Hydrophobic Tagged Dihydrofolate Reductase for Creating Misfolded Glycoprotein Mimetics

In the endoplasmic reticulum (ER), nascent glycoproteins that have not acquired the native conformation are either repaired or sorted for degradation by specific quality‐control systems composed by various proteins. Among them, UDP‐glucose:glycoprotein glucosyltransferase (UGGT) serves as a folding sensor in the ER. However, the molecular mechanism of its recognition remains obscure. This study used pseudo‐misfolded glycoproteins, comprising a modified dihydrofolate reductase with artificial pyrene–cysteine moiety on the protein surface (pDHFR) and Man₉GlcNAc₂‐methotrexate (M9‐MTX). All five M9‐MTX/pDHFR complexes, with a pyrene group at different positions, were found to be good substrates of UGGT, irrespective of the site of pyrene modification. These results suggest UGGT's mode of substrate recognition is fuzzy, thus allowing various glycoproteins to be accommodated in the folding cycle.     

High‐pressure microfluidisation pretreatment disaggregate peanut protein isolates to prepare antihypertensive peptide fractions

High‐pressure microfluidisation (HPM) pretreatment was applied to increase in vitro antihypertensive activity of peanut peptide fractions (PPF). The morphology of protein in aqueous dispersion revealed that peanut protein isolate (PPI) disaggregated at relatively low pressure (≤120 MPa) and re‐aggregated at relatively high pressures (150–210 MPa). The treated pressure of 120 MPa could lead to the most disaggregation of PPI. Small peptides contents, trichloroacetic acid‐nitrogen soluble index (TCA‐NSI) and degree of hydrolysis (DH) of peanut protein hydrolysates (PPH) all reached the highest at 120 MPa. Consequently, it possessed the highest angiotensin converting enzyme (ACE) and renin inhibitory activity. The highest surface hydrophobicity occurred at 120 MPa pretreatment samples. Thirty‐nine oligopeptides at 120 MPa pretreatment were identified by ultra‐performance liquid chromatography‐quadrupole time‐of‐flight (UPLC‐Q‐TOF) mass spectrometer combined with Progenesis QI for Proteomics software compared with 29 and 35 at control and 210 MPa, respectively. This meant that disaggregation of PPI at 120 MPa resulted in the release of new hydrophobic peptide.     

Development of lateral flow immunoassay for rapid control and quantification of the presence of the colorant Sudan I in spices and seafood

The lateral flow immunoassay for colorant Sudan I detection was developed using the monoclonal antibody to Sudan I conjugated with gold nanoparticles. Conditions of the assay were optimized for both qualitative control and quantification of Sudan I in food matrixes with minimal dilution of the extracting media (final content of methanol – 50%). In the case of photometric registration, the limit of Sudan I detection in extracts was 2.5 ng/mL (which corresponds to 10 μg/kg of food and spices), and the working range of determined concentrations was 13–240 ng/mL. The average error of measurements varied from 5.5 to 16.9%. The visual limit of detection, which is the minimum concentration causing absence of visible coloration at the test zone, was 1 μg/mL. The assay efficiency was confirmed by its application to determine Sudan I content in highly colored spices (turmeric, curry) and seafood (caviar, mussels, fish). Thus, the developed assay is suitable as a rapid, inexpensive tool for revealing Sudan I quantification by mobile laboratories and in out-of-laboratory conditions.     

Production of soya milk containing low flatulence‐causing oligosaccharides in a packed bed reactor using immobilised α‐galactosidase

Peanut α‐galactosidase was immobilised in calcium alginate beads and used to hydrolyse the flatulence‐causing oligosaccharides, raffinose and stachyose, in soya milk in batch and in packed bed reactor with recycle. The immobilised enzyme exhibited a slightly lower activity than the free enzyme. The activity yield of immobilised α‐galactosidase was 75.1% and the immobilisation yield was 82.6%. Batch hydrolysis using immobilised enzyme at 55 °C resulted in 96% reduction in the oligosaccharides after 12 h. For the continuous process, a packed bed reactor with recycle was used. More than 98% of the oligosaccharides were hydrolysed after 6 h of reaction at 55 °C. The immobilised enzyme also proved to be stable up to three repeated hydrolysis reactions.     

Functional Peptide Monolayers at Interfaces

Amphiphilic peptides can be designed to form ordered supramolecular structures at hydrophilic-hydrophobic interfaces. These systems rely on the ability of peptides to fold into certain secondary structures at interfaces. This review focuses on the design of amphiphilic β-sheet peptide assemblies in monolayers at interfaces, and their relevance to inducing mineralization and interactions with specific ions. In addition, the review discusses recent studies demonstrating the applicability of designed amphiphilic β-sheet peptides to detection of specific small molecules and to elucidating intermolecular interactions relevant to drug delivery and enzyme catalysis systems.     

层状氢氧化氨基苯甲酸锌固定化辣根过氧化物酶及其催化性质

利用层状氢氧化氨基苯甲酸锌对辣根过氧化物酶(HRP)进行固定化实验,通过紫外分析探讨了固定化效果与HRP质量浓度、体系p H的关系。利用Lineweaver-Burk双倒数做图法测定了固定化HRP的动力学常数,考察了固定化HRP的储存稳定性和重复利用性。实验结果表明,在体系p H=8.0、HRP质量浓度为1.0 mg/m L的条件下,层状氢氧化氨基苯甲酸锌对HRP的饱和固载量为100 mg/g,最大比活力为60.27 U/mg。固定化HRP的米氏常数(Km)、催化常数(Kcat)、酶活性(X)分别为3.23 mmol/L,42.5 s-1,57.5 U/mg。与游离HRP相比,固定化HRP的Km提高了44.2%,Kcat下降了74.0%,X值下降了74.4%。储存40 d后,固定化HRP的活性为游离HRP的5.35倍。循环利用8次后,固定化HRP的活性保留率仍为79.6%。