Synthesis of l-dopa by escherichia intermedia cells immobilized in a polyacrylamide gel

Summary Escherichia intermedia cells were immobilized by entrapment in a polyacrylamide gel and used for l-dopa synthesis from pyrocatechol, pyruvate and ammonia. An immobilized cell preparation containing 75 mg cells/g gel retained 45%–50% of the activity of free cells. The effect of temperature, pH and substrate concentration of the initial rate of l-dopa synthesis was very similar for free and immobilized cells. Substrate inhibition was observed for pyrocatechol, pyruvate and ammonia. In a batch reactor, 5.4 g·l^-1 l-dopa was obtained, with 100% conversion yield of pyrocatechol and l-dopa productivity of 0.18 g·l^-1·h^-1. The use of a pyrocatechol-borate complex decreased by-product formation and catalyst inactivation.     

Molecular breeding of a Brevibacterium lactofermentum l-phenylalanine producer using a cloned prephenate dehydratase gene

Summary The prephenate dehydratase gene was cloned from a mutant of Brevibacterium lactofermentum, AJ11957 that produced enzyme free from feedback inhibition. The recombinant plasmids pPH11 and pPH14 complemented a phenylalanine auxotroph of B. lactofermentum, A-15, provided the transformant with the desensitized enzyme and caused an increased level of the enzyme compared to that of a wild strain. Plasmid pPH14 was introduced into l-phenylalanine producers genetically induced from B. lactofermentum; MF358 and FP-1 excreting l-tyrosine and anthranilate, respectively, as by-products. Both transformants predominantly accumulated l-phenylalanine at the expense of by-product formation. Co-existence of pPH14 and pTAR16, a recombinant plasmid expressing desensitized 3-deoxy-d-arabino-hepturosonate-7-phosphate synthase had a marked effect on further improvement in l-phenylalanine productivity, accompanied by an increase in the corresponding enzyme activity. The parent, MF358, accumulating 5.5 g/l l-phenylalanine, 6.8 g/l l-tyrosine and 0.3 g/l anthranilate turned into a potent l-phenylalanine producer producing 18.2 g/l l-phenylalanine and 1.0 g/l l-tyrosine by-product. Offprint requests to: Hisao Ito     

<Emphasis Type="SmallCaps">L</Emphasis>-Tyrosine production by deregulated strains of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The excretion of the aromatic amino acid l-tyrosine was achieved by manipulating three gene targets in the wild-type Escherichia coli K12: The feedback-inhibition-resistant (fbr) derivatives of aroG and tyrA were expressed on a low-copy-number vector, and the TyrR-mediated regulation of the aromatic amino acid biosynthesis was eliminated by deleting the tyrR gene. The generation of this l-tyrosine producer, strain T1, was based only on the deregulation of the aromatic amino acid biosynthesis pathway, but no structural genes in the genome were affected. A second tyrosine over-producing strain, E. coli T2, was generated considering the possible limitation of precursor substrates. To enhance the availability of the two precursor substrates phosphoenolpyruvate and erythrose-4-phosphate, the ppsA and the tktA genes were over-expressed in the strain T1 background, increasing l-tyrosine production by 80% in 50-ml batch cultures. Fed-batch fermentations revealed that l-tyrosine production was tightly correlated with cell growth, exhibiting the maximum productivity at the end of the exponential growth phase. The final l-tyrosine concentrations were 3.8 g/l for E. coli T1 and 9.7 g/l for E. coli T2 with a yield of l-tyrosine per glucose of 0.037 g/g (T1) and 0.102 g/g (T2), respectively.     

Biotransformation of benzeldehyde to l-phenylacetylcarbinol,an intermediate in l-ephedrine production,by immobilized Candida utilis

Biotransformation of benzaldehyde to l-phenylacetylcarbinol (l-PAC) as a key intermediate for l-ephedrine synthesis has been evaluated using immobilized Candida utilis. During biotransformation, the benzaldehyde level and respiratory quotient significantly affected both l-PAC and by-product benzyl alcohol formation. By controlling the benzaldehyde level at 2 g/l, maintaining a respiratory quotient of 5–7 and pulse feeding glucose, a final concentration of 15.2 g/l l-PAC was achieved in a fed-batch process. This compares with previous published results of 10–12 g/l in batch culture and 10 g/l l-PAC in a semicontinuous process with immobilized Saccharomyces cerevisiae. In a single stage continuous process with immobilized C. utilis, the steady state l-PAC concentration was significantly reduced because of the sustained toxic effects of benzaldehyde.     

Comparative study of d-xylose conversion to ethanol by immobilized growing or non-growing cells of the yeast Pachysolen tannophilus

Summary The direct conversion of d-xylose to ethanol was investigated using immobilized growing and non-growing cells of the yeast Pachysolen tannophilus. Both preparations produced ethanol from d-xylose, however the d-xylose conversion to ethanol was much better with immobilized growing cells. Ethanol concentration up to 22.9 g/l and ethanol yield of 0.351 g/g of d-xylose were obtained in batch fermentation by immobilized growing cells whereas only 17.0 g/l and 0.308 g/g of d-xylose were obtained by immobilized non-growing cells. With continuous systems, immobilized growing cells were necessary for the long-term operation, since a steady state ethanol concentration of 17.7 g/l was maintained for only one week by immobilized non-growing cell reactor. With simultaneous control of aeration rate and concentrations of nitrogen sources in feed medium, immobilized growing cells of P. tannophilus showed excellent performance. At a residence time of 25 h, the immobilized cell reactor produced 26.9 g/l of ethanol from 65 g/l of d-xylose in feed medium.     

Applicability of pectate-entrapped <Emphasis Type="Italic">Lactobacillus casei</Emphasis> cells for <Emphasis Type="SmallCaps">l</Emphasis>(+) lactic acid production from whey

Lactic acid is a versatile organic acid, which finds major application in the food, pharmaceuticals, and chemical industries. Microbial fermentation has the advantage that by choosing a strain of lactic acid bacteria producing only one of the isomers, an optically pure product can be obtained. The production of l(+) lactic acid is of significant importance from nutritional viewpoint and finds greater use in food industry. In view of economic significance of immobilization technology over the free-cell system, immobilized preparation of Lactobacillus casei was employed in the present investigation to produce l(+) lactic acid from whey medium. The process conditions for the immobilization of this bacterium using calcium pectate gel were optimized, and the developed cell system was found stable during whey fermentation to lactic acid. A high lactose conversion (94.37%) to lactic acid (32.95 g/l) was achieved with the developed immobilized system. The long-term viability of the pectate-entrapped bacterial cells was tested by reusing the immobilized bacterial biomass, and the entrapped bacterial cells showed no decrease in lactose conversion to lactic acid up to 16 batches, which proved its high stability and potential for commercial application.     

Strategies for the improvement of salidroside production in cell suspension cultures of Rhodiola sachalinensis

Strategies of elicitation and precursor feeding were applied to improve salidroside production in cell suspension cultures of Rhodiola sachalinensis. Of the seven elicitors examined, that extracted from Aspergillus niger was the most effective, increasing the salidroside content by five-fold when added on the day of inoculation 40 mg carbohydrate is medium. Three possible precursors for salidroside synthesis, l-phenylalanine, l-tyrosol and l-tyrosine were added to the cultures. A high content of salidroside (1.440%) was attained with an initial l-tyrosol concentration of 0.5 mm in the medium. Combined application of the two strategies resulted in a significantly high salidroside content of 1.734%, corresponding to a salidroside yield of 200 mg/l. Received: 5 November 1996 / Revision received: 17 February 1997 / Accepted 11 April 1997     

Inhibitory action of serine on growth of bacteria of the genusBacillus on mineral synthetic media

l-Serine added to minimal synthetic media stops the growth ofBacillus subtilis, B. megaterium, B. mycoides andB. pantothenticus. All tested subspecies ofBacillus thuringiensis appear resistant to this amino acid. Serine acts bacteriostatically onB. subtilis strain B 003 and this effect depends on the concentrations of both the amino acid and the plated bacterium. Growth of serine-sensitiveBacillus strains can be restored by simultaneous addition of some other amino acids (e.g. l-threonine,l-arginine,l-aspartate orl-alanine) to the minimal media. This alleviating effect depends on the kind of amino acid. Some amino acids (e.g. l-threonine,l-tyrosine orl-tryptophan) are only effective when the serine concentration is not higher than 125 μmol/L, others (l-arginine,l-proline,l-alanine,l-aspartate orl-glutamate) are effective even when the serine concentration is as high as 500 μmol/L.     

Identification and optimization of tyrosine hydroxylase activity in Mucuna pruriens DC. var. utilis

Tyrosine hydroxylase, an iron containing tetrahydrobiopterin dependent monooxygenase (tyrosine 3-monooxygenase; EC 1.14.16.2), catalyzes the rate-limiting step in which l-dopa is formed from the substrate l-tyrosine. l-Dopa concentration and activity of l-tyrosine hydroxylase enzyme were measured in roots, stem, leaves, pods, and immature seeds of Mucuna pruriens. Immature seeds contained maximum l-dopa content and mature leaves possessed maximum catalytic activity of tyrosine hydroxylase. Tyrosine hydroxylase from leaf homogenate was characterized as a 55 kDa protein by SDS-PAGE and Western-blot analysis with monoclonal mouse IgG2a tyrosine hydroxylase antibody. The conditions for maximum tyrosine hydroxylase activity from the leaf extract were optimized with respect to temperature, pH, cofactor 6-MPH₄, and divalent metal ions. The tyrosine hydroxylase from leaf extract possessed a K _m value of 808.63 μM for l-tyrosine at 37°C and pH 6.0. The activity of the enzyme was slightly inhibited at 2,000 μM l-tyrosine. Higher concentrations of the cofactor 6-MPH₄, however, completely inhibited the synthesis of l-dopa. Tyrosine hydroxylase converted specific monophenols such as l-tyrosine (808.63 μM) and tyramine (K _m 1.1 mM) to diphenols l-dopa and dopamine, respectively. Fe(II) activated the enzyme while higher concentration of other divalent metals reduced its activity. For the first time, tyrosine hydroxylase from M. pruriens is being reported in this study.     

Bioconversion of L-tyrosine to L-DOPA by a novel bacterium Bacillus sp. JPJ

l-DOPA is an amino acid derivative and most potent drug used against Parkinson’s disease, generally obtained from Mucuna pruriens seeds. In present communication, we have studied the in vitro production of l-DOPA from l-tyrosine by novel bacterium Bacillus sp. JPJ. This bacterium produced 99.4% of l-DOPA from l-tyrosine in buffer (pH 8) containing 1 mg ml⁻¹ cell mass incubated at 40°C for 60 min. The combination of CuSO₄ and l-ascorbic acid showed the inducing effect at concentrations of 0.06 and 0.04 mg ml⁻¹, respectively. The activated charcoal 2 mg ml⁻¹ was essential for maximum bioconversion of l-tyrosine to l-DOPA and the crude tyrosinase activity was 2.7 U mg⁻¹ of tyrosinase. Kinetic studies showed significant values of Y _p/s (0.994), Q _s (0.500) and q _s (0.994) after optimization of the process. The production of l-DOPA was confirmed by analytical techniques such as HPTLC, HPLC and GC–MS. This is the first report on rapid and efficient production of l-DOPA from l-tyrosine by bacterial source which is more effective than the plant, fungal and yeast systems.     

Cloning and expression of a tyrosinase from <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>: application in <Emphasis Type="SmallCaps">l</Emphasis>-DOPA biotransformation

Amplification of the tyrosinase gene (melO) from the genomic DNA of Aspergillus oryzae NCIM 1212 yielded a 1.6-kb product. This gene was cloned into pYLEX1, and the resulting pTyro-YLEX1 vector was transformed in Yarrowia lipolytica strain Po1g. A clone displaying the highest specific activity for tyrosinase (10.94 U/mg) was used for obtaining the complementary DNA (cDNA) and for protein expression studies. cDNA sequence analysis indicated the splicing of an intron present in the melO gene by Po1g. Native polyacrylamide gel electrophoresis, acidification at pH 3.0 followed by activity staining with l-DOPA indicated the expression of an active tyrosinase. The clone over-expressing the tyrosinase transformed l-tyrosine to l-DOPA. On optimization of conditions for the biotransformation (pH 4.0, temperature 60°C and with 3.5 mg of biomass), 0.4 mg/ml of l-DOPA was obtained.     

Hyper-production of l-trytophan via fermentation with crystallization

A stable and fast l-tryptophan producer, AGX1757, was isolated from Escherichia coli W3110 trpAE1 trpR tnaA, which carried pSC101-trpI15·14. Cells of AGX1757 did not lose the composite plasmid during fermentation. Whenever a fed-batch culture of AGX1757 attained an l-tryptophan concentration of about 30 g/l, indole began to appear in the broth. The emergence of indole was caused by inhibition of tryptophan synthase due to accumulated l-tryptophan. Hence, the production rate of l-tryptophan sharply decreased. A higher solubility of l-tryptophan in the supernatant of culture broth (about 32 g/l) than that in the initial medium (about 22 g/l) was attributed to some unknown interaction between l-tryptophan and certain macromolecular material(s) coming from the bacterial cells. An addition of non-ionic detergents into the supernatant was effective for decreasing the solubility of l-tryptophan, hence causing crystallization of l-tryptophan. Pluronic L-61 was supplied from outside to an extent of 0.5% in terms of wt% concentration at around 45 h of fermentation when the l-tryptophan accumulated reached about 25 g/l. This addition actually caused crystallization of l-tryptophan and, as a result, the inhibitory effect of tryptophan synthase by l-tryptophan accumulated in the broth could be alleviated. Thus far, further fermentation became possible. l-Tryptophan of more than 50 g/l was finally produced by feeding solutions of both glucose and anthranilic acid. Correspondence to: H. Tsunekawa     

Continuous production of l-serine by immobilized growing Corynebacterium glycinophilum cells

Summary Auxotrophic mutant cells of Corynebacterium glycinophilum with high l-serine production activity were immobilized by entrapment with various gel materials, such as synthetic prepolymers and natural polysaccharides. The entrapped cells were used for estimation of l-serine productivity in a medium supplemented with glycine as a precursor. Based on the above criteria, including cell growth in gels and cell leakage from gels, calcium alginate was the most suitable gel material. Continuous l-serine fermentation with calcium alginate-entrapped growing cells was successfully achieved in an air-bubbled reactor for at least 13 days.     

Isolation,identification and characterisation of a soil bacterium producing an enzyme with l-phenylalanine oxidase activity

A gram-positive, mesophilic bacterium which assimilated l-phenylalanine but which failed to utilise l-tyrosine was isolated from soil. The isolate, identified as a strain of Bacillus carotarum, converted l-phenylalanine to phenylpyruvate with the initial step catalysed by an inducible, intracellular enzyme which possessed l-phenylalanine oxidase activity. Phenylalanine oxidase has not been previously reported in Gram-positive bacteria, although there are a few examples of non-specific l-amino acid oxidases with activity towards l-phenylalanine. The isolate grew abundantly on complex media but failed to synthesise significant amounts of the enzyme in the absence of l-phenylalanine. The highest enzyme levels were achieved in a chemically defined minimal salts medium containing the amino acid at 10 g/l as the primary carbon and energy source.     

From scratch to value: engineering <Emphasis Type="Italic">Escherichia coli</Emphasis> wild type cells to the production of <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine and other fine chemicals derived from chorismate

Recombinant strains of Escherichia coli K-12 for the production of the three aromatic amino acids (l-phenylalanine, l-tryptophan, l-tyrosine) have been constructed. The largest demand is for l-phenylalanine (l-Phe), as it can be used as a building block for the low-calorie sweetener, aspartame. Besides l-Phe, an increasing number of shikimic acid pathway intermediates can be produced from appropriate E. coli mutants with blocks in this pathway. The last common intermediate, chorismate, in E. coli not only serves for production of aromatic amino acids but can also be used for high-titer production of non-aromatic compounds, e.g., cyclohexadiene-transdiols. In an approach to diversity-oriented metabolic engineering (metabolic grafting), platform strains with increased flux through the general aromatic pathway were created by suitable gene deletions, additions, or rearrangements. Examples for rational strain constructions for l-phenylalanine and chorismate derivatives are given with emphasis on genetic engineering. As a result, l-phenylalanine producers are available, which were derived through several defined steps from E. coli K-12 wild type. These mutant strains showed l-phenylalanine titers of up to 38 g/l of l-phenylalanine (and up to 45.5 g/l using in situ product recovery). Likewise, two cyclohexadiene-transdiols could be recovered.     

Immobilization of <Emphasis Type="Italic">Escherichia coli</Emphasis> cells with <Emphasis Type="Italic">cis</Emphasis>-epoxysuccinate hydrolase activity for <Emphasis Type="SmallCaps">d</Emphasis>(−)-tartaric acid production

Immobilization of cis-epoxysuccinate hydrolase-containing E. coli for d(−)-tartaric acid production was screened by various methods. The highest recovery of activity was obtained by entrapment in κ-carrageenan gel. 23.6 g biomass/l and 43.4 g κ-carrageenan/l were the best immobilization conditions optimized by response surface methodology with 83% yield (114 U/g). Cell autolysis was observed after immobilization. Immobilized cells showed high pH (5–10) stability, thermal (up to 65°C) stability, conversion rate (>99.5%), enantioselectivity (ee > 99.6%), and were less affected by metal ions and surfactants compared with free cells. Conversion rate for immobilized cells preserved 93% after 10 repeated batches (5% for free cells).     

Preparation of optically active β-hydroxy-α-amino acid by immobilized <Emphasis Type="Italic">Escherichia coli</Emphasis> cells with serine hydroxymethyl transferase activity

In this research, an improved method for preparation of optically pure β-hydroxy-α-amino acids, catalyzed by serine hydroxymethyl transferase with threonine aldolase activity, is reported. Using recombinant serine hydroxymethyl transferase (SHMT), an enzymatic resolution process was established. A series of new substrates, β-phenylserine, β-(nitrophenyl) serine and β-(methylsulfonylphenyl) serine were used in the resolution process catalyzed by immobilized Escherichia coli cells with SHMT activity. It was observed that the K _m for l-threonine was 28-fold higher than that for l-allo-threonine, suggesting that this enzyme can be classified as a low-specificity l-allo-threonine aldolase. The results also shows that SHMT activity with β-phenylserine as substrate was about 1.48-fold and 1.25-fold higher than that with β-(methylsulfonylphenyl) serine and β-(nitrophenyl) serine as substrate, respectively. Reaction conditions were optimized by using 200 mmol/l β-hydroxy-α-amino acid, and 0.1 g/ml of immobilized SHMT cells at pH 7.5 and 45°C. Under these conditions, the immobilized cells were continuously used 10 times, yielding an average conversion rate of 60.4%. Bead activity did not change significantly the first five times they were used, and the average conversion rate during the first five instances was 84.1%. The immobilized cells exhibited favourable operational stability.     

Enzymatic synthesis of l-β-chloroalanine using amino acid dehydrogenase

l--Chloroalanine is a useful intermediate for the synthesis of several l-amino acids. Conditions for synthesizing optically pure l--chloroalanine from 3-chloropyruvate using alanine dehydrogenase (AlaDH), leucine dehydrogenase and phenylalanine dehydrogenase with a regeneration of NADH by formate dehydrogenase (FDH) were investigated. The enzymatic reaction was carried out at neutral pH because of a chemical instability of 3-chloropyruvate on the alkaline side. Commercially available AlaDH from Bacillus stearothermophilus IFO 12550 showed the highest activity for the production of l--chloroalanine at pH 7.5. The K _m and V _max values for 3-chloropyruvate of AlaDH were calculated to be 300 units/mg and 62.5 mm, respectively. Although 3-chloropyruvate had no inhibitory effect on AlaDH, it acted as a non-competitive inhibitor with FDH. 3-Chloropyruvate was added into the reaction mixture in a stepwise manner to avoid the inhibition. l--Chloroalanine was produced with high chemical (>90%) and optical yields (100% enantiometric excess) and at a high concentration (43 g/l).     

The effect of various amino acids and drugs on thepara-andmeta-hydroxyphenylacetic acid concentrations in the mouse caudate nucleus

Injection ofl-p-tyrosine (800 mg/kg, 2 h) increased the mouse striatalpara-hydroxyphenylacetic acid (p-HPAA) concentrations. A smaller dose ofd,l-m-tyrosine (20 mg/kg, 2h) produced a larger increase in mouse striatalmeta-hydroxyphenylacetic acid (m-HPAA) concentrations. The administration ofl-phenylalanine to mice caused a slight increase in thep-HPAA concentrations in the corpus striatum after 2h while a larger dose ofl-phenylalanine (800 mg/kg) produced a greater increase. Eight hours followingl-phenylalanine injection,p-HPAA concentrations were still elevated. Withd-phenylalanine a significant increase was observed at eight hours after drug administration.Two drugs which reduce dopamine synthesis, -methyl-para-tyrosine and apomorphine, decreasedm-HPAA striatal concentrations without affectingp-HPAA concentrations. From these results, it is proposed that tyrosine hydroxylase activity determinesp-HPAA concentrations by regulatingp-tyrosine availability. This enzyme may also synthesizem-tyrosine which is subsequently decarboxylated to formm-tyramine and then oxidatively deaminated to formm-HPAA.