Comparative Cold Shock Expression and Characterization of Fungal Dye-Decolorizing Peroxidases.

Dye-decolorizing peroxidases (DyPs) from Auricularia auricula-judae, Bjerkandera adusta, Pleurotus ostreatus and Marasmius scorodonius (Basidiomycota) were expressed in Escherichia coli using the cold shock-inducible expression system pCOLD I DNA. Functional expression was achieved without the addition of hemin or the co-expression of any chaperones. The presence or absence of the native signal sequence had a strong impact on the success of the expression, but the effect was not consistent for the different DyPs. While BaDyP and AajDyP were stable at 50 °C, the more thermolabile MsP2 and PoDyp, upon catalytic intervention, lend themselves to more rapid thermal inactivation. The bleaching of norbixin (E 160b) using MsP2 was most efficient at pH 4.0, while BaDyP and AajDypP worked best in the weakly acidic to neutral range, indicating a choice of DyPs for a broad field of applications in different food matrices.

Functional expression of a valencene dioxygenase from Pleurotus sapidus in E. coli.

Valencene dioxygenase (ValOx) from the edible basidiomycete Pleurotus sapidus converted the sesquiterpene (+)-valencene to the valuable grapefruit flavour (+)-nootkatone and to nootkatols through intermediate hydroperoxides. Expression of the enzyme was carried out in the cytosol and periplasm of Escherichia coli. The heterologous production led to high yields of inclusion bodies. The poor yield of soluble recombinant protein was improved by various strategies including cold shock expression, chaperone co-expression, and employment of mutant E. coli strains. Up to 60 mg of the biologically active, soluble ValOx was produced by cold shock under control of the cspA promoter at 8 °C in the BL21(DE3)Star strain and co-expression of the E. coli trigger factor. The recombinant enzyme, purified using the N-terminal His tag, showed the catalytic properties of the wild-type enzyme, as was confirmed by the LC-MS analysis of hydroperoxide intermediates and GC-MS analysis of the volatile products.

Volatile flavours in raw egg yolk of hens fed on different diets.

BACKGROUND: Recent studies have suggested that the composition of lipophilic components of egg yolk is influenced by the feed. The aim of the present study was to isolate volatile flavours from egg yolk after different feeding trials using solvent extraction and thin layer high-vacuum distillation. The resulting aroma extract was analysed by various gas chromatographic techniques. Chickens were either fed with laying meal, laying meal plus cabbage and onion or laying meal plus rapeseed oil or held in free-range. RESULTS: The predominating odour impressions were described as onion-like. Comparing all analytical and sensory data of the flavour extracts, there were minimal differences among the respective samples. Free-range eggs contained fewer volatile compounds than the other samples, whereas rapeseed oil supplementation caused an enrichment of sulfur compounds. CONCLUSION: While data from gas chromatography/flame ionisation detection, gas chromatography/mass spectrometry and gas chromatography/olfactometry were less conclusive, the results from sulfur-specific analysis using gas chromatography/flame photometric detection showed a considerable effect. However, because of the low abundance of sulfur compounds in the yolk, these differences are not expected to be perceivable by the consumer.

Lactic fermentation to improve the aroma of protein extracts of sweet lupin (Lupinus angustifolius).

Lupin protein extracts (LPE) are prone to the emission of a beany off-flavour during storage, which confines its application in foods. Fermentation of LPE using several lactic acid bacteria was conducted to reduce off-flavour formation in stored samples. The aroma profile of untreated LPE was compared to those of fermented protein extracts (LPEF). Hexanal and n-hexanol were used as indicator substances of progressing lipid oxidation. The most powerful odourants were evaluated by GC-olfactometry-flavour dilution analysis and identified according to their mass spectra, odour descriptions, and retention indices. Twenty two volatile substances with dilution factors equal to or higher than 100 were determined in both LPE and LPEF, amongst them n-pentanal, n-hexanal, 1-pyrroline, dimethyl trisulfide, 1-octen-3-one, 3-octen-2-one, 1-octen-3-ol, and ß-damascenone. The aroma profile was significantly modified by the fermentation process and the off-flavours were reduced and/or masked by newly formed compounds.

Generation of norisoprenoid flavors from carotenoids by fungal peroxidases.

To biotechnologically produce norisoprenoid flavor compounds, two extracellular peroxidases (MsP1 and MsP2) capable of degrading carotenoids were isolated from the culture supernatants of the basidiomycete Marasmius scorodonius (garlic mushroom). The encoding genes were cloned from genomic DNA and cDNA libraries, and databank homology searches identified MsP1 and MsP2 as members of the so-called "DyP-type" peroxidase family. Wild type enzymes and recombinant peroxidases expressed in Escherichia coli were employed for the release of norisoprenoids from various terpenoid precursor molecules. Carotenes, xanthophylls, and apocarotenals were subjected to the enzymatic degradation. Released volatile products were characterized by GC-FID and GC-MS, whereas nonvolatile breakdown products were analyzed by means of HPLC-DAD and HPLC-MS. C13 norisoprenoids together with C10 products proved to be the main volatile degradation products in each case.

Heterologous expression of the msp2 gene from Marasmius scorodonius.

For the heterologous expression of the msp2 gene from the edible mushroom Marasmius scorodonius in Escherichia coli the cDNA encoding the extracellular Msp2 peroxidase was cloned into the pBAD III expression plasmid. Expression of the protein with or without signal peptide was investigated in E. coli strains TOP10 and LMG194. Different PCR products were amplified for expression of the native target protein or a protein with a signal peptide. Omitting the native stop codon and adding six His-residues resulted in a fusion protein amenable to immune detection and purification by immobilised metal affinity chromatography. In E. coli the recombinant protein was produced in high yield as insoluble inclusion bodies. The influence of different parameters on MsP2 refolding was investigated. Active enzyme was obtained by glutathione-mediated oxidation in a medium containing urea, Ca(2+), and hemin.

Functional expression of the lipase gene Lip2 of Pleurotus sapidus in Escherichia coli.

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.

Novel peroxidases of Marasmius scorodonius degrade beta-carotene.

Two extracellular enzymes (MsP1 and MsP2) capable of efficient beta-carotene degradation were purified from culture supernatants of the basidiomycete Marasmius scorodonius (garlic mushroom). Under native conditions, the enzymes exhibited molecular masses of approximately 150 and approximately 120 kDa, respectively. SDS-PAGE and mass spectrometric data suggested a composition of two identical subunits for both enzymes. Biochemical characterisation of the purified proteins showed isoelectric points of 3.7 and 3.5, and the presence of heme groups in the active enzymes. Partial amino acid sequences were derived from N-terminal Edman degradation and from mass spectrometric ab initio sequencing of internal peptides. cDNAs of 1,604 to 1,923 bp, containing open reading frames (ORF) of 508 to 513 amino acids, respectively, were cloned from a cDNA library of M. scorodonius. These data suggest glycosylation degrees of approximately 23% for MsP1 and 8% for MsP2. Databank homology searches revealed sequence homologies of MsP1 and MsP2 to unusual peroxidases of the fungi Thanatephorus cucumeris (DyP) and Termitomyces albuminosus (TAP).