Novel biodegradable polyesters derived from carbohydrates

This thesis concerns the synthesis of new biodegradable polyesters from carbohydrates. All the new products have been characterised using nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry; some have been studied using X-ray crystallography. In Chapter 1, which is the introduction, some of the leading biodegradable polymers are introduced, in particular attention is paid to polyesters which are synthesised from carbohydrates. In addition, the standard polymerisation methods for the polyester synthesis - ring opening polymerisation and step polymerisation - are described. Chapter 2 details the synthesis and polymerisation of acetic acid 5-acetoxy-6-oxo-tetrahydro-pyran-2-yl methyl ester. Three different initiating systems have been used for ring opening polymerisations; the kinetics and the polymer product of the polymerisation are discussed. L-Lactide was used in copolymerisations with acetic acid 5-acetoxy-6-oxo-tetrahydro-pyran-2-yl methyl ester; the copolymers show distinct thermal properties and accelerated degradation rates compared with poly(L-Lactide) (PLLA). Chapter 3 involves applying functionalised carbohydrates, with one free hydroxyl group, as the co-initiator for the ring opening polymerisation (ROP) of L-lactide. The polymerisations were well controlled with linear relationships between the percentage lactide conversion and the polymer’s molecular weight. The carbohydrates form the functionalised end group of the PLLA, which, in turn, improve the hydrophilicity of the resulting PLLA. These end-group functionalised polylactides have been used as the matrix for human-osteoblast-derived osteosarcoma cells (SaOS-2 cells) culturing, and these studies proved that the carbohydrate end groups were non toxic. Chapter 4 describes the synthesis of a series of other carbohydrate lactones derived from D-glucono-1,5-lactone, D-xylose and 2-deoxy-Dribose; the lactones include 2,3,4-tri-O-benzyl-D-xylonolactone, 6-methyl-2-oxotetrahydro- 2H-pyran-3-yl acetate, (S)-6-(benzyloxymethyl)-tetrahydropyran-2-one. The ROP of these lactones resulted in formation of low molecular weight oligomers, except for 2,3,4-tri-O-benzyl-D-xylonolactone, which can not be polymerised. Chapter 5 describes the overall conclusions resulting from the experiments described in the thesis. Chapter 6 is the experimental section and thus provides a detailed description of the synthesis of all the compounds prepared in the thesis and their analytical data. The Appendices include two papers that have already been published, describing some of the work in the thesis; important additional NMR spectra and MALDI-ToF spectra; and the complete X-ray crystallography data

Characterisation, cloning and production of industrially interesting enzymes : gluconolactone oxidase of Penicillium cyaneo-fulvum and gluconate 5-dehydrogenase of Gluconobacter suboxydans

The work covered in this thesis is focused on the development of technology for bioconversion of glucose into D-erythorbic acid (D-EA) and 5-ketogluconic acid (5-KGA). The task was to show on proof-of-concept level the functionality of the enzymatic conversion or one-step bioconversion of glucose to these acids. The feasibility of both studies to be further developed for production processes was also evaluated. The glucose - D-EA bioconversion study was based on the use of a cloned gene encoding a D-EA forming soluble flavoprotein, D-gluconolactone oxidase (GLO). GLO was purified from Penicillium cyaneo-fulvum and partially sequenced. The peptide sequences obtained were used to isolate a cDNA clone encoding the enzyme. The cloned gene (GenBank accession no. AY576053) is homologous to the other known eukaryotic lactone oxidases and also to some putative prokaryotic lactone oxidases. Analysis of the deduced protein sequence of GLO indicated the presence of a typical secretion signal sequence at the N-terminus of the enzyme. No other targeting/anchoring signals were found, suggesting that GLO is the first known lactone oxidase that is secreted rather than targeted to the membranes of the endoplasmic reticulum or mitochondria. Experimental evidence supports this analysis, as near complete secretion of GLO was observed in two different yeast expression systems. Highest expression levels of GLO were obtained using Pichia pastoris as an expression host. Recombinant GLO was characterised and the suitability of purified GLO for the production of D-EA was studied. Immobilised GLO was found to be rapidly inactivated during D-EA production. The feasibility of in vivo glucose - D-EA conversion using a P. pastoris strain co-expressing the genes of GLO and glucose oxidase (GOD, E.C. 1.1.3.4) of A. niger was demonstrated. The glucose - 5-KGA bioconversion study followed a similar strategy to that used in the D-EA production research. The rationale was based on the use of a cloned gene encoding a membrane-bound pyrroloquinoline quinone (PQQ)-dependent gluconate 5-dehydrogenase (GA 5-DH). GA 5-DH was purified to homogeneity from the only source of this enzyme known in literature, Gluconobacter suboxydans, and partially sequenced. Using the amino acid sequence information, the GA 5-DH gene was cloned from a genomic library of G. suboxydans. The cloned gene was sequenced (GenBank accession no. AJ577472) and found to be an operon of two adjacent genes encoding two subunits of GA 5-DH. It turned out that GA 5-DH is a rather close homologue of a sorbitol dehydrogenase from another G. suboxydans strain. It was also found that GA 5-DH has significant polyol dehydrogenase activity. The G. suboxydans GA 5-DH gene was poorly expressed in E. coli. Under optimised conditions maximum expression levels of GA 5-DH did not exceed the levels found in wild-type G. suboxydans. Attempts to increase expression levels resulted in repression of growth and extensive cell lysis. However, the expression levels were sufficient to demonstrate the possibility of bioconversion of glucose and gluconate into 5-KGA using recombinant strains of E. coli. An uncharacterised homologue of GA 5-DH was identified in Xanthomonas campestris using in silico screening. This enzyme encoded by chromosomal locus NP_636946 was found by a sequencing project of X. campestris and named as a hypothetical glucose dehydrogenase. The gene encoding this uncharacterised enzyme was cloned, expressed in E. coli and found to encode a gluconate/polyol dehydrogenase without glucose dehydrogenase activity. Moreover, the X. campestris GA 5-DH gene was expressed in E. coli at nearly 30 times higher levels than the G. suboxydans GA 5-DH gene. Good expressability of the X. campestris GA-5DH gene makes it a valuable tool not only for 5-KGA production in the tartaric acid (TA) bioprocess, but possibly also for other bioprocesses (e.g. oxidation of sorbitol into L-sorbose). In addition to glucose - 5-KGA bioconversion, a preliminary study of the feasibility of enzymatic conversion of 5-KGA into TA was carried out. Here, the efficacy of the first step of a prospective two-step conversion route including a transketolase and a dehydrogenase was confirmed. It was found that transketolase convert 5-KGA into TA semialdehyde. A candidate for the second step was suggested to be succinic dehydrogenase, but this was not tested. The analysis of the two subprojects indicated that bioconversion of glucose to TA using X. campestris GA 5-DH should be prioritised first and the process development efforts in future should be focused on development of more efficient GA 5-DH production strains by screening a more suitable production host and by protein engineering.Erytorbiinihappo on C-vitaminiinin stereoisomeeri, jota käytetään elintarvikkeissa antioksidanttina. Erytorbiinihappoa tuotetaan teollisuudessa moniportaisella prosessilla, joka on samankaltainen kuin C-vitamiinin valmistuksessa käytettävä Reichstein-prosessi. Luonnossa ainoastaan Penicillium-homeiden on havaittu tuottavan erytorbiinihappoa. Synteesireitti glukoosista erytorbiinihapoksi on lyhyt, sisältäen vain kaksi entsyymiä, glukoosioksidaasin, joka hapettaa glukoosin glukono-1,5-laktoniksi (joka vesiliuoksessa spontaanisti tasapainottuu glukono-1,4-laktonin ja glukonihapon kanssa) ja glukonolaktonioksidaasin, joka hapettaa glukono-1,5- ja glukono-1,4-laktonin edelleen erytorbiinihapoksi. 1960- ja 1970-luvuilla kehiteltiin menetelmiä erytorbiinihapon tuottamiseksi Penicillium-homeilla, mutta kannattavaa prosessia ei ole onnistuttu rakentamaan. Tartarihappo on toinen elintarviketeollisuuden kannalta melko keskeinen orgaaninen happo, jota käytetään paitsi happamuuden säätelyaineena myös raaka-aineena emulgointiaineiden valmistuksessa. Tartarihappoa tuotetaan nykyisin pääasiassa viiniteollisuuden sivutuotteena. Koska viinintuotannon vuotuinen sadonvaihtelu heijastuu voimakkaasti myös tartarihapon markkinahintaan, uusia menetelmiä hapon tuottamiseksi on etsitty vakaamman hintatason takaamiseksi. Tartarihappoa voidaankin valmistaa 5-ketoglukonihaposta hapettamalla esimerkiksi jalometallien tai vanadaatin avulla. 5-ketoglukonihappoa voidaan valmistaa glukoosista fermentoimalla käyttäen 5-ketoglukonihappoa tuottavia Glukonobacter- tai Acetobacter-kantoja. Tässä väitöstutkimuksessa on selvitetty mahdollisuuksia tuottaa erytorbiinihappoa, 5-ketoglukonihappoa ja tartarihappoa geneettisesti muokatuilla tuotantomikrobeilla. Työssä on tuotettu ja puhdistettu erytorbiinihapon ja 5-ketoglukonihapon synteesejä katalysoivat entsyymit glukonolaktonioksidaasi ja glukonaatti-5-dehydrogenaasi. Osittaisen aminohapposekvenssien avulla entsyymejä koodaavat geenit on kloonattu cDNA- ja genomikirjastoista. Kumpaakin entsyymiä on ylituotettu heterologisessa isäntämikrobissa (Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris). Rekombinanttisesti tuotettuja entsyymejä on karakterisoitu ja niiden sekä keinotekoisen metaboliareitin omaavien tuotantomikrobien soveltuvuutta kyseisten orgaanisten happojen teolliseen tuotantoprosessiin on arvioitu. Sekä erytorbiinihappoa että 5-ketoglukonihappoa tuotettiin sekä rekombinanttisten entsyymien että geneettisesti modifioitujen tuotantoisäntien avulla. Glukonolaktonioksidaasia ylituotettiin 72-kertaisesti P. pastoriksessa verrattuna villityypin Penicillium cyaneo-fulvum-kantaan. Rekombinanttisesti tuotettu glukonolaktonioksidaasi immobilisoitiin ja immobilisoidulla entsyymillä onnistuttiin tuottamaan erytorbiinihappoa glukonihaposta. P. pastoris-kanta, jossa ylituotettiin sekä Aspergillus nigerin glukoosioksidaasia että glukonolaktonioksidaasia, konvertoi fermantaatiossa glukoosia erytorbiinihapoksi 6% saannolla. Tutkimusessa glukonolaktonioksidaasin havaittiin olevan erittyvä entsyymi, toisin kuin aiemmin on luultu ja se onkin toistaiseksi ainoa tunnettu erittyvä sokerilaktonioksidaasi. Geenisekvenssin perusteella havaitiin, että P. cyaneo-fulvumin glukonolaktonioksidaasi on ainoastaan kaukainen sukulainen muille tunnetuille sokerilaktonioksidaaseille muodostaen oman erittyvien laktonioksidaasien alaryhmän. Glukonaatti-5-dehydrogenaasin ylituotto E. colissa osoittautui vaikeaksi ja rekombinanttista entsyymiä onnistuttiin tuottamaan ainoastaan lähes samalla tasolla kuin villityypin Gluconobacter suboxydansissa. Koska E. colissa ei kuitenkaan ole sivuaktiivisuutena glukonaatti-2-dehydrogenaasia, ovat 5-ketoglukonaatin maksimisaannot rekombinanttikannalla teoriassa parempia kuin G. oxydanssissa, jolla fermentoitaessa osa glukonihaposta päätyi 2-ketoglukonihapoksi. Geneettisesti modifioidulla E. colilla onnistuttiin konvertoimaan 75% glukonihaposta 5-ketoglukonihapoksi. Glukonaatti-5-dehydrogenaasin geenisekvekssin avulla löytyi kuitenkin mielenkiintoinen homologinen karakterisoimaton entsyymi Xanthomonas campestriksesta. Tämän entsyymin geenin kloonaus ja ylituotto onnistui selvästi paremmin ollen 25-kertainen villityyppiin verrattuna. Karakterisoitaessa entsyymi osoittautuikin glukonaatti-5-dehydrogenaasiksi eikä todennäköiseksi glukoosidehydrogenaasiksi, kuten se oli sekvensointiprojektin yhteydessä GenBank:iin nimetty. Kumpikin entsyymi osoittautui kuitenkin omaavan huomattavasti laajemman substraattispesifisyyden, pystyen käyttämään substraattinaan glukonihapon lisäksi monia erilaisia polyoleja, kuten sorbitoli ja arabitoli. Suurimmat aktiivisuudet mitattiin käyttäen substraattina 2,3-butandiolia. Entsyymien substraattispesifisyys vastaa hyvin empiirisiin havaintoihin perustuvaa Bertrand-Hudsonin sääntöä sekundääristen polyolien hapettamisesta etikkahappobakteereilla. Luultavasti glukonaatti-5-dehydrogenaasi vastaa pääosin tästä säännöstä. Laajasta substraattispesifisyydestä johtuen glukonaatti-5-dehydrogenaasin teolliset sovellutukset ovat paljon aiemmin oletettua laajemmat ja sille voi löytyä lisää sovelluksia mm. C-vitamiinin ja joidenkin hienokemikaalien teollisessa tuotannossa

Triblock copolyesters derived from lactic acid and glucose: synthesis, nanoparticle formation and simulation

ABA triblock copolyesters were synthesized by ring-opening polymerization (ROP) of l-lactide in solution initiated by a telechelic d-glucose-based polyester macroinitiator. The macroinitiator with a number-average molecular weight about 2500 g mol-1 was synthesized by non-stoichiometric polycondensation in the melt of 2,4:3,5-di-O-methylene-d-glucitol and dimethyl succinate. Two triblock copolyesters of Mn ranging between ~6000 and ~9000 g mol-1, and differing in the length of the polylactide blocks were prepared. These copolyesters started to decompose when heated at ~220 °C and degraded slowly upon aqueous incubation under physiological conditions. They did not display any perceivable crystallinity and showed a single glass transition temperature (Tg) around 60 °C with the higher value corresponding to the larger content in glucitol units. The copolyesters were able to form nanoparticles with average diameters of ~100–130 nm and satisfactory dispersity. The effect of the block lengths on size, ¿-potential values and physical stability of the nanoparticles was evaluated. A molecular dynamics simulation study allowed modelling the two-phase structure of the nanoparticles and evidenced the preference of the glucose-based block to be peripherally located.Peer ReviewedPostprint (author's final draft

Electrochemical Reactivity at Free and Supported Gold Nanocatalysts Surface

This chapter presents an overview on size, structure, morphology, composition as well as the effect of the support on the electrocatalytic properties of gold nanoparticles (AuNPs). It was found that the electrocatalytic properties of unsupported AuNPs strongly depend on their size and shape. Consequently, the electrocatalytic properties of AuNPs can be tuned. Furthermore, to design high-performance electrocatalysts with minimal precious metal content and cost, the direct immobilization of metal NPs onto carbon-based substrates during their synthesis constitutes another elegant alternative and has been thoroughly examined. These “easy-to-use” supports as scaffolds for AuNPs, namely carbon black, carbon paper, etc., offer beneficial contributions. Indeed, thanks to their high available surface area, good electronic conductivity and synergistic effect between the chemical species present on their surface and the loaded NPs, carbon-based supports enable maximizing the efficient utilization of the catalysts toward drastic enhancement in both activity and durability. We also examined different judicious combinations of (electro)analytical techniques for the unambiguous determination of the reaction product(s) over the Au-based nanocatalysts, using glucose as model molecule given its importance in electrocatalysis. The performances of carbon-supported AuNPs as anode materials in direct glucose fuel cell in alkaline medium were also discussed

Direct growth of ternary copper nickel cobalt oxide nanowires as binder-free electrode on carbon cloth for nonenzymatic glucose sensing

A new binder free electrode based on ternary copper nickel cobalt oxide nanowires grown on the carbon cloth (CuNiCoO4 NWs@ carbon cloth) was prepared and characterized by field emission scanning electron microscopy (FE-SEM), x-ray diffraction (XRD), Energy-dispersive X-ray spectroscopy (EDX), and cyclic voltammetry (CV). The prepared electrocatalyst was directly used for electrochemical sensing of glucose without using enzyme. The effect of different parameters such as potential scan rate, switching potential, and glucose concentration on the electrochemical oxidation of glucose was investigated. The results showed that such an electrode presents excellent catalytic activity toward the oxidation of glucose in aqueous alkaline solution. Under optimum conditions, the potential application of the electrode was evaluated by applying it to the analytical determination of glucose concentration. The results revealed that the electrocatalytic current increased linearly with the glucose concentration in the range from 0.02 to 1.4 mM with a low detection limit of 6.5 μM and good sensitivity of as high as 1782 μA mM−1 cm−2 . Selectivity investigations demonstrate that the CuNiCoO4 NWs@CC electrode could be used for selective detection of glucose in the presence of interfering species. Real sample analysis shows reasonable RSD values implying negligible matrix effect in determination of glucose in human serum samples

Platinum-based nanodendrites as glucose oxidase-mimicking surrogates

Catalytic conversion of glucose represents an interesting field of research with multiple applications. From the biotechnology point of view, glucose conversion leads to the fabrication of different added-value by-products. In the field of nanocatalytic medicine, the reduction of glucose levels within the tumor microenvironment (TME) represents an appealing approach based on the starvation of cancer cells. Glucose typically achieves high conversion rates with the aid of glucose oxidase (GOx) enzymes or by fermentation. GOx is subjected to degradation, possesses poor recyclability and operates under very specific reaction conditions. Gold-based materials have been typically explored as inorganic catalytic alternatives to GOx in order to convert glucose into building block chemicals of interest. Still, the lack of sufficient selectivity towards certain products such as gluconolactone, the requirement of high fluxes of oxygen or the critical size dependency hinder their full potential, especially in liquid phase reactions. The present work describes the synthesis of platinum-based nanodendrites as novel enzyme-mimicking inorganic surrogates able to convert glucose into gluconolactone with outstanding selectivity values above 85%. We have also studied the enzymatic behavior of these Pt-based nanozymes using the Michaelis–Menten and Lineweaver–Burk models and used the main calculation approaches available in the literature to determine highly competitive glucose turnover rates for Pt or Pt–Au nanodendrites

Synthesis and characterization of lactones by Azotobacter chroococcum

The current paper deals with new metabolites of different groups produced by the Azotobacter chroococcum XU1 strain. Until now, a wide variety of secondary metabolites were documented for this species, but some compounds are being reported for the first time. These compounds—representatives of lactones. An important finding within this survey was the production of lactones, namely 1,5-D-gluconolactone and D, L-mevalonic acid lactone. It is interesting to note that the strain produced 1,5-D-gluconolactone as a response to the substrate modification (C-source): in the D-glucose supplemented Ashby, the major compound was 1,5-D-gluconolactone instead of EPS (which is produced in the sucrose supplemented Ashby)

The synthesis of azido and amino carbasugars

06.03.2018 tarihli ve 30352 sayılı Resmi Gazetede yayımlanan “Yükseköğretim Kanunu İle Bazı Kanun Ve Kanun Hükmünde Kararnamelerde Değişiklik Yapılması Hakkında Kanun” ile 18.06.2018 tarihli “Lisansüstü Tezlerin Elektronik Ortamda Toplanması, Düzenlenmesi ve Erişime Açılmasına İlişkin Yönerge” gereğince tam metin erişime açılmıştır.Öncelikle, azido ve amino karbaşekerlerin sentez yöntemine hazır olarak elde edilebilen anhidrit bileşiğinden 83 çıkılarak bir kaç adımda sentezlenen anahtar bileşiğin 90 oluşumu ile başlandı. 90 nolu bileşik etanol içinde azid iyonu ile reaksiyonu 91 ve 92 nolu bileşikleri vermiştir. Bu iki bileşiğin hidrolizi sonucunda 93 ve 94 nolu bileşikler oluşmuştur. Daha sonra 94 nolu bileşiğin hidrojenasyonuyla 95 nolu bileşik elde edilmiştir. İkinci aşamada 92 nolu bileşikteki furan halkası sülfamik asit içinde reaksiyonu ile açılıp asetillenmesiyle 96 nolu bileşik elde edilmiştir. Daha sonra bu bileşiğin hidrolizi 97 nolu bileşiği vermiştir. Son olarak 97 nolu bileşiğin hidrojenasyonu sonucu 98 nolu bileşik elde edilmiştir.Firstly the method of preparing azido and amino carbasugars with their derivatives began with the formation of the key compound 90 which synthesized after several steps from readily available anhydride compound 83. The compound 90 was reacted with azide ion in etharnol to give compounds 91 and 92, the compounds 91 and 92 were hydrolized to produce compounds 93 and 94.finally a compound 94 was hydrogenized to afford compound 95. Secondly furan ring of the compound 92 was cleaved by the acetylation reaction in sulfamic acid to produce compound 96 and then hydrolized to give compound 97. Lastly the compound 97 was hydrogenized to afford compound 98

Determination of gluconic acid in wine using high pressure liquid chromatography with pulsed amperometric detection

The paper establishes a new HPLC method for measurement of the total content of gluconic acid (GA) in wine using a pulsed amperometric detector equipped with a gold electrode cell and an anion exchange column. A NaOH solution was used at a flow rate of 1.5 ml·min-1 as a mobile phase under isocratic conditions. Analysis took 30 min. The linearity of the method was shown up to 500 mg·l-1, with a LOQ of 2.0 mg·l-1 (10σ). Precision (as RSD%) was under 2.5 % in the usual range of GA concentration in natural wines. Accuracy was checked in comparison with the usual enzymatic method.