Abstract:This paper presents an enzymatic process for sorbitol and gluconic acid production using cells of Zymomonas mobilis permeabilized with CTAB. Equimolar solutions of glucose and fructose (from 96.0 to 422.2 g/L) were used. In a batch reactor, conversions of 97% were attained after 15 to 20 hours of reaction. The effect of the initial concentration of the substrates was evaluated in experiments using 20% more and 20% less glucose than fructose. It was observed that the reaction performed with more fructose reache… Show more
“…Consequently, we concluded that the quantification of enzyme activity based on rates of hydroxide consumption is very reliable, as described and recommended by several authors [9,10,19,22]. In addition, preliminary tests carried out by our group with standard solutions prepared with known enzyme compositions indicated that the precision of reaction rate measurements was better than 1.0 9 10 -4 g l -1 h -1 , which is much lower than the precision reported here when distinct fermentation runs and reaction tests were performed.…”
Section: Resultssupporting
confidence: 57%
“…As the reaction between glucose and fructose is equimolar, the use of the hydroxide mass consumed during the reaction to estimate the concentration of all reagents and products has been well documented [9,10,19,22]. In order to check if this assumption remains valid for the reaction of fructose and lactose, we used a second, independent methodology to quantify lactose, as described in the previous section.…”
Section: Resultsmentioning
confidence: 99%
“…Following the addition of cetyltrimethylamonium bromide (CTAB) (0.04 g CTAB .g cell -1 ) [16,22], the suspension was homogenized, kept under constant stirring for 30 min at room temperature, and centrifuged one more time. Permeabilized cells were resuspended in water, with the pH adjusted to 6.2.…”
The proper determination of experimental errors in bioprocesses can be very important because experimental errors can exert a major impact on the analysis of experimental results. Despite this, the effect of experimental errors on the analysis of bioprocess data has been largely overlooked in the literature. For this reason, we performed detailed statistical analyses of experimental errors obtained during the production of lactobionic acid and sorbitol in a system utilizing as catalyst the GFOR (glucose-fructose oxidoreductase) enzyme from permeabilized cells of the bacteria Zymomonas mobilis. The magnitude of the experimental errors thus obtained were then correlated with the process operation conditions and with the composition of the culture media used for bacterial growth. It is shown that experimental errors can depend very significantly on the operation conditions and affect the interpretation of available experimental data. More specifically, in this study, experimental errors depended on the nutritional supplements added to the cultivation medium, the inoculation process, and the reaction time, which may be of fundamental importance for actual process development. The results obtained also indicate, for the first time, that GFOR activity can be affected by the composition of the medium in which cells are cultivated.
“…Consequently, we concluded that the quantification of enzyme activity based on rates of hydroxide consumption is very reliable, as described and recommended by several authors [9,10,19,22]. In addition, preliminary tests carried out by our group with standard solutions prepared with known enzyme compositions indicated that the precision of reaction rate measurements was better than 1.0 9 10 -4 g l -1 h -1 , which is much lower than the precision reported here when distinct fermentation runs and reaction tests were performed.…”
Section: Resultssupporting
confidence: 57%
“…As the reaction between glucose and fructose is equimolar, the use of the hydroxide mass consumed during the reaction to estimate the concentration of all reagents and products has been well documented [9,10,19,22]. In order to check if this assumption remains valid for the reaction of fructose and lactose, we used a second, independent methodology to quantify lactose, as described in the previous section.…”
Section: Resultsmentioning
confidence: 99%
“…Following the addition of cetyltrimethylamonium bromide (CTAB) (0.04 g CTAB .g cell -1 ) [16,22], the suspension was homogenized, kept under constant stirring for 30 min at room temperature, and centrifuged one more time. Permeabilized cells were resuspended in water, with the pH adjusted to 6.2.…”
The proper determination of experimental errors in bioprocesses can be very important because experimental errors can exert a major impact on the analysis of experimental results. Despite this, the effect of experimental errors on the analysis of bioprocess data has been largely overlooked in the literature. For this reason, we performed detailed statistical analyses of experimental errors obtained during the production of lactobionic acid and sorbitol in a system utilizing as catalyst the GFOR (glucose-fructose oxidoreductase) enzyme from permeabilized cells of the bacteria Zymomonas mobilis. The magnitude of the experimental errors thus obtained were then correlated with the process operation conditions and with the composition of the culture media used for bacterial growth. It is shown that experimental errors can depend very significantly on the operation conditions and affect the interpretation of available experimental data. More specifically, in this study, experimental errors depended on the nutritional supplements added to the cultivation medium, the inoculation process, and the reaction time, which may be of fundamental importance for actual process development. The results obtained also indicate, for the first time, that GFOR activity can be affected by the composition of the medium in which cells are cultivated.
“…The Chemical Engineering Program’s Bioprocesses Laboratory at COPPE, Federal University of Rio de Janeiro, Brazil, has used the bacterium Zymomonas mobilis for years to study the production of various substances, like ethanol, glyconic acid, sorbitol, lactobionic acid, and L-asparaginase [ 11 – 16 ]. The L-asparaginase obtained from Z .…”
L-asparaginase is an enzyme used as a chemotherapeutic agent, mainly for treating acute lymphoblastic leukemia. In this study, the gene of L-asparaginase from Zymomonas mobilis was cloned in pET vectors, fused to a histidine tag, and had its codons optimized. The L-asparaginase was expressed extracellularly and intracellularly (cytoplasmically) in Escherichia coli in far larger quantities than obtained from the microorganism of origin, and sufficient for initial cytotoxicity tests on leukemic cells. The in silico analysis of the protein from Z. mobilis indicated the presence of a signal peptide in the sequence, as well as high identity to other sequences of L-asparaginases with antileukemic activity. The protein was expressed in a bioreactor with a complex culture medium, yielding 0.13 IU/mL extracellular L-asparaginase and 3.6 IU/mL intracellular L-asparaginase after 4 h of induction with IPTG. The cytotoxicity results suggest that recombinant L-asparaginase from Z. mobilis expressed extracellularly in E.coli has a cytotoxic and cytostatic effect on leukemic cells.
Equimolar amounts of lactobionic acid and sorbitol may be obtained in a reaction catalyzed by the enzymes glucose-fructose oxidoreductase and glucono-δ-lactonase, which are found in the periplasm of Zymomonas mobilis. These reactions are generally conducted using immobilized bacterial cells, and the cell treatment and immobilization steps are costly and time-consuming. This study evaluated alternatives to simplify the preparation of calcium alginate-immobilized biocatalyst and its application in different operation modes and types of reactors. It was possible to eliminate cell permeabilization with cetyltrimethylammonium bromide, and the reticulation of Z. mobilis cells with glutaraldehyde sufficed to inhibit the fermentative metabolism of carbohydrates by the bacterium, with accumulation of bioconversion products. When the process was carried out in a mechanically stirred reactor in batch mode, 530 mmol L of products were obtained in 24 h. The process was also tested in fed-batch mode so as to use of a larger amount of lactose, since it could not be used in the batch because of its low solubility in water. Under this condition, final products concentration reached 745 mmol L within 42 h. Similar results were obtained for reactions conducted in a pneumatically stirred reactor in batch and fed-batch modes, proving the potential use of this process in several industrial settings.
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