Role of the Sortase A in the Release of Cell-Wall Proteinase PrtS in the Growth Medium of Streptococcus thermophilus 4F44.

Growth of the lactic acid bacterium Streptococcus thermophilus in milk depends on its capacity to hydrolyze proteins of this medium through its surface proteolytic activity. Thus, strains exhibiting the cell envelope proteinase (CEP) PrtS are able to grow in milk at high cellular density. Due to its LPNTG motif, which is possibly the substrate of the sortase A (SrtA), PrtS is anchored to the cell wall in most S. thermophilus strains. Conversely, a soluble extracellular PrtS activity has been reported in the strain 4F44. It corresponds, in fact, to a certain proportion of PrtS that is not anchored to the cell wall but rather is released in the growth medium. The main difference between PrtS of strain 4F44 (PrtS4F44) and other PrtS concerns the absence of a 32-residue imperfect duplication in the prodomain of the CEP, postulated as being required for the maturation and correct subsequent anchoring of PrtS. In fact, both mature (without the prodomain at the N-terminal extremity) and immature (with the prodomain) forms are found in the soluble PrtS4F44 form along with an intact LPNTG at their C-terminal extremity. Investigations we present in this work show that (i) the imperfect duplication is not implied in PrtS maturation; (ii) the maturase PrtM is irrelevant in PrtS maturation which is probably automaturated; and (iii) SrtA allows for the PrtS anchoring in S. thermophilus but the SrtA of strain 4F44 (SrtA4F44) displays an altered activity.

N-acylation of L-amino acids in aqueous media: Evaluation of the catalytic performances of Streptomyces ambofaciens aminoacylases.

N-acylated amino acids are widely used as surfactants and/or actives in cosmetics and household formulations. Their industrial production is based on the use of the Schotten-Baumann chemical and unselective reaction. Faced to the growing demand for greener production processes, selective enzymatic synthesis in more environment-friendly conditions starts to be considered as a potential alternative. This study concerns the use of the aminoacylases from Streptomyces ambofaciens to selectively catalyse aminoacid acylation reaction by fatty acids in aqueous medium. The results demonstrated that, when using undecylenoic acid as acyl donor, these aminoacylases properly catalyse the acylation of 14 of the 20 proteogenic l-amino acids tested on their α amino group with a great variability depending on the nature of the amino acid (polar or not, positively/negatively charged, aromatic or not…). More precisely, the following 9 amino acids were shown to be preferentially acylated by S. ambofaciens aminoacylases as follows: lysine > arginine > leucine > methionine > phenylalanine > valine > cysteine > isoleucine > threonine. Different fatty acids were used as acyl donors and, in most cases, the fatty acid length influenced the conversion yield. The kinetic study of α-lauroy-lysine synthesis showed a positive influence of lysine concentration with Vmax and Km of 3.7 mM/h and 76 mM, respectively. Besides, the lauric acid had an inhibitory effect on the reaction with Ki of 70 mM. The addition of cobalt to the reaction medium led to a more than six-fold increase of the reaction rate. These results, achieved with the aminoacylases from S. ambofaciens represent an improved enzyme-based N-acylated amino acids production in order to provide an alternative way to the Schotten-Baumann chemical reaction currently used in the industry.

Enzymatic synthesis of geranyl acetate in packed bed reactor in supercritical carbon dioxide under various pressure-temperature conditions and reactor configurations.

Data in this paper describes the catalytic performances, expressed in terms of conversion %, of geraniol and acetic acid to geranyl acetate, using the immobilized lipase B from Candida antarctica in packed bed reactors (PBR) using supercritical CO2 as a solvent. Readers will find data related to different Figures or equations of the article as well as supplementary data that will help to make the difference between flowrates of CO2 in a liquid state and corresponding flowrates of supercritical CO2 for various CO2 pressure and temperature combinations.

An aminoacylase activity from Streptomyces ambofaciens catalyzes the acylation of lysine on α-position and peptides on N-terminal position.

The presence of aminoacylase activities was investigated in a crude extract of Streptomyces ambofaciens ATCC23877. First activities catalyzing the hydrolysis of N-α or ε-acetyl-L-lysine were identified. Furthermore, the acylation of lysine and different peptides was studied and compared with results obtained with lipase B of Candida antarctica (CALB). Different regioselectivities were demonstrated for the two classes of enzymes. CALB was able to catalyze acylation only on the ε-position whereas the crude extract from S. ambofaciens possessed the rare ability to catalyze the N-acylation on the α-position of the lysine or of the amino-acid in N-terminal position of peptides. Two genes, SAM23877_1485 and SAM23877_1734, were identified in the genome of Streptomyces ambofaciens ATCC23877 whose products show similarities with the previously identified aminoacylases from Streptomyces mobaraensis. The proteins encoded by these two genes were responsible for the major aminoacylase hydrolytic activities. Furthermore, we show that the hydrolysis of N-α-acetyl-L-lysine could be attributed to the product of SAM23877_1734 gene.

Enzymatic modification of polysaccharides: Mechanisms, properties, and potential applications: A review.

Polysaccharides are natural biopolymers found in almost all living organisms. They are used extensively in various industrial applications, such as food, adhesives, pharmaceuticals, and cosmetics. In many cases, their practical use is limited because of their weak solubility in neutral pH, their unsuitable hydrophilic/hydrophobic balance. In this context, chemical or enzymatic modification of their structure appears as a relevant way, to improve their properties, and thus to enlarge the field of their potential applications. Taking into account the reduction of the input energy and the environmental impact, and due to high specificity and selectivity properties, enzymatic bioprocesses have been investigated as attractive alternatives to toxic and non-specific chemical approaches. This review discusses the methods of enzymatic functionalization of four well-known polysaccharides, chitosan, cellulose, pectin and starch. Particular emphasis was placed on the methods, the reaction types and the enzymes implicated in the modification such as laccases, peroxidases lipases, tyrosinases, and transglutaminases. The impact of functionalization on the properties and the applications of polysaccharide derivatives were described.

Enzymatic production of bioactive docosahexaenoic acid phenolic ester.

Docosahexaenoic acid (DHA) is increasingly considered for its health benefits. However, its use as functional food ingredient is still limited by its instability. In this work, we developed an efficient and solvent-free bioprocess for the synthesis of a phenolic ester of DHA. A fed-batch process catalyzed by Candida antarctica lipase B was optimised, leading to the production of 440 g/L vanillyl ester (DHA-VE). Structural characterisation of the purified product indicated acylation of the primary OH group of vanillyl alcohol. DHA-VE exhibited a high radical scavenging activity in acellular systems. In vivo experiments showed increased DHA levels in erythrocytes and brain tissues of mice fed DHA-VE-supplemented diet. Moreover, in vitro neuroprotective properties of DHA-VE were demonstrated in rat primary neurons exposed to amyloid-ß oligomers. In conclusion, DHA-VE synergized the main beneficial effects of two common natural biomolecules and therefore appears a promising functional ingredient for food applications.

Oxidative stability of DHA phenolic ester.

Docosahexaenoic acid vanillyl ester (DHA-VE) was synthesized from docosahexaenoic acid ethyl ester (DHA-EE) and vanillyl alcohol by a solvent-free alcoholysis process catalysed by Candida antarctica lipase B. Oxidative stability of pure DHA-VE and the crude reaction medium consisting of 45% DHA-VE and 55% DHA-EE were compared with that of DHA-EE under various storage conditions. Oxidation progress was followed by determination of conjugated dienes and FTIR measurements. Analyses showed that DHA-EE was rapidly oxidised under all storage conditions in comparison with DHA-VE and crude reaction medium, whatever the temperature and the storage time. The grafting of vanillyl alcohol appeared as a powerful way to stabilize DHA against oxidation. Thanks to their stability, both DHA-VE and the crude reaction medium, allowing the production of the ester, offer huge potential as functional ingredients.

Solubilities inferred from the combination of experiment and simulation. Case study of quercetin in a variety of solvents.

A strategy to infer solubilities from the combination of experiment and all-atom simulations is presented. From a single experimental estimate, the solubility of a substrate can be predicted in various environments from the related free energies of solvation. In the case of quercetin, the methodology was shown to reproduce the experimental solubilities in chloroform, water, acetonitrile, acetone, and tert-amyl alcohol within 0.5 log unit. The reliability of the estimates is markedly correlated to the accuracy of the experimental measure and to both the accuracy and precision of the computed free energies of solvation.

Enzymatic acylation of flavonoids: effect of the nature of the substrate, origin of lipase, and operating conditions on conversion yield and regioselectivity.

The conversion yield at equilibrium, the initial rate, and the regioselectivity of the enzymatic acetylation of aglycone flavonoids (quercetin, naringenin, hesperetin, and chrysin) were investigated and compared to those obtained with a glycosylated one (isoquercitrin). The effects of a wide range of operating conditions were quantified. Fourier transform infrared spectrometry (FT-IR), NMR, and high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) analyses showed that for glycosylated flavonoids, in the presence of Candida antarctica (CAL-B), the acetylation occurred on the 2''-OH, 3''-OH, and 6''-OH of the glucose part, while with Pseudomonas cepacea lipase (PSL-C) acetylation takes place on 6''-OH of the sugar and 4'-OH of the B-ring. For aglycone flavonoids, the acetylation occurred only with PSL-C on 4'-OH, 3'-OH, and 7-OH hydroxyls. The conversion yield and the number and the relative proportions of the synthesized products were found dependent on the nature of the enzyme, the molar ratio, and the flavonoid structure. The initial rate was affected only by the origin of the enzyme.