Continuous production of NADP by immobilized Brevibacterium ammoniagenes cells.

Whole cells of Brevibacterium ammoniagenes IAM 1645 having the polyphosphate NAD-kinase were successfully immobilized in a polyacrylamide gel lattice. The immobilized cells were activated by treatment with organic solvents or detergents. The pH optimum of the immobilized cells for the production of NADP was 7.0, and divalent metal ions were required to maintain the elevated activity of polyphosphate NAD-kinase. Highly pure NADP was continuously produced in high yield by the immobilized cell column. The half-life of this column was about eight days.     

Study of an enzyme membrane reactor with immobilized fumarase for production of L-malic acid

The conversion of fumaric acid into L-malic acid by fumarase immobilized in a membrane reactor was analyzed experimentally. The enzyme was entrapped in asymmetric capillary membranes made of polysulfone. The performance of the reactor was evaluated in terms of conversion degree, reaction rate, and stability. The influence of operating conditions, such as amount of immobilized enzyme, substrate concentration, residence time, and axial flow rate, were investigated. The kinetic parameters K(m), V(max), and k(+2) were also measured. The stability of the immobilized enzyme was very good, showing no activity decay during more than 2 weeks of continuous operation.     

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.     

Thermal regulation of fatty acid synthetase from Brevibacterium ammoniagenes.

The fatty acid composition of Brevibacterium ammoniagenes was affected by the temperature of growth. As the growth temperature was lowered, the proportion of unsaturated fatty acids increased. The fatty acid synthetase obtained from B. ammoniagense produced oleic acid as well as saturated fatty acids. The ratio of unsaturated to saturated fatty acids synthesized by this enzyme in vitro was dependent on the temperature of the enzyme reaction but not on the growth temperature of B. ammoniagenes from which the enzyme was prepared. These results suggest that the changes of composition in cellular fatty acids reflect the temperature dependence of the fatty acid synthetase.     

Enzymatic synthesis of L-malic acid from fumaric acid using immobilized Escherichia coli cells

Optimal conditions were chosen for cultivation of Escherichia coli 85 cells with a rather high fumarate-hydratase activity on a cheap medium containing no edible raw material. An active biocatalyst for the synthesis of L-malic acid from fumaric acid was obtained based on E. coli 85 cells immobilized in carrageenan. The enzymatic synthesis of L-malic acid from potassium fumarate was kinetically studied and optimized. Some thermodynamic parameters of fumaric acid hydration into malic acid were determined. A technique for assaying the reaction mixture was developed that involved high performance liquid chromatography.     

Continuous production ofL-malic acid by immobilizedBrevibacterium ammoniagenes cells

Summary Continuous production ofL-malic acid from fumaric acid using immobilized microbial cells was investigated. Several microorganisms having fumarase activity were immobilized into a polyacrylamide gel lattice. Among the microorganisms tested, immobilizedBrevibacterium ammoniagenes IAM 1645 showed the highest enzyme activity, but produced an unwanted by-product, succinic acid. Conditions for suppression of this side reaction were investigated, and bile extract treatment of immobilized cells was found to be effective.The bile extract treatment of immobilized cells also resulted in a marked increase of reaction rate forL-malic acid formation.No difference was observed between the native enzyme and immobilized cells in optimal pH and temperature of the enzyme reaction.The effect of temperature on the reaction rate and the stability of fumarase activity of an immobilized cell column were investigated under conditions of continuous enzyme reaction. The decay of enzyme activity during continuous enzyme reaction was expressed by an exponential relationship. Half-life of the fumarase activity of the immobilized cell column at 37°C was calculated to be 52.5 days.Presented at the Annual Meeting of the Society of Fermentation Technology, Japan, Osaka, Japan, October 30, 1975.     

Enzymatic bases for the fatty acid positioning in phospholipids of Brevibacterium ammoniagenes

Positional distribution of fatty acids in phospholipids from Brevibacterium ammoniagenes was analyzed to find that phosphatidylethanolamine consisted mainly of 1-saturated acyl 2-unsaturated acyl species while phosphatidylglycerol consisted mainly of 1-unsaturated acyl 2-saturated acyl species. Three acyltransferase systems were characterized in a membrane preparation--the acylations of glycerophosphate, 1-acyl-glycerophosphate, and 2-acyl-glycerophosphate--which appeared to be catalyzed by different enzymes. The distribution of fatty acids in the phosphatidylethanolamine molecule was not correlated simply with the specificities of these enzymes, but the relatively high specificity for palmitoyl-CoA of the glycerophosphate acyltransferase system to form 2-acyl-glycerophosphate, followed the relatively high specificity for oleoyl-CoA of the 2-acyl-glycerophosphate acyltransferase system, provided a basis for producing the major molecular species of phosphatidylglycerol.     

Temperature- and growth-phase-dependent changes in membrane fatty acid compositions of Brevibacterium ammoniagenes

Fatty acids newly synthesized by Brevibacterium ammoniagenes grown at different temperatures were analyzed. The assay temperature, not the growth temperature, was found to be the major factor affecting the unsaturated/saturated ratio of newly synthesized fatty acids in logarithmic-phase cells. However, in the stationary-phase cells the growth temperature also affected the product profile significantly; cells grown at 7 degrees C produced relatively more oleate and stearate and less palmitate and hexadecenoate when shifted up to 37 degrees C than did cells grown and assayed at 37 degrees C. The unsaturated/saturated ratio as well as average chain length of fatty acids also varied along with the progress of isothermal growth phase. These changes in fatty acid product profiles observed in vivo could be mimicked in vitro assays of the fatty acid synthetase by changing malonyl-CoA concentrations. Our results suggest that the malonyl-CoA concentration is a factor which, in addition to temperature, determines growth-phase-dependent and growth-temperature-dependent changes in the unsaturated/saturated ratios of fatty acids.     

Immobilization of Brevibacterium flavum with carrageenan and its application for continuous production of l-malic acid

Extensive experiments were carried out to improve the productivity ofl-malic acid from fumaric acid using Brevibacterium flavum immobilized with carrageenan. The most favourable preparation for the continuous production ofl-malic acid was obtained when 16 g of B. flavum cells was entrapped in 100 ml 3.4% carrageenan gel. However, the immobilized cells produced an unwanted by-product, succinic acid. Treatment of the immobilized cells with 0.6% bile extract suppressed the side reaction and gave the highest operational stability of fumarase activity. By the immobilization of intact cells, the optimal temperature of the enzyme reaction shifted to 10°C higher, the optimal pH became broader, and the operational stability of fumarase activity increased. The effect of temperature on the stability of fumarase activity in the immobilized cell column was investigated under conditions of continuous enzyme reaction. The decay of fumarase activity during continuous enzyme reaction was expressed by an exponential relationship. The productivity of the immobilized B. flavum using carrageenan was as high as 5.2 times that of the conventional immobilized B. ammoniagenes using polyacrylamide.     

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%。     

Effect of antibiotics on 5'-inosinic acid biosynthesis by a Brevibacterium ammoniagenes mutant

The effect of streptomycin, erythromycin, kanamycin and penicillin on the biosynthesis of 5'-inosinic acid (IMP) by the mutant strain Brevibacterium ammoniagenes was studied. It has been found that the efficiency of antibiotic action depends not only in its concentration but on the age of the culture. When the antibiotics were introduced into the culture broth at the beginning of fermentation, they inhibited the culture growth and accumulation of IMP in the cultural medium. Only after 36-72 hours of cultivation the addition of the antibiotics stimulated the biosynthesis. All the antibiotics tested when adding at the definite for each of them period of fermentation and at the definite concentration stimulated the accumulation of IMP. The stimulating effect appears to be connected with an increase in permeability. A considerable increase in the number of anormalous elongated and swollen cells and, as a rule, in the protein content of the cultural supernatant indicates the fact. Streptomycin and kanamycin were the most efficient antibiotics, as they increased the IMP yield from 10.4 to 17.5 g/l.     

Purification and characterization of FAD synthetase from Brevibacterium ammoniagenes

The bifunctional enzyme FAD synthetase from Brevibacterium ammoniagenes was purified by a method involving ATP-affinity chromatography. The final preparation was more than 95% pure. The apparent molecular weight of the enzyme was determined as 38,000 and the isoelectric point as 4.6. Although previous attempts to separate the enzymatic activities had failed, ATP:riboflavin 5'-phosphotransferase and ATP:FMN-adenylyltransferase activities in B. ammoniagenes were believed to be located on two separate proteins with similar properties, possibly joined in a complex. The following evidence, however, suggests the presence of both activities on a single polypeptide chain. The two activities copurify in the same ratio through the purification scheme as presented. Only a single band could be detected when aliquots from the final purification step were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, nondenaturing gel electrophoresis, and isoelectric focusing. Edman degradation of the protein yielded a single N-terminal sequence.     

Identification and functional differentiation of two type I fatty acid synthases in Brevibacterium ammoniagenes.

The fatty acid synthase (FAS) from Brevibacterium ammoniagenes is a homohexameric multienzyme complex that catalyzes the synthesis of both saturated and unsaturated fatty acids. By immunological screening of a B. ammoniagenes expression library, an fas DNA fragment was isolated and subsequently used to clone the entire gene together with its flanking sequences. Within 10,525 bp of sequenced DNA, the 9,189-bp FAS coding region was identified, corresponding to a protein of 3,063 amino acids with a molecular mass of 324,910 Da. This gene (fasA) encodes, at its 5' end, the same amino acid sequence as is observed with purified B. ammoniagenes FAS. A second reading frame encoding another B. ammoniagenes FAS variant (FasB) had been identified previously. Both sequences are colinear and exhibit 61 and 47% identity at the DNA and protein levels, respectively. By using specific antibodies raised against a unique peptide sequence of FasB, this enzyme was shown to represent only 5 to 10% of the cellular FAS protein. Insertional inactivation of the FasB coding sequence causes no defective phenotype, while fasA disruptants require oleic acid for growth. Correspondingly, oleate-dependent B. ammoniagenes cells obtained by ethyl methanesulfonate mutagenesis were complemented by transformation with fasA DNA but not with fasB DNA. The data indicate that B. ammoniagenes contains two related though differently expressed type I FASs. FasA represents the bulk of cellular FAS protein and catalyzes the synthesis of both saturated and unsaturated fatty acids, while the minor variant, FasB, cannot catalyze the synthesis of oleic acid.     

Organic acid production by Basidiomycetes. 3. Cultural conditions for L-malic acid production

Cultural conditions were examined for the purpose of increasing yields of l-malic acid by the Basidiomycetes Schizophyllum commune and Merulius tremellosus, which have the ability to produce this acid as a main product in CaCO(3)-containing medium in shaken culture. The most favorable nitrogen sources selected were 0.3% (NH(4))(2)SO(4) and 0.18% NH(4)Cl. Effective combinations of inorganic salts in the medium were 0.1% KH(2)PO(4), 0.05% MgSO(4).7H(2)O, and 0.05% KCl, and suitable concentrations of glucose were 5 to 10%. Several nonionic surface-active agents promoted the filamentous mycelial growth of these strains and increased acid production. In particular, Tween 80 in 0.3% concentration markedly stimulated malic acid production by S. commune, and yields greater than 50% based on available glucose, were obtained after 10 to 14 days. Acid production by M. tremellosus was stimulated most with 0.5% Carbowax 4000 (polyethylene glycol), and the resultant yields were more than 40%.     

Acetic acid production by immobilized acetobacter cells

Summary Using a pulsed gas and liquid flow and with cells directly adsorbed on to a suitably-formed support, aerobic transformations can be carried out in a fixed-cell reactor with significant gain in efficiency. Immobilized cells of Acetobacter on cordierite can produce acetic acid at a high rate which, at different dilution rates, may be limited either by product inhibition or by oxygen transfer requirements.     

Stereochemical studies of hydrogen incorporation from nucleotides with fatty acid synthetase from Brevibacterium ammoniagenes.

The biosynthesis of fatty acids from malonyl-CoA and acetyl-CoA was investigated with an enzyme preparation which was purified 100-fold from Brevibacterium ammoniagenes. Fatty acids synthesized in the presence of D2O and stereospecifically deuterated NADPH and NADH were isolated and analyzed by mass chromatography to examine the localization of deuterium in the molecule. The following results were obtained: 1) HB hydrogen of NADPH was used for beta-ketoacyl reductase. 2) HB hydrogen of NADH was used for enoyl reductase. 3) Hydrogen atoms from water were found on the even-numbered methylene carbon atoms (2-hydrogen atoms per carbon atom) and some were also found on the odd-numbered methylene carbon atoms. 4) Hydrogen atoms from NADPH was found on the odd-numbered methylene carbon atoms (1 hydrogen per carbon). 5) Hydrogen atoms from NADH was also found on the odd-numbered methylene carbon atoms, but the number of incorporated hydrogen atoms was less than expected. The exchange of HB hydrogen of NADH with water catalyzed by enoyl reductase was suspected. 6) The exchange of methylene hydrogen atoms of malonyl-CoA with protons of water was suggested by 13C NMR analysis.     

Purification and Properties of Pantothenate Kinase from Brevibacterium ammoniagenes IFO 12071

Pantothenate kinase (ATP: pantothenate 4′-phosphotransferase, EC 2.7.1.33) was purified about 200-fold from the cell extract of Brevibacterium ammoniagenes IFO 12071 by ammonium sulfate fractionation, DEAE-cellulose chromatography, and Sephadex G-150 gel filtration. The purified enzyme gave a single band on polyacrylamide gel electrophoresis. The molecular weight was calculated approximately 45,000. The enzyme catalyzed the formation of pantothenic acid 4′-phosphate and ADP from pantothenate and ATP in the presence of Mg²⁺ ATP could be substituted for, partly, by ITP, GTP, and UTP. The enzyme phosphorylated not only pantothenate, but also pantothenoylcysteine, pantetheine, and pantothenyl alcohol. Apparent Km values were 6.7×10^?5 m for pantothenate, 3.5×10^?5 m for ATP, and 10^?3 m for Mg²⁺. The reaction was inhibited by the intermediates of CoA biosynthesis, of which CoA itself was a most effective inhibitor. Other properties of the enzyme were also investigated.     

Engineering analysis of continuous production of L-aspartic acid by immobilized Escherichia coli cells in fixed beds

The reaction mechanism and decay behavior of aspartase activity for immobilized Escherichia coli cells were investigated by using a sectional packed column. Reaction within the immobilized cell column proceeded at zero-order on substrate solutions ranging in concentration from 0.1 to 1.0M, and the initial reaction rate was found to be 1.556 × 10^?2 mol/min/liter of immobilized cells. The effect of temperature on the reaction rate constant was investigated. The Arrhenius plot was straight line at temperatures below 43°C, and the activation energy for immobilized cells was calculated to be 12.36 kcal/mol. Asparatase activity in the immobilized cell column decayed exponentially and uniformly in all sections of a column. Its half-life was approximately 120 days. The rate of formation of L-aspartic acid was shown to be independent of column dimensions.