Continuous culture system for production of biopolymer levan using erwinia herbicola

The optimal production of the fructan biopolymer levan by the bacterium Erwinia herbicola was investigated, including variations in nitrogen, carbon and phosphorous sources, pH, incubation time, culture yields up to 19% by weight produced based on conversion of sucrose as the carbon source when grown in a continuous culture system and processed by tangential flow filtration. Product identity was confirmed with gas chromatography (GC) and (13)C nuclear magnetic resonance (NMR). Gel permeation chromatography (GPC) and low-angle laser light scattering (LALLS) determination of the molecular weight of the product showed a significant difference in molecular weight values dependent on the method of analysis. Analysis by GPC resulted in molecular weight one order of magnitude lower than LALLS independent of sample, underscoring the unusual nature of this biopolymer.     

Microbial fructosyltransferases and the role of fructans

Microbial fructosyltransferases are polymerases that are involved in microbial fructan (levan, inulin and fructo-oligosaccharide) biosynthesis. Structurally, microbial fructosyltransferase proteins share the catalytic domain of glycoside hydrolases 68 family and are grouped in seven phylogenetically related clusters. Fructosyltransferase-encoding genes are organized in operons or in clusters associated with other genes related to carbohydrate metabolism or fructosyltransferase secretion. Fructosyltransferase gene expression is mainly regulated by two-component systems or phosphorelay mechanisms that respond to sucrose availability or other environmental signals. Microbial fructans are involved in conferring resistance to environmental stress such as water deprivation, nutrient assimilation, biofilm formation, and as virulence factors in colonization. As a result of the biological and industrial importance of fructans, fructosyltransferases have been the subject of extensive research, conducted to improve their enzymatic activity or to elucidate their biological role in nature.     

Arabidopsis AtcwINV3 and 6 are not invertases but are fructan exohydrolases (FEHs) with different substrate specificities

The genome of Arabidopsis thaliana contains six putative cell-wall type invertase genes (AtcwINV1-6). Heterologous expression of AtcwINV1, 3 and 6 cDNAs in Pichia pastoris revealed that the enzymes encoded by AtcwINV3 and 6 did not show invertase activity. Instead, AtcwINV3 is a 6-FEH and AtcwINV6 is a fructan exohydrolase (FEH) that can degrade both inulin and levan-type fructans. For AtcwINV6 it is proposed to use the term (6&1) FEH. In contrast, AtcwINV1 is a typical invertase. FEH activity was also detected in crude extracts of different parts of Arabidopsis. To verify that the FEH activity of AtcwINV3 and 6 were not artefacts of the heterologous expression system, the protein corresponding to AtcwINV3 was isolated from whole Arabidopsis plants and indeed showed only 6-FEH activity and no invertase activity. Although no fructans can be detected in Arabidopsis plants, it is shown that kestoses (trimers) can be synthesized in crude leaf extracts. The putative physiological significance of FEH in so-called non-fructan plants is discussed.     

Biochemical and Economical Aspects of Levan Synthesis by Zymomonas Mobilis

With flocculent cells of Zymomonas mobilis levan was produced in a continuous upflow-tower fermentation with a productivity of up to 16 gl^-1h^-1. A large-scale process basing on whole Zymomonas cells is thought to be economical, if levan and ethanol production can be carried out simultaneously. Levan sucrase as the enzyme responsible for levan biosynthesis was purified and partially characterized.     

Levan production using mutant strains of Zymomonas mobilis in different culture conditions

Several levan hyperproducing mutants of Zymomonas mobilis strains were selected by mutagenesis with N-methyl-N-nitro-nitrosoguanidine and caffeine. Highest levan production (41 g l^–1) was obtained with a mutant strain HL 29 in a culture medium containing 200 g sucrose l^–1 and 0.5 g (NH₄)₂SO₄ l^–1 stored at 7 °C for 29 days. This is the first report describing the levan synthesis by Z. mobilis at 7 °C.     

The intracellular sucrase (SacA) of Zymomonas mobilis is not involved in sucrose assimilation

The intracellular sucrase SacA from Zymomonas mobilis was purified to homogeneity from a recombinant E. coli strain containing the SacA gene under an expression system. The protein was monomeric with a molecular mass of 58 kDa. The sucrase activity was maximal at 25 °C and thermal stability of the purified protein was low (50% recovery after 30 min at 46 °C ). The activation energy was low at 33 kJ mol^–1. Maximum activity was at pH 6.5. Activity was strongly inhibited (>99%) by SH blocking reagents and reducing agents slightly (10–60%) increased the activity of purified SacA. The sucrase showed a low K _M (42 mM) and k _cat (125 s^–1) which indicated its very low efficiency for sucrose hydrolysis. A mutant strain of Z. mobilis not able to grow on sucrose was isolated. This strain (ZM4S) lacked the two sucrases SacB and SacC but SacA was present in the intracellular fraction. Therefore, SacA alone is unable to allow growth Z. mobilis on sucrose.     

Effects of oil reservoir conditions on the production of water‐insoluble Levan by Bacillus licheniformis

Bacillus licheniformis produced a water‐insoluble levan which has potential application as a selective plugging agent in microbial enhanced oil recovery (MEOR). The microorganism grew on sucrose, glucose, and fructose but produced levan only on sucrose. Plugging may thus be selectively controlled in the reservoir by substrate manipulation. B. licheniformis and a crude preparation of its extracellular enzymes were evaluated for their ability to produce levan under reservoir conditions. Oil reservoirs which have a temperature of less than 55°C, a pH between 6 and 9, a pressure less than 500 atm, and a salt concentration of 4% or less are potentially suitable. Examples of such reservoir conditions are found in Lloydminster on the Alberta‐Saskatchewan border, one of the largest Canadian oil reserves.     

Amphotericin B Resistance Assay for the Detection of Cholesterol Biosynthesis Inhibitors

Three different types of cholesterol biosynthesis inhibitors, lovastatin, ketoconazole, and 25-hydroxycholesterol, showed their respective resistance against amphotericin B (AmB) cytotoxicity in Chinese hamster ovary (CHO)-K1 cells. The negative correlation between the acquisition of AmB resistance and the decrease of cellular cholesterol content by the inhibitor was confirmed.     

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.