Extraction and Activity of O-acetylserine(thiol)lyase (OASTL) from Microalga Chlorella sorokiniana

O-acetylserine(thiol)lyase (OASTL) is an enzyme catalysing the reaction of inorganic sulphide with O-acetylserine to form the S-containing amino acid L-cysteine. Here we describe an improved protocol to evaluate the activity of this enzyme from the microalga Chlorella sorokiniana. It is a colorimetric assay based on the reaction between cysteine, the product of OASTL activity, and ninhydrin reagent, which forms a thiazolidine (Thz)

Determination of Reduced and Total Glutathione Content in Extremophilic Microalga Galdieria phlegrea

Glutathione is an important molecule involved in the primary and secondary metabolism of all organisms. The Glutathione redox status is an indicator of the cellular redox state. Therefore, it is important to have precise methods on hand to determine the glutathione redox status in the cell. In this protocol, we describe an improved spectrophotometric method to estimate the content of reduced (GSH) and oxidized (GSSG) forms of glutathione in the extremophilic microalga Galdieria phlegrea

Effect of bicarbonate on growth of the oleaginous microalga Botryococcus braunii

The effect of bicarbonate, produced by the enzymatic hydration of CO2 from postcombustion fumes, was investigated on Botryococcus braunii growth. The NaHCO3, supplied to cultures in the role of inorganic carbon source is proposed as a more eco-sustainable alternative to gaseous CO2. The salt was provided to the cultures at the final concentration of 0.5-1.5-2.5 g L-1. The growth rate was considered for specific time intervals (T0-T5, T5-T10 and T0-T10) showing values significantly higher in the culture supplemented with 2.5 g L-1 bicarbonate. The doubling times were also considered in all experimental cultures showing a faster doubling for the period T0÷T5. The increase in pH drives the increase in growth in the experimental conditions in which the salt was added. The results suggest that bicarbonate is able to promote the algal growth, therefore it can be considered a valid alternative to CO2 gas

Affinity purification of O-acetylserine(thiol)lyase from chlorella sorokinianaby recombinant proteins from Arabidopsis thaliana

In the unicellular green alga Chlorella sorokiniana (211/8 k), the protein O-acetylserine(thiol)lyase (OASTL), representing the key-enzyme in the biosynthetic cysteine pathway, was isolated and purified to apparent homogeneity. The purification was carried out in cells grown in the presence of all nutrients or in sulphate (S) deprived cells. After 24 h of S-starvation, a 17-fold increase in the specific activity of OASTL was measured. In order to enable the identification of OASTL proteins from non-model organisms such as C. sorokiniana, the recombinant his-tagged SAT5 protein from Arabidopsis thaliana was immobilized by metal chelate chromatography. OASTL proteins from C. sorokiniana were affinity purified in one step and activities were enhanced 29- and 41-fold, from S-sufficient and S-starved (24 h) cells, respectively. The successful application of SAT/OASTL interaction for purification confirms for the first time the existence of the cysteine synthase complexes in microalgae. The purified proteins have apparent molecular masses between 32–34 kDa and are thus slightly larger compared to those found in other vascular plants. The enhanced OASTL activity in S-starved cells can be attributed to increased amounts of plastidic and the emergence of cytosolic OASTL isoforms. The results provide proof-of-concept for the biochemical analysis of the cysteine synthase complex in diverse microalgal species

Effects of sulphate-deficiency on cysteine metabolism in the green alga Chlorella sorokiniana

Plants, bacteria and fungi have the ability to assimilate inorganic sulphur and incorporate it into inorganic compounds. Animals on the contrary, do not assimilate inorganic sulphur; they require methionine as an essential amino acid for their source of sulphur nutrient. The amino acid cysteine (Cys) is the first committed molecule in plant metabolism containing sulphur and it is the sulphide donor for the generation of methionine, glutathione (GSH), phytochelatins, iron-sulphur clusters, vitamins, cofactors, and multiple secondary metabolites; that’s why the regulation of Cys-biosynthesis is critically important. The aim of this research was to investigate the effects of sulphur-deficiency on plant cysteine metabolism and to characterize the O-acetylserine(thiol)lyase (OASTL), a key-enzyme in cysteine biosynthesis

Non-Protein Thiol Compounds and Antioxidant Responses Involved in Bryophyte Heavy-Metal Tolerance

Heavy-metal pollution represents a problem which has been widely discussed in recent years. The biological effects of heavy metals have been studied in both animals and plants, ranging from oxidative stress to genotoxicity. Plants, above all metal-tolerant species, have evolved a wide spectrum of strategies to counteract exposure to toxic metal concentrations. Among these strategies, the chelation and vacuolar sequestration of heavy metals are, after cell-wall immobilization, the first line of defence that prevent heavy metals from interacting with cell components. Furthermore, bryophytes activate a series of antioxidant non-enzymatic and enzymatic responses to counteract the effects of heavy metal in the cellular compartments. In this review, the role of non-protein thiol compounds and antioxidant molecules in bryophytes will be discussed

Fungal Contamination in Microalgal Cultivation: Biological and Biotechnological Aspects of Fungi-Microalgae Interaction

In the last few decades, the increasing interest in microalgae as sources of new biomolecules and environmental remediators stimulated scientists' investigations and industrial applications. Nowadays, microalgae are exploited in different fields such as cosmeceuticals, nutraceuticals and as human and animal food supplements. Microalgae can be grown using various cultivation systems depending on their final application. One of the main problems in microalgae cultivations is the possible presence of biological contaminants. Fungi, among the main contaminants in microalgal cultures, are able to influence the production and quality of biomass significantly. Here, we describe fungal contamination considering both shortcomings and benefits of fungi-microalgae interactions, highlighting the biological aspects of this interaction and the possible biotechnological applications