A casein solution was hydrolyzed with Alcalase 2.4 L (EC 3.4.21.62) and the recombinantly produced aminopeptidases PepX (EC 3.4.14.11) and PepN (EC 3.4.11.2) from Lactobacillus helveticus ATCC 12046 in various combinations to analyze the synergistic effect of these peptidases during casein hydrolysis. The sequential application of PepX or PepN after prehydrolysis with Alcalase resulted in an relative degree of hydrolysis (rDH) increase of 1.12- or 2.00-fold, respectively, compared to only using Alcalase. By a combined application of PepX and PepN the rDH increased ~ 2.32-fold. Using Alcalase, PepX and PepN simultaneously from the beginning the rDH increased ~ 2.42-fold. Compared to the single application of PepX or PepN after an Alcalase treatment, the combined usage led to an increased amount of small peptides (< 1.1 kDa) and free amino acids. After the sequential application of first Alcalase and then PepX and PepN, only 14 peptides, which originated mainly from the C-terminal end of the β-casein chain remained. Even the opioid precursor peptide VYPFPGPIPN [β-casein, ƒ(59–68); V-β-casomorphine-9], generated by the Alcalase treatment was fully hydrolyzed after adding PepX and PepN. Therefore, the synergistic effect of PepX and PepN during casein hydrolysis was confirmed. The simultaneous application of Alcalase, PepX and PepN from the beginning showed similar results as the sequential application, but only three remaining peptides were observed by the mass spectrometric analysis. Additionally, the hydrolysis time was reduced from 16 h (sequential approach) to 6.5 h (simultaneous approach). This indicated a further synergism between Alcalase and the two aminopeptidases.

The aim of this study was to determine the optimal conditions for operating an enzyme membrane reactor system to obtain a high yield of amino acids and peptides released from wheat gluten with Flavourzyme™. The optimal operating conditions were determined using multivariate analysis. A fractional factorial design made up of 27 runs amended by eight axial points was followed in a bench-scale enzyme membrane reactor system (V = 2 L), and responses were recorded over 8 h. Optimal conditions determined were a temperature of 50 ± 1 °C and a Flavourzyme™ activity of 116 ± 4 nkatLeu-pNA/mL. Modeling resulted in an optimal substrate feed rate of 19.2 g/L/h and a permeate flux of 57 L/m2/h. The enzymatic wheat gluten hydrolysis in an enzyme membrane reactor was conducted for 72 h with an average total product space–time yield of 12.6 ± 1.3 g/L/h (n = 4) and a free amino groups space–time yield of 4.4 ± 0.4 g/L/h (n = 4), and the substrate utilization was 0.82 ± 0.01 (g/g) (n = 4). The resulting product–enzyme ratios were 7.7 ± 0.6 mgTP/nkatLeu-pNA (n = 4) and 3.0 ± 0.2 mgFAA/nkatLeu-pNA (n = 4). The productivity of the enzyme membrane reactor improved compared to the reference batch process and doubled compared to previously proposed enzyme membrane reactor processes for the hydrolysis of wheat gluten.

Heat-resistant enzymes of psychrotolerant microorganisms are associated with the spoilage of UHT-milk and milk products. In this study, we investigated the extracellular peptidase, esterase and lipase activity after submerged cultivation at 6 degrees C of 231 recently isolated microorganisms in milk medium. In contrast to the widely used agar diffusion tests for secretion of hydrolases, here a more realistic liquid screening approach was used. The advantages of the latter are the possibility of quantifying the enzyme activity in volumetric units and the opportunity to simulate the growth conditions of the microorganisms to the storage conditions of raw milk. The majority of enzymatically active isolates belonged to Gram-negative bacteria, especially the genus Pseudomonas. Surprisingly, among them, twelve novel Pseudomonas species were discovered. In this study, we demonstrated that numerous raw milk isolates, including bacteria and yeasts, produce extracellular enzymes that may cause spoilage problems for the dairy industry. (C) 2015 Elsevier Ltd. All rights reserved.

The refrigerated storage of raw milk selects for psychrotolerant microorganisms, many of which produce peptidases and lipases. Some of these enzymes are heat resistant and are not sufficiently inactivated by pasteurisation or even ultra-high temperature (UHT) treatment. In the current study, 20 different raw cow's milk samples from single farms and dairy bulk tanks were analysed close to delivery to the dairies or close to processing in the dairy for their cultivable microbiota as well as the lipolytic and proteolytic potential of the isolated microorganisms.Altogether, 2906 isolates have been identified and assigned to 169 species and 61 genera. Pseudomonas, Lactococcus and Acinetobacter were the most abundant genera making up 62% of all isolates, whereas 46 genera had an abundance of <1% and represent only 6.6%. Of all isolates, 18% belong to hitherto unknown species, indicating that a large fraction of the milk microbiota is still unexplored. The potential of the isolates to produce lipases or peptidases followed in many cases a genus or group specific pattern. All isolates identified as members of the genus Pseudomonas exhibited mainly lipolytic and proteolytic activity or solely proteolytic activity. On the other hand, nearly all isolates of the genus Acinetobacter were lipolytic but not proteolytic. Only 37% of all tested lactic acid bacteria (LAB) showed enzymatic activity at 6°C and the type of activity was proteolytic in 97% of these cases.

A secreted peptidase from Pseudomonas panacis was identified and purified. Genome sequencing of the producer strain allowed identification of the peptidase as AprA based on a comparison to peptide sequences of mass spectra obtained from the purified enzyme. The amino acid sequence of the 49.4 kDa peptidase was 98% similar to the metallopeptidase AprX from a Pseudomonas fluorescens strain. The peptidase showed maximum activity at pH 8 and 40 degrees C and withstood general ultra-high temperature (UHT) processing (138 degrees C for 18 s) in skim milk, with 88.0 +/- 7.7% of the initial enzyme activity remaining after heating. The peptidase showed considerable enzyme activity under storage conditions of UHT milk. The potential for spoilage of milk might during storage was verified by adding very low enzyme activities to UHT-treated milk. The addition of 1 pkat mL(-1) peptidase activity resulted in a destabilisation of the milk during four weeks storage. (C) 2015 Elsevier Ltd. All rights reserved.

Microbial lipases may be produced during milk storage and processing. This can lead to organoleptic changes in the corresponding dairy products. Thus, monitoring of lipase activity in milk is desirable. Turbidity of milk prevents a direct photometric measurement of lipase activity using chromophore- or fluorophore-based assays. Laborious pretreatments or alternative analytical methods normally have to be used. With the method for the determination of lipolytic activity (MeDeLi) proposed here, it is possible to measure lipase activity directly in the natural milk utilizing tailored fluorometric substrates. Only a defatting step is carried out initially for the MeDeLi. Then, the conversion of added lipase substrate is carried out in the unmodified milk without addition of any solutions or any enzyme extraction procedure which may influence the enzyme activity. Thereafter, the milk sample is treated with two solutions to remove the turbidity of milk by dissolution. A valid and sensitive fluorometric measurement is then possible. The applicability of the MeDeLi was demonstrated in comparison with tests published previously: The limit of detection for lipolytic activity measured by MeDeLi was the lowest, with 41 pkat/mL. Raw milk, milk products, and spoiled milk samples were also investigated with the MeDeLi.

The production of hydrolases from cereals has been examined in order to investigate food-derived enzymes as an alternative source to microbial enzymes for the use in food processes. For that, the influence of temperature on the pretreatment, imbibition and germination of barley and wheat grains was determined by measuring the β-glucosidase, β-galactosidase and lipase activities using a design of experiments. The evaluation of the statistical model showed an increase of the β-glucosidase activity with low imbibition and low germination temperature for barley grains and low imbibition and high germination temperature for wheat grains. The maximum β-glucosidase activity in wheat extracts was (585±151) nkat per g of dry mass (dm), while in barley extracts it was (109±15) nkat per g of dm. The maximum β-galactosidase activities in barley and wheat extracts were (34±12) and (63±23) nkat per g of dm, respectively. The maximum lipase activities of (6.7±0.1) and (4.6±4.4) nkat per g of dm in barley and wheat extracts, respectively, were rather low compared to the glycosidase activities. The extracts were also tested for other hydrolase activities (e.g. peptidase and α-amylase activities). The insights obtained enable the basis for the potential use of cereal hydrolases in food processing, which might be attractive to consumers.

A X-prolyl-dipeptidyl aminopeptidase (PepX, EC 3.4.14.11) from Lactobacillus helveticus ATCC 12046 was automatically purified to homogeneity after cultivation in MRS broth. The purification of PepX lasted only 6 h and the PepX activity yield was 56%. The specific activity was determined at 370 nkat(H-Ala-pro-pNA) mg(-1) and the purification factor was 162-fold. A gas chromatographic assay was established for the determination of the PepX activity using original tripeptide substrates. The kinetic parameters of purified PepX were analyzed using different original tripeptide substrates with the structure Xxx-Pro-Yyy. Alanine at position P-2 (Xxx) or P-1' (Yyy) was retained, while the corresponding amino acid at P-1' or P-2 (serine, tyrosine, aspartic acid, or arginine) was varied. We analyzed the influence of the position of the different amino acids (P2 or P-1') and the side chain group (serine: polar and uncharged; tyrosine: hydrophobic; aspartic acid: negatively charged; arginine: positively charged) on the PepX activity and the kinetic parameters. Significant differences were observed by comparing the kinetic parameters of PepX using original tripeptides as substrates with the chromogenic peptide H-Ala-Pro-pNA as a substrate; for instance, a K-m-value of 0.54 mM and a V-max-value of 4.76 nkat mL(-1) were determined using the tripeptide Ser-Pro-Ala as a substrate. The kinetic parameters for the chromogenic peptide H-Ala-Pro-pNA were 1.53 mM and 2.61 nkat mL(-1) for K-m and V-max, respectively. (C) 2014 Elsevier B.V. All rights reserved.

Bacillus lentus alkaline peptidase (BLAP) was used for casein (CN) hydrolysis in an enzyme membrane reactor (EMR) because it was found that BLAP was competitively inhibited by its products. The employed membranes had different molecular weight cut-offs (MWCO 1, 5 and 10 kDa). It was shown that the productivity of the EMR could be significantly improved (28 %) in comparison with batch hydrolysis under the same conditions after 20 h. All resulting EMR peptide mixtures showed a homogenous peptide pattern in HPLC–UV analysis. The obtained peptide mixtures exhibited Angiotensin-I-converting enzyme (ACE) inhibitory and antioxidative activity. The ACE inhibition of the peptide mixtures was dependent on the MWCO of the membranes. The resulting apparent IC 50 values were 115, 131 and 420 μg ml −1 for the 1, 5 and 10 kDa MWCO membranes, respectively. In kinetic studies, a mixed-type inhibition was observed for the three peptide mixtures. The radical scavenging activity was determined with the ABTS assay, and IC 50 values between 20 and 25 μg ml −1 were obtained for the generated peptide mixtures. In addition, the identified VYPFPGPIPN peptide exhibited ACE inhibition and antioxidant activity with IC 50 values of 325 and 6.2 μM, respectively. The peptide YQEPVLGPVRGPFPIIV exhibited radical scavenging activity with an IC 50 value of 5.2 μM.

Demicellization of a 90 mM sodium dodecyl sulfate (SDS) solution in water at 10, 22, and 30 °C was studied by isothermal titration calorimetry (ITC). ΔH of the demicellization process was strongly temperature dependent, having an exothermic progression (-20.4 ± 0.9 kJ∕mol, max) at 10 °C and an endothermic one (3.7 ± 1.2 kJ∕mol, max) at 30 °C. ΔH for micelle dilution followed a slightly endothermic progression (0.9 ± 0.5 kJ∕mol at 30 °C, 0.7 ± 1.3 kJ∕mol at 22 °C, and 0.0 ± 0.5 kJ∕mol at 10 °C) at all studied temperatures. No differences in ΔH for micelle dilution and demicellization was observed at 22 °C. The temperature dependence of ΔH measured by ITC could be related to hydrophobic interactions. Therefore, ITC was shown to be a useful tool to describe the thermodynamics of demicellization processes and in addition to determine alterations in ΔH caused by changes in hydrophobic and steric∕electrostatic interactions.

Highly purified proteins are essential for the investigation of the functional and biochemical properties of proteins. The purification of a protein requires several steps, which are often time-consuming. In our study, the Angiotensin-I-Converting-Enzyme (ACE; EC 3.4.15.1) was solubilised from pig lung without additional detergents, which are commonly used, under mild alkaline conditions in a Tris-HCl buffer (50mM, pH 9.0) for 48h. An automation of the ACE purification was performed using a multi-step protocol in less than 8h, resulting in a purified protein with a specific activity of 37Umg(-1) (purification factor 308) and a yield of 23.6%. The automated ACE purification used an ordinary fast-protein-liquid-chromatography (FPLC) system equipped with two additional switching valves. These switching valves were needed for the buffer stream inversion and for the connection of the Superloop™ used for the protein parking. Automated ACE purification was performed using four combined chromatography steps, including two desalting procedures. The purification methods contained two hydrophobic interaction chromatography steps, a Cibacron 3FG-A chromatography step and a strong anion exchange chromatography step. The purified ACE was characterised by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE. The estimated monomer size of the purified glycosylated ACE was determined to be ∼175kDa by SDS-PAGE, with the dimeric form at ∼330kDa as characterised by a native PAGE using a novel activity staining protocol. For the activity staining, the tripeptide l-Phe-Gly-Gly was used as the substrate. The ACE cleaved the dipeptide Gly-Gly, releasing the l-Phe to be oxidised with l-amino acid oxidase. Combined with peroxidase and o-dianisidine, the generated H(2)O(2) stained a brown coloured band. This automated purification protocol can be easily adapted to be used with other protein purification tasks.

The presented work introduces a novel method to immobilize enzymes either purified or directly out of a crude extract onto magnetic particles in the micrometer range. This method is based on the creation of a fusion protein consisting of the enzyme of choice and a mutant dehalogenase. The dehalogenase gene is commercially available from the company Promega under the name HaloTag(TM). When the fusion protein is contacted with magnetic beads having chemically synthesized, chloroalkane ligands on their surface, the dehalogenase and the ligand undergo a covalent coupling leading to stable and spatially defined immobilization. The principle was proved with a lipase fused to the HaloTag(TM) gene and magnetic poly(methyl)methacrylate beads as carriers. The solubility of the tagged lipase was strongly increased by fusion of the malE gene at the N-terminal end of the HaloTag(TM) lipase gene. This tripartite protein was purified on amylose resin and used for immobilization. About 13 µg protein could be immobilized per 1 mg of beads within a few minutes. Due to the defined binding site, no activity loss was observed in the course of the immobilization. The resulting enzyme carrier was tested with the same beads up to six times for lipase activity over a storage period of 36 days at 8 °C. No loss of activity was found during this time.

Circular dichroism (CD) was used to characterize the secondary structure of penicillin G acylase upon covalent immobilization on silica nanoparticles. Covalent immobilization was achieved by functionalizing the silica nanoparticles with glutardialdehyde and coupling to the free NH(2) groups of the enzyme (lysine and arginine side chains). The loading of the covalently bound enzyme was increased up to saturation, which was reached at 54.6 mg immobilized enzyme per g silica nanobeads. For structural characterization of the commercially available enzyme its exact molecular mass was determined by mass spectrometry in order to enable precise evaluation of the CD data. The fraction of secondary structure elements of the free and immobilized enzyme were estimated from the respective CD spectra using standard algorithms (CONTINLL, CDSSTR, SELCON3). The fractions obtained by the different algorithms for the free enzyme agreed well with one another and also with data from X-ray diffraction described in the literature. Interestingly, the secondary structure fractions found for the immobilized enzyme were very similar to the free enzyme and nearly constant over all experiments. These results indicate that even a loading of up to 55.8 mg/g (enzyme per silica nanoparticles) causes only slight structural changes. However, the specific activity determined by a kinetic assay decreased by around 60%, when increasing the loading from 14.9 to 55.8 mg/g. Because of the fact that we found no major changes in the secondary structure, diffusion limitation seems to be the main reason for the decline of the specific activity.