Simple purification method for a recombinantly expressed native His-tag-free aminopeptidase A from Lactobacillus delbrueckii.

The aminopeptidase A (PepA; EC 3.4.11.7) is an intracellular exopeptidase present in lactic acid bacteria. The PepA cleaves glutamyl/aspartyl residues from the N-terminal end of peptides and can, therefore, be applied for the production of protein hydrolysates with an increased amount of these amino acids, which results in a savory taste (umami). The first PepA from a lactobacilli strain was recombinantly expressed in Escherichia coli in a recently published study and harbored a C-terminal His6-tag for easier purification. Due to the fact that a His-tag might influence the properties of an enzyme, a simple purification method for the non-His-tagged PepA was required. Surprisingly, the PepA precipitated at a very low ammonium sulfate concentration of 5%. Unusual for a precipitating step, the purity of PepA was over 95% and the obtained activity yield was 110%. The high purity allows biochemical characterization and kinetic investigation. As a result, the optimum pH (6.0-6.5) and temperature (60-65 °C) were comparable to the His6-tag harboring PepA; the KM value was at 0.79 mM slightly lower compared to 1.21 mM, respectively. Since PepA is a homo dodecamer, it has a high molecular mass of approximately 480 kDa. Therefore, a subsequent preparative size-exclusion chromatography (SEC) step seemed promising. The PepA after SEC was purified to homogeneity. In summary, the simple two-step purification method presented can be applied to purify high amounts of PepA that will allow the performance of experiments in the future to crystalize PepA for the first time.

A fusion protein consisting of the exopeptidases PepN and PepX-production, characterization, and application.

Nowadays, general and specific aminopeptidases are of great interest, especially for protein hydrolysis in the food industry. As shown previously, it is confirmed that the general aminopeptidase N (PepN; EC 3.4.11.2) and the proline-specific peptidase PepX (EC 3.4.14.11) from Lactobacillus helveticus ATCC 12046 show a synergistic effect during protein hydrolysis which results in high degrees of hydrolysis and reduced bitterness. To combine both activities, the enzymes were linked and a fusion protein called PepN-L1-PepX (FUS-PepN-PepX) was created. After production and purification, the fusion protein was characterized. Some of its biochemical characteristics were altered in favor for an application compared to the single enzymes. As an example, the optimum temperature for the PepN activity increased from 30 °C for the single enzyme to 35 °C for FUS-PepN. In addition, the temperature stability of PepX was higher for FUS-PepX than for the single enzyme (50 % compared to 40 % residual activity at 50 °C after 14 days, respectively). In addition, the disulfide bridge-reducing reagent ß-mercaptoethanol did not longer inactivate the FUS-PepN activity. Furthermore, the K M values decreased for both enzyme activities in the fusion protein. Finally, it was found that the synergistic hydrolysis performance in a casein hydrolysis was not reduced for the fusion protein. The increase of the relative degree of hydrolysis of a prehydrolyzed casein solution was the same as it was for the single enzymes. As a benefit, the resulting hydrolysate showed a strong antioxidative capacity (ABTS-IC50 value: 5.81 µg mL(-1)).

A Novel Glutamyl (Aspartyl)-Specific Aminopeptidase A from Lactobacillus delbrueckii with Promising Properties for Application.

Lactic acid bacteria (LAB) are auxotrophic for a number of amino acids. Thus, LAB have one of the strongest proteolytic systems to acquit their amino acid requirements. One of the intracellular exopeptidases present in LAB is the glutamyl (aspartyl) specific aminopeptidase (PepA; EC 3.4.11.7). Most of the PepA enzymes characterized yet, belonged to Lactococcus lactis sp., but no PepA from a Lactobacillus sp. has been characterized so far. In this study, we cloned a putative pepA gene from Lb. delbrueckii ssp. lactis DSM 20072 and characterized it after purification. For comparison, we also cloned, purified and characterized PepA from Lc. lactis ssp. lactis DSM 20481. Due to the low homology between both enzymes (30%), differences between the biochemical characteristics were very likely. This was confirmed, for example, by the more acidic optimum pH value of 6.0 for Lb-PepA compared to pH 8.0 for Lc-PepA. In addition, although the optimum temperature is quite similar for both enzymes (Lb-PepA: 60°C; Lc-PepA: 65°C), the temperature stability after three days, 20°C below the optimum temperature, was higher for Lb-PepA (60% residual activity) than for Lc-PepA (2% residual activity). EDTA inhibited both enzymes and the strongest activation was found for CoCl2, indicating that both enzymes are metallopeptidases. In contrast to Lc-PepA, disulfide bond-reducing agents such as dithiothreitol did not inhibit Lb-PepA. Finally, Lb-PepA was not product-inhibited by L-Glu, whereas Lc-PepA showed an inhibition.

Enzymatic production of lactulose and epilactose in milk.

The enzymatic production of lactulose was described recently through conversion of lactose by a thermophilic cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE). In the current study, we examined the application of CsCE for lactulose and epilactose production in milk (1.5% fat). The bioconversions were carried out in stirred reaction vessels at 2 different temperatures (50 and 8°C) at a scale of 25 mL volume. At 50°C, 2 highly different CsCE amounts were investigated for the time course of formation of lactulose and epilactose. The conversion of milk lactose (initial lactose content of 48.5 ± 2.1 g/L) resulted in a final yield of 57.7% (28.0 g/L) lactulose and 15.5% (7.49 g/L) epilactose in the case of the approximately 9.5-fold higher CsCE amount (39.5 µkat epilactose, 50°C) after 24 h. Another enzymatic lactose conversion was carried out at low 8°C, an industrially relevant temperature for milk processing. Although the CsCE originated from a thermophilic microorganism, it was still applicable at 8°C. This enzymatic lactose conversion resulted in 56.7% (27.5 g/L) lactulose and 13.6% (6.57 g/L) epilactose from initial milk lactose after 72 h. The time courses of lactose conversion by CsCE suggested that first epilactose formed and afterward lactulose via epilactose. To the best of our knowledge, this is the first time that an enzyme has produced lactulose directly in milk in situ at industrially relevant temperatures.

New bioreactor-coupled rapid stopped-flow sampling technique for measurements of metabolite dynamics on a subsecond time scale.

Knowledge of concentrations of intracellular metabolites is important for quantitative analysis of metabolic networks. As far as the very fast response of intracellular metabolites in the millisecond range is concerned, the frequently used pulse technique shows an inherent limitation. The time span between the disturbance and the first sample is constrained by the time necessary to obtain a homogeneous distribution of the pertubation within the bioreactor. For determination of rapid changes, a novel sampling technique based on the stopped-flow method has been developed. A continuous stream of biosuspension leaving the bioreactor is being mixed with a glucose solution in a turbulent mixing chamber. Through computer-aided activation of sequentially positioned three-way valves, different residence times and thus reaction times can be verified. The application of this new sampling method is illustrated with examples including measurements of adenine nucleotides and glucose-6-phosphate in Saccharomyces cerevisiae as well as measurements related to the PTS system in Escherichia coli.