RESUMO
A bacterial trehalose phosphorylase (TPase; EC 2.4.1.64) was purified from the culture supernatant of Bacillus stearothermophilus SK-1 to apparent homogeneity, and some properties were investigated. Furthermore, a gene from SK-1 responsible for the TPase was cloned by Southern hybridization with a degenerate oligonucleotide probe synthesized on the basis of the N-terminal sequence of the purified enzyme. The Mr of the enzyme was estimated to be 150,000 by gel filtration and 83,000 by SDS-PAGE, so the enzyme is likely to be a homodimer. The enzyme had optimum activity at pH 7.0-8.0 or nearby and the optimum temperature was about 75 degrees C. The deduced amino acid sequence of the SK-1 TPase encodes a theoretical protein with a Mr of 87,950. Alignment of amino acid sequences with a maltose phosphorylase from Lactobacillus brevis the crystal structure and active site of which had been analyzed suggested that these two phosphorylases evolved from a common ancestor. The Escherichia coli cells harboring the plasmid containing the cloned TPase gene had about 100 times the activity of SK-1.
Assuntos
Geobacillus stearothermophilus/enzimologia , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Escherichia coli , Geobacillus stearothermophilus/genética , Glucosiltransferases/química , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Cinética , Dados de Sequência Molecular , Peso Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , TemperaturaRESUMO
The maltose phosphorylase (MPase) gene of Bacillus sp. strain RK-1 was cloned by PCR with oligonucleotide primers designed on the basis of a partial N-terminal amino acid sequence of the purified enzyme. The MPase gene consisted of 2,655 bp encoding a theoretical protein with a Mr of 88,460, and had no secretion signal sequence, although most of the MPase activity was detected in the culture supernatant of RK-1. This cloned MPase gene and the trehalose phosphorylase (TPase) gene from Bacillus stearothermophilus SK-1 were efficiently expressed intracellularly under the control of the Bacillus amyloliquefaciens alpha-amylase promoter in Bacillus subtilis. The production yields were estimated to be more than 2 g of enzyme per liter of medium, about 250 times the production of the original strains, in a simple shake flask. About 60% of maltose was converted into trehalose by the simultaneous action of both enzymes produced in B. subtilis.
