Cloning of the Thermostable α-Amylase Gene From <I>Pyrococcus woesei</I> in <I>Escherichia coli</I> : Isolation and Some Properties of the Enzyme

Abstract

Pyrococcus woesei (DSM 3773) alpha-amylase gene was cloned into pET21d(+) and pYTB2 plasmids, and the pET21d(+)alpha-amyl and pYTB2alpha-amyl vectors obtained were used for expression of thermostable alpha-amylase or fusion of alpha-amylase and intein in Escherichia coli BL21(DE3) or BL21(DE3)pLysS cells, respectively. As compared with other expression systems, the synthesis of alpha-amylase in fusion with intein in E. coli BL21(DE3)pLysS strain led to a lower level of inclusion bodies formation-they exhibit only 35% of total cell activity-and high productivity of the soluble enzyme form (195,000 U/L of the growth medium). The thermostable alpha-amylase can be purified free of most of the bacterial protein and released from fusion with intein by heat treatment at about 75 degrees C in the presence of thiol compounds. The recombinant enzyme has maximal activity at pH 5.6 and 95 degrees C. The half-life of this preparation in 0.05 M acetate buffer (pH 5.6) at 90 degrees C and 110 degrees C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following incubation for 2 h at 120 degrees C. Maltose was the main end product of starch hydrolysis catalyzed by this alpha-amylase. However, small amounts of glucose and some residual unconverted oligosaccharides were also detected. Furthermore, this enzyme shows remarkable activity toward glycogen (49.9% of the value determined for starch hydrolysis) but not toward pullulan.

Efficient solubilization, purification of recombinant extracellular α-amylase from pyrococcus furiosus expressed as inclusion bodies in Escherichia coli

“…The PFA puriWed by alkaline method showed a half-life of 3 h at 120°C and retained over 10% of its maximal activity even after 40 min of incubation at 128°C (Fig. 5b), which were similar to the results reported before [7,8,10].…”

“…Results have shown that coexpression of PFA with thioredoxin in E. coli at 18°C is able to diminish inclusion body formation [5], and fusion expression of PFA with intein in E. coli also lead to a lower level of inclusion body formation [7]. However, all these soluble expressions resulted in the lower levels of total recombinant protein production.…”

Enzimas termoestáveis: fontes, produção e aplicação industrial

“…O uso direto dos organismos hipertermófilos para produção das enzimas tem se mostrado inviável, em função das dificuldades de cultivos dos mesmos e baixa expressão dos genes envolvidos. A alternativa que se busca é a expressão desses genes em organismos mais adaptados a processos fermentativos industriais, como Escherichia coli ou leveduras 11,90 . Os fungos, embora sejam organismos eucarióticos e, portanto, com termofilia moderada, são também bons produtores de amilases termoestáveis.…”

Transcriptional and Biochemical Analysis of Starch Metabolism in the Hyperthermophilic Archaeon Pyrococcus furiosus

“…For example, the recombinant form of a putative extracellular ␣-amylase (PF0477), which produces maltooligosaccharides from starch, has been characterized (9, 25), and an ␣-amylase closely related to PF0477 was characterized from the related species, P. woesei (13,18,27). In addition, an extracellular starch-hydrolyzing amylopullulanase has been characterized from P. furiosus and from the related hyperthermophile Thermococcus litoralis (7).…”