3a) The estimated half-life (t1/2) for EcSTH activity was 5 h at

3a). The estimated half-life (t1/2) for EcSTH activity was 5 h at 50 °C, with the enzyme still retaining 10% activity after incubation for 16 h (Fig. 3b). The prolonged storage of enzymes is a particular concern in many industrial applications. We explored the stability of EcSTH at 4 °C and at room temperature (25 °C) over a period of 25 days. The activity of purified EcSTH was unchanged at 4 °C, while the enzyme retained 65%

of the initial activity at 25 °C (Fig. 3c). It was reported that the storage at −80, −20 °C and high temperature could cause an aggregation of STHs from A. vinelandii and E. coli (van den Broek et al., 1971), which may reduce enzyme activity during storage. We conclude that 4 °C is an ideal temperature

IDH inhibitor clinical trial for STH storage. The apparent kinetic constants for reducing thio-NAD+ to thio-NADH were determined from initial velocity studies and calculated using the Lineweaver–Burk plot (Table 1). The Km for thio-NAD+ by EcSTH (133.2 μM) was higher than that of A. vinelandii STH (75 μM) reported by van den Broek & Veeger (1971), but lower than A. vinelandii STH (250 μM) reported by Chung (1970). The Km for NADPH by EcSTH was 68.29 μM, which was slightly higher than that of A. vinelandii STH (40 μM) (van den Broek & Veeger, 1971). The maximum turnover rates (kcat) of CHIR-99021 in vitro EcSTH are 259.5 and 167.9 s−1 for thio-NAD+ and NADPH, respectively (Table 1). The catalytic efficiency (kcat/Km) of EcSTH towards NADPH is 1.25 times that with thio-NAD+ (Table 1). Substrate inhibition was observed at high concentrations

of NADPH (Fig. 4a), but not of thio-NAD+ (Fig. 4b). Similar results were obtained from A. vinelandii STH (van den Broek & Veeger, 1971). However, the activity of Pseudomonas aeruginosa STH was strongly activated by NADPH (Widmer & Kaplan, 1977; Boonstra et al., 1999). The effects of metal ions, adenine nucleotides, a reducer, a chelating agent and a nonaqueous solvent were determined using two methods (Table 2). The results show that the EcSTH activity is not find more affected by monovalent metal ions, but is inhibited by most divalent metal ions (Mn2+, Co2+, Zn2+, Ni2+), except Mg2+ and Ca2+. No activity was detected in the presence of 2 mM Cu2+. All monovalent metal ions and most divalent metal ions had no effect on EcSTH activity after preincubation for 30 min, although Zn2+, Ni2+ and Cu2+ caused about 90%, 10% and 30% of activity loss, respectively. In an earlier study, the activity of A. vinelandii STH was increased 10–20-fold by Ca2+ and Mg2+ at an alkaline pH (Voordouw et al., 1980). Our work demonstrates that metal ions are not needed for catalysis by STH. EcSTH activity is strongly activated by adenine nucleotides and is increased by 75%, 71% and 53% in the presence of ATP, ADP and AMP, respectively. However, after preincubation for 30 min, this activation is significantly decreased to 1–18% of the original activity (Table 2).

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