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Using a Recombinant Metagenomic Lipase for Enantiomeric Separation of Pharmaceutically Important Drug Intermediates
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Rakesh Kumar, Uttam Chand Banerjee, Jagdeep Kaur
Since the active site has been proposed to contain a conserved serine, PMSF was used as an inhibitor. To our surprise, PMSF did not inhibit the LipR1 lipase activity (Table 3.3). We observed temperature dependent inhibition with PMSF. At 55°C, LipR1 protein loses its 40–50% activity even with 1 mM of PMSF. Lipases have been shown to remain active in presence of PMSF in some cases (Li and Zhang, 2005; Soliman et al., 2007). It is very likely that active site serine is somehow inaccessible to PMSF yet at higher temperature some conformational changes in the protein makes the catalytic serine accessible to the inhibitor. Contrary to this DEPC, a histidine modifier inhibited the enzyme activity at very low concentration suggesting the easy accessibility of the catalytic histidine residue to DEPC. Eserine, an esterase’s inhibitor did not affect the enzyme activity, which suggested that it is a true lipase.
Evaluating the Effectiveness of Metal Pollution Controls in a Smelter by Using Metallothionein and Other Biochemical Responses in Fish
Published in Michael C. Newman, Alan W. McIntosh, Metal Ecotoxicology, 2020
John F. Klaverkamp, Michael D. Dutton, Henry S. Majewski, Robert V. Hunt, Laurie J. Wesson
In 1986, pooled samples of liver and kidney of suckers from each lake were homogenized using a Polytron in two volumes of 10 nM phosphate buffer, pH 7.6, with 5 nM 2-mercaptoethanol, 0.02% (w/v) sodium azide, and 0.15 M KCl. Phenylmethylsulfonylfluoride (PMSF) was added to the mixture (0.1 M) as a protease inhibitor. Homogenates were centrifuged at 30,000 g for 30 min at 4°C. The surface lipid layer was removed from the surface of the supernatant.
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.
Purification, biochemical, and thermal properties of fibrinolytic enzyme secreted by Bacillus cereus SRM-001
Published in Preparative Biochemistry and Biotechnology, 2018
Manoj Kumar Narasimhan, Selvarajan Ethiraj, Tamilarasan Krishnamurthi, Mathur Rajesh
The effect of inhibitors was studied with 5 mM EDTA, 5 mM 2-mercaptoethanol, and 5 mM PMSF. EDTA chelates metal ions inhibiting metalloproteases. 2-Mercaptoethanol reduces disulfide bonds to disrupt the activity of enzymes. However, it may also reduce the active site thiol residues enhancing cysteine proteases activity. PMSF irreversibly sulfonates the active site serine residue to inhibit serine protease activity. The results show that residual FEA was found to be unaffected in the presence of EDTA and 2-mercaptoethanol, suggesting that isolated enzyme is neither metalloprotease nor cysteine protease, respectively, and this also indicates that disulfide bonds are not necessary for enzyme activity (Table 2). The results also show that incubation with PMSF leads to inhibition of FEA, suggesting that the isolated enzyme is a serine protease. It is interesting to note that several investigators have also reported that most fibrinolytic enzymes are serine proteases.[525354]