1887

Abstract

Summary: The 90 kDa glucosaminidase protein was purified to apparent homogeneity from vegetative cells of AC327, and then the corresponding gene was cloned into in two inactive forms by standard procedures. Nucleotide sequencing of the glucosaminidase region revealed a monocistronic operon, (designated ) encoding a 95.6 kDa protein, comprising 880 amino acid residues, which has a typical signal peptide. Moreover, another monocistronic operon (designated ), encoding a 35.4 kDa protein, was found upstream of the glucosaminidase gene. Expression of a fusion gene, driven by regulatory sequences, was observed during the exponential growth phase. The introduction of a null mutation greatly reduced (by about 95%) the expression of the fusion. Amino acid sequence analysis of the glucosaminidase showed two types of direct repeats, each type being present twice, in the N-terminal-to-central region of the glucosaminidase: these repeats probably represent the cell-wall-binding domain. Zymographic analysis revealed that the 90 kDa glucosaminidase is partly processed to several smaller proteins (35-39 kDa), retaining lytic activity. Processing of these proteins occurred between the N-terminal cell-wall-binding and C-terminal catalytic domains of the glucosaminidase, the site being located between the 569th and 606th codons of the glucosaminidase. Serial deletions from the N-terminus of the glucosaminidase revealed that the loss of more than one repeating unit drastically reduces its lytic activity toward cell walls. The gene product, in either an intact or a truncated form, was found to be lethal for , and the N-terminally truncated glucosaminidase proteins, produced in , were very unstable. The partially purified glucosaminidase from was found to be very unstable at low ionic strength at 37 °C, but this instability was overcome by the addition of either SDS-purified cell wall or protease inhibitor (PMSF) to the enzyme or after purification of the glucosaminidase to apparent homogeneity.

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1995-10-01
2021-10-18
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