Technical communication: A simple and economically viable medium for the growth of Agrobacterium radiobacter for the production of d-amino acid

A simple and cost effective medium for the growth and d-amino acid production by Agrobacterium radiobacter from hydantoin is reported. The effectiveness of this medium is compared with other synthetic media.     

Effect of cell membrane of Agrobacterium radiobacter on enhancing D-amino acids production from racemic hydantoins

An interesting phenomenon was observed that the existence of the intact cell membrane can enhance the D-amino acids production from D,L-5-substituted hydantoins by reacting with the whole cells of Agrobacterium radiobacter. Two intracellular enzymes were involved in the reaction process. The first enzyme D-hydantoinase converted hydantoins to carbamoyl derivatives which were further converted to D-amino acids by D-amidohydrolase. The amount of D-amino acids produced from hydantoins by the intact cells were 1.8–2.4 fold higher than the toluene treated cells. In addition, by using the intact cells the amount of D-amino acids produced from hydantoins was about 10 fold higher than that produced directly from carbamoyl derivatives. The relatively lower permeability of cell membrane to the reaction intermediate carbamoyl derivatives was confirmed by a simple mathematical model to be the main factor for the better performance of the intact cells for D-amino acid production. Besides, the low intracellular enzymes activities also contributed to the effect of intact cell membrane on enhancing the D-amino acid production.     

The relationship between glucose transport and the production of succinoglucan exopolysaccharide by Agrobacterium radiobacter

Agrobacterium radiobacter NCIB 11883 was grown in ammonia-limited continuous culture at low dilution rate with glucose as the carbon source. Under these conditions the organism produced an extracellular succinoglucan polysaccharide and transported glucose using the same periplasmic glucose-binding proteins (GBP1 and GBP2) as during glucose-limited growth. Transition from glucose- to ammonia-limited growth was accompanied by a very rapid decrease in glucose uptake capacity, whereas the glucose-binding proteins were diluted out much more slowly (t1/2 approximately 1 h and 14 h respectively). Although the rate of glucose uptake and the concentrations of GBP1 and GBP2 were much lower during ammonia limitation, the activities of enzymes involved in the early stages of glucose metabolism and in the production of succinoglucan precursors were essentially unchanged. Glucose transport was also investigated in two new strains of A. radiobacter which had been isolated following prolonged growth under glucose limitation. Glucose uptake by strain AR18 was significantly less repressed during ammonia limitation compared with either the original parent strain or strain AR9, and this was reflected both in its relatively high concentration of GBP1 and in its significantly higher rate of succinoglucan synthesis. Flux control analysis using 6-chloro-6-deoxy-D-glucose as an inhibitor of glucose transport showed that the latter was a major kinetic control point for succinoglucan production. It is concluded that glucose uptake by A. radiobacter, particularly via the GBP1-dependent system, is only moderately repressed during ammonia-limited growth and that the organism avoids the potentially deleterious effects of accumulating excess glucose by converting the surplus into succinoglucan.     

Identification, sequencing and mutagenesis of the gene for a d-carbamoylase from Agrobacterium radiobacter

Abstract A clone positive for d-carbamoylase activity (2.7 kb Hin dIII- Bam H1 DNA fragment) was obtained by screening a genomic library of Agrobacterium radiobacter in Escherichia coli . This DNA fragment contains an open reading frame of 912 bp which is predicted to encode a peptide of 304 amino acids with a calculated molecular mass of 34247 Da. The d-carbamoylase gene. named cauA , was placed under the control of T7 RNA-dependent promoter and expressed in E. coli BL21 (DE3). After induction with isopropyl-thio-β-d-galactopyranoside, the synthesis of d-carbamoylase in E. coli reached about 40% of the total protein. The expressed protein was shown to possess a molecular mass, on SDS-PAGE, of 36 kDa and showed an enhanced allowed us to establish that a Pro¹⁴→Leu¹⁴ exchange leads to an inactive enzyme species, while a Cys²⁷⁹→Ser²⁷⁹ exchange did not impair the functional properties of the enxyme.     

Box-Behnken design in the development of optimized complex medium for phenol degradation using Pseudomonas putida (NICM 2174)

The biodegradation of phenol by Pseudomonas putida (NICM 2174), a potential biodegradent of phenol has been investigated for its degrading potential under different operating conditions. Box-Behnken design has been employed to study the effect of different experimental variables. Four variables of maltose (0.25, 0.5, 0.75?g/l), phosphate (3, 12.5, 22?g/l), pH (7, 8, 9) and temperature (30?°C, 32?°C, 34?°C) were used to identify the significant effects and interactions in the batch studies. A second order polynomial regression model, has been developed using the experimental data. It was found that the degrading potential of Pseudomonas putida (NICM 2174) was strongly affected by the variations in maltose, phosphate, pH and temperature. The experimental values were in good agreement with the predicted values, the correlation coefficient was found to be 0.9980. Optimum conditions of the variables for the growth of Pseudomonas putida (NICM2174) and for maximum biodegradation of phenol are maltose (0.052?g/l), phosphate (8.97?g/l), pH (7.9) and temperature (31.5?°C).     

Exploring the Enantioselective Mechanism of Halohydrin Dehalogenase from Agrobacterium radiobacter AD1 by Iterative Saturation Mutagenesis

Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) shows great potential in producing valuable chiral epoxides and β-substituted alcohols. The wild-type (WT) enzyme displays a high R-enantiopreference toward most aromatic substrates, whereas no S-selective HheC has been reported to date. To obtain more enantioselective enzymes, seven noncatalytic active-site residues were subjected to iterative saturation mutagenesis (ISM). After two rounds of screening aspects of both activity and enantioselectivity (E), three outstanding mutants (Thr134Val/Leu142Met, Leu142Phe/Asn176His, and Pro84Val/Phe86Pro/Thr134Ala/Asn176Ala mutants) with divergent enantioselectivity were obtained. The two double mutants displayed approximately 2-fold improvement in R-enantioselectivity toward 2-chloro-1-phenylethanol (2-CPE) without a significant loss of enzyme activity compared with the WT enzyme. Strikingly, the Pro84Val/Phe86Pro/Thr134Ala/Asn176Ala mutant showed an inverted enantioselectivity (from an E_R of 65 [WT] to an E_S of 101) and approximately 100-fold-enhanced catalytic efficiency toward (S)-2-CPE. Molecular dynamic simulation and docking analysis revealed that the phenyl side chain of (S)-2-CPE bound at a different location than that of its R-counterpart; those mutations generated extra connections for the binding of the favored enantiomer, while the eliminated connections reduced binding of the nonfavored enantiomer, all of which could contribute to the observed inverted enantiopreference.     

Tyrosine residues serve as proton donor in the catalytic mechanism of epoxide hydrolase from Agrobacterium radiobacter

Epoxide hydrolase from Agrobacterium radiobacter catalyzes the hydrolysis of epoxides to their diols via an alkyl-enzyme intermediate. The recently solved X-ray structure of the enzyme shows that two tyrosine residues (Tyr152 and Tyr215) are positioned close to the nucleophile Asp107 in such a way that they can serve as proton donor in the alkylation reaction step. The role of these tyrosines, which are conserved in other epoxide hydrolases, was studied by site-directed mutagenesis. Mutation of Tyr215 to Phe and Ala and mutation of Tyr152 to Phe resulted in mutant enzymes of which the k(cat) values were only 2-4-fold lower than for wild-type enzyme, whereas the K(m) values for the (R)-enantiomers of styrene oxide and p-nitrostyrene oxide were 3 orders of magnitude higher than the K(m) values of wild-type enzyme, showing that the alkylation half-reaction is severely affected by the mutations. Pre-steady-state analysis of the conversion of (R)-styrene oxide by the Y215F and Y215A mutants showed that the 1000-fold elevated K(m) values were mainly caused by a 15-40-fold increase in K(S) and a 20-fold reduction in the rate of alkylation. The rates of hydrolysis of the alkyl-enzyme intermediates were not significantly affected by the mutations. The double mutant Y152F+Y215F showed only a low residual activity for (R)-styrene oxide, with a k(cat)/K(m) value that was 6 orders of magnitude lower than with wild-type enzyme and 3 orders of magnitude lower than with the single tyrosine mutants. This indicates that the effects of the mutations were cumulative. The side chain of Gln134 is positioned in the active site of the X-ray structure of epoxide hydrolase. Mutation of Gln134 to Ala resulted in an active enzyme with slightly altered steady-state kinetic parameters compared to wild-type enzyme, indicating that Gln134 is not essential for catalysis and that the side chain of Gln134 mimics bound substrate. Based upon this observation, the inhibitory potential of various unsubstituted amides was tested, resulting in the identification of phenylacetamide as a competitive inhibitor with an inhibition constant of 30 microM.     

Characterization of 5-aminolevulinate synthase from Agrobacterium radiobacter,screening new inhibitors for 5-aminolevulinate dehydratase from Escherichia coli and their potential use for high 5-aminolevulinate production

The hemA gene encoding 5-aminolevulinate synthase (ALAS) from Agrobacterium radiobacter zju-0121 showed 92.6% homology with that from A. radiobacter ATCC4718 and contained several rare codons. To enhance the expression of this gene, Escherichia coli Rosetta(DE3), which is a rare codon optimizer strain, was used as the host to construct an efficient recombinant strain. And the encoded protein was over-expressed as fusion protein and was purified by affinity purification on Ni-NTA agarose and by gel filtration chromatography on Sephadex G-25 Medium resin. The recombinant protein was partly characterized, and d-glucose, d-fructose, d-xylose, d-mannose, l-arabinose, d-galactose, lactose, sucrose and maltose were detected to have no distinct inhibition on this recombinant ALAS. Meanwhile, 20 mM d-glucose or d-xylose inhibited about 20% activity of ALA dehydratase (ALAD) from Escherichia coli Rosetta(DE3). Combining d-xylose as a new inhibitor for ALAD with d-glucose in fed-batch culture and based on the optimal culture system using Rosetta(DE3)/pET28a-hemA, the yield of ALA achieved was 7.3 g/l (56 mM) under the appropriate conditions in the fermenter.     

Application of agro-industrial by-products for riboflavin production by Eremothecium ashbyii NRRL 1363

Riboflavin production is significantly determined by the type and initial concentration of the carbon and nitrogen sources and also by other flavinogenic stimulants. Using an optimum carbon and nitrogen concentration, an industrial fermentation medium has been designed with molasses as the carbon source and peanut seed cake as the nitrogen source. In addition the stimulatory effect of some of the low-cost agro-industrial by-products on riboflavin yield was investigated. Received: 10 March 1996 / Received revision: 25 June 1996 / Accepted: 14 July 1996     

Protein engineering of epoxide hydrolase from Agrobacterium radiobacter AD1 for enhanced activity and enantioselective production of (R)-1-phenylethane-1,2-diol

DNA shuffling and saturation mutagenesis of positions F108, L190, I219, D235, and C248 were used to generate variants of the epoxide hydrolase of Agrobacterium radiobacter AD1 (EchA) with enhanced enantioselectivity and activity for styrene oxide and enhanced activity for 1,2-epoxyhexane and epoxypropane. EchA variant I219F has more than fivefold-enhanced enantioselectivity toward racemic styrene oxide, with the enantiomeric ratio value (E value) for the production of (R)-1-phenylethane-1,2-diol increased from 17 for the wild-type enzyme to 91, as well as twofold-improved activity for the production of (R)-1-phenylethane-1,2-diol (1.96 +/- 0.09 versus 1.04 +/- 0.07 micromol/min/mg for wild-type EchA). Computer modeling indicated that this mutation significantly alters (R)-styrene oxide binding in the active site. Another three variants from EchA active-site engineering, F108L/C248I, I219L/C248I, and F108L/I219L/C248I, also exhibited improved enantioselectivity toward racemic styrene oxide in favor of production of the corresponding diol in the (R) configuration (twofold enhancement in their E values). Variant F108L/I219L/C248I also demonstrated 10-fold- and 2-fold-increased activity on 5 mM epoxypropane (24 +/- 2 versus 2.4 +/- 0.3 micromol/min/mg for the wild-type enzyme) and 5 mM 1,2-epoxyhexane (5.2 +/- 0.5 versus 2.6 +/- 0.0 micromol/min/mg for the wild-type enzyme). Both variants L190F (isolated from a DNA shuffling library) and L190Y (created from subsequent saturation mutagenesis) showed significantly enhanced activity for racemic styrene oxide hydrolysis, with 4.8-fold (8.6 +/- 0.3 versus 1.8 +/- 0.2 micromol/min/mg for the wild-type enzyme) and 2.7-fold (4.8 +/- 0.8 versus 1.8 +/- 0.2 micromol/min/mg for the wild-type enzyme) improvements, respectively. L190Y also hydrolyzed 1,2-epoxyhexane 2.5 times faster than the wild-type enzyme.     

The consequence of stimulating glucose dehydrogenase activity by the addition of PQQ on metabolite production by Agrobacterium radiobacter NCIB 11883

Summary Agrobacterium radiobacter NCIB 11 883 does not produce gluconate under conditions of glucose excess in batch or continuous culture. However, the addition of micromolar concentrations of pyrrolo quinoline quinone (PQQ) to fermentation media resulted in rapid excretion of gluconate by batch and continuous cultures. This rapid dehydrogenation of glucose was found in cells grown under carbon and nitrogen limitation and is constitutive which suggests that the only reason why this activity is not normally expressed is due to the inability of the organism to synthesize the prosthetic group (PQQ) of the glucose dehydrogenase enzyme.Although the addition of PQQ to batch and continuous cultures caused a very rapid specific rate of gluconate production (0.6–1.1 g gluconate g^-1 dry wt. h^-1) the rate of exopolysaccharide production remained unaltered. Indeed, when the rates of substrate and oxygen uptake are corrected for the rate of gluconate production in the presence of PQQ there appears to be little physiological consequence as a result of this oxidation.     

Characterization of the genes for two protocatechuate 3, 4-dioxygenases from the 4-sulfocatechol-degrading bacterium Agrobacterium radiobacter strain S2

The genes for two different protocatechuate 3,4-dioxygenases (P34Os) were cloned from the 4-sulfocatechol-degrading bacterium Agrobacterium radiobacter strain S2 (DSMZ 5681). The pcaH1G1 genes encoded a P34O (P34O-I) which oxidized protocatechuate but not 4-sulfocatechol. These genes were part of a protocatechuate-degradative operon which strongly resembled the isofunctional operon from the protocatechuate-degrading strain Agrobacterium tumefaciens A348 described previously by D. Parke (FEMS Microbiol. Lett. 146:3-12, 1997). The second P34O (P34O-II), encoded by the pcaH2G2 genes, was functionally expressed and shown to convert protocatechuate and 4-sulfocatechol. A comparison of the deduced amino acid sequences of PcaH-I and PcaH-II, and of PcaG-I and PcaG-II, with each other and with the corresponding sequences from the P34Os, from other bacterial genera suggested that the genes for the P34O-II were obtained by strain S2 by lateral gene transfer. The genes encoding the P34O-II were found in a putative operon together with two genes which, according to sequence alignments, encoded transport proteins. Further downstream from this putative operon, two open reading frames which code for a putative regulator protein of the IclR family and a putative 3-carboxymuconate cycloisomerase were identified.     

Conditions for the production of Agrocin 84 byAgrobacterium radiobacter K84

Summary A chemically defined medium was developed that supported the growth ofAgrobacterium radiobacter K84 and the production of agrocin 84. Various supplements were investigated for their effect on growth rate and production of agrocin 84 using a well-diffusion assay method. Mannitol was found to be a better substrate for growth ofA. radiobacter K84 compared to the other sugar alcohols and sugars tested. By contrast,d-fructose was the better substrate for the production of agrocin 84. Biotin supplementation stimulated production of agrocin 84 but did not eliminate the diauxic lag seen with the basal medium. The opine, octopine, inhibited growth ofA. radiobacter K84 and production of agrocin 84 as did coenzyme B₁₂. By contrast, the cytokinin, isopentenyl adenosine, was marginally stimulatory to production, as was vitamin B₁₂. Acetosyringone supplementation had a negligible effect on growth rate and production of agrocin 84.     

Exploring the thermostable properties of halohydrin dehalogenase from Agrobacterium radiobacter AD1 by a combinatorial directed evolution strategy

As a crucial factor for biocatalysts, protein thermostability often arises from a combination of factors that are often difficult to rationalize. In this work, the thermostable nature of halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) was systematically explored using a combinatorial directed evolution approach. For this, a mutagenesis library of HheC mutants was first constructed using error-prone PCR with low mutagenesis frequency. After screening approximately 2000 colonies, six mutants with eight mutation sites were obtained. Those mutation sites were subsequently combined by adopting several rounds of iterative saturation mutagenesis (ISM) approach. After four rounds of saturation mutagenesis, one best mutant ISM-4 with a 3400-fold improvement in half-life (t _1/2) inactivation at 65 °C, 18 °C increase in apparent T _m value, and 20 °C increase in optimum temperature was obtained, compared to wild-type HheC. To the best of our knowledge, the mutant represents the most thermostable HheC variant reported up to now. Moreover, the mutant was as active as wild-type enzyme for the substrate 1,3-dichloro-2-propanol, and they remained most enantioselectivity of wild-type enzyme in the kinetic resolution of rac-2-chloro-1-phenolethanol, exhibiting a great potential for industrial applications. Our structural investigation highlights that surface loop regions are hot spots for modulating the thermostability of HheC, the residues located at these regions contribute to the thermostability of HheC in a cooperative way, and protein rigidity and oligomeric interface connections contribute to the thermostability of HheC. All of these essential factors could be used for further design of an even more thermostable HheC, which, in turn, could greatly facilitate the application of the enzyme as a biocatalyst.

     

Immobilization of organophosphohydrolase OpdA from Agrobacterium radiobacter by overproduction at the surface of polyester inclusions inside engineered Escherichia coli

Organophosphorus pesticides (OP) are highly toxic and are widely used as insecticides. Bacterial organophosphohydrolases which hydrolyze a variety of OPs have been considered for the clean-up of polluted environments. This study describes the engineering of Escherichia coli towards the overproduction of the organophosphohydrolase (OpdA) from Agrobacterium radiobacter at the surface of polyester inclusions. The OpdA was N-terminally fused via a designed linker region to the C-terminus of polyester inclusion-forming enzyme PhaC of Ralstonia eutropha. The PhaC-L-OpdA fusion protein was overproduced by using the strong T7 promoter and when coexpressed with genes phaA (encoding β-ketothiolase) and phaB (encoding acetoacetyl-CoA reductase) from R. eutropha this led to formation of polyester inclusions abundantly displaying OpdA. These OpdA beads showed organophosphohydrolase activity of 1,840 U/g wet polyester beads or 4,412 U/g protein. Steady state kinetics revealed that when compared with free OpdA the k(cat) (s(-1)) of 139 of immobilized OpdA was reduced by about 16.5-fold while the K(M) (M) of 2.5 × 10(-4) was increased by 1.6-fold. The immobilized OpdA showed increased temperature stability. Moreover, the stability of OpdA immobilized to polyester beads was assessed by incubating OpdA beads at 25°C for up to 11 days and no significant loss in enzyme activity was detected. The application performance of the OpdA beads with respect to hydrolysis of OPs in contaminated environments was demonstrated in wool scour spiked with fluorescent coumaphos. This study demonstrated a new strategy toward the efficient recombinant production of immobilized organophosphohydrolase, the OpdA, suitable for bioremediation applications.     

Development of a chemically defined medium for the production of enterolysin A from Enterococcus faecalis B9510

Aim

This study aimed to develop a simplified chemically defined medium that could sustain the growth and bacteriocin (enterolysin A) production by Enterococcus faecalis B9510.

Methods and Results

The nutritional requirements of E. faecalis B9510 in a chemically defined medium were determined by single omission experiments. It was observed that eight amino acids (arginine, glycine, histidine, isoleucine, leucine, methionine, tryptophan and valine), three B vitamins (nicotinic acid, Ca‐pantothenic acid and pyridoxal) and magnesium sulphate were essential for growth. Based on this information, a Simplified Defined Medium (SDM) was formed consisting of 26 components. Comparison of SDM with M‐17 showed that growth and bacteriocin production in SDM was similar to that in M‐17. The bacteriocin from SDM was then purified by ultrafiltration. The retentate of ultrafiltration step was analysed by SDS‐PAGE and the results showed a single active band in the gel, which was excised and analysed by mass spectrometry, which indicated that the active band was enterolysin A, a cell wall degrading bacteriocin.

Conclusions

A simplified defined medium can be formulated for the growth and bacteriocin production by Enterococcus faecalis, whose efficiency is comparable with that of a complex commercial medium.

Significance and Impact of the Study

The development of such a medium can be useful for bacteriocin production and subsequent purification in a simplified manner and, therefore, helpful in the identification of novel bacteriocins.     

Response surface optimization of medium components for citric acid production by Aspergillus niger NRRL 567 grown in peat moss

Aspergillus niger NRRL 567 was grown in an inert support material for citric acid production. Optimization of the medium components, including ethanol, methanol, phytate, olive oil and surfactant was carried out using "one-factor-at-a-time" and central composite design (CCD) methods. Optimization using "one-factor-at-a-time" was performed and the supplement of ethanol and methanol between 15 and 30 g/kg dry peat moss (DPM) enhanced citric acid production while higher levels than 30 g/kg DPM had an inhibitory effect on citric acid production at 48 and 72 h of incubation. Based on the results of "one-factor-at-a-time" optimization, phytate, olive oil and methanol were the selected additives to test the effect on citric acid production using CCD. The three variables were identified to have significant effects on citric acid production and the maximum citric acid production of 354.8 g/kg DPM was resulted from the combination of 19 g phytate/kg DPM, 49 g olive oil/kg DPM and 37 g methanol/kg DPM at 120 h. Maximum citric acid production in optimized condition by CCD represented about a 2.7-fold increase compared to that obtained from control before optimization.     

Structure and function of the 3-carboxy-cis,cis-muconate lactonizing enzyme from the protocatechuate degradative pathway of Agrobacterium radiobacter S2

3-carboxy-cis,cis-muconate lactonizing enzymes participate in the protocatechuate branch of the 3-oxoadipate pathway of various aerobic bacteria. The gene encoding a 3-carboxy-cis,cis-muconate lactonizing enzyme (pcaB1S2) was cloned from a gene cluster involved in protocatechuate degradation by Agrobacterium radiobacter strain S2. This gene encoded for a 3-carboxy-cis,cis-muconate lactonizing enzyme of 353 amino acids - significantly smaller than all previously studied 3-carboxy-cis,cis-muconate lactonizing enzymes. This enzyme, ArCMLE1, was produced in Escherichia coli and shown to convert not only 3-carboxy-cis,cis-muconate but also 3-sulfomuconate. ArCMLE1 was purified as a His-tagged enzyme variant, and the basic catalytic constants for the conversion of 3-carboxy-cis,cis-muconate and 3-sulfomuconate were determined. In contrast, Agrobacterium tumefaciens 3-carboxy-cis,cis-muconate lactonizing enzyme 1 could not, despite 87% sequence identity to ArCMLE1, use 3-sulfomuconate as substrate. The crystal structure of ArCMLE1 was determined at 2.2 A resolution. Consistent with the sequence, it showed that the C-terminal domain, present in all other members of the fumarase II family, is missing in ArCMLE1. Nonetheless, both the tertiary and quaternary structures, and the structure of the active site, are similar to those of Pseudomonas putida 3-carboxy-cis,cis-muconate lactonizing enzyme. One principal difference is that ArCMLE1 contains an Arg, as opposed to a Trp, in the active site. This indicates that activation of the carboxylic nucleophile by a hydrophobic environment is not required for lactonization, unlike earlier proposals [Yang J, Wang Y, Woolridge EM, Arora V, Petsko GA, Kozarich JW & Ringe D (2004) Biochemistry43, 10424-10434]. We identified citrate and isocitrate as noncompetitive inhibitors of ArCMLE1, and found a potential binding pocket for them on the enzyme outside the active site.     

Amino acids and preimplantation development of the mouse in protein-free potassium simplex optimized medium

Development of outbred CF1 mouse zygotes in vitro was studied in a chemically defined, protein-free medium both with and without amino acids. The addition of amino acids to protein-free potassium simplex optimized medium (KSOM) had little effect on the proportion of embryos that developed at least to the zona-enclosed blastocyst stage. In contrast, amino acids stimulated very significantly, in a dilution-dependent way, the proportion of blastocysts that at least partially or completely hatched. Amino acids also stimulated cell proliferation in both the trophectoderm and inner cell mass (ICM) cells, at rates that favored proliferation of cells in the ICM; had no effect on the incidence of cell death (oncosis or apoptosis); and improved development of the basement membranes, which form on the blastocoelic surface of the trophectoderm and between the primitive endoderm and the primitive ectoderm. Thus, KSOM, supplemented with amino acids but containing no protein supplements, supports development of a newly fertilized ovum to the late blastocyst stage, in which its normal, three-dimensional structure is preserved and in which the ICM has been partitioned into the primitive ectoderm and primitive endoderm.     

Enantioconvergent production of (R)-1-phenyl-1,2-ethanediol from styrene oxide by combining the Solanum tuberosum and an evolved Agrobacterium radiobacter AD1 epoxide hydrolases

Soluble epoxide hydrolase (EH) from the potato Solanum tuberosum and an evolved EH of the bacterium Agrobacterium radiobacter AD1, EchA-I219F, were purified for the enantioconvergent hydrolysis of racemic styrene oxide into the single product (R)-1-phenyl-1,2-ethanediol, which is an important intermediate for pharmaceuticals. EchA-I219F has enhanced enantioselectivity (enantiomeric ratio of 91 based on products) for converting (R)-styrene oxide to (R)-1-phenyl-1,2-ethanediol (2.0 +/- 0.2 micromol/min/mg), and the potato EH converts (S)-styrene oxide primarily to the same enantiomer, (R)-1-phenyl-1,2-ethanediol (22 +/- 1 micromol/min/mg), with an enantiomeric ratio of 40 +/- 17 (based on substrates). By mixing these two purified enzymes, inexpensive racemic styrene oxide (5 mM) was converted at 100% yield to 98% enantiomeric excess (R)-1-phenyl-1,2-ethanediol at 4.7 +/- 0.7 micromol/min/mg. Hence, at least 99% of substrate is converted into a single stereospecific product at a rapid rate.