A new versatile peroxidase with extremophilic traits over-produced in MicroTom cell cultures.

Peroxidases are widespread key antioxidant enzymes that catalyse the oxidation of electron donor substrates in parallel with the decomposition of H2O2. In this work, a novel tomato peroxidase, named SAAP2, was isolated from MicroTom cell cultures, purified, and characterised. The enzyme was identified with 64% sequence coverage as the leprx21 gene product (suberization-associated anionic peroxidase 2-like) from Solanum lycopersicum, 334 amino acids long. Compared to other plant peroxidases, SAAP2 was more active at elevated temperatures, with the optimal temperature and pH at 90 °C and 5.0, respectively. Furthermore, the enzyme retained more than 80% of its maximal activity over the range of 70-80 °C and the presence of NaCl (1.0-4.5 M). It also exhibited broad pH versatility (65% relative activity over the pH range 2.0-7.0), acid-tolerance (80% residual activity after 22 h at pH 2.0-7.0), high thermostability (50% residual activity after 2 h at 80 °C) and proteolytic resistance. SAAP2 exhibited exceptional resistance under thermo-acidic conditions compared to the horseradish peroxidase benchmark, suggesting that it may find potential applications as a supplement or anti-pollution agent in the food industry.

Recombinant Expression of Archaeal Superoxide Dismutases in Plant Cell Cultures: A Sustainable Solution with Potential Application in the Food Industry.

Superoxide dismutase (SOD) is a fundamental antioxidant enzyme that neutralises superoxide ions, one of the main reactive oxygen species (ROS). Extremophile organisms possess enzymes that offer high stability and catalytic performances under a wide range of conditions, thus representing an exceptional source of biocatalysts useful for industrial processes. In this study, SODs from the thermo-halophilic Aeropyrum pernix (SODAp) and the thermo-acidophilic Saccharolobus solfataricus (SODSs) were heterologously expressed in transgenic tomato cell cultures. Cell extracts enriched with SODAp and SODSs showed a remarkable resistance to salt and low pHs, respectively, together with optimal activity at high temperatures. Moreover, the treatment of tuna fillets with SODAp-extracts induced an extension of the shelf-life of this product without resorting to the use of illicit substances. The results suggested that the recombinant plant extracts enriched with the extremozymes could find potential applications as dietary supplements in the nutrition sector or as additives in the food preservation area, representing a more natural and appealing alternative to chemical preservatives for the market.

An Oenothera biennis Cell Cultures Extract Endowed with Skin Anti-Ageing Activity Improves Cell Mechanical Properties.

Skin aging is a very well-known process setting a gradual worsening of skin mechanical features due to a decline in the production of the extra-cellular matrix machinery and to a concurrent change in the contraction process. To slow this progression, it is crucial to induce the expression of several proteins able to promote elastic fibers formation and tissue repair. Here, the Oenothera biennis cell culture aqueous extract has been investigated from a chemical point of view and then it was tested in vitro, in cell, and in ex vivo experiments as adjuvant in counteracting skin aging. Accordingly, it has been shown that the Oenothera biennis extract was able, by increasing MYLK gene expression, to promote matrix collagen contraction, actin polymerization, and the production of essential ECM proteins.

The extraordinary resistance to UV radiations of a manganese superoxide dismutase of Deinococcus radiodurans offers promising potentialities in skin care applications.

An overproduction of free radicals or reactive oxygen species, often due to environmental factors, can alter the DNA structure and irreversibly modify proteins and lipids in the living cells. The superoxide anion (O2-) is one of the strongest oxidant molecules produced under oxidative stress conditions but it can be neutralized by the action of the enzymes SuperOxide Dismutases (SODs). In all the human tissues, SODs are essential for the prevention of serious diseases and the protection against oxidative stress damages. In the dermo-cosmetic sector, SODs have found promising applications, but their use is limited due to the loss of activity following the addition of the enzyme in the skin care formulas and the exposure of the skin to UV radiations and heat. Extremophile organisms, which proliferate in extreme physical and/or geochemical conditions, represent a potential source of stable SOD enzymes, able to function even in harsh conditions of high temperature, acid pH and long UV exposures. In the present study we investigated on a Mn-SOD deriving from the extremophilic bacterium Deinococcus radiodurans and, after its expression in E.coli, the Mn-SOD was characterized in terms of chemical and physical properties. Its extraordinary features in terms of UV resistance prompted us to investigate further about its potential applications in the dermo-cosmetic sector. It was expressed in Solanum lycopersicum (tomato) cell cultures with the main goal of developing a new ingredient, capable of keeping its ROS neutralizing activity once exposed to UV radiations and even when added to skin care formulas.

Targeting the diuretic hormone receptor to control the cotton leafworm, Spodoptera littoralis.

The cotton leafworm, Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae), is one of the most devastating pests of crops worldwide. Several types of treatments have been used against this pest, but many of them failed because of the rapid development of genetic resistance in the different insect populations. G protein coupled receptors have vital functions in most organisms, including insects; thus, they are appealing targets for species-specific pest control strategies. Among the insect G protein coupled receptors, the diuretic hormone receptors have several key roles in development and metabolism, but their importance in vivo and their potential role as targets of novel pest control strategies are largely unexplored. With the goal of using DHR genes as targets to control S. littoralis, we cloned a corticotropin-releasing factor-like binding receptor in this species and expressed the corresponding dsRNA in tobacco plants to knock down the receptor activity in vivo through RNA interference. We also expressed the receptor in mammalian cells to study its signaling pathways. The results indicate that this diuretic hormone receptor gene has vital roles in S. littoralis and represents an excellent molecular target to protect agriculturally-important plants from this pest.

A tomato stem cell extract, containing antioxidant compounds and metal chelating factors, protects skin cells from heavy metal-induced damages.

Heavy metals can cause several genotoxic effects on cells, including oxidative stress, DNA sequence breakage and protein modification. Among the body organs, skin is certainly the most exposed to heavy metal stress and thus the most damaged by the toxic effects that these chemicals cause. Moreover, heavy metals, in particular nickel, can induce the over-expression of collagenases (enzymes responsible for collagen degradation), leading to weakening of the skin extracellular matrix. Plants have evolved sophisticated mechanisms to protect their cells from heavy metal toxicity, including the synthesis of metal chelating proteins and peptides, such as metallothioneins and phytochelatins (PC), which capture the metals and prevent the damages on the cellular structures. To protect human skin cells from heavy metal toxicity, we developed a new cosmetic active ingredient from Lycopersicon esculentum (tomato) cultured stem cells. This product, besides its high content of antioxidant compounds, contained PC, effective in the protection of skin cells towards heavy metal toxicity. We have demonstrated that this new product preserves nuclear DNA integrity from heavy metal damages, by inducing genes responsible for DNA repair and protection, and neutralizes the effect of heavy metals on collagen degradation, by inhibiting collagenase expression and inducing the synthesis of new collagen.

A mixture of peptides and sugars derived from plant cell walls increases plant defense responses to stress and attenuates ageing-associated molecular changes in cultured skin cells.

Small peptides and aminoacid derivatives have been extensively studied for their effect of inducing plant defense responses, and thus increasing plant tolerance to a wide range of abiotic stresses. Similarly to plants, these compounds can activate different signaling pathways in mammalian skin cells as well, leading to the up-regulation of anti-aging specific genes. This suggests the existence of analogous defense response mechanisms, well conserved both in plants and animal cells. In this article, we describe the preparation of a new mixture of peptides and sugars derived from the chemical and enzymatic digestion of plant cell wall glycoproteins. We investigate the multiple roles of this product as potential "biostimulator" to protect plants from abiotic stresses, and also as potential cosmeceutical. In particular, the molecular effects of the peptide/sugar mixture of inducing plant defense responsive genes and protecting cultured skin cells from oxidative burst damages were deeply evaluated.

The Chitinase A from the baculovirus AcMNPV enhances resistance to both fungi and herbivorous pests in tobacco.

Biotechnology has allowed the development of novel strategies to obtain plants that are more resistant to pests, fungal pathogens and other agents of biotic stress. The obvious advantages of having genotypes with multiple beneficial traits have recently fostered the development of gene pyramiding strategies, but less attention has been given to the study of genes that can increase resistance to different types of harmful organisms. Here we report that a recombinant Chitinase A protein of the Autographa californica nuclear polyhedrosis virus (AcMNPV) has both antifungal and insecticide properties in vitro. Transgenic tobacco plants expressing an active ChiA protein showed reduced damages caused by fungal pathogens and lepidopteran larvae, while did not have an effect on aphid populations. To our knowledge, this is the first report on the characterisation and expression in plants of a single gene that increases resistance against herbivorous pests and fungal pathogens and not affecting non-target insects. The implications and the potential of the ChiA gene for plant molecular breeding and biotechnology are discussed.

AcMNPV ChiA protein disrupts the peritrophic membrane and alters midgut physiology of Bombyx mori larvae.

Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) chitinase A (ChiA) is a protein which promotes the final liquefaction of infected host larvae. The potential of this viral molecule as a new tool for insect control is explored here. The ChiA gene was isolated from the AcMNPV genome by PCR and expressed in E. coli. The recombinant protein, purified by affinity chromatography, showed both exo- and endo-chitinase activities and produced perforations on the peritrophic membrane (PM) of Bombyx mori larvae which increased in number and in size, in a dose-dependent manner. This structural alteration resulted into a significant increase of PM permeability to methylene blue and to the small neuropeptide proctolin. When the fifth instar larvae of B. mori were fed on a artificial diet supplemented with the recombinant ChiA, 100% mortality was observed at a dose of 1 microg/g of larval body weight (LW), while at sub-lethal doses of 0.56 microg/g LW, a reduced larval growth was recorded. These results indicate that AcMNPV-ChiA may offer interesting new opportunities for pest control.