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D Kluepfel

Publications and source records attributed to D Kluepfel.

At least 37 records · Page 2Linked to original sources
PubMed1996

Improved Production of Mannanase by Streptomyces lividans.

Replacement of the natural promoter of the (beta)-mannanase gene of Streptomyces lividans by lacp resulted in a 15-fold increase in enzyme production over that of the previously reported clone S. lividans IAF36, a clone carrying multiple copies of manA, and a 350-fold increase over that of the wild-type strain S. lividans 1326. In addition, the use of lacp in the shuttle vector pIAF199 allowed synthesis of the enzymes on carbon sources that did not contain mannan, such as xylan and whey, which offers interesting possibilities for industrial production of the enzyme.

Journal Article↗
PubMed1995

Analysis of DNA flanking the xlnB locus of Streptomyces lividans reveals genes encoding acetyl xylan esterase and the RNA component of ribonuclease P.

Nucleotide sequencing revealed the gene (axeA) encoding acetyl xylan esterase (AxeA) downstream from xlnB in the Streptomyces lividans DNA insert of plasmid pIAF42. AxeA consists of a catalytic- and a substrate-binding domain separated by a Gly-rich linker. The N terminus showed no significant homology with published esterases and acetyl xylan esterases, but some homology was found with the xylanases XylA and XylD and the NodB protein of Rhizobium species which is involved in the biosynthesis of root nodulation factors. The C terminus of AxeA is highly homologous to the C-termini of xylanases XlnB and TFXA, corresponding to the xylan-binding domain of these enzymes. Furthermore, the RNaseP RNA component was found immediately upstream from xlnB gene.

Acetylesterase↗
PubMed1995

A cellulase/xylanase-negative mutant of Streptomyces lividans 1326 defective in cellobiose and xylobiose uptake is mutated in a gene encoding a protein homologous to ATP-binding proteins.

The uptake of monosaccharides (glucose and xylose) and disaccharides (cellobiose and xylobiose) was evaluated in the Streptomyces lividans mutant strain 10-164. The pleiotropic mutation had no effect on glucose uptake; however, the Vmax of xylose uptake was decreased 10-fold as compared to the wild-type strain, S. lividans 1326, and the transport system of cellobiose and xylobiose, the putative inducers of the cellulase and xylanase genes, was completely abolished resulting in a cellulase/xylanase-negative mutant. An accumulation of xylose and glucose in culture media was observed when the mutant was grown on xylobiose and cellobiose, respectively. Cell-associated beta-glucosidase and low levels of extracellular beta-glucosidase were detected in both strains. When gluconolactone, a beta-glucosidase inhibitor, was added to the medium there was no uptake of cellobiose or release of glucose by the mutant strain, whereas the uptake of cellobiose by the wild-type strain was not significantly affected. It is thus proposed that the active transport system for cellobiose and xylobiose is affected in mutant strain 10-164. Glucose and xylose production from disaccharide hydrolysis are due to beta-glucosidase and beta-xylosidase activities, which sustain the growth of the mutant strain. Clones complementing the mutation were isolated from a gene bank constructed using mutant strain 10-164. The msiK gene codes for MsiK, a 40 kDa multiple sugar import protein, which belongs to the family of ATP-binding proteins. The mutation is located in the B site which is responsible for ATP binding. This protein probably provides energy to the xylose and disaccharide transport system as a result of the hydrolysis of ATP.

Adenosine Triphosphatases↗
PubMed1995

Production and secretion of proteins by streptomycetes.

Streptomycetes produce a large number of extracellular enzymes as part of their saprophytic mode of life. Their ability to synthesize enzymes as products of their primary metabolism could lead to the production of many proteins of industrial importance. The development of high-yielding expression systems for both homologous and heterologous gene products is of considerable interest. In this article, we review the current knowledge on the various factors that affect the production and secretion of proteins by streptomycetes and try to evaluate the suitability of these bacteria for the large-scale production of proteins of industrial importance.

Amino Acid Sequence↗
PubMed1994

Purification and characterization of an alpha-L-arabinofuranosidase from Streptomyces lividans 66 and DNA sequence of the gene (abfA).

The gene encoding an alpha-L-arabinofuranosidase (abfA) was homologously cloned in Streptomyces lividans and its DNA sequence was determined. The enzyme was purified from the cytoplasm of the hyperproducing clone S. lividans IAF116. Its M(r) was estimated by gel filtration and found to be approx. 380,000. Since SDS/PAGE indicated a native protein of M(r) 69,000, it can be concluded that the native protein consists of several subunits of that size. The pI value was 4.6. The kinetic constants determined with p-nitrophenyl alpha-L-arabinofuranoside as substrate were a Vmax of 180 units/mg of protein and a Km of 0.6 mM. The specific activity of the purified enzyme on this substrate was 153 units/mg of protein. Optimal enzyme activity was obtained at 60 degrees C and pH 6.0. The enzyme cleaved p-nitrophenyl alpha-L-arabinofuranoside, but had no activity on a variety of other p-nitrophenyl glycosides, except on p-nitrophenyl beta-D-xylopyranoside. The enzyme showed no activity on oat-spelts (Avena sativa) xylan or arabinogalactan, but acted on beet (Beta) arabinan or arabinoxylan. Hydrolysis occurred on arabino-oligoxylosides obtained from oat-splets xylan after digestion with xylanases. Since S. lividans normally does not secrete arabinofuranosidase, this enzyme may play a role in the assimilation of arabinose moieties from arabinose-containing xylo-oligosaccharides generated by beta-xylosidases or xylanases.

Amino Acid Sequence↗
PubMed1994

Crystal structure, at 2.6-A resolution, of the Streptomyces lividans xylanase A, a member of the F family of beta-1,4-D-glycanases.

The crystal structure of the 32-kDa catalytic domain of the Streptomyces lividans xylanase A was solved by molecular isomorphous replacement methods and subsequently refined at 2.6-A resolution to a conventional crystallographic R factor of 0.21. This is the first successful structure determination of a member of the F family of endo-beta-1,4-D-glycanases. Unlike the recently determined xylanases of the G family (Wakarchuk, W. W., Campbell, R. L., Sung, W. L., Davoodi, J., and Yaguchi, M. (1994) Protein Sci. 3, 467-475), where the catalytic domains have a unique beta-sheet structure, the 32-kDa domain of the S. lividans xylanase A is folded into a complete (alpha/beta)8 barrel, the first such fold observed among beta-1,4-D-glycanases. The active site is located at the carbonyl end of the beta barrel. The crystal structure supports the earlier assignment of Glu-128 and Glu-236 as the catalytic amino acids (Moreau, A., Roberge, M., Manin, C., Shareck, F., Kluepfel, D., and Morosoli, R. (1994) Biochem. J., in press).

Binding Sites↗
PubMed1994

Identification of two acidic residues involved in the catalysis of xylanase A from Streptomyces lividans.

On the basis of similarities between known xylanase sequences of the F family, three invariant acidic residues of xylanase A from Streptomyces lividans were investigated. Site-directed-mutagenesis experiments were carried out in Escherichia coli after engineering the xylanase A gene to allow its expression. Replacement of Glu-128 or Glu-236 by their isosteric form (Gln) completely abolished enzyme activity with xylan and p-nitrophenyl beta-D-cellobioside, indicating that the two substrates are hydrolysed at the same site. These two amino acids probably represent the catalytic residues. Immunological studies, which showed that the two mutants retained the same epitopes, indicate that the lack of activity is the result of the mutation rather than misfolding of the protein. Mutation D124E did not affect the kinetic parameters with xylan as substrate, but D124N reduced the Km 16-fold and the Vmax. 14-fold when compared with the wild-type enzyme. The mutations had a more pronounced effect with p-nitrophenyl beta-D-cellobioside as the substrate. Mutation D124E increased the Km and decreased the Vmax. 5-fold each, while D124N reduced the Km 4.5-fold and the Vmax. 75-fold. The mutations had no effect on the cleavage mode of xylopentaose.

Base Sequence↗
PubMed1994

Alteration of the cleavage mode and of the transglycosylation reactions of the xylanase A of Streptomyces lividans 1326 by site-directed mutagenesis of the Asn173 residue.

The amino acid replacement of Asn173 by Asp in the xylanase A (Xln A) of Streptomyces lividans significantly altered its enzymic properties. A time-course hydrolysis of xylan showed that the altered xylanase ([N173D] Xln A) initially produced larger amounts of xylose (X1), xylobiose (X2) and xylotriose (X3) than Xln A, but less xylotetraose (X4). The bond-cleavage frequencies were determined for both enzymes using xylopentaose (X5), xylotetraose (X4) and xylotriose (X3) labelled at the reducing end of the molecule. Xln A hydrolysed X5, yielding 56% X2 and 44% X3, while [N173D]Xln A liberated 90% X2 and only 10% X3. Both enzymes hydrolysed X4 into 100% X2 and X3 into 100% X1. Transglycosylation reactions were detected in HPLC hydrolysis patterns using high substrate concentrations, where larger products than the starting substrates were formed. Their subsequent degradation also affected the yield of hydrolysis products. Using X5 as substrate, products from xylohexaose (X6) up to xylooligosides larger than xylooctaose (X8) were synthesized by Xln A, while [N173D]Xln A produced only a small amount of xyloheptaose (X7) and X8. Xln A hydrolysed X5 into an equivalent amount of X4 and X2 and 1.5-fold more X3. However, [N173D]Xln A yielded the same amount of X3 and X2 but half as much X4. With X4 as substrate, Xln A synthesized twofold more X7 and X6 than [N173D]Xln A. Xln A liberated 1.4-fold more X3 than X2, while [N173D]Xln A yielded twofold more X2 than X3. Xln A liberated almost fourfold more X2 than X1 from X3, while [N173D]Xln A produced only twofold more X2 than X1. These results indicated that the negative charge introduced by the mutation greatly affected the transglycosylation reaction catalysed by this xylanase.

Amino Acid Sequence↗
PubMed1994

Increase in catalytic activity and thermostability of the xylanase A of Streptomyces lividans 1326 by site-specific mutagenesis.

The xylanase A gene from Streptomyces lividans was modified by site-directed mutagenesis, selecting for mutations that improved the catalytic activity and thermostability of the enzyme. Mutant notation uses the one-letter abbreviation for amino acids. The first and the last letters represent, respectively, the residue to be changed and the replacing residue. The number indicates the position of the substitution. The mutant enzymes F155Y, R156E, R156K, and N173D were respectively 28, 10, 50, and 25% more active than the wild-type enzyme. In addition, the half-lives at 60 degrees C of the R156E and N173D xylanases were respectively 6 and 40 min longer than that of the wild-type enzyme even in the absence of substrate. The favorable single mutations were combined to generate the double mutants E156/173D and K156/173D, which were 22 and 47% less active than the wild type. However, the activity half-life of the E156/173D enzyme at 60 degrees C was twice that of the xylanase A. The pH-activity profiles of all the mutant xylanases were similar to that of the wild-type enzyme.

Amino Acid Sequence↗
PubMed1994

Effects of disruption of xylanase-encoding genes on the xylanolytic system of Streptomyces lividans.

Wild-type Streptomyces lividans produced the three xylanases (XlnA, XlnB, and XlnC) when xylan, xylan hydrolysates obtained by the action of XlnA, XlnB, and XlnC, or purified small xylo-oligosaccharides (xylobiose [X2], xylotriose [X3], xylotetraose [X4], and xylopentaose [X5]) were used as the carbon source. The three xylanase genes of S. lividans (xlnA, xlnB, and xlnC) were disrupted by using vectors that integrate into the respective genes. Disruption of one or more of the xln genes resulted in reduced growth rates and reduced total xylanase activities when the strain was grown in xylan. The greatest effect was observed when xlnA was disrupted. In medium containing xylan, disruption of xlnA did not affect expression of xlnB and xlnC; disruption of xlnB did not affect expression of xlnA but affected expression of xlnC; and disruption of xlnC did not affect expression of xlnA but affected expression of xlnB. A fraction of XlnB or XlnC hydrolytic products (those with a degree of polymerization greater than 11 [X11]) was found to stimulate expression of xlnB and xlnC in strains disrupted in xlnC and xlnB, respectively, whereas lower-molecular-weight fractions as well as purified small xylo-oligosaccharides did not. The stimulating molecule(s) lost its effect when it was hydrolyzed further by XlnA. A mechanism of transglycosylation reactions by the S. lividans xylanases is postulated to be involved in the regulation of xln genes.

Base Sequence↗
PubMed1993

Mode of action of three endo-beta-1,4-xylanases of Streptomyces lividans.

The mode of action of three genetically distinct endo-beta-1,4-xylanases (EXs) of Streptomyces lividans, XlnA, XlnB and XlnC, belonging to two different xylanase families, was investigated on a variety of polysaccharide and oligosaccharide substrates. Viscosimetric measurements showed that all three enzymes have about the same endo-acting character. Occurrence of multiple pathways of substrate degradation at high concentration of beta-1,4-xylooligosaccharides suggested that all three enzymes were retaining glycanases. The enzymes differed considerably in their mode of action on various heteroxylans and on rhodymenan. XlnA hydrolyzed all tested polysaccharides to a higher degree than XlnB or XlnC, through liberation of smaller hydrolysis products, both linear or branched. XlnA performed much better than XlnB or XlnC, particularly on acetylxylan, liberating large amounts of short acetylated and non-acetylated fragments. XlnB and XlnC liberated from acetylxylan only limited amounts of larger acetylated fragments. XlnA exhibited also much higher catalytic efficiency than the other two EXs on short beta-1,4-xylooligosaccharides. The kinetic parameters and bond-cleavage frequencies determined for xylotriose, xylotetraose and xylopentaose using 1-3H-reducing-end-labelled compounds suggested that the substrate binding site of XlnA is smaller and differently organized than those in XlnB or XlnC. In contrast to XlnB and XlnC, XlnA also exhibited significant aryl-beta-xylosidase activity. No distinctive catalytic properties of either XlnB or XlnC were found which were not inherent also to XlnA. High-molecular-mass EXs of the XlnA type show much greater catalytic versatility due than low-molecular-mass EXs of the XlnB or XlnC type.

Carbohydrate Sequence↗
PubMed1993

Beta-mannanase of Streptomyces lividans 66: cloning and DNA sequence of the manA gene and characterization of the enzyme.

The gene coding for a beta-mannanase was cloned homologously from Streptomyces lividans and its DNA sequence was determined. The fully secreted enzyme was isolated and purified from culture filtrates of the hyperproducing clone S. lividans IAF36 grown in mineral salt media containing galactomannan as the main carbon source. It had a molecular mass of 36 kDa and a specific activity of 876 i.u./mg of protein. Under the assay conditions used, the optimal enzyme activity was obtained at 58 degrees C and a pH of 6.8. The pI was 3.5. The kinetic constants of this mannanase determined with galactomannan as substrate were a Vmax. of 205 i.u./mg of enzyme and a Km of 0.77 mg/ml. Data from SDS/PAGE and Western blotting show that the cloned enzyme was identical to that of the wild-type strain.

Amino Acid Sequence↗
PubMed1992

Purification and characterization of an endoglucanase from Streptomyces lividans 66 and DNA sequence of the gene.

The endoglucanase isolated from culture filtrates of Streptomyces lividans IAF74 was shown to have an Mr of 46,000 and a pI of 3.3. The specific enzyme activity of 539 IU/mg, determined by the reducing assay method on carboxymethyl cellulose, is among the highest reported in the literature. The cellulase showed typical endo-type activity when reacting on oligocellodextrins. Optimal enzyme activity was obtained at 50 degrees C and pH 5.5. The kinetic constants for this endoglucanase, determined with carboxymethyl cellulose as the substrate, were a Vmax of 24.9 IU/mg of enzyme and a Km of 4.2 mg/ml. Activity was found against neither methylumbelliferyl- nor p-nitrophenyl-cellobiopyranoside nor with xylan. The DNA sequence contains one possible reading frame validated by the N terminus of the mature purified protein. However, neither ATG nor GTG starting codons were identified near the ribosome-binding site. A putative TTG codon was found as a good candidate for the start codon. Comparison of the primary amino acid sequence of the endoglucanase of S. lividans revealed that the N terminus contains a bacterial cellulose-binding domain. The catalytic domain at the C terminus showed similarity to endoglucanases from a Bacillus sp. Thus, the endoglucanase CelA belongs to family A of cellulases as described before (N. R. Gilkes, B. Henrissat, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, Microbiol. Rev. 55:303-315, 1991.

Amino Acid Sequence↗
PubMed1992

A novel shuttle vector for Streptomyces spp. and Escherichia coli as a tool in site-directed mutagenesis.

This paper describes the construction and utilization of a novel shuttle vector for Streptomyces spp. and Escherichia coli as a useful vector in site-directed mutagenesis. The shuttle vector pIAFS20 (6.7 kb) has the following features: a replicon for Streptomyces spp., isolated from plasmid pIJ702; the thiostrepton-resistance gene as a selective marker in Streptomyces; the ColE1 origin, allowing replication in E. coli; and the ampicillin-resistance gene as a selective marker in E. coli. Vector pIAFS20 also contains the phage f1 intergenic region, which permits production of single-stranded DNA in E. coli after superinfection with helper phage M13K07. Moreover, the lac promoter is located in front of the multiple cloning sites cassette, allowing eventual expression of the cloned genes in E. coli. After mutagenesis and screening of the mutants in E. coli, the plasmids can be readily used to transform Streptomyces spp. As a demonstration, a 3.2-kb DNA fragment containing the gene encoding the xylanase A from Streptomyces lividans 1326 was inserted into pIAFS20, and the promoter region of this gene served as a target for site-directed mutagenesis. The two deletions reported here confirm the efficiency of this new vector as a tool in mutagenesis.

Base Sequence↗
PubMed1991

Sequences of three genes specifying xylanases in Streptomyces lividans.

The entire nucleotide (nt) sequences of three genes (xlnA, xlnB and xlnC) of Streptomyces lividans encoding three distinct xylanases (Xln) have been determined. The nt sequences were confirmed by comparing the deduced amino acid (aa) sequences with the ones derived from the N-terminal aa sequences of the mature purified proteins. The N-terminus of the XlnA showed some homology with either the N-termini or the C-termini of eight other Xln and of two exo-glucanases. The N-terminus of XlnB is homologous to that of XlnC and to Xln of seven other microorganisms.

Amino Acid Sequence↗
PubMed1990

Purification and characterization of a new xylanase (xylanase B) produced by Streptomyces lividans 66.

A new extracellular xylanase produced by Streptomyces lividans 66 was isolated from a genetically engineered clone of that strain. This enzyme, named xylanase B, has an Mr of 31,000 and acts specifically on xylan as an endo-type xylanase producing short-chain xylo-oligosaccharides. The activity is optimal at pH 6.5 and at a temperature of 55 degrees C, which is similar to that of the previously characterized xylanase A. Xylanase B is glycosylated and has a pI of 8.4; its Km and Vmax. values are 3.71 mg/ml and 1.96 mmol/mg of enzyme respectively. Specific antibodies raised against xylanase A show no cross-reaction with xylanase B; however, the anti-(xylanase B) antibodies react slightly with xylanase A. A comparison of the hydrolysis products obtained from oat-spelts xylan with both enzymes show that xylanase A preferentially degrades short-chain oligo-xylosides, whereas xylanase B acts on the longer, water-insoluble, molecules.

Blotting, Western↗
PubMed1990

Cloning of a second xylanase-encoding gene of Streptomyces lividans 66.

A second xylanase-encoding gene (xln) of Streptomyces lividans 66 was cloned by functional complementation of a xylanase/endocellulase-negative double mutant of S. lividans, using the multicopy plasmid pIJ702. Three clones contained a common 3.1-kb DNA fragment encoding the biosynthesis of a 31-kDa xylanase and a 22-kDa protein which was immunorelated. The xylanase represented at least 80% of the total secreted proteins. These three clones also secreted a small amount of the previously reported 43-kDa xylanase which was detected only by using specific antibodies. Most likely, the DNA fragment contained the complete structural xln gene and some regulatory sequences, since the enzymatic activity was repressed by glucose. In the in vitro transcription-translation system, the plasmid pIAF42 encoded an immunoprecipitable 35-kDa xylanase indicating the presence of a 4-kDa signal peptide.

Bacterial Proteins↗