Serine substitution for cysteine residues in levansucrase selectively abolishes levan forming activity

Levansucrase is responsible for levan formation during sucrose fermentation of Zymomonas mobilis, and this decreases the efficiency of ethanol production. As thiol modifying agents decrease levan formation, a role for cysteine residues in levansucrase activity has been examined using derivatives of Z. mobilis levansucrase that carry serine substitutions of cysteine at positions 121, 151 or 244. These substitutions abolished the levan forming activity of levansucrase whilst only halving its activity in sucrose hydrolysis. Thus, polymerase and hydrolase activities of Z. mobilis levansucrase are separate and have different requirements for the enzyme's cysteine residues.     

Enhanced levan production using chitin-binding domain fused levansucrase immobilized on chitin beads

Levan is a homopolymer of fructose which can be produced by the transfructosylation reaction of levansucrase (EC 2.4.1.10) from sucrose. In particular, levan synthesized by Zymomonas mobilis has found a wide and potential application in the food and pharmaceutical industry. In this study, the immobilization of Z. mobilis levansucrae (encoded by levU) was attempted for repeated production of levan. By fusion levU with the chitin-binding domain (ChBD), the hybrid protein was overproduced in a soluble form in Escherichia coli. After direct absorption of the protein mixture from E. coli onto chitin beads, levansucrase tagged with ChBD was found to specifically attach to the affinity matrix. Subsequent analysis indicated that the linkage between the enzyme and chitin beads was substantially stable. Furthermore, with 20% sucrose, the production of levan was enhanced by 60% to reach 83 g/l using the immobilized levansucrase as compared to that by the free counterpart. This production yield accounts for 41.5% conversion yield (g/g) on the basis of sucrose. After all, a total production of levan with 480 g/l was obtained by recycling of the immobilized enzyme for seven times. It is apparent that this approach offers a promising way for levan production by Z. mobilis levansucrase immobilized on chitin beads.     

An influence of ethanol and temperature on products formation by different preparations of Zymomonas mobilis extracellular levansucrase

The ethanol and temperature effects on the ratio between Zymomonas mobilis 113S extracellular levansucrase activities were studied using fermentation broth supernatant, ??levan?Clevansucrase?? sediment precipitated by ethanol and highly purified enzyme. The fructooligosaccharide (FOS) production at different temperatures in the presence of ethanol was investigated. An ethanol increases FOS biosynthesis activity part of levansucrase. Especially, this effect was pronounced at lower temperatures (35?C40?°C) and using purified levansucrase. The inverse relationship between temperature and ratio synthetic activity/total activity of levansucrase was found. The FOS composition containing mostly 1-kestose, 6-kestose, and neokestose obtained in the presence of different ethanol concentrations was found relative constant, while the changes in the sucrose concentration and temperature gave slight changes in the ratio between 1-kestose and 6-kestose.     

Identification and characterisation of the extracellular sucrases of Zymomonas mobilis UQM 2716 (ATCC 39676)

An investigation was conducted to isolate, and characterise the extracellular sucrases of Zymomonas mobilis UQM 2716. Levansucrase (EC 2.4.1.10) was the only extracellular sucrase produced by this organism. This enzyme was responsible for sucrose hydrolysis, levan formation, and oligosaccharide production. It had a molecular mass of 98 kDa, a Michaelis constant (K _m) of 64 mm, and a pH optimum of 5.5. It was inhibited by glucose, but not by fructose, ethanol, sorbitol, NaCl, TRIS or ethylenediaminetetraacetic acid (EDTA). The formation of levan was the principal reaction catalysed by this enzyme at low temperatures. However, levan formation was thermolabile, being irreversibly lost when levansucrase was heated to 35°C. S This did not effect sucrose hydrolysis or oligosaccharide formation, which were optimal at 45°C. Sucrose concentration greatly influenced the type of acceptor molecule used in the transfructosylation reactions catalysed by levansucrase. At low sucrose concentration, the predominant reaction catalysed was the hydrolysis of sucrose to free glucose and fructose. At high sucrose concentrations, oligosaccharide production was the major reaction catalysed.     

Flame-burned stainless steel wire spheres as carriers of Zymomonas mobilis cells and extracellular levansucrase

In the present work, the use of flame-burned WS as carriers of Z. mobilis and extracellular levansucrase and the effect of the cell fixation method by dehydration on system productivity were investigated. Lyophilization and convective drying of Z. mobilis biomass at 30°C to a moisture content of 10–14% gave the best results for the repeated batch fermentations of a sucrose medium to obtain levan and ethanol. Significant correlation between the product formation and the concentration of free cells in the fermentation medium was established. Clearly, the cells were weakly bound to the newly generated WS and were washed out into the medium during fermentation. Here the hypothesis is presented that components excreted from damaged cells during dehydration can intensify the reactivation of damaged living cells and influence the interactions between the cells and the wire surface. The passive immobilization of extracellular levansucrase in oxidized WS was also observed. The superiority of oxidized WS in comparison with non-treated WS is related to an increase in the number of OH groups. The potential regeneration of WS by burning after the termination of fermentation cycles was also considered.     

Production of Levansucrase from Bacillus subtilis NRC 33a and Enzymic Synthesis of Levan and Fructo-Oligosaccharides

Bacillus subtilis NRC33a was able to produce both inducible and constitutive extracellular levansucrase, respectively, using sucrose and glucose as carbon source. The optimal production of the levansucrase was at 30°C. The effect of different nitrogen sources showed that baker’s yeast with 2% concentration gave the highest levansucrase activity. Addition of 0.15 g/L MgSO₄ was the most favorable for levansucrase production. The enzymic synthesis of levan was studied using 60% acetone fraction. The results indicated that high enzyme concentrations produced increasing amounts of levan, and hence conversion of fructose to levan reached 84% using 1000 μg/ml enzyme protein. Sucrose concentration was the most effective factor controlling the molecular weight of the synthesized levan. The conversion of fructose to levan was maximal at 30°C. The time of reaction clearly affected the conversion of fructose to levan, which reached its maximum productivity at 18 hours (92%). Identification of levan indicated that fructose was the building unit of levan.     

Enzymic synthesis of levan and fructo-oligosaccharides by <Emphasis Type="Italic">Bacillus circulans</Emphasis> and improvement of levansucrase stability by carbohydrate coupling

Bacillus circulans was able to produce extracellular levansucrase using sucrose as carbon source optimally at 35°C. The enzymic synthesis of levan and fructo-oligosaccharides was studied using a 50% ethanol fraction of crude extract. The molecular weight of the synthesized levan was markedly affected by sucrose concentration, the molecular weight of levan decreased with increased sucrose concentration up to 32% whereby fructo-oligosaccharides were isolated. Temperature and the reaction time clearly affected the conversion of fructose to levan with molecular weight values ranging from 10 to 38 kDa. Identification of levan indicated that fructose was the building unit of the levan obtained. Thermal and pH stabilities of B. circulans levansucrase could be improved by enzyme glycosylation using sodium metaperiodate treatment. Chemical modification provides additional points of attachment of the enzyme to the support which offered the modified enzyme greater stabilization than did the free enzyme. The modified enzyme exhibited thermal tolerance up to 50°C, where it retained 88.25% of its activity, while the free enzyme only retained 64.55% of its original activity. The half-life significantly increased from 130 min for the free enzyme to 347 min for the modified enzyme at 50°C, however, it increased from 103 min for the free enzyme to 210 min for the modified enzyme at 60°C. Other properties i.e., the response to some metal ions as well as the ability to convert higher substrate levels and tolerance to an extension of the reaction periods were also improved upon modification. Obviously, the results obtained outlined the conditions leading to the formation of important high or low molecular weight or levan and fructo-oligosaccharides suitable for different industrial applications.     

Sugar Beet Juice Fermentation by Zymomonas mobilis Attached to Stainless Steel Wire Spheres

The fermentation of sugar beet juice as well as juice syrup medium by Zymomonas mobilis inoculum attached to stainless steel wire spheres was investigated. A semi‐synthetic sucrose medium enriched with mineral salts and yeast extract was used as the control. It was established that raw sugar beet juice ensured good Zymomonas mobilis culture growth and slightly decreased ethanol synthesis applying both flame‐burned and TiCl₄‐treated wire spheres as carriers (Q_x = 0.05—0.06 g/l × h; Q_eth = 1.02—1.22 g/l × h). High ethanol yield was also observed in juice medium (Y = 0.45‐0.46 g/g), however, levan synthesis with this medium decreased. The application of juice syrup brought about less growth effect and ethanol synthesis as compared to juice medium. The use of semi‐synthetic sucrose medium resulted in high levan production (Q_lev = 0.6—0.7 g/l × h), however, reduced ethanol production by 40%. In conclusion, sugar beet juice or syrup is recommendable for the preparation of Zymomonas mobilis inoculum. The levan production stage has to be realized using an optimized semi‐synthetic sucrose medium. The installed wire spheres filled with inocula provided the possibility for a repeated batch fermentation process, which could be recommended for both juice and semi‐synthetic sucrose medium fermentation.     

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.     

Production of levan using recombinant levansucrase immobilized on hydroxyapatite

Levansucrase of Zymomonas mobilis was immobilized onto the surface of hydroxyapatite by ionic binding. Optimum conditions for the immobilization were: pH 6.0, 4 h of immobilization reaction time, and 20 U of enzyme/g of matrix. The enzymatic and biochemical properties of the immobilized enzyme were similar to those of the native enzyme, especially towards the effect of salts and detergents. The immobilized enzyme showed sucrose hydrolysis activity higher as that of the native enzyme, but levan formation activity was 70% of the native enzyme. HPLC analysis of levan produced by immobilized enzyme showed the presence of two different types of levan: high-molecular-weight levan and low-molecular-weight levan. The proportion of low-molecular-weight levan to total levan produced by the immobilized enzyme was much higher than that with the native enzyme, indicating that immobilized levansucrase could be applied to produce low-molecular-weight levan. Immobilized levansucrase retained 65% of the original activity after 6 times of repeated uses and 67% of the initial activity after 40 d when stored at 4 °C.     

Gene cloning, characterization, and heterologous expression of levansucrase from Bacillus amyloliquefaciens

Although levan produced by Bacillus amyloliquefaciens is known to have efficient immunostimulant property which gives 100% survival of common carp when infected with Aeromonas hydrophila, no detailed reports are available describing kinetic studies of d-glucose production and levan formation. In this study, we cloned and characterized the enzymatic kinetics using levansucrase expressed in Escherichia coli. Optimum pH for d-glucose production and levan formation was 6.0 and 8.0, respectively, whereas optimum temperature was 30°C and 4°C, respectively. The K _m and V _max values for levansucrase were calculated to be 47.81 mM sucrose and 57.47 μmole/min mg protein, respectively. Prominent expression of levansucrase was obtained through xylose induction in Bacillus megaterium, where most of the His₆-tagged protein was secreted into the culture broth, giving levansucrase activity of 12,906 U/l. Response-surface methodology (RSM) was further employed to optimize the fermentation conditions and improve the level of levansucrase production. Maximum levansucrase activity of 20,251 U/l was obtained in 12 h of fermentation carried out at 28°C, starting induction with 0.735% xylose when A ₆₀₀ was 1.2, which was 1.6- and 62-fold higher than those obtained in the nonoptimized conditions for the recombinant strain and the native strain, respectively.     

Sucrose medium osmolality as a regulator of anabolic and catabolic parameters in Zymomonas culture

This study focuses on the growth of Zymomonas mobilis strain 113 S and its ethanol and levan production under the conditions of increasing sucrose medium osmolality caused by NaCl, KCl, sorbitol or maltose. The increase in medium osmolality (700–1,500 mosml/kg) was accompanied by the inhibition of growth (growth rate, biomass yield) and ethanol production (specific productivity and yield) In contrast, levan synthesis was less affected or even stimulated and, as a consequence, levan specific productivity was increased significantly. A decrease in the anabolic growth parameters correlated with a parallel inhibition of glucose-6-P dehydrogenase and alcohol dehydrogenase (isoenzyme ADH II) activities. A significant inverse linear relationship (r = ? 0.932, 1 ? P = 0.01) was observed between the values of the specific productivities of ethanol and levan. This relationship was confirmed independently by a controlled reduction of growth and ethanol productivity (3.75–4.75 mM sodiumbisulphite as an acceptor of acetaldehyde formed in the pyruvate decarboxylase reaction). As further support of this relationship, a significant inverse correlation was observed between levan specific productivity and ATP concentration in Zymomonas mobilis cells, most probably demonstrating that a reduced level of energetic metabolism is favourable for levan production.     

Levan production by Zymomonas mobilis cells. Attached to plaited spheres

In this work, an immobilization method for polymer-levan production by a non-flocculating Z mobilis culture was developed. The extent of cell attachment to the stainless steel wire surface, culture growth and product synthesis were described. It was established that during short-term passive immobilization of non-flocculation Z mobilis cells on a stainless steel wire surface, sufficient amounts of biomass for proper levan and ethano fermentation could not be obtained. Adherence of cells was improved by pressing the paste-like biomass within stainless steel spheres knitted from wire with subsequent dehydration. Biomass fixed in metal spheres was used for repeated batch fermentation of levan. The activation period of cells within wire spheres (WS) was 48 h in duration. During this time, cell growth stabilized at production levels of ethanol and levan of Q_eth = 1.238 g/l × h and q_eth = 0.47 g/l × h; Q_eth = 0.526 g/l × h and q_eth = 0.20 g/l × h. Five stable fermentation cycles were realized using one wire sphere inoculum, and maintaining a stable ratio of 2.4 of biomass suspended in the medium to biomass fixed in the sphere. Using fixed Z mobilis biomass in the WS, the total amount of inoculum could be reduced for batch fermentation. Large plaited wire spheres with biomass may have potential in fermentation in viscous systems, including levan production.     

Optimal conditions for levan formation by an overexpressed recombinant levansucrase

Summary A genetically modified levansucrase, which contained His-affinity tag in its C-terminal, was constructed by PCR reaction using two synthetic primers. This modified protein was produced up to 30 % in total cell protein of E. coli, and was purified by a one-step affinity chromatography. The optimum pH for levan production was pH 5 and the optimum temperature was 0 °C. The higher velocity of levan formation within shorter enzyme reaction times was achieved by increasing the levels of enzyme concentration. The optimal sucrose concentration for levan production was around 20 %. Under these conditions, more than 50 g levan/l was produced.     

The sacB and sacC genes encoding levansucrase and sucrase form a gene cluster in Zymomonas mobilis

Summary The Zymomonas mobilis gene sacB that encodes the extracellular levansucrase was cloned and expressed in Escherichia coli. The gene product exhibited both sucrose hydrolysis activity and levan forming capability. Sub-cellular fractionation of E. coli carrying pLSS41 revealed that about 95% of the total sucrase activity was detected in the cytoplasmic fraction. The levansucrase gene was overexpressed (about hundred fold) in E. coli under T7 polymerase expression system. Nucleotide sequence analysis of this gene revealed an open reading frame of 1269 bp long coding for a protein of 423 amino acids with a molecular mass of 46.7 KDa. The deduced amino acid sequence was identical to the N-terminal amino acids of protein A51 of Z. mobilis ZM4. Therefore, the product of sacB is levansucrase. This is the first extracellular enzyme of Z. mobilis sequenced which does not possess a signal sequence. This gene is located 198 bp upstream of sacC gene encoding for the extracellular sucrase forming a gene cluster     

Enzymatic synthesis of levan by Zymomonas mobilis levansucrase overexpressed in Escherichia coli

Summary Levansucrase gene from Zymomonas mobilis was expressed efficiently in Escherichia coli and the overproduced recombinant levansucrase amounted to 40% of the total cell protein. Using E. coli lysate, levan was synthesized in a sucrose-based medium enzymatically with the conversion yields of up to 46% from fructose liberated in 25 hrs of incubation. More levan was formed at lower temperatures in the reaction mixture, whereas higher temperatures were favoured for the accumulation of free fructose or short chain oligosaccharides.     

Fermentation characteristics of levansucrase mutants of Zymomonas mobilis

Two mutants, Ls1 and Ls2, of Zymomonas mobilis B-806 unable to produce levan were isolated. With native gel electrophoresis and zymogram analysis it was confirmed that the mutants did not synthesize active levansucrase (E2). However, they produced intracellular sucrase (E1) and extracellular invertase (E3). Comparison of these mutants with the parent strain for alcohol production on glucose, fructose and sucrose (100 g/l each) media revealed that the final ethanol concentration achieved in sucrose medium was only about 5 g/l higher with the mutants than with the wild type. The ethanol yield of the mutants increased from 0.48 g/g to 0.50 g/g on sucrose medium.     

Disruption of the Zymomonas mobilis extracellular sucrase gene (sacC) improves levan production

AIMS: Disruption of the extracellular Zymomonas mobilis sucrase gene (sacC) to improve levan production. METHODS AND RESULTS: A PCR-amplified tetracycline resistance cassette was inserted within the cloned sacC gene in pZS2811. The recombinant construct was transferred to Z. mobilis by electroporation. The Z. mobilis sacC gene, encoding an efficient extracellular sucrase, was inactivated. A sacC defective mutant of Z. mobilis, which resulted from homologous recombination, was selected and the sacC gene disruption was confirmed by PCR. Fermentation trials with this mutant were conducted, and levansucrase activity and levan production were measured. In sucrose medium, the sacC mutant strain produced threefold higher levansucrase (SacB) than the parent strain. This resulted in higher levels of levan production, whilst ethanol production was considerably decreased. CONCLUSIONS: Zymomonas mobilis sacC gene encoding an extracellular sucrase was inactivated by gene disruption. This sacC mutant strain produced higher level of levan in sucrose medium because of the improved levansucrase (SacB) than the parent strain. SIGNIFICANCE AND IMPACT OF THE STUDY: The Z. mobilis CT2, sacC mutant produces high level of levansucrase (SacB) and can be used for the production of levan.     

The effect of pH,temperature and sucrose concentration on high productivity continuous ethanol fermentation using Zymomonas mobilis

Summary A flocculent strain of Zymomonas mobilis was used for ethanol production from sucrose. Using a fermentor with cell recycle (internal and external settler) high sugar conversion and ethanol productivity were obtained. At a dilution rate of 0.5 h^-1 (giving 96% sugar conversion) the ethanol productivity, yield and concentrations respectively were 20 g/l/h, 0.45 g/g and 40 g/l using a medium containing 100 g/l sucrose. At a sucrose concentration of 150 g/l, the ethanol concentration reached 60 g/l. The ethanol yield was 80% theoretical due to levan and fructo-oligomer formation. No sorbitol was detected. This fermentation was conducted at a range of conditions from 30 to 36°C and from pH 4.0 to 5.5.     

The effect of chemical treatment of stainless steel wire surfaces on Zymomonas mobilis cell attachment and product synthesis

The attachment, growth and product synthesis of non-flocculating Zymomonas mobilis cell, fixed in stainless steel wire spheres (WS), were investigated. The carrier surface was activated by treatment with titanium (IV) chloride (TiCl₄) and γ-aminopropyltriethoxysilane (AS) in an attempt to raise the efficiency in the immobilization of the cells. System productivity for ethanol and levan production, using cells immobilized on a modified stainless steel in the batch fermentation of a sucrose medium, rose as a result of increased biomass compared to the productivity of cells fixed on untreated (control) metal surfaces. Stabilized ethanol synthesis was demonstrated in the course of four cycles (each cycle 48 h) of repeated fermentations with a stainless steel carrier treated with AS, and three cycles when TiCl₄ was used. Levan synthesis decreased after three cycles with cells immobilized on a silanized surface. System productivity for ethanol and levan production after the fourth cycle in experiments with TiCl₄-activated, silanized and unmodified carriers were Q_eth = 1.01, 1.06 and 0.27 g/l × h; Q_lev = 0.32, 0.29 and 0.12 g/l × h, respectively. However, the specific productivity of biomass for product synthesis was higher in fermentation systems with untreated stainless steel surfaces, probably due to some loss of physiological activity of cells attached to a modified carrier. Investigations of throughly washed activated stainless steel wire surfaces, by scanning electron microscopy after immobilization, showed significant attachment of cells to the carriers. A polymer layer covered the wire surface treated with TiCl₄ after fermentations. This may be explained as the binding of extracellular polysaccharide, such as the fructose-polymer levan and yeast extract components, to the modified support via chelation. After four fermentations, craters and holes in the polymer layer were evident, probably as a result of CO₂ formation. A small number of cells appeared on this layer. In view of the good ethanol formation during all fermentation cycles, it is probably that active Z. mobilis cells remained under the polymer layer. Wire treatment with AS resulted in the formation of long filamentous cells during fermentation and some disturbance of cellular fission. This may be partly explained by strong electrostatic interactions between the positively charged carrier surface and the predominately negatively charged surface of Z mobilis cells. However, this did not significantly affect other cellular functions. The surface of the wire treated with AG was practically without a polymer layer.