Nielsen, Connie

a-Amylaser tilhører en familie af enzymer, der katalyserer hydrolysen af a-D-(1,4) glykosidbindinger i stivelse og lignende forbindelser. a-Amylaser er et af de vigtigste industrielle enzymer i dag og det benyttes i mange forskellige industrier, som for eksempel detergent industrien. Industrielle amylaser produceres ved fermentering af genetisk modificerede bakterier, hvor det oprindelige amylase-gen er blevet manipuleret til at kode for et enzym med forbedret ydeevne. På trods af at den benyttede ekspressions-celle kun indeholder kopier af dette ene amylase-gen identificeres mange forskellige a-amylaser med forskellige pI værdier i det endelige produkt. Dette indikerer at enzymet bliver modificeret.  Formålet med dette Ph.D. projekt er at identificere forskellene mellem de mange amylaseformer og undersøge om disse observerede modifikationer har en indflydelse på enzymets aktivitet og vaskeegenskaber. Fokus i dette projekt er på Bacillus a-amylaser, der benyttes i detergent industrien.  Ph.D.-afhandlingen begynder med en introduktion af Bacillus a-amylaser, de modifikationer der kan forårsage de observerede pI ændringer og de massespektrometriske metoder, der benyttes i projektet til identifikation af disse modifikationer. Derefter følger et metode afsnit og til sidst præsenteres og diskuteres de opnåede resultater. Prøver fra fermenteringen viste, at de mange amylaseformer dannes allerede tidligt i løbet af denne proces. Karakterisering af de mange amylaseformer fra tre forskellige Bacillus amylaser viste at vaske egenskaberne blev forringet for formerne med de lavere pI værdier. Fraktioner indeholdende Bacillus licheniformis a-amylase (BLA) former med forskellige pI niveauer blev karakteriseret grundigere. Dette viste at fraktionerne indeholdende BLA formerne med de laveste pI værdier også havde lavere katalytisk effektivitet. Modifikationer, der forårsager de forskellige BLA former, blev identificeret ved brug af tandemmassespektrometri (MS/MS). Glykeringer er en af de modifikationer der forventes at være med til at forårsage de forskellige former. Disse modifikationer kan være svære at identificere ved brug af de gængse fragmenteringsmetoder i MS/MS såsom higher-energy C-trap dissociation (HCD), da de kan kløve modifikationen af under fragmenteringen. Derfor blev HCD sammenlignet med en mindre hård fragmenteringsmetode kaldet electron transfer dissociation (ETD), som vil være bedre til at identificere glykeringer, da den ikke skulle kløve labile modifikationer af. Begge fragmenteringsmetoder viste sig brugbare til identifikation af glykeringer, men ETD identificerede flere glykeringssites og gav generelt en bedre sekvensdækning for de glykerede peptider.  MS/MS analyser af de forskellige BLA former ved brug af både HCD og ETD fragmentering identificerede modifikationerne deamideringer, glykeringer og carbamyleringer. Disse modifikationer vil alle forårsage de fald i pI-værdier, som blev observeret for amylaserne. Modifikationerne blev kun observeret på aminosyrer på BLA’s overflade på nær nogle enkelte deamideringer, der var i området lige omkring det aktive site. Nogle af de modificerede aminosyrer er involverede i enzymets stabilitet, aktivitet og substratbinding. Nogle af modifikationerne har derfor sandsynligvis en indflydelse på disse faktorer.  Indflydelsen af disse modifikationer på BLA blev undersøgt for nogle specifikke sites. Dette blev gjort ved karakterisering af BLA varianter med site specifikke mutationer. Kinetiske analyser af BLA varianter med mutationer, der ligner deamideringer i det aktive site og i substrat bindingskløften, viste at disse enkelt mutationer havde betydning for de kinetiske parametre kcat og KM, bestemt på amylose og maltoheptaose. Disse BLA varianter viste også generelt forbedrede vaskeegenskaber. Modifikationer på Lys- og Arg-sidekæderne, der kunne være involveret i binding af længere substrater eller en del af sekundære bindings sites, blev også analyseret ved site specifikke mutationer til aminosyrer med neutrale sidekæder af nogenlunde samme størrelse. Derved kunne effekten af at fjerne mulige glykerings sites vurderes. Disse mutationer havde ingen indflydelse på de kinetiske parametre bestemt på substraterne amylose og maltoheptaose, men de havde derimod en generel positiv effekt på BLA’s vaskeegenskaber.a-Amylases are a family of enzymes which catalyse the hydrolysis of a-D-(1,4) glucosidic linkages in starch and related compounds. a-Amylases are some of the most important industrial enzymes and they are used in a number of different industries one of these being the detergent industry. The commercial a-amylases are produced by fermentations of genetically modified bacteria, where the native gene has been manipulated to code for an enzyme with improved performance characteristics. Despite the genome only contains copies of the same a-amylase gene, multiple different a-amylase forms varying in pI value can be identified when analysing the final enzyme product, hereby indicating that the protein is modified.  The purpose of the present Ph.D. project is to identify differences between the multiple forms of a-amylase derived from the same gene and determine if or how these various modifications affect the activity and wash performance. The focus of the present project is on a-amylases fromBacillus species used in the detergent industry. The thesis begins with an introduction to Bacillus a-amylases, modifications which could cause the multiple forms varying in pI value and mass spectrometry (MS) methods used for the characterisation of these modifications. This is followed by a chapter concerning the methods used, followed by a chapter discussing the results obtained during the project. Studies of the multiple forms of a-amylases revealed that the multiple forms occur during the fermentation, where all the forms were observed already early on in the process. Characterisation of the wash performance of the multiple forms from three different Bacillus amylases showed tendencies of decreasing wash performance with decreasing pI values. A more thorough characterisation of pools of Bacillus licheniformis a-amylase (BLA) varying in pI revealed that the pools containing the forms with the lowest pI values had slightly lower catalytic efficiency on maltoheptaose and amylose.  Identification of the modifications responsible for the multiple forms of BLA was performed by tandem mass spectrometry (MS/MS). One of the modifications suspected to be responsible for the multiple forms, i.e. glycations, can be difficult to identify by the fragmentation methods normally used in MS/MS, such as higher-energy C-trap dissociation (HCD) as they tend to cleave of the modification. Therefore, HCD was compared with the soft fragmentations method electron transfer dissociation (ETD), which should be better at identifying these modifications because it leaves the modifications intact. Both methods were able to identify glycation sites on a model protein, though ETD identified more sites and provided higher sequence coverage for the glycated peptides.  MS/MS analysis by HCD and ETD of the multiple forms of BLA identified deamidations, glycations and carbamylations as the modifications responsible for the forms varying in pI. The modified sites were mainly located on the surface of BLA with a few deamidations in the area around the active site. The modifications were in some cases observed on amino acids involved in stability, activity and substrate binding. Therefore, some of the multiple forms of BLA will most likely differ in stability, activity and performance due to these modifications.  Finally the influence of these modified sites was evaluated by characterisation of BLA variants with single site specific mutations. The BLA variants with mutations mimicking deamidations in the active site and in the substrate binding cleft were found to influence the kinetic parameters (kcat and KM) in different ways. The wash performance was also influenced by these mutations though these effects were mainly positive. Modified Lys and Arg residues suspected to be involved in substrate binding of longer substrates or secondary binding sites were evaluated by site specific mutations to amino acids with neutral side chains of approximately the same size . Hereby, evaluating the effect of removing possible carbohydrate interaction sites. This did not have any effect on the kinetic parameters but it generally resulted in increased wash performances.</p

Online Research Database In Technology

Amalyse modifications during production and application

http://orbit.dtu.dk/ws/files/77537590/Connie_Nielsen_2c_aflevering_af_ph.d._afhandling..PDF

Amalyse modifications during production and application

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