Human ß-defensin-2 production from S. cerevisiae using the repressible MET17 promoter.

BACKGROUND: Baker's yeast Saccharomyces cerevisiae is a proven host for the commercial production of recombinant biopharmaceutical proteins. For the manufacture of heterologous proteins with activities deleterious to the host it can be desirable to minimise production during the growth phase and induce production late in the exponential phase. Protein expression by regulated promoter systems offers the possibility of improving productivity in this way by separating the recombinant protein production phase from the yeast growth phase. Commonly used inducible promoters do not always offer convenient solutions for industrial scale biopharmaceutical production with engineered yeast systems. RESULTS: Here we show improved secretion of the antimicrobial protein, human ß-defensin-2, (hBD2), using the S. cerevisiae MET17 promoter by repressing expression during the growth phase. In shake flask culture, a higher final concentration of human ß-defensin-2 was obtained using the repressible MET17 promoter system than when using the strong constitutive promoter from proteinase B (PRB1) in a yeast strain developed for high-level commercial production of recombinant proteins. Furthermore, this was achieved in under half the time using the MET17 promoter compared to the PRB1 promoter. Cell density, plasmid copy-number, transcript level and protein concentration in the culture supernatant were used to study the effects of different initial methionine concentrations in the culture media for the production of human ß-defensin-2 secreted from S. cerevisiae. CONCLUSIONS: The repressible S. cerevisiae MET17 promoter was more efficient than a strong constitutive promoter for the production of human ß-defensin-2 from S. cerevisiae in small-scale culture and offers advantages for the commercial production of this and other heterologous proteins which are deleterious to the host organism. Furthermore, the MET17 promoter activity can be modulated by methionine alone, which has a safety profile applicable to biopharmaceutical manufacturing.

Design of a Novel Peptide with Esterolytic Activity toward PET by Mimicking the Catalytic Motif of Serine Hydrolases.

Serine hydrolases have become increasingly important for recycling PET plastics. However, their properties are inherently constrained by their 3D structure, which in turn limits the conditions for their application. Considering peptides as catalysts for industrial depolymerization processes can help us to escape some of these limitations. In this article, a 25 amino acid thermostable peptide, HSH-25, was designed to depolymerize PET. The peptide incorporates a His-Ser-His motif, inspired by the catalytic triad found in the serine hydrolase family, into a ß-hairpin fold. Stability of the fold was investigated by molecular dynamics simulations. Esterolytic activity of the peptide toward model substrates was detected within a pH range from pH 7 to pH 9.5. Degradation of polymeric PET substrates was confirmed by atomic force microscopy imaging on spin-coated PET thin films.

De novo design of a polycarbonate hydrolase.

Enzymatic degradation of plastics is currently limited to the use of engineered natural enzymes. As of yet, all engineering approaches applied to plastic degrading enzymes retain the natural $\alpha /\beta $-fold. While mutations can be used to increase thermostability, an inherent maximum likely exists for the $\alpha /\beta $-fold. It is thus of interest to introduce catalytic activity toward plastics in a different protein fold to escape the sequence space of plastic degrading enzymes. Here, a method for designing highly thermostable enzymes that can degrade plastics is described. With the help of Rosetta an active site catalysing the hydrolysis of polycarbonate is introduced into a set of thermostable scaffolds. Through computational evaluation, a potential PCase was selected and produced recombinantly in Escherichia coli. Thermal analysis suggests that the design has a melting temperature of >95$^{\circ }$C. Activity toward polycarbonate was confirmed using atomic force spectroscopy (AFM), proving the successful design of a PCase.

EstB from Burkholderia gladioli: a novel esterase with a beta-lactamase fold reveals steric factors to discriminate between esterolytic and beta-lactam cleaving activity.

Esterases form a diverse class of enzymes of largely unknown physiological role. Because many drugs and pesticides carry ester functions, the hydrolysis of such compounds forms at least one potential biological function. Carboxylesterases catalyze the hydrolysis of short chain aliphatic and aromatic carboxylic ester compounds. Esterases, D-alanyl-D-alanine-peptidases (DD-peptidases) and beta-lactamases can be grouped into two distinct classes of hydrolases with different folds and topologically unrelated catalytic residues, the one class comprising of esterases, the other one of beta-lactamases and DD-peptidases. The chemical reactivities of esters and beta-lactams towards hydrolysis are quite similar, which raises the question of which factors prevent esterases from displaying beta-lactamase activity and vice versa. Here we describe the crystal structure of EstB, an esterase isolated from Burkholderia gladioli. It shows the protein to belong to a novel class of esterases with homology to Penicillin binding proteins, notably DD-peptidase and class C beta-lactamases. Site-directed mutagenesis and the crystal structure of the complex with diisopropyl-fluorophosphate suggest Ser75 within the "beta-lactamase" Ser-x-x-Lys motif to act as catalytic nucleophile. Despite its structural homology to beta-lactamases, EstB shows no beta-lactamase activity. Although the nature and arrangement of active-site residues is very similar between EstB and homologous beta-lactamases, there are considerable differences in the shape of the active site tunnel. Modeling studies suggest steric factors to account for the enzyme's selectivity for ester hydrolysis versus beta-lactam cleavage.

The Thermal Stability of the Fusarium solani pisi Cutinase as a Function of pH.

We have investigated the thermal stability of the Fusarium solani pisi cutinase as a function of pH, in the range from pH 2-12. Its highest enzymatic activity coincides with the pH-range at which it displays its highest thermal stability. The unfolding of the enzyme as a function of pH was investigated by microcalorimetry. The ratio between the calorimetric enthalpy (DeltaH(cal)) and the van't Hoff enthalpy (DeltaH(v)) obtained, is far from unity, indicating that cutinase does not exhibit a simple two state unfolding behaviour. The role of pH on the electrostatic contribution to the thermal stability was assessed using TITRA. We propose a molecular interpretation for the pH-variation in enzymatic activity.

Backbone and sidechain 1H, 13C and 15N resonance assignments of the human brain-type fatty acid binding protein (FABP7) in its apo form and the holo forms binding to DHA, oleic acid, linoleic acid and elaidic acid.

In this manuscript, we present the backbone and side chain assignments of human brain-type fatty acid binding protein, also known as FABP7, in its apo form and in four different holo forms, bound to DHA, oleic acid, linoleic acid and elaidic acid.

Production of Fusarium solani f. sp. pisi cutinase in Fusarium venenatum A3/5.

Fusarium venenatum A3/5 was transformed using the Aspergillus niger expression plasmid, pIGF, in which the coding sequence for the F. solani f. sp. pisi cutinase gene had been inserted in frame, with a KEX2 cleavage site, with the truncated A. niger glucoamylase gene under control of the A. niger glucoamylase promoter. The transformant produced up to 21 U cutinase l(-1) in minimal medium containing glucose or starch as the primary carbon source. Glucoamylase (165 U l(-1) or 8 mg l(-1)) was also produced. Both the transformant and the parent strain produced cutinase in medium containing cutin.

The interaction of Fusarium solani pisi cutinase with long chain spin label esters.

We here present a study of the interaction between the Fusarium solani pisi cutinase mutant S120A and spin-labeled 4,4-dimethyloxazoline-N-oxyl-(DOXYL)-stearoyl-glycerol substrates in a micellar system. The interaction is detected by NMR measuring changes in chemical shift for 1H and 15N as well as relaxation parameters for backbone 1H (T1) and 15N (T1, T2) atoms as well as for side chain methyl groups 1H (T1). The detected interaction shows a weak binding of cutinase to the lipid micelles. Structural and mobility changes are located inside and around the active site, its flanking loops, and the oxyanion hole, respectively. Relaxation changes in the amino acid pairs Ser 92, Ala 93 and Thr 173, Gly 174 positioned at the edge of each of the active site flanking loops make these residues prime candidates for hinges, allowing for structural rearrangement during substrate binding. The cutinase mutant S120A used carries a 15 amino acid pro-peptide; the significance of this pro-peptide was so far undetermined. We show here that the pro-peptide is affected by the presence of the micellar substrate. Relaxation enhancements indicative of spatial proximity between the DOXYL group in the lipid chain and some hydrophobic residues surrounding the active site could be found.

Quantization of pH: evidence for acidic activity of triglyceride lipases.

Here we present a study of lipolytic activity of lipases from Fusarium solani pisi (cutinase), Rhizomucor miehei, Pseudomonas cepacia, and Humicola lanuginosa. Their activities toward triolein provide clear evidence for considerable enzymatic activity under acidic conditions. The activity was followed using Fourier transform infrared attenuated total reflection (FTIR-ATR) and nuclear magnetic resonance (NMR). Using these approaches, all the lipases that were studied exhibited lipolytic activity down to pH 4. The common model for the catalytic activity of the F. solani pisi cutinase, and lipases in general, requires the deprotonation of the active site histidine. Measurements using (13)C NMR spectroscopy showed a pK(a) value in the absence of substrate that is not consistent with the detected acid activity. We propose a novel model for the electrostatics in the active site of cutinase that could explain the observed acidic activity. The active site is essentially covered with the lipid surface during catalysis, thus preventing chemical communication between the active site and the bulk solvent. We propose that the classical definition of pH in bulk solution is not applicable to the active site environment of a lipase when the active site is inaccessible to solvent. In small restricted volumes, the pH must be quantized, and since much of the biological world is dependent on compartmentalization of processes in small volumes, it becomes relevant to investigate when this mechanism comes into play. We have made a quantitative assessment of how large the restricted volume can be and still lead to quantization of pH.