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BACKGROUND
Various neutral and alkaline peptidases are commercially available for use in protein hydrolysis under neutral to alkaline conditions. However, the hydrolysis of proteins under acidic conditions by applying fungal aspartic peptidases (FAPs) has not been investigated in depth so far. The aim of this study, thus, was to purify a FAP from the commercial enzyme preparation, ROHALASE® BXL, determine its biochemical characteristics, and investigate its application for the hydrolysis of food and animal feed proteins under acidic conditions.
RESULTS
A Trichoderma reesei derived FAP, with an apparent molecular mass of 45.8 kDa (sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SDS-PAGE) was purified 13.8-fold with a yield of 37% from ROHALASE® BXL. The FAP was identified as an aspartate protease (UniProt ID: G0R8T0) by inhibition and nano-LC-ESI-MS/MS studies. The FAP showed the highest activity at 50°C and pH 4.0. Monovalent cations, organic solvents, and reducing agents were tolerated well by the FAP. The FAP underwent an apparent competitive product inhibition by soy protein hydrolysate and whey protein hydrolysate with apparent Ki-values of 1.75 and 30.2 mg*mL−1, respectively. The FAP showed promising results in food (soy protein isolate and whey protein isolate) and animal feed protein hydrolyses. For the latter, an increase in the soluble protein content of 109% was noted after 30 min.
CONCLUSION
Our results demonstrate the applicability of fungal aspartic endopeptidases in the food and animal feed industry. Efficient protein hydrolysis of industrially relevant substrates such as acidic whey or animal feed proteins could be conducted by applying fungal aspartic peptidases. © 2022 Society of Chemical Industry.
A novel peptidyl-lys metalloendopeptidase (Tc-LysN) from Tramates coccinea was recombinantly expressed in Komagataella phaffii using the native pro-protein sequence. The peptidase was secreted into the culture broth as zymogen (~38 kDa) and mature enzyme (~19.8 kDa) simultaneously. The mature Tc-LysN was purified to homogeneity with a single step anion-exchange chromatography at pH 7.2. N-terminal sequencing using TMTpro Zero and mass spectrometry of the mature Tc-LysN indicated that the pro-peptide was cleaved between the amino acid positions 184 and 185 at the Kex2 cleavage site present in the native pro-protein sequence. The pH optimum of Tc-LysN was determined to be 5.0 while it maintained ≥60% activity between pH values 4.5—7.5 and ≥30% activity between pH values 8.5—10.0, indicating its broad applicability. The temperature maximum of Tc-LysN was determined to be 60 °C. After 18 h of incubation at 80 °C, Tc-LysN still retained ~20% activity. Organic solvents such as methanol and acetonitrile, at concentrations as high as 40% (v/v), were found to enhance Tc-LysN’s activity up to ~100% and ~50%, respectively. Tc-LysN’s thermostability, ability to withstand up to 8 M urea, tolerance to high concentrations of organic solvents, and an acidic pH optimum make it a viable candidate to be employed in proteomics workflows in which alkaline conditions might pose a challenge. The nano-LC-MS/MS analysis revealed bovine serum albumin (BSA)’s sequence coverage of 84% using Tc-LysN which was comparable to the sequence coverage of 90% by trypsin peptides.
This review provides an overview on the production and analysis techniques of antioxidative peptides from food proteins. Regarding the production of antioxidative peptides, interlinked factors must be considered. Depending on the protein substrate, different peptidases or peptidase systems containing multiple enzymes as well as a specific production process must be chosen. The antioxidative peptides might be produced in a batch process including multiple pre- and post-treatments, besides the hydrolyses with peptidases itself. As an alternative, the potential of continuous production systems is discussed in this review. Furthermore, robust analyses tools are needed to gain control of the process and final product properties. With no standardized methodology available for antioxidative peptide evaluation, pros and cons of various strategies for peptide separation and antioxidative measurement are discussed in this review. Therefore, this review provides a roadmap for antioxidative peptide generation from various sources for research and development as well as for potential industrial use.
Phytases are widely used food and feed enzymes to improve phosphate availability and reduce anti-nutritional factors. Despite the benefits, enzyme usage is restricted by the harsh conditions in a gastrointestinal tract (pH 2–6) and feed pelleting conditions at high temperatures (60–90 °C). The commercially available phytase Quantum® Blue has been immobilized as CLEAs using glutardialdehyde and soy protein resulting in a residual activity of 33%. The influence of the precipitating agent, precipitant concentration, cross-linker concentration and cross-linking time, sodium borohydride as well as the proteic feeders gluten, soy protein and bovine serum albumin (BSA) has been optimized. The best conditions were 90% (v/v) ethyl lactate as precipitating reagent, 100 mM glutardialdehyde and a soy protein concentration of 227 mg/L with a cross-linking time of 1 h. The intrinsically stable phytase remained its high thermal stability and temperature optimum. The phytase-CLEA achieved a 425% increase of residual activity under harsh acidic conditions between pH 2.2 and 3.5 compared to the free enzyme. The free and immobilized phytase were deployed in an in vitro assay simulating the acidic conditions in the gizzard of poultry at pH 2. The hydrolysis of phytate was monitored using a novel high-performance thin-layer chromatography (HPTLC) analysis and DAD scanner to study the InsPx fingerprint. All lower inositol phosphate pools (InsP1–InsP6) and free phosphate were separated and analyzed. The phytase-CLEA efficiently released 80% of the total phosphate within 180 min, whereas the free enzyme only released 6% in the same time under the same conditions.
Enzyme‐assisted HPTLC method for the simultaneous analysis of inositol phosphates and phosphate
(2023)
Background
The analysis of myo‐inositol phosphates (InsPx) released by phytases during phytic acid degradation is challenging and time‐consuming, particularly in terms of sample preparation, isomer separation, and detection. However, a fast and robust analysis method is crucial when screening for phytases during protein engineering approaches, which result in a large number of samples, to ensure reliable identification of promising novel enzymes or target variants with improved characteristics, for example, pH range, thermal stability, and phosphate release kinetics.
Results
The simultaneous analysis of several InsPx (InsP1‐InsP4 and InsP5 + 6) as well as free phosphate was established on cellulose HPTLC plates using a buffered mobile phase. Inositol phosphates were subsequently stained using a novel enzyme‐assisted staining procedure. Immobilized InsPx were hydrolyzed by a phytase solution of Quantum® Blueliquid 5G followed by a molybdate reagent derivatization. Resulting blue zones were captured by DAD scan. The method shows good repeatability (intra‐day and intra‐lab) with maximum deviations of the Rf value of 0.01. The HPTLC method was applied to three commercially available phytases at two pH levels relevant to the gastrointestinal tract of poultry (pH 5.5 and pH 3.6) to observe their phytate degradation pattern and thus visualize their InsPx fingerprint.
Conclusion
This HPTLC method presents a semi‐high‐throughput analysis for the simultaneous analysis of phytic acid and the resulting lower inositol phosphates after its enzymatic hydrolysis and is also an effective tool to visualize the InsPx fingerprints and possible accumulations of inositol phosphates.