A highly stable Cu/Zn superoxide dismutase from Withania somnifera plant: gene cloning, expression and characterization of the recombinant protein

Chen

South African Journal of Botany

et al. 2015

a b s t r a c t Edited by E BalazsKeywords: Pepper Cytoplasmic male sterility Protein Identification Two dimension difference gel electrophoresis Pepper (Capsicum annuum L.) is a globally important and often cross-pollinated vegetable that exhibits significant heterosis. Though cytoplasmic male sterility (CMS) has an important value for pepper hybrid production, its molecular mechanism remains unclear. To explore the molecular mechanism of CMS in pepper at the proteome level, we examined the anther proteomes of a cytoplasmic male sterile line FS1030A and its maintainer line FS1030B by transmission electronic microscopy (TEM) during abortion at the microspore mother cell stage. Two dimension difference gel electrophoresis (2D-DIGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses were used to determine differentially expressed proteins. About 1070 anther protein spots were detected on each gel, and 13 protein spots were expressed differently between FS1030A and FS1030B (p b 0.05). Twelve of the thirteen protein spots (92.3%) were successfully identified by NCBInr as nine proteins, namely down-regulated triosephosphate isomerase, actin, glutathione S-transferase (GST), Cu/Zn superoxide dismutase (Cu/Zn-SOD), and up-regulated branched-chain α-keto acid dehydrogenase E3 subunit, ketol-acid reductoisomerase (KARI), aspartic protease, and nascent polypeptides. The low-expression of triosephosphate isomerase involved in glycolysis impacts oxidative phosphorylation significantly and ultimately results in a reduction in ATP production, and over-expression of aspartic protease blocking normal programmed cell death (PCD) played important roles in the occurrence of CMS-FS1030A. This is the first study on pepper CMS using a proteomic approach. These findings provide a good basis for further studies of CMS in pepper.

Section: Discussionsupporting

confidence: 76%

Identification of proteins associated with cytoplasmic male sterility in pepper ( Capsicum annuum L.)

Chen

South African Journal of Botany

et al. 2015

Zeitschrift Für Naturforschung C

“…However, about 49% of the activity were lost when the pH was above 9, which was similar to the other reported Cu/Zn-SODs in plant species (Sheng et al, 2004;He et al, 2008). This pH tolerance range of JcCu/Zn-SOD was much broader than that of Cu/Zn-SOD from some other species (Madanala et al, 2011). As shown in Fig.…”

Section: Thermostability Ph Stability and Effects Of Various Reagensupporting

confidence: 88%

“…This was different from other Cu/Zn-SODs. The molecular mass of many homodimeric plant Cu/Zn-SODs is in the range of 28 − 34 kDa (Haddad and Yuan, 2005;Madanala et al, 2011).…”

Section: Determination Of Molecular Mass and Isoelectric Pointmentioning

confidence: 99%

Cloning, Expression, and Purification of a Cu/Zn Superoxide Dismutase from Jatropha curcas

Chen

Gao²,

Cai³

et al. 2013

We report cDNA cloning, expression, purifi cation, and characterization of a novel Cu/ Zn superoxide dismutase (SOD) from Jatropha curcas leaves. The full-length cDNA of this SOD contained a 496-bp open-reading frame (ORF) encoding 162 amino acid residues. The recombinant plasmid containing the SOD coding sequence was introduced into Escherichia coli, and the SOD was expressed as a fusion protein. The recombinant SOD was purifi ed from a high-density fed-batch culture using a combination of immobilized metal ion affi nity chromatography (IMAC) and Sephadex G25 desalting chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-fl ight mass spectrometry (MALDI-TOF MS) analysis indicated that the recombinant SOD was a monomeric protein with a molecular mass of approximately 16.4 kDa. Isoelectric focusing showed that this SOD was a basic protein with pI values of 7. 04, 7.33, 8.62, and 8.77. The activity of the SOD was stable at 70 °C for 10 min, and in a broad pH range from 4 to 9. The presence of urea (up to 8 M), guanidinium chloride (up to 6 M), and 2-mercaptoethanol (up to 8 mM) had little effect on the activity. The activity decreased gradually with increasing concentrations of imidazole, hydrogen peroxide, and ethylenediaminetetraacetic acid (EDTA). Atomic absorption spectrometry showed the presence of 0.239 copper and 0.258 zinc atoms, respectively, in the SOD polypeptide.

“…Also, the enhanced SOD on S. tuberosa roots is thermostable till 40°C higher than the purified SOD from wheat seedlings (Lai et al, ). Cu‐Zn SODs from various sources showed varied level of resistance to heat denaturation, for example, the irreversible heat inactivation of Cu–Zn SOD from Radix lithospermi and Anas platyrhynchos domestica showed resistance till 60 and 70°C (Haddad & Yuan, ), whereas from P. atrosanguinea (Kumar, Dutt, Bagler, Ahuja, & Kumar, ), Jatropha curcas (Kaur et al, ) , Trigonella foenum‐graecum, Myristica fragrans, Cuminum cyminum (Mahapatra, Kanagarethinam, & Chaudhary, ) , Zingiber officinale (Chaudhary, Kaur, Jabalia, & Bansal, ), and W. somnifera (Madanala et al, ) show high resistance up to 80°C. It has been noted that seeds from Radix lethospermi has highly unstable thermal properties, unlike Cu–Zn SODs reported to be resistant to thermal inactivation (Roe et al, ; Tainer, Getzoff, Beem, Richardson, & Richardson, ).…”

Section: Resultsmentioning

confidence: 99%

Juglans regia and Ribes nigrum as potential nutraceuticals: Source of thermostable superoxide dismutase enzyme

et al. 2019

In the present study, superoxide dismutase (SOD) extracted from dry fruits; Juglans regia (Walnut; W) and Ribes nigrum (Munakka; M) was partially purified into 0%–40% and 40%–80% fractions based on ammonium sulfate saturation levels. The partially purified fractions (0%–40%) exhibited purification level of 3.09‐ (W) and 3.22‐ (M) fold with specific activity 79.32 Umg−1 (W) and 125.23 Umg−1 (M). SOD from both the sources was found to be thermally stable, that is, 80°C (W) and 70°C (M). Kinetic studies showed Km values to be 3.33 mM (W) and 2.86 mM (M), whereas the activation energy (Ea) calculated as 24.52 KJ mol−1 (W) and 26.25 KJ mol−1 (M). Na+, Mn2+, and Ba2+ ions acted as potential inhibitors, whereas Fe2+ stimulated SOD from both the sources. Among these metal ions, Na+ exhibited uncompetitive inhibition in both cases; with Ki values of 0.7 mM (W) and 0.9 mM (M), suggesting the more prominent binding affinity and effectiveness. Practical applications Awareness need to be created among people for multifactorial health benefits of nutraceuticals in day‐to‐day life. Nutritional consumption from fruits, nuts, and vegetables safeguard against various maladies like cardiovascular diseases, diabetes, and cancers. Superoxide dismutase (EC 1.15.1.1) is a standout among the most critical metal‐containing enzymes that act as a main line of defense against oxidative stress. Antioxidant‐based drugs and formulations have been developed in the recent years and research is emphasized on its impact on oxidative stress levels. In this study, Juglans regia (W) and Ribes nigrum (M) were found to have thermostable SOD enzyme with excellent antioxidant properties. Thermal stability of an enzyme improves its significance making it industry friendly with therapeutically vital products, alongside their utilization as supplement in numerous therapeutic formulations.