Cloning, expression and characterization of a lipase gene (lip3) from Pseudomonas aeruginosa LST-03.

A lipase gene (lip3) was cloned from the Pseudomonas aeruginosa strain LST-03 (which tolerates organic solvents) and expressed in Escherichia coli. The cloned sequence includes an ORF consisting of 945 nucleotides, encoding a protein of 315 amino acids (Lip3 lipase, 34.8 kDa). The predicted Lip3 lipase belongs to the class of serine hydrolases; the catalytic triad consists of the residues Ser-137, Asp-258, and His-286. The gene cloned in the present study does not encode the LST-03 lipase, a previously isolated solvent-stable lipase secreted by P. aeruginosa LST-03, because the N-terminal amino acid sequence of the Lip3 lipase differs from that of the LST-03 lipase. Although the effects of pH on the activity and stability of the Lip3 lipase, and the temperature optimum of the enzyme, were similar to those of the LST-03 lipase, the relative activity of the Lip3 lipase at lower temperatures (0-35 degrees C) was higher than that of the LST-03 lipase. In the absence of organic solvents, the half-life of the Lip3 lipase was similar to that of the LST-03 lipase. However, in the presence of most of the organic solvents tested in this study (the exceptions were ethylene glycol and glycerol), the stability of the Lip3 lipase was lower than that of the LST-03 lipase.

Theoretical investigation of fructose 1,6-diphosphate production and simultaneous ATP regeneration by conjugated enzymes in an ultrafiltration hollow-fiber reactor.

The performance of an ultrafiltration hollow-fiber reactor, in which enzymatic synthesis of fructose 1,6-diphosphate (FDP) from glucose and enzymatic ATP regeneration are performed simultaneously, was analyzed theoretically. The reaction system consists of three-step synthetic reactions catalyzed by glucokinase (GK), phosphoglucose isomerase and phosphofructokinase, and the ATP regeneration reaction catalyzed by acetate kinase. Based on a simple analytical model developed previously in which the liquid flowing in a tube was assumed to be plug flow and the radial concentration gradients in the tubes and shell side space were both neglected, a computer program was developed to calculate the concentration profiles of all the components along the flow direction in the tubes and shell side space of the reactor. From the FDP concentrations at the reactor outlet calculated under various operational conditions, reactor performances such as the FDP yield and the ATP recycle number were determined. The calculation showed the interesting phenomenon that under some conditions the FDP yield was higher when GK concentration was lower.

Experimental investigation of fructose 1,6-diphosphate production and simultaneous ATP regeneration by conjugated enzymes in an ultrafiltration hollow-fiber reactor.

The enzymatic synthesis of fructose 1,6-diphosphate (FDP) from glucose and the enzymatic ATP regeneration were performed simultaneously in an ultrafiltration hollow-fiber reactor using both the purified enzymes and the crude cell extract of Bacillus stearothermophilus. The process consisted of the three-step synthetic reactions catalyzed by glucokinase, phosphoglucose isomerase and phosphofructokinase, and the ATP regeneration reaction catalyzed by acetate kinase. The experimental results of the yield and the recycle number agreed well with the theoretical predictions calculated using a computer program developed in our preceding study. This proved the validity of this computer program for predicting or analyzing the reactor performance.