Leticia Luciana Torres

• PhD in Biological Sciences
• Research Associate at University College London

Plasmids of the ColE1 family are among the most frequently used in molecular biology. They were adopted early for many biotechnology applications, and as models to study plasmid biology. Their mechanism of replication is well understood, involving specific interactions between a plasmid encoded sense-antisense gene pair (RNAI and RNAII). Due to suc...

Phi29 DNA polymerase (DNAP) is the replicative enzyme of the Bacillus subtilis bacteriophage Phi29. Its extraordinary processivity and its ability to perform isothermal amplification of DNA are central to many molecular biology applications, including high‐sensitivity detection and large‐scale production of DNA. We present here Phi29 DNAP as an eff...

Biocontainment comprises any strategy applied to ensure that harmful organisms are confined to controlled laboratory conditions and not allowed to escape into the environment. Genetically engineered microorganisms (GEMs), regardless of the nature of the modification and how it was established, have potential human or ecological impact if accidental...

Life on Earth is incredibly diverse. Yet, underneath that diversity, there are a number of constants and highly conserved processes: all life is based on DNA and RNA; the genetic code is universal; biology is limited to a small subset of potential chemistries. A vast amount of knowledge has been accrued through describing and characterizing enzymes...

A homologue of the Escherichia coli penicillin acylase is encoded in the genomes of several thermophiles, including in different Thermus thermophilus strains. Although the natural substrate of this enzyme is not known, this acylase shows a marked preference for penicillin K over penicillin G. Three-dimensional models were created in which the catal...

Penicillin acylases (PACs) are enzymes of industrial relevance in the manufacture of β-lactam antibiotics. Development of a PAC with a longer half-life under the reaction conditions used is essential for the improvement of the operational stability of the process. A gene encoding a homologue to Escherichia coli PAC was found in the genome of the th...

Optimum pH of HIS6::TthPAC. The TthPAC enzymatic activity was assayed at 65°C in the presence of 2.5 mM PenK and in 20 mM Britton-Robinson buffer at pH 3, 4, 5, 6, 7, 8 or 9.

Comparison of the kinetic data of TthPAC with other penicillin acylases. Data were measured at 40°C unless otherwise stated.

Chaperone elimination after co-expression with HIS6:: Tth PAC. Tth PAC was co-expressed with GroEL/ES in E. coli BL21 cells. Total soluble protein fraction was incubated in the absence (lanes 1–4) or in the presence of 5 mM ATP/10 mM Cl2Mg (lanes 5–8) [41] for 2 h at 4°C. In order to separate the Tth PAC from the detached GroEL/ES a 0-, 30-, 45- or...

Heterologous expression of the chimeric protein SpEco-PACTthin E. coli cells.TthPAC α- and β-subunit were immunodetected in the cytoplasmic fraction (upper and middle panels) and in the periplasmic fraction (lower pannel) of E. coli cells from BL21 strain(1, A and G); Rosetta-gami2 strain (2, B and H); BL21 strain co-expressing GroEL/ES and trigger...

Sequence alignment of PKAs and characterized PGAs. TthPKA, Thermus thermophilus HB27 PKA [TTC1972]; AutaPKA, Actinoplanes utahensis PKA [P29958]; SlavPKA, Streptomyces lavendulae PKA [AY611030]; EcoPGA, Escherichia coli PGA [P06875]; AfaePGA, Alcaligenes faecalis [ADD11517]; PretPGA, Providencia rettgeri PGA [AAP86197]; KcitPGA, Kluyvera citrophila...

Thermal inactivation course of TthPAC. Solutions containing 0.08 mg/ml of purified TthPAC were incubated in 50 mM phosphate buffer pH 7.5 at 65 and 75°C. The residual hydrolytic activity was determined using 5 mM penicillin K as substrate.

Table S2. Primers used in this work.

A promiscuous but very enantioselective (-)-γ-lactamase activity in the kinetic resolution of the Vince lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) was detected in the Pseudomonas fluorescens esterase I (PFEI). The lactamase activity was increased 200-fold by the introduction of a point mutation and resulted as enantioselective as the Microbacteriu...

Higher plants and cyanobacteria metabolize sucrose (Suc) by a similar set of enzymes. Suc synthase (SuS, A/UDP-glucose: D: -fructose 2-α-D: -glucosyl transferase) catalyzes a reversible reaction. However, it is in the cleavage of Suc that this enzyme plays an important role in vivo, providing sugar nucleotides for polysaccharide biosynthesis. In cy...

A metabolic pathway for biosynthesis of the nonreducing disaccharide mannosylfructose (β-fructofuranosyl-α-mannopyranoside), an important osmolyte in Agrobacterium tumefaciens, was discovered. We have identified and functionally characterized two ORFs that correspond to genes (named mfpsA and mfppA) encoding the rare enzymes mannosylfructose-phosph...