Discovery of oxidative enzymes for food engineering : Tyrosinase and sulfhydryl oxidase

Enzymes offer many advantages in industrial processes, such as high specificity, mild treatment conditions and low energy requirements. Therefore, the industry has exploited them in many sectors including food processing. Enzymes can modify food properties by acting on small molecules or on polymers such as carbohydrates or proteins. Crosslinking enzymes such as tyrosinases and sulfhydryl oxidases catalyse the formation of novel covalent bonds between specific residues in proteins and/or peptides, thus forming or modifying the protein network of food. In this study, novel secreted fungal proteins with sequence features typical of tyrosinases and sulfhydryl oxidases were iden-tified through a genome mining study. Representatives of both of these enzyme families were selected for heterologous produc-tion in the filamentous fungus Trichoderma reesei and biochemical characterisation. Firstly, a novel family of putative tyrosinases carrying a shorter sequence than the previously characterised tyrosinases was discovered. These proteins lacked the whole linker and C-terminal domain that possibly play a role in cofactor incorporation, folding or protein activity. One of these proteins, AoCO4 from Aspergillus oryzae, was produced in T. reesei with a production level of about 1.5 g/l. The enzyme AoCO4 was correctly folded and bound the copper cofactors with a type-3 copper centre. However, the enzyme had only a low level of activity with the phenolic substrates tested. Highest activity was obtained with 4-tert-butylcatechol. Since tyrosine was not a substrate for AoCO4, the enzyme was classified as catechol oxidase. Secondly, the genome analysis for secreted proteins with sequence features typical of flavin-dependent sulfhydryl oxidases pinpointed two previously uncharacterised proteins AoSOX1 and AoSOX2 from A. oryzae. These two novel sulfhydryl oxidases were produced in T. reesei with production levels of 70 and 180 mg/l, respectively, in shake flask cultivations. AoSOX1 and AoSOX2 were FAD-dependent enzymes with a dimeric tertiary structure and they both showed activity on small sulfhydryl compounds such as glutathione and dithiothreitol, and were drastically inhibited by zinc sulphate. AoSOX2 showed good stabil-ity to thermal and chemical denaturation, being superior to AoSOX1 in this respect. Thirdly, the suitability of AoSOX1 as a possible baking improver was elucidated. The effect of AoSOX1, alone and in combi-nation with the widely used improver ascorbic acid was tested on yeasted wheat dough, both fresh and frozen, and on fresh water-flour dough. In all cases, AoSOX1 had no effect on the fermentation properties of fresh yeasted dough. AoSOX1 nega-tively affected the fermentation properties of frozen doughs and accelerated the damaging effects of the frozen storage, i.e. giving a softer dough with poorer gas retention abilities than the control. In combination with ascorbic acid, AoSOX1 gave harder doughs. In accordance, rheological studies in yeast-free dough showed that the presence of only AoSOX1 resulted in weaker and more extensible dough whereas a dough with opposite properties was obtained if ascorbic acid was also used. Doughs containing ascorbic acid and increasing amounts of AoSOX1 were harder in a dose-dependent manner. Sulfhydryl oxidase AoSOX1 had an enhancing effect on the dough hardening mechanism of ascorbic acid. This was ascribed mainly to the produc-tion of hydrogen peroxide in the SOX reaction which is able to convert the ascorbic acid to the actual improver dehydroascorbic acid. In addition, AoSOX1 could possibly oxidise the free glutathione in the dough and thus prevent the loss of dough strength caused by the spontaneous reduction of the disulfide bonds constituting the dough protein network. Sulfhydryl oxidase AoSOX1 is therefore able to enhance the action of ascorbic acid in wheat dough and could potentially be applied in wheat dough baking.Rihmamaisia sieniä tai niiden tuottamia entsyymejä voidaan käyttää hyväksi monien elintarvikkeiden, kuten leipomotuotteiden, juustojen ja jogurttien valmistuksessa. Monet sienet on havaittu turvallisiksi elintarvikkekäytössä, ja esimerkiksi Aspergillus oryzae sientä on käytetty Aasiassa soijakastikkeen ja alkoholijuomien kuten saken valmistuksessa yli 2000 vuotta. Tässä tutkimuksessa analysoitiin rihmasienten genomeja, ja löydettiin A. oryzaen genomista geenit, jotka koodittavat kolmea uutta, elintarvikeprosesseissa mahdollisesti sovellettavissa olevaa entsyymiä. Nämä entsyymit tuotettiin toisessa rihmasienessa, Trichoderma reeseissä, ja tuotetut entsyymit puhdistettiin ja niiden ominaisuuksia tutkittiin. Yhtä entsyymeistä testattiin leivonnassa, ja se pystyi parantamaan vehnätaikinan ominaisuuksia yhdessä C-vitamiinin kanssa. Tietyt entsyymit, kuten tyrosinaasit ja sulfhydryylioksidaasit pystyvät muokkaamaan ruuan rakennetta liittämällä yhteen ruoka-aineen sisältämiä erilaisia molekyylejä. Näitä entsyymejä kutsutaan ristisilloittaviksi entsyymeiksi. Tässä työssä löydettiin genomianalyysillä uusi pienempien tyrosinaasientsyymien ryhmä, jossa entsyymeiltä puuttuu muille tyrosinaaseille tyypillinen C-terminaalinen osa. Tämän osan merkityksestä entsyymin toiminnalle on vallalla erilaisia käsityksiä. Työssä tuotettiin T. reeseissä ryhmän yksi edustaja, AoCO4. Entsyymi oli aktiivinen ja sitoi aktiiviseen keskukseensa kaksi kupariatomia kuten tyrosinaasit yleensä. Entsyymillä oli kuitenkin varsin matala aktiivisuus testattuja substraatteja kohtaan. Se ei hapettanut tyrosiinia, ja siksi se luokiteltiin katekolioksidaasien ryhmään, jonka jäsenet ovat sekvensseiltään tyrosinaasien kanssa hyvin samankaltaisia. A. oryzaesta löydettiin genomianalyysin perusteella myös kaksi uutta sulhydryylioksidaasia, AoSOX1 ja AoSOX2. Nämä entsyymit olivat väriltään keltaisia, koska ne sitovat flaviinin kofaktorinaan. Niillä todettiin olevan aktiivisuutta pieniä sulfhydryyliyhdisteitä kuten glutationia ja aminohappo kysteiiniä kohtaan. AoSOX2 oli lämpötilan ja pH:n suhteen stabiilimpi kuin AoSOX1. AoSOX1-sulfhydryylioksidaasin kykyä parantaa vehnätaikinan ominaisuuksia tutkittiin pelkällä entsyymillä ja yhdessä C-vitamiinin kanssa. AoSOX1 ei vaikuttanut hiivaa sisältävien pakastamattomien taikinoiden kohoamiseen, mutta sillä oli selvä vaikutus pakastettuihin taikinoihin sekä yksin että yhdessä C-vitamiinin kanssa. Jotta ilmiötä voitaisiin ymmärtää paremmin, tehtiin kokeita yksinkertaisemmilla hiivattomilla taikinoilla. Niissä AoSOX1 yksin pehmensi taikinaa, kun taas yhdessä C-vitamiinin kanssa entsyymi lisäsi vitamiinin taikinaa kovettavaa vaikutusta. Näin osoitettiin, että AoSOX1:tä voidaan käyttää hyödyllisenä vehnätaikinoiden valmistuksen tehoaineena

Secreted fungal sulfhydryl oxidases: sequence analysis and characterisation of a representative flavin-dependent enzyme from Aspergillus oryzae

<p>Abstract</p> <p>Background</p> <p>Sulfhydryl oxidases are flavin-dependent enzymes that catalyse the formation of de novo disulfide bonds from free thiol groups, with the reduction of molecular oxygen to hydrogen peroxide. Sulfhydryl oxidases have been investigated in the food industry to remove the burnt flavour of ultraheat-treated milk and are currently studied as potential crosslinking enzymes, aiming at strengthening wheat dough and improving the overall bread quality.</p> <p>Results</p> <p>In the present study, potential sulfhydryl oxidases were identified in the publicly available fungal genome sequences and their sequence characteristics were studied. A representative sulfhydryl oxidase from <it>Aspergillus oryzae</it>, AoSOX1, was expressed in the fungus <it>Trichoderma reesei</it>. AoSOX1 was produced in relatively good yields and was purified and biochemically characterised. The enzyme catalysed the oxidation of thiol-containing compounds like glutathione, D/L-cysteine, beta-mercaptoethanol and DTT. The enzyme had a melting temperature of 57°C, a pH optimum of 7.5 and its enzymatic activity was completely inhibited in the presence of 1 mM ZnSO4.</p> <p>Conclusions</p> <p>Eighteen potentially secreted sulfhydryl oxidases were detected in the publicly available fungal genomes analysed and a novel proline-tryptophan dipeptide in the characteristic motif CXXC, where X is any amino acid, was found. A representative protein, AoSOX1 from <it>A. oryzae</it>, was produced in <it>T. reesei </it>in an active form and had the characteristics of sulfhydryl oxidases. Further testing of the activity on thiol groups within larger peptides and on protein level will be needed to assess the application potential of this enzyme.</p

Enzyme-catalyzed protein crosslinking

The process of protein crosslinking comprises the chemical, enzymatic, or chemoenzymatic formation of new covalent bonds between polypeptides. This allows (1) the site-directed coupling of proteins with distinct properties and (2) the de novo assembly of polymeric protein networks. Transferases, hydrolases, and oxidoreductases can be employed as catalysts for the synthesis of crosslinked proteins, thereby complementing chemical crosslinking strategies. Here, we review enzymatic approaches that are used for protein crosslinking at the industrial level or have shown promising potential in investigations on the lab-scale. We illustrate the underlying mechanisms of crosslink formation and point out the roles of the enzymes in their natural environments. Additionally, we discuss advantages and drawbacks of the enzyme-based crosslinking strategies and their potential for different application

Characterization of sulfhydryl oxidase from Aspergillus tubingensis

Background: Despite of the presence of sulfhydryl oxidases (SOXs) in the secretomes of industrially relevant organisms and their many potential applications, only few of these enzymes have been biochemically characterized. In addition, basic functions of most of the SOX enzymes reported so far are not fully understood. In particular, the physiological role of secreted fungal SOXs is unclear. Results: The recently identified SOX from Aspergillus tubingensis (AtSOX) was produced, purified and characterized in the present work. AtSOX had a pH optimum of 6.5, and showed a good pH stability retaining more than 80% of the initial activity in a pH range 4-8.5 within 20 h. More than 70% of the initial activity was retained after incubation at 50 degrees C for 20 h. AtSOX contains a non-covalently bound flavin cofactor. The enzyme oxidised a sulfhydryl group of glutathione to form a disulfide bond, as verified by nuclear magnetic resonance spectroscopy. AtSOX preferred glutathione as a substrate over cysteine and dithiothreitol. The activity of the enzyme was totally inhibited by 10 mM zinc sulphate. Peptide-and protein-bound sulfhydryl groups in bikunin, gliotoxin, holomycin, insulin B chain, and ribonuclease A, were not oxidised by the enzyme. Based on the analysis of 33 fungal genomes, SOX enzyme encoding genes were found close to nonribosomal peptide synthetases (NRPS) but not with polyketide synthases (PKS). In the phylogenetic tree, constructed from 25 SOX and thioredoxin reductase sequences from IPR000103 InterPro family, AtSOX was evolutionary closely related to other Aspergillus SOXs. Oxidoreductases involved in the maturation of nonribosomal peptides of fungal and bacterial origin, namely GliT, HlmI and DepH, were also evolutionary closely related to AtSOX whereas fungal thioreductases were more distant. Conclusions: AtSOX (55 kDa) is a fungal secreted flavin-dependent enzyme with good stability to both pH and temperature. A Michaelis-Menten behaviour was observed with reduced glutathione as a substrate. Based on the location of SOX enzyme encoding genes close to NRPSs, SOXs could be involved in the secondary metabolism and act as an accessory enzyme in the production of nonribosomal peptides.Peer reviewe

Chromogenic culture media complements diagnostic cytology in the visual identification of pathogenic skin bacteria in dogs and cats

In dogs and cats, bacterial skin infections (pyoderma and otitis externa) are a common cause for visiting the veterinary clinic. The most frequent skin pathogens are Staphylococcus pseudintermedius, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, often requiring different therapeutic antibiotic protocols. Unfavorably, existing diagnostics based on cytology cannot reveal bacterial species but only bacterial shapes such as cocci or rods. This microscopic limitation could be overcome by clinical translation of affordable chromogenic media, which enable species identification based on bacterial colonies growing in different colors and sizes. In this study, we determined how well inexperienced general veterinary clinicians identified bacterial pathogens from the skin and ears on two commercial (Chromatic™ MH and Flexicult® Vet) and one custom-made Mueller Hinton agar-based chromogenic medium. For this purpose, four veterinarians evaluated 100 unique samples representing 10 bacterial species. On average, clinicians correctly identified between 72.1 and 86.3% of bacterial species. Colony colors developed quickly on the Chromatic™ MH medium, leading to the highest 81.6% identification accuracy after 24 h incubation. However, Flexicult® Vet exhibited the highest accuracy of 86.3% after prolonged 48 h incubation. Evaluators easily recognized bacteria displaying uniquely colored colonies like green-brown Pseudomonas aeruginosa, blue Enterococcus faecalis, orange-brown Proteus spp., and red Escherichia coli. Oppositely, staphylococci shared uncharacteristically pale pink colonies causing misidentifications among the genus, deteriorating overall accuracy by around 10 percentage points (from 90.9%). Another reason for identification errors was the evaluators’ inexperience, reflected in not recognizing colony size differences. For example, although Streptococcus canis exhibited the tiniest colonies, the species was frequently mistaken for other cocci. Finally, around 10% of errors were negligence-related slips due to unconsidered sample history. To conclude, the introduction of chromogenic media into veterinary clinics can significantly complement diagnostics in skin inflammations by identifying pathogen species in around 80% of cases. The extra information may help in therapeutic dilemmas on antibiotics and standard antimicrobial susceptibility testing. Additional personnel training and evaluation help by visuals, flowcharts, checklists, and, if necessary, microbiologists could further improve identification accuracy

Colorectal Cancer Stage at Diagnosis Before vs During the COVID-19 Pandemic in Italy

IMPORTANCE Delays in screening programs and the reluctance of patients to seek medical attention because of the outbreak of SARS-CoV-2 could be associated with the risk of more advanced colorectal cancers at diagnosis. OBJECTIVE To evaluate whether the SARS-CoV-2 pandemic was associated with more advanced oncologic stage and change in clinical presentation for patients with colorectal cancer. DESIGN, SETTING, AND PARTICIPANTS This retrospective, multicenter cohort study included all 17 938 adult patients who underwent surgery for colorectal cancer from March 1, 2020, to December 31, 2021 (pandemic period), and from January 1, 2018, to February 29, 2020 (prepandemic period), in 81 participating centers in Italy, including tertiary centers and community hospitals. Follow-up was 30 days from surgery. EXPOSURES Any type of surgical procedure for colorectal cancer, including explorative surgery, palliative procedures, and atypical or segmental resections. MAIN OUTCOMES AND MEASURES The primary outcome was advanced stage of colorectal cancer at diagnosis. Secondary outcomes were distant metastasis, T4 stage, aggressive biology (defined as cancer with at least 1 of the following characteristics: signet ring cells, mucinous tumor, budding, lymphovascular invasion, perineural invasion, and lymphangitis), stenotic lesion, emergency surgery, and palliative surgery. The independent association between the pandemic period and the outcomes was assessed using multivariate random-effects logistic regression, with hospital as the cluster variable. RESULTS A total of 17 938 patients (10 007 men [55.8%]; mean [SD] age, 70.6 [12.2] years) underwent surgery for colorectal cancer: 7796 (43.5%) during the pandemic period and 10 142 (56.5%) during the prepandemic period. Logistic regression indicated that the pandemic period was significantly associated with an increased rate of advanced-stage colorectal cancer (odds ratio [OR], 1.07; 95%CI, 1.01-1.13; P = .03), aggressive biology (OR, 1.32; 95%CI, 1.15-1.53; P &lt; .001), and stenotic lesions (OR, 1.15; 95%CI, 1.01-1.31; P = .03). CONCLUSIONS AND RELEVANCE This cohort study suggests a significant association between the SARS-CoV-2 pandemic and the risk of a more advanced oncologic stage at diagnosis among patients undergoing surgery for colorectal cancer and might indicate a potential reduction of survival for these patients