Lipase-Catalyzed Condensation Reaction of 4-Nitrobenzaldehyde with Acetyl Acetone in Aqueous–Organic Cosolvent Mixtures and in Nearly Anhydrous Media

<div><p></p><p>The nature of the product(s) in lipase-catalyzed reaction of acetyl acetone with 4-nitrobenzaldehyde was found to depend upon the source of lipase and the reaction medium. Mucor javanicus lipase was found to give 70% aldol with 80% enantiomeric excess in anhydrous <i>t</i>-amyl alcohol. A 2:2 adduct was formed by the dimerization of the aldol along with an unsaturated cyclic ether as the side products in varying proportions depending upon the reaction medium and the lipase used.</p> <p>[Supplementary materials are available for this article. Go to the publisher's online edition of <i>Synthetic Communications</i>® for the following free supplemental resource(s): Full experimental and spectral details.]</p> </div

HPAEC analysis of <i>Ct</i>43Ara<i>f</i> reaction mixture showing released sugars.

<p><b>A</b>) L-arabinose, <b>B</b>) D-xylose, <b>C</b>) rye arabinoxylan, <b>D</b>) wheat arabinoxylan and <b>E</b>) oat spelt xylan. The reaction was carried out at pH 5.7, 50°C for 30 min.</p

The molecular architecture of <i>Ct</i>43Ara<i>f</i> shows modular structure with an N-terminal family 43 glycoside hydrolase (<i>Ct</i>GH43) catalytic module (903 bp), a C-terminal family 6 carbohydrate binding module (<i>Ct</i>CBM6B, 402 bp) and another family 6 Carbohydrate binding module (<i>Ct</i>CBM6A, 405 bp) sandwiched between these two modules.

<p>The molecular architecture of <i>Ct</i>43Ara<i>f</i> shows modular structure with an N-terminal family 43 glycoside hydrolase (<i>Ct</i>GH43) catalytic module (903 bp), a C-terminal family 6 carbohydrate binding module (<i>Ct</i>CBM6B, 402 bp) and another family 6 Carbohydrate binding module (<i>Ct</i>CBM6A, 405 bp) sandwiched between these two modules.</p

Substrate specificity of <i>Ct</i>43Ara<i>f</i> and <i>Ct</i>GH43 from <i>C. thermocellum.</i>

<p><i>All the assays were performed at 50°C using 20 mM sodium phosphate (pH 5.7) buffer for ^aCt43Araf and 20 mM sodium acetate (pH 5.4) buffer for ^bCtGH43. The assays were performed in triplicates. The incubation time and other conditions for reducing sugar estimation were as same as described in the Materials and Methods section.</i></p>*<p><i>/**It was prepared by Megazyme (Ireland) via controlled enzymatic hydrolysis of de branched sugar beet arabinan as described in the manufacturer’s instructions.</i></p><p>ND = <i>No activity detected.</i></p

Kinetic properties and catalytic efficiencies of <i>Ct</i>43Ara<i>f</i> and <i>Ct</i>GH43 from <i>C. thermocellum.</i>

<p><i>The assays with natural substrates were carried out with 20 mM sodium phosphate buffer (pH 5.7) for <b>^a</b>Ct43Araf and sodium acetate buffer (pH 5.4) for <b>^b</b>CtGH43 at 50°C. The assays were performed in triplicates. The incubation time and other conditions for reducing sugar estimation were as same as described in the Materials and Methods section. <b>^c</b>The assays with synthetic pNP-glycosides were carried out in 20 mM sodium phosphate buffer pH 5.7.</i></p

Protein-melting analysis displaying normal melting curve (–), melting curve in presence of 10 mM Ca²⁺ ions (–), and melting curve in presence of 10 mM Ca²⁺ ions and 10 mM EGTA (–•–), A) Melting-profile of <i>Ct</i>43Ara<i>f</i> and B) melting profile of truncated derivative, <i>Ct</i>GH43.

<p>Protein-melting analysis displaying normal melting curve (–), melting curve in presence of 10 mM Ca²⁺ ions (–), and melting curve in presence of 10 mM Ca²⁺ ions and 10 mM EGTA (–•–), A) Melting-profile of <i>Ct</i>43Ara<i>f</i> and B) melting profile of truncated derivative, <i>Ct</i>GH43.</p

Thin layer chromatography analysis of reaction products of <i>Ct</i>43Ara<i>f</i>.

<p>Dark spots on TLC plate show the standards L-arabinose, D-xylose and cellobiose (spots 1, 2 and 3, respectively) while spots 4, 5 and 6 represent hydrolyzed products from rye arabinoxylan, wheat arabinoxylan and oat spelt xylan, respectively, showing that only L-arabinose is released as the breakdown product.</p

The percentage of secondary structure contents of <i>Ct</i>GH43 protein as estimated from far-UV CD spectra.

*<p><i>Secondary structure prediction using PSIPRED VIEW software.</i></p

Maximum effect on enzyme activity of <i>Ct</i>43Ara<i>f</i> and <i>Ct</i>GH43 from <i>Clostridium thermocellum</i> at maximum concentration of metal ions and chelating agents.

<p><i>No additives were added in control and the activity was taken as 100%.</i></p

A) SDS-PAGE (13%) showing over-expression and purification of <i>Ct</i>43Ara<i>f</i>.

<p>Lane 1: Page Ruler protein marker, Lane 2: uninduced <i>Ct</i>43Ara<i>f</i> cells, Lane 3: Induced <i>Ct</i>43Ara<i>f</i> cells, Lane 4: Cell free extract, Lanes 5: Purified <i>Ct</i>43Ara<i>f</i> (63 kDa approx.), <b>B</b>) Effect of pH and temperature on <i>Ct</i>43Ara<i>f</i> activity, where (•) represents pH profile and (▾) represents temperature profile, <b>C</b>) pH and thermal stability analysis of <i>Ct</i>43Ara<i>f,</i> where (▾) represents pH stability and (•) represents thermal stability profile.</p