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Production, Purification, and Application of the Microbial Enzymes
Published in Pankaj Bhatt, Industrial Applications of Microbial Enzymes, 2023
Anupam Pandey, Ankita H. Tripathi, Priyanka H. Tripathi
The extraction of the intracellular enzyme is more tedious as they require breakage of the cell wall. Various mechanical (e.g., bead milling, homogenization, ultra-sonication, osmotic shock) and chemical (e.g., acid and alkali treatments) techniques are the most familiar processes for breaking the cell wall. The enzymatic hydrolysis of the cell wall uses lytic enzymes, such as lyase. Esterase is an effective technique that can be used alone or in combination with physical and chemical treatments. After breaking the cell wall, enzymes present in the supernatant can be easily separated using centrifugation from unbroken cell pellets and large insoluble molecules. Secreted proteins that act as enzymes can be separated directly from the cell pellet using centrifugation for lab scale and by filtration for pilot scale purification. A cocktail of various protease inhibitors, such as EDTA, leupeptin, pepstatin A, and PMSF, has been used to prevent enzyme inactivation and degradation during protein extraction. The protocol for the purification of enzymes is largely based upon the need of the enzyme. For example, the alkaline protease, which is used as an ingredient in the detergent industry, requires partial fractionation compared to streptokinase, which has application in therapeutics needs 99% purity (Burgess, 2009).
Protocols for Key Steps in the Development of an Immunoassay
Published in Richard O’Kennedy, Caroline Murphy, Immunoassays, 2017
Caroline Murphy, Richard O’Kennedy
Protease inhibitors can be added to prevent protein degradation [16]. Serine protease inhibitors, for example, target proteases that break down peptide bonds in proteins where serine serves as the active site. Some examples of protease inhibitors (and their working concentrations) include: Serine proteases inhibitors – Phenylmethylsulphonyl fluoride (PMSF) 0.1–1 mM– Benzamidine 1 mM– Aprotinin 5 μg mL−1Acid protease inhibitor – Pepstatin A 1 μg mL−1Thiol protease inhibitor – Leupeptin 1 μg mL−1
Applications of Carbon Nanotubes in Biosensing and Nanomedicine
Published in James E. Morris, Kris Iniewski, Graphene, Carbon Nanotubes, and Nanostructures, 2017
In addition to small biomolecules, CNTs have also been used for the fabrication of bio-sensors for macrobiomolecules such as protein, DNA, etc. Yu and coworkers reported the use of CNTs for immunoassay [108]. In their work, CNTs were used both as electrodes that coupled primary antibodies (Ab1) and as “vectors” that hosted secondary antibodies (Ab2) and HRP. Amplified sensing signals resulted from the large surface area of CNTs, which can bind a large number of Ab1 on the electrode and a large number of HRP in the vectors. After the formation of the sandwich structure (Figure 8.8), the CNT-based electrode can be used for the detection of prostate-specific antigen (PSA) by measuring the electrochemical voltage derived from the reaction between the added H2O2 and the HRP on the CNTs. This approach could increase the detection sensitivity for PSA some 10–100 times compared with the commercial clinical immunoassays presently available [108]. Another CNT-based immunoassay via formation of the sandwich structure has also been reported [18,109–113]. Usually, CNTs were used for electrode modification and binding Ab1, and then on the other end of the sandwich, the signals were amplified by nanostructured materials or enzymes. Mahmoud and coworkers have developed biosensors for HIV-1 protease (HIV-1 PR) using CNT-based electrodes [114]. First, a gold electrode was modified with thiolated CNTs and gold nanoparticles. Thiol-modified ferrocene-pepstatin was then bound to the nanoparticles. The pepstatin can bind the protease molecule, which decreases the signal and shifts the oxidation potential for ferrocene by blocking penetration of the supporting electrolyte (Figure 8.9). An estimated detection limit of this electrode is ca. 0.8 pM [114]. Another strategy is to use electrochemical impedance spectroscopy to investigate the same electrode [115]. When protease binds to the ferrocene-pepstatin, the charge transfer resistance of the electrode is changed. These approaches can be used to perform competitive assays for protease inhibitor drugs because if the protease is bound to a drug, it will not bind the electrode. CNTs have also been used as a label for signal amplification on the far end of the sandwich structure in the immunoassay. For example, Lai and coworkers fabricated a GOx-functionalized Au nanoparticle/CNT nanocomposite as a label for signal amplification [116]. For the fabrication of the biosensor, first, colloidal Prussian blue, Au nanoparticles, and antibody 1 were coated layer by layer on carbon electrodes. Then, the GOx-functionalized nanocomposites modified with antibody 2 were used for the fabrication of a sandwich-type immunoassay. The signal was obtained by detecting the produced H2O2 by GOx-catalyzed reaction. The sensor exhibits detection limits of 1.4 and 2.2 pg ml–1 for carcinoembryonic antigen and α-fetoprotein, respectively [116].
Biochemical characterization of a partially purified protease from Aspergillus terreus 7461 and its application as an environmentally friendly dehairing agent for leather industry
Published in Preparative Biochemistry & Biotechnology, 2021
Emmly Ernesto de Lima, Daniel Guerra Franco, Rodrigo Mattos Silva Galeano, Nelciele Cavalieri de Alencar Guimarães, Douglas Chodi Masui, Giovana Cristina Giannesi, Fabiana Fonseca Zanoelo
Protease was strongly inhibited by PMSF, a synthetic serine protease inhibitor, widely used in biochemical studies to investigate the enzymatic mode of action.[48] Inhibition occurs due to sulfonation of the serine residue at the active site of the enzyme.[49,50] These results are similar to those described for A. terreus IJIRA 6.2[39], H. rhossiliensis OWVT-1[40], A. flavus[16] and A. brasiliensis.[36] On the other hand, the protease activity was not affected by the presence of the inhibitor pepstatin A, described in the literature as a competitive and reversible inhibitor of aspartic proteases.[51] Studies with the protease of P. aurantiogriseum have shown inhibition by PMSF and pepstatin A in 100 and 55%, respectively, suggesting that it is a serine protease with aspartic residues on its active site.[9] The data reported in this work confirm that the protease from A. terreus belongs to the class of serine proteases.
Evaluation of the milk clotting properties of an aspartic peptidase secreted by Rhizopus microsporus
Published in Preparative Biochemistry & Biotechnology, 2020
Ronivaldo Rodrigues da Silva, Tatiane Beltramini Souto, Nathalia Gonsales da Rosa, Lilian Caroline Gonçalves de Oliveira, Maria Aparecida Juliano, Luiz Juliano, Jose C. Rosa, Hamilton Cabral
Inhibition by Pepstatin A suggested the presence of aspartic acid at the active site. This characteristic, in addition to peptide sequencing, which identified homology with other peptidases from R. microsporus, are consistent with the enzyme belonging to the aspartic peptidase group. Analysis of peptide sequences of this peptidase through an NCBI database search revealed homology with other rhizopuspepsins of similar predicted molecular mass to R. microsporus, such as a putative rhizopuspepsin with mass of 27,522.20 Da and theoretical pI: 4.7 (GenBank: CEG74337.1); rhizopuspepsin II with a mass of 34,239.07 Da and theoretical pI: 4.60 (PRF: 1402278B); rhizopuspepsin with a mass of 37,102.38 Da and theoretical pI: 5.12 (GenBank: AAA33879.1); and rhizopuspepsin II with a mass of 41,351.52 Da and theoretical pI: 5.94 (XP_023470209.1).