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The gene encoding a novel xyloglucanase (Xeg) belonging to family 74 glycoside hydrolases was isolated from a Jonesia sp. strain through functional screening in Escherichia coli. The encoded xyloglucanase is a protein of 972 aminoacyl residues with a 23 residue aminoterminal signal peptide. Over-expression of Xeg in B. subtilis or E. coli failed. In contrast, Xeg was successfully over-expressed and secreted in Streptomyces lividans TK24. To this end Xeg was fused C-terminally to the secretory signal peptide of the subtilisin inhibitor protein (vsi) from Streptomyces venezuelae. The native Xeg signal peptide derived from Jonesia sp. is only poorly functional in S. lividans. Under optimal growth conditions, significant amounts of mature Xeg (100-150 mg/l) are secreted in the spent growth media. A protocol to rapidly purify Xeg to homogeneity from culture supernatants was developed. Biophysical and biochemical analyses indicate that the enzyme is intact, stable and fully functional. Xeg is the longest heterologous polypeptide shown to be secreted from S. lividans. This study further validates use of S. lividans for production of active heterologous proteins and demonstrates that heterologous polypeptides of up to 100 kDa are also tractable by this system.  相似文献   

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Extracellular chitinases of Streptomyces peucetius and a chitinase overproducing mutant, SPVI, were purified to homogeneity by ion exchange and gel filtration chromatography. The purified enzyme has a molecular mass of 42 kDa on SDS-PAGE, and the N-terminal amino acid sequence of the protein from the wild type showed homology to catalytic domains (Domain IV) of several other Streptomyces chitinases such as S. lividans 66, S. coelicolor A3(2), S. plicatus, and S. thermoviolaceus OPC-520. Purified SPVI chitinase cross-reacted to anti-chitinase antibodies of wild-type S. peucetius chitinase. A genomic library of SPVI constructed in E. coli using lambda DASH II was probed with chiC of S. lividans 66 to screen for the chitinase gene. A 2.7 kb fragment containing the chitinase gene was subcloned from a lambda DASH II clone, and sequenced. The deduced protein had a molecular mass of 68 kDa, and showed domain organization similar to that of S. lividans 66 chiC. The N-terminal amino acid sequence of the purified S. peucetius chitinase matched with the N-terminus of the catalytic domain, indicating the proteolytic processing of 68 kDa chitinase precursor protein to 42 kDa mature chitinase containing the catalytic domain only. A putative chiR sequence of a two-component regulatory system was found upstream of the chiC sequence.  相似文献   

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Extracellular amylase in Streptomyces lividans was undetectable in starch-supplemented medium. However, S. lividans produced fivefold-higher levels of amylase than Streptomyces griseus IMRU 3570 when transformed with the S. griseus amy gene. Two major proteins of 57 and 50 kDa with amylase activity accumulated in the culture broths of the donor S. griseus and S. lividans transformed with the amy gene. Both proteins were also present in protoplast lysates in the same relative proportion; they gave a positive reaction with antibodies against the 57-kDa amylase. They did not differ in substrate specificity or enzyme kinetics. The two amylases were purified to homogeneity by a two-step procedure. Both proteins showed the same amino-terminal sequence of amino acids, suggesting that both proteins are derived from the same gene. The deduced signal peptide has 28 amino acids with two positively charged arginines near the amino-terminal end. When an internal NcoI fragment was removed from the amy gene, the resulting S. lividans transformants did not synthesize any of the two amylase proteins and showed no reaction in immunoblotting. Formation of the 50-kDa protein was observed when pure 57-kDa amylase was treated with supernatants of protoplast lysates but not when it was treated with membrane preparations, indicating that the native 57-kDa amylase could be processed intracellularly.  相似文献   

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A gene pknA, coding for an eukaryotic-type protein Ser/Thr kinase, was cloned from the Streptomyces coelicolor A3(2) chromosome. The PknA protein kinase, containing the C-terminal eukaryotic-type kinase domain with an N-terminal extension, was expressed in Escherichia coli and Streptomyces lividans. The affinity purified MBP-PknA fusion protein was assayed for kinase activity that showed its ability to autophosphorylate in vitro in the presence of [gamma-32P]ATP. The activity was Mn2+ dependent. The preautophosphorylated kinase phosphorylated at least two proteins (sizes 30 and 32 kDa) in the S. coelicolor J1501 cell-free extracts of all developmental stages. The larger of them was also phosphorylated in vitro by an endogenous protein kinase in late stages extracts, but not earlier. Although Mn2+ dependent protein phosphorylation has previously been described in Streptomyces, this is the first report of a gene encoding such an enzyme in this genus.  相似文献   

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We have shown previously that a gene encoding a homologue to the eukaryotic dolichol-phosphate-D-mannose, protein O-D-mannosyltransferase, was required for (phi)C31 infection of Streptomyces coelicolor. Here we show that a gene encoding the homologue to dolichol-phosphate-mannose synthase is also essential for phage sensitivity. These data confirm the role of glycosylation in the phage receptor for (phi)C31 in S. coelicolor.  相似文献   

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A 1400-base DNA fragment, which contains the gene encoding the extracellular active-site serine beta-lactamase of Streptomyces albus G previously cloned into Streptomyces lividans [Dehottay et al. (1986) Gene 42, 31-36], was sequenced. The gene codes for a 314-amino-acid precursor, the N-terminal region of which has the characteristics of a signal peptide. The beta-lactamase as excreted by the host strain S. lividans PD6 has a ragged N-terminus, indicating either the presence of a leader peptidase of poor specificity or the action of an aminopeptidase. The primary structure (as deduced from the nucleotide sequence) was confirmed by amino acid sequencing of a 16-residue stretch at the amino terminus of the protein, a 12-residue stretch containing the active-site serine [De Meester et al. (1987) Biochem. J. 244, 427-432] and a 23-residue stretch obtained by trypsin digestion of the protein. The beta-lactamase belongs to class A, has three half-cystine residues (one of which occurs on the amino side of the active-site serine) and is inactivated by thiol reagents. Putative ribosome binding site and terminator region were identified.  相似文献   

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Upstream of the Streptomyces coelicolor A3(2) chitinase G gene, a small gene (named chb3) is located whose deduced product shares 37% identical amino acids with the previously described CHB1 protein from Streptomyces olivaceoviridis. The chb3 gene and its upstream region were cloned in a multicopy vector and transformed into the plasmid-free Streptomyces lividans TK21 strain. The CHB3 protein (14.9 kDa) was secreted by the S. lividans TK21 transformant during growth in the presence of glucose, N-acetylglucosamine, yeast extract, and chitin. The protein was purified to homogeneity using anionic exchange, hydrophobic interaction chromatographies, and gel filtration. In contrast to CHB1, CHB3 targets alpha-chitin, beta-chitin, and chitosan at pH 6.0 but does so relatively loosely. The ecological implications of the divergence of substrate specificity of various types of chitin-binding proteins are described.  相似文献   

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通过三步亚克隆 ,将单点突变葡萄糖异构酶 ( GIG1 38P)基因及其调控序列插入链霉菌质粒p IJ40 83,构建重组表达质粒 p IJ40 83- GI1 .用重组质粒转化变铅青链霉菌 TK54原生质体 ,经硫链丝菌素抗性 ( Th R)筛选 ,获得重组菌株 TK54/p IJ40 83- GI1 .酶活力测定和 SDS- PAGE分析表明 ,GIG1 38P基因在变铅青链霉菌中得到高效表达 ,GI1粗酶液比活力为 1 5U/mg,GI1表达量约占菌体可溶性蛋白的 2 5% .同时也研究了重组质粒的遗传稳定性 .重组菌株在无选择压力条件下经液体连续传代培养 ,GI1比活力和 GI1表达量在 2 0 0 h传代时间中呈平缓下降趋势  相似文献   

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