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Nucleotide sequence of the celA gene encoding a cellodextrinase of Ruminococcus flavefaciens FD-1 总被引:3,自引:0,他引:3
Summary The nucleotide sequence of a 3.6 kb DNA fragment containing a cellodextrinase gene (celA) fromRuminococcus flavefaciens FD-1 was determined. The gene was expressed from its own regulatory region inEscherichia coli and a putative consensus promoter sequence was identified upstream of a ribosome binding site and a TTG start codon. The complete amino acid sequence of the CeIA enzyme (352 residues) was deduced and showed no significant homology to cellulases from other oganisms. Two lysozymetype active sites were found in the amino-terminal third of the enzyme. InE. coli the cloned CeIA protein was translocated into the periplasm. The lack of a typical signal sequence, and the results of transposonphoA mutagenesis experiments indicated that CeIA is secreted by a mechanism other than a leader peptide.Abbreviations CMCase
carboxymethylcellulase
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celA
gene coding for CeIA
- CelA
cellodextrinase
- ORF
open reading frame
- phoA
gene encoding alkaline phosphatase
- pNPC
p-nitrophenyl--d-cellobioside 相似文献
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Eldie Berger Winsome A. Jones David T. Jones David R. Woods 《Molecular & general genetics : MGG》1990,223(2):310-318
Summary The nucleotide sequence of a 2.314 kb DNA segment containing a gene (cedl) expressing cellodextrinase activity from Butyrivibrio fibrisolvens H17c was determined. The B. fibrisolvens H17c gene was expressed from a weak internal promoter in Escherichia coli and a putative consensus promoter sequence was identified upstream of a ribosome binding site and a GTG start codon. The complete amino acid sequence (547 residues) was deduced and homology was demonstrated with the Clostridium thermocellum endoglucanase D (EGD), Pseudomonas fluorescens var. cellulose endoglucanase (EG), and a cellulase from the avocado fruit (Persea americana). The ced1 gene product Cedl showed cellodextrinase activity and rapidly hydrolysed short-chain cellodextrins to yield either cellobiose or cellobiose and glucose as end products. The Cedl enzyme released cellobiose from p-nitrophenyl--d-cellobioside and the enzyme was not inhibited by methylcellulose, an inhibitor of endoglucanase activity. Although the major activity of the Cedl enzyme was that of a cellodextrinase it also showed limited activity against endoglucanase specific substrates [carboxymethylcellulose (CMC), lichenan, laminarin and xylan]. Analysis by SDS-polyacrylamide gel electrophoresis with incorporated CMC showed a major activity band with an apparent M
r of approximately 61000. The calculated M
r of the ced1 gene product was 61023.Abbreviations Ap
ampicillin
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ced1
gene coding for Ced1
- Ced1
cellodextrinase from B. fibrisolvens
- CMC
carboxymethylcellulose
- LB
Luria Bertani
- ORF
open reading frame
- pNPC
p-nitrophenyl--d-cellobioside
- PC
phosphate citrate
- HCA
hydrophobic cluster analysis 相似文献
3.
Abstract A cellulase gene from Ruminococcus flavefaciens FD-1 had previously been cloned in Escherichia coli . The product of this gene, CelA, was purified from E. coli and characterised. This 39 kDa cellulase is antigenically related, and of similar mass, to a protein in R. flavefaciens . The enzyme has cellodextrinase activity with predominantly exo-type action. CelA activity was optimal at pH 6.5 and 41°C, and was inhibited in the presence of divalent metal cations. The K m and V max were determined as 0.68 mM and 1.89 μmol min−1 mg−1 of CelA, respectively. Cellobiose was the major end product of cellodextrin hydrolysis, and our results suggest that celluboise is competitive inhibitor of CelA. 相似文献
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