Klonierung, Expression, biochemische Charakterisierung und Oligosaccharid-synthese durch [beta]-Galactosidase aus Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 und Bifidobacterium breve DSM 20231

Im Mittelpunkt dieser Arbeit stand die Anwendung von Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 und Bifidobacterium breve DSM 20213 für die Produktion des Enzyms -Galactosidase, die biochemische Charakterisierung dieser Enzyme sowie die Bildung von Galactooligosacchariden (GOS) und Heterooligosacchariden (HeOS). Während GOS bereits als Präbiotika sehr gut etabliert sind, kann auch angenommen werden, dass HeOS, die mittels dieser Enzyme aus probiotischen Organismen gebildet werden, ebenfalls präbiotische Eigenschaften aufweisen und speziell das Wachstum von Lactobazillen bzw. Bifidobakterien im Darm fördern. Das lacZ Gen aus L. bulgaricus, welches für die -Galactosidase kodiert, wurde mittels verschiedener induzierbarer Vektoren in L. plantarum WCFS1 überexprimiert, während die beiden -Galactosidasen aus B. breve, BbregalI and BbregalII, in Escherichia coli heterolog produziert wurden, wobei hier Koexpression der Chaperone GroEL/GroES notwendig war. Diese drei rekombinanten -Galactosidasen wurden anschließend im Detail biochemisch charakterisiert. Lbulgal, BbregalI und BbregalII zeigten sehr hohe Transgalactosylierungsaktivität mit Laktose als Substrat; die maximalen Ausbeuten an GOS lagen bei etwa 50, 33 und 44% relativ zu den gesamten Zuckern bei Anwendung von 200 g/L Laktose als Ausgangssubstrat. Die wichtigsten Transgalaktosylierungsprodukte von BbregalI und BbregalII waren -D-Galp-(1-6)-D-Glc und -D-Galp-(1-3)-D-Lac, während Lbulgal primär -D-Galp-(16)-D-Glc und -D-Galp-(16)-Lac bildete. Die drei Enzyme wurden ebenfalls bezüglich ihrer Eignung, auf bestimmte Zuckerakzeptoren (D-Galactose, L-Fucose, GlcNAc and GalNAc) Galaktosylreste zu übertragen, untersucht. Diese Eigenschaft wurde mittels des Partitionsverhältnisses kNu/kwater quantifiziert. Für Lbulgal und BbregalII war dieses Verhältnis für GlcNAc als Akzeptor etwa 2 bzw. 6 Mal höher als für Glucose und Lactose, was darauf hinweist, dass GlcNAc einen ausgezeichneter Akzeptor für den Galactosylrest darstellt. Bei Verwendung von Laktose als Galactosyldonor und GlcNAc als Akzeptor konnte schließlich mit beiden Enzymen -D-Galp-(1-6)-GlcNAc als Hauptprodukt in sehr guten Ausbeuten erhalten. Die Struktur dieses Produktes wurde auch mittels NMR bestätigt.The study focused on the use of Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 and Bifidobacterium breve DSM 20213 for the production and characterization of -galactosidase as well galacto- oligosaccharides (GOS) and hetero-oligosaccharides (HeOS) synthesis. It is anticipated that GOS and HeOS produced by these -galactosidases will be used for the specific proliferation of these bacterial genera in the gut, thus they can be considered prebiotic. The lacZ gene from L. bulgaricus was cloned into different inducible lactobacillal expression vectors for overexpression in the host strain L. plantarum WCFS1 while the two -galactosidases, BbregalI and BbregalII, from B. breve were overexpressed in Escherichia coli with co-expression of the chaperones GroEL/GroES. The three recombinant -galactosidases were purified to electrophoretic homogeneity and further characterized. When used for lactose conversion in transferase mode, Lbulgal, BbregalI and BbregalII showed very high transgalactosylation activity; the maximum yields of GOS was approximately 50, 33, and 44% of total sugars, respectively when using an initial concentration of 200 g/L lactose. The predominant transgalactosylation products of BbregalI and BbregalII are -D-Galp-(1-6)-D-Glc and -D-Galp-(1-3)-D-Lac while that of Lbulgal are -D-Galp-(16)-D-Glc and -D-Galp-(16)-Lac. Lbulgal, BbregalI, and BbregalII were also investigated with respect to their propensity to transfer galactosyl moieties onto lactose, D-glucose and D-galactose, L-fucose, GlcNAc and GalNAc under defined, initial-velocity conditions. Galactosyl transfer from Lbulgal or BbregalII to GlcNAc occurs with a partitioning ratios kNu/kwater that are 2 and 6 times those for the reactions of the galactosylated enzymes with glucose and lactose, respectively. Using lactose as galactosyl donor and GlcNAc as acceptor, Lbulgal and BbregalII synthesized -D-Galp-(1-6)-GlcNAc as the major product. The structure of this product was confirmed by NMR. These results indicate that these enzymes can be of interest for synthesis of both prebiotic GOS and HeOS.eingereicht von Sheryl Lozel ArreolaAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Univ. für Bodenkultur, Diss., 2014OeBB(VLID)193037

Transferase Activity of Lactobacillal and Bifidobacterial β‑Galactosidases with Various Sugars as Galactosyl Acceptors

The β-galactosidases from Lactobacillus reuteri L103 (<i>Lreu</i>βgal), Lactobacillus delbrueckii subsp. <i>bulgaricus</i> DSM 20081 (<i>Lbul</i>βgal), and Bifidobacterium breve DSM 20281 (<i>Bbre</i>βgal-I and <i>Bbre</i>βgal-II) were investigated in detail with respect to their propensity to transfer galactosyl moieties onto lactose, its hydrolysis products d-glucose and d-galactose, and certain sugar acceptors such as <i>N</i>-acetyl-d-glucosamine (GlcNAc), <i>N</i>-acetyl-d-galactosamine (GalNAc), and l-fucose (Fuc) under defined, initial velocity conditions. The rate constants or partitioning ratios (<i>k</i>_Nu/<i>k</i>_water) determined for these different acceptors (termed nucleophiles, Nu) were used as a measure for the ability of a certain substance to act as a galactosyl acceptor of these β-galactosidases. When using <i>Lbul</i>βgal or <i>Bbre</i>βgal-II, the galactosyl transfer to GlcNAc was 6 and 10 times higher than that to lactose, respectively. With lactose and GlcNAc used in equimolar substrate concentrations, <i>Lbul</i>βgal and <i>Bbre</i>βgal-II catalyzed the formation of <i>N</i>-acetyl-allolactosamine with the highest yields of 41 and 24%, respectively, as calculated from the initial GlcNAc concentration

Two β-Galactosidases from the Human Isolate <i>Bifidobacterium breve</i> DSM 20213: Molecular Cloning and Expression, Biochemical Characterization and Synthesis of Galacto-Oligosaccharides

<div><p>Two β-galactosidases, β-gal I and β-gal II, from <i>Bifidobacterium breve</i> DSM 20213, which was isolated from the intestine of an infant, were overexpressed in <i>Escherichia coli</i> with co-expression of the chaperones GroEL/GroES, purified to electrophoretic homogeneity and biochemically characterized. Both β-gal I and β-gal II belong to glycoside hydrolase family 2 and are homodimers with native molecular masses of 220 and 211 kDa, respectively. The optimum pH and temperature for hydrolysis of the two substrates <i>o</i>-nitrophenyl-β-D-galactopyranoside (<i>o</i>NPG) and lactose were determined at pH 7.0 and 50°C for β-gal I, and at pH 6.5 and 55°C for β-gal II, respectively. The <i>k</i>_cat/<i>K</i>_m values for <i>o</i>NPG and lactose hydrolysis are 722 and 7.4 mM⁻¹s⁻¹ for β-gal I, and 543 and 25 mM⁻¹s⁻¹ for β-gal II. Both β-gal I and β-gal II are only moderately inhibited by their reaction products D-galactose and D-glucose. Both enzymes were found to be very well suited for the production of galacto-oligosaccharides with total GOS yields of 33% and 44% of total sugars obtained with β-gal I and β-gal II, respectively. The predominant transgalactosylation products are β<i>-</i>D-Gal<i>p</i>-(1→6)-D-Glc (allolactose) and β<i>-</i>D-Gal<i>p-</i>(1→3)-D-Lac, accounting together for more than 75% and 65% of the GOS formed by transgalactosylation by β-gal I and β-gal II, respectively, indicating that both enzymes have a propensity to synthesize β<i>-</i>(1→6) and β<i>-</i>(1→3)-linked GOS. The resulting GOS mixtures contained relatively high fractions of allolactose, which results from the fact that glucose is a far better acceptor for galactosyl transfer than galactose and lactose, and intramolecular transgalactosylation contributes significantly to the formation of this disaccharide.</p></div

pH (A and B) and temperature (C and D) optimum of β-galactosidase activity for <i>B. breve</i> β-gal I (•) and β-gal II (○) using <i>o</i>NPG (A and C) and lactose (B and D) as substrate.

<p>Values are the mean of two independent experiments and the standard deviation was always less than 5%.</p

Formation and degradation of individual GOS formed by <i>B. breve</i> β-gal I (A) and β-gal II (B) during lactose conversion.

<p>Reaction conditions: initial lactose concentration of 200 g L⁻¹ in 50 mM sodium phosphate buffer (pH 6.5) with 1 mM Mg²⁺ and 30°C and 1.0 U_Lac mL⁻¹ β-gal I or 2.5 U_Lac mL⁻¹ β-gal II. Symbols: (•) D-Gal<i>p</i>-(1→6)-D-Glc; (▪) D-Gal<i>p</i>-(1→6)-D-Gal; (▴) Gal<i>p</i>-(1→3)-D-Gal; (Δ) D-Gal<i>p</i>-(1→3)-D-Glc; (□) D-Gal<i>p</i>-(1→3)-D-Lac; (○) D-Gal<i>p</i>-(1→4)-Lac, (+) D-Gal<i>p</i>-(1→6)-D-Lac.</p

Stability of <i>β</i>-galactosidases from <i>B. breve</i> at different temperatures in the absence of MgCl₂ as well as in the presence of 1 and 10 mM MgCl₂.

<p>Data given are the inactivation constants <i>k</i>_in and the half-life times of activity <i>τ</i>_1/2. Values are the mean of two independent experiments and the standard deviation was always less than 5%.</p

β-Galactosidase activities in cell-free extracts of recombinant <i>E. coli</i> expressing <i>B. breve</i> β-gal I or β-gal II with and without coexpression of chaperones^a.

a<p>Values are the mean of two cultivations.</p>b<p><i>o</i>NPG was used to determine enzyme activity.</p>c<p>The expression factors are calculated as the ratios of the volumetric β-galactosidase activities obtained from the expressions with chaperones and without chaperones.</p

pH stability of the β-galactosidases from <i>B. breve</i> β-gal I (•) and β-gal II (○) incubated at 37°C in Britton-Robinson buffer over a pH range of pH 5.0–9.0 for 4 h (solid lines) and 10 h (dashed lines).

<p>The residual activity was measured after 4(B) and <i>o</i>NPG was used as substrate for the enzyme assay. Values are the mean of two independent experiments and the standard deviation was always less than 5%.</p