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Chitin deacetylase, the enzyme that catalyzes the hydrolysis of acetamido groups of N-acetylglucosamine in chitin, has been purified to homogeneity from mycelial extracts of the fungus Mucor rouxii and further characterized. The enzyme exhibits a low pI (-3). Its apparent molecular mass was determined to be =75 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and -80 kDa by size-exclusion chromatography, suggesting that the enzyme exists as a monomer. Carbohydrate analysis of purified chitin deacetylase revealed that the enzyme is a high-mannose glycoprotein and that its carbohydrate content is -30% by weight. Chitin deacetylase is active on several chitinous substrates and chitin derivatives. The enzyme requires at least four N-acetylglucosamine residues (chitotetraose) for catalysis, and it is inhibited by carboxylic acids, particularly acetic acid. When glycol chitin (a water-soluble chitin derivative) was used as substrate, the optimum temperature for enzyme activity was determined to be -.50°C and the optimum pH was -4.5.

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To study the in vivo role of TFIID in the transcriptional regulation of hepatic genes, we generated mice with liver-specific disruption of the TAF10 gene. Inactivation of TAF10 in hepatocytes resulted in the dissociation of TFIID into individual components. This correlated with the downregulation of most hepatocyte-specific genes during embryonic life and a defect in liver organogenesis. Unexpectedly, however, the transcription of less than 5% of active genes was affected by TAF10 inactivation and TFIID disassembly in adult liver. The extent of changes in transcription of the affected genes was dependent on the timing of their activation during liver development, relative to that of TAF10 inactivation. Furthermore, TFIID dissociation from promoters leads to the re-expression of several postnatally silenced hepatic genes. Promoter occupancy analyses, combined with expression profiling, demonstrate that TFIID is required for the initial activation or postnatal repression of genes, while it is dispensable for maintaining ongoing transcription.

Chitin deacetylase (EC 3.5.1.41) hydrolyzes the N-acetamido groups of N-acetyl-D-glucosamine residues in chitin. A cDNA to the Mucor rouxu mRNA encoding chitin deacetylase was isolated, characterized, and sequenced. Protein sequence comparisons revealed significant imilarities of the fungal chitin deacetylase to rhizobial nodB proteins and to an uncharacterized protein encoded by a Bacillus stearothermophilus open reading frame. These data suggest the functional homology of these evolutionarily distant proteins. NodB is a chitooligosaccharide deacetylase essential for the biosynthesis of the bacterial nodulation signals, termed Nod factors. The observed similarity of chitin deacetylase to the B. stearothermophius gene product suggests that this gene encodes a polysaccharide deacetylase.Chitin exists in the cell wall of several Zygomycetes species in its deacetylated form, referred to as chitosan (1). A chitosan layer has also been identified in the spore wall of Saccharomyces cerevisiae (2). The biosynthesis of chitosan in fungi proceeds by the coordinated action of chitin synthase (EC 2.4.1.16) and chitin deacetylase (EC 3.5.1.41). The former enzyme synthesizes chitin by polymerization of N-acetylglucosaminyl residues from UDP-N-acetylglucosamine, whereas the latter hydrolyzes the N-acetamido groups on the nascent chitin chains (3,4).Chitosan is a biopolymer with unique properties favorable for a broad variety of industrial and biomedical applications (5-7). Presently, chitosan is produced by the thermochemical deacetylation of crab chitin. To develop an alternative enzymatic process for chitosan production, we have initiated a study of fungal chitin deacetylases.Chitin deacetylase from the fungus Mucor rouxii has been purified to homogeneity (8). The enzyme is an acidic glycoprotein of =75 kDa with =30% (wt/wt) carbohydrate content. Further biochemical characterization revealed that the enzyme has a very stringent speciflcity for (31-4-linked N-acetylglucosamine homopolymers, requires at least four residues (chitotetraose) for catalysis, and can achieve extensive deacetylation on chitin polymers.We report here the cloning of a cDNA that encodes M. the AZAP vector, and the packaging extracts were bought from Stratagene. The Sequenase 2.0 kit was purchased from United States Biochemical. Immobilon-P and nitrocellulose membranes were purchased from Millipore. Restriction enzymes were obtained from Minotech. Synthetic oligonucleotides were obtained from the Institute of Molecular Biology and Biotechnology Microchemistry Facility.Growth of M. rouxiu Mycelia. M. rouxii was grown with vigorous shaking at 28°C in a medium containing 0.3% yeast extract, 1% peptone, and 2% glucose; the pH was adjusted to 4.5 with H2SO4 (1). The medium was inoculated with 108 spores per liter, and the mycelia were harvested at early growth phase by fitration.Enzyme Purification. Chitin deacetylase was purified to homogeneity from mycelial extracts of the fungus M. rouxii as described (8).Protein Sequencing. The amino-terminal seq...