Academic literature on the topic 'Settore CHIM/11 - Chimica e Biotecnologia delle Fermentazioni'

Il siero di latte e la scotta sono effluenti provenienti rispettivamente dal processo di trasformazione del latte in formaggio e ricotta. Il siero di latte contiene minerali, lipidi, lattosio e proteine; la scotta contiene principalmente lattosio. Il siero può essere riutilizzato in diversi modi, come l'estrazione di proteine o per l’alimentazione animale, mentre la scotta è considerata solamente un rifiuto. Inoltre, a causa degli ingenti volumi di siero prodotti nel mondo, vengono a crearsi seri problemi ambientali e di smaltimento. Destinazioni alternative di questi effluenti, come le trasformazioni biotecnologiche, possono essere un modo per raggiungere il duplice obiettivo di migliorare il valore aggiunto dei processi agroindustriali e di ridurre il loro impatto ambientale. In questo lavoro sono state studiate le condizioni migliori per produrre bioetanolo dal lattosio del siero e della scotta. Kluyveromyces marxianus è stato scelto come lievito lattosio-fermentante. Sono state effettuate fermentazioni su scala di laboratorio aerobiche e anaerobiche in batch, fermentazioni semicontinue in fase dispersa e con cellule immobilizzate in alginato di calcio,. Diverse temperature sono state testate per migliorare la produzione di etanolo. Le migliori prestazioni, per entrambe le matrici, sono state raggiunte a basse temperature (28°C). Anche le alte temperature sono compatibili con buone rese di etanolo nelle fermentazioni con siero. Ottimi risultati si sono ottenuti anche con la scotta a 37°C e a 28°C. Le fermentazioni semicontinue in fase dispersa danno le migliori produzioni di etanolo, in particolare con la scotta. Invece, l'uso di cellule di lievito intrappolate in alginato di calcio non ha migliorato i risultati di processo. In conclusione, entrambi gli effluenti possono essere considerati adatti per la produzione di etanolo. Le buone rese ottenute dalla scotta permettono di trasformare questo rifiuto in una risorsa.
Whey and scotta are effluents coming from cheese and ricotta processing respectively. Whey contains minerals, lipids, lactose and proteins; scotta contains mainly lactose. Whey can be reused by several ways, such as protein extraction or animal feeding, while nowadays scotta is just considered a waste; moreover, due to very high volumes of whey produced in the world, it poses serious environmental problems for disposal. Alternative destinations of these effluents, such as biotechnological transformations, can be a way to reach both goals of improving the added value of agroindustrial processes and reducing their environmental impact. In this work we investigated the way to produce bioethanol from lactose of whey and scotta and to optimize the fermentation yields. Kluyveromyces marxianus var. marxianus was chosen as lactose-fermenting yeast. Batch aerobic and anaerobic fermentations and semicontinuous fermentations in dispersed phase and in immobilized phase were carried out of whey, scotta at a laboratory scale. Different temperatures were also tested in order to try to improve the ethanol production. The best performances for both matrices were reached at low temperatures (28°C). High temperatures are also compatible with good ethanol yields in whey fermentations. Very good results are also obtained with scotta at 37°C and at 28°C. Semicontinuous fermentations in dispersed phase gave the best fermentation performances, in particular with scotta. Instead, the use of yeast cells entrapped in calcium alginate did not improve the process results. Then both effluents can be considered suitable for ethanol production. The good yields obtained from scotta allow to transform this waste in a source.

Il siero di latte e la scotta sono effluenti provenienti rispettivamente dal processo di trasformazione del latte in formaggio e ricotta. Il siero di latte contiene minerali, lipidi, lattosio e proteine; la scotta contiene principalmente lattosio. Il siero può essere riutilizzato in diversi modi, come l'estrazione di proteine o per l’alimentazione animale, mentre la scotta è considerata solamente un rifiuto. Inoltre, a causa degli ingenti volumi di siero prodotti nel mondo, vengono a crearsi seri problemi ambientali e di smaltimento. Destinazioni alternative di questi effluenti, come le trasformazioni biotecnologiche, possono essere un modo per raggiungere il duplice obiettivo di migliorare il valore aggiunto dei processi agroindustriali e di ridurre il loro impatto ambientale. In questo lavoro sono state studiate le condizioni migliori per produrre bioetanolo dal lattosio del siero e della scotta. Kluyveromyces marxianus è stato scelto come lievito lattosio-fermentante. Sono state effettuate fermentazioni su scala di laboratorio aerobiche e anaerobiche in batch, fermentazioni semicontinue in fase dispersa e con cellule immobilizzate in alginato di calcio,. Diverse temperature sono state testate per migliorare la produzione di etanolo. Le migliori prestazioni, per entrambe le matrici, sono state raggiunte a basse temperature (28°C). Anche le alte temperature sono compatibili con buone rese di etanolo nelle fermentazioni con siero. Ottimi risultati si sono ottenuti anche con la scotta a 37°C e a 28°C. Le fermentazioni semicontinue in fase dispersa danno le migliori produzioni di etanolo, in particolare con la scotta. Invece, l'uso di cellule di lievito intrappolate in alginato di calcio non ha migliorato i risultati di processo. In conclusione, entrambi gli effluenti possono essere considerati adatti per la produzione di etanolo. Le buone rese ottenute dalla scotta permettono di trasformare questo rifiuto in una risorsa.
Whey and scotta are effluents coming from cheese and ricotta processing respectively. Whey contains minerals, lipids, lactose and proteins; scotta contains mainly lactose. Whey can be reused by several ways, such as protein extraction or animal feeding, while nowadays scotta is just considered a waste; moreover, due to very high volumes of whey produced in the world, it poses serious environmental problems for disposal. Alternative destinations of these effluents, such as biotechnological transformations, can be a way to reach both goals of improving the added value of agroindustrial processes and reducing their environmental impact. In this work we investigated the way to produce bioethanol from lactose of whey and scotta and to optimize the fermentation yields. Kluyveromyces marxianus var. marxianus was chosen as lactose-fermenting yeast. Batch aerobic and anaerobic fermentations and semicontinuous fermentations in dispersed phase and in immobilized phase were carried out of whey, scotta at a laboratory scale. Different temperatures were also tested in order to try to improve the ethanol production. The best performances for both matrices were reached at low temperatures (28°C). High temperatures are also compatible with good ethanol yields in whey fermentations. Very good results are also obtained with scotta at 37°C and at 28°C. Semicontinuous fermentations in dispersed phase gave the best fermentation performances, in particular with scotta. Instead, the use of yeast cells entrapped in calcium alginate did not improve the process results. Then both effluents can be considered suitable for ethanol production. The good yields obtained from scotta allow to transform this waste in a source.

This PhD research project was aimed at the development of new biocatalytic processes to produce natural sugars by selection and characterisation of new enzymes able to produce fructooligosaccharides. Biochemical studies were performed to obtain information on the mechanism of action and understand the structural elements that define the activity. After the development of the biotransformation conditions, a continuous production of FOS was studied and a cheap separation method of the transformation products was also assessed, in order to obtain FOS in purified form. Mass spectrometry studies were performed on the purified enzymes after purification from wild-type strains. After a screening for FOS production from sucrose two microorganisms were chosen for their activity up to 30 % (w/w) conversion and differences in FOS mixture production: CF215 = Cladosporium cladosporioides CK1 = Penicilium sizovae CF215 produce a mixture similar to the commercial product Actilight®, while CK1 produce for almost kestose (GF3). Under the optimised biotransformation conditions the maximum accumulation of FOS was 56 % (w/w) and 31 % (w/w) for CF215 and CK1 respectively. We were able to isolate and characterise seven different carbohydrates such as 1-kestose, 1-nystose, 1-fructofuranosylnystose, 6-kestose, neo-kestose and neo-nystose for CF215 while CK1 produce only 1-kestose, 1-nystose, 1-fructofuranosylnystose and 6-kestose. Another oligosaccharides was isolated and fully characterised from CF215 mixture, named blastose (Fru-β(26)-Glc). An immobilization study was carried using the DALGEEs (Dried Alginate Entrapped Enzymes) method on the mycelium of CF215 strain. The maximum accumulation of FOS using DALGEEs mycelium was 51 % (w/w), reached in common buffer and seawater. With this cheap technique we develop a continuous FOS production using the facilities of Flow chemistry. The reactor, filled with DALGEEs and celite, was stable for months and the maximum accumulation of FOS was 52% (w/w). At this flow stream of FOS mixture we added a batch step to purify the FOS from glucose that represent the 26 % (w/w) of entire mixture. Glucose Oxidase from Novozymes® named Glyzyme® MONO 10.000 BG was employed and the result was the reduction of glucose from 26 % (w/w) to 3% (w/w). This purification step was added for two reasons: to obtain a cheap and fast method for FOS purification from glucose and to simplify the blastose HPLC purification. 56 mg of purified blastose were obtained and utilised to perform a pioneer study of blastose prebiotic action. The growth of 5 different lactobacillus strains (Lactobacillus paracasei DG, Lactobacillus rhamnosus GG, Lactobacillus paracasei SHIROTA, Lactobacillus johnsonii LC1 and Lactobacillus reuteri ATCC55730) were followed with the addiction of different carbohydrates as only carbon source (glucose, Actilight®, inulin, blastose). The best results were reached with Lactobacillus johnsonii LC1 where the Vmax using 0.5 % (w/v) of blastose was higher than glucose 0.5 % (w/v) (1.125 ± 0.023 1/h and 0.521 ± 0.054 1/h respectively). In the second part of this PhD project the purification of the enzymes involved in FOS formation was achieved after several chromatographic steps. The molecular weight (MW) of the two proteins was ≈50 kDA for the enzyme from Cladosporium cladosporioides (monomeric) and ≈75 kDa for the one from Penicilium sizovae (monomeric). The enzyme from C. cladosporioides was biochemically characterised and shown a Km of 129 ± 6 mM, Vmax of 2.83 ± 0.04 U/mL, Kcat of 2.88 ± 0.04 1/s and a Kcat/Km of 22.3 ± 1.4 1/M*s with sucrose and a Km of 268 ± 6 mM, Vmax of 0.0328 ± 0.003 U/mL, Kcat of 0.0334 ± 0,003 1/s and Kcat/Km of 0.124 ± 0.014 1/M*s with 1-kestose. A mass spectrometry MALDI-TOF analysis study was performed on the protein, showing a MW of 61178 Da. A trypsin digestion was performed and the fragments analysed but we didn’t found match in databases. The molecular study of the protein was stopped until protein sequence elucidation.

This PhD thesis is divided into four chapters that report the development of different microbial biocatalysts aimed at the valorization of food and biotechnological industrial by-products. The experimental parts are preceded by an introduction aimed to give a background on the impact of biocatalysis on green chemistry and, more generally on circular economy, together with a systematic review of the main molecular biology approaches employed to genetically engineer acetic acid bacteria. The second chapter reports the optimization of a biocatalytic system for the regioselective hydroxylation of different terpenes employing a Mycobacterium sp. CYP153A6 monoxygenase. In chapter 3 and in chapter 4 is described the development of different recombinant acetic acid bacteria strains, aimed at the production of highly added value products, such as perillic acid, starting from limonene, a cheap and highly available substrate derived from the agro-food industry. Limonene was employed as a pure compound (chapter 3) or via fermentation of orange peel wastes (chapter 4). The last chapter deals with the isolation and characterization of a bacterial cellulose (BC) producer strain, namely Komagataeibacter rhaeticus ENS9b, able to produce BC from acetate and crude glycerol, a by-product from the biodiesel production process.

Questa tesi descrive le biotrasformazioni di acidi biliari con ceppi derivanti da campionamenti in Italia e in Ecuador di microrganismi appartenenti al phylum degli Attinobatteri. Dal campionamento fatto in Italia lo screening di 86 ceppi (Capitolo 2) ha permesso di individuarne uno particolarmente interessante, che dopo caratterizzazione, è risultato essere Pseudomonas alcaliphila, Questo ceppo ha prodotto in rese quantitative il 12-idrossi-androstane-1,4-diene-3,17-dione (12-HADD) 2a (95%) partendo dall’acido desossicolico 1a. Risultati analoghi si sono ottenuti con l’acido colico 1b, l’acido chenodesossicolico 1c e l’acido iodesossicolico 1d che hanno fornito rispettivamente 7,12-HADD 2b (23%), 7-HADD 2c (52%) e 6-HADD 2d (83%) (Capitolo 3) . Vengono riportate inoltre, le biotrasformazioni di acido colico, acido desossicolico, acido iodesossicolico con batteri isolati dal macello rurale di Cayambe (provincia di Pichincha, Ecuador) (Capitolo 4) che hanno portato alla sintesi di bendigoli e altri metaboliti. I ceppi più attivi sono stati caratterizzati e appartengono al genere Pseudomonas e Rhodococcus. L’acido colico 1a ha fornito il 3-cheto derivato 2a (45%) e il 3-cheto-4-ene derivato 3a (45%) con P. mendocina ECS10, il 3,12-dicheto-4-ene derivato 4a (60%) con Rh. erythropolis ECS25 e il 9,10-secosteroide 6 (15%) con Rh. erythropolis ECS12. Il bendigolo F 5a (20%) è stato ottenuto con P. fragi ECS22. Dalla biotrasformazione di acido desossicolico 1b si è ottenuto con P. prosekii ECS1 e Rh. erythropolis ECS25 il 3-cheto derivato 2b (20% e 61% rispettivamente), mentre il 3-cheto-4-ene derivato 3b è stato ottenuto con P. prosekii ECS1 e P. mendocina ECS10 (22 e 95% rispettivamente). P. fragi ECS9 ha dato inoltre il bendigolo A 8b (80%). Infine, dalla biotrasformazione di acido iodesossicolico (1c) con P.mendocina ECS10 si è ottentuto il 3-cheto derivato 2c (50%) e con Rh. erythropolis ESC12 il 6-idrossi-3-cheto-23,24-dinor-5-colan-22-oico 9c (66%). Il bendigolo G 5c (13%) è stato ottenuto dalla biotrasformazione con P. prosekii ECS1 e il bendigolo H 8c con P. prosekii ECS1 e Rh. erythropolis ESC12 (20 e 16% rispettivamente).
This research describes the biotransformation of bile acids with microbial strains belonging to the phylum Actinobacter, sampled in Italy and Ecuador. From the 86 Italian samples (Chapter 2) a promising isolate was characterized and identified as Pseudomonas alcaliphila. This strain produced high yields of 12-hydroxy-androstane-1,4-diene-3,17-dione (12-HADD) 2a (95%) from deoxycholic acid 1a. Similar results were obtained with cholic 1b, chenodeoxycholic 1c and hyodeoxycholic acid 1d; which yielded 7,12-HADD 2b (23%), 7-HADD 2c (52%) e 6-HADD 2d (83%) (Chapter 3). In addition, information concerning biotransformations of cholic, deoxycholic, and hyodeoxycholic acids, performed with Ecuadorian strains isolated from the rural slaughterhouse at Cayambe (Pichincha province, Ecuador) is presented in Chapter 4. In this case, biotransformations produced bendigoles and other metabolites. The most promising samples belong to genus Pseudomonas and Rhodococcus. Cholic acid 1a produced 3-keto derivative 2a (45%) 3-keto-4-ene derivative 3a (45%) with P. mendocina ECS10, 3,12-diketo-4-ene derivative 4a (60%) with Rh. erythropolis ECS25 and 9,10-secosteroid 6 (15%) with Rh. erythropolis ECS12. Bendigole F 5a (20%) was obtained with P. fragi ECS22. Biotransformation of deoxycholic acid 1b with P. prosekii ECS1 and Rh. erythropolis ECS25 produced 3-keto derivative 2b (20% e 61% respectively), whereas 3-keto-4-ene derivative 3b was obtained with P. prosekii ECS1 and P. mendocina ECS10 (22 e 95% respectively). P. fragi ECS9 also produced bendigole A 8b (80%). Finally, biotransformation of hyodeoxycholic (1c) with P.mendocina ECS10 produced 3-keto derivative 2c (50%) and 6-hydroxy-3-keto-23,24-dinor-5-cholan-22-oico 9c (66%) with Rh. erythropolis ESC12. Bendigole G 5c (13%) was obtained from biotransformation with P. prosekii ECS1 and bendigole H 8c with P. prosekii ECS1 and Rh. erythropolis ESC12 (20 e 16% respectively).

The present thesis has the aim of develop and find new and more environmental friendly synthetic routes for the synthesis of active pharmaceutical ingredients (APIs) and pharmaceutically interesting intermediates, exploiting the advantages of the combination between flow chemistry and biocatalysis. Indeed, biocatalytic processes in continuous flow reactors have attracted attention in recent years for carrying out continuous manufacturing systems with high level of intensification. Flow processing has the potential to accelerate heterogeneous biotransformations due to biocatalyst high local concentration and enhanced mass transfer, making large-scale production more economically feasible in significantly smaller equipment with a substantial decrease in reaction time, from hours to a few minutes, and improvement in space–time yield, with increases of up to 650-fold as compared to batch processes. Moreover, biocatalyst stability is enhanced by working in an environment where harsh mixing is avoided. Overall, these features result in reduced inventory, waste and energy requirements of the flow biocatalytic process, as compared to the conventional batch mode. In particular, I focused my attention on redox reactions, since for these the traditional chemical procedures and reagents are far from being sustainable and environmental friendly. For example, for oxidative reactions the most used chemical reactives are Chromium VI (a well known cancerogenic agent), Dess-Martin periodinane (a potential explosive reagent) and the Swern reagent, a thiol based compound that produces dimethyl sulphide as co-product. Moreover, the traditional chemical methods are not able to reach the selectivity and specificity that is possible to achieve with biocatalytical systems. Briefly, the projects I was involved in during my PhD and that are present in the thesis are: 1. Development of a new synthetic route to obtain Captopril, using both chemical and biocatalyzed reactions and exploiting the advantages of flow chemistry, that allows to perform continuous synthesis; 2. Development of a flow based biocatalyzed oxidation with immobilized whole cells of Acetobacter aceti in order to obtain enantiomerically pure mono-carboxylic acids, starting from the corresponding diols; 3. Stereoselective reduction of ketones and di-ketones, in order to obtain enantiomerically pure mono-alcohol products, using together two enzymes (ketoreductase from Pichia glucozyma and a glucodehydrogenase from Bacillus megaterium) in a Flow Chemistry pcked bed reactor; 4. Stereoselective reduction of 2,2-disubstituted 1,3-cyclopenta- and 1,3 cyclohexanediones using both whole cells and a purified ketoreductase from Pichia glucozyma, to obtain enantiomerically pure mono-alcohols products, that can be important intermediates in the synthesis of various steroids. 5. Use of an immobilized transaminase from Halomonas elongata able to perform transaminations in both directions (from amine to aldehydes, and from aldehydes to amine) with the Flow Reactor technology; To reach the goal, I both used whole cells and purified enzymes as biocatalysts, either in a free or immobilized form, and I exploited many advantages of continuous flow technology, as for example downstream processes (i.e., in-line acidifications, extractions and purifications) that allowed me to in-line purify the products, thus avoiding the traditional work-up procedures, reducing the operational times and the amount of organic solvents used. In almost all cases, important results were achieved, as faster kinetics, cleaner procedures that required less purification steps, complete stereo- and regioselectivity, higher conversions and productivities compared to batch procedures, increased stability of the biocatalyst, that could be used for several cycles, thus reducing the waste.

Two new biocatalyst-tool have been studied: starting from interesting whole-cell application in the agro, food and zootechnical field, the hydrolytic activity was related to two carboxylesterases that have been purified until electrophoretic omogeneity. The enzyme from Kluyveromyces marxianus CBS 1553 was also studied from catalytic and biochemical point of view: the overall activity and stability over a wide range of condition indicate that this enzyme has the potential to be exploited in biotechnological applications.

In this PhD thesis the first application of acetylacetoin synthase (AAS), by B. licheniformis DSM 13, as a biocatalyst for the stereoselective formation of C-C bonds is described. AAS, a thiamine diphosphate (ThDP) dependent enzyme, catalyzes the condensation of dialkyl or alkyl-aryl-1,2-diketones into the corresponding α-hydroxy-β-diketones with the elimination of a carboxylic acid. The reactions were carried out using a single α-diketone as donor and acceptor (homo-coupling) or two different α-diketones (cross-coupling). The AAS enzymatic reaction of a new C-C bond formation is highly chemo-, regio- and enantioselective. The α-hydroxy-β-diketones obtained from the reactions of homo- and cross-coupling were reduced with acetylacetoin reductase (AAR), a dehydrogenase obtained from the same bacterium. The combined use of AAS and AAR allowed the preparation of a new range of optically pure α-alkyl-α,β-dihydroxyketones starting from commercial α-diketones. The stereochemistry of the enantiopure syn-α-alkyl-α,β-dihydroxyketones was assigned on the basis of NOE experiments, while their absolute configuration was determined transforming one of these compounds in the natural product (+)-citreodiol. The absolute configuration of α-alkyl-α, β-dihydroxyketones confirmed the S-stereospecificity of the AAR-reduction and R-stereospecificity of AAS homo and cross-coupling reactions. On the basis of the AAS activity, an alternative synthetic biomimetic route, reminiscent the ThDP-dependent enzymes activity, was studied. Both thiamine hydrochloride and its simplified analogue, thiazolium salt, act as pre-catalysts coupled with an appropriate basis and are able to activate α-diketones such as acyl-anion equivalents that can be transferred to enable ketonic acceptors as α-diketones and α-ketoesters. These carboligation reactions have been optimized in catalytic conditions using polyethylene glycol (PEG400), an eco-friendly reaction medium, that made easier the reaction workup allowing, in addition, the catalyst recycling. A further synthetic application of AAS was the chemo-enzymatic synthesis of the natural aroma of green tea. The chirality of this compound, closely related to its organoleptic properties, is actually studied in our laboratories. The versatility of AAS as biocatalyst for C-C bond forming reactions and the raised interest by its particular applications in organic synthesis promped us to purify the enzyme, with the ultimate goal of identifying the gene encoding for AAS in the genome of B. licheniformis DSM 13.