Continuous production of L-malic acid by immobilized cells

l-Malic acid is used extensively in the pharmaceutical industry and as a food additive. It is now produced on an industrial scale by the enzymatic conversion of fumaric acid using immobilized cells of Brevibacterium flavum. Recent improvements to this system, especially the use of x-carrageenan supports, have resulted in a continuous process capable of yielding 30 tonnes of l-malic acid per month.     

产L-苹果酸重组大肠杆菌的构建

基于产琥珀酸重组大肠杆菌E.coli B0013-1050的琥珀酸合成途径,利用Red同源重组技术结合Xer/dif重组系统敲除富马酸酶基因fumB、fumC,苹果酸酶基因maeB,构建L-苹果酸合成途径,最终得到重组大肠杆菌E.coli2030,该菌株在15 L发酵罐中,产L-苹果酸12.5 g/L,葡萄糖-苹果酸转化率为52.1%,同时对发酵产物中主要杂酸丙酮酸和琥珀酸的生产原因进行了初步的探讨与分析。为进一步提高L-苹果酸的转化率,整合表达来源于黄曲霉的苹果酸脱氢酶基因,构建重组菌E.coli 2040,在15 L发酵罐中产L-苹果酸14 g/L,葡萄糖-苹果酸转化率提高到60.3%。     

Stability of the biocatalysts of L-aspartic acid synthesis based on immobilized Escherichia coli cells

Experiments were carried out to investigate the process of a continuous enzymatic synthesis of L-aspartic acid from ammonium fumarate in uniform filling flow reactors. Escherichia coli (Soviet strain 85) cells immobilized in polyacrylamide gel granules reinforced by a solid carrier were used as biocatalysts. The conditions, under which a high aspartase activity of the biocatalyst and a stable hydrodynamic performance of the reactor were maintained, were determined. The main kinetic characteristics of a continuous performance of the reactor for 150 days were obtained.     

Conversion of fumaric acid to L-malic by sol-gel immobilized Saccharomyces cerevisiae in a supported liquid membrane bioreactor

Conversion of fumaric acid (FA) to L-malic acid (LMA) was carried out in a bioreactor divided by two supported liquid membranes (SLMs) into three compartments: Feed, Reaction, and Product. The Feed/Reaction SLM, made of tri-n-octylphosphine oxide (vol 10%) in ethyl acetate, was selective toward the substrate, fumaric acid (S(FA/LMA) = 10). The Reaction/Product SLM, made of di(2-ethylhexyl) phosphate (vol 10%) in dichloromethane, was selective toward the product, L-malic acid (S(LMA/FA) = 680). Immobilized yeast engineered to overproduce the enzyme fumarase [E.C. 4.2.1.2] was placed in the Reaction compartment and served as the catalyst. The yeast was immobilized in small glasslike beads of alginate-silicate sol-gel matrix. The construction of the bioreactor ensured unidirectional flow of the substrate from the Feed to the Reaction and of the product from the Reaction to the Product compartments, with the inorganic counterion traveling in the opposite direction. The conversion of almost 100%, above the equilibrium value of ca. 84% and higher than that for the industrial process, 70%, was achieved. In contrast to the existing industrial biocatalytic process resulting in L-malic acid salts, direct production of the free acid is described.     

Stability of biocatalysts on the basis of carrageenan-immobilized Escherichia coli during continuous synthesis of L-malic acid

Continuous enzymatic synthesis of L-malic acid from potassium fumarate in packed-bed flow reactors was investigated. Carrageenan-immobilized Escherichia coli cells were used as a biocatalyst. The operational stability of the biocatalyst fumarase activity was studied, and conditions for preserving high activity of the biocatalyst were determined.     

Metabolic engineering of Escherichia coli for the production of fumaric acid

Fumaric acid is a naturally occurring organic acid that is an intermediate of the tricarboxylic acid cycle. Fungal species belonging to Rhizopus have traditionally been employed for the production of fumaric acid. In this study, Escherichia coli was metabolically engineered for the production of fumaric acid under aerobic condition. For the aerobic production of fumaric acid, the iclR gene was deleted to redirect the carbon flux through the glyoxylate shunt. In addition, the fumA, fumB, and fumC genes were also deleted to enhance fumaric acid formation. The resulting strain was able to produce 1.45 g/L of fumaric acid from 15 g/L of glucose in flask culture. Based on in silico flux response analysis, this base strain was further engineered by plasmid‐based overexpression of the native ppc gene, encoding phosphoenolpyruvate carboxylase (PPC), from the strong tac promoter, which resulted in the production of 4.09 g/L of fumaric acid. Additionally, the arcA and ptsG genes were deleted to reinforce the oxidative TCA cycle flux, and the aspA gene was deleted to block the conversion of fumaric acid into L ‐aspartic acid. Since it is desirable to avoid the use of inducer, the lacI gene was also deleted. To increase glucose uptake rate and fumaric acid productivity, the native promoter of the galP gene was replaced with the strong trc promoter. Fed‐batch culture of the final strain CWF812 allowed production of 28.2 g/L fumaric acid in 63 h with the overall yield and productivity of 0.389 g fumaric acid/g glucose and 0.448 g/L/h, respectively. This study demonstrates the possibility for the efficient production of fumaric acid by metabolically engineered E. coli. Biotechnol. Bioeng. 2013; 110: 2025–2034. © 2013 Wiley Periodicals, Inc.     

Enzymatic synthesis of isoamyl acetate using immobilized lipase from Rhizomucor miehei

The effects of important reaction parameters for enhancing isoamyl acetate formation through lipase-catalyzed esterification of isoamyl alcohol were investigated in this study. Increase in substrate (acid) concentration led to decrease in conversions. A critical enzyme concentration of 3 g l(-1) was detected for a substrate concentration of 0.06 M (each of alcohol and acid). Solvents with partition coefficient higher than 1000 (log P>3.0) supported enzyme activity to give high conversions. Acetic acid at higher concentrations could not be esterified easily probably owing to its role in lowering the microaqueous pH of the enzyme. Extraneous water/buffer addition decreased the isoamyl acetate yields slightly ( approximately 10%) at 0.005-0.01% v/v of the reaction mixture and drastically (>40%) at above 0.01% v/v. Buffer saturation of the organic solvent employed improved esterification (upto two-fold), particularly at moderately higher substrate concentrations (>0.18 M). Employing acetic anhydride instead of acetic acid resulted in a two-fold increase in the yields (at 0.25 M substrate). Use of excess nucleophile (alcohol) concentration by increasing the alcohol/acid molar ratio resulted in higher conversions in shorter duration (upto eight-fold even at 1.5 M acetic acid). Yields above 80% were achieved with substrate concentrations as high as 1.5 M and more than 150 g l(-1) isoamyl acetate concentrations were obtained employing a relatively low enzyme concentration of 10 g l(-1). The operational stability of lipase was also observed to be reasonably high enabling ten reuses of the biocatalyst.     

Enzymatic synthesis of esters using an immobilized lipase

Various esters were synthesized in nearly anhydrous hexane from alcohols and carboxylic acids using a lipase from Candida cylindracea. The enzyme was immobilized on a nylon support and protein loadings as high as 10 mg/g were obtained. The activity of the immobilized enzyme was maximum in a range of temperatures from 25 to 37 degrees C. Ethylpropionate was formed from ethanol and propionic acid at a rate of 0.017 mol/h g immobilized protein. Different esters were formed at comparable rates and equilibrium conversions could generally be approached in less than 10 h in a batch reaction system. The immobilized lipase catalyst was quite stable and retained about one third of the initial activity after repeated experiments during the course of 72 days. A stirred tank continuous flow reactor was used successfully for the continuous production of esters.     

Enzymatic synthesis of sialic acid derivative by immobilized lipase from Candida antarctica

The effects of important reaction parameters on the enhancement of sialic acid derivative lipophilic properties through the lipase-catalyzed esterification of N-acetyl neuraminic acid methyl ester are investigated in this study. It is found that the lipase Novozym 435 from Candida antarctica is particularly useful in the preparation of sialic acid methyl ester monononanoate (SAMEMN). The optimum temperature for the SAMEMN synthesis reaction using Novozym 435 is 60 °C, and nonanoic anhydride is found to be the best substrate among all acyl donors. The Novozym 435-catalyzed esterification of N-acetyl neuraminic acid methyl ester gave a maximum yield of 87.7% after 6 h in acetonitrile at 60 °C. Because the novel method developed is simple, yet effective, it could potentially be used industrially for the production of sialic acid derivatives.     

Cell mass synthesis and degradation by immobilized Escherichia coli

Escherichia coli K-12 cells were grown in a confined volume using microporous hollow fiber membranes. The local cell concentrations in the reactors were above 400 g dry mass/L, in excess of the predicted limit based on the specific volume of free cells determined by tracer exclusion. Cell mass synthesis and degradation rates in these reactors were measured using radioisotope labeling with (35)S. Net accumulation of cell material persisted at these high cell densities. The rates of substrate uptake and cell growth were predicted from the theory of reaction and diffusion assuming that kinetics of cell metabolism are identical for free-living and immobilized cells. This theory was tested by comparison of overall rates and by the size of the region in which cell growth occurred, measured by autoradiography. A yield coefficient of 4 +/- 1 mol sulfur/mol glucose was measured, in agreement with the value determined for free-living cells in similar conditions. Cell growth occurs in a thin layer (10-30 mum), at a rate similar to the growth rate for free cells. Volume expansion by the cells as a consequence of proliferation induces convection of cell mass out of the growth region into a region of the reactor filled with starving cells, which then accumulate in the reactor. The combination of mass-balance and spatial distribution measurements made possible by the use of radioisotope labeling enables a direct test for mass transfer limitations, the determination of the intrinsic cell kinetics, and noninvasive measurements of cell growth in immobilized cell reactors.     

Enzymatic synthesis of isopropyl myristate using immobilized lipase from Bacillus cereus MTCC 8372

A purified alkaline thermo-tolerant bacterial lipase from Bacillus cereus MTCC 8372 was immobilized on a Poly (MAc-co-DMA-cl-MBAm) hydrogel. The hydrogel showed approximately 94% binding capacity for lipase. The immobilized lipase (2.36 IU) was used to achieve esterification ofmyristic acid and isopropanol in n-heptane at 65 degrees C under continuous shaking. The myristic acid and isopropanol when used at a concentration of 100 mM each in n-heptane resulted in formation of isopropyl myristate (66.0 +/- 0.3 mM) in 15 h. The reaction temperature below or higher than 65 degrees C markedly reduced the formation of isopropyl myristate. Addition of a molecular sieve (3 A x 1.5 mm) to the reaction mixture drastically reduced the ester formation. The hydrogel bound lipase when repetitively used to perform esterification under optimized conditions resulted in 38.0 +/- 0.2 mM isopropyl myristate after the 3rd cycle of esterification.     

Fatty acid synthesis in Escherichia coli

1. Fatty acid formation by cells of a strain of Escherichia coli has been studied in the exponential, post-exponential and stationary phases of growth. 2. During the exponential phase of growth, the metabolic quotient (mmumoles of fatty acid synthesized/mg. dry wt. of cells/hr.) for each fatty acid in the extractable lipid was constant. 3. The newly synthesized fatty acid mixtures produced during this phase contained hexadecanoic acid (41%), hexadecenoic acid (31%), octadecenoic acid (21%) and the C(17)-cyclopropane acid, methylenehexadecanoic acid (4%). 4. As the proportion of newly synthesized material increased, changes in the fatty acid composition of the cells during this period were towards this constant composition. 5. Abrupt changes in fatty acid synthesis occurred when exponential growth ceased. 6. In media in which glycerol, or SO(4) (2-) or Mg(2+), was growth-limiting there was a small accumulation of C(17)-cyclopropane acid in cells growing in the post-exponential phase of growth. 7. Where either NH(4) (+) or PO(4) (3-) was growth-limiting and there were adequate supplies of glycerol, Mg(2+) and SO(4) (2-), there was a marked accumulation of C(17)-cyclopropane acid and C(19)-cyclopropane acid appeared. 8. Under appropriate conditions the metabolic quotient for C(17)-cyclopropane acid increased up to sevenfold at the end of exponential growth. Simultaneously the metabolic quotients of the other acids fell. 9. A mixture of glycerol, Mg(2+) and SO(4) (2-) stimulated cyclopropane acid formation in resting cells.     

Phosphatidic acid synthesis in Escherichia coli

The kinetic properties of acyl-coenzyme A (CoA): l-alpha-glycerol-phosphate trans-acylase (EC 2.3.1.15) from Escherichia coli were studied. At 10 C, a temperature at which the reaction was proportional to time and enzyme concentration, the enzyme had an apparent K(m) of 60 mum for l-alpha-glycerol-phosphate. The curve describing the velocity of the reaction as a function of palmitoyl-CoA concentration was sigmoid but the plot of v(-1) versus [S](-3) gave a straight line. A K(m) of about 11 mum was calculated for palmitoyl-CoA. Adenosine triphosphate specifically inhibited the reaction, being a noncompetitive inhibitor in respect to l-alpha-glycerol phosphate. Inhibition only occurred with high concentrations of palmitoyl-CoA, and maximal inhibition was 60%.     

Biosynthesis of iminodiacetic acid from iminodiacetonitrile by immobilized recombinant Escherichia coli harboring nitrilase

Iminodiacetic acid (IDA) is widely used as an intermediate in the manufacture of chelating agents, glyphosate herbicides and surfactants. In the current work, the fragment with the length of 1,110 bp encoding the Acidovorax facilis nitrilase was obtained. The recombinant nitrilase expressed in Escherichia coli BL21 (DE3) was successfully used in the production of IDA from iminodiacetonitrile. To improve the stability of operation, the recombinant cells were entrapped in polyvinyl alcohol (PVA) and sodium alginate (SA) copolymer. The maximum relative nitrilase activity with 98.1% was further observed at 1.0% SA, 8.0% PVA, 1.0% CaCl(2), and 5.0% wet cells, under conditions of 1.0% iminodiacetonitrile in distilled water and a temperature of 40°C, respectively. The entrapped cells facilitated easy separation and good recycling compared with free cells. Moreover, the immobilized cells showed good operation and storage stability. This report is the first to describe IDA preparation using immobilized recombinant E. coli harboring nitrilase.     

Production of D-tagatose at high temperatures using immobilized Escherichia coli cells expressing L-arabinose isomerase from Thermotoga neapolitana

Escherichia coli cells expressing l-arabinose isomerase from Thermotoga neapolitana (TNAI) were immobilized in calcium alginate beads. The resulting cell reactor (2.4 U, t _1/2 = 43 days at 70°C) in a continuous recycling mode at 70°C produced 49 and 38 g d-tagatose/l from 180 and 90 g d-galactose/l, respectively, within 12 h.     

Factors affecting the synthesis of penicillins by the immobilized penicillin acylase from Escherichia coli

The effect of pH, temperature, reactant concentration, and reaction time has been investigated for the synthesis of N-benzhydryl-N′-acetamidopiperazyl-6-penicillanic acid and N-benzyl-N′-acetamidopiperazyl-6-penicillanic acid from 6-aminopenicillanic acid by the immobilized penicillin acylase from Escherichia coli. The synthesis of penicillins from carboxylic acids proceeds most rapidly at pH 5; with ethyl ester derivatives of carboxylic acids the pH optimum is higher (6–7). The most rapid synthesis of penicillins was obtained with ethyl ester derivatives of carboxylic acids. The optimum temperatures were 25–35°C.