Identification, molecular and biochemical characterization of a novel thermoactive and thermostable glucoamylase from Thermoanaerobacter ethanolicus.

PURPOSE: We identified a new glucoamylase (TeGA) from Thermoanaerobacter ethanolicus, a thermophilic anaerobic bacterium. Structural studies suggest that TeGA belongs to the family 15 of glycosylhydrolases (GH15). METHODS: The expression of this enzyme was optimized in E. coli (BL21) cells in order to have the highest amount of soluble protein (around 3 mg/l of culture medium). RESULTS: TeGA showed a high optimum temperature of 75 °C. It also showed one of the highest specific activities reported for a bacterial glucoamylase (75.3 U/mg) and was also stable in a wide pH range (3.0-10.0). Although the enzyme was preferentially active with maltose, it was also able to hydrolyze different soluble starches such as those from potato, corn or rice. TeGA showed a high thermostability up to around 70 °C, which was increased in the presence of PEG8000, and also showed to be stable in the presence of moderate concentrations of ethanol. CONCLUSION: We propose that TeGA could be suitable for use in different industrial processes such as biofuel production and food processing.

CBM20CP, a novel functional protein of starch metabolism in green algae.

Ostreococcus tauri is a picoalga that contains a small and compact genome, which resembles that of higher plants in the multiplicity of enzymes involved in starch synthesis (ADP-glucose pyrophosphorylase, ADPGlc PPase; granule bound starch synthase, GBSS; starch synthases, SSI, SSII, SSIII; and starch branching enzyme, SBE, between others), except starch synthase IV (SSIV). Although its genome is fully sequenced, there are still many genes and proteins to which no function was assigned. Here, we identify the OT_ostta06g01880 gene that encodes CBM20CP, a plastidial protein which contains a central carbohydrate binding domain of the CBM20 family, and a coiled coil domain at the C-terminus that lacks catalytic activity. We demonstrate that CBM20CP has the ability to bind starch, amylose and amylopectin with different affinities. Furthermore, this protein interacts with OsttaSSIII-B, increasing its binding to starch granules, its catalytic efficiency and promoting granule growth. The results allow us to postulate a functional role for CBM20CP in starch metabolism in green algae. KEY MESSAGE: CBM20CP, a plastidial protein that has a modular structure but lacks catalytic activity, regulates the synthesis of starch in Ostreococcus tauri.

Characterization of SdGA, a cold-adapted glucoamylase from Saccharophagus degradans.

We investigated the structural and functional properties of SdGA, a glucoamylase (GA) from Saccharophagus degradans, a marine bacterium which degrades different complex polysaccharides at high rate. SdGA is composed mainly by a N-terminal GH15_N domain linked to a C-terminal catalytic domain (CD) found in the GH15 family of glycosylhydrolases with an overall structure similar to other bacterial GAs. The protein was expressed in Escherichia coli cells, purified and its biochemical properties were investigated. Although SdGA has a maximum activity at 39 °C and pH 6.0, it also shows high activity in a wide range, from low to mild temperatures, like cold-adapted enzymes. Furthermore, SdGA has a higher content of flexible residues and a larger CD due to various amino acid insertions compared to other thermostable GAs. We propose that this novel SdGA, is a cold-adapted enzyme that might be suitable for use in different industrial processes that require enzymes which act at low or medium temperatures.

Applications of Bioinformatics to Plant Biotechnology.

Bioinformatics encompasses many tools and techniques that today are essential for all areas of research in the biological sciences. New databases with a wealth of information about genomes, proteins, metabolites, and metabolic pathways appear almost daily. Particularly, for scientists who carry out research in plant biology, the amount of information has multiplied exponentially due to the large number of databases available for many individual plant species. In this sense, bioinformatics together with next generation sequencing and 'omics' approaches, can provide tools for plant breeding and the genetic engineering of plants. In addition, these technologies enable a better understanding of the processes and mechanisms that can lead to plants with increased tolerance to different abiotic stress conditions and resistance to pathogen attack, as well as the development of crop varieties with improved nutritional quality of seeds and fruits.

Identification and characterization of a novel starch branching enzyme from the picoalgae Ostreococcus tauri.

Starch branching enzyme is a highly conserved protein from plants to algae. This enzyme participates in starch granule assembly by the addition of α-1,6-glucan branches to the α-1,4-polyglucans. This modification determines the structure of amylopectin thus arranging the final composition of the starch granule. Herein, we describe the function of the Ot01g03030 gene from the picoalgae Ostreococcus tauri. Although in silico analysis suggested that this gene codes for a starch debranching enzyme, our biochemical studies support that this gene encodes a branching enzyme (BE). The resulting 1058 amino acids protein has two in tandem carbohydrate binding domains (CBMs, from the CBM41 and CBM48 families) at the N-terminal (residues 64-403) followed by the C-terminal catalytic domain (residues 426-1058). Analysis of the BE truncated isoforms show that the CBMs bind differentially to whole starch, amylose or amylopectin. Furthermore, both CBMs seem to be essential for BE activity, as no catalytic activity was detected in the truncated enzyme comprising only by the catalytic domain. Our results suggest that the Ot01g03030 gene codifies for a functional BE containing two CBMs from CBM41 and CBM48 families which are critical for enzyme function and regulation.

Identification of a novel starch synthase III from the picoalgae Ostreococcus tauri.

Hydrosoluble glycogen is the major energy storage compound in bacteria, archaea, fungi, and animal cells. In contrast, photosynthetic eukaryotes have evolved to build a highly organized semicrystalline granule of starch. Several enzymes are involved in polysaccharide synthesis, among which glycogen or starch synthase catalyze the elongation of the α-1,4-glucan chain. Ostreococcus tauri, accumulates a single starch granule and contains three starch synthase III (SSIII) isoforms, known as OsttaSSIII-A, OsttaSSIII-B and OsttaSSIII-C. After amino acids sequence analysis we found that OsttaSSIII-C lacks starch-binding domains, being 49% identical to the catalytic region of the SSIII from Arabidopsis thaliana and 32% identical to the entire Escherichia coli glycogen synthase. The recombinant, highly purified OsttaSSIII-C exhibited preference to use as a primer branched glycans (such as rabbit muscle glycogen and amylopectin), rather than amylose. Also, the enzyme displayed a high affinity toward ADP-glucose. We found a marked conservation of the amino acids located in the catalytic site, and specifically determined the role of residues R270, K275 and E352 by site-directed mutagenesis. Results show that these residues are important for OsttaSSIII-C activity, suggesting a strong similarity between the active site of the O. tauri SSIII-C isoform and other bacterial glycogen synthases.

Functional demonstrations of starch binding domains present in Ostreococcus tauri starch synthases isoforms.

BACKGROUND: Starch-binding domains are key modules present in several enzymes involved in polysaccharide metabolism. These non-catalytic modules have already been described as essential for starch-binding and the catalytic activity of starch synthase III from the higher plant Arabidopsis thaliana. In Ostreococcus tauri, a unicellular green alga of the Prasinophyceae family, there are three SSIII isoforms, known as Ostta SSIII-A, SSIII-B and SSIII-C. RESULTS: In this work, using in silico and in vitro characterization techniques, we have demonstrated that Ostta SSIII-A, SSIII-B and SSIII-C contain two, three and no starch-binding domains, respectively. Additionally, our phylogenetic analysis has indicated that OsttaSSIII-B, presenting three N-terminal SBDs, is the isoform more closely related to higher plant SSIII. Furthermore, the sequence alignment and homology modeling data gathered showed that both the main 3-D structures of all the modeled domains obtained and the main amino acid residues implicated in starch binding are well conserved in O. tauri SSIII starch-binding domains. In addition, adsorption assays showed that OsttaSSIII-A D2 and SSIII-B D2 domains are the two that make the greatest contribution to amylose and amylopectin binding, while OsttaSSIII-B D1 is also important for starch binding. CONCLUSIONS: The results presented here suggest that differences between OsttaSSIII-A and SSIII-B SBDs in the number of and binding of amino acid residues may produce differential affinities for each isoform to polysaccharides. Increasing the knowledge about SBDs may lead to their employment in biomedical and industrial applications.