Uck-Han Chun

Comparison of characteristics of levan produced by different preparations of levansucrase from Zymomonas mobilis

The characteristics of levan formation by different preparations of levansucrase (free and immobilized enzyme and toluene-permeabilized whole cells), derived from recombinant levansucrase from Zymomonas mobilis expressed in Escherichia coli, were investigated. The maximal yield of levan by the three preparations were similar and were about 70–80% on a fructose-released basis with sucrose as nutrient at 100 g l−1. Immobilized enzyme and toluene-permeabilized whole cells produced low molecular weight levan (2–3 × 106), as determined by HPLC while high molecular weight levan (>6 × 106) was the major product with the free levansucrase. The size of levan can thus be controlled by immobilized levansucrase and toluene-permeabilized whole cells in high yield.

Continuous pollution monitoring using Photobacterium phosphoreum

Abstract Photobacterium phosphoreum is a marine bacterium which is used extensively as a bioluminescent indicator of pollutants, where the presence of toxicants diminishes light output. To evaluate the utility of cell immobilisation in continuous toxicity testing, the sensitivity of P. phosphoreum to five gelling agents was evaluated relative to the retention of bioluminescence in 3% NaCl-glycerol suspensions. Following storage at 4°C, the control cultures retained light output for up to 2 weeks before significant decline; alginate-glycerol suspensions were stable for up to 4 weeks and bioluminescence was detectable for up to 6 weeks. Cells stored in agar were no more stable than the control, whereas cells gelled in agarose and low-melting point agarose showed a significant decline in bioluminescence within 2 weeks of storage. Bioluminescence was totally retained in alginate-glycerol suspensions stored at −80°C for up to 12 weeks. P. phosphoreum was successfully immobilised in strontium alginate and showed a dose-related response to four of the five heavy metal ions, SDS and pentachlorophenol tested when responses were followed over a time-course. A flow-through system for Sr-alginate immobilised cells was developed and conditions for operation were optimised. When cells were exposed to a pulse of 4-nitrophenol or salicylate then the nutrient feed continued, bioluminescence declined in response (pulse of 4–6 min) to these pollutants then recovered to a new stable rate of decline which was faster than the pre-exposure rate. These results demonstrate the potential of using immobilised P. phosphoreum in a continuous flow-through system for real-time environmental monitoring of water quality.

Levan production by use of the recombinant levansucrase immobilized on titanium-activated magnetite

Abstract Levansucrase from Zymomonas mobilis expressed in Escherichia coli was immobilized onto the surface of magnetite. Optimum conditions for the immobilization were, pH 4.0; immobilization reaction time of 30 min and 8 U of enzyme per gram of matrix. Immobilization yield was 75% under optimized conditions. The maximum production yield of levan by the immobilized levansucrase was about 42% on a fructose released basis with sucrose as substrate at 100 g/l. Thermal stability of the levansucrase increased by immobilization procedure. Furthermore, immobilized enzyme showed the less polymerizing activity in levan formation, producing the low-molecular weight (MW) levan. The molecular weight of levan can be controlled by using the immobilized levansucrase in high yield. Immobilized levansucrase retained 61% of the original activity after five repeated uses.

Improvement of the process for sorbitol production with Zymomonas mobilis immobilised in κ-carrageenan

Abstract The production of sorbitol by permeabilised cells of Zymomonas mobilis immobilised in κ-carrageenan has been investigated. Permeabilised cells were treated with cetyltrimethylammoniumbromide (CTAB) prior to immobilisation to cross-link the enzymes of the glucose-fructose oxidoreductase (GFOR). Rigidity of the immobilised beads was increased two-fold with 90% conversion efficiency by addition of 40% (w/v) polyols (glycerol 25 g + propylene glycol 5 g) to 60% (w/v) distilled water containing 2·5% (w/v) κ-carrageenan as final concentration, prior to immobilisation, κ-Carrageenan beads entrapping permeabilised cells were dried to improve bead rigidity and storage stability. During a semi-batch process for 72 h with dry beads, there was an improvement of the loss of enzyme activity (less than 10%). In a batch process, the K m, fructose value for the free cells, wet beads and dry κ-carrageenan beads were 71·7, 72·4 and 116·7 g litre −1 , respectively. Higher productivity was obtained with a two-stage continuous packed bed reactor with both wet and dry κ-carrageenan beads.

Effects of dietary fructan on cecal enzyme activities in rats

In this study, we have attempted to determine the effects of dietary fructose polymers (fructan), high molecular-weight β-(2,6)-linked levan, and low-molecular-weight β-(2,1)-linked inulin, on two intestinal enzymes (β-glucuronidase and β-glucosidase). As a preliminary experiment, when intestinal microflora were cultured in anaerobic media harboring levan or its oligosaccharides, bacterial cell growth was observed in the levanoligosaccharide-supplemented media, but not in the levan-supplemented media, indicating that levan’s size is important for the utilization by intestinal bacteria of levan as an energy source. In our animal study, the intake of a levan-rich diet was determined to significantly attenuate the activity of the harmful enzyme β-glucuronidase, but did not affect the activity of β-glucosidase.

l-Sorbose production from glucose and fructose using Zymomonas mobilis and Gluconobacter suboxydans in a two-stage fed-batch reactor

A biological conversion of L-sorbose from the mixture of glucose and fructose was studied. D-Sorbitol was produced efficiently from fructose by permeabilized Zymomonas mobilis cells and then converted to L-sorbose by Gluconobacter suboxydans. The process for the production of L-sorbose from D-sorbitol by the resting cells of G. suboxydans was also optimized with respect to the substrate concentration, oxygen tension and cell density of the reaction medium. Gluconate produced from glucose by Z. mobilis seemed to act as an inhibitor in the conversion reaction of D-sorbitol into L-sorbose by G. suboxydans. A two-stage fed-batch process, therefore, was developed to minimize the inhibitory effect of gluconate on the conversion reaction.

Immobilization ofPhotobacterium phosphoreum for monitoring of toxic substances

A new sensing system based on the immobilization of luminescent bacteria,Photobacterium phosphoreum, was proposed for continuous real-time monitoring of pollutants. The response curves demonstrate thatPhotobacterium phosphoreum immobilized on the strontium alginate was very sensitive to seven reference chemicals used. The significant inhibitory concentrations for bioluminescence emission were 5 ppm for Pb(NO3)2, NiCl2, CdCl2, 50 ppm for NaAsO2, 0.1 ppm for HgCl2, 0.5 ppm for pentachlorophenol and less than 5 ppm for SDS, respectively. The alginate mixed-cells (AMC) retained their luminescence during experimental period (29 days) under storage condition of − 80°C. The variables affecting performance of continuous flow through monitoring (CFTM) were optimized in order to ensure stability and efficiency. The flow through cell with strontium-alginate immobilized luminescent bacteria was tested with salicylate and 4-nitrophenol and a rapid response of luminescence was recorded by time drivemode in bioluminescence spectrometer after exposure to both toxicants.

Production of Sorbitol with Cetyltrimethylammoniumbromide Permeabilized Cells of Zymomonas mobilis

Zymomonas mobilis has been known to produce ethanol at fast rated). The formation of sorbitol by this bacteria has been evaluated over the last few years when Zymomonas mobilis was cultured on sucrose or a mixture of glucose and fructose(2,3). The mechanism of sorbitol formation with Zymomonas mobilis was found to be that the reduction of fructose to sorbitol is coupled with the dehydrogenation of glucose to gluconolatone(4). Gluconolactone is thenfurther hydrolyzed to gluconate by a gluconolactonase(5). The newly found enzyme complex that is responsible for conversion of fructose to sorbitol has been described as glucose-fructose oxidoreductase with a tightly coupled cofactor, NADP(5). This cofactor is nondialyzable. Only permeabilized cells produce sorbitol and gluconate by the gluconate kinase in the Entner Doudoroff pathway(6). A continous process employing cells treated with toluene and immobilized in alginate has been developed and some loss of enzyme activity was observed after 120 h due to leakage of oxidoreductase from the permeabilized cells or further proteolysis by toluene during the process (7)