Isolation, Characterization and Application of Protease Enzyme from Marine Bacteria

Protease constitutes the major group of catalytic enzymes which is involved in hydrolyzing peptide bond of proteins. Marine sediment sample were collected and protease producing bacterial isolates were identified by using casein as a substrate. The organisms were characterized by biochemical test and identified as Bacillus sp. In order to check for the production of protease enzyme, quantitative protease assay and Lowry’s method of protein estimation was carried out. The crude extract of protease was subjected for blood stain removal activity and the enzyme proved to be efficient which removed the stain in 15 min. The purpose of the current study is to isolate, identify, characterize and to carry out applications of protease enzyme from marine bacteria isolated from mangrove sediment samples.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp3 papain-like protease

The COVID-19 pandemic has emerged as the biggest life-threatening disease of this century. Whilst vaccination should provide a long-term solution, this is pitted against the constant threat of mutations in the virus rendering the current vaccines less effective. Consequently, small molecule antiviral agents would be extremely useful to complement the vaccination program. The causative agent of COVID-19 is a novel coronavirus, SARS-CoV-2, which encodes at least nine enzymatic activities that all have drug targeting potential. The papain-like protease (PLpro) contained in the nsp3 protein generates viral non-structural proteins from a polyprotein precursor, and cleaves ubiquitin and ISG protein conjugates. Here we describe the expression and purification of PLpro. We developed a protease assay that was used to screen a custom compound library from which we identified dihydrotanshinone I and Ro 08-2750 as compounds that inhibit PLpro in protease and isopeptidase assays and also inhibit viral replication in cell culture-based assays.

Development of a Bio-Layer Interferometry-Based Protease Assay Using HIV-1 Protease as a Model

Proteolytic enzymes have great significance in medicine and the pharmaceutical industry and are applied in multiple fields of life sciences. Therefore, cost-efficient, reliable and sensitive real-time monitoring methods are highly desirable to measure protease activity. In this paper, we describe the development of a new experimental approach for investigation of proteolytic enzymes. The method was designed by the combination of recombinant fusion protein substrates and bio-layer interferometry (BLI). The protease (PR) of human immunodeficiency virus type 1 (HIV-1) was applied as model enzyme to set up and test the method. The principle of the assay is that the recombinant protein substrates immobilized to the surface of biosensor are specifically cleaved by the PR, and the substrate processing can be followed by measuring change in the layer thickness by optical measurement. We successfully used this method to detect the HIV-1 PR activity in real time, and the initial rate of the signal decrease was found to be proportional to the enzyme activity. Substrates representing wild-type and modified cleavage sites were designed to study HIV-1 PR’s specificity, and the BLI-based measurements showed differential cleavage efficiency of the substrates, which was proven by enzyme kinetic measurements. We applied this BLI-based assay to experimentally confirm the existence of extended binding sites at the surface of HIV-1 PR. We found the measurements may be performed using lysates of cells expressing the fusion protein, without primary purification of the substrate. The designed BLI-based protease assay is high-throughput-compatible and enables real-time and small-volume measurements, thus providing a new and versatile approach to study proteolytic enzymes.

Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp3 Papain-like Protease

The COVID-19 pandemic has emerged as the biggest life-threatening disease of this century. Whilst vaccination should provide a long-term solution, this is pitted against the constant threat of mutations in the virus rendering the current vaccines less effective. Consequently, small molecule antiviral agents would be extremely useful to complement the vaccination program. The causative agent of COVID-19 is a novel coronavirus, SARS-CoV-2, which encodes at least nine enzymatic activities that all have drug targeting potential. The papain-like protease (PLpro) contained in the nsp3 protein generates viral non-structural proteins from a polyprotein precursor, and cleaves ubiquitin and ISG protein conjugates. Here we describe the expression and purification of PLpro. We developed a protease assay that was used to screen a custom chemical library from which we identified Dihydrotanshinone I and Ro 08-2750 as compounds that inhibit PLpro in protease and isopeptidase assays and also inhibit viral replication in cell culture-based assays.

Dinitrosyl Iron Complexes (DNICs) as Inhibitors of the SARS-CoV-2 Main Protease

By repurposing DNICs designed for other medicinal purposes, the possibility of protease inhibition was investigated in silico using AutoDock 4.2.6 (AD4) and in vitro via a FRET protease assay. AD4...

Isolation of Protease Inhibitors from Cyanobacteriaum Microcystis sp. Strain BM 25 Which Inhibits Major Digestive Enzyme in Daphnia magna

Cyanobacterial mass developments in eutrophic ponds and lakes are a major concern for lake management, as many cyanobacteria produce a huge variety of toxic secondary metabolites, e.g. microcystins. The aim of this research was to culture a strain of the cyanobacterium Microcystis sp strain BM25, to observe its biomass production and to isolate and purify protease inhibitors from this cyanobacterial biomass. Different secondary metabolites were isolated following a standard bioassay-guideline.  Isolation was performed, with an enzymatic protease assay as bioassay. High performance liquid chromatography was used to identify different fractions of secondary metabolite from the strain BM25. Moreover, protease homogenates were isolated from Daphnia magna in order to test the inhibitors against naturally occurring major digestive proteases trypsin and chymotrypsin. It was measured that 60% MeOH and the 80% MeOH C18-SPE fraction inhibits chymotrypsin activity 98% (6 nmol pNA min-1 mg-1) and 99 % (4 nmol pNA min-1 mg-1), respectively. In contrast, trypsin activity was not inhibited by methanolic extracts of this cyanobacterium strain.

Protein degradability of grassland forage under simulated rotational spring grazing

A cutting experiment was conducted to analyze the changes in the crude protein (CP) fraction content and in the estimated ruminal protein degradability of forage, obtained in conditions of simulated rotational spring grazing on permanent grassland. The field trial was conducted on permanent pasture during 2015 and included three cuttings as a simulated rotational spring grazing. For determination of protein degradability of pasture forage, the fractionation of the CP according to Cornell Net Carbohydrate and Protein System (CNCPS v6.5) and the Streptomyces griseus protease assay were used. Relative to CP, no significant differences were found among cuts for ammonia N content (A1 fraction) and for protein fraction C which is completely unavailable to the animals. Values for soluble true protein (A2 fraction) and cell wall-associated protein, which is acid detergent soluble (B2), were significantly increased (p<0.05) while a significant reduction (p<0.05) of the moderately degradable protein (B1) content was determined during the growing season. The lower rumen degradable protein (RDP) content of grassland herbage was obtained in the second cut which was significant (p<0.05) according to the CNCPS procedure. Obtained high solubility and degradability of CP in pasture require adequate content of readily available carbohydrates in rations for grazing ruminants to provide efficient utilization of consumed protein.