Jutta Schmitt

Rational evolution of a medium chain-specific cytochrome P-450 BM-3 variant.

The single mutant F87A of cytochrome P-450 BM-3 from Bacillus megaterium was engineered by rational evolution to achieve improved hydroxylation activity for medium chain length substrates (C8-C10). Rational evolution combines rational design and directed evolution to overcome the drawbacks of these methods when applied individually. Based on the X-ray structure of the enzyme, eight mutation sites (P25, V26, R47, Y51, S72, A74, L188, and M354) were identified by modeling. Sublibraries created by site-specific randomization mutagenesis of each single site were screened using a spectroscopic assay based on omega-p-nitrophenoxycarboxylic acids (pNCA). The mutants showing activity for shorter chain length substrates were combined, and these combi-libraries were screened again for mutants with even better catalytic properties. Using this approach, a P-450 BM-3 variant with five mutations (V26T, R47F, A74G, L188K, and F87A) that efficiently hydrolyzes 8-pNCA was obtained. The catalytic efficiency of this mutant towards omega-p-nitrophenoxydecanoic acid (10-pNCA) and omega-p-nitrophenoxydodecanoic acid (12-pNCA) is comparable to that of the wild-type P-450 BM-3.

Directed evolution of a bacterial α-amylase: Toward enhanced pH-performance and higher specific activity

α‐Amylases, in particular, microbial α‐amylases, are widely used in industrial processes such as starch liquefaction and pulp processes, and more recently in detergency. Due to the need for α‐amylases with high specific activity and activity at alkaline pH, which are critical parameters, for example, for the use in detergents, we have enhanced the α‐amylase from Bacillus amyloliquefaciens (BAA). The genes coding for the wild‐type BAA and the mutants BAA S201N and BAA N297D were subjected to error‐prone PCR and gene shuffling. For the screening of mutants we developed a novel, reliable assay suitable for high throughput screening based on the Phadebas assay. One mutant (BAA 42) has an optimal activity at pH 7, corresponding to a shift of one pH unit compared to the wild type. BAA 42 is active over a broader pH range than the wild type, resulting in a 5‐fold higher activity at pH 10. In addition, the activity in periplasmic extracts and the specific activity increased 4‐ and 1.5‐fold, respectively. Another mutant (BAA 29) possesses a wild‐type‐like pH profile but possesses a 40‐fold higher activity in periplasmic extracts and a 9‐fold higher specific activity. The comparison of the amino acid sequences of these two mutants with other homologous microbial α‐amylases revealed the mutation of the highly conserved residues W194R, S197P, and A230V. In addition, three further mutations were found K406R, N414S, and E356D, the latter being present in other bacterial α‐amylases.

P450 in biotechnology : zinc driven ω-hydroxylation of p-nitrophenoxydodecanoic acid using P450 BM-3 F87A as a catalyst

Cytochrome P450 enzymes require the delivery of two electrons to the heme protein for their enzymatic function. NADPH or NADH are usually used as reduction equivalents. In the absence of a substrate, NADPH may inactivate P450 enzymes. Furthermore, it is expensive, making it unsuitable for the preparative synthesis of fine chemicals. Approaches for replacing NADPH with an electrochemically generated reduction by using platinum-electrodes and different mediators are known. In the present study, NADPH was substituted by the mediator cobalt(III)sepulchrate and zinc dust that serves as an electron source. The mutated fatty acid hydroxylase P450 BM-3 F87A from Bacillus megaterium was chosen as a catalyst, since it shows a three-fold higher sensitivity and a nearly five-fold higher activity for p-nitrophenoxydodecanoic acid (12-pNCA) than the wild-type enzyme. The formation of p-nitrophenolate can easily be monitored using a photometer at 410 nm. The turnover rate of the zinc/cobalt(III)sepulchrate system reaches 20% of the NADPH activity. Compared to the electrochemical approaches the activity is at least 77% higher (turnover 125 eq min-1). The presented alternative cofactor system can be used instead of NADPH or expensive electrochemical devices (platinum electrodes) for fine chemical synthesis.

DNA isolation from soil samples for cloning in different hosts

Abstract Many protocols to extract DNA directly from soil samples have been developed in recent years. We employed two extraction methods which differed in the method of lysis and compared these methods with respect to yield, purity and degree of shearing. The main focus was on the specific isolation of DNA from different microorganisms, especially DNA from actinomycetes, as these cells are very difficult to lyse, in contrast to non-actinomycetes. Thus, we used both methods to isolate DNA from Pseudomonas, Arthrobacter and Rhodococcus and from soil spiked with the respective microorganisms. Both methods rendered high DNA yields with a low degree of shearing, but differed in the type of cells that were lysed. By one protocol (utilizing enzymatic lysis) only DNA from the Gram-negative Pseudomonas strain could be obtained whereas, by the other protocol (utilizing mechanical lysis), all microorganisms that were used could be lysed and DNA extracted from them. Using a combination of both protocols, DNA from those organisms could be obtained selectively. Furthermore, one of the protocols was modified, resulting in higher DNA yield and purity.

High-yield expression of the recombinant, atrazine-specific Fab fragment K411B by the methylotrophic yeast Pichia pastoris

In this report, we describe the high-yield secretory expression ( approximately 40 mg x l(-1)) of pure, atrazine-specific Fab fragments (K411B) from Pichia pastoris that was achieved by co-integration of the genes encoding the heavy and light chains (both under the control of the alcohol oxidase promoter) into the genome of the yeast cells. Antibody-expressing clones were selected by SDS-PAGE and ELISA and fed-batch fermentations were carried out in a 5-l scale. Both chains of the Fab were successfully expressed upon methanol induction and almost no other proteins were secreted into the media. Approximately 30% of the two chains formed the active Fab fragment containing the intermolecular disulphide bond, as determined by Western blot analysis under non-reducing conditions. Crude culture supernatant was used to study the binding properties of the Fab fragment toward different s-triazines by means of competitive ELISA: the IC50 value for the detection of atrazine was determined from the standard curve as 3 microg x l(-1), which is one magnitude higher than the value obtained with the parental mAb K4E7 but equals that obtained when the same Fab fragment was expressed in Escherichia coli cells. In addition, the cross-reactivity pattern of the Fab from Pichia is comparable to that of E. coli and to the parental mAb K4E7.

Cloning, Functional Expression, and Characterization of Recombinant Pig Liver Esterase

The N‐terminal amino acid sequence of pig liver esterase (PLE) from a commercial sample was determined and shown to match closely to a published sequence encoding a proline‐β‐naphthylamidase from pig liver. Next, mRNA isolated from pig liver was transcribed into cDNA and primers deduced from the N‐terminal sequence were used to clone the 1698 base pairs of PLE cDNA. Initial attempts to express the cDNA in Escherichia coli and Pichia pastoris with different expression vectors and secretion signal sequences failed. Only after deletion of the putative C‐terminal sequence His‐Ala‐Glu‐Leu, usually considered as an endoplasmic reticulum retention signal, could heterologous expression of PLE be readily achieved in the methylotrophic yeast P. pastoris. Recombinant PLE (rPLE) was secreted into the medium and exhibited a specific activity of approximately 600 U mg−1 and a Vmax/Km value of 139 μmol min−1 mM−1 with p‐nitrophenyl acetate as a substrate. Activity staining of renatured sodium dodecylsulfate–polyacrylamide gels gave a single band with esterolytic activity for rPLE, whereas several bands are visible in crude commercial PLE preparations. This was confirmed by native gels, which also show that rPLE is active as a trimer. Biochemical characterization of the recombinant enzyme and comparison with properties of commercial PLE preparations as well as with published data confirmed that we expressed a single PLE isoenzyme which showed a high preference for proline‐β‐naphthylamide. This is a substrate specificity for the so‐called γ subunit of PLE. The optimum pH value and temperature for the recombinant PLE were 8.0 and 60 °C, respectively. The determined molecular weight of the secreted enzyme was approximately 61–62 kDa, which closely matches the calculated value of 62.419 kDa. The active site residues are located at Ser203, His448, and Asp97, and the typical consensus sequence motif for hydrolases was found around the active site serine (Gly‐Glu‐Ser‐Ala‐Gly).

Fluorophor-linked immunosorbent assay: a time- and cost-saving method for the characterization of antibody fragments using a fusion protein of a single-chain antibody fragment and enhanced green fluorescent protein

A novel assay, referred to as fluorophor-linked immunosorbent assay (FLISA), for the characterization of single-chain antibody fragments (scFvs) is described. The principle of the method is the fusion of an scFv to enhanced green fluorescent protein (EGFP). The scFv domain, which binds to the immobilized hapten, can be detected by measuring the fluorescence of the EGFP domain. The time-consuming binding of secondary antibodies and enzyme reaction, necessary for enzyme-linked immunosorbent assays (ELISAs) are not required. Consequently, the assay time of 1.5 h needed to complete the FLISA is much shorter than that of comparable ELISAs, which require about 5 h. This renders the FLISA suitable for applications where a short assay time is essential, such as screening of mutant libraries of scFvs in directed evolution experiments or monitoring of the amount of functionally expressed recombinant protein during production processes. In contrast to a comparable ELISA, the FLISA showed no saturation when determining the relative amount of functional scFv. The amount of the soluble fraction of cell extracts from Escherichia coli expressing the fusion protein and the normalized fluorescence signal showed a linear correlation with R(2)>0.99. The usefulness of a competitive FLISA for the detection of analytes is shown exemplarily by the detection of s-triazines with the s-triazine-specific scFv K411B.

Identification of factors impeding the production of a single-chain antibody fragment in Escherichia coli by comparing in vivo and in vitro expression

The atrazine-specific single-chain variable antibody fragments (scFv) K411B was produced by expression in either the cytoplasm or the periplasm of Escherichia coli BL21(DE3). For periplasmic production, the pelB leader was N-terminally fused to scFv, whereas the unfused variant resulted in cytoplasmic expression. The extent of protein accumulation differed significantly. Expression of scFv with leader was 2.3 times higher than that of the protein without leader. This was further investigated by generating the respective translation profiles using coupled in vitro transcription/translation assays, the results of which were in agreement. This comparative approach was also applied to functionality: Periplasmic expression and in vitro expression resulted in only 10% correctly folded scFv, indicating that the oxidizing environment of the periplasm did not increase proper folding. Thus, the data obtained in vitro confirmed the findings observed in vivo and suggested that the discrepancy in expression levels was due to different translation efficiencies. However, the in vivo production of scFv with enhanced green fluorescent protein (EGFP) fused C-terminally (scFv-EGFP) was only successful in the cytoplasm, although in vitro the expression with and without the leader rendered the same production profile as for scFv. This indicated that neither the translation efficiency nor the solubility but other factors impeded periplasmic expression of the fusion protein.

Detection of 4-chlorobenzoate using immobilized recombinant Escherichia coli reporter strains

Abstract A test for the detection of 4-chlorobenzoic acid (4-CBA) based on reporter microorganisms is developed. A 1.7-kb DNA fragment, upstream of the dehalogenase operon from Arthrobacter sp. strain SU, is fused to the promoterless luciferase operon of Vibrio fischeri . This reporter construct is introduced into two different Escherichia coli strains (UTL2 and RFM443), which specifically respond to the presence of 4-CBA with light emission. The development and optimization of an alginate-based immobilization procedure of the microorganisms in microtiter plates is described. In this format, 4-CBA can be detected in the concentration range of 113 μM–3.6 mM. The membrane leaky mutant UTL2 of E. coli transformed with the reporter construct improves the detection limit to 28 μM when 150 mM KNO 3 is added to the medium.