The crystals of beta-amylase from Bacillus cereus belong to space group P21 with the following cell dimensions: a = 57.70 A, b = 92.87 A, c = 65.93 A, and beta =101.95 degrees. The structures of free and maltose-bound beta-amylases were determined by X-ray crystallography at 2.1 and 2.5 A with R-factors of 0.170 and 0.164, respectively. The final model of the maltose-bound form comprises 516 amino acid residues, four maltose molecules, 275 water molecules, one Ca2+, one acetate, and one sulfate ion. The enzyme consists of a core (beta/alpha)8-barrel domain (residues 5-434) and a C-terminal starch-binding domain (residues 435-613). Besides the active site in the core where two maltose molecules are bound in tandem, two novel maltose-binding sites were found in the core L4 region and in the C-terminal domain. The structure of the core domain is similar to that of soybean beta-amylase except for the L4 maltose-binding site, whereas the C-terminal domain has the same secondary structure as domain E of cyclodextrin glucosyltransferase. These two maltose-binding sites are 32-36 A apart from the active site. These results indicate that the ability of B. cereus beta-amylase to digest raw starch can be attributed to the additional two maltose-binding sites.

2-Aminophenol 1,6-dioxygenase was purified from the cell extracts of Pseudomonas sp. AP-3 grown on 2-aminophenol. The product from 2-aminophenol by catalysis of the purified enzyme was identified as 2-aminomuconic 6-semialdehyde by gas chromatographic and mass spectrometric analyses. The molecular mass of the native enzyme was 140 kDa based on gel filtration. It was dissociated into molecular mass subunits of 32 (␣-subunit) and 40 kDa (␤-subunit) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the dioxygenase was a heterotetramer of ␣ 2 ␤ 2 . The genes coding for the ␣-and ␤-subunits of the enzyme were cloned and sequenced. Open reading frames of the genes (amnA and amnB) were 816 and 918 base pairs in length, respectively. The amino acid sequences predicted from the open reading frames of amnA and amnB corresponded to the NH 2 -terminal amino acid sequences of the ␣-subunit (AmnA) and ␤-subunit (AmnB), respectively. The deduced amino acid sequences of AmnB showed identities to some extent with HpaD (25.4%) and HpcB (24.4%) that are homoprotocatechuate 2,3-dioxygenases from Escherichia coli W and C, respectively, belonging to class III in the extradiol dioxygenases. On the other hand, AmnA had identity (23.3%) with only AmnB among the enzymes examined.Dioxygenases catalyzing the fission of benzene rings are key enzymes in the metabolic pathways of aromatic compounds by microorganisms. Most of these kinds of previously reported dioxygenases attack monocyclic aromatic compounds with two adjacent hydroxyl groups such as catechol and protocatechuic acid and open the benzene rings through the intradiol or extradiol fission reaction (1, 2). However, some bacteria have been reported to synthesize dioxygenases that cleave the benzene rings of hydroquinone (3-5) and gentisic acid (6, 7).In our investigations on the microbial metabolism of anilines, we isolated several microorganisms capable of growing on 2-aminophenol as the sole carbon, nitrogen, and energy source. When one isolate, Pseudomonas sp. AP-3, grows with this substrate, it synthesizes an enzyme acting on 2-aminophenol. This enzyme was partially purified with a 103-fold increase in the specific activity from its cell extracts. We proposed that the enzyme is a dioxygenase catalyzing the ring fission of 2-aminophenol with the consumption of 1 mol of O 2 per mol of substrate (8).Our aim was to advance the purification of 2-aminophenol 1,6-dioxygenase from Pseudomonas sp. AP-3 and elucidate the molecular and catalytic properties of the purified enzyme. Because the product from 2-aminophenol by catalysis of the enzyme is rapidly and nonenzymatically converted into picolinate (8, 9), the real product has remained unverified. Furthermore, we attempted the cloning and sequencing of the gene of the dioxygenase, which would determine the category of this enzyme in the dioxygenase groups.Recently, Lendenmann and Spain (10) reported the purification and characterization of the 2-aminophenol 1,6-dioxygenase from nitrobenzene-degrading Pseudomo...

Tannase (tannin acyl hydrolase, EC 3 .1.1.20) was purified from the culture broth of Candida sp. by rivanol fractionation and chromatography of columns of ECTEOLA-cellulose , Sepharose 6B and Sephadex G-200. The purified tannase was homogeneous on SDS-poly

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