Flavonoid substrates converted with <i>Nh</i>KHS.

<p>Flavonoid substrates converted with <i>Nh</i>KHS.</p

Kinetic characterization of <i>Nh</i>KHS with the substrate XN.

<p>Reaction mixtures contained 0.05 mg mL^-1 of purified enzyme in 50 mM sodium citrate, pH 6.0, with 2% ethanol (v/v) and the XN concentration was varied from 0.125 to 3.0 mM. Reactions were incubated for 2 min at 35°C and 150 rpm in triplicates. (A) Michaelis-Menten plot. Kinetic parameters were determined directly from the regression data of a nonlinear hyperbolic curve fit. (B) Summary of kinetic constants for XN hydration by <i>Nh</i>KHS.</p

Formation of HO-KV from KV catalyzed by <i>Nh</i>KHS.

<p><i>P</i>. <i>pastoris</i> culture supernatants were incubated with KV for 3 h at 35°C and product formation was analyzed via HPLC-UV at 371 nm. Insets represent mass spectra of selected peaks at respective retention times determined by HPLC-MS in positive SIM mode. The retention time of the substrate was determined by analysis of a 2 mM authentic KV standard (A). No product formation was observed using the culture supernatant of a <i>P</i>. <i>pastoris</i> WT control (B). Inset of (A & B) represent the mass spectrum of the KV peak at a retention time of 3.1 min. The retention time of the product was determined by analysis of a 0.5 mM authentic HO-KV standard (C). HO-KV was formed using the culture supernatant of <i>PpKHS</i>Alpha expressing <i>Nh</i>KHS (D). Insets of (C & D) represent the mass spectrum of the product peak at a retention time of 1.4 min.</p

Recombinant expression, purification and biochemical characterization of kievitone hydratase from <i>Nectria haematococca</i> - Fig 1

<p><b>Analysis of secreted protein and purification of <i>Nh</i>KHS from <i>P</i>. <i>pastoris</i> culture supernatant (A).</b> Proteins were precipitated from the culture supernatant of <i>P</i>. <i>pastoris</i> wild type strain (lane 1) and <i>PpKHS</i>Alpha strain (lane 2) with the chloroform/methanol method. Four hundred <b>μ</b>L of cell culture were precipitated. KHS was purified via Ni-NTA affinity chromatography. Ten <b>μ</b>L of flow through (lane 4), washing fractions with 10 mM imidazole (lanes 5 and 6) and 50 mM imidazole (lanes 7 and 8), as well as 4 <b>μ</b>L of concentrated, pooled elution fractions (lane 9) were loaded onto the gel. PageRuler^TM Prestained Protein Ladder was used as molecular weight standard (Lane 3). <b><i>In vitro</i> deglycosylation of purified KHS with Endo<i>H</i></b>_{<b><i>f</i></b>} <b>(B).</b> Deglycosylation reactions were incubated for 0.5–2.5 h.</p

Formation of HO-XN from XN catalyzed by <i>Nh</i>KHS.

<p><i>P</i>. <i>pastoris</i> culture supernatants were incubated with XN for 3 h at 35°C and product formation was analyzed via HPLC-UV at 371 nm. Insets represent mass spectra of selected peaks at respective retention times determined by HPLC-MS in positive SIM mode. The retention time of the substrate was determined by analysis of a 2 mM authentic XN standard (A). No product formation was observed using the culture supernatant of a <i>P</i>. <i>pastoris</i> WT control (B). Insets of (A & B) represent the mass spectrum of the XN peak at a retention time of 4.3 min. The retention time of the product was determined by analysis of a 0.5 mM authentic HO-XN standard (C). HO-XN was formed using the culture supernatant of <i>PpKHS</i>Alpha expressing <i>NhKHS</i> (D). Insets of (C & D) represent the mass spectrum of the product peak at a retention time of 2.8 min.</p