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.

Juverhälsoproblem orsakade av Streptococcus agalactiae på stora mjölkgårdar med mjölkningsrobot

Streptococcus agalactiae –juverinflammation var tidigare ett stort problem i många länder, inklusive Finland. I och med förbättrad mjölkningshygien och antibiotikabehandlingar har bakterien så gott som eradikerats från mjölkbesättningarna. Nu verkar bakterien i viss mån ha kommit tillbaka till våra stora mjölkgårdar. Avhandlingens experimentella del utfördes på en mjölkgård, med ca 180 mjölkande och tre mjölkningsrobotar, som haft problem med Str. agalactiae. Man hoppades utreda hur stort problemet på gården var samt möjliga smittovägar. Man undersökte också möjligheten att använda mjölkningsrobotens automatiska provtagningsutrustning för provtagning av bakteriella prov. PCRmetoden jämfördes med konventionell odling vid diagnostik av juverinflammationer orsakade av Str. agalactiae. På gården gick man igenom anteckningar samt hälso- och seminkort för att få en bild över situationen. Man gjorde en uppföljning av mjölkningen för tolv kor vid den ena mjölkningsroboten. Man tog 47 stycken kospecifika mjölkprov samt ett prov från mjölktanken. Mjölkprov i tre serier både mjölkade för hand och direkt från mjölkuppsamlaren på mjölkningsroboten togs. Man tog sammanlagt 23 renlighetsprov från mjölkningsroboten, tre från den automatiska provtagningsutrustningen samt två från djurskötarnas händer. Från den automatiska provtagningsutrustningen togs även ett genomsköljningsprov. Av mjölkprov som tidigare tagits på gården hade man hittat Str. agalactiae i ca 17%. I denna studie hittades Str. agalactiae i tre kospecifika mjölkprov, vilket motsvarar en prevalens på ca 2%. Vid uppföljningen av mjölkningarna upptäcktes inget alarmerande, men spenarnas hälsa samt tommjölkningar är något som bör följas upp. Av renlighetsproven hittades Str. agalactiae i ett prov taget från borsthållaren. Svaren från mjölkproven tagna i serier tyder på att den automatiska provtagningsutrustningen inte går att använda till bakteriella prov, eftersom mjölken från en Str. agalactiae –infekterad ko verkar påverka resultatet också hos följande kor. Resultatet är väntat, eftersom mjölkprov alltid skall tas aseptiskt och det går inte med den automatiska provtagningsutrustningen så som den i dagsläget är utvecklad. Från sju av nio mjölkprov, där man hittat Str. agalactiae med PCR-metoden, hittades bakterien också med konventionell odling. Från tankmjölksprovet kunde man inte hitta Str. agalactiae med konventionell odling. PCR-metoden verkar enligt den här studien vara mer känslig att upptäcka Str. agalactiae jämfört med konventionell odling.