Purification and Characterization of a Novel Redox-Regulated Isoform of Myrosinase (β-Thioglucoside Glucohydrolase) from <i>Lepidium latifolium</i> L.

Bhat, Rohini; Kaur, Tarandeep; Khajuria, Manu; Vyas, Ruchika; Vyas, Dhiraj

doi:10.1021/acs.jafc.5b04468

Cited by 13 publications

(9 citation statements)

References 46 publications

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“…With the purification process of myrosinase produced in E. coli BL21(DE3)-myr and S. cerevisiae MGY70-myr, an enzyme preparation was obtained with a specific activity close to 6.4 and 5.2 (U/mg), respectively, unlike that obtained by other authors for myrosinases from other origins, such as myrosinase purified from Lepitidium latifolium leaves, with a specific activity of 3.12 (U/mg) [ 16 ] and myrosinase from a different origin than vegetable, obtained from the Brevicoryne brassicae aphid, with a specific activity of 0.9 (U/mg) [ 19 ]. These results were shown to be significantly lower than those obtained in this work for both enzymes, and this is because the purification processes are carried out by means of protein precipitation with ammonium sulfate, gel filtration and affinity chromatography with concanavalin A resins.…”

Section: Discussionmentioning

confidence: 99%

“…Similar kinetic parameters such as K m , V max and k cat have been described in other recombinant myrosinases produced in A. thaliana [ 15 , 44 ] and C. papaya [ 32 ]. In other myrosinases obtained from plants, a lower catalytic efficiency has been observed, as in the case of the enzymes of A. rusticana and Lepidium latifolium L [ 13 , 16 ].…”

Section: Discussionmentioning

confidence: 99%

“…Several research groups have studied the biochemical and kinetic characteristics of myrosinases obtained from different sources [ 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 ]. Structural studies using X-ray diffraction have made it possible to elucidate the crystal structure of myrosinase from Sinapis alba , where it was revealed that the monomeric subunit folds to form a highly glycosylated dimer stabilized by Zn 2+ , with a catalytic site in each monomer of the protein, and in turn each monomer folds with an α and β barrel-like structure.…”

Section: Introductionmentioning

confidence: 99%

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Broccoli Myrosinase cDNA Expression in Escherichia coli and Saccharomyces cerevisiae

2022

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Myrosinases (EC 3.2.1.147) are enzymes known for the generation of hydrolysis products that have a potential beneficial effect on human health. Their reaction mechanisms are widely studied, in order to improve and optimize secondary metabolite production processes. In this work, kinetic and biochemical properties of the broccoli myrosinase enzyme produced from its cDNA cloned in Escherichia coli and Saccharomyces cerevisiae were investigated. The results revealed that the thermal stability of the enzyme produced in S. cerevisiae was slightly higher (30 to 60 °C) than that of myrosinase produced in E. coli (20 to 50 °C). The effect of pH on the enzymatic activity was similar in both enzymes, with pH 3 being the optimum value under the reaction conditions used. The kinetic behavior of both enzymes was adjusted to the Michaelis–Menten model. The catalytic efficiency was up to 4 times higher in myrosinase produced in S. cerevisiae, compared to myrosinase produced in E. coli. The glycosylations present in the enzyme would be related to the formation of a dimeric quaternary structure and would not play an essential role in enzymatic activity, since both enzymes were biologically active. These results will probably allow the development of strategies for the production of bioactive metabolites of medical interest.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Broccoli Myrosinase cDNA Expression in Escherichia coli and Saccharomyces cerevisiae

2022

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“…Myrosinase activity was determined by evaluating the rate of sinigrin degradation as described earlier (Bhat et al 2015). Briefly, leaves (50 mg) were crushed in prechilled pestle and mortar using liquid nitrogen and mixed with 1 ml of 25 mM potassium phosphate extraction buffer (pH 7.0) containing 1 mM EDTA, 2 mM DTT, 1 mM PMSF, 0.05% Triton‐X and 5% PVPP (w/v), followed by centrifugation at 16 000 g for 30 min.…”

Section: Methodsmentioning

confidence: 99%

Effect of temperature and insect herbivory on the regulation of glucosinolate‐myrosinase system in Lepidium latifolium

Bhat

Faiz

Ali

et al. 2020

Physiologia Plantarum

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The glucosinolate-myrosinase (GLS-MYR) system is an important component of plant-insect interactions. However, there is no report on its performance in field conditions where the plants are subjected to both abiotic and biotic pressures simultaneously. We investigated the GLS-MYR system in a Himalayan ecotype of Lepidium latifolium that is recognized for its adaptive potential in field conditions. In order to understand the independent contribution of temperature and Pieris brassicae herbivory on the components of the GLS-MYR system, different conditions were simulated in the growth chamber. During field conditions, the final GLS hydrolysis products were found to be regulated by the metabolic GLS levels, the temperature conditions, and the density of insect interactions. These factors influence the expression of the hydrolyzing and specifier proteins, which further affects the GLS hydrolysis products.Our results suggest that the production of hydrolysis products is differentially affected under field conditions. While allyl isothiocyanate is significantly (P ≤ 0.05) affected by temperature but not insect density, 1-cyano-2,3-epithiopropane is not affected by either. The study shows that the outcome of the GLS-MYR system in a plant is a consequence of the combinatorial effect of ecophysiological factors and the insect interactions that eventually decide the performance of a plant in an environment. | INTRODUCTIONLepidium latifolium (perennial pepperweed) is an ecologically important plant belonging to the family Brassicaceae. Although native to southern Europe and Asia (Bhat et al. 2016, Schultze-Motel 1986, it has shown widespread presence in most parts of the world, including the western United States, coastal New England, Mexico, Canada, and Australia (Young et al. 1995). The wide ecological amplitude of Lepidium latifolium suggests that it is a reservoir of several novel physiological and biochemical mechanisms that help this plant to adapt to a diverse set of environmental conditions. We have shown a responsive redox mechanism where the glutathione plays an important role in the induction of antioxidant enzymes having higher thiol content (Kaur, Bhat, et al. 2013). Dynamic photosynthetic machinery to mitigate higher photosynthetic efficiency and light-dependent xanthophyll cycle turnover has also been shown to suggest the wider adaptability of this plant in the Himalayas (Bhat et al. 2016). Lepidium latifolium has also been recognized as the highest source of sinigrin having nutraceutical importance (Kaur, Hussain, et al. 2013).Sinigrin is an allyl glucosinolate, a constituent of the glucosinolate-myrosinase system (GLS-MYR) that forms the important mechanism during plant-insect interactions in Brassicaceae (Hopkins et al. 2009). GLS-MYR is an enzyme-substrate complex that is

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“…In normal, GLs in cell are separated from MYR (Bhat, Kaur, Khajuria, Vyas, & Vyas, 2015). Only when the cells are broken, they will be combined and then react, resulting in the production of SF and other metabolites (Braschi, Leoni, Cinti, Palmieri, & Gessa, 2011; Okunade, Ghawi, Methven, & Niranjan, 2015).…”

Section: Introductionmentioning

confidence: 99%

Selection and microencapsulation of myrosinase enzyme from broccoli sprouts of different varieties and characteristics evaluation

Wang

et al. 2020

J Food Process Engineering

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Myrosinase (MYR) can hydrolyze glucosinolates rich in crucifer to produce sulforaphane, which has significant anticancer effect. In this study, MYR activity in broccoli seeds and sprouts of different varieties were firstly compared. Then, after optimization for the microencapsulation condition of MYR, the characteristics of free and encapsulated MYR enzyme were evaluated and compared. Broccoli variety and germination time both significantly affected the specific MYR activity, total protein content and total MYR activity in sprouts. The optimum condition for encapsulating MYR was the sodium alginate addition of 5.29 ml, CaCl 2 concentration of 0.75%, the hardening time of 29.98 min, and the crosslinking time of 47.20 min. Encapsulated MYR with the specific activity of 2.25 U/mg was obtained (54.6% of free MYR). Encapsulated MYR could retain its activity under wider pH range and higher temperature than free MYR. Encapsulated MYR also kept higher activity during storage at room temperature. Supplement of encapsulated MYR was favorable for SF production in broccoli sprouts during storage. This research would be beneficial for producing more sulforaphane from broccoli sprouts as raw materials of functional foods. Practical Applications The function of sulforaphane has been widely accepted. However, products currently on the market contain only its precursor of glucosinolate, which is extremely inefficient in converting to SF in vivo. Encapsulated myrosinase have better temperature and pH tolerance, which can ensure the conversion of Gls to SF before oral intake. It is a good choice to provide encapsulated myrosinase and glucosinolates together to consumer. 1 | INTRODUCTION Myrosinase (EC 3.2.3.1,MYR), also known as β-thioglucoside hydrolase, is a hydrolase that degrades glucosinolates (GLs) in cruciferous plants (Bernardi, Finiguerra, Rossi, & Palmieri, 2003; Martinez-Ballesta & Carvajal, 2015). Under certain conditions, it can decompose GLs to produce glucose, sulfate, isothiocyanates, acetonitrile, thiocyanate and other substances. Sulforaphane (SF), as one kind of isothiocyanates, is the most valuable hydrolyzate and it possesses significant anticancer effect (Atwell et al., 2015; Giacoppo et al., 2015). Epidemiological studies including vitro cell and animal studies demonstrated that SF is the crucial factors for preventing the development of cancer including lung tumor, mammary cancer, breast cancer and bladder cancer, and so forth (Mokhtari et al., 2018). Great attention has been paid on this bioactive compound throughout the world in the past decades.

show abstract

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

Cited by 13 publications

References 46 publications

Broccoli Myrosinase cDNA Expression in Escherichia coli and Saccharomyces cerevisiae

Broccoli Myrosinase cDNA Expression in Escherichia coli and Saccharomyces cerevisiae

Effect of temperature and insect herbivory on the regulation of glucosinolate‐myrosinase system in Lepidium latifolium

Selection and microencapsulation of myrosinase enzyme from broccoli sprouts of different varieties and characteristics evaluation

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