Fermentazione con Bacillus Stearothermophilus: produzione di 2,3-butandiolo, studio della via metabolica ed applicazioni biocatalitiche

The here presented PhD regarded fermentation with the bacteria Bacillus stearothermophilus ATCC 2027 evaluating its potential production of 2,3-butanediol, a metabolic product with wide applications, evaluating also the metabolic pathway and the involved enzyme. B. steraothermophilus was previously successfully used in our Laboratory in biocatalysis obtaining kinetic resolution of chiral alcohols via oxidation e stereo selective reduction of 1,2-diketons to S,Sdiols. Growing substrate analysis evidenced bacteria capacity to produce significant amounts of 2,3- butanediol (2,3-BD) and its precursor acetoin (AC) using sucrose as carbon source. 2,3-butanediol is widely used in many fields, from alimentary to polymers industries, beside its use as bio-fuel or additive in fuels, increasing the relative importance of its production. The first aim of my PhD activity was to verify real quality of 2,3-BD and AC produced by B. stearothermophilus sucrose’s fermentation, and to flowingly screen other mono- and disaccharides as carbon sources. Experiments were conducted at fixed sucrose concentration (40, 30, 20, 10 gr/l) and results indicated fermentation with 30 gr/l of sucrose as the best in terms of yield (about 100%) and carbon consume (residual about 1 gr/l). This result was compared with those obtained using other carbon sources as glucose, fructose, xylose, maltose, lactose and cellobiose, and cane molasses, chosen in relation to their high production in agroindustrial processes. Obtained results evidenced B. stearothermophilus complete sucrose fermentation and an only partial fermentation of its constituents monosaccharides, fructose and glucose. The other sugars give small amounts of the two metabolites. An important point of the present research activity was the comprehension of biochemical mechanisms allowing microorganism production of the cited metabolites. Gaschromatographic endproducts analysis with a chiral column demonstrated B. stearothermophilus production of high quantity of (R)-acetoin, (2R,3R)-butanediol and meso-butanediol. Consequently, a study on the cited bacteria metabolic pathway was developed adopting methods previously used with other microorganisms. Similarly to other cogeneric bacteria, Bacillus sterarothermophilus presents two metabolic pathways to produce butanediol. The firs, more diffused, “catabolic way” starts from pyruvate derived from glicolysis and by the way of three enzymatic steps (condensation, decarboxylation and reduction) produces 2,3-butanediol. The second, less diffused, indicated as “butanediol cycle” starts from diacetil and produces butanediol by the way of three enzymatic reactions (condensation, reduction, hydrolysis). The present research evidenced a new S-stereo specific acetoin-reductase (AC-reductase) that in association with the enzyme diacetil acetoin reductase (BSDR) previously used in biocatalysis, produces meso-butanediol. “Butanediol cycle” was confirmed by the presence of an acetil acetoin synthetase (AAC-synthetase) capable of acetilacetoin (AAC) production from diacetil. While AC-reductase was partially purified, AAC-synthetase was used raw in biocatalysis. Other 1,2 dichetons, beside diacetil, were considered as possible starting-products to obtain α- hydroxy-dichetons variably substituted. 3,4-hexanedione and 1-phenyl-2,3-propanedione were used obtaining respectively 4-hydroxy-4-ethyl-3,5-heptanedione and 1-phenil-2-hydroxy-2-methyl-1,3- butanedione. Obtained results evidenced AAC-synthetase efficiency in this catalysis showing an almost total conversion in the first case (82%) and a lower product yield in the second one (45%) with optical purity of chiral product about 40%. The present PhD research activity allowed also publications on fermentation1, metabolic pathway2 and the formation of C-C linkage mediated by AAC-synthetase.3 References 1. P. P. GIOVANNINI, M. MANTOVANI, A. MEDICI, P. PEDRINI– Productions of 2,3-butandiol by Bacillus sterothermophilus: fermentation and metabolic pathway. Proceedings of IBIC 2008, Chem. Eng. Transactions, 14, 281-286 (2008). 2. P.P. GIOVANNINI, M. MANTOVANI, M. FOGAGNOLO, S. MAIETTI, A. MEDICI, P. PEDRINI – Bacillus stearothermophilus fermentation: the enzymatic route to 3R-hydroxy-2-butanone and meso-and 2R,3Rbutanediol. Journal of Molecular Catalysis B: enzymatic, in stampa. 3. P.P. GIOVANNINI, M. MANTOVANI, A. MEDICI, P. PEDRINI - Enzymatic Carbon-Carbon Bond Formation: Synthesis of a-Hydroxy-1,3-Diketones from the Corresponding 1,2-Diketones. Organic Letters, in stampa.