Accumulation of chlorophyll and essential oils in photomixotrophic cell cultures of Citrus sp

Heterotrophically or photomixotrophically initiated callus cultures of Citrus paradisi, C. limon and C.aurantifolia were grown on different nutrient media and under different light regimes. Calli of C.paradisi that contained > 140 mg chlorophyll per kg wet weight accumulated about 40 volatile mono- and sesquiterpene hydrocarbons, oxigenated terpenes and aliphatic aldehydes. Upon five subcultivations the best yielding callus contained about 5% (186 mg × kg-1 wet wt) of the volatiles found in peel tissue (exo/mesocarp section), and about the twentyfold amount of that found in the fleshy endocarp. The composition of the essential oils from most of the cell cultures equalled grapefruit peel oil, but was shifted to a more fruit flesh-like composition, after the concentration of gellan gum in the medium was increased from 3 to 9 g per L. C. limon produced 11 monoterpenes and n-nonanal (40 mg × kg-1 wet wt max.), and C.aurantifolia yielded limonene only (4.4 mg × kg-1 wet wt max.). For all of the indicated species chlorophyll content and accumulation of volatiles were positively correlated. Addition of exogenous valencene to suspended cells of C.paradisi led to a stable concentration of the conversion product nootkatone. This stably maintained level suggested that a decreased catabolism of available carbon sources might have accounted for the significant accumulation of essential oil constituents

Formation of Aliphatic and Aromatic α-Hydroxy Ketones by Zygosaccharomyces bisporus

The wild-type yeast strain Zygosaccharomyces bisporus CBS 702 produced a-hydroxy-ketones (acyloins) from amino acid precursors after transamination to the corresponding 2-oxo acids. The key enzyme of the subsequent decarboxylation and C-C bond forming reaction, pyruvate decarboxylase (PDC), was examined for its substrate- and stereo-specific-ity. A wide variety of saturated and unsaturated aliphatic and aromatic aldehydes was successfully converted to acyloins. 19 of the biotransformation products identified had not been reported as natural substances before. Product yields were strongly affected by substrate structure. © 2000, Verlag der Zeitschrift für Naturforschung. All rights reserved

Hydrolysis of chlorogenic acid in apple juice using a p‐coumaryl esterase of Rhizoctonia solani

BACKGROUND: Apple juice is rich in polyphenolic compounds, especially in chlorogenic acid. A sour and bitter taste has been attributed to the compound. Chlorogenic acid in coffee powder was quickly hydrolysed by a p‐coumaryl esterase of Rhizoctonia solani (RspCAE) at its optimal pH of 6.0. It was unknown, however, if RspCAE would also degrade chlorogenic acid under the strongly acidic conditions (pH 3.3) present in apple juice. RESULTS: Treatment of apple juice with RspCAE led to a chlorogenic acid degradation from 53.38 ± 0.94 mg L−1 to 21.02 ± 1.47 mg L−1. Simultaneously, the caffeic acid content increased from 6.72 ± 0.69 mg L−1 to 19.33 ± 1.86 mg/L−1. The aroma profile of the enzymatically treated sample and a control sample differed in only one volatile. Vitispirane had a higher flavour dilution factor in the treated juice. Sensory analysis showed no significant difference in the taste profile ( p < 0.05). CONCLUSION: These results demonstrated a high stability and substrate specificity of RspCAE. An increase in caffeic acid and a concurrent decrease in chlorogenic acid concentration may exert a beneficial effect on human health

A three-enzyme-system to degrade curcumin to natural vanillin

The symmetrical structure of curcumin includes two 4-hydroxy-3-methoxyphenyl substructures. Laccase catalyzed formation of a phenol radical, radical migration and oxygen insertion at the benzylic positions can result in the formation of vanillin. As vanillin itself is a preferred phenolic substrate of laccases, the formation of vanillin oligomers and polymers is inevitable, once vanillin becomes liberated. To decelerate the oligomerization, one of the phenolic hydroxyl groups was protected via acetylation. Monoacetyl curcumin with an approximate molar yield of 49% was the major acetylation product, when a lipase from Candida antarctica (CAL) was used. In the second step, monoacetyl curcumin was incubated with purified laccases of various basidiomycete fungi in a biphasic system (diethyl ether/aqueous buffer). A laccase from Funalia trogii (LccFtr) resulted in a high conversion (46% molar yield of curcumin monoacetate) to vanillin acetate. The non-protected vanillin moiety reacted to a mixture of higher molecular products. In the third step, the protecting group was removed from vanillin acetate using a feruloyl esterase from Pleurotus eryngii (PeFaeA) (68% molar yield). Alignment of the amino acid sequences indicated that high potential laccases performed better in this mediator and cofactor-free reaction

Purification, characterisation and cDNA sequencing of pyruvate decarboxylase from Zygosaccharomyces bisporus

Cells of the wild-type yeast strain Zygosaccharomyces bisporus CBS 702 form alpha-hydroxy ketones from aromatic amino acid precursors during fermentation, Pyruvate decarboxylase (PDC, E.C. 4.1.1.1), the key enzyme of this biotransformation catalysing the nonoxidative decarboxylation of pyruvate and other 2-oxo-acids, was purified and characterised. The active enzyme is homotetrameric (alpha(4)) with a molecular mass of about 244 kDa, Activation of PDC by its substrate pyruvate results in a sigmoidal dependence of the reaction rate from substrate concentration (apparent K-m value 1.73 mM; Hill coefficient 2.10). A cDNA library was screened using a PCR-based procedure, and a 1856 bp cDNA of PDC was identified and sequenced. The cDNA encodes a polypeptide of 563 amino acid residues (monomeric unit), Sequence alignments demonstrate high homologies (> 80%) to PDC genes from Saccharomyces cerevisiae, Kluyveromyces lactis and Kluyveromyces marxianus.DF

A comparison of cell wall disruption techniques for the isolation of intracellular metabolites from Pleurotus and Lepista sp.

Different techniques were compared for their effectiveness in the disruption of the rigid cell walls of Basidiomycetes, Grinding under liquid nitrogen, stirred glass bead milling and enzymatic cell lysis were applied to the mycelia of Pleurotus sapidus and Lepista irina grown submerged. Each of the disruption procedures was evaluated by testing the quantity and quality of released intracellular metabolites: DNA, RNA, enzymes, and secondary metabolites. The most suitable method for nucleic acid isolation was grinding under liquid nitrogen, while bead mill homogenization was the superior technique for isolation of active enzymes. A new effective method is proposed for isolation of secondary metabolites with the aid of bead milling of fungal mycelia. © 2006 Verlag der Zeitschrift für Naturforschung

Regio- and stereoselective fungal oxyfunctionalisation of limonenes

Selective transformations of limonene by asco- and basidiomycetes were investigated. On the shake flask scale, Penicillium citrinum hydrated R-(+)-limonene to α-terpineol [83% regioselectivity (rs), more than 80 mg l-1 product yield], and Gongronella butleri catalysed the terminal oxidation to yield perillyl alcohol (60% rs, 16 mg l-1). On the laboratory bioreactor scale, Penicillium digitatum produced a peak concentration of 506 mg α-terpineol l-1 in the fed-batch mode, equivalent to a theoretical yield of 67%, and no volatile by-products were found. Fusarium proliferatum transformed R-(+)-limonene enantiospecifically to cis-(+)-carveol (98.6% ee, more than 35 mg l-1 product yield) and S-(-)-limonene predominantly to trans-(-)-carveol (96.3% ee). Pleurotus sapidus selectively dehydrogenised the accumulating trans-(-)-carveol to the corresponding enantiopure R-(-)-carvone. The results show that a careful selection of strain and bioprocess parameters may improve both the yield and the optical purity of a desired product

GC/MS Analysis of Some Bioactive Constituents from Carthamus lanatus L.

Sterols, triterpenes, volatiles, polar and other constituents in aerial parts of Carthamus lanatus were analyzed by gas chromatography-mass spectrometry. Over 90 compounds were identified most of them new for the species. Sitosterol and stigmasterol were the most abundant of 10 sterols identified in the sterol fraction. Taraxasterol, α- and β-amyrine prevailed in the triterpene fraction. Volatiles, sterols and a fraction of the dichloromethane extract showed strong cytotoxicity (Artemia salina assay)

An extracellular carboxylesterase from the basidiomycete Pleurotus sapidus hydrolyses xanthophyll esters

An extracellular enzyme capable of efficient hydrolysis of xanthophyll esters was purified from culture supernatants of the basidiomycete Pleurotus sapidus. Under native conditions, the enzyme exhibited a molecular mass of 430 kDa, and SDS-PAGE data suggested a composition of eight identical subunits. Biochemical characterisation of the purified protein showed an isoelectric point of 4.5, and ideal hydrolysis conditions were observed at pH 5.8 and 40 degrees C. Partial amino acid sequences were derived from N-terminal Edman degradation and from mass spectrometric ab initio sequencing of internal peptides. An 1861-bp cDNA containing an open reading frame of 1641 bp was cloned from a cDNA library that showed ca. 40% homology to Candida rugosa lipases. The P sapidus carboxylesterase represents the first enzyme of the lipase/esterase family from a basidiomycetous fungus that has been characterised at the molecular level

Production of natural colorants by liquid fermentation with Chlorociboria aeruginascens and Laetiporus sulphureus and prospective applications

The replacement of potentially hazardous synthetic dyes with natural dyes and pigments are of great interest for a sustainable economy. In order to obtain cost-efficient, environmentally friendly and competitive products, improvements in the cultivation and extraction of pigment-producing organisms and in dyeing processes are necessary. In our study, we were able to scale up the production of xylindein by Chlorociboria aeruginascens from 3 to 70 L bioreactor cultivations. We have identified important bioprocess parameters like low shear stress (150 rpm, tip speed <0.5 m/s) for optimal pigment yield (4.8 mg/L/d). Additionally, we have demonstrated the potential of laetiporic acid production by Laetiporus sulphureus in various cultivation systems and media, achieving dried biomass concentrations of almost 10 g/L with a 7 L bioreactor cultivation after 17 days. Extractions performed at 70°C and 15 min incubation time showed optimal results. To the best of our knowledge, we have described for the first time the use of this pigment in silk dyeing, which results in a brilliant hue that cannot easily be produced by other natural pigments. © 2020 The Authors. Engineering in Life Sciences published by Wiley-VCH Gmb