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.

Mechanism-based inhibition of CYP2C8 by gemfibrozil in humans : characterisation of time and dose relationships

Drug-drug interactions may cause serious, even fatal clinical consequences. Therefore, it is important to examine the interaction potential of new chemical entities early in drug development. Mechanism-based inhibition is a pharmacokinetic interaction type, which causes irreversible loss of enzyme activity and can therefore lead to unusually profound and long-lasting consequences. The in vitro in vivo extrapolation (IVIVE) of drug-drug interactions caused by mechanism-based inhibition is challenging. Consequently, many of these interactions have remained unrecognised for many years. The concomitant use of the fibrate-class lipid-lowering agent gemfibrozil increases the concentrations of some drugs and their effects markedly. Even fatal cases of rhabdomyolysis occurred in patients administering gemfibrozil and cerivastatin concomitantly. One of the main mechanisms behind this effect is the mechanism-based inhibition of the cytochrome P450 (CYP) 2C8 enzyme by a glucuronide metabolite of gemfibrozil leading to increased cerivastatin concentrations. Although the clinical use of gemfibrozil has clearly decreased during recent years, gemfibrozil is still needed in some special cases. To enable safe use of gemfibrozil concomitantly with other drugs, information concerning the time and dose relationships of CYP2C8 inhibition by gemfibrozil should be known. This work was carried out as four in vivo clinical drug-drug interaction studies to examine the time and dose relationships of the mechanism-based inhibitory effect of gemfibrozil on CYP2C8. The oral antidiabetic drug repaglinide was used as a probe drug for measuring CYP2C8 activity in healthy volunteers. In this work, mechanism-based inhibition of the CYP2C8 enzyme by gemfibrozil was found to occur rapidly in humans. The inhibitory effect developed to its maximum already when repaglinide was given 1-3 h after gemfibrozil intake. In addition, the inhibition was shown to abate slowly. A full recovery of CYP2C8 activity, as measured by repaglinide metabolism, was achieved 96 h after cessation of gemfibrozil treatment. The dose-dependency of the mechanism-based inhibition of CYP2C8 by gemfibrozil was shown for the first time in this work. CYP2C8 activity was halved by a single 30 mg dose of gemfibrozil or by twice daily administration of less than 30 mg of gemfibrozil. Furthermore, CYP2C8 activity was decreased over 90% by a single dose of 900 mg gemfibrozil or twice daily dosing of approximately 100 mg gemfibrozil. In addition, with the application of physiological models to the data obtained in the dose-dependency studies, the major role of mechanism-based inhibition of CYP2C8 in the interaction between gemfibrozil and repaglinide was confirmed. The results of this work enhance the proper use of gemfibrozil and the safety of patients. The information related to time-dependency of CYP2C8 inhibition by gemfibrozil may also give new insights in order to improve the IVIVE of the drug-drug interactions of new chemical entities. The information obtained by this work may be utilised also in the design of clinical drug-drug interaction studies in the future.

Requirements for information management system in agricultural contracting

Menneinä vuosikymmeninä maatalouden työt ovat ensin koneellistuneet voimakkaasti ja sittemmin mukaan on tullut automaatio. Nykyään koneiden kokoa suurentamalla ei enää saada tuottavuutta nostettua merkittävästi, vaan työn tehostaminen täytyy tehdä olemassa olevien resurssien käyttöä tehostamalla. Tässä työssä tarkastelun kohteena on ajosilppuriketju nurmisäilörehun korjuussa. Säilörehun korjuun intensiivisyys ja koneyksiköiden runsas määrä ovat työnjohdon kannalta vaativa yhdistelmä. Työn tavoitteena oli selvittää vaatimuksia maatalouden urakoinnin tueksi kehitettävälle tiedonhallintajärjestelmälle. Tutkimusta varten haastateltiin yhteensä 12 urakoitsijaa tai yhteistyötä tekevää viljelijää. Tutkimuksen perusteella urakoitsijoilla on tarvetta tietojärjestelmille.Luonnollisesti urakoinnin laajuus ja järjestelyt vaikuttavat asiaan. Tutkimuksen perusteella keskeisimpiä vaatimuksia tiedonhallinnalle ovat: • mahdollisimman laaja, yksityiskohtainen ja automaattinen tiedon keruu tehtävästä työstä • karttapohjaisuus, kuljettajien opastus kohteisiin • asiakasrekisteri, työn tilaus sähköisesti • tarjouspyyntöpohjat, hintalaskurit • luotettavuus, tiedon säilyvyys • sovellettavuus monenlaisiin töihin • yhteensopivuus muiden järjestelmien kanssa Kehitettävän järjestelmän tulisi siis tutkimuksen perusteella sisältää seuraavia osia: helppokäyttöinen suunnittelu/asiakasrekisterityökalu, toimintoja koneiden seurantaan, opastukseen ja johtamiseen, työnaikainen tiedonkeruu sekä kerätyn tiedon käsittelytoimintoja. Kaikki käyttäjät eivät kuitenkaan tarvitse kaikkia toimintoja, joten urakoitsijan on voitava valita tarvitsemansa osat ja mahdollisesti lisätä toimintoja myöhemmin. Tiukoissa taloudellisissa ja ajallisissa raameissa toimivat urakoitsijat ovat vaativia asiakkaita, joiden käyttämän tekniikan tulee olla toimivaa ja luotettavaa. Toisaalta inhimillisiä virheitä sattuu kokeneillekin, joten hyvällä tietojärjestelmällä työstä tulee helpompaa ja tehokkaampaa.