Oreste Ghisalba

Synthesis of both enantiomeric forms of 2-substituted 1,3-propanediol monoacetates starting from a common prochiral precursor, using enzymatic transformations in aqueous and in organic media

Abstract A direct entry to both enantiomeric forms c and ent-c based on enzyme catalyzed transformations of prochiral compounds of type a and b is described. The catalysts used are carboxyl esterase preparations obtained from crude porcine pancreas lipase.

Biodegradation of chemical waste by specialized methylotrophs, an alternative to physical methods of waste disposal

Abstract Microbial waste treatment systems were established for the disposal of chemical wastes by using specialized methylotrophs in pure, mixed or enriched cultures. Three typical mother liquors originating from the production of large-scale chemicals were selected as model wastes: a monomethyl sulfate containing mother liquor from a methylation process, a trimethylethylammonium chloride containing mother liquor from a dealkylation process and a N,N-dimethyl-formamide containing mother liquor. The microorganisms enriched and isolated for the biodegradation (mineralization) of the three model wastes were characterized and found to be bacteria of the genera Pseudomonas (trimethylethylammonium chloride utilizers and N,N-dimethylformamide utilizers) and Hyphomicrobium (monomethyl sulfate utilizers). Degradation pathways for monomethyl sulfate, trimethylethylammonium chloride and N,N-dimethylformamide are proposed. Some of the postulated novel enzymes were detected in cell-free extracts. The substrate spectra of our new species of specialized methylotrophs were investigated in order to evaluate their potential use for the biodegradation of selected organic solvents (Cl- and C2-compounds) currently used in synthesis processes (acetamide, acetonitrile, ethanol etc.). Pilot fermentations with pure substrates and model wastes are described and technical aspects and requirements of specialized microbial waste treatment systems are discussed.

A genetic approach to the biosynthesis of the rifamycin-chromophore in Nocardia mediterranei. III. Isolation and identification of an early aromatic ansamycin-precursor containing the seven-carbon amino starter-unit and three initial acetate/propionate-units of the ansa chain.

A number of rifamycin non-producing UV-mutants derived from Nocardia mediterranei strains N813 (rifamycin B producer) and A10 (aro--mutant excreting shikimate derived from strain N813) were found to accumulate an identical complex of aromatic components instead of rifamycin B. The main component of this aromatic complex, product P8/1-OG, was isolated from six of these P--mutant strains and identified spectroscopically as a very early precursor in the biosynthesis of rifamycins.

Optimization of a process for the production of (R)-2-hydroxy-4-phenylbutyric acid — an intermediate for inhibitors of angiotensin converting enzyme

(R)-2-Hydroxy-4-phenylbutyric acid, an intermediate in the manufacture of inhibitors of angiotensin converting enzyme, can be produced continuously in an enzyme membrane reactor by enzymatic reduction of its corresponding alpha-keto acid. D-Lactate dehydrogenase (D-LDH) from Staphylococcus epidermidis was chosen as the most appropriate enzyme to carry out the NADH-dependent reduction. Formate dehydrogenase (FDH) was used for NADH regeneration. Detailed kinetic measurements and a mathematical model for the coupled enzyme reactions were applied to calculate the optimal conditions for continuous production of the alpha-hydroxy acid. A mass of 1 kg [corrected] (R)-2-hydroxy-4-phenylbutyric acid was synthesized in a 220 ml enzyme membrane reactor over a period of 4 weeks. A mean space-time-yield of 165 g l-1 d-1 was achieved at low enzyme consumptions of 150 U kg-1 alpha-hydroxy acid for FDH and D-LDH.

CMP-N-acetyl neuraminic-acid synthetase from Escherichia coli: fermentative production and application for the preparative synthesis of CMP-neuraminic acid

In an optimized sorbitol/yeast extract/mineral salt medium up to 12 U/l CMP-N-acetyl-neuraminic-acid (Neu5Ac) synthetase was produced by Escherichia coli K-235 in shake-flask culture. A colony mutant of this strain, E. coli K-235/CS1, was isolated with improved enzyme formation: in shake flasks with a yield of up to 20.8 U/l and 54 mU/mg protein in the cell extract. With this strain 26500 U CMP-Neu5Ac synthetase was produced with a high specific activity (0.128 U/mg) by fed-batch fermentation on 230-1 scale. On a 10-l scale the enzyme yield was 191 U/l culture medium. The enzyme was partially purified by precipitation with polyethyleneglycol resulting in a three- to fourfold enrichment and a recovery rate of more than 80%; most of the CTP hydrolysing enzymes were removed. The native synthetase was deactivated completely by incubation at 45°C for 10 min, but could be stabilized remarkably by glycerol and different salts. The enzyme was used for the preparative synthesis of CMP-Neu5Ac with a conversion yield of 87% based on CTP.

A GENETIC APPROACH TO THE BIOSYNTHESIS OF THE RIFAMYCINCHROMOPHORE IN NOCARDIA MEDITERRANEI

The mutant under study, designated A8, is derived from a Nocardia mediterranei strain, N813, which is a high rifamycin B producer. A8 is auxotrophic for aromatic amino acids and produces much less rifamycin B than the parent. A mixture of pentoses with D (--) ribulose as the main product is accumulated in the fermentation broth of this mutant. It was shown to be affected in its transketolase activity as no formation of D-sedoheptulose -7P from pentose-phosphates could be detected in vitro using crude extracts. The only pathway so far known which is derived from D-sedoheptulose-7P is the shikimate pathway leading to aromatic amino acids and vitamins. Biochemical and genetic investigations with mutant A8, which is defective in both the biosynthesis of rifamycins and the biosynthesis of shikimate pathway products, show that the seven-carbon amino unit of the rifamycin-chromophore must be derived from an intermediate of the shikimate pathway.

Chemo-enzymatic approaches for the creation of novel chiral building blocks and reagents for pharmaceutical applications

This paper describes three recent examples of innovative biocatalyst applications and technology developments from our laboratory giving access to useful chiral building blocks (examples 1 and 2) and chiral reagents (example 3). 1. Preparative to larger scale synthesis of enantiomerically pure chiral arylalkylamines using whole cell biotransformations with microorganisms containing novel enantioselective amidohydrolases. 2. Preparative to larger scale chemo-enzymatic synthesis of d- and l-tert-leucines using the very cheap commercial bulk enzyme Alcalase. 3. Preparative scale chemo-enzymatic synthesis of rare and expensive inositol phosphates, including 5′-TAMRA-labeled (P1-tethered) d-PIP3, using commercial lipase preparations.

Biodegradation and utilization of quaternary alkylammonium compounds by specialized methylotrophs

Dealkylations in organic syntheses are often carried out with trimethylamine (TMA) as the alkylacceptor. In such reactions an ethyl group is transferred from an alkyldonor (e.g. a substituted diethyl phosphate or diethyl thiophosphate) to TMA and a trimethylethylammonium salt (TMEA) is formed in stoichiometric quantities. The production of large-scale chemicals such as agrochemicals or dyestuffs involving deethylations (dealkylations) with trimethylamine thus yields large volumes of mother liquors containing trimethylethylammonium salt (or trimethylalkylammonium salt). The regeneration of TMA from TMEA is unfortunately rather difficult to achieve. Alkaline hydrolysis of TMEA at elevated temperature yields TMA as the main product but also other methylated amines and by-products (stoichiometry rather unclear). The purification of TMA by distillation of such a hydrolysate is very expensive (much energy is needed for cooling). One of the methods best suited for its disposal is the incineration of mother liquors containing TMEA. Since biodegradation of trimethylethylammonium salts would be an alternative to physical or chemical methods of disposal we searched for TMEA degrading microorganisms. Trimethylethylammonium salts are (at least partially) Cl-compounds. We therefore decided to investigate and isolate methylotrophs (Cl-utilizing microorganisms). The various Cl-compounds utilized according to the literature by specialized methylotrophs as the sole source of carbon and energy have been mentioned in the preceding publication 4. For the degradation of tetramethylammonium chloride, a homologue of TMEA, only two microorganisms have been described so far: Bacterium 5H25 and Bacillus PM62. However, many of the methylotrophs reported to utilize methylamine, dimethylamine and/ or trimethylamine have never been tested for their ability to grow on tetramethylammonium salts. Mackrell and Walker 6 were able to enrich cultures of microorganisms growing on 10 mM tetramethylammonium chloride as the sole carbon source. These microorganisms could be adapted to grow on 10 mM trimethylethylammonium chloride. While no attempts were made to isolate and characterize the TMEAdegrading organisms from these enrichment cultures, it was suggested that pseudomonads might be involved. Concentrations higher than 10 mM tetramethylammonium chloride or TMEA were not tested (10 mM TMEA-chloride _-__ 1.23 g/l). Evaluation of methylotrophs from culture collections

Chemo-enzymatic Synthesis of both Enantiomers of myo-Inositol 1,3,4,5-tetrakisphosphate.

D-Ins(1,3,4,5)P4 and unnatural L-Ins(1,3,4,5)P4 were prepared in gram-quantities from D- and L-2,6-di-O-benzyl-myo-inositol by a chemical phosphorylation and deprotection step in high yield and purity without extensive purification. The optically pure benzyl derivatives were obtained by enzyme-catalyzed resolution of racemic 2,6-di-O-benzyl-myo-inositol under acyl-transfer conditions in vinyl acetate as the acyl donor. The lipase of Candida antarctica only acetylated regio- and enantio-selectively the L-enantiomer, providing exclusively L-5-acetyl-2,6-di-O-benzyl-myo-inositol, whereas the D-enantiomer remained unchanged.

Recombinant Lactobacillus leichmannii ribonucleosidetriphosphate reductase as biocatalyst in the preparative synthesis of 2′-deoxyribonucleoside-5′-triphosphates

Abstract Recombinant Lactobacillus leichmannii ribonucleosidetriphosphate reductase has been purified and evaluated as a biocatalyst for the preparative synthesis of 2′-deoxyribonucleoside-5′-triphosphates. The addition of expensive 2′-deoxyribonucleoside-5′-triphosphates as allosteric effectors of ribonucleosidetriphosphate reductase was not necessary due to high concentrations of inorganic salts in the reaction mixture. Good conversion of the tested ribonucleoside-5′-triphosphate substrates ATP, CTP, GTP, ITP, and UTP was observed. From a variety of reducing agents 1,4-dithio- dl -threitol (DTT), 1,4-dithioerythritol (DTE), bis-(2-mercaptoethyl)-sulfone, and 1,3-propanedithiol showed to be the most effective reducing agents for re-reduction of the active center thiols of ribonucleosidetriphosphate reductase. The kinetic parameters of ribonucleosidetriphosphate reductase with respect to affinity of ribonucleoside-5′-triphosphate substrates, the cofactor 5′-deoxyadenosylcobalamin, and the reducing agents DTT or 1,3-propanedithiol under the employed reaction conditions were determined. Substrate inhibition was not observed. Preparative gram-scale 2′-reductions of ribonucleoside-5′-triphosphates proceeded to completion.