Leopoldo Güereca

Properties of levansucrase fromBacillus circulans

A polysaccharide-producing bacterium was isolated from cane sugar. It was identified asBacillus circulans and produced levansucrase at pH and temperature optima of 5–7 and 40°C respectively. The enzyme is extracellular and inducible with sucrose. It possesses initial hydrolytic and transferase activities that can be altered by modifying reaction conditions. Levansucrase was recovered from the fermentation broth by extraction with polyethylene glycol (1500 Da). Further purification resulted in an enzyme with a molecular mass of 52 kDa and a pI of 4.7. At high sucrose concentration (300 mM), the transferase activity but not the hydrolase activity were inhibited. Levan increased the transferase activity but had no effect on the hydrolytic activity. The levansucrase had high transferase activity with maltose, galactose and lactose and moderate activity towards sorbitol and glycerol.

Structural and functional studies of α-helix 5 region from Bacillus thuringiensis Cry1Ab δ-endotoxin

Abstract The crystal insecticidal proteins from Bacillus thuringiensis are modular proteins comprised of three domains connected by single linkers. Domain I is a seven α-helix bundle, which has been involved in membrane insertion and pore formation activity. Site-directed mutagenesis has contributed to identify regions that might play an important role in the structure of the pore-forming domain within the membrane. There are several evidences that support that the hairpin α4-α5 inserts into the membrane in an antiparallel manner, while other helices lie on the membrane surface. We hypothesized that highly conserved residues of α5 could play an important role in toxin insertion, oligomerization and/or pore formation. A total of 15 Cry1Ab mutants located in six conserved residues of Cry1Ab, Y153, Y161, H168, R173, W182 and G183, were isolated. Eleven mutants were located within helix α5, one mutant was located in the loop α4-α5 and three mutants, W182P, W182I and G183C, were located in the loop α5-α6. Their effect on binding, K + permeability and toxicity against Manduca sexta larvae was analyzed and compared. The results provide direct evidence that some residues located within α5 have an important role in stability of the toxin within the insect gut, while some others also have an important role in pore formation. The results also provide evidence that conserved residues within helix α5 are not involved in oligomer formation since mutations in these residues are able to make pores in vitro.

The oligomeric state of Bacillus thuringiensis Cry toxins in solution.

The molecular mass of different Cry toxins produced by Bacillus thuringiensis bacteria was estimated by size-exclusion chromatography and non-denaturing polyacrylamide gel electrophoresis at neutral and alkaline pH in order to assess the existence of oligomers in solution. We found that Cry1Aa, Cry1Ac, Cry1C, Cry1D and Cry3A toxins exist in solution as a mixture of monomer and high molecular mass aggregates with an apparent molecular mass greater than 600 kDa, that depend on the time elapsed between toxin activation and analysis. Aggregation of toxins by disulfide bonds is unlikely because aggregates are also observed in samples incubated with DTT. These data show that the Cry toxins studied do not form oligomers of less than ten subunits in solution and suggest that oligomer formation may occur after the toxin binds to the receptor and inserts into the membrane.

Influence of the germline sequence on the thermodynamic stability and fibrillogenicity of human lambda 6 light chains

Light chain‐associated amyloidosis is a fatal disease characterized by the aggregation and pathologic deposition of monoclonal light chain‐related fragments as amyloid fibrils in organs or tissues throughout the body. Notably, it has been observed that proteins encoded by the λ variable light chain (VL) gene segment 6a are invariably associated with amyloid deposition; however, the contribution of the gene to this phenomenon has not been established. In this regard, we have determined the thermodynamic stability and kinetics of in vitro fibrillogenesis of a recombinant (r) VL protein, designated 6aJL2, which contains the predicted sequences encoded by the 6a and JL2 germline genes. Additionally, we studied a 6a mutant (6aJL2‐Arg25Gly), that is present in ∼25% of all amyloid‐associated λ6 light chains. Remarkably, the wild‐type 6aJL2 protein was more stable than were all known amyloidogenic κ and λ light chains for which stability parameters are available; more importantly, it was even more so (and less fibrillogenic) than the only clinically proven nonamyloidogenic λ6 protein, Jto. Conversely, the mutated 6aJL2‐R25G molecule was considerably less stable and more fibrillogenic than was the native 6aJL2. Our data indicate that the propensity of λ6 light chains to form amyloid can not be attributed to thermodynamic instability of the germline‐encoded Vλ6 domain, but rather, is dependent on sequence alterations that render such proteins amyloidogenic. Proteins 2008.

Penicillin acylase extraction by osmotic shock

Penicillin acylase was extracted from E. Coli by osmotic shock. The process was optimized by factorial design, scaled up and integrated to a purification process in order to compare it with purification processes reported in the literature. A specific parameter PPEF (Purification Processes Evaluation Factor) was defined for this purpose.

Biochemical characterization of the lipolytic activity of Pseudomonas aeruginosa IGB 83

Physiological regulation of the extracellular lipolytic activity of the Pseudomonas aeruginosa isolate IGB 83 was investigated by growing the bacteria with different carbon sources. Glycerol and triacylglycerols induce lipolytic activity, while glucose or acetate do not. The optimum temperature for the extracellular lipolytic activity was found to be 55°C while maximal activity was found at pH 10. The activity half-life at pH 8·5 and 55°C was 13 min. The lipase was purified from the culture supernatant and had an apparent molecular weight of 58 kD and an isoelectric point of 8. The biochemical characterization of the alkaline and thermoresistant lipolytic activity and a comparison with the reported characteristics of other enzymes clearly shows that this enzyme constitutes a novel lipase.

Identification and isolation of human insulin A and B chains by high-performance liquid chromatography

A method for the isolation, identification and quantification of human insulin A and B chains by high-performance liquid chromatography (HPLC) is described. These chains were isolated from a peptide mixture produced by E. coli with modified genes obtained by genetic engineering. The method is based on the use of hydrophilic reagents, forming ion pairs in a reversed-phase column. Because some undesirable effects resulting from the use of phosphoric acid were observed, especially with the B chain, a new HPLC method was developed for each of the two human insulin chains. The use of trifluoroacetic acid as a counter ion for the A chain and of formic acid for the B chain led to the rapid isolation and purification of each chain by HPLC. The advantage of this method is that it provides a highly pure product, which was identified by polyacrylamide gel electrophoresis and amino acid analysis.

A new expression vector for the production of fused proteins in Escherichia coli

SummaryThe construction of a new expression vector for fused proteins production in Escherichia coli is reported. This new vehicle uses the trp promoter-operator control region for the high level expression of a DNA fragment that codes for the amino terminal fragment of the cI λ repressor protein. This truncated polypeptide is accumulated as inclusion bodies that are easily purified. To probe the benefits of the system, synthetic DNA that codes for the human insulin B chain, was cloned at the end of the DNA coding region for the cI truncated peptide. The hybrid peptide thus produced after induction, allowed an easy and reproducible purification of active insulin B chain.

Design of an aqueous two-phase system for the purification of ICP from Bacillus thuringiensis

Abstract A method is described for the purification of Bacillus thuringiensis protein crystals based on an aqueous two-phase system distribution, composed of potassium phosphate-polyethylene glycol-water. This method is simple, one step, fast, inexpensive and efficient compared with other techniques. It has been used successfully to purify and characterize the protein crystals from several strains of Bacillus thuringiensis .

Updating knowledge on new medically important scorpion species in Mexico

&NA; The increment in the number of scorpion envenoming cases in Mexico is mainly associated to the rapid growth of the urban areas, and consequently, to the invasion of natural habitats of these arachnids. On the other hand, there is a great diversity of scorpion species, so it is indispensable to identify those of medical importance, which we now know are many more than the 7–8 previously reported as dangerous to humans. Because different LD50 values have been reported for the venom of the same species, probably due to variations in the experimental conditions used, in this work we determined the LD50 values for the venoms of 13 different species of scorpions using simple but systematic procedures. This information constitutes a referent on the level of toxicity of medically important scorpion species from Mexico and establishes the bases for a more comprehensive assessment of the neutralizing capacity of current and developing antivenoms. HighlightsThe total diversity of Mexican toxic scorpions has not been determined.We demonstrated that there are at least 13 medically important species.Systematic determination of venom LD50 revealed different levels of toxicity.These results will help to develop new anti‐venoms.