Clostridium thermohydrosulfuricum 39E, a gram-positive thermophilic anaerobic bacterium, produced a cyclodextrin (CD)-degrading enzyme, cyclodextrinase (CDase) (EC 3.2.1.54). The enzyme was purified to homogeneity from Escherichia coli cells carrying a recombinant multicopy plasmid that contained the gene encoding for thermophilic CDase. The purified enzyme was a monomer with an Mr of 66,000 ± 2,000. It showed the highest activity at pH 5.9 and 65°C. The enzyme hydrolyzed a-, j3-, and 'y-CD and linear maltooligosaccharides to yield maltose and glucose. The Km values for a-, 0-, and fy-CD were 2.5, 2.1, and 1.3 mM, respectively. The rates of hydrolysis for polysaccharides (starch, amylose, amylopectin, and pullulan) were less than 5% of the rate of hydrolysis for a-CD. The entire nucleotide sequence of the CDase gene was determined. The deduced amino acid sequence of CDase, consisting of 574 amino acids, showed some similarities with those of various amylolytic enzymes.Cyclodextrins (CDs) are cyclic oligosaccharides consisting of a-1,4-linked 6-, 7-, or 8-glucopyranose units usually referred to as a-, 1-, or -y-CDs, respectively. One of the remarkable properties of the CDs is their resistance to hydrolysis by the common starch-splitting enzymes. Among the bacterial amylases, only those from Bacillus subtilis (27), Pseudomonas sp. (14), and Flavobacterium sp. (2) catalyze the hydrolysis of CDs; for the first of these enzymes, CDs are poor substrates, while the latter two enzymes degrade CDs faster than starch. Until now, only four strains, B. macerans (7), B. coagulans (16), B. sphaericus (29), and a Bacillus sp. (43), have been known to produce cyclomaltodextrinase (EC 3.2.1.54, cyclomaltodextrin hydrolase, decycling) (CDase), an enzyme which effectively hydrolyzes CDs and linear maltodextrins. In contrast to the extensive information concerning amylolytic enzymes, studies on CDases have been confined to these four enzymes. To our best knowledge, nothing to date has been reported about the gene structure of CDases.In the course of our studies on thermophilic bacteria, we recently characterized a thermophilic CDase from Clostnidium thermohydrosulfuricum 39E (33). This enzyme was partially purified (about 205-fold), and many of its properties were determined. However, because the CDase was not purified to homogeneity, important molecular and kinetic properties of the enzyme remain unknown. In the present study, we report on the purification and detailed characterization of C thermohydrosulfuricum 39E CDase from Esch-

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In Escherichia coli, amino acid starvation results in the coordinate inhibition of a variety of metabolic activities, including fatty acid and phospholipid biosynthesis. By using primer extension analysis we identified the fabH promoter responsible for transcription of the fabH, fabD and fabG genes encoding fatty acid biosynthetic enzymes. The response of the fabH promoter to amino acid starvation was determined in vivo. Transcripts originating from the fabH promoter were quantified by employing a ribonuclease protection assay. The fabH promoter was subject to relA-dependent stringent control and was repressed approximately 4-fold upon amino acid starvation. The results suggest that inhibition of transcription initiation of lipid biosynthetic genes in starved cells contributes to the stringent control of lipid biosynthesis.

The amylopullulanase gene (apu) of the thermophilic anaerobic bacterium Thermoanaerobacterium saccharolyticum B6A-RI was cloned into Escherichia coli. The complete nucleotide sequence of the gene was determined. It encoded a protein consisting of 1,288 amino acids with a signal peptide of 35 amino acids. The enzyme purified from E. coli was a monomer with an Mr of 142,000 + 2,000 and had the same catalytic and thermal characteristics as the native glycoprotein from T. saccharolyticum B6A. Linear alignment and the hydrophobic cluster analysis were used to compare this amylopullulanase with other amylolytic enzymes. Both methods revealed strictly conserved amino acid residues among these enzymes, and it is proposed that Asp-594, Asp-700, and Glu-623 are a putative catalytic triad of the T. saccharolyticum B6A-RI amylopullulanase.

The amino acid sequences of cyclomaltodextrinase (CDase) from Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosuIfuricum 39E) and other amylolytic enzymes were compared by using linear alignment and hydrophobic cluster analysis. Two Asp and one Glu residue, which were considered to be the catalytic residues of the compared enzymes according to crystallographic or protein engineering experiments, were also conserved in CDase. ASPIRE, Asp4*' and GIu'~~ of the CDase were individually replaced by means of site-directed mutagenesis. The mutant enzymes completely lost activity, suggesting that these residues play an important role in catalysis.

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By using insertional mutagenesis we demonstrated that the rpmF gene encoding ribosomal protein L32, theplsX gene encoding a protein involved in membrane lipid synthesis and several fatty acid biosynthetic genes (fabH, fabD and fabG) are cotranscribed. Organization of these genes into an operon may play a role in the coordinate regulation of the synthesis of ribosomes and the cell membranes.

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