Sebastián Cerminati

High-level production of Bacillus cereus phospholipase C in Corynebacterium glutamicum.

Enzymatic oil degumming (removal of phospholipids) using phospholipase C (PLC) is a well-established and environmentally friendly process for vegetable oil refining. In this work, we report the production of recombinant Bacillus cereus PLC in Corynebacterium glutamicum ATCC 13869 in a high cell density fermentation process and its performance in soybean oil degumming. A final concentration of 5.5g/L of the recombinant enzyme was achieved when the respective gene was expressed from the tac promoter in a semi-defined medium. After treatment with trypsin to cleave the propeptide, the mature enzyme completely hydrolyzed phosphatidylcholine and phosphatidylethanolamine, which represent 70% of the phospholipids present in soybean oil. The results presented here show the feasibility of using B. cereus PLC for oil degumming and provide a manufacturing process for the cost effective production of this enzyme.

Dissecting the Metal Selectivity of MerR Monovalent Metal Ion Sensors in Salmonella

Two homologous transcription factors, CueR and GolS, that belong to the MerR metalloregulatory family are responsible for Salmonella Cu and Au sensing and resistance, respectively. They share similarities not only in their sequences, but also in their target transcription binding sites. While CueR responds similarly to Au, Ag, or Cu to induce the expression of its target genes, GolS shows higher activation by Au than by Ag or Cu. We showed that the ability of GolS to distinguish Au from Cu resides in the metal-binding loop motif. Here, we identify the amino acids within the motif that determine in vivo metal selectivity. We show that residues at positions 113 and 118 within the metal-binding loop are the main contributors to metal selectivity. The presence of a Pro residue at position 113 favors the detection of Cu, while the presence of Pro at position 118 disfavors it. Our results highlight the molecular bases that allow these regulators to coordinate the correct metal ion directing the response to a particular metal injury.

Selective detection of gold using genetically engineered bacterial reporters

Salmonella typhimurium harbours a Au‐resistance system whose expression is controlled by GolS, a transcriptional regulator of the MerR family that selectively detects Au with high sensitivity. We developed both Salmonella and genetically engineered Escherichia coli strains as Au‐selective whole‐cell biosensors by coupling the strictly regulated GolS‐dependent golB promoter to the gfp reporter gene. The bio‐reporters were evaluated under different laboratory conditions and calibrated for their use as selective Au detectors. Due to the intrinsic characteristics of the regulatory protein, the transgenic E. coli sensor exhibits low background, high signal‐to‐noise ratio, and improved sensitivity for detection of Au ions in a wide range of concentrations (up to 470 nM) with a calculated detection limit of ∼33 nM (6 µg L−1 or parts per billion) Au(I). The fluorescent Au‐sensing bacteria exhibit also minimal interference by chemically related metals such as Cu or Ag that are commonly found in Au deposits. These highly specific and sensitive Au detectors might allow the development of rapid and robust screening tools to improve discovery and extraction procedures. Biotechnol. Bioeng. 2011;108: 2553–2560.

The CpxR/CpxA system contributes to Salmonella gold-resistance by controlling the GolS-dependent gesABC transcription

Several regulatory systems contribute to bacterial resistance to heavy metals controlling the expression of factors required to eliminate the intoxicant and/or to repair the damage caused by it. In Salmonella, the response to Au ions is mediated by the specific metalloregulator GolS that, among other genes, controls the expression of the RND-efflux pump GesABC. In this work, we demonstrate that CpxR/CpxA, a main cell-envelope stress-responding system, promotes gesABC transcription in the presence of Au ions at neutral pH. Deletion of either cpxA or cpxR, or mutation of the CpxR-binding site identified upstream of the GolS-operator in the gesABC promoter region reduces but does not abrogate the GolS- and Au-dependent activation of gesABC. Au also triggers the activation of the CpxR/CpxA system and deletion of the cpxRA operon severely reduces survival in the presence of the toxic metal. Our results indicate that the coordinated action of GolS and CpxR/CpxA contribute to protecting the cell from severe Au damage.

The production, properties, and applications of thermostable steryl glucosidases

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.

The βγ-crystallin domain of Lysinibacillus sphaericus phosphatidylinositol phospholipase C plays a central role in protein stability

Abstractβγ-crystallin has emerged as a superfamily of structurally homologous proteins with representatives across all domains of life. A major portion of this superfamily is constituted by microbial members. This superfamily has also been recognized as a novel group of Ca2+-binding proteins with a large diversity and variable properties in Ca2+ binding and stability. We have recently described a new phosphatidylinositol phospholipase C from Lysinibacillus sphaericus (LS-PIPLC) which was shown to efficiently remove phosphatidylinositol from crude vegetable oil. Here, the role of the C-terminal βγ-crystallin domain of LS-PIPLC was analyzed in the context of the whole protein. A truncated protein in which the C-terminal βγ-crystallin domain was deleted (LS-PIPLCΔCRY) is catalytically as efficient as the full-length protein (LS-PIPLC). However, the thermal and chemical stability of LS-PIPLCΔCRY are highly affected, demonstrating a stabilizing role for this domain. It is also shown that the presence of Ca2+ increases the thermal and chemical stability of the protein both in aqueous media and in oil, making LS-PIPLC an excellent candidate for use in industrial soybean oil degumming.