Dieter Kluepfel

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.

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.

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

Centre de Recherche en Microbiologie Appliquée, Institut Armand‐Frappier, Université du Québec, Ville de Laval, Québec H7N 4Z3, Canada.

Characterization of cellulase and xylanase activities of Streptomyces lividans

SummaryThe production of cellulases and of xylanase by Streptomyces lividans 1326 was studied under different growth conditions. The strain grew between 18°C and 46°C and is therefore thermotolerant. Submerged cultures of the microorganism, when grown on a defined salt medium containing xylan as main carbon source, exhibited an overall cellulolytic activity as determined by the filter paper test. S. lividans produced optimal levels of extracellular β-1,4-glucan-glucanohydrolase (1 IU/ml) and large amounts of β-1,4-xylanxylanohydrolase (50 IU/ml) at 40°C. A better production of both enzymes was observed when xylan instead of cellulose was used as substrate.The stability of the enzyme was found to be significantly greater than those of the cellulases and xylanases produced by other streptomycetes. The optimal incubation temperatures for the enzyme assays were 55°C and 60°C for CM-cellulase and xylanase respectively and optimal pH values were found in the range of pH 6–7.

Cloning of the xylanase gene of Streptomyces lividans

The xylanase (xln) gene of Streptomyces lividans 1326 was cloned by functional complementation of the xylanase-negative and beta-1,4-glucan-glucanohydrolase-negative double mutant of S. lividans using the multicopy plasmid pIJ702. Three clones had a common 2-kb DNA fragment as determined by restriction mapping and Southern hybridization. These clones secreted a xylanase of Mr 43,000 which reacted with specific anti-xylanase antibodies and corresponded exactly to the enzyme previously isolated from the wild-type strain. The DNA fragment likely carried the full structural gene, the xln promoter and also the regulatory sequence, since the xylanase activity was inducible by xylan. Enzyme levels of up to 380 IU/ml of culture filtrate were obtained.

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

Abstract The mode of action of three genetically distinct endo-β-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 β-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 β-1,4-xylooligosaccharides. The kinetic parameters and bond-cleavage frequencies determined for xylotriose, xylotetraose and xylopentaose using 1- 3 H-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-β-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.

Substrate specificity and mode of action of acetylxylan esterase from Streptomyces lividans

The substrate specificity of purified acetylxylan esterase (AcXE) from Streptomyces lividans was investigated on partially and fully acetylated methyl glycopyranosides. The enzyme exhibited deacetylation regioselectivity on model compounds which provided insights pertaining to its function in acetylxylan degradation. The enzyme catalyzed double deacetylation of methyl 2,3,4‐tri‐O‐acetyl‐β‐d‐xylopyranoside and methyl 2,3,4,6‐tetra‐O‐acetyl‐β‐d‐glucopyranoside at positions 2 and 3. Two methyl xylopyranoside diacetates, which had a free hydroxyl group at position 2 or 3, i.e. the derivatives that most closely mimic monoacetylated xylopyranosyl residues in acetylxylan, were deacetylated 1 to 2 orders of magnitude faster than methyl 2,3,4‐tri‐O‐acetyl‐β‐d‐xylopyranoside and methyl 2,3‐di‐O‐acetyl‐β‐d‐xylopyranoside. These observations explain the double deacetylation. The second acetyl group is released immediately after the first one is removed from the fully acetylated methyl β‐d‐xylo‐ and ‐glucopyranoside. The results suggest that in acetylxylan degradation the enzyme rapidly deacetylates monoacetylated xylopyranosyl residues, but attacks doubly acetylated residues much more slowly. Evidence is also presented that the St. lividans enzyme could be the first real substrate‐specific AcXE.

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.

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.

Cloning of a second xylanase-encoding gene of Strettomyces 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.