A novel protein glutaminase from Bacteroides helcogenes-characterization and comparison.

Deamidation is a promising tool to improve solubility and other functional properties of food proteins. One possibility of protein deamidation is the use of a protein glutaminase (PG; EC 3.5.1.44), an enzyme that catalyzes the deamidation of internal glutamine residues in proteins to glutamic acid residues. The PG from Chryseobacterium proteolyticum is the only one described in literature to date and is commercially available (Amano Enzyme Inc., Japan; PGA). Based on a similarity search, we discovered a predicted, uncharacterized protein from Bacteroides helcogenes and this protein was verified as a PG. After recombinant production and purification, the novel PG (BH-PG) was biochemically characterized and compared with PGA. Some advantageous characteristics for potential application of BH-PG compared with PGA were the higher temperature stability (residual activity after 24 h of incubation at 50 °C was 87% for BH-PG and 2% for PGA), an optimum pH value at acidic conditions (pH 5.5) and less product inhibition by ammonia that is released during the deamidation of proteins (residual activity after adding 40 mM ammonia was 77% for BH-PG and 27% for PGA). Finally, the applicability of BH-PG and PGA was compared by gluten deamidation experiments. Consequently, the final solubility of the nearly insoluble food protein gluten was 94% after BH-PG treatment, whereas the solubility was around 66% when using PGA.

Enzymatic production of emulsifying whey protein hydrolysates without the need of heat inactivation.

BACKGROUND: One possible way to modify the emulsifying properties of whey proteins is by enzymatic hydrolysis. However, most studies covering the influence of the hydrolysis on whey proteins used a heating step (>65 °C) to inactivate the enzyme. This leads to irreversible product changes, like protein denaturation and increased viscosity. Here, the objective was to investigate the single effect of hydrolysis on the emulsifying properties of whey proteins under conditions without a temperature step for enzyme inactivation. Therefore, two acidic peptidase preparations (Maxipro AFP, Protease AP-30L) differing in their peptidase composition were investigated and applied at 45 °C and pH 2.75. The enzyme inactivation was realized by a simple shift to pH 7.0. RESULTS: After the pH shift, no activity or further hydrolysis was measurable. For the products, no differences (assuming P > 0.05) regarding the emulsifying properties were detected between the two peptidase preparations used. The emulsifying properties of the whey protein isolate hydrolysates produced increased (i.e. half-life >71%) until a degree of hydrolysis of 1.1%. This indicated that the endopeptidase (aspergillopepsin I) present in both preparations was determining the emulsifying properties. As a plus, the presence of exopeptidases in Protease AP-30L compared with Maxipro AFP reduced the bitterness of the hydrolysate (-50%). CONCLUSION: The application of acidic endo- and exopeptidases enables the production of emulsifying whey protein isolate hydrolysates at high protein concentrations (≥10%) without a commonly used heat inactivation step. The presence of exopeptidases in acidic peptidase preparations is favorable, due to the improved taste. © 2019 Society of Chemical Industry.

Two Heat Resistant Endopeptidases from Pseudomonas Species with Destabilizing Potential during Milk Storage.

In the current study, the extracellular endopeptidases from Pseudomonas lundensis and Pseudomonas proteolytica were investigated. The amino acid sequence identity between both endopeptidases is 68%. Both endopeptidases were purified to homogeneity and partially characterized. They were classified as metallopeptidases with a maximum activity at pH 10.0 ( P. lundensis) or 8.5 ( P. proteolytica) at 35 °C. Both remained active in skim milk with 39.7 ± 2.4% and 24.5 ± 3.3%, respectively, of the initial enzyme activity after UHT processing (138 °C for 20 s), indicating the relevance for milk destabilization. The transition points in buffer were determined at 50 °C ( P. lundensis) and 43 °C ( P. proteolytica) using circular dichroism spectroscopy. The loss of the secondary structure at different temperatures was correlated with residual peptidase activities after heat treatment. The ability to destabilize UHT milk was proven by supplementation of skim milk with endopeptidase and storage for 4 weeks.

Improving the colloidal and sensory properties of a caseinate hydrolysate using particular exopeptidases.

Enzymatic hydrolysis with endopeptidases can be used to modify the colloidal properties of food proteins. In this study, sodium caseinate was hydrolyzed with Sternzym BP 25201, containing a thermolysin-like endopeptidase from Geobacillus stearothermophilus as the only peptidase, to a DH of 2.3 ± 1%. The hydrolysate (pre-hydrolysate) obtained was increased in its foam (+35%) and emulsion stability (+200%) compared to untreated sodium caseinate but showed a bitter taste. This hydrolysate was further treated with the exopeptidases PepN, PepX or PepA, acting on the N-terminus of peptides. Depending on the specificity of the exopeptidase used, changes regarding the hydrolysate properties (hydrophobicity, size), colloidal behavior (emulsions, foams) and taste were observed. No changes regarding the bitterness but further improvements regarding the colloidal stability (foam: +69%, emulsion: +29%) were determined after the application of PepA, which is specific for the hydrophilic amino acids Asp, Glu and Ser. By contrast, treatment with the general aminopeptidase PepN resulted in a non-bitter product, with no significant changes regarding the colloidal properties compared to the pre-hydrolysate (p < 0.05). Similar results to those for PepN (reduced bitterness compared to the pre-hydrolysate, enhanced colloidal stability compared to sodium caseinate) were also obtained using commercial Flavourzyme, which was reduced in its endopeptidase activity (exo-flavourzyme). In conclusion, the modifications obtained with the applied exopeptidases offer a potent tool for researchers and the industry to produce non-bitter protein hydrolysates with increased colloidal properties.

Influence of the metal ion on the enzyme activity and kinetics of PepA from Lactobacillus delbrueckii.

The aminopeptidase A (PepA; EC 3.4.11.7) belongs to the group of metallopeptidases with two bound metal ions per subunit (M1M2(PepA)) and is specific for the cleavage of N-terminal glutamic (Glu) and aspartic acid (Asp) and, in low amounts, serine (Ser) residues. Our group recently characterized the first PepA from a Lactobacillus strain. However, the characterization was performed using synthetic para-nitroaniline substrates and not original peptide substrates, as was done in the current study. Prior to the characterization using original peptide substrates, the PepA purified was converted to its inactive apo-form and eight different metal ions were tested to restore its activity. It was found that five of the metal ions were able to reactivate apo-PepA: Co2+, Cu2+, Mn2+, Ni2+ and Zn2+. Interestingly, depending on the metal ion used for reactivation, the activity and the pH and temperature profile differed. Exemplarily, MnMn(PepA), NiNi(PepA) and ZnZn(PepA) had an activity optimum using MES buffer (50mM, pH 6.0) and 60°C, whereas the activity optimum changed to Na/K-phosphate-buffer (50mM, pH 7.0) and 55°C for CuCu(PepA). However, more important than the changes in optimum pH and temperature, the kinetic properties of PepA were affected by the metal ion used. While all PepA variants could release N-terminal Glu or Asp, only CoCo(PepA), NiNi(PepA) and CuCu(PepA) could release Ser from the particular peptide substrate. In addition, it was found that the enzyme efficiency (Vmax/KM) and catalytic mechanism (positive cooperative binding (Hill coefficent; n), substrate inhibition (KIS)) were influenced by the metal ion. Exemplarily, a high cooperativity (n>2),KIS value >20mM and preference for N-terminal Glu were detected for CuCu(PepA). In summary, the results suggested that an exchange of the metal ion can be used for tailoring the properties of PepA for specific hydrolysis requirements.

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 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.

Novel high-performance metagenome ß-galactosidases for lactose hydrolysis in the dairy industry.

The industrially utilised ß-galactosidases from Kluyveromyces spp. and Aspergillus spp. feature undesirable kinetic properties in praxis, such as an unsatisfactory lactose affinity (KM) and product inhibition (KI) by galactose. In this study, a metagenome library of about 1.3 million clones was investigated with a three-step activity-based screening strategy in order to find new ß-galactosidases with more favourable kinetic properties. Six novel metagenome ß-galactosidases (M1-M6) were found with an improved lactose hydrolysis performance in original milk when directly compared to the commercial ß-galactosidase from Kluyveromyces lactis (GODO-YNL2). The best metagenome candidate, called "M1", was recombinantly produced in Escherichia coli BL21(DE3) in a bioreactor (volume 35 L), resulting in a total ß-galactosidase M1 activity of about 1100 µkatoNPGal,37 °C L(-1). Since milk is a sensitive and complex medium, it has to be processed at 5-10 °C in the dairy industry. Therefore, the ß-galactosidase M1 was tested at 8 °C in milk and possessed a good stability (t1/2=21.8 d), a desirably low apparent KM,lactose,8 °C value of 3.8±0.7 mM and a high apparent KI,galactose,8 °C value of 196.6±55.5 mM. A lactose hydrolysis process (milk, 40 nkatlactose mLmilk,8 °C(-1)) was conducted at a scale of 0.5L to compare the performance of M1 with the commercial ß-galactosidase from K. lactis (GODO-YNL2). Lactose was completely (>99.99%) hydrolysed by M1 and to 99.6% (w/v) by K. lactis ß-galactosidase after 25 h process time. Thus, M1 was able to achieve the limit of <100 mg lactose per litre milk, which is recommended for dairy products labelled as "lactose-free".