Increase in the production of ß-carotene in recombinant Escherichia coli cultured in a chemically defined medium supplemented with amino acids.

Escherichia coli DH5α strain was selected as the recombinant host, and a chemically defined medium supplemented with amino acids was used instead of a complex medium for the efficient production of ß-carotene. In a fed-batch culture using glycerol with a chemically defined medium supplemented with amino acids, the concentration, specific content, and productivity of ß-carotene were 2,470 mg/l, 72 mg/g cells, and 77 mg/l h after 32 h, respectively. These values were, respectively, 43, 33, and 26 % higher than those obtained using the complex medium. This is the highest ß-carotene production that has been reported among the recombinant cells to date.

Characterization of a recombinant thermostable D-lyxose isomerase from Dictyoglomus turgidum that produces D-lyxose from D-xylulose.

A putative D-lyxose isomerase from Dictyoglomus turgidum was purified with a specific activity of 19 U/mg for D-lyxose isomerization by heat treatment and affinity chromatography. The native enzyme was estimated as a 42 kDa dimer by gel-filtration chromatography. The activity of the enzyme was highest for D-lyxose, suggesting that it is a D-lyxose isomerase. The maximum activity of the enzyme was at pH 7.5 and 75°C in the presence of 0.5 mM Co(2+), with a half-life of 108 min, K(m) of 39 mM, and k(cat) of 3,570 1/min. The enzyme is the most thermostable D-lyxose isomerase among those characterized to date. It converted 500 g D-xylulose/l to 380 g D-lyxose/l after 2 h. This is the highest concentration and productivity of D-lyxose reported thus far.

Enhancement of retinal production by supplementing the surfactant Span 80 using metabolically engineered Escherichia coli.

The optimal temperature and pH for retinal production using metabolically engineered Escherichia coli in a 7-l fermentor were found to be 30°C and 7.0, respectively. The agitation speed was a critical factor for retinal production. The optimal agitation speed was 400 rpm (oxygen transfer coefficient, k(L)a, = 92 1/h) in batch culture and 600 rpm (k(L)a=148 1/h) in a fed-batch culture of glycerol. Span 80 was selected as a surfactant for retinal production in metabolically engineered E. coli because Span 80 had proven the most effective for increased retinal production among the tested surfactants. Under the optimal conditions in the fed-batch culture with 5 g/l Span 80, the cell mass and the concentration, content, and productivity of retinal were 24.7 g/l, 600 mg/l, 24.3mg/g-cells, and 18 mg l(-1)h(-1) after 33 h, respectively. They were 1.2-, 2.7-, 2.3-, and 2.7-fold higher than those in the fed-batch culture without Span 80, respectively. The concentration and productivity of retinal in this study were the highest ever reported. The hydrophilic portion of Span 80 (sorbitan) did not affect cell growth and retinal production, but the hydrophobic portion (oleic acid) stimulated cell growth. However, oleic acid plus sorbitan did not stimulate retinal production. Thus, Span 80, as a linked compound of oleic acid and sorbitan produced by esterification, proved to be an effective surfactant for the enhancement of retinal production.

Ginsenoside F1 production from ginsenoside Rg1 by a purified ß-glucosidase from Fusarium moniliforme var. subglutinans.

Fusarium moniliforme var. subglutinans was selected from among 100 strains of fungi for producing ginsenoside F(1) from ginsenoside Rg(1). The enzyme responsible was purified as a single 85 kDa band with a specific activity of 136 U mg(-1). It hydrolysed glucose-linked ginsenosides Rb(1), Rd and Rg(1) but not for other monosaccharide-linked ginsenosides, Rb(2), Rc, R(1), and Re. Under the optimum conditions of pH 6.0, 50 °C, 30 U l(-1) of enzyme, and 5 mg Rg(1) ml(-1), 4 mg F(1) ml(-1) was produced after 4 h, with a molar yield of 100% and a productivity of 1 g l(-1) h(-1). This represents the highest productivity and conversion yield of F(1) yet reported.

Improvement in the thermostability of D-psicose 3-epimerase from Agrobacterium tumefaciens by random and site-directed mutagenesis.

The S213C, I33L, and I33L S213C variants of D-psicose 3-epimerase from Agrobacterium tumefaciens, which were obtained by random and site-directed mutagenesis, displayed increases of 2.5, 5, and 7.5°C in the temperature for maximal enzyme activity, increases of 3.3-, 7.2-, and 29.9-fold in the half-life at 50°C, and increases of 3.1, 4.3, and 7.6°C in apparent melting temperature, respectively, compared with the wild-type enzyme. Molecular modeling suggests that the improvement in thermostability in these variants may have resulted from increased putative hydrogen bonds and formation of new aromatic stacking interactions. The immobilized wild-type enzyme with and without borate maintained activity for 8 days at a conversion yield of 70% (350 g/liter psicose) and for 16 days at a conversion yield of 30% (150 g/liter psicose), respectively. After 8 or 16 days, the enzyme activity gradually decreased, and the conversion yields with and without borate were reduced to 22 and 9.6%, respectively, at 30 days. In contrast, the activities of the immobilized I33L S213C variant with and without borate did not decrease during the operation time of 30 days. These results suggest that the I33L S213C variant may be useful as an industrial producer of D-psicose.

Characterization of a recombinant cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus and its application in the production of mannose from glucose.

A putative N-acyl-D-glucosamine 2-epimerase from Caldicellulosiruptor saccharolyticus was cloned and expressed in Escherichia coli. The recombinant enzyme was identified as a cellobiose 2-epimerase by the analysis of the activity for substrates, acid-hydrolyzed products, and amino acid sequence. The cellobiose 2-epimerase was purified with a specific activity of 35 nmol min(-1) mg(-1) for D-glucose with a 47-kDa monomer. The epimerization activity for D-glucose was maximal at pH 7.5 and 75°C. The half-lives of the enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were 142, 71, 35, 18, and 4.6 h, respectively. The enzyme catalyzed the epimerization reactions of the aldoses harboring hydroxyl groups oriented in the right-hand configuration at the C2 position and the left-hand configuration at the C3 position, such as D-glucose, D-xylose, L-altrose, L-idose, and L-arabinose, to their C2 epimers, such as D-mannose, D-lyxose, L-allose, L-gulose, and L-ribose, respectively. The enzyme catalyzed also the isomerization reactions. The enzyme exhibited the highest activity for mannose among monosaccharides. Thus, mannose at 75 g l(-1) and fructose at 47.5 g l(-1) were produced from 500 g l(-1) glucose at pH 7.5 and 75°C over 3 h by the enzyme.