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Wim J Quax

Publications and source records attributed to Wim J Quax.

At least 37 records · Page 2Linked to original sources
PubMed2004

Mutational analysis of a key residue in the substrate specificity of a cephalosporin acylase.

beta-Lactam acylases are crucial for the synthesis of semisynthetic cephalosporins and penicillins. Unfortunately, there are no cephalosporin acylases known that can efficiently hydrolyse the amino-adipic side chain of Cephalosporin C. In a previous directed evolution experiment, residue Asn266 of the glutaryl acylase from Pseudomonas SY-77 was identified as being important for substrate specificity. In order to explore the function of this residue in substrate specificity, we performed a complete mutational analysis of position 266. Codons for all amino acids were introduced in the gene, 16 proteins that could be functionally expressed in Escherichia coli were purified to homogeneity and their catalytic parameters were determined. The mutant enzymes displayed a broad spectrum of affinities and activities, pointing to the flexibility of the enzyme at this position. Mutants in which Asn266 was changed into Phe, Gln, Trp and Tyr displayed up to twofold better catalytic efficiency (k(cat)/K(m))than the wild-type enzyme when adipyl-7-aminodesacetoxycephalosporanic acid (adipyl-7-ADCA) was used as substrate, due to a decreased K(m). Only mutants SY-77(N266H) and SY-77(N266M) showed an improvement of both catalytic parameters, resulting in 10- and 15-times higher catalytic efficiency with adipyl-7-ADCA, respectively. Remarkably, the catalytic activity (k(cat)) of SY-77(N266M) when using adipyl-7-ADCA as substrate was as high as when glutaryl-7-aminocephalosporanic acid (glutaryl-7-ACA) was used, and approaches commercially interesting activity. SY-77(N266Q), SY-77(N266H) and SY-77(N266M) mutants showed a modest improvement in hydrolysing Cephalosporin C. Since these mutants also have a good catalytic efficiency when adipyl-7-ADCA is used and are still active towards glutaryl-7-ACA, they can be regarded as broad substrate acylases. These results demonstrate that the combination of directed evolution for the identification of important positions, together with saturation mutagenesis for finding the optimal amino acid, is a very effective method for finding improved biocatalysts.

Adipates↗
PubMed2004

Characterization of the interaction between human complement protein C4 and a single-chain variable fragment antibody by capillary electrophoresis and surface plasmon resonance.

Immunoaffinity capillary electrophoresis and surface plasmon resonance have been used for the characterization of the interaction between two large-sized proteins, the human complement protein C4 and the single-chain variable fragment C43. The rather high kinetic rate constants as determined by surface plasmon resonance pointed out that a capillary electrophoresis method had to be applied, in which the labeled C4 is preincubated with C43 before injection and the same concentration of C43 is included in the running buffer. Analysis of the concentration dependence of the small mobility shift of the fluorescent C4 signal upon binding of C43 resulted in a dissociation constant that was comparable to the one obtained with surface plasmon resonance. This study is one of the few examples where capillary electrophoresis is successfully used to characterize the interaction between large proteins.

Antibodies↗
PubMed2004

Improved beta-lactam acylases and their use as industrial biocatalysts.

Whereas the beta-lactam acylases are traditionally used for the hydrolytic processing of penicillin G and cephalosporin C, new and mutated acylases can be used for the hydrolysis of alternative fermentation products as well as for the synthesis of semisynthetic beta-lactam antibiotics. Three-dimensional structural analyses and site-directed mutagenesis studies have increased the understanding of the catalytic mechanism of these enzymes. The yield of hydrolysis and synthesis has been greatly improved by process design, including immobilization of the enzyme and the use of alternative reaction media. Significant advances have also been made in the resolution of racemic mixtures by means of stereoselective acylation/hydrolysis using beta-lactam acylases.

Amidohydrolases↗
PubMed2004

Proteomics of protein secretion by Bacillus subtilis: separating the "secrets" of the secretome.

Secretory proteins perform a variety of important "remote-control" functions for bacterial survival in the environment. The availability of complete genome sequences has allowed us to make predictions about the composition of bacterial machinery for protein secretion as well as the extracellular complement of bacterial proteomes. Recently, the power of proteomics was successfully employed to evaluate genome-based models of these so-called secretomes. Progress in this field is well illustrated by the proteomic analysis of protein secretion by the gram-positive bacterium Bacillus subtilis, for which approximately 90 extracellular proteins were identified. Analysis of these proteins disclosed various "secrets of the secretome," such as the residence of cytoplasmic and predicted cell envelope proteins in the extracellular proteome. This showed that genome-based predictions reflect only approximately 50% of the actual composition of the extracellular proteome of B. subtilis. Importantly, proteomics allowed the first verification of the impact of individual secretion machinery components on the total flow of proteins from the cytoplasm to the extracellular environment. In conclusion, proteomics has yielded a variety of novel leads for the analysis of protein traffic in B. subtilis and other gram-positive bacteria. Ultimately, such leads will serve to increase our understanding of virulence factor biogenesis in gram-positive pathogens, which is likely to be of high medical relevance.

Bacillus subtilis↗
PubMed2003

Analysis of a substrate specificity switch residue of cephalosporin acylase.

Residue Phe375 of cephalosporin acylase has been identified as one of the residues that is involved in substrate specificity. A complete mutational analysis was performed by substituting Phe375 with the 19 other amino acids and characterising all purified mutant enzymes. Several mutations cause a substrate specificity shift from the preferred substrate of the enzyme, glutaryl-7-ACA, towards the desired substrate, adipyl-7-ADCA. The catalytic efficiency ( [Formula: see text] (cat)/ [Formula: see text] (m)) of mutant SY-77(F375C) towards adipyl-7-ADCA was increased 6-fold with respect to the wild-type enzyme, due to a strong decrease of [Formula: see text] (m). The [Formula: see text] (cat) of mutant SY-77(F375H) towards adipyl-7-ADCA was increased 2.4-fold. The mutational effects point at two possible mechanisms by which residue 375 accommodates the long side chain of adipyl-7-ADCA, either by a widening of a hydrophobic ring-like structure that positions the aliphatic part of the side chain of the substrate, or by hydrogen bonding to the carboxylate head of the side chain.

Adipates↗
PubMed2003

Extracellular lipases from Bacillus subtilis: regulation of gene expression and enzyme activity by amino acid supply and external pH.

Bacillus subtilis secretes two lipases LipA and LipB into the culture medium. Both enzyme genes were differentially expressed depending on the growth conditions as determined by activity assays and Western blotting in B. subtilis mutant strains lipA, lipB, and the corresponding lipA/lipB double mutant. In minimal medium, LipA was produced at wild-type level in a lipB mutant, however, no LipB protein was detected in a lipA mutant. Interestingly, LipA was produced and secreted at wild-type level in rich medium, but the enzyme remained enzymatically inactive, presumably being caused by a shift of the growth medium to acid pH. Furthermore, expression of the lipase genes was studied using transcriptional fusions with the lacZ reporter gene. The expression of lipA was repressed by high amino acid concentrations, whereas the lipB gene expression remained unaffected.

Amino Acids↗
PubMed2003

Genome engineering reveals large dispensable regions in Bacillus subtilis.

Bacterial genomes contain 250 to 500 essential genes, as suggested by single gene disruptions and theoretical considerations. If this view is correct, the remaining nonessential genes of an organism, such as Bacillus subtilis, have been acquired during evolution in its perpetually changing ecological niches. Notably, approximately 47% of the approximately 4,100 genes of B. subtilis belong to paralogous gene families in which several members have overlapping functions. Thus, essential gene functions will outnumber essential genes. To answer the question to what extent the most recently acquired DNA contributes to the life of B. subtilis under standard laboratory growth conditions, we initiated a "reconstruction" of the B. subtilis genome by removing prophages and AT-rich islands. Stepwise deletion of two prophages (SPbeta, PBSX), three prophage-like regions, and the largest operon of B. subtilis (pks) resulted in a genome reduction of 7.7% and elimination of 332 genes. The resulting strain was phenotypically characterized by metabolic flux analysis, proteomics, and specific assays for protein secretion, competence development, sporulation, and cell motility. We show that genome engineering is a feasible strategy for functional analysis of large gene clusters, and that removal of dispensable genomic regions may pave the way toward an optimized Bacillus cell factory.

Bacillus Phages↗
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↗
PubMed2003

A phytochemical study of lignans in whole plants and cell suspension cultures of Anthriscus sylvestris.

In the roots of Anthriscus sylvestris 12 different lignans were detected. Arctigenin, dimethylmatairesinol, dimethylthujaplicatin, podophyllotoxin, 7-hydroxyyatein and 7-hydroxyanhydropodorhizol have not been previously reported to be present in A. sylvestris. In the cell suspension cultures, which were initiated for this study, trace amounts of deoxypodophyllotoxin could be detected. With these cell suspension cultures we carried out feeding experiments using deoxypodophyllotoxin, yatein and, anhydropodorhizol. Yatein had a toxic effect on the cell cultures and was, like anhydropodorhizol, not converted into any detectable product. Deoxypodophyllotoxin, in contrast, was converted into podophyllotoxin, yielding significantly higher concentration than measured in whole plants.

Apiaceae↗
PubMed2003

The bioconversion process of deoxypodophyllotoxin with Linum flavum cell cultures.

The in vitro cell suspension culture of Linum flavum is able to convert high amounts of the 2,7'-cyclolignan deoxypodophyllotoxin into 6-methoxypodophyllotoxin 7- O-glucoside. We studied this conversion in detail by monitoring the intermediates and side-products after feeding different concentrations of deoxypodophyllotoxin. At a low concentration (0.1 mM) deoxypodophyllotoxin is rapidly converted into 6-methoxypodophyllotoxin 7- O-glucoside, 6-methoxypodophyllotoxin and traces of beta-peltatin and podophyllotoxin. The feeding of 0.5 and 2.0 mM also shows a rapid conversion into 6-methoxypodophyllotoxin 7- O-glucoside, but a delayed formation of 6-methoxypodophyllotoxin and beta-peltatin. By using different extraction methods we delivered proof in favour of the hypothesis that a part of the deoxypodophyllotoxin after uptake is temporarily stored as beta-peltatin glucoside.

Cells, Cultured↗
PubMed2003

Lignan profiles of indoor-cultivated Anthriscus sylvestris.

In a previous study we have shown that different populations of Anthriscus sylvestris (L.) Hoffm. (Apiaceae) yield significantly different lignan profiles. In this study we collected the seeds of A. sylvestris from 4 locations, one in England and three in The Netherlands. The seeds germinated and were grown under identical laboratory conditions. After 5 months the plants were harvested and the lignan profile in the roots and aerial parts was analysed using GC-MS. The plants from the seeds of the 4 locations showed several significant phenotypic differences in, for instance, dry-weight. However the 4 groups did not differ significantly in their lignan profile in their roots. The lignans in the roots of A. sylvestris, deoxypodophyllotoxin, yatein and anhydropodorhizol reached, on average, equal amounts in all 4 groups. Within the 4 groups, however, a large quantitative variation in lignan profile between the individual plants was observed, pointing to within-population genetic differences between individual plants.

Apiaceae↗
PubMed2002

Functional genomic analysis of the Bacillus subtilis Tat pathway for protein secretion.

Protein secretion from Bacillus species is a major industrial production tool with a market of over $1 billion per year. However, standard export technologies, based on the well-characterised general secretory (Sec) pathway, are frequently inapplicable for the production of proteins. The recently discovered twin-arginine translocation (Tat) pathway offers additional potential to transport proteins. Here we review the use of functional genomic and proteomic approaches to explore the Tat pathway of Bacillus subtilis. The properties of Tat pathway components and the twin-arginine signal peptides that direct proteins into this pathway are discussed. Where appropriate, a comparison is made with Tat systems from other organism, such as Escherichia coli. Recent findings with the latter organism in particular provide proof-of-principle that the Tat pathway can be exploited for the production of Sec-incompatible proteins.

Amino Acid Sequence↗
PubMed2002

Selective contribution of the twin-arginine translocation pathway to protein secretion in Bacillus subtilis.

The availability of the complete genome sequence of Bacillus subtilis has allowed the prediction of all exported proteins of this Gram-positive eubacterium. Recently, approximately 180 secretory and 114 lipoprotein signal peptides were predicted to direct protein export from the cytoplasm. Whereas most exported proteins appear to use the Sec pathway, 69 of these proteins could potentially use the Tat pathway, as their signal peptides contain RR- or KR-motifs. In the present studies, proteomic techniques were applied to verify how many extracellular B. subtilis proteins follow the Tat pathway. Strikingly, the extracellular accumulation of 13 proteins with potential RR/KR-signal peptides was Tat-independent, showing that their RR/KR-motifs are not recognized by the Tat machinery. In fact, only the phosphodiesterase PhoD was shown to be secreted in a strictly Tat-dependent manner. Sodium azide-inhibition of SecA strongly affected the extracellular appearance of de novo synthesized proteins, including the lipase LipA and two other proteins with predicted RR/KR-signal peptides. The SecA-dependent export of pre-LipA is particularly remarkable, because its RR-signal peptide conforms well to stringent criteria for the prediction of Tat-dependent export in Escherichia coli. Taken together, our observations show that the Tat pathway makes a highly selective contribution to the extracellular proteome of B. subtilis.

Amino Acid Sequence↗
PubMed2002

Altering the substrate specificity of cephalosporin acylase by directed evolution of the Beta -subunit.

Using directed evolution, we have selected an adipyl acylase enzyme that can be used for a one-step bioconversion of adipyl-7-aminodesacetoxycephalosporanic acid (adipyl-7-ADCA) to 7-ADCA, an important compound for the synthesis of semisynthetic cephalosporins. The starting point for the directed evolution was the glutaryl acylase from Pseudomonas SY-77. The gene fragment encoding the beta-subunit was divided into five overlapping parts that were mutagenized separately using error-prone PCR. Mutants were selected in a leucine-deficient host using adipyl-leucine as the sole leucine source. In total, 24 out of 41 plate-selected mutants were found to have a significantly improved ratio of adipyl-7-ADCA versus glutaryl-7-ACA hydrolysis. Several mutations around the substrate-binding site were isolated, especially in two hot spot positions: residues Phe-375 and Asn-266. Five mutants were further characterized by determination of their Michaelis-Menten parameters. Strikingly, mutant SY-77(N266H) shows a nearly 10-fold improved catalytic efficiency (k(cat)/K(m)) on adipyl-7-ADCA, resulting from a 50% increase in k(cat) and a 6-fold decrease in K(m), without decreasing the catalytic efficiency on glutaryl-7-ACA. In contrast, the improved adipyl/glutaryl activity ratio of mutant SY-77(F375L) mainly is a consequence of a decreased catalytic efficiency toward glutaryl-7-ACA. These results are discussed in the light of a structural model of SY-77 glutaryl acylase.

Mutagenesis↗
PubMed2002

Volatile components from Anthriscus sylvestris (L.) Hoffm.

The volatile components of fresh leaves and roots from Anthriscus sylvestris (L.) Hoffm., obtained through hydrodistillation, were analysed by GC and GC-MS. This was compared to dichloromethane extracts of both fresh and dried leaf and root material. The monoterpene fraction (69-70%) dominated, while beta-phellandrene (39-45%) was the main component in both the leaf and the root oil. Other components in the leaf oil were beta-myrcene (17%), sabinene (6.2%), Z-beta-ocimene (5.4%) and benzene acetaldehyde (4.1%). In the roots we found Z-beta-ocimene (16.9%) and alpha-pinene (4.6%) as other major components. These principle constituents of both essential oils were also present in the dichloromethane extracts of the fresh and dried leaves and the roots, although in much smaller percentages. Comparing hydrodistillation of fresh plant material with a dichloromethane extract, the latter yielded a considerably lower amount of constituents. In addition, air drying and freeze drying resulted in a significant loss of volatile constituents as compared to fresh material (dichloromethane extract).

Apiaceae↗
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

Thiol-disulfide oxidoreductases are essential for the production of the lantibiotic sublancin 168.

Thiol-disulfide oxidoreductases are required for disulfide bond formation in proteins that are exported from the cytoplasm. Four enzymes of this type, termed BdbA, BdbB, BdbC, and BdbD, have been identified in the Gram-positive eubacterium Bacillus subtilis. BdbC and BdbD have been shown to be critical for the folding of a protein required for DNA uptake during natural competence. In contrast, no function has been assigned so far to the BdbA and BdbB proteins. The bdbA and bdbB genes are located in one operon that also contains the genes specifying the lantibiotic sublancin 168 and the ATP-binding cassette transporter SunT. Interestingly sublancin 168 contains two disulfide bonds. The present studies demonstrate that SunT and BdbB, but not BdbA, are required for the production of active sublancin 168. In addition, the BdbB paralogue BdbC is at least partly able to replace BdbB in sublancin 168 production. These observations show the unprecedented involvement of thiol-disulfide oxidoreductases in the synthesis of a peptide antibiotic. Notably BdbB cannot complement BdbC in competence development, showing that these two closely related thiol-disulfide oxidoreductases have different, but partly overlapping, substrate specificities.

ATP-Binding Cassette Transporters↗