolysed trimyristate (C14). The differences in the abilities of these two enzymes to hydrolyse p-nitrophenyl esters were observed as well. The order of extracellular lipase hydrolysis relation velocity was as follows: p-nitrophenyl decanoate > p-nitrophenyl caprylate > p-nitrophenyl laurate > p-nitrophenyl palmitate > p-nitrophenyl stearate. The cell-bound lipase indicates preference for p-nitrophenyl palmitate. The most striking differences in the ratios between the activity of both lipases (extracellular : cell-bound) towards different fatty acid methyl esters were 2.2 towards methyl hexanoate and 0.46 towards methyl stearate (C18). The Michaelis constant (K(m) ) and maximum reaction rate (V(max) ) for p-nitrophenyl palmitate hydrolysis of cell-bound lipase were significantly higher (K(m) 2.462 mM and V(max) 0.210 U/g/min) than those of extracellular lipase (K(m) 0.406 mM and V(max) 0.006 U/g/min).

The Use of Sour and Sweet Whey in Producing Compositions with Pleasant Aromas Using the Mold Galactomyces geotrichum: Identification of Key Odorants.

Fermented products with a pleasant aroma and with strong honey, rose, and fruit odor notes were developed through the biotransformation of a medium containing sour or sweet whey with the addition of l-phenylalanine by the Galactomyces geotrichum mold. In order to obtain the strong honey-rose aroma, G. geotrichum strains were screened and fermentation conditions were optimized to achieve a preferable ratio (>1) of phenylacetaldehyde to 2-phenylethanol by the Ehrlich pathway. This allowed post-fermentation products with the ratio of concentrations of phenylacetaldehyde to 2-phenylethanol being 1.7:1. Additionally, the use of gas chromatography-olfactometry (GC-O) analysis and the calculation of odor activity values (OAVs) allowed 10 key odorants to be identified in post-fermentation products. The highest OAVs were found for phenylacetaldehyde with a honey odor in both sour and sweet whey cultures (3010 and 1776, respectively). In the variant with sour whey, the following compounds with the highest OAVs were 3-methyl-1-butanol (131), 3-(methylthio)-propanal (119), 3-methylbutanal (90), dimethyl trisulfide (71), 2,3-butanedione (37), and 2-phenylethanol (29). In the post-fermentation product with sweet whey, the following compounds with the highest OAVs were 3-(methylthio)-propanal (112), dimethyl trisulfide (69), and 2,3-butanedione (41).

Determination of metabolites of Geotrichum citri-aurantii treated with peppermint oil using liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry.

Sour rot is a leading disease of citrus fruit caused by the postharvest pathogen Geotrichum citri-aurantii. It has been reported that essential oils can be used as substitutes for synthetic fungicides to control the pathogen. In this study, changes in metabolites and antifungal effects of G. citri-aurantii treated with peppermint oil (PO) were investigated. The inhibition rate of the mycelial growth increased as the PO concentration increased, and 6 µl PO/disk resulted in a radial growth inhibition of 79.2%. The electrical conductivity of G. citri-aurantii treated with PO increased compared to the control. By comparing the metabolic profiles of treated and untreated G. citri-aurantii cells, a total of 53 distinct metabolites 9 were up-regulated and 44 were down-regulated were found, including 16 lipid metabolites, 6 carbohydrate metabolites, 2 amino acid metabolites, 5 alcohols, 2 glycoside metabolites, and 3 ketone metabolites, etc, and these metabolites are involved in 25 major metabolic pathways. PRACTICAL APPLICATIONS: Chemical fungicides can effectively control G. citri-aurantii during fruit postharvest period. However, synthetic chemical fungicides have gradually led to buildup of resistance of fungil, which seriously causes the frequent of food-borne diseases. PO extracted from natural plants can be used as natural additive in many foods due to their antioxidant, antibacterial, and antifungal properties. Therefore, PO can be considered as a promising bacteriostatic agent for the defense of G. citri-aurantii during fruit postharvest period.

[Cloning and overexpression of lipase gene from Geotrichum candidum Y162].

By means of bioinformatics, we aligned nucleotide sequence of reported lipase gene from Geotrichum. Primers were designed based on the conservative nucleotide sequence, and the lipase gene of G. candidum Y162 was cloned for the first time in China. Nucleotide sequencing revealed that the open reading frame has 1692 nucleotides without any introns, encoding 563 amino acid residues including a signal sequence of 19 amino acid residues, which is 86% identical to lipase I of G. fermentans. Subsequently, we cloned the lipase gene into expression vector pPIC9K, and then transformed into Pichia pastoris GS115. Cultures of recombined P. pastoris accumulated active enzyme in the supernatant to levels of 55 U/mL after induction for 96 hours in shake flasks. The purified lipase exhibited maximum activity at 50 degrees C and pH 8.0, and was stable between pH 6.0 and 10.0 and below 60 degrees C. Lipase activity was compatible with the presence of organic solvents such as methanol, n-heptane, hexane, cyclohexane, glycerol, benzene and diethyl ether. Lipase showed hydrolysis preference for triacylglycerol substrates containing cis-9 unsaturated fatty acid. The results suggest that the lipase could be a candidate for industrial applications.

Structure determination of neoefrapeptins A to N: peptides with insecticidal activity produced by the fungus Geotrichum candidum.

The structures of neoefrapeptins A to N, peptides with insecticidal activity, were elucidated. They showed a close similarity to efrapeptin. However, all neoefrapeptins contained the very rare amino acid 1-amino-cyclopropane-carboxylic acid and some of them also contained (2S,3S)-3-methylproline. The neoefrapeptins are the first case, in which these amino acids are found as building blocks for linear peptides. They were identified by comparison of the silylated hydrolyzate to reference material by GC/MS (EI-mode). The sequence was elucidated using mass spectrometry (ESI+ mode). Full scan spectra showed two fragments in high yield, even under mild ionization conditions. MS/MS spectra of these two fragments yielded fragment rich spectra from which the sequence of the compounds was determined almost completely. The proteolytic cleavage with the proteinase papain yielded products that allowed to prove the rest of the sequence and the identity of the C-terminus to efrapeptin. The proteolytic cleavage products allowed furthermore to determine the position of the isobaric amino acids, pipecolic acid and 3-methylproline in neoefrapeptin F, as well as the location of R-isovaline and S-isovaline. Papain digestion was such established as a tool for structure elucidation of peptides rich in alpha,alpha-dialkylated amino acids. CD spectra suggested a 3(10) helical structure for neoefrapeptins A and F.

Molecular characterization of a Galactomyces geotrichum lipase, another member of the cholinesterase/lipase family.

Geotrichum candidum secretes several lipase isoenzymes, differing in their selectively towards esters of long chain fatty acids with a cis-9 double bond. One group shows an absolute selectively towards these fatty acid esters, the other group has a more relaxed specificity and will also hydrolyze other long chain fatty acid esters. Galactomyces geotrichum secrets a lipase that has the same specificity as the latter group. The corresponding lipase gene was cloned from Galactomyces geotrichum. From an alignment of our enzymes' primary structure with those of different strains of Geotrichum candidum, remarkable conservation is evident and it is not yet possible to identify residues/structures responsible for differences in fatty acid specificity. Comparison of the GCL/GGL family with a variety of lipases from other sources, indicated that they are more related to mammalian than microbial lipases.

Enzymatic versus spontaneous S-methyl thioester synthesis in Geotrichum candidum.

The synthesis of short chain S-methyl thioesters was investigated in Geotrichum candidum strain GcG. The results indicated the involvement of an enzymatic reaction in this microorganism that led to the synthesis of S-methyl thioacetate (MTA) when methanethiol and acetyl-CoA were used as substrates. MTA was generated from these substrates by enzymatic or spontaneous reactions, whose relative importance depended largely on pH and temperature. For longer chain acyl-CoA compounds (C3 to C6), thioester synthesis was primarily spontaneous. Short chain fatty acid activation by a CoA residue probably is a prerequisite for the synthesis of S-methyl thioesters.

Fungal cell-wall galactomannans isolated from Geotrichum spp. and their teleomorphs, Dipodascus and Galactomyces.

The alkali-extracted water-soluble galactomannan F1SS isolated from the cell wall of two species each of Geotrichum, Galactomyces, and Dipodascus have been studied by methylation analysis and NMR spectroscopy, and their structure is established as the following: [carbohydrate structure: see text]

Ustilipids, acylated beta-D-mannopyranosyl D-erythritols from Ustilago maydis and Geotrichum candidum.

Ustilago species produce an extracellular oil that shows activity in various pharmaceutical assays. We isolated several complexes of this heterogeneous glycolipid from cultures of Ustilago maydis DSM 11494 and Geotrichum candidum ST 002515, and determined the chemical structures of these new compounds, termed ustilipids, on the basis of NMR experiments, mass spectra, and fatty acid analyses. They all possess a 4-O-beta-D-mannopyranosyl D-erythritol basic framework, the configuration of which was confirmed, after initial solvolysis, by a single-crystal X-ray structure analysis. All the investigated ustilipids and related compounds are similarly constructed: the three hydroxy groups of the erythritol side chain are free in all cases, whereas the hydroxy groups of the mannose residue are for the most part acylated. Medium-chain fatty acids have for the first time been detected as components of glycolipids produced by Ustilaginales. While the 2-hydroxy group of the mannose residue is esterified with a C2-C8 carboxylate side chain, the 3-hydroxy group is in all cases esterified by a longer, C12-C20 fatty acid residue. The oxygen functionalities at the 4 and 6 positions are either acetylated or present as free hydroxy groups. Ustilipids antagonize dopamine D3 receptors in micromolar quantities; other members of this class of compounds have been found to possess an inhibitory action on the neurotensin receptor. The hemolytic activity of ustilipids is low.