Heterogeneous Nucleation of Protein Crystals on Fluorinated Layered Silicate

Here, we describe an improved system for protein crystallization based on heterogeneous nucleation using fluorinated layered silicate. In addition, we also investigated the mechanism of nucleation on the silicate surface. Crystallization of lysozyme using silicates with different chemical compositions indicated that fluorosilicates promoted nucleation whereas the silicates without fluorine did not. The use of synthesized saponites for lysozyme crystallization confirmed that the substitution of hydroxyl groups contained in the lamellae structure for fluorine atoms is responsible for the nucleation-inducing property of the nucleant. Crystallization of twelve proteins with a wide range of pI values revealed that the nucleation promoting effect of the saponites tended to increase with increased substitution rate. Furthermore, the saponite with the highest fluorine content promoted nucleation in all the test proteins regardless of their overall net charge. Adsorption experiments of proteins on the saponites confirmed that the density of adsorbed molecules increased according to the substitution rate, thereby explaining the heterogeneous nucleation on the silicate surface

Two Forms of NAD-Dependent d-Mandelate Dehydrogenase in Enterococcus faecalis IAM 10071

Two forms of NAD-dependent d-mandelate dehydrogenase (d-ManDHs) were purified from Enterococcus faecalis IAM 10071. While these two enzymes consistently exhibited high activity toward large 2-ketoacid substrates that were branched at the C3 or C4 position, they gave distinctly different K(m) and V(max) values for these substrates and had distinct molecular weights by gel electrophoresis and gel filtration

Double reciprocal plot for the phosphorolysis of Sop₃ with different concentrations of inorganic phosphate.

<p>Concentrations of inorganic phosphate were 0.5(open triangle), 1.0 mM (filled square), 2.0 mM (open square), 3.0 mM (filled circle), and 5.0 mM (open circle). The kinetic parameters are as follows: <i>k</i>_cat = 21±1 (s⁻¹), <i>K</i>_mA = 0.66±0.14 (mM), <i>K</i>_mB = 1.3±0.2 (mM), and <i>K</i>_iA = 3.3±0.6 (mM), where A represents Sop₃ and B is Pi. Grafit version 7.0.3 was used to perform non-linear regression and calculation of values.</p

TLC analysis of reaction products from acceptors and Glc1<i>P</i>.

<p>(A) Reaction products from monosaccharides and Glc1<i>P</i>. Reactions were performed with 2 mg/ml of Lin1839r in the presence of 50 mM monosaccharides and 50 mM Glc1<i>P</i> for 1 h. (B) Time course of the reaction products from glucose and Glc1<i>P</i>. Substrates used were 50 mM glucose and 50 mM Glc1<i>P</i>. The enzyme concentration used was 2 mg/ml. (C) Reaction products after reaction for 2 h using 10 mM Sop₂ and 20 mM Glc1<i>P</i> as substrates. (D) Time course of the reaction products from laminaribiose and Glc1<i>P</i>. The substrates used were 10 mM laminaribiose and 10 mM Glc1<i>P</i>. The enzyme concentration used was 0.5 mg/ml. (A, C) Presence and absence of Lin1839r are represented with ‘+’ and ‘−’, respectively. (B, D) M, marker. Numbers under a line represent reaction time.</p

Reaction scheme for the Lin1839 protein.

<p>Reaction scheme for the Lin1839 protein.</p