Immobilization of Lepidium draba peroxidase on a novel Zn-MOF nanostructure.

In the present study, ultrasound irradiation was utilized to synthesize a novel zinc metal-organic framework (MOF). Scanning electron microscopic images, exhibited homogenous morphology with a nano-sized distribution of the Zn-MOF structure as also confirmed by X-ray diffraction patterns. Following, physical immobilization of Lepidium draba peroxidase (LDP) were optimized on the Zn-MOF in phosphate buffer (50 mM, pH 6.5), ratio amount of MOF/enzyme; 7/1 after shaking for 15 min at 25 °C, with high protein loading of 109.9 mg/g and immobilization yield of 93.3%. Immobilized enzyme (IE) exhibited more than 330% enhanced specific activity and also exhibited more than 150% specific affinity to its substrate (3,3',5,5'-tetramethylbenzidine) with respect to the free enzyme (FE). Optimum temperature of the IE was obtained at 20 °C while its was 25 °C for the FE, and thermostability of the IE augmented at temperature of 30 °C and 40 °C by the factors of 104 and 108% respectively. pH stability under neutral and basic condition and storage stability of the IE improved with respect to the FE as well as its structural stability (Tm; 73 °C for IE vs. 63 °C for FE). Furthermore, immobilization is accompanied with alteration on the enzyme structure as revealed by the intrinsic and extrinsic fluorescence spectra.

Improvement of kinetic properties and thermostability of recombinant Lepidium draba peroxidase (LDP) upon exposed to osmolytes.

In this study, effects of different concentrations of glycine and D-sorbitol were analyzed on the activity and thermostability of recombinant Lepidium draba peroxidase (LDP). Based on the results, activity of the enzyme increased in the presence of various concentrations of these osmolytes. Maximum activity was detected for the enzyme in the presence of 300 mM glycine and 600 mM sorbitol. In presence of the aforementioned doses of osmolytes, enzyme affinity for substrate (3,3',5,5'-tetramethylbenzidine and H2O2) and Vmax increased. According to the results, enzyme stability improved against temperature and H2O2. Furthermore, structural changes of the enzyme upon exposure to the osmolytes were revealed by the use of far-UV circular dichroism and fluorescence methods. The results showed, whereas the secondary structure of the enzyme was not significantly changed upon exposed to the osmolytes, the fluorescence studies revealed microenvironment of the aromatic residues dramatically affected by them. Overall, it may be speculated, structural changes of the enzyme upon exposed to the osmolytes, lead to the improvement of its kinetic properties and stability that can be benefit for using of it in in vitro applications.

Optimization of in vitro refolding conditions of recombinant Lepidium draba peroxidase using design of experiments.

The main objective of this study was to optimize the in vitro refolding conditions of the recombinant Lepidium draba peroxidase (LDP). Initially, the effects of various factors were investigated on LDP refolding yield using one-factor-at-a-time (OFAT) method. Based on the OFAT results, optimum concentrations for LDP refolding were 2 M urea, 2 mM CaCl2, 0.42 mM l-glutathione oxidized (GSSG), 0.20 mg/ml protein, and 12 µM hemin as well as pH 7. Secondly, according to the OFAT results, design of experiments (DOE) was applied for investigation of the interactions between factors including protein (P), urea (U), CaCl2 (C), and GSSG (G). The results showed the possible interaction between PC, PG, PU, and GU. Lastly, response surface methodology (RSM) was used for final refolding conditions optimization. The final optimized refolding conditions for LDP were conducted as 2 M urea, 1 mM CaCl2, 0.70 mM GSSG, 0.07 mM DTT, 0.15 mg/ml protein which they obtained from RSM results and 12 µM hemin, and pH 7 according to the results of OFAT method. Overall, under optimal conditions, 23.4 mg active refolded LDP per liter of expression medium was obtained. So, the refolding yield was calculated to be approximately 48%.

Heterologous Expression, Purification and Characterization of a Peroxidase Isolated from Lepidium draba.

Peroxidase is one of the most widely used enzymes in biotechnology and medicine. In the current study, cDNA encoding peroxidase from Lepidium draba (LDP) was cloned and expressed in Escherichia coli BL21 (DE3) cells in the form of inclusion bodies (IBs). To achieve purified active enzyme, IBs were solubilized before being purified and refolded. The deduced amino acid sequence (308) of the LDP gene (924 bp) revealed 88.96% identity to horseradish peroxidase C1A (HRP C1A). The results of basic local alignment search tool (BLAST) and phylogenetic analysis of the protein sequence showed that this enzyme belongs to the neutral group of class III plant peroxidases. According to sequence analysis and structural modeling, critical amino acids in heme and calcium binding domain as well as cysteine residues were conserved as HRP C1A except for calcium binding domain where valine228 was replaced with isoleucine. The far-UV circular dichroism (CD) results were confirmed by homology modeling data showing the enzyme consists mainly of α-helices as other plant peroxidases. Overall, according to the results of catalytic activity and refolding yield, LDP can be introduced as a novel peroxidase for medical and biotechnology applications.

In silico locating the immune-reactive segments of Lepidium draba peroxidase and designing a less immune-reactive enzyme derivative.

Peroxidases have broad applications in industry, environmental as well as pharmaceutical and diagnosis. Recently applicability of peroxidases in cancer therapy was mentioned. In the present study, a horseradish peroxidase homologue from Lepidium draba was subjected to in silico analyzes aiming at identifying and locating immune-reactive regions. A derivative sequence with decreased immunogenicity and increased stability also suggested. The tertiary structure of the enzyme was predicted. The functional and structural importance of residues was annotated as well as the conservatory status of each residue. The immune-dominant regions of protein were predicted with various software. N-terminal 4 residues, NFSHTGL (186-192), PRNGN (210-214), PLVRAYADGTQKFFN (261-275), and last 4 residues in C-terminal were predicted to be the consensus immunogenic segments of L. draba peroxidase. The modifications were applied to wild type sequence in order to mitigate its immune-reactiveness. The modifications were based on predicted energetic status of residues and naturally occurred amino acids in each position of the enzyme sequence, extracted from alignment file of 150 homologous peroxidases. The new enzyme derivative is predicted to be less immune-reactive and more stable. Thus the sequence is better suited to therapeutic applications.

Dynamics of glucosinolate-myrosinase system during Plutella xylostella interaction to a novel host Lepidium latifolium L.

Plutella xylostella L. is a notorious pest of cruciferous crops causing worldwide losses of $4-5 billion per year. Developing classical biological control to this pest include an introduction of host plants that act as natural enemies showing deviation from the preference-performance regimen in the evolutionary ecology of plant-insect interactions. The present study was designed to understand the role of glucosinolate-myrosinase system during P. xylostella interactions with a novel host. Adult moth preference and larval performance study were conducted on a novel host Lepidium latifolium L. (LL) that has high sinigrin content and was compared with its laboratory host Arabidopsis thaliana (AT). The glucosinolate-myrosinase system was studied in a time course experiment during larval feeding in choice and no-choice experiments. Adult moths visit and prefers LL over AT for oviposition. Conversely, LL leaves were not preferred and proved detrimental for P. xylostella larvae. Aliphatic and indolic glucosinolates were found to decrease significantly (p≤0.05) in AT during initial 12h of P. xylostella challenge, whereas, they were not affected in LL. Also, MYB transcription factor expression and myrosinase activity in LL do not suggest a typical host response to a specialist insect. This preference-performance mismatch of P. xylostella on LL mediated by glucosinolate pattern suggests that this novel plant could be utilized in P. xylostella management.

Purification and Characterization of a Novel Redox-Regulated Isoform of Myrosinase (ß-Thioglucoside Glucohydrolase) from Lepidium latifolium L.

Myrosinase (ExPASy entry EC 3.2.1.147) is involved in the hydrolysis of glucosinolates to isothiocyanates, nitriles, and thiocyanates that are responsible for various ecological and health benefits. Myrosinase was purified from the leaves of Lepidium latifolium, a high-altitude plant, to homogeneity in a three-step purification process. Purified enzyme exists as dimer in native form (∼160 kDa) with a subunit size of ∼70 kDa. The enzyme exhibited maximum activity at pH 6.0 and 50 °C. With sinigrin as substrate, the enzyme showed Km and Vmax values of 171 ± 23 µM and 0.302 µmol min(-1) mg(-1), respectively. The enzyme was found to be redox-regulated, with an increase in Vmax and Kcat in the presence of GSH. Reduced forms of the enzyme were found to be more active. This thiol-regulated kinetic behavior of myrosinase signifies enzyme's strategy to fine-tune its activity in different redox environments, thus regulating its biological effects.

Calcium promotes activity and confers heat stability on plant peroxidases.

In this paper we demonstrate how peroxidase (PO) activities and their heat stability correlate with the availability of free Ca(2+) ions. Calcium ions work as a molecular switch for PO activity and exert a protective function, rendering POs heat stable. The concentration ranges of these two activities differ markedly. POs are activated by µM Ca(2+) concentration ranges, whereas heat stabilization is observed in the nM range. This suggests the existence of different Ca(2+) binding sites. The heat stability of POs depends on the source plant species. Terrestrial plants have POs that exhibit higher temperature stability than those POs from limnic and marine plants. Different POs from a single species can differ in terms of heat stability. The abundance of different POs within a plant is dependent on age and developmental stage. The heat stability of a PO does not necessarily correlate with the maximum temperature the source species is usually exposed to in its natural habitat. This raises questions on the role of POs in the heat tolerance of plants. Consequently, detailed investigations are needed to identify and characterize individual POs, with regard to their genetic origin, subcellular expression, tissue abundance, developmental emergence and their functions in innate and acquired heat tolerance.

Propriedades físico-químicas do amido isolado, estudo de parâmetros enzimáticos durante o armazenamento e caracterização de enzimas amilolíticas em raízes de maca (Lepidium meyenii Walp) / Physico-chemical properties of isolated starch, enzymatic parameters during storage, and characterization of amylolytic enzymes of maca (Lepidium meyenii Walp. ) root

A maca (Lepidium meyenii Walpers) é uma planta herbácea bienal da família Brassicae, cultivada principalmente na região dos Andes da América do Sul. A parte subterrânea vem sendo consumida por muito tempo devido a seu valor nutricional e energético, mas é mais conhecida no mercado peruano e internacional por alegadas propriedades terapêuticas. Esta raiz apresenta até 76% de carboidratos, dos quais 30% é amido. Este trabalho teve como objetivos estudar: as propriedades físico-químicas e funcionais do amido isolado; os parâmetros enzimáticos durante o armazenamento e a purificação parcial de enzimas amilolíticas. Em relação às propriedades do amido, este apresentou um teor de amilose de 20% valor semelhante aos encontrados em raízes e tubérculos similares. A turbidez das suspensões de amido apresentou estabilidade durante o armazenamento. A temperatura de gelatinização e a viscosidade da pasta foram a 45,7° e 46°C, respectivamente. Com base nos dados obtidos, o amido de maca seria indicado para alimentos que requeiram temperaturas moderadas no processamento, não sendo apropriado para o emprego em alimentos congelados. Os parâmetros enzimáticos medidos tais como teor de amido total, teor de açúcares solúveis, atividade amilolítica total, atividade de α e β amilases, não mostraram diferenças significativas entre as medidas durante um período de armazenamento de 16 dias. As microscopias eletrônicas de varredura (MEV) dos grânulos de amido mostraram grãos íntegros com superfícies lisas, com algumas depressões ao redor dos grânulos os quais poderiam indicar o inicio de ataque enzimático, ou fraturas na purificação. Em relação à purificação de enzimas amilolíticas, foi possível separar uma fração ativa com a carboximetilcelulose (CMC) seguida de cromatografia liquida de alta resolução (CLAE) que permitiu a separação de duas frações protéicas, analisadas por eletroforese SDS-PAGE e eletroforese bidimensional (2D). Os polipeptídeos identificados no gel 2D apresentaram...

Maca (Lepidium meyenii Walpers) is a biennial herbaceous plant from Brassicae family, grown mainly in the Andes of South America. The underground part has been consumed for a long time due to its nutritional value and energy, but is best known in the Peruvian and international market for alleged therapeutic properties. This root has up to 76% carbohydrates, of which 30% is starch. This work aimed to study: the physico-chemical properties of isolated starch, the enzymatic parameters during storage and partial purification of amylases. In relation to the properties of starch, the amylose content showed a 20% value similar to those found in roots and tubers alike. The turbidity of starch suspensions was stable during storage. The gelatinization temperature and viscosity of the paste were 45.7 ° and 46 ° C, respectively. Based on data obtained from the starch of litter would be given to foods that require moderate temperatures in processing and is not suitable for use in frozen foods. The enzymatic parameters measured such as total starch content, soluble sugars, total amylolytic activity, activity of α and β amylases, showed no significant differences between the measures over a storage period of 16 days. Electronic microscopy (SEM) of starch granules showed grains with smooth surfaces, with some depressions around the granules which could indicate the beginning of enzymatic attack, or fractures in the purification. Regarding the purification of amylases was possible to separate an active fraction with carboxymethylcellulose (CMC) followed by high-resolution liquid chromatography (HPLC) which allowed the separation of two protein fractions, analyzed by SDS-PAGE and two-dimensional electrophoresis (2D ). The polypeptides had a molecular mass between 22 and 27 kDa and isoelectric points ranging from 4.8 to 7.3.