Improved Foods Using Enzymes from Basidiomycetes

Within the kingdom of fungi, the division Basidiomycota represents more than 30,000 species, some with huge genomes indicating great metabolic potential. The fruiting bodies of many basidiomycetes are appreciated as food (“mushrooms”). Solid-state and submerged cultivation processes have been established for many species. Specifically, xylophilic fungi secrete numerous enzymes but also form smaller metabolites along unique pathways; both groups of compounds may be of interest to the food processing industry. To stimulate further research and not aim at comprehensiveness in the broad field, this review describes some recent progress in fermentation processes and the knowledge of fungal genetics. Processes with potential for food applications based on lipases, esterases, glycosidases, peptidases and oxidoreductases are presented. The formation and degradation of colourants, the degradation of harmful food components, the formation of food ingredients and particularly of volatile and non-volatile flavours serve as examples. In summary, edible basidiomycetes are foods—and catalysts—for food applications and rich donors of genes to construct heterologous cell factories for fermentation processes. Options arise to support the worldwide trend toward greener, more eco-friendly and sustainable processes

Cross-Linking of Wheat Bran Arabinoxylan by Fungal Laccases Yields Firm Gels

The native extractable arabinoxylans (AX) from wheat bran were cross-linked by the commercial laccase C (LccC) and self-produced laccases from Funalia trogii (LccFtr) and Pleurotus pulmonarius (LccPpu) (0.04 U/µg FA, each). Dynamic oscillation measurements of the 6% AX gels demonstrated a storage modulus of 9.4 kPa for LccC, 9.8 kPa for LccFtr, and 10.0 kPa for LccPpu. A loss factor ≤ 0.6 was recorded in the range from 20 to 80 Hz for all three laccases, and remained constant for four weeks of storage, when LccFtr and LccPpu were used. Arabinoxylan gel characteristics, including high water holding capacity, swelling ratio in saliva, and heat resistance indicated a covalently cross-linked network. Neither the mediator compounds caffeic acid and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), nor citrus pectin, enhanced the elastic properties of the gels. Using laccases as an oxidant provided gels with a solid and stable texture, comparable in firmness to traditional gelatin gels. Thus, AX gels can be presented in the vegan, halal, and kosher food markets. They may also find use in pharmaceutical and other industrial applications

Mutagenesis of the l-Amino Acid Ligase RizA Increased the Production of Bioactive Dipeptides

The l-amino acid ligase RizA from B. subtilis selectively synthesizes dipeptides containing an N-terminal arginine. Many arginyl dipeptides have salt-taste enhancing properties while Arg-Phe has been found to have an antihypertensive effect. A total of 21 RizA variants were created by site-directed mutagenesis of eight amino acids in the substrate binding pocket. The variants were recombinantly produced in E. coli and purified by affinity chromatography. Biocatalytic reactions were set up with arginine and four amino acids differing in size and polarity (aspartic acid, serine, alanine, and phenylalanine) and were analyzed by RP-HPLC with fluorescence detection. Variant T81F significantly improved the yield in comparison to wild type RizA for aspartic acid (7 to 17%), serine (33 to 47%) and alanine (12 to 17%). S84F increased product yield similarly for aspartic acid (7 to 17%) and serine (33 to 42%). D376E increased the yield with alanine (12 to 19%) and phenylalanine (11 to 26%). The largest change was observed for S156A, which showed a yield for Arg-Phe of 40% corresponding to a 270% increase in product concentration. This study expands the knowledge about positions governing the substrate specificity of RizA and may help to inform future protein engineering endeavors

Co-Immobilization of RizA Variants with Acetate Kinase for the Production of Bioactive Arginyl Dipeptides

The biocatalytic system comprised of RizA and acetate kinase (AckA) combines the specific synthesis of bioactive arginyl dipeptides with efficient ATP regeneration. Immobilization of this coupled enzyme system was performed and characterized in terms of activity, specificity and reusability of the immobilisates. Co-immobilization of RizA and AckA into a single immobilisate conferred no disadvantage in comparison to immobilization of only RizA, and a small addition of AckA (20:1) was sufficient for ATP regeneration. New variants of RizA were constructed by combining mutations to yield variants with increased biocatalytic activity and specificity. A selection of RizA variants were co-immobilized with AckA and used for the production of the salt-taste enhancers Arg-Ser and Arg-Ala and the antihypertensive Arg-Phe. The best variants yielded final dipeptide concentrations of 11.3 mM Arg-Ser (T81F_A158S) and 11.8 mM Arg-Phe (K83F_S156A), the latter of which represents a five-fold increase in comparison to the wild-type enzyme. T81F_A158S retained more than 50% activity for over 96 h and K83F_S156A for over 72 h. This study provides the first example of the successful co-immobilization of an l-amino acid ligase with an ATP-regenerating enzyme and paves the way towards a bioprocess for the production of bioactive dipeptides

Cross-Linking of Fibrex Gel by Fungal Laccase: Gel Rheological and Structural Characteristics

Sugar beet fibre (fibrex) is an abundant side-stream from the sugar refining industry. A self-produced laccase from Funalia trogii (LccFtr) (0.05 U/µg FA) successfully cross-linked fibrex to an edible gel. Dynamic oscillation measurements of the 10% fibrex gels showed a storage modulus of 5.52 kPa and loss factors ≤ 0.36 in the range from 20 to 80 Hz. Comparing storage stability of sweetened 10% fibrex gels with sweetened commercial 6% gelatin gels (10% and 30% d-sucrose) indicated a constant storage modulus and loss factors ≤ 0.7 during four weeks of storage in fibrex gels. Loss factors of sweetened gelatin gels were ≤0.2, and their storage modulus decreased from 9 to 7 kPa after adding d-sucrose and remained steady for four weeks of storage. Fibrex gel characteristics, including high water holding capacity, swelling ratio in saliva, and heat resistance are attributed to a covalently cross-linked network. Vanillin, as a mediator, and citrus pectin did not enhance covalent cross-links and elastic properties of the fibrex gels. Thus, laccase as an oxidative agent provided gels with a solid and stable texture. Fibrex gels may find uses in pharmaceutical and other industrial applications, which require a heat-resistant gel that forms easily at room temperature. They also represent an ethical alternative for manufacturing vegan, halal, and kosher food

A DyP-type peroxidase of pleurotus sapidus with alkene cleaving activity

Alkene cleavage is a possibility to generate aldehydes with olfactory properties for the fragrance and flavor industry. A dye-decolorizing peroxidase (DyP) of the basidiomycete Pleurotus sapidus (PsaPOX) cleaved the aryl alkene trans-anethole. The PsaPOX was semi-purified from the mycelium via FPLC, and the corresponding gene was identified. The amino acid sequence as well as the predicted tertiary structure showed typical characteristics of DyPs as well as a non-canonical Mn2+-oxidation site on its surface. The gene was expressed in Komagataella pfaffii GS115 yielding activities up to 142 U/L using 2,20-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) as substrate. PsaPOX exhibited optima at pH 3.5 and 40 ◦C and showed highest peroxidase activity in the presence of 100 µM H2O2 and 25 mM Mn2+. PsaPOX lacked the typical activity of DyPs towards anthraquinone dyes, but oxidized Mn2+ to Mn3+. In addition, bleaching of β-carotene and annatto was observed. Biotransformation experiments verified the alkene cleavage activity towards the aryl alkenes (E)-methyl isoeugenol, α-methylstyrene, and trans-anethole, which was increased almost twofold in the presence of Mn2+. The resultant aldehydes are olfactants used in the fragrance and flavor industry. PsaPOX is the first described DyP with alkene cleavage activity towards aryl alkenes and showed potential as biocatalyst for flavor production

Sesquiterpene Cyclases from the Basidiomycete Cerrena unicolor

Hundreds of terpenoids have been isolated from Basidiomycota, among them are volatile mono- and sesquiterpenes with amazing sensory qualities, representing a promising alternative to essential oils from endangered plant species. Sesquiterpene synthases (STS) appear to be an abundant class of enzymes in these fungi. The basidiomycete Cerrena unicolor, a known sesquiterpene producer, was in silico screened for sesquiterpene cyclases via homology Basic Local Alignment Search Tool searches. Cyclase genes identified were cloned and heterologously expressed in Escherichia coli Bl21 using pCOLD I as the expression vector. Ten cyclases were successfully produced and purified, and their identity was confirmed using amino acid sequencing of tryptic peptides by nano-liquid chromatography-high resolution-electrospray ionization-tandem mass spectrometry. Gas chromatography/mass spectrometry analysis was applied to characterize these cyclases according to the formation of sesquiterpene hydrocarbons and oxidized terpenoids. Bioinformatic characterization and phylogenetic determination allowed for the classification of these diverse fungal enzymes. A representative single and a multi-product STS, respectively, were further analyzed for their dependency from divalent metal cations as a cofactor for the catalytic activity

Impact of Agro-Industrial Side-Streams on Sesquiterpene Production by Submerged Cultured Cerrena unicolor

The quality and harvest of essential oils depend on a large number of factors, most of which are hard to control in an open-field environment. Therefore, Basidiomycota have gained attention as a source for biotechnologically produced terpenoids. The basidiomycete Cerrena unicolor (Cun) was cultivated in submerged culture, and the production of sesquiterpenoids was analyzed via stir bar sorptive extraction (SBSE), followed by thermo-desorption gas chromatography coupled with mass spectrometry (TDS-GC-MS). Identification of aroma-active sesquiterpenoids was supported by GC, coupled with an olfactory detection port (ODP). Following the ideal of a circular bioeconomy, Cun was submerged (up-scalable) cultivated, and supplemented with a variety of food industrial side-streams. The effects of the different supplementations and of pure fatty acids were evaluated by liquid extraction and analysis of the terpenoids via GC-MS. As sesquiterpenoid production was enhanced by the most by lipid-rich side-streams, a cultivation with 13C-labeled acetate was conducted. Data confirmed that lipid-rich side-streams enhanced the sesquiterpene production through an increased acetyl-CoA pool

Recombinant Production of Arginyl Dipeptides by l-Amino Acid Ligase RizA Coupled with ATP Regeneration

Arginyl dipeptides like Arg-Ser, Arg-Ala, and Arg-Gly are salt-taste enhancers and can potentially be used to reduce the salt content of food. The l-amino acid ligase RizA from B. subtilis selectively synthesizes arginyl dipeptides. However, industrial application is prevented by the high cost of the cofactor adenosine triphosphate (ATP). Thus, a coupled reaction system was created consisting of RizA and acetate kinase (AckA) from E. coli providing ATP regeneration from acetyl phosphate. Both enzymes were recombinantly produced in E. coli and purified by affinity chromatography. Biocatalytic reactions were varied and analyzed by RP-HPLC with fluorescence detection. Under optimal conditions the system produced up to 5.9 g/L Arg-Ser corresponding to an ATP efficiency of 23 g Arg-Ser per gram ATP. Using similar conditions with alanine or glycine as second amino acid, 2.6 g/L Arg-Ala or 2.4 g/L Arg Gly were produced. The RizA/AckA system selectively produced substantial amounts of arginyl dipeptides while minimizing the usage of the expensive ATP

Co-Oxidative Transformation of Piperine to Piperonal and 3,4-Methylenedioxycinnamaldehyde by a Lipoxygenase from Pleurotus sapidus

The valuable aroma compound piperonal with its vanilla-like olfactory properties is of high interest for the fragrance and flavor industry. A lipoxygenase (LOXPsa1) of the basidiomycete Pleurotus sapidus was identified to convert piperine, the abundant pungent principle of black pepper (Piper nigrum), to piperonal and a second volatile product, 3,4-methylenedioxycinnamaldehyde, with a vanilla-like odor through an alkene cleavage. The reaction principle was co-oxidation, as proven by its dependence on the presence of linoleic or α-linolenic acid, common substrates of lipoxygenases. Optimization of the reaction conditions (substrate concentrations, reaction temperature and time) led to a 24-fold and 15-fold increase of the piperonal and 3,4-methylenedioxycinnamaldehyde concentration using the recombinant enzyme. Monokaryotic strains showed different concentrations of and ratios between the two reaction products