Andres Aguirre

Phospholipids play a central role in all living organisms. Phospholipases, the enzymes aimed at modifying phospholipids, are consequently widespread in nature and play diverse roles, from lipid metabolism and cellular signaling in eukaryotes to virulence and nutrient acquisition in microbes. Phospholipases catalyze the hydrolysis of one or more ester or phosphodiester bonds of glycerophospholipids. The use of phospholipases with industrial purposes has constantly increased over the last 30 years. This demand is rapidly growing given the ongoing improvements in protein engineering and the reduction of enzymes manufacturing costs, making them suitable for industrial use. Here, a general overview of phopholipases A, B, C, and D and their industrial application is presented along with potential new uses for these enzymes. We draw attention to commercial phospholipases used to improve the emulsifying properties of products in the baking, egg, and dairy industries. On the other hand, the improvement of oil degumming by phospholipases is thoroughly analyzed. Moreover, recent developments in enzymatic biodiesel production and the use of phospholipases for the synthesis of phospholipids with pharmaceutical or nutritional value are reviewed.

Extremophilic microorganisms are a rich source of enzymes, the enzymes which can serve as industrial catalysts that can withstand harsh processing conditions. An example is thermostable β-glucosidases that are addressing a challenging problem in the biodiesel industry: removing steryl glucosides (SGs) from biodiesel. Steryl glucosidases (SGases) must be tolerant to heat and solvents in order to function efficiently in biodiesel. The amphipathic nature of SGs also requires enzymes with an affinity for water/solvent interfaces in order to achieve efficient hydrolysis. Additionally, the development of an enzymatic process involving a commodity such as soybean biodiesel must be cost-effective, necessitating an efficient manufacturing process for SGases. This review summarizes the identification of microbial SGases and their applications, discusses biodiesel refining processes and the development of analytical methods for identifying and quantifying SGs in foods and biodiesel, and considers technologies for strain engineering and process optimization for the heterologous production of a SGase from Thermococcus litoralis. All of these technologies might be used for the production of other thermostable enzymes. Structural features of SGases and the feasibility of protein engineering for novel applications are explored.

Biodiesels produced from vegetable oils have a major quality problem due to the presence of steryl glucosides (SGs), which form precipitates that clog filters and cause engine failures. Recently, we described an enzymatic process for removing SGs from biodiesel. However, industrial adoption of this technology was hindered by the cost of the steryl glucosidase (SGase) enzyme used. Here we report the development and validation at the pilot scale of a cost-efficient process for manufacturing the SGase. First, we tested various low-cost carbon sources for the Escherichia coli producing strain, ultimately developing a fed-batch fermentation process that utilizes crude glycerol as a feedstock. Next, we designed an efficient process for isolating the SGase. That process uses a novel thermolysis approach in the presence of a non-ionic detergent, centrifugation to separate the solids, and ultrafiltration to concentrate and formulate the final product. Our cost analysis indicates that on a large scale, the dose of enzyme required to eliminate SGs from each ton of biodiesel will have a manufacturing cost below $1. The new process for manufacturing the SGase, which will lead to biodiesels of a higher quality, should contribute to facilitate the global adoption of this renewable fuel. Our technology could also be used to manufacture other thermostable proteins in E. coli.

Biodiesels produced from transesterification of vegetable oils have a major problem in quality due to the presence of precipitates, which are mostly composed of steryl glucosides (SGs). We have recently described an enzymatic method for the efficient removal of SGs from biodiesel, based on the activity of a thermostable β-glycosidase from Thermococcus litoralis. In the present work, we describe the development of an Escherichia coli-based expression system and a high cell density fermentation process. Strain and process engineering include the assessment of different promoters to drive the expression of a codon-optimized gene, the co-expression of molecular chaperones and the development of a high cell density fermentation process. A 200-fold increase in the production titers was achieved, which directly impacts on the costs of the industrial process for treating biodiesel.

Biodiesels produced from transesterification of vegetable oils have a major quality problem due to the presence of precipitates, which need to be removed to avoid clogging of filters and engine failures. These precipitates have been reported to be mostly composed of steryl glucosides (SGs), but so far industrial cost-effective methods to remove these compounds are not available. Here we describe a novel method for the efficient removal of SGs from biodiesel, based on the hydrolytic activity of a thermostable β-glycosidase obtained from Thermococcus litoralis. A steryl glucosidase (SGase) enzyme from T. litoralis was produced and purified from Escherichia coli cultures expressing a synthetic gene, and used to treat soybean-derived biodiesel. Several optimization steps allowed for the selection of optimal reaction conditions to finally provide a simple and efficient process for the removal of SGs from crude biodiesel. The resulting biodiesel displayed filterability properties similar to distilled biodiesel according to the total contamination (TC), the cold soak filtration test (CSFT), filter blocking tendency (FBT), and cold soak filter blocking tendency (CSFBT) tests. The process was successfully scaled up to a 20 ton reactor, confirming its adaptability to industrial settings. The results presented in this work provide a novel path for the removal of steryl glucosides from biodiesel using a cost-effective, environmentally friendly and scalable enzymatic process, contributing to the adoption of this renewable fuel.

Steryl glucosides (SG) are common contaminants in biodiesel that form precipitates, which form and cause problems due to fouling during transport and storage. Therefore, their quantification is necessary to assess the quality of this fuel. The methods currently available for SG analysis require expensive instrumentation, need a previous concentration step by solid‐phase extraction (SPE) or are of limited use for the quantitative assessment. We developed an enzymatic method for SG quantification in biodiesel samples based on the hydrolysis of the glucoside catalyzed by a broadly specific beta glucosidase and the subsequent determination of the glucose released by the reaction. The method is non‐expensive, sensitive and was adapted to 96‐well format fluorescence plate reader, making it useful for the parallel assay of multiple samples. The enzymatic assay presented here represent a valuable tool for both quality control and the development of improved biodiesel production and purification procedures.

Biodiesels are mostly produced from lipid transesterification of vegetable oils, including those from soybean, jatropha, palm, rapeseed, sunflower, and others. Unfortunately, transesterification of oil produces various unwanted side products, including steryl glucosides (SG), which precipitate and need to be removed to avoid clogging of filters and engine failures. So far, efficient and cost-effective methods to remove SGs from biodiesel are not available. Here we describe for the first time the identification, characterization and heterologous production of an enzyme capable of hydrolyzing SGs. A synthetic codon-optimized version of the lacS gene from Sulfolobus solfataricus was efficiently expressed and purified from Escherichia coli, and used to treat soybean derived biodiesel containing 100 ppm of SGs. After optimizing different variables, we found that at pH 5.5 and 87 °C, and in the presence of 0.9 % of the emulsifier polyglycerol polyricinoleate, 81 % of the total amount of SGs present in biodiesel were hydrolyzed by the enzyme. This remarkable reduction in SGs suggests a path for the removal of these contaminants from biodiesel on industrial scale using an environmentally friendly enzymatic process.

Salmonella enterica serovar Typhimurium is an intracellular bacterial pathogen which can colonize a variety of hosts, including human, causing syndromes that vary from gastroenteritis and diarrhea to systemic disease.

ADP‐ribosylation of host cell proteins is a common mode of cell intoxication by pathogenic bacterial toxins. Antibodies induced by immunization with inactivated ADP‐ribosylating toxins provide efficient protection in case of some secreted toxins, e.g. , diphtheria and pertussis toxins. However, other ADP‐ribosylating toxins, such as Salmonella SpvB toxin, are secreted directly from the Salmonella ‐containing vacuole into the cytosol of target cells via the SPI‐2 encoded bacterial type III secretion system, and thus are inaccessible to conventional antibodies. Small‐molecule ADP‐ribosylation inhibitors are fraught with potential side effects caused by inhibition of endogenous ADP‐ribosyltransferases. Here, we report the development of a single‐domain antibody from an immunized llama that blocks the capacity of SpvB to ADP‐ribosylate actin at a molar ratio of 1:1. The single‐domain antibody, when expressed as an intrabody, effectively protected cells from the cytotoxic activity of a translocation‐competent chimeric C2IN‐C/SpvB toxin. Transfected cells were also protected against cytoskeletal alterations induced by wild‐type SpvB‐expressing strains of Salmonella. This proof of principle paves the way for developing new antidotes against intracellular toxins.—Al‐zogaray, V., Danquah, W., Aguirre, A., Urrutia, M., Berguer, P., García Vescovi, E., Haag, F., Koch‐Nolte, F., Goldbaum, F. A. Single‐domain llama antibodies as specific intracellular inhibitors of SpvB, the actin ADP‐ribosylating toxin of Salmonella typhimurium. FASEB J. 25, 526–534 (2011). www.fasebj.org

Bacterial survival in diverse and changing environments relies on the accurate interplay between different regulatory pathways, which determine the design of an adequate adaptive response. The proper outcome depends on a precise gene expression profile generated from the finely tuned and concerted action of transcriptional factors of distinct regulatory hierarchies. Salmonella enterica serovar Typhimurium harbors multiple regulatory systems that are crucial for the bacterium to cope with harsh extra- and intracellular environments. In this work, we found that the expression of Salmonella RstA, a response regulator from the two-component system family, was able to downregulate the expression of three RpoS-controlled genes ( narZ , spvA , and bapA ). Furthermore, this downregulation was achieved by a reduction in RpoS cellular levels. The alternative sigma factor RpoS is critical for bacterial endurance under the most-stressful conditions, including stationary-phase entrance and host adaptation. Accordingly, RpoS cellular levels are tightly controlled by complex transcriptional, translational, and posttranslational mechanisms. The analysis of each regulatory step revealed that in Salmonella , RstA expression was able to promote RpoS degradation independently of the MviA-ClpXP proteolytic pathway. Additionally, we show that RstA is involved in modulating Salmonella biofilm formation. The fact that the RpoS-modulated genes affected by RstA expression have previously been demonstrated to contribute to Salmonella pathogenic traits, which include biofilm-forming capacity, suggests that under yet unknown conditions, RstA may function as a control point of RpoS-dependent pathways that govern Salmonella virulence.