Evaluation of ultrasound velocity measurements for estimating protease activities using casein as substrate

Abstract

Ultrasonic resonator technology (URT) was compared with the well established UV-Vis/ninhydrin assay to estimate protease activities in defined buffer systems. Hydrolysis of casein was measured using subtilisin, trypsin, halophilic protease from Haloferax mediterranei and Bacillus lentus alkaline protease. Sensitivity, reproducibility, working range as well as the limit of detection and the limit of quantification were comparable for both methods. Salt concentrations (0.5 M NaCl) interfered with the URT method. The quantification of protease activity by URT was possible when the product concentration measured by the UV-Vis/ninhydrin assay was correlated to the corresponding ultrasonic velocity signals.

Control of Tetranychus urticae Koch by extracts of three essential oils of chamomile, marjoram and Eucalyptus

“…The decrease in proteinase enzyme which is involved in the biological system of defense proves the presence of proteinase inhibitor in the extracts as cited by Manns, Merijn and Azzouz et al [18,24,25] who suggest that the extracts can induce defense gene expression of proteinase inhibitor activity. Proteinase inhibitors are proteins that inhibit digestive enzymes in the gut of arthropod herbivores, which can reduce their growth, reproduction.…”

“…Acid phosphatase and alkaline phosphatase were determined according to the method described by Manns et al [18] . Protease activity was determined according to ElSharabasy [19] by casein digestion methods.…”

Microbial proteases and their applications

“…For example, prior information on the structure of the enzyme is needed to perform ELISA-based assays ( Blair and McDowell, 1995 ). For ultrasonic resolver technology, a different analytical method is needed and must be first correlated to the corresponding ultrasonic velocity signals in advance ( Born et al, 2010 ). These methods will not be introduced in detail here.…”

Advances in Analysis of Milk Proteases Activity at Surfaces and in a Volume by Acoustic Methods

“…The “Method 2” calibration involves the parallel ultrasonic velocity measurement with a discontinuous orthogonal method that measures the degree of hydrolysis at each time point. Orthogonal methods that have been utilized in the calibration of ultrasonic profiles of protein hydrolysis include the conjugation of fluorescent dye, boron-dipyrromethene (BODIPY), with casein aggregates for fluorescence measurements [ 110 ], ninhydrin [ 110 , 111 ] and TNBS methods [ 73 , 104 ] for UV-Vis measurements of the hydrolysis of bovine casein by trypsin and β-lactoglobulin by α-chymotrypsin and β-casein by trypsin, respectively. The TNBS method is considered a milder and more reliable reference method to ultrasonic calibration than ninhydrin.…”

“…The ultrasonic attenuation data at 15 MHz showed the highest change due to the ultrasonic wave scattering effect, which is a function of the particle radius. Born et al [ 111 ] ultrasonically monitored the trypsin hydrolysis of β-casein in phosphate buffer using ultrasonic resonance technology (URT). The ultrasonic method is a single-frequency measurement at 10 MHz.…”