Amino Acid Sequence and Molecular Structure of an Alkaline Amylopullulanase from Bacillus That Hydrolyzes α-1,4 and α-1,6 Linkages in Polysaccharides at Different Active Sites

Yuji Hatada,Kazuaki Igarashi,Katsuya Ozaki,Katsutoshi Ara,Jun Hitomi,Tohru Kobayashi,Shuji Kawai,Tomoyoshi Watabe,Susumu Ito

doi:10.1074/jbc.271.39.24075

Yuji Hatada, Kazuaki Igarashi + Show 7 more

Open Access

https://doi.org/10.1074/jbc.271.39.24075

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Abstract

An amylopullulanase from alkalophilic Bacillus sp. KSM-1378 hydrolyzes both alpha-1,6 linkages in pullulan and alpha-1,4 linkages in other polysaccharides, with maximum activity in each case at an alkaline pH, to generate oligosaccharides (Ara, K., Saeki, K., Igarashi, K., Takaiwa, M., Uemura, T., Hagihara, H., Kawai, S., and Ito, S. (1995) Biochim. Biophys. Acta 1243, 315-324). Here, we report the molecular cloning and sequencing of the gene for and the structure of this enzyme and show that its dual hydrolytic activities are associated with two independent active sites. The structural gene contained a single, long open reading frame of 5,814 base pairs, corresponding to 1,938 amino acids that included a signal peptide of 32 amino acids. The molecular mass of the extracellular mature enzyme (Glu33 through Leu1938) was calculated to be 211,450 Da, a value close to the 210 kDa determined for the amylopullulanase produced by Bacillus sp. KSM-1378. The amylase and the pullulanase domains were located in the amino-terminal half and in the carboxyl-terminal half of the enzyme, respectively, being separated by a tandem repeat of a sequence of 35 amino acids. Four regions, designated I, II, III, and IV, were highly conserved in each catalytic domain, and they included a putative catalytic triad Asp550-Glu579-Asp645 for the amylase activity and Asp1464-Glu1493-Asp1581 for the pullulanase activity. The purified enzyme was rotary shadowed at a low angle and observed by transmission electron microscopy; it appeared to be a "castanet-like" or "bent dumbbell-like" molecule with a diameter of approximately 25 nm.

Highlights

We have found and characterized some unique debranching enzymes, such as an alkaline pullulanase (Ara et al, 1992), an alkali-resistant neopullulanase (Igarashi et al, 1992), and an alkaline isoamylase (EC 3.2.1.68) (Ara et al, 1994), in strains of alkalophilic Bacillus, and these enzymes can be used as effective additives in dishwashing and laundry detergents under
This APase is unique in that it efficiently hydrolyzes the ␣-1,6 linkages of pullulan, as well as the ␣-1,4 linkages of amylose, amylopectin, and glycogen at alkaline pH values, whereas other APases, the type that have frequently been found in cultures of some thermophiles, are all active at acid or neutral pH
After transformation of E. coli cells with the ligation mixture, an attempt was made to identify amylase- and pullulanasepositive transformants by the formation of halos on starchazure and red pullulan agar plates, respectively. No transformants with both amylase and pullulanase activities nor any amylase-positive transformants were obtained under the cloning conditions that employed the E. coli pBR322 system

Summary

Introduction

The purified intact APase (0.5 mg) was partially digested with papain (5 milliunits; Sigma) at 25 °C for 10 min in 2 ml of 10 mM Tris-HCl buffer (pH 8.0) to generate the two fragments, designated the 114-kDa amylose-hydrolyzing polypeptide and the 102-kDa pullulan-hydrolyzing polypeptide (Ara et al, 1996). To determine the amino-terminal sequence of APase, the purified preparation of enzyme described above was further purified by nondenaturing PAGE (10% (w/v) acrylamide, 0.5-mm thickness).

Results

Conclusion