Production of lactobionic acid and sorbitol from lactose/fructose substrate using GFOR/GL enzymes from Zymomonas mobilis cells: a kinetic study

In this work, we have investigated the kinetics of the biotechnological production of lactobionic acid (LBA) and sorbitol by the catalytic action of glucose-fructose oxidoreductase (GFOR) and glucono-δ-lactonase (GL) enzymes. The cells of bacterium Zymomonas mobilis ATCC 29191 containing this enzymatic complex were submitted to permeabilization and reticulation procedures. The effect of the concentration of substrates on the rate of product formation using a mobilized cell system was investigated. The application of higher fructose concentration seems to not affect the initial rate of formation of the bionic acid. Under conditions of low initial concentration of lactose, the experimental kinetic data of the bi-substrate reaction were modelled by assuming a rate equation of the classical ping-pong mechanism. The found kinetic parameters displayed a low affinity of the GFOR enzyme for both substrates. The enzymatic system did not exhibit normal Michaelis-Menten kinetics in response to a change of concentration of lactose, when fructose was held constant, presenting a sigmoid relationship between initial velocity and substrate concentration. A rate equation based on Hill kinetics was used to describe the kinetic behaviour of this enzyme-substituted reaction at higher lactose concentrations. The results from batch experiments using immobilized cells within Ca-alginate beads revealed that there is no pronounced occurrence of mass transfer limitations on LBA production for beads with 1.2 mm in average diameter. This discussion aids for defining the best operating conditions to maximize the productivity for LBA and sorbitol in this bioconversion and also for reducing the complexity of downstream separation processes.     

Cloning of cDNAs encoding sorbitol dehydrogenase-2a and b, enzymatic characterization, and up-regulated expression of the genes in Bombyx mori diapause eggs exposed to 5 °C

We previously cloned a cDNA for sorbitol dehydrogenase (SDH1) from Bombyx mori. In the present study we cloned two additional cDNAs encoding SDHs (designated as SDH2a and SDH2b). The amino acid sequences of SDH2ab were almost the same and had higher similarity to the SDHs of other organisms than to B. mori SDH1. The SDH2ab and SDH1genes were located in tandem within about 40 kbp on chromosome 21. SDH2ab mRNAs increased after exposing diapause eggs to 5 °C for 40 days, beginning at 2 days post-oviposition, to break diapause. However, they were at very low levels in diapausing eggs incubated at 25 °C continuously from oviposition. These changes in expression pattern of SDH2ab mRNA were almost the same as that of SDH1 mRNA. To understand whether SDH1 and SDH2 were responsible for the SDH activity seen in diapause eggs exposed to 5 °C for more than 60 days, we expressed a His-tagged SDH2a fusion protein in Escherichia coli and examined its enzymatic parameters. The maximum activity of SDH2a observed at pH 8.4∼9.0, and the Km value for sorbitol was 12.6 mM, similar to the kinetic properties of other SDHs. Due to the significantly higher similarity between SDH2a and b, they were thought to have similar kinetic properties. Therefore, we purified SDH from B. mori diapause-terminated eggs exposed to 5 °C for 300 days which showed higher SDH activity using two-step affinity chromatography. The highly purified SDH showed a higher Km value (125 mM) for sorbitol, being similar to the value (136 mM) determined previously from Eadie-Hofstee plots using egg crude extract as an enzyme source; additionally, the plots showed one slope indicating one Km value. Moreover, in silico analysis indicated that no SDH genes other than SDH1 and 2ab are present in B. mori genomic DNA. These results suggest that SDH1 activity may be responsible for the majority of the increased SDH activity seen in diapause eggs after acclimation to 5 °C rather than SDH2ab. Further, the relative sequence divergence among these genes is consistent with the idea/hypothesis that the original SDH gene was first duplicated into SDH1 and SDH2, and then SDH2 was duplicated into the SDH2a and SDH2b genes.     

Plasmodium falciparum: stage-specific lactate production in synchronized cultures

Sequential treatment with gelatin followed by sorbitol-produced Plasmodium falciparum cultures that remained synchronous for ? 144 hr. The schizont stage was associated with more lactate production than the other asexual erythrocytic stages of the parasite (11–21 vs 4–7 neq/hr-10⁶ parasitized red blood cells [PRBC]). Lactate production in asynchronous cultures was relatively constant at 8–11 neq/hr-10⁶ PRBC and was directly proportional to parasitemia (P < 0.001).     

Sorbitol synthesis by an engineered Lactobacillus casei strain expressing a sorbitol-6-phosphate dehydrogenase gene within the lactose operon

Sorbitol is claimed to have important health-promoting effects and Lactobacillus casei is a lactic acid bacterium relevant as probiotic and used as a cheese starter culture. A sorbitol-producing L. casei strain might therefore be of considerable interest in the food industry. A recombinant strain of L. casei was constructed by the integration of a d-sorbitol-6-phosphate dehydrogenase-encoding gene (gutF) in the chromosomal lactose operon (strain BL232). gutF expression in this strain followed the same regulation as that of the lac genes, that is, it was repressed by glucose and induced by lactose. (13)C-nuclear magnetic resonance analysis of supernatants of BL232 resting cells demonstrated that, when pre-grown on lactose, cells were able to synthesize sorbitol from glucose. Inactivation of the l-lactate dehydrogenase gene in BL232 led to an increase in sorbitol production, suggesting that the engineered route provides an alternative pathway for NAD(+) regeneration.     

Export of the periplasmic NADP-containing glucose-fructose oxidoreductase of Zymomonas mobilis

Glucose-fructose oxidoreductase (GFOR) of the gram-negative bacterium Zymomonas mobilis is a periplasmic enzyme with the tightly bound cofactor NADP. The preprotein carries an unusually long N-terminal signal sequence of 52 amino acid residues. A sorbitol-negative mutant strain (ACM3963) was found to be deficient in GFOR activity and was used for the expression of plasmid-borne copies of the wild-type gfo gene or of alleles encoding alterations in the signal sequence of the pre-GFOR protein. Z. mobilis cells with the wild-type gfo allele translocated pre-GFOR, at least partially, via the Sec pathway since CCCP (carboxylcyanide-m-chlorophenylhydrazone; uncoupler of proton motive force) or sodium azide (inhibitor of SecA) abolished the processing of GFOR. A gfo allele with the hydrophobic region of the signal sequence removed (residues 32–46; Δ32–46) led to a protein that was no longer processed, but showed full enzymatic activity (180 U/mg) and had the cofactor NADP firmly bound. A deletion in the n-region of the signal sequence (residues 2–20; Δ2–20) or exchange of the entire GFOR signal sequence with the signal sequence of gluconolactonase of Z. mobilis led to active and processed GFOR. Strain ACM3963 could not grow in the presence of high sugar concentrations (1 M sucrose) unless sorbitol was added. The presence of the plasmid-borne gfo wild-type allele or of the Δ2–20 deletion led to the restoration of growth on media with 1 M sucrose, whereas the presence of the Δ32–46 deletion led to a growth behavior similar to that of strain ACM3963, with no sorbitol formation from sucrose. Received: 14 December 1995 / Accepted: 4 March 1996     

Effect of sorbitol concentration on regeneration of embryogenic calli in upland rice varieties (Oryza sativa L.)

This study describes the impact of sorbitol on plantlets regeneration frequency (PRF) of four rice cultivars (japonica, Oryza sativa L.) both of which mature and immature embryo-derived calli were investigated. The variance analysis results showed that PRF of the three elite upland rice cultivars, Handao297 (HD297), Handao502 (HD502), Handao65 (HD65) and one lowland rice cultivar Zhongzuo93 (ZZ93) were significantly increased with addition of appropriate amount of sorbitol in culture media. Supplementing appropriate sorbitol in the media of a continous culture from induction and maintenance to regeneration for mature embryo-derived calli could improve PRF dramatically, originally from 27.6% up to a maximum of 71.8%. Especially to low regenerative capacity (LRC) cultivar HD65, the PRF was increased over 7-fold (from 9.7% to 74.0%). The optimum concentrations of sorbitol for calli induction, subculture and differentiation were 5, 20 and 40 g/l, respectively. Adding sorbitol, only in maintenance media at concentration of 20 g/l, also enhanced the PRF greatly in all the cultivars from 27.6% to 43.3%. Similar results were observed when incorporating with maltose in regenerating media both in immature and mature embryo-derived calli. The optimal concentration was 25 g/l sorbitol + 20 g/l maltose and 20 g/l sorbitol + 25 g/l maltose, respectively. HD297 appeared to be the most responsive genotype compared to other cultivars in PRF, 99.2% in immature embryo-derived calli and 76.8% in mature embryo-derived calli. The results and relevant conclusions might be valuable to establish an efficient plant regeneration system from somatic embryogenesis culture in upland rice.     

Molecular dynamics studies of the conformation of sorbitol

Molecular dynamics simulations of a 3 molal aqueous solution of d-sorbitol (also called d-glucitol) have been performed at 300 K, as well as at two elevated temperatures to promote conformational transitions. In principle, sorbitol is more flexible than glucose since it does not contain a constraining ring. However, a conformational analysis revealed that the sorbitol chain remains extended in solution, in contrast to the bent conformation found experimentally in the crystalline form. While there are 243 staggered conformations of the backbone possible for this open-chain polyol, only a very limited number were found to be stable in the simulations. Although many conformers were briefly sampled, only eight were significantly populated in the simulation. The carbon backbones of all but two of these eight conformers were completely extended, unlike the bent crystal conformation. These extended conformers were stabilized by a quite persistent intramolecular hydrogen bond between the hydroxyl groups of carbon C-2 and C-4. The conformational populations were found to be in good agreement with the limited available NMR data except for the C-2–C-3 torsion (spanned by the O-2–O-4 hydrogen bond), where the NMR data support a more bent structure.