Biotechnological applications of Candida antarctica lipase A: State-of-the-art

The yeast Candida antarctica produces two different lipases, lipases A and B. While lipase B (CAL-B) is probably the mostly employed hydrolase in the biocatalysis field, the use of the lipase A (CAL-A) has been rather scarce and consequently its tridimensional structure has not been elucidated yet. However, CAL-A is a useful biocatalyst with many different applications that have been described especially in the last few years. Its attractiveness results from its unique features among hydrolases: the high thermostability, allowing operation at T > 90 °C; the ability to accept tertiary and sterically hindered alcohols, which has recently been attributed to the existence of a specific aminoacidic sequence in the active site; the sn-2 recognition in hydrolysis of triglycerides; the selectivity towards trans-fatty acids; the stability in the acidic pH range. Furthermore, it is considered to be an excellent biocatalyst for the asymmetric synthesis of amino acids/amino esters, due to its chemoselectivity towards amine groups. Considering all these aspects, in the present review, the origin, the properties and the applications of the CAL-A are briefly described and discussed, pointing out the unique characteristics of this biocatalyst.     

Turn stabilization in short peptides by C(alpha)-methylated alpha-amino acids

The crystal-state conformations of three protected tripeptides, four tetrapeptides, and one pentapeptide, heavily based on the chiral C(alpha)-methylated alpha-amino acids Iva, (alpha Me)Nva, and (Me)Val, were assessed by X-ray diffraction analyses. The eight peptide sequences are as follows: Z-(D-Iva)2-D-Val-OMe, Z-D-Iva-L-Iva-Gly-OtBu, Z-L-Pro-D-Iva-L-Iva-Gly-OtBu, Z-L-Pro-L-Iva-D-Iva-Gly-OtBu, Z-Aib-[L-(alpha Me)Nva]2-OtBu, Ac-[L-(alpha Me)Val]3-D-(alpha Me)Val-OtBu, Z-[L-(alpha Me)Val]4-OH, and Z-L-Ala-[L-(alpha Me)Nva]4-OtBu. Two independent molecules were observed in the asymmetric units of Z-D-Iva-L-Iva-Gly-OtBu and Z-Aib-[L-(alpha Me)Nva]2-OtBu, while three independent molecules were seen in Z-L-Ala-[L-(alpha Me)Nva]4-OtBu. All peptides are folded in a single or multiple beta-turn conformations. Interestingly: (i) a water bridge within the N-terminal beta-turn is seen in Z-L-Pro-L-Iva-D-Iva-Gly-OtBu (dihydrate), and (ii) the hydroxyl group of the C-terminal carboxyl functionality of Z-[L-(alpha Me)Val]4-OH generates an oxy-analogue of a beta-turn. The N-terminal beta-turn is missing in molecules A and B, but it does occur, although poorly stabilized, in molecule C, of Z-L-Ala-[L-(alpha Me)Nva]4-OtBu.     

Defense and resistance-inducing activities in tobacco of the sulfated beta-1,3 glucan PS3 and its synergistic activities with the unsulfated molecule

Laminarin, a beta-1,3 glucan with single beta-glucose branches at position 6, was chemically sulfated to produce PS3 with a degree of sulfation of 2.4. PS3 has previously been shown to activate the salicylic acid (SA) signaling pathway in infiltrated tobacco and Arabidopsis thaliana leaf tissues. Here, we investigated whether PS3 induces systemic defense and resistance responses in tobacco. Using a radiolabeled compound, it was first demonstrated that PS3 remains strictly localized to the infiltrated tissues. PS3 is also resistant to beta-glucanase degradation. In transgenic PR1-beta-glucuronidase (GUS) tobacco plants, PS3 causes a strong increase in GUS activity in treated tissues but none in untreated leaves. PS3-infiltrated tissues challenged with tobacco mosaic virus (TMV) 8 d after elicitor application show a decrease in both the lesion number and the lesion size, whereas treatment with laminarin, the unsulfated native glucan, affected only the lesion number. PS3 does not induce systemic acquired resistance to TMV. PS3 and laminarin show synergistic effects in promoting the oxidative burst in tobacco cell suspensions and in increasing the expression of genes encoding O-methyltransferases of the phenylpropanoid pathway in tobacco plants. No synergistic effect was observed on the expression of either the SA-dependent acidic PR1 gene or the ethylene-dependent basic PR5 gene in tobacco plants.     

GROWTH STIMULATION OF ALEXANDRIUM TAMARENSE (DINOPHYCEAE) BY HUMIC SUBSTANCES FROM THE MANICOUAGAN RIVER (EASTERN CANADA)1

In the St. Lawrence Estuary, annual recurrent blooms of the toxic dinoflagellate Alexandrium tamarense L. Balech are associated with brackish waters. Riverine inputs are suspected to favor bloom development by increasing water column stability and/or by providing growth stimulants such as humic substances (HS). A 17‐day culture experiment was conducted to evaluate the importance of HS as growth factors for A. tamarense. Nonaxenic cultures were exposed to four HS extracts from three different sources: humic and fulvic acids isolated from the Manicouagan River, Quebec, Canada; humic acids from the Suwannee River, Georgia, United States; and a desalted alkaline soil extract. For each extract, four concentrations were tested as supplements to the artificial Keller medium, a nitrate‐rich algal culture medium. Additions of HS from all sources significantly enhanced the overall growth rates relative to the controls. Concentrations of HS, estimated by UV spectrophotometry, remained constant throughout the exponential growth phase, suggesting that the HS were acting mainly as growth promoters during our experiment. Dose–response curves indicated that HS could increase the growth rate of A. tamarense even at low concentrations, such as those encountered in the St. Lawrence Estuary. Our results support the hypothesis that HS from the Manicouagan River plume can stimulate the development of toxic dinoflagellate blooms.     

Virulence of Salmonella enteritidis phage type 4 is related to the possession of a 38 MDa plasmid

Nine strains of Salmonella enteritidis phage type 4 were examined for virulence in BALB/c mice. The possession of a 38 MDa plasmid was necessary for full virulence. Strains carrying this plasmid had LD50 values of less than 20 bacteria whilst plasmid-free strains had LD50 values of greater than 10(6) bacteria when challenged intraperitoneally. Pathogenesis of disease involved the widespread distribution of bacteria throughout the tissues. Possession of the 38 MDa plasmid could not be linked with the ability of strains to express novel outer membrane proteins, to produce toxins affecting Vero, Y1, HeLa, Henle or HEp-2 cells, or to invade HEp-2 cells. Furthermore, the 38 MDa plasmid did not encode an aerobactin-mediated iron uptake system or the production of a haemolysin. Strains of S. enteritidis PT4 isolated in 1967, 1978 or 1979 and possessing the 38 MDa plasmid showed the same virulence properties as the current plasmid-carrying strains. This suggests that the enhanced virulence of the current strains for poultry is unlikely to be the result of changes in the 38 MDa plasmid.     

The Flexibility of the Nucleic Acids: (II) The Calculation of Internal Energy and Applications to Mononucleotide Repeat DNA

Abstract

Results concerning the flexibility of mononucleotide repeat DNA are presented using a novel methodology, denoted “SIR”, to describe continuous changes in the structure of the nucleic acid. This methodology, combined with internal energy calculations and analytical energy gradients allows us to determine optimal conformations of poly(dG)?poly(dC) and poly (dA)?poly(dT) in both the A and B forms, taking into account the influence of the solvent medium and explicit counterions. Subsequently, several different types of distorsion of these optimal structures are investigated. It is shown that excellent correlation with experimental results concerning coupled changes in structural variables is obtained and several new correlations are also detected.     

Synthesis and biological evaluation of potential threonine synthase inhibitors: Rhizocticin A and Plumbemycin A

Rhizocticins and Plumbemycins are natural phosphonate antibiotics produced by the bacterial strains Bacillus subtilis ATCC 6633 and Streptomyces plumbeus, respectively. Up to now, these potential threonine synthase inhibitors have only been synthesized under enzymatic catalysis. Here we report the chemical stereoselective synthesis of the non-proteinogenic (S,Z)-2-amino-5-phosphonopent-3-enoic acid [(S,Z)-APPA] and its use for the synthesis of Rhizocticin A and Plumbemycin A. In this work, (S,Z)-APPA was synthesized via the Still–Gennari olefination starting from Garner’s aldehyde. The Michaelis–Arbuzov reaction was used to form the phosphorus–carbon bond. Oligopeptides were prepared using liquid phase peptide synthesis (LPPS) and were tested against selected bacteria and fungi.     

Validation of Horne and Ostberg morningness-eveningness questionnaire in a middle-aged population of French workers

As suggested by the authors, the Horne and Ostberg morning/evening questionnaire (MEQ) has never been adapted to evaluate a nonstudent population. The purpose of this study was to validate this MEQ in a sample of middle-aged workers by modifying only the cutoffs. It was administered in 566 non-shift-workers aged 51.2 to 3.2 years who presented no sleep disorders. According to the Home and Ostberg classification, the sample consisted of 62.1% morning type, 36.6% neither type, and 2.2% evening type. Multiple correspondence analysis, which determines the principal components, was performed on all MEQ items. Then an ascending hierarchical classification was applied to determine 3 clusters from these principal components. On the basis of these 3 clusters, new cutoffs were determined: evening types were considered as scoring under 53 and morning types above 64, thus giving 28.1% morning type, 51.7% neither type, and 20.2% evening type. As an external validation, eveningness was associated with later bedtime and waking-up time (more pronounced at the weekend), greater need for sleep, larger daily sleep debt, greater morning sleepiness, and ease of returning to sleep in the early morning. A positive correlation between age and morningness was again found. This study confirms that "owls" are not rare in a middle-aged sample. We conclude that this adapted MEQ could be useful when investigating age-related changes in sleep.     

Synthesis and properties of new bolaform and macrocyclic galactose-based surfactants obtained by olefin metathesis

A series of galactose-based surfactants with various structures likely to display new interesting properties were synthesized. Four monocatenary surfactants were elaborated by microwave-assisted galactosylation of undecanol or 10-undecenol. These compounds were slightly soluble in water. Their tensioactive properties were determined at 45 degrees C. Olefin metathesis was used to synthesize the two single-chain bolaforms from undec-10-enyl galactopyranosides; two pseudomacrocyclic bolaforms were prepared by grafting two carbamates at O-4 and O-4' sugar positions of the single-chain bolaforms. These four surfactants are insoluble in water and undergo monolayer compression. Cyclization of these bolaforms by olefin metathesis led to macrocyclic surfactant analogues of archaeobacterial membrane components.