Surface properties of bacteria from activated sludge in relation to bioflocculation.

Two bacterial stains were isolated from the activated sludge and identified as Leucobacter sp. and Alcaligenesfaecalis by 16S rDNA sequencing. Pure cultures of these two strains, representing well or poorly settled bacteria, were used to investigate the mechanism of bioflocculation in activated sludge. Based on the analyses of the characteristics of cells hydrophobicity, zeta-potential, flocculation ability and extracellular polymeric substance (EPS) composition under different growth stages, it was found that the ratio of cell EPS protein had the highly influence on zeta-potential and hydrophobicity, which were important factors to bioflocculation. Cellulase and Proteinase K could destroy the extracellular biopolymer and resulted in a decrease in the hydrophobicity and zeta-potential. However, in our study, the flocculation characteristics exhibited differently in relation to cellulase and Proteinase K. Flocculation of cells treated with cellulase and Proteinase K decreased sharply, and then recovered quickly in cellulase treatment, while cells treated with Proteinase K showed no sign of recovery. This reveals that the presence of protein in extracellular biopolymer plays an important role to the bioflocculation of cells.

Stereoselective nitrile hydrolysis by immobilized whole-cell biocatalyst.

The present work attempts to deal with the stability and reusability aspect of nitrilase from Alcaligenes faecalis for the production of (R)-(-)-mandelic acid. Four entrapment matrixes were screened to search for a suitable support, and alginate was found to have significant process advantages over its other counterparts. Thermodynamic analysis allowed us to account for decreased enantioselectivity (E) as a result of immobilization. The system was also characterized based on the Thiele modulus (phi). Efficient reusability of the biocatalyst up to 35 batches was achieved by immobilization as compared to 9 batches for free cells, and cross-linking extended it further to 40 batches. Finally, synthetic utility of the immobilized biocatalyst was demonstrated on a preparative scale to produce 640 g of (R)-(-)-mandelic acid with 97% enantiomeric excess (ee).

Characterization of a novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis.

A novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis has been isolated and characterized. The protein was extracted from the cells with 1 m NaCl, which required 1.5-fold, single-step purification to yield near-homogeneous preparations. In solution, the protein exists as homomeric aggregates, of mean diameter 21.6 nm, consisting of 22-kDa subunits. MS/MS data for peptides obtained by trypsin digestion of the thiosterase did not match any peptide from Escherichia coli thioesterases or any other thioesterases in the database. The thioesterase was associated exclusively with the surface of cells as revealed by ultrastructural studies using electron microscopy and immunogold labeling. It hydrolyzed saturated and unsaturated fatty acyl-CoAs of C12 to C18 chain length with Vmax and Km of 3.58-9.73 micromol x min(-1) x (mg protein)(-1) and 2.66-4.11 microm, respectively. A catalytically important histidine residue is implicated in the active site of the enzyme. The thioesterase was active and stable over a wide range of temperature and pH. Maximum activity was observed at 65 degrees C and pH 10.5, and varied between 60% and 80% at temperatures of 25-70 degrees C and pH 6.5-10. The thioesterase also hydrolyzed p-nitrophenyl esters of C2 to C12 chain length, but substrate competition experiments demonstrated that the long-chain acyl-CoAs are better substrates for thioesterase than p-nitrophenyl esters. When assayed at 37 and 20 degrees C, the affinity and catalytic efficiency of the thioesterase for palmitoleoyl-CoA and cis-vaccenoyl-CoA were reduced approximately twofold at the lower temperature, but remained largely unaltered for palmitoyl-CoA.