Markus Pietzsch

Substrate-dependent enantioselectivity of a novel hydantoinase from Arthrobacter aurescens DSM 3745 : Purification and characterization as new member of cyclic amidases

A hydantoinase from Arthrobacter aurescens DSM 3745 has been purified to homogeneity with a yield of 77% using a three-step purification procedure. The active enzyme is a tetramer consisting of four identical subunits, each with a molecular mass of 49670 Da as determined by mass spectrometry. The N-terminal amino acid sequence of the enzyme indicates sequence identities to cyclic amidases involved in the nucleotide metabolism as the D-hydantoinase from Agrobacterium radiobacter (53%), the D-selective dihydropyrimidinase from Bacillus stearothermophilus (38%), the allantoinase from Rana catesbeiana (26%), as well as to the catalytic subunit of the urease from Helicobacter pylori (50%). However, all studies based on substrate-dependent growth, induction and catalytic behavior documented the novelty of the bacterial hydantoinase and that its physiological role is not related to any of these enzymes or known metabolic pathways. Its substrate specificity differs from hydantoinases listed in Enzyme Nomenclature and is rather more predominant for the cleavage of aryl- than for alkyl-hydantoin derivatives. It is shown that the stereoselectivity of this enzyme depends on the substrate used for bioconversion: although it is strictly L-selective for the cleavage of D,L-5-indolylmethylhydantoin, it appears to be D-selective for the hydrolysis of D,L-methylthioethylhydantoin. Due to these findings we conclude that this novel bacterial hydantoinase should be classified as a new member of the EC-group 3.5.2 of cyclic amidases.

Enzymatically Catalyzed HES conjugation Using Microbial Transglutaminase: Proof of Feasibility

Polymer-drug and polymer-protein conjugates are promising candidates for the delivery of therapeutic agents. PEGylation, using poly(ethylene glycol) for the conjugation, is now the gold standard in this field, and some PEGylated proteins have successfully reached the market. Hydroxyethyl starch (HES) is a water-soluble, biodegradable derivative of starch that is currently being investigated as a substitute for PEG. So far, only chemical methods have been suggested for HES conjugation; however, these may have detrimental effects on proteins. Here, we report an enzymatic method for HES conjugation using a recombinant microbial transglutaminase (rMTG). The latter catalyzes the acyl transfer between the gamma-carboxamide group of a glutaminyl residue (acyl donors) and a variety of primary amines (acyl acceptors), including the amino group of lysine. HES was modified with N-carbobenzyloxy glutaminyl glycine (Z-QG) and hexamethylene diamine (HMDA) to act as acyl donor and acyl acceptor, respectively. Using (1)H NMR, the degree of modification with Z-QG and HMDA was found to be 4.6 and 3.9 mol%, respectively. Using SDS-PAGE, it was possible to show that the modified HES successfully coupled to test compounds, proving that it is accepted as a substrate by rMTG. Finally, the process described in this study is a simple, mild approach to produce fully biodegradable polymer-drug and polymer-protein conjugates.

Cloning, nucleotide sequence and expression of a new l-N-carbamoylase gene from Arthrobacter aurescens DSM 3747 in E. coli

An L-N-carbamoyl amino acid amidohydrolase (L-N-carbamoylase) from Arthrobacter aurescens DSM 3747 was cloned in E. coli and the nucleotide sequence was determined. After expression of the gene in E. coli the enzyme was purified to homogeneity and characterized. The enzyme was shown to be strictly L-specific and exhibited the highest activity in the hydrolysis of beta-aryl substituted N alpha-carbamoyl-alanines as e.g. N-carbamoyl-tryptophan. Carbamoyl derivatives of beta-alanine and charged aliphatic amino acids were not accepted as substrates. The N-carbamoylase of A. aurescens DSM 3747 differs from all known enzymes with respect to its substrate specificity although amino acid sequence identity scores of 35-38% to other N-carbamoylases have been detected. The enzyme consists of two subunits of 44,000 Da, and has an isoelectric point of 4.3. The optima of temperature and pH were determined to be 50 degrees C and pH 8.5 respectively. At 37 degrees C the enzyme was completely stable for several days.

Purification and characterization of a highly selective epoxide hydrolase from Nocardia sp. EH1

A highly enantioselective, soluble epoxide from Nocardia sp. EH1 was purified to homogeneity via a four-step procedure: (i) hydrophobic interaction chromatography on Phenyl Sepharose CL-4B, (ii) anion exchange chromatography on SOURCE 30Q, followed by (iii) a second hydrophobic interaction chromatography on Phenyl Sepharose HP, and finally (iv) gel-filtration on Superdex 75 HR 10/30. The pure protein was shown to be a monomer of integral of 34 kDa possessing an optimum pH of 8-9. Neither UV-absorbing cofactors nor metal ions were required for activity. In contrast to whole-cell activity, the partially purified enzyme proved to be considerably less stable. Stabilization was achieved by addition of non-ionic detergents such as Tween 80 or Triton X-100, causing a shift of the temperature optimum from 35 to 40 degrees C. Both effects combined led to an enhancement of the relative activity of up to approximately 150% of that of the native enzyme.

Chiral silicon groups as auxiliaries for enantioselective synthesis: access to optically active silanes by biotransformation and the enantiospecific preparation of (R)-(+)-1-phenylethanol

Abstract The synthesis of the optically active alkoxymethyl-substituted acetylsilanes (+)-3 and (−)-b was achieved by the bioreduction of (±)-3 with resting cells of Trigonopsis variabilis to the diastereoisomeric alcohols (+)-4 and (+)-5. These two compounds were separated by chromatography and separately reoxidized to the desired optically active silyl ketones. As a simple example of the use of chiral alkoxymethyl-substituted silyl groups as auxiliaries for the synthesis of enantiomerically enriched silicon-free compounds, the chelate controlled 1,2-addition of phenyl lithium to (−)-3 and the stereospecific conversion of the corresponding major addition product to (R)-(+)-1-phenylethanol (+)-10 is presented.

Mechanical disruption of Arthrobacter sp. DSM 3747 in stirred ball mills for the release of hydantoin-cleaving enzymes

Abstract The release of three intracellular enzymes from Arthrobacter sp. DSM 3747 has been investigated. Previous experiments showed that the yield of enzyme activity could be maximized by mechanical cell disintegration in a stirred ball mill. In this paper experiments for a further biochemical engineering optimization of the disintegration step are presented. The influences of operational parameters of the stirred ball mill on the disintegration result have been detected. It could be shown tha the disintegration of the investigated organism is a function of specific energy input for high cell concentrations in the suspension together with bead sizes between 0.2 and 0.8 mm. The utilization of input energy for cell rupture reaches its maximum if the disintegration rate correlates with specific energy. This result is the basis for scale-up. In those cases where the disintegration rate does not correlate with energy input due to low concentrations or large bead diameters the influence of the nondimensional mixing time or stress frequency on cell rupture has been experimentally proved.

Random mutagenesis of a recombinant microbial transglutaminase for the generation of thermostable and heat-sensitive variants

Recombinant microbial transglutaminase (rMTG), an enzyme useful for the cross-linking or the posttranslational modification of (therapeutic) proteins, was optimized by random mutagenesis for the first time. A screening method was developed which, in addition to state-of-the-art procedures, includes a proteolytic activation step of the expressed soluble pro-enzyme. The library of 5,500 clones was screened for variants with increased thermostability and heat-sensitivity, respectively. Mutant enzymes were overproduced, isolated and characterized. After just one round of mutagenesis, nine variants with a single amino acid exchange showed a remarkably increased thermostability at 60 degrees C. The exchange of a serine residue close to the N-terminus against proline resulted in an rMTG mutant (S2P) with 270% increased half-life. Seven variants exhibited an increased heat-sensitivity at 60 degrees C of which one mutant (G25S) retained its specific activity between 10 and 40 degrees C. The mutations responsible for the increased thermostability and the heat-sensitivity were identified and assigned to the three-dimensional (3D) structure. All single point mutations related to changed thermal properties of rMTG are located in the N-terminal domain (i.e. the left side wall of the active site cleft of the front view of the MTG as defined by the literature [Kashiwagi, T., Yokoyama, K., Ishikawa, K., Ono, K., Ejima, D., Matsui, H., Suzuki, E., 2002. Crystal structure of microbial transglutaminase from Streptoverticillium mobaraense. J. Biol. Chem. 277, 44252-44260] showing the importance of this part of the protein.

Royalactin is not a royal making of a queen

Royalactin is not a royal making of a queen Anja Buttstedt;Christian Ihling;Markus Pietzsch;Robin Moritz; Nature

Synthesis of planar chiral [2.2]paracyclophanes by biotransformations: kinetic resolution of 4-formyl-[2.2]paracyclophane by asymmetric reduction

Abstract The synthesis of enantiomerically pure (S)-4-formyl-[2.2]paracyclophane 1 (>99% ee) and (R)-4-hydroxymethyl-[2.2]paracyclophane 2 (>78% ee) was achieved by bioreduction of (RS)- 1 with a yield of 49 and 34% respectively. From several microorganisms screened only a strain of the yeast Saccharomyces cerevisiae (DSM 11285) showed a stereospecific reduction of this planar chiral substrate (E>100). Despite the high enantiomeric ratio, it is necessary to maintain the conversion at almost 50% in order to obtain a high enantiomeric excess of both substrate and product of the reduction reaction. Tween 80 together with methanol was found to be the most suitable cosolvent mixture which enhances the solubility of the substrate and does not effect the biocatalyst. For the calculation of E the enantiomeric excesses of substrate and product were measured at various conversions by chiral gas chromatography. Commercially available alcohol dehydrogenases such as HLADH, YADH and TBADH were tested for the desired reaction too, but found to be completely inactive.

A d-specific hydantoin amidohydrolase: properties of the metalloenzyme purified from Arthrobacter crystallopoietes

Abstract A d -specific hydantoinase has been purified to homogeneity from Arthrobacter crystallopoietes DSM 20117 with a yield of 5% related to the crude extract. The active enzyme is a tetramer of 257 kDa consisting of four identical subunits, each with a molecular mass of 60 kDa. Incubation of the enzyme with the metal-chelating agent EDTA had no inhibitory effect, while 8-hydroxyquinoline-5-sulfonic acid resulted in a complete and irreversible inactivation. The purified enzyme contains zinc as cofactor, which could be detected by subjection to direct analysis using inductive/coupled plasma-atomic emission spectrometry. The hydantoinase has a wide substrate specificity for the d -selective cleavage of 5-monosubstituted hydantoin derivatives with aliphatic and aromatic side chains. The Vmax-value for phenylhydantoin is 217 U/mg, the Km-value is 8 mM. Dihydrouracil was found to be a natural substrate (Vmax=35 U/mg). The N-terminal amino acid sequence of the enzyme shows distinct homologies to other metal-dependent cyclic amidases involved in the nucleotide metabolism especially to dihydropyrimidinases as well as to ureases, l - and unselective hydantoinases. Due to these findings, this enzyme has to be considered as a possible link in the evolution to related l -selective and unselective hydantoinases from the genus of Arthrobacter.