A chemoenzymatic strategy for site-selective functionalization of native peptides and proteins.

The emergence of new therapeutic modalities requires complementary tools for their efficient syntheses. Availability of methodologies for site-selective modification of biomolecules remains a long-standing challenge, given the inherent complexity and the presence of repeating residues that bear functional groups with similar reactivity profiles. We describe a bioconjugation strategy for modification of native peptides relying on high site selectivity conveyed by enzymes. We engineered penicillin G acylases to distinguish among free amino moieties of insulin (two at amino termini and an internal lysine) and manipulate cleavable phenylacetamide groups in a programmable manner to form protected insulin derivatives. This enables selective and specific chemical ligation to synthesize homogeneous bioconjugates, improving yield and purity compared to the existing methods, and generally opens avenues in the functionalization of native proteins to access biological probes or drugs.

Functional expression of a penicillin acylase from the extreme thermophile Thermus thermophilus HB27 in Escherichia coli.

BACKGROUND: Penicillin acylases (PACs) are enzymes of industrial relevance in the manufacture of ß-lactam antibiotics. Development of a PAC with a longer half-life under the reaction conditions used is essential for the improvement of the operational stability of the process. A gene encoding a homologue to Escherichia coli PAC was found in the genome of the thermophilic bacterium Thermus thermophilus (Tth) HB27. Because of the nature of this PAC and its complex maturation that is crucial to reach its functional heterodimeric final conformation, the overexpression of this enzyme in a heterologous mesophilic host was a challenge. Here we describe the purification and characterization of the PAC protein from Tth HB27 overexpressed in Escherichia coli. RESULTS: Fusions to a superfolder green fluorescent protein and differential membrane solubilization assays indicated that the native enzyme remains attached through its amino-terminal end to the outer side of the cytoplasmic membrane of Tth cells. In order to overexpress this PAC in E. coli cells, a variant of the protein devoid of its membrane anchoring segment was constructed. The effect of the co-expression of chaperones and calcium supplementation of the culture medium was investigated. The total production of PAC was enhanced by the presence of DnaK/J and GrpE and even more by trigger factor and GroEL/ES. In addition, 10 mM calcium markedly improved both PAC specific and volumetric activities. Recombinant PAC was affinity-purified and proper maturation of the protein was confirmed by SDS-PAGE and MALDI-TOF analysis of the subunits. The recombinant protein was tested for activity towards several penicillins, cephalosporins and homoserine lactones. Hydrophobic acyl-chain penicillins were preferred over the rest of the substrates. Penicillin K (octanoyl penicillin) was the best substrate, with the highest specificity constant value (16.12 mM-1.seg-1). The optimum pH was aprox. 4 and the optimum temperature was 75 °C. The half-life of the enzyme at this temperature was 9.2 h. CONCLUSIONS: This is the first report concerning the heterologous expression of a pac gene from a thermophilic microorganism in the mesophilic host E. coli. The recombinant protein was identified as a penicillin K-deacylating thermozyme.

Preparation of cross-linked enzyme aggregates by using bovine serum albumin as a proteic feeder.

Addition of bovine serum albumin (BSA) as a proteic feeder facilitates obtaining cross-linked enzyme aggregates (CLEAs) in cases where the protein concentration in the enzyme preparation is low and/or the enzyme activity is vulnerable to the high concentration of glutaraldehyde required to obtain aggregates. CLEAs of Pseudomonas cepacia lipase and penicillin acylase were prepared. CLEA of lipase prepared in the presence of BSA retained 100% activity whereas CLEA prepared without BSA retained only 0.4% activity of the starting enzyme preparation. Lipase CLEA showed 12-fold increase in activity over free enzyme powder when the CLEA was used in transesterification of tributyrin. For the transesterification of Jatropha oil, while free enzyme powder required 8 h and 50 mg lipase to obtain 77% conversion, CLEA required only 6 h and 6.25 mg lipase to obtain 90% conversion. In the case of penicillin acylase, 86% activity could be retained in CLEA prepared with BSA whereas CLEA made without BSA retained only 50% activity. CLEA prepared without BSA lost 20% activity after 8 h at 45 degrees C whereas CLEA with BSA retained full activity. CLEA prepared with BSA showed Vmax/Km of 36.3 min-1 whereas CLEA prepared without BSA had Vmax/Km of 17.4 min-1 only. Scanning electron microscopy analysis showed that CLEAs prepared in the presence of BSA were less amorphous and closer in morphology to CLEAs of other enzymes described in the literature.