Biomedical subjects

Raymond M D Verhaert

Publications and source records attributed to Raymond M D Verhaert.

4 recordsLinked to original sources

PubMed2003

Binding of phage displayed Bacillus subtilis lipase A to a phosphonate suicide inhibitor.

Phage display can be used as a protein engineering tool to select proteins with desirable binding properties from a library of randomly constructed mutants. Here, we describe the development of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has marked properties for the preparation of pharmaceutically relevant chiral compounds. The lipase gene was cloned upstream of the phage g3p encoding sequence and downstream of a modified g3p signal sequence. Consequently, the enzyme was displayed at the surface of bacteriophage fd as a fusion to its minor coat protein g3p. The phage-bound lipase was correctly folded and fully enzymatically active as determined from the hydrolysis of p-nitrophenylcaprylate with K(m)-values of 0.38 and 0.33 mM for the phage displayed and soluble lipase, respectively. Both soluble lipase and lipase expressed on bacteriophages reacted covalently with a phosphonate suicide inhibitor. The phage does not hamper lipase binding, since both soluble and phage-bound lipase have a similar half-life of inactivation of approximately 5 min. Therefore, we conclude that the Bacillus lipase can be functionally expressed on bacteriophages as a fusion to the phage coat protein g3p. The specific interaction with the suicide inhibitor offers a fast and reproducible method for the future selection of mutant enzymes with an enantioselectivity towards new substrates.

Bacillus Phages↗

PubMed2002

Phage display selects for amylases with improved low pH starch-binding.

Directed evolution of secreted industrial enzymes is hampered by the lack of powerful selection techniques. We have explored surface display to select for enzyme variants with improved binding performance on complex polymeric substrates. By a combination of saturation mutagenesis and phage display we selected alpha-amylase variants, which have the ability to bind starch substrate at industrially preferred low pH conditions. First we displayed active alpha-amylase on the surface of phage fd. Secondly we developed a selection system that is based on the ability of alpha-amylase displaying phages to bind to cross-linked starch. This system was used to probe the involvement of specific beta-strands in substrate interaction. Finally, a saturated library of alpha-amylase mutants with one or more amino acid residues changed in their Cbeta4 starch-binding domain was subjected to phage display selection. Mutant molecules with good starch-binding and hydrolytic capacity could be isolated from the phage library by repeated binding and elution of phage particles at lowered pH value. Apart from the wild type alpha-amylase a specific subset of variants, with only changes in three out of the seven possible positions, was selected. All selected variants could hydrolyse starch and heptamaltose at low pH. Interestingly, variants were found with a starch hydrolysis ratio at pH 4.5/7.5 that is improved relative to the wild type alpha-amylase. These data demonstrate that useful alpha-amylase mutants can be selected via surface display on the basis of their binding properties to starch at lowered pH values.

Amino Acid Sequence↗

PubMed2002

Directed evolution of a glutaryl acylase into an adipyl acylase.

Semi-synthetic cephalosporin antibiotics belong to the top 10 of most sold drugs, and are produced from 7-aminodesacetoxycephalosporanic acid (7-ADCA). Recently new routes have been developed which allow for the production of adipyl-7-ADCA by a novel fermentation process. To complete the biosynthesis of 7-ADCA a highly active adipyl acylase is needed for deacylation of the adipyl derivative. Such an adipyl acylase can be generated from known glutaryl acylases. The glutaryl acylase of Pseudomonas SY-77 was mutated in a first round by exploration mutagenesis. For selection the mutants were grown on an adipyl substrate. The residues that are important to the adipyl acylase activity were identified, and in a second round saturation mutagenesis of this selected stretch of residues yielded variants with a threefold increased catalytic efficiency. The effect of the mutations could be rationalized on hindsight by the 3D structure of the acylase. In conclusion, the substrate specificity of a dicarboxylic acid acylase was shifted towards adipyl-7-ADCA by a two-step directed evolution strategy. Although derivatives of the substrate were used for selection, mutants retained activity on the beta-lactam substrate. The strategy herein described may be generally applicable to all beta-lactam acylases.

Amidohydrolases↗

PubMed2001

Peptide display on live MS2 phage: restrictions at the RNA genome level.

The potential of the RNA phage MS2 to accommodate extra amino acids in its major coat protein has been examined. Accordingly, a pentapeptide was encoded in the genome as an N-terminal extension. In the MS2 crystal structure, this part of the coat protein forms a loop that extends from the outer surface of the icosahedral virion. At the RNA level, the insert forms a large loop at the top of an existing hairpin. This study shows that it is possible to maintain inserts in the coat protein of live phages. However, not all inserts were genetically stable. Some suffer deletions, while others underwent adaptation by base substitutions. Whether or not an insert is stable appears to be determined by the choice of the nucleic acid sequence used to encode the extra peptide. This effect was not caused by differential translation, because coat-protein synthesis was equal in wild-type and mutants. We conclude that the stability of the insert depends on the structure of the large RNA hairpin loop, as demonstrated by the fact that a single substitution can convert an unstable loop into a stable one.

Base Sequence↗