Immobilization and Stabilization of Beta-Xylosidases from Penicillium janczewskii

β-Xylosidases are critical for complete degradation of xylan, the second main constituent of plant cell walls. A minor β-xylosidase (BXYL II) from Penicillium janczewskii was purified by ammonium sulfate precipitation (30% saturation) followed by DEAE-Sephadex chromatography in pH 6.5 and elution with KCl. The enzyme presented molecular weight (MW) of 301 kDa estimated by size exclusion chromatography. Optimal activity was observed in pH 3.0 and 70–75 °C, with higher stability in pH 3.0–4.5 and half-lives of 11, 5, and 2 min at 65, 70, and 75 °C, respectively. Inhibition was moderate with Pb⁺² and citrate and total with Cu⁺², Hg⁺², and Co⁺². Partially purified BXYL II and BXYL I (the main β-xylosidase from this fungus) were individually immobilized and stabilized in glyoxyl agarose gels. At 65 °C, immobilized BXYL I and BXYL II presented half-lives of 4.9 and 23.1 h, respectively, therefore being 12.3-fold and 33-fold more stable than their unipuntual CNBr derivatives (reference mimicking soluble enzyme behaviors). During long-term incubation in pH 5.0 at 50 °C, BXYL I and BXYL II glyoxyl derivatives preserved 85 and 35% activity after 25 and 7 days, respectively. Immobilized BXYL I retained 70% activity after 10 reuse cycles of p-nitrophenyl-β-D-xylopyranoside hydrolysis.

     

Full enzymatic hydrolysis of commercial sucrose laurate by immobilized-stabilized derivatives of lipase from Thermomyces lanuginosa

Sucrose laurate is a detergent that is useful for various biochemical applications because it is a green compound and is easily degradable after hydrolysis with a lipase or esterase. One problem observed in the process of sucrose laurate degradation is that most commercial detergent preparations are impure, necessitating the hydrolysis of all of the sucrose esters present in the preparation, all of them with detergent properties. In this article, a highly active catalyst, which is able to perform the hydrolysis of commercial sucrose laurate, is presented. The use of glyoxyl agarose preparations of a previously aminated Thermomyces lanuginosa lipase (TLL) enabled complete hydrolysis, in less than 30 min, of all of the compounds that comprise the mixture. In addition, this derivative is stable in the presence of 20% ethanol, which is necessary to prevent microbial contamination.     

Enzyme immobilization strategies for the design of robust and efficient biocatalysts

Different strategies for the preparation of efficient and robust immobilized biocatalysts are here reviewed. Different physico-chemical approaches are discussed.i.- The stabilization of enzyme by any kind of immobilization on pre-existing porous supports.ii.- The stabilization of enzymes by multipoint covalent attachment on support surfaces.iii.- Additional stabilization of immobilized-stabilized enzyme by physical or chemical modification with polymers.These three strategies can be easily developed when enzymes are immobilized in pre-existing porous supports. In addition to that, these immobilized-stabilized derivatives are optimal to develop enzyme reaction engineering and reactor engineering. Stabilizations ranging between 1000 and 100,000 folds regarding diluted soluble enzymes are here reported.     

Microextraction sample preparation techniques in forensic analytical toxicology

Sample preparation is a critical step in forensic analytical toxicology. Different extraction techniques are employed with the goals of removing interferences from the biological samples, such as blood, tissues and hair, reducing matrix effects and concentrating the target analytes, among others. With the objective of developing faster and more ecological procedures, microextraction techniques have been expanding their applications in the recent years. This article reviews various microextraction methods, which include solid‐based microextraction, such as solid‐phase microextraction, microextraction by packed sorbent and stir‐bar sorptive extraction, and liquid‐based microextraction, such as single drop/hollow fiber‐based liquid‐phase microextraction and dispersive liquid–liquid microextraction, as well as their applications to forensic toxicology analysis. The development trend in future microextraction sample preparation is discussed.     

Regio-selective deprotection of peracetylated sugars via lipase hydrolysis

Purified lipases (via interfacial activation on hydrophobic supports) from different microbial extracts have been evaluated in the regio-selective hydrolysis of peracetylated sugars (peracetylated glucose, ribose and sucrose). Among the enzymes tested, lipases from Candida rugosa (CRL) and from Pseudomonas fluorescens (PFL) exhibited the best properties in these reactions.Then, we have prepared two different immobilized lipase preparations obtained by interfacial activation on hydrophobic supports or by covalent attachment on glutaraldehyde agarose. Interfacially activated lipases exhibited a higher activity than covalently attached enzymes (even by a 100-fold factor), giving the higher yields of mono deacetylated sugars (in some instances by more than a threefold factor) in short reaction times. In the hydrolysis of 1,2,3,5-tetra-O-acetyl-β-d-ribofuranose catalyzed by PFL adsorbed on octyl agarosa, hydrolyzed mainly the 3 position (30% of yield) while the CRL gave the hydrolysis only in position 5 (about 50% of yield).Depending on the enzyme immobilized preparation, we have been able also to obtain selective hydrolysis of 1,2,3,4,6-penta-O-acetyl-α/β-d-glucopyranose obtaining a free hydroxyl group in position 1, 4 or 6. Moreover, selective hydrolysis in the 4′ position of peracetylated sucrose was achieved when the hydrolysis is performed with CRL immobilized on octyl-agarose (yield was 77%).     

Measurement of the slope of the π0-electromagnetic form factor

Using a magnetic spectrometer, we have studied 30 000 π⁰ → e⁺e^?γ events coming from K_π2⁺ decays in flight. We find a positive value for the slope of the neutral-pion electromagnetic form factor: a = +0.10 ± 0.03 (or a = +0.05 ± 0.03 without radiative corrections). This value is higher than the predictions of vector meson models.     

Observation of the π0 → e+e− decay

We have observed for the first time the very rare π⁰ → e⁺e^? decay. The tagged π⁰'s come from K⁺ → π⁺π⁰ decays in flight. We find the branching ratio to be (2.23_?1.1^+2.4) × 10^?7 (90% C.L.). This value is well above the QED unitary limit and comes in rough agreement with a second-order electromagnetic process mediated by vector mesons.     

A study of 225 000 τ+ decays

In this article, results of the analysis of 225 000 τ⁺ decays in flight recorded at the CERN-PS with an electronic detector are presented. A quadratic fit of the matrix element |M|² ∝ 1 + aY + bX² + cY² gives the following values for the parameters: a = 02814 ± 0.0082; b = ?0.099 ± 0.019; c = ?0.001 ± 0.023.     

Measurement of the polarization in the forward peak of π−p→K0λ at 5 GeV/c

The π^?p→K⁰λ polarization has been measured at 5 GeV/c in the range 0<?t<1.4 (GeV/c)². The polarization is small for ?t?0.4 (GeV/c)², becoming negative at the higher values of ?t.     

Measurement of the polarization parameter of the reactionsπ− p → π0n and ηn at 5 and 8 GeV/c

Measurements of the polarization parameter of the reactions π^?p → π⁰n and π^?p → ηn at 4.90 and 7.85 GeV/c and for a squared four-momentum transfer ?t ranging from 0.1 to 2.0 (GeV/c)² have been achieved by using a butanol polarized proton target and detecting only the two γ's from the neutral meson decay. The background due to events involving bound protons has been separately measured and subtracted out. A large positive polarization has been found for the reaction π^?p → π⁰n. It is consistent with other pion-nucleon scattering data connected by isospin conservation. The polarization for the reaction π^?p → ηn is not significantly different from zero within the large experimental errors.