A novel maltotetraose-forming alkaline α-amylase from an alkalophilic Bacillus strain, GM8901

Abstract

Abstract An alkalophilic bacterium, Bacillus sp. strain GM8901, grown at pH 10.5 and 50°C, produced five alkaline amylases in culture broth. At an early stage of the bacterial growth, amylase I (amyl I) was produced initially and then, as cultivation progressed, four alkaline amylases, Amyl II, Amyl III, Amyl IV, and Amyl V, were produced from proteolytic degradation of Amyl I. A serine protease present in the culture medium was believed to be involved in Amyl I degradation. We purified Amyl I from the culture supernatant, by ammonium sulfate precipitation, heparin-Sepharose CL-6B column chromatography, phenyl-Toyopearl column chromatography, and Mono Q HR5/5 high-performance liquid chromatography. The molecular weight of Amyl I was estimated to be about 97,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amyl I had an extremely high optimal pH of 11.0 to 12.0 and was stable in a broad pH range of 6.0 to 13.0. Amyl I had an optimal temperature of 60°C and was stable up to 50°C. Thermostability was increased in the presence of Ca2+ and soluble starch. The enzyme required metal ions such as Ca2+, Mg2+, Cu2+, Co2+, Ag+, Zn2+, and Fe2+ for its enzyme activity and was inhibited by 1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride. According to the mode of action of Amyl I on starch, Amyl I was classified as an exo- and α-amylase. Amyl I produced maltotetraose predominantly from starch via intermediates such as maltohexaose and maltopentaose. The gene encoding the maltotetraose-forming alkaline amylase (Amyl I) was cloned from Bacillus sp. strain GM8901 into Escherichia coli JM83. The nucleotide sequence of the cloned 2.8 kb DNA revealed that it contains one open reading frame of 2,554 nucleotides without a translational stop codon. The deduced amino acid sequence for these 2,554 nucleotides is 848 amino acids including a signal peptide of 29 residues at its NH2-terminal end. The deduced amino acid sequence of Amyl I contains four conserved regions that constitute that active center of α-amylases. However, The deduced amino acid sequence of the mature Amyl I showed relatively low homology, 20–40%, with those of the saccharifying type α-amylases and the liquefying type α-amylases. The conserved regions and homologous region of Amyl I with other known α-amylases were restricted in the NH2-terminal part (about 450 amino acids) of Amyl I and the remaining COOH-terminal part (about 400 amino acids) showed no homology with other known amylases. When the amino acid sequence of amyl I was compared with those of exo-α-amylases and alkaline amylases, homologous regions which seemed to be involved in the alkalophilicity or the exo-cleavage mode of Amyl I were not found.