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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.
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.