Enantioselective synthesis of (R)-phenylephrine by Serratia marcescens BCRC10948 cells that homologously express SM_SDR.

A short-chain dehydrogenase/reductase from Serratia marcescens BCRC10948, SM_SDR, has been cloned and expressed in Escherichia coli for the bioconversion of 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) to (R)-phenylephrine[(R)-PE]. However, only 5.11mM (R)-PE was obtained from 10mM HPMAE after a 9h conversion in the previous report. To improve the biocatalytic efficiency, the homologous expression of the SM_SDR in S. marcescens BCRC10948 was achieved using the T5 promoter for expression. By using 2% glycerol as carbon source, we found that 8.00±0.15mM of (R)-PE with more than 99% enantiomeric excess was produced from 10mM HPMAE after 12h conversion at 30°C and pH 7.0. More importantly, by using 50mM HPMAE as the substrate, 23.78±0.84mM of (R)-PE was produced after a 12h conversion with the productivity and the conversion yield of 1.98mmol (R)-PE/lh and 47.50%, respectively. The recombinant S. marcescens cells could be recycled 6 times for the production of (R)-PE, and the bioconversion efficiency remained at 85% when compared to that at the first cycle. Our data indicated that a high conversion efficiency of HPMAE to (R)-PE could be achieved using S. marcescens BCRC10948 cells that homologously express the SM_SDR.

Structure-guided design of Serratia marcescens short-chain dehydrogenase/reductase for stereoselective synthesis of (R)-phenylephrine.

Bioconversion is useful to produce optically pure enantiomers in the pharmaceutical industry, thereby avoiding problems with side reactions during organic synthesis processes. A short-chain dehydrogenase/reductase from Serratia marcescens BCRC 10948 (SmSDR) can stereoselectively convert 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) into (R)-phenylephrine [(R)-PE], which is marketed medically as a nasal decongestant agent. The whole-cell conversion process for the synthesis of (R)-PE using SmSDR was reported to have an unexpectedly low conversion rate. We reported the crystal structure of the SmSDR and designed profitable variants to improve the enzymatic activity by structure-guided approach. Several important residues in the structure were observed to form hydrophobic clusters that stabilize the mobile loops surrounding the pocket. Of these, Phe98 and Phe202 face toward each other and connect the upper curvature from the two arms (i.e., the α7 helix and loopß4-α4). The mutant structure of the double substitutions (F98YF202Y) exhibited a hydrogen bond between the curvatures that stabilizes the flexible arms. Site-directed mutagenesis characterization revealed that the mutations (F98Y, F98YF202Y, and F98YF202L) of the flexible loops that stabilize the region exhibited a higher transformation activity toward HPMAE. Together, our results suggest a robust structure-guided approach that can be used to generate a valuable engineered variant for pharmaceutical applications.

Stereoselective synthesis of (R)-phenylephrine using recombinant Escherichia coli cells expressing a novel short-chain dehydrogenase/reductase gene from Serratia marcescens BCRC 10948.

(R)-Phenylephrine [(R)-PE] is an α1-adrenergic receptor agonist and is widely used as a nasal decongestant to treat the common cold without the side effects of other ephedrine adrenergic drugs. We identified a short-chain dehydrogenase/reductase (SM_SDR) from Serratia marcescens BCRC 10948 that was able to convert 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) into (R)-PE. The SM_SDR used NADPH and NADH as cofactors with specific activities of 17.35±0.71 and 5.57±0.07mU/mg protein, respectively, at 30°C and pH 7.0, thereby indicating that this enzyme could be categorized as an NADPH-preferring short-chain dehydrogenase/reductase. Escherichia coli strain BL21 (DE3) expressing SM_SDR could convert HPMAE into (R)-PE with more than 99% enantiomeric excess. The productivity and conversion yield were 0.57mmolPE/lh and 51.06%, respectively, using 10mM HPMAE. Fructose was the most effective carbon source for the conversion of HPMAE to (R)-PE.

Crystal structures of lysine-preferred racemases, the non-antibiotic selectable markers for transgenic plants.

Lysine racemase, a pyridoxal 5'-phosphate (PLP)-dependent amino acid racemase that catalyzes the interconversion of lysine enantiomers, is valuable to serve as a novel non-antibiotic selectable marker in the generation of transgenic plants. Here, we have determined the first crystal structure of a lysine racemase (Lyr) from Proteus mirabilis BCRC10725, which shows the highest activity toward lysine and weaker activity towards arginine. In addition, we establish the first broad-specificity amino acid racemase (Bar) structure from Pseudomonas putida DSM84, which presents not only the highest activity toward lysine but also remarkably broad substrate specificity. A complex structure of Bar-lysine is also established here. These structures demonstrate the similar fold of alanine racemase, which is a head-to-tail homodimer with each protomer containing an N-terminal (α/ß)(8) barrel and a C-terminal ß-stranded domain. The active-site residues are located at the protomer interface that is a funnel-like cavity with two catalytic bases, one from each protomer, and the PLP binding site is at the bottom of this cavity. Structural comparisons, site-directed mutagenesis, kinetic, and modeling studies identify a conserved arginine and an adjacent conserved asparagine that fix the orientation of the PLP O3 atom in both structures and assist in the enzyme activity. Furthermore, side chains of two residues in α-helix 10 have been discovered to point toward the cavity and define the substrate specificity. Our results provide a structural foundation for the design of racemases with pre-determined substrate specificity and for the development of the non-antibiotic selection system in transgenic plants.

Purification and characterization of an aspartic protease from the Rhizopus oryzae protease extract, Peptidase R

Background Aspartic proteases are a subfamily of endopeptidases that are useful in a variety of applications, especially in the food processing industry. Here we describe a novel aspartic protease that was purified from Peptidase R, a commercial protease preparation derived from Rhizopus oryzae. Results An aspartic protease sourced from Peptidase R was purified to homogeneity by anion exchange chromatography followed by polishing with a hydrophobic interaction chromatography column, resulting in a 3.4-fold increase in specific activity (57.5 × 10³ U/mg) and 58.8% recovery. The estimated molecular weight of the purified enzyme was 39 kDa. The N-terminal sequence of the purified protein exhibited 63-75% identity to rhizopuspepsins from various Rhizopus species. The enzyme exhibited maximal activity at 75°C in glycine-HCl buffer, pH 3.4 with casein as the substrate. The protease was stable at 35°C for 60 min and had an observed half-life of approximately 30 min at 45°C. Enzyme activity was not significantly inhibited by chelation with ethylenediamine tetraacetic acid (EDTA), and the addition of metal ions to EDTA-treated protease did not significantly change enzyme activity, indicating that proteolysis is not metal ion-dependent. The purified enzyme was completely inactivated by the aspartic protease inhibitor Pepstatin A. Conclusion Based on the observed enzyme activity, inhibition profile with Pepstatin A, and sequence similarity to other rhizopuspepsins, we have classified this enzyme as an aspartic protease.

Genome sequence of the lytic bacteriophage P1201 from Corynebacterium glutamicum NCHU 87078: evolutionary relationships to phages from Corynebacterineae.

P1201 is a lytic corynephage of Corynebacterium glutamicum NCHU 87078. Its genome consists of a linear double-stranded DNA molecule of 70,579 base pairs, with 3'-protruding cohesive ends of ten nucleotides. We have identified 69 putative open reading frames, including three apparent genes (thymidylate synthase, terminase, and RNR alpha subunit genes) that are interrupted by an intein. Protein-splicing activities of these inteins were demonstrated in Escherichia coli. Three structural proteins including major capsid and major tail proteins were separated by SDS-PAGE and identified by both LC-MS-MS and N-terminal sequence analyses. Bioinformatics analysis indicated that only about 8.7% of its putative gene products shared substantial protein sequence similarity with the lytic corynephage BFK20 from Brevibacterium flavum, the only corynephage whose genome had been sequenced to date, revealing that the P1201 genome is distinct from BFK20. The mosaic-like genome of P1201 indicates extensive horizontal gene transfer among P1201, Gordonia terrae phage GTE5, mycobacteriophages, and several regions of Corynebacterium spp. genomes.