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.     

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.     

SZ蛋白酶对羊毛的减量和防毡缩性能的影响

在用蛋白酶对羊毛织物进行改性处理的研究中，获得一种对羊毛具有独特处理效果的蛋白酶ＳＺ。本文主要就ＳＺ蛋白酶对羊毛的减量特性及其防毡性能的影响进行研究。ＳＺ蛋白酶采用Ｐｅｒｚｙｍ工艺对羊毛进行减量处理，可有效地防止羊毛毡缩。减量处理对羊毛的损伤较小，实际应用可以被接受。研究表明，羊毛的减量防毡缩处理并不需要完全去除鳞片，对于有效的减量，去除２／３的鳞片可获得最佳的减量处理效果。     

Use of commercial protease preparations to reduce protein and lipid content of maize starch

A method was developed to produce pure maize starch from maize flour using a protease processing step, and additional salt washing and sulphite steeping steps. A range of commercial protease enzymes were evaluated for this purpose. The laboratory scale procedure that was developed, using one protease in particular (Promod P25P, thermolysin), produced relatively pure starch (<0.45% protein). Using the same procedure, but applying to starches which had been produced in advance using traditional wet milling, starch protein contents could be reduced further by 25–50% with the lipid content reduced by up to 25%. The amount of starch damage was minimal using this approach (<1%). This procedure could be developed industrially for a ‘greener approach’ to starch extraction – although it may still be necessary to incorporate sulphite steeping stages to facilitate protein solubilisation and extraction.