This diploma thesis deals with the characterization of rheological properties and biodegradation of a poly(3-hydroxy-co-4-hydroxybutyrate), a copolymer produced by a strain of Cupriavidus malaysiensis. The theoretical part focuses on the rheology and degradation of polymers in general. It also contains the basic characteristics of polyhydroxyalkanoates and deals in more details with the description of the studied copolymer, its properties, synthesis, decomposition and possibilities of its applications. In the experimental part, the given copolymer was investigated in its native form and also in the form of copolymer mixtures filaments, which were enriched with additives (stabilizers and plasticizers). In terms of rheology, the samples showed pseudoplastic behaviour, which was slightly limited by the plasticization of the material. The highest thermal stability observed as a function of changes in complex viscosity over time was observed in a sample of a copolymer mixture containing stabilizers and plasticizer Citrofol BII. The plasticization also caused significant changes in thermal properties, especially crystallinity, which decreased compared to the native copolymer. In vitro degradation studies of samples in the form of films prepared by dissolving copolymer mixtures in chloroform were performed in phosphate buffer with lipase, in simulated body fluid and in synthetic gastric juice. The course of the process itself was characterized by the method of determining the molecular weight (SEC-MALLS) and the weight loss over time (gravimetrically). The results showed that the analysed samples are subject to degradation in all used environments. The most considerable molecular weight loss after 105 days (76 %) was observed in the sample containing stabilizers and plasticizer Citrofol BII in the environment of synthetic gastric juice. The most extensive gravimetric weight loss was attributed to the sample with stabilizers and without plasticizers, in the environment of phosphate buffer with lipase, specifically by 79%.

This diploma thesis studied the ability of Cupriavidus malaysiensis, Delftia acidovorans and Azohydromonas lata to produce poly(3-hydroxybutyrate-co-4-hydroxybutyrate), [P(3HB-co-4HB)], by using -butyrolactone and 1,4-butanediol as carbon substrates. The objective of this work was the production and characterisation of isolated polyhydroxyalkanoates (PHA). The theoretical part deals with the basic description and classification of polyhydroxyalkanoates. Next, the biosyntheses of the most investigated PHAs were described. The practical section of the work discusses and presents the output of the cultivation of five bacterial strains selected for the production of [P(3HB-co-4HB)], namely, Cupriavidus malaysiensis (DSM 19379), Delftia acidovorans (DSM 39), Delftia acidovorans (CCM 2410), Delftia acidovorans (CCM 283) and Azohydromonas lata (CCM 4448). The effect of the modified cultivation conditions for each of the used bacteria on the PHA production yields was discussed. The produced biomass after the cultivation was characterised spectrophotometrically, gravimetrically and by gas chromatography. Polymers were isolated from the biomass by the extraction in chloroform. The isolated polymers were characterised from the viewpoint of chemical composition, molecular weight and thermal properties by using Attenuated total reflection infrared spectroscopy, Size exclusion chromatography, Differential scanning calorimetry and Thermogravimetric analysis.

Growing concerns of the negative effects on the environment and dependency of fossil fuelsare major driving forces for finding novel sustainable production pathways for plastic.Metabolic engineering has emerged as a powerful tool to enable microorganisms to producenon-native metabolites. The aim of this project was recombinant production of 4-hydroxybutyrate (4-HB) by expressing two enzymes in the model organism Escherichia coli.α-ketoglutarate decarboxylase (SucA) from Mycobacterium smegmatis followed by 4-hydroxybutyrate dehydrogenase (4-HBd) from Clostridium kluyveri was expressed inEscherichia coli. Results showed that the genes were successfully transformed and expressedin E. coli and after protein purification a concentration of 0.9 g/L SucA and 9.8 g/L 4-HBdwas achieved. Furthermore, some protein activity was detected by a coupled reaction withSucA and 4-HBd. When the enzymes got coupled together a change in NADH concentrationcould be detected spectrophotometrically. The enzymes were also tested for substratespecificity by using substrates with various carbon chain lengths and a decrease in NADHconcentration was seen. However, a decrease in the negative control for the experiments wasalso seen indicating a breakdown of NADH over time rather than consumption. Therefore, noconclusion could be drawn about the promiscuity of the enzymes. Lastly a single plasmidssystem was tested where both the genes were ligated on the same plasmid (pCDF duet) andexpressed successfully in E. coli Bl21DE3. / Ökad oro för miljön samt behovet av fossila resurser för produktion av plaster har gjort detnödvändigt att skapa nya och mer hållbara produktions vägar. Genetisk modifikation av olikaorganismer har utvecklats som ett starkt redskap för att få mikroorganismer att framställametaboliter som de normalt inte producerar. Målet med detta projekt var rekombinantproduktion av gamma hydroxibutansyra (4-HB) genom att uttrycka två enzym i modellorganismen Escherichia coli. Dessa enzym bestod av α-ketoglutarat dekarboxylas (SucA) frånMycobacterium smegmatis samt 4-hydroxybutyrate dehydrogenas (4-HBd) från Clostridiumkluyveri. Resultaten visade att proteinerna lyckades utryckas i E. coli med en koncentration av0,9 g/L SucA och 9,8 g/L 4-HBd som uppnåddes efter rening. Utöver detta detekterades ävenviss enzymaktivitet genom att kopplad enzymreaktion mellan 4-HBd och SucA och mätakonsumtionen av NADH spektrofotometriskt över tid. Enzymen testades även försubstratspecificitet genom att köra reaktionen med substrat med olika längd på kolkedjan. Dåkunde en minskning i NADH koncentrationen ses men det gjordes det även för de negativakontrollerna vilket indikerar nedbrytning av NADH och inte konsumtion av NADH. Ingaslutsatser angående enzymens substratspecificitet kunde därför dras. Det sista som gjordes varatt sätta in båda generna i ett en plasmidsystem där båda generna sattes in på samma plasmid(pCDF duet) och uttrycktes framgångsrikt i E. coli Bl21DE3.