ABSTRACT
The Erwinia chrysanthemi (strain 3937) celY gene encoding the minor endoglucanase (EGY) was sequenced. The analysis of the upstream region allowed us to identify an in vivo active promoter recognized by the NtrA (sigma 54) holoenzyme. No similarity was found between the predicted amino acid (aa) sequences of EGY and either the Er. chrysanthemi major endoglucanase, EGZ, or the Er. carotovora CelS endoglucanase. In contrast, a very high level of identity, both at the nucleotide and the predicted aa levels, was found between celY and an EG-encoding gene from Cellulomonas uda, a Gram + bacterium taxonomically distant from Er. chrysanthemi. By comparing the molar G + C% of the cellulase-encoding genes and that of Er. chrysanthemi and C. uda chromosomal DNAs, we speculate that celY was transferred from Er. chrysanthemi to C. uda.
Subject(s)
Cellulase/genetics , Dickeya chrysanthemi/genetics , Genes, Bacterial , Transfection , Amino Acid Sequence , Base Sequence , Biological Evolution , Cloning, Molecular , Dickeya chrysanthemi/enzymology , Molecular Sequence Data , Multigene Family , Promoter Regions, Genetic , Sequence Homology, Nucleic Acid , Species SpecificityABSTRACT
Nucleotide sequencing of the celZ gene encoding the extracellular endoglucanase Z of Erwinia chrysanthemi indicated the presence of an open reading frame encoding 428 amino acids. The mature protein appeared to be extended by a signal peptide of 43 amino acids; this sequence is unusually long and positively charged (+5). It was shown to function as a signal peptide by fusing it to a truncated phoA gene encoding Escherichia coli alkaline phosphatase. Comparison of the encoded sequence with those of the endoglucanases of Bacillus subtilis and alkalophilic Bacillus revealed the existence of a region of extensive homology occurring in all three proteins at about the same distance from the NH2-terminal end. These regions may be involved in substrate binding and/or catalytic sites.
Subject(s)
Bacillus subtilis/genetics , Bacillus/genetics , Cellulase/genetics , Erwinia/genetics , Alkaline Phosphatase/genetics , Amino Acid Sequence , Bacillus/enzymology , Bacillus subtilis/enzymology , Base Sequence , DNA Restriction Enzymes , Erwinia/enzymology , Escherichia coli/enzymology , Escherichia coli/genetics , Genes , Genes, Bacterial , Molecular Sequence Data , Plasmids , Sequence Homology, Nucleic AcidABSTRACT
An endoglucanase (1,4-beta-D-glucan glucanohydrolase, EC 3.2.1.4) from a new cellulolytic thermophilic bacterium was purified to apparent homogeneity after being separated from a xylanase. Little carbohydrate was associated with the endoglucanase. A molecular weight of 91,000 and 99,000 was determined by SDS-polyacrylamide gel electrophoresis and by gel filtration of the native enzyme on Ultrogel ACA 34. The optimal pH was approximately 6.4 and the enzyme was isoelectric at pH 3.85. The enzyme was found highly thermostable: it retained 50 per cent of its activity after 1 hour at 85 degrees C. Hydrolysis of CMC took place with a rapid decrease in viscosity but a slow liberation of reducing sugars, indicating endo-enzyme activity. It showed little capacity to hydrolyze highly ordered cellulose. Cellobiose inhibited the activity of the endoglucanase. None of the metal ions tested stimulated the activity. The enzyme was completely inactivated by 1 mM Hg2+ and was inhibited by thiol reagents.
