The properties of synthetic peptides, including potency, stability, and bioavailability, are strongly influenced by modification of the peptide chain termini. Unfortunately, generally applicable methods for selective and mild C-terminal peptide functionalization are lacking. In this work, we explored the peptide amidase from Stenotrophomonas maltophilia as a versatile catalyst for diverse carboxy-terminal peptide modification reactions. Because the scope of application of the enzyme is hampered by its mediocre stability, we used computational protein engineering supported by energy calculations and molecular dynamics simulations to discover a number of stabilizing mutations. Twelve mutations were combined to yield a highly thermostable (Δ T m = 23 °C) and solvent-compatible enzyme. Protein crystallography and molecular dynamics simulations revealed the biophysical effects of mutations contributing to the enhanced robustness. The resulting enzyme catalyzed the selective C-terminal modification of synthetic peptides with small nucleophiles such as ammonia, methylamine, and hydroxylamine in various organic (co)­solvents. The use of a nonaqueous environment allowed modification of peptide free acids with >85% product yield under thermodynamic control. On the basis of the crystal structure, further mutagenesis gave a biocatalyst that favors introduction of larger functional groups. Thus, the use of computational and rational protein design provided a tool for diverse enzymatic peptide modification.

c 1,2,3-Trichloropropane (TCP) is a toxic compound that is recalcitrant to biodegradation in the environment. Attempts to isolate TCP-degrading organisms using enrichment cultivation have failed. A potential biodegradation pathway starts with hydrolytic dehalogenation to 2,3-dichloro-1-propanol (DCP), followed by oxidative metabolism. To obtain a practically applicable TCPdegrading organism, we introduced an engineered haloalkane dehalogenase with improved TCP degradation activity into the DCP-degrading bacterium Pseudomonas putida MC4. For this purpose, the dehalogenase gene (dhaA31) was cloned behind the constitutive dhlA promoter and was introduced into the genome of strain MC4 using a transposon delivery system. The transposon-located antibiotic resistance marker was subsequently removed using a resolvase step. Growth of the resulting engineered bacterium, P. putida MC4-5222, on TCP was indeed observed, and all organic chlorine was released as chloride. A packed-bed reactor with immobilized cells of strain MC4-5222 degraded >95% of influent TCP (0.33 mM) under continuous-flow conditions, with stoichiometric release of inorganic chloride. The results demonstrate the successful use of a laboratory-evolved dehalogenase and genetic engineering to produce an effective, plasmid-free, and stable whole-cell biocatalyst for the aerobic bioremediation of a recalcitrant chlorinated hydrocarbon.

Fluorinated organic compounds are of growing industrial importance, with applications such as agrochemicals, pharmaceuticals, and performance materials (23,24,28,41). The safe use of such compounds, as well as appropriate disposal and treatment of wastes, will benefit from knowledge about their biodegradation. However, little information is available about the microbial metabolism of fluorinated organic compounds compared to other halogenated chemicals. Most studies on the bacterial degradation of fluorinated organics describe fluorobenzoic acids, which under aerobic conditions can be converted into the corresponding fluorocatechols (3, 17, 33). Papers about the degradation of fluorophenols have also appeared (11,25,51).4-Fluorocinnamic acid (4-FCA) is used in industry for the synthesis of flavors and pharmaceuticals (8), and polymers of 4-FCA are applied in electronics (14). It was proposed that under aerobic conditions in nonacclimated industrial activated sludge, 4-FCA could be converted into 4-fluorobenzoic acid (4-FBA) via the formation of 4-fluoroacetophenone (4-FAP) (8, 32). In another study, using activated sludge from a wastewater treatment plant, 4-FCA was suggested to be transformed into an epoxide that is converted to 4-FAP. This compound would then be converted into 4-FBA, but no products were detected from the breakdown of 4-FBA (5).The conversion of nonhalogenated cinnamic acids to benzoic acids, such as the transformation of ferulic acid to vanillic acid, has been described previously (2,4,20,31,39). Cinnamic acid, coumaric acid, and ferulic acid are transformed by Streptomyces setonii (47) and Rhodopseudomonas palustris to benzoic acid or the corresponding derivatives (19). Alcanivorax borkumensis MBIC 4326 (9) and Papillibacter cinnamivorans (7) transformed cinnamic acid into benzoic acid. The metabolism of these compounds thus proceeds with side chain degradation prior to ring cleavage. Side chain degradation is carried out either by ␤-oxidation or by direct deacetylation mechanisms, which leads to elimination of two carbon units from the unsaturated side chain in bacteria, yeasts, and fungi (40).Since no clear information on the degradation route of 4-FCA is available, we have isolated two pure bacterial strains to study the complete microbial metabolism of the compound, and in this paper, we propose a degradation pathway. MATERIALS AND METHODSGrowth conditions. Cells of strains G1 and H1 were grown aerobically at 30°C under rotary shaking or in a fermentor. Growth medium (MMY) contained (per liter) 5.37 g of Na 2 HPO 4 ⅐ 12H 2 O, 1.36 g of KH 2 PO 4 , 0.5 g of (NH 4 ) 2 SO 4 , and 0. Enrichment and isolation of 4-FCA-and 4-FBA-degrading organisms. Soil samples collected from a site in the Netherlands contaminated mainly with chlorobenzene and halogenated aliphatic compounds were used as the initial inocula for the 4-FCA and 4-FBA enrichment cultures. Flasks contained 40 ml MMY and 5 mM 4-FCA or 5 mM 4-FBA as the sole source of carbon and energy. The cultures were incubated at room temperatur...

ABSTRACTAPseudomonas putidastrain (MC4) that can utilize 2,3-dichloro-1-propanol (DCP) and several aliphatic haloacids and haloalcohols as sole carbon and energy source for growth was isolated from contaminated soil. Degradation of DCP was found to start with oxidation and concomitant dehalogenation catalyzed by a 72-kDa monomeric protein (DppA) that was isolated from cell lysate. ThedppAgene was cloned from a cosmid library and appeared to encode a protein equipped with a signal peptide and that possessed high similarity to quinohemoprotein alcohol dehydrogenases (ADHs), particularly ADH IIB and ADH IIG fromPseudomonas putidaHK. This novel dehalogenating dehydrogenase has a broad substrate range, encompassing a number of nonhalogenated alcohols and haloalcohols. With DCP, DppA exhibited akcatof 17 s−1.1H nuclear magnetic resonance experiments indicated that DCP oxidation by DppA in the presence of 2,6-dichlorophenolindophenol (DCPIP) and potassium ferricyanide [K3Fe(CN)6] yielded 2-chloroacrolein, which was oxidized to 2-chloroacrylic acid.

Chemoenzymatic peptide synthesis is a rapidly developing technology for cost effective peptide production on a large scale. As an alternative to the traditional C!N strategy, which employs expensive N-protected building blocks in each step, we have investigated an N!C extension route that is based on activation of a peptide C-terminal amide protecting group to the corresponding methyl ester. We found that this conversion is efficiently catalysed by Stenotrophomonas maltophilia peptide amidase in neat organic media. The system excludes the possibility of internal peptide cleavage as the enzyme lacks intrinsic protease activity. The produced peptide methyl ester was used for peptide chain extension in a kinetically controlled reaction by a thermostable protease.