Fluorescence quenching, structural and unfolding studies of a purified cysteine protease, ZCPG from Zingiber montanum rhizome

Jamir, Kizukala; Seshagirirao, Kottapalli

doi:10.1016/j.ijbiomac.2017.08.019

Cited by 11 publications

(9 citation statements)

References 15 publications

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“…Nevertheless, the loop content in AP is 7.2% less than in NP I. Ordered structures (α‐helix and β‐strand) have been reported to be helpful for stabilizing an enzyme structure suffering from thermal denaturation . Concurrently, loop regions in the spatial structure of proteins are apt to unfold and destabilize because of their great flexibility under the externally detrimental conditions .…”

Section: Resultsmentioning

confidence: 99%

“…Ordered structures ( -helix and -strand) have been reported to be helpful for stabilizing an enzyme structure suffering from thermal denaturation. 22,23 Concurrently, loop regions in the spatial structure of proteins are apt to unfold and destabilize because of their great flexibility under the externally detrimental conditions. 21,24 Therefore, the more stable spatial structure of AP could be due to its higher contents of -helix and -strand and smaller content of loop regions than those in NP I.…”

Section: Secondary Structure Acidic Amino Acid Residues and Hydrophomentioning

confidence: 99%

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Separation, biochemical characterization and salt‐tolerant mechanisms of alkaline protease from Aspergillus oryzae

et al. 2019

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BACKGROUND The salt tolerance of proteases secreted by Aspergillus oryzae 3.042 closely relates to the utilization of raw materials and the quality of soy sauce. However, little is known about the salt‐tolerant proteases and their salt‐tolerant mechanisms. RESULTS In this study, we isolated and identified a salt‐tolerant alkaline protease (AP, approximately 29 kDa) produced by A. oryzae 3.042. It was considered as a metal‐ion‐independent serine protease. The optimum and stable pH values were both pH 9.0 and the optimum temperature was 40 °C. Over 20% relative activity of AP remained in the presence of 3.0 mol L−1 NaCl after 7 days, but its Km and Vmax were only mildly influenced by the presence of 3.0 mol L−1 NaCl, indicating its outstanding salt tolerance. Furthermore, AP was more stable than non‐salt‐tolerant protease at high salinity. The salt‐tolerant mechanisms of AP could be due to more salt bridges, higher proportion of ordered secondary structures and stronger hydrophobic amino acid residues in the interior. CONCLUSIONS The above results are vital for maintaining, activating and/or modulating the activity of AP in high‐salt environments. They would also provide theoretical guidance for the modification of AP and the engineering of A. oryzae 3.042 so as to secrete more AP. © 2018 Society of Chemical Industry

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Section: Resultsmentioning

confidence: 99%

Section: Secondary Structure Acidic Amino Acid Residues and Hydrophomentioning

confidence: 99%

Separation, biochemical characterization and salt‐tolerant mechanisms of alkaline protease from Aspergillus oryzae

et al. 2019

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“…The UV‐visible absorption spectra (Fig. 1A) contain two absorption peaks at 220 and 275 nm, which represented the peptide groups and aromatic side chains, respectively 24 . With rising pH values, the intensity of the absorption peak at 275 nm increased first (pH 4 to 5) and decreased later (pH 5 to 8), which may be a result of the extension of aromatic side chains at a certain pH value, and then the hydrogen bond between protease and substrate can be established by more effective interactions 9 .…”

Section: Resultsmentioning

confidence: 99%

Effects of temperature and pH on the structure of a metalloprotease from Lactobacillus fermentumR6 isolated from Harbin dry sausages and molecular docking between protease and meat protein

et al. 2021

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BACKGROUND Microbial protease can interact with meat protein in fermented meat products at a certain pH and temperature. To investigate the effects of various pH values and temperatures on the structural characteristics of Lactobacillus fermentum R6 protease, which was isolated from Harbin dry sausages, spectroscopy techniques and molecular dynamics were utilized to evaluate structural changes. RESULTS The protease exhibited a stable spatial structure at pH 7 and 40 °C, and the extension of the protease structure was also promoted. Although the structure of the protease could be changed or destroyed by pH 8 and 70 °C, it was mainly determined by the changes of secondary and tertiary structures such as α‐helix, β‐sheet, β‐turn and random coil. In addition, carbonyl vibration, ‐NH vibration, C–H stretching vibration and disulphide bonds were present in L. fermentum R6 protease under various pH and temperature conditions. Molecular docking showed that the protease can interact with myosin light chain, myosin heavy chain, actin and myoglobin. CONCLUSION The protease can maintain stable structure and interact with meat protein, which reflected certain application prospects in the fermentation of Harbin dry sausages. © 2021 Society of Chemical Industry

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“…The decrease in fluorescence intensity at pH 3 may be due to the presence of the molten globule (MG) intermediate state in the protease 1147. Similarly, lipase-3646 [24] and cysteine protease ZCPG [63] were shown that exhibit the behavior of the MG state at pH 3 and pH 2, respectively. The overall results were in agreement with those obtained for temperature and pH stability and the MD simulation in the presence of selected surfactants.…”

Section: Plos Onementioning

confidence: 94%

Structural and biochemical characterization of a novel thermophilic Coh01147 protease

et al. 2020

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Proteases play an essential role in living organisms and represent one of the largest groups of industrial enzymes. The aim of this work was recombinant production and characterization of a newly identified thermostable protease 1147 from thermophilum indigenous Cohnella sp. A01. Phylogenetic tree analysis showed that protease 1147 is closely related to the cysteine proteases from DJ-1/ThiJ/PfpI superfamily, with the conserved catalytic tetrad. Structural prediction using MODELLER 9v7 indicated that protease 1147 has an overall α/β sandwich tertiary structure. The gene of protease 1147 was cloned and expressed in Escherichia coli (E. coli) BL21. The recombinant protease 1147 appeared as a homogenous band of 18 kDa in SDS-PAGE, which was verified by western blot and zymography. The recombinant protein was purified with a yield of approximately 88% in a single step using Ni-NTA affinity chromatography. Furthermore, a rapid one-step thermal shock procedure was successfully implemented to purify the protein with a yield of 73%. Using casein as the substrate, K m , and k cat , k ca t/K m values of 13.72 mM, 3.143 × 10 −3 (s-1), and 0.381 (M-1 S-1) were obtained, respectively. The maximum protease activity was detected at pH = 7 and 60˚C with the inactivation rate constant (kin) of 2.10 × 10-3 (m-1), and half-life (t 1/2) of 330.07 min. Protease 1147 exhibited excellent stability to organic solvent, metal ions, and 1% SDS. The protease activity was significantly enhanced by Tween 20 and Tween 80 and suppressed by cysteine protease specific inhibitors. Docking results and molecular dynamics (MD) simulation revealed that Tween 20 interacted with protease 1147 via hydrogen bonds and made the structure more stable. CD and fluorescence spectra indicated structural changes taking place at 100˚C, very basic and acidic pH, and in the presence of Tween 20. These properties make this newly characterized protease a potential candidate for various biotechnological applications.

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Fluorescence quenching, structural and unfolding studies of a purified cysteine protease, ZCPG from Zingiber montanum rhizome

Cited by 11 publications

References 15 publications

Separation, biochemical characterization and salt‐tolerant mechanisms of alkaline protease from Aspergillus oryzae

Separation, biochemical characterization and salt‐tolerant mechanisms of alkaline protease from Aspergillus oryzae

Effects of temperature and pH on the structure of a metalloprotease from Lactobacillus fermentumR6 isolated from Harbin dry sausages and molecular docking between protease and meat protein

Structural and biochemical characterization of a novel thermophilic Coh01147 protease

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