Design and Characterization of a New pVII Combinatorial Phage Display Peptide Library for Protease Substrate Mining Using Factor VII Activating Protease (FSAP) as Model

We describe a novel, easy and efficient combinatorial phage display peptide substrate-mining method to map the substrate specificity of proteases. The peptide library is displayed on the pVII capsid of the M13 bacteriophage, which renders pIII necessary for infectivity and efficient retrieval, in an unmodified state. As capture module, the 3XFLAG was chosen due to its very high binding efficiency to anti-FLAG mAbs and its independency of any post-translational modification. This library was tested with Factor-VII activating protease (WT-FSAP) and its single-nucleotide polymorphism variant Marburg-I (MI)-FSAP. The WT-FSAP results confirmed the previously reported Arg/Lys centered FSAP cleavage site consensus as dominant, as well as reinforcing MI-FSAP as a loss-of-function mutant. Surprisingly, rare substrate clones devoid of basic amino acids were also identified. Indeed one of these peptides was cleaved as free peptide, thus suggesting a broader range of WT-FSAP substrates than previously anticipated.     

Further characterisation of protease inhibitors from pigeon pea (cajanus cajan (l) millsp) seeds

Cajanus trypsin inhibitor (CTI) and Cajanus trypsin-chymotrypsin inhibitor (CTCI) previously purified from cv TAT-10 were further characterised. The modification of the inhibitors revealed the presence of lysine at the trypsin reactive site in both CTI and CTCI. Modification of tyrosine at the reactive site of CTCI did not abolish chymotrypsin inhibition suggesting the presence of leucine or phenylalanine as reported in other chymotrypsin inhibitors. CTCI did not contain tryptophan. Dissociation constants for the inhibitors with bovine trypsin were in the region of 0.69 nmol (CTCI) and 0.029 nmol (CTI). Although the protease inhibitors lost their inhibitory activity on exposure to 2-mercaptoethanol they remained attached to the enzyme. The inhibitors were not very effective against the protease from Helicoverpa armigera which is a serious field pest of Cajanus. Dissociation constants for the inhibitors with the larval enzyme were in the region of 100 nmol.     

Structure-Activity Relationships of Benzamides and Isoindolines Designed as SARS-CoV Protease Inhibitors Effective against SARS-CoV-2

Inhibition of coronavirus (CoV)-encoded papain-like cysteine proteases (PL^pro) represents an attractive strategy to treat infections by these important human pathogens. Herein we report on structure-activity relationships (SAR) of the noncovalent active-site directed inhibitor (R)-5-amino-2-methyl-N-(1-(naphthalen-1-yl)ethyl) benzamide ( 2 b ), which is known to bind into the S3 and S4 pockets of the SARS-CoV PL^pro. Moreover, we report the discovery of isoindolines as a new class of potent PL^pro inhibitors. The studies also provide a deeper understanding of the binding modes of this inhibitor class. Importantly, the inhibitors were also confirmed to inhibit SARS-CoV-2 replication in cell culture suggesting that, due to the high structural similarities of the target proteases, inhibitors identified against SARS-CoV PL^pro are valuable starting points for the development of new pan-coronaviral inhibitors.     

‘Lipase and protease production of dairy Penicillium sp. on milk‐protein‐based solid substrates’

The lipolytic and proteolytic activity of Penicillium camemberti PC TT033 and Penicillium roqueforti PR G3, cultured on the whey solids or simulated cheese media, were compared under several pH reaction conditions. Lipolytic activity was higher when both strains had been cultured on the whey medium than on the simulated cheese medium, whereas proteolytic activity was less influenced by the culture medium. The relationship between the reaction pH and these enzyme activities was dependent on the culture medium, which suggested that the expression level and balance of isozyme rely on the culture substrate.     

Synthesis and characterization of a triple enzyme-inorganic hybrid nanoflower (TrpE@ihNF) as a combination of three pancreatic digestive enzymes amylase,protease and lipase

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Liquid-liquid extraction of protease from cold-adapted yeast Rhodotorula mucilaginosa L7 using biocompatible and biodegradable aqueous two-phase systems

This work aimed to optimize the extraction of an extracellular protease produced by the cold-adapted yeast Rhodotorula mucilaginosa L7 using aqueous two-phase systems (ATPS) comprising polyethylene glycol (PEG) and sodium citrate or sodium tartrate. First, the biocompatibility of the phase forming agents was assessed. The results obtained with PEG-2000, PEG-4000, and PEG-6000 demonstrated that even at large PEG concentrations (32 wt%) the protease maintains its activity after 3 h of reaction, whereas an increase in salt concentration provokes a gradual decrease in protease stability. Subsequently, the partitioning of the protease in both types of ATPS was assessed, evaluating the effect of temperature, molecular weight, and concentration of PEG on protease purification, using two 2³-full factorial designs. The best partitioning conditions were obtained in PEG-6000/sodium tartrate-based ATPS, at 30ºC (with a yield of 81.09 ± 0.66% and a purification factor of 2.51 ± 0.03). Thus, considering the biodegradable characteristics of the system, the PEG/sodium tartrate ATPS is a viable and economic low-resolution step in protease purification, with a strong potential for future industrial application.     

Effect of different treatments for the destabilization of coconut milk emulsion

Coconut milk is an emulsion which is stabilized by naturally occurring proteins. The main objective of the present work is to explore different methods employing thermal, pH, chilling, enzyme treatments and combination of enzyme treatments followed by chilling and thawing for effective destabilization of the coconut milk emulsion. Stability of emulsion is evaluated by measuring the creaming index and observed for the changes in structure of oil droplets, using phase contrast microscope. Combination of treatments (enzyme treatment at 37 °C followed by chilling and thawing) of coconut milk emulsion has resulted in highest yield of 94.5%. Physico-chemical properties and fatty acid compositions are evaluated for coconut oil obtained by combination of treatments and compared with that of commercial coconut oil. It is found that the oil obtained by combination of treatments is low with respect to free fatty acids and peroxide value and high in lauric acid content.     

酶法制备小分子大豆肽的蛋白酶筛选以及酶解条件优化

本研究对制备小分子大豆肽的蛋白酶进行筛选,并对酶解条件进行优化。综合考虑酶量对产物的水解度、平均分子量及游离氨基酸的影响,确定酶解的最佳条件为:温度55℃,pH值8.5,底物浓度为10%,分别加入1.5%的木瓜蛋白酶、菠萝蛋白酶、无花果蛋白酶和3942中性蛋白酶,调pH值至4.5,再分别加入2.5%的Fromase真菌蛋白酶和537酸性蛋白酶。结果显示,多酶复合水解可提高蛋白质降解率,并尽可能降低多肽的相对分子质量。     

Comparison of composition, enzyme activity and selected functional properties of potato proteins isolated from potato juice with two different expanded bed resins

Protein concentrates prepared by expanded bed adsorption (EBA) chromatography from industrial potato juice (PJ) were analysed for chemical composition, color, enzyme activities, thermal properties and selected functional properties (solubility and emulsifying stability). Two EBA multi-modal resins, MIMO I-45 and MIMO 1300 (UpFront Chromatography), were employed under various pH conditions resulting in four potato protein concentrates, A-D. Concentrate B contained an electrophoretically pure protease inhibitor fraction (20-21 kDa), whereas concentrate A, C and D contained both patatin (41 kDa) and protease inhibitors. The potato protein concentrates were explored for the presence of transitions from native to denatured states using differential scanning calorimetry (DSC). Concentrate C had lower heat of transition (ΔH) and T-onset than the other concentrates. The concentrate containing protease inhibitors exhibited the highest denaturation temperature and enthalpy. All concentrates differed significantly (P < 0.05) in color brightness, with concentrate B and D emerging as the brightest. The solubility of the concentrates was evaluated at pH 6 and pH 4.5. All concentrates had lower solubility at pH 4.5 than at pH 6 (70-80%). The stability of emulsions (1% protein, 20% oil, 0.08% xanthan gum) against creaming was analysed with a new method based on the Single electrode Capacitance Probe (SeCaP) technology. Small differences among concentrates were observed by the new SeCaP method.