Identification of toxic substances in phenol-acetone wastewater on activated sludge and selective toxicity removal performance with ferrous pretreatment.

We investigated the effects of toxic wastewater generated during the production of phenol-acetone on activated sludge and tested pretreatment methods to selectively remove the toxicity. We found that the microbial activity in the activated sludge was inhibited by the wastewater, in which cumene hydroperoxide (CHP) with a medium effective concentration (EC50) of 225 mg L-1 was the main toxic substance. We tested one pretreatment method with ferrous iron to selectively remove the CHP. The CHP decomposition process, which mainly produced acetophenone, was very quick. The CHP was selectively transformed into low-toxicity organics, and a maximum of 92% was removed when 1.08 mmol L-1 of ferrous iron was added, for a reaction time of 10 min, a pH of 5, and a temperature of 25 °C, and the resulting wastewater only slightly inhibited the oxygen uptake rate of activated sludge. The acclimation of activated sludge was accelerated, and a COD removal rate of more than 85% was achieved within a week. Our results confirm that ferrous iron provides a cost-effective method to selectively remove toxins from wastewater.

Asymmetric synthesis of (S)-4-chloro-3-hydroxybutanoate by sorbose reductase from Candida albicans with two co-existing recombinant Escherichia coli strains.

An NADPH-dependent sorbose reductase from Candida albicans was identified to catalyze the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE). The activity of the recombinant enzyme toward COBE was 6.2 U/mg. The asymmetric reduction of COBE was performed with two coexisting recombinant Escherichia coli strains, in which the recombinant E. coli expressing glucose dehydrogenase was used as an NADPH regenerator. An optical purity of 99% (e.e.) and a maximum yield of 1240 mM (S)-4-chloro-3-hydroxybutanoate were obtained under an optimal biomass ratio of 1:2. A highest turnover number of 53,900 was achieved without adding extra NADP(+)/NADPH compared with those known COBE-catalytic systems.

Development of a substrate-coupled biocatalytic process driven by an NADPH-dependent sorbose reductase from Candida albicans for the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate.

A substrate-coupled biocatalytic process was developed based on the reactions catalyzed by an NADPH-dependent sorbose reductase (SOU1) from Candida albicans in which ethyl 4-chloro-3-oxobutanoate (COBE) was reduced to (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE], while NADPH was regenerated by the same enzyme via oxidation of sugar alcohols. (S)-CHBE yields of 1,140, 1,150, and 780 mM were obtained from 1,220 mM COBE when sorbitol, mannitol, and xylitol were used as co-substrates, respectively. Optimization of COBE and sorbitol proportions resulted in a maximum yield of (S)-CHBE (2,340 mM) from 2,500 mM COBE, and the enantiomeric excess was 99.6 %. The substrate-coupled system driven by SOU1 maintained a stable pH and a robust intracellular NADPH circulation; thus, pH adjustment and addition of extra coenzymes were unnecessary.

A novel reductase from Candida albicans for the production of ethyl (S)-4-chloro-3-hydroxybutanoate.

A novel NADPH-dependent reductase (CaCR) from Candida albicans was cloned for the first time. It catalyzed asymmetric reduction to produce ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE). It contained an open reading frame of 843 bp encoding 281 amino acids. When co-expressed with a glucose dehydrogenase in Escherichia coli, recombinant CaCR exhibited an activity of 5.7 U/mg with ethyl 4-chloro-3-oxobutanoate (COBE) as substrate. In the biocatalysis of COBE to (S)-CHBE, 1320 mM (S)-CHBE was obtained without extra NADP+/NADPH in a water/butyl acetate system, and the optical purity of the (S)-isomer was higher than 99% enantiomeric excess.