Cloning and expression of a thermophilic α-amylase gene from Bacillus stearothermophilus in Escherichia coli

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Abstract
A 6.4 Kb HindIII fragment of Bacillus stearothermophilus DY-5 DNA cloned in Escherichia coli using pBR322 as a vector was shown to direct the synthesis of a thermophilic α-amylase. In attempts to reduce the size of the insert, the α-amylase gene was shown to be contained in a 3.1 Kb HindIII-BamHI fragment of the donor strain DNA. The α-amylase gene was stably maintained and expressed efficiently in E. coli. The enzymic properties of α-amylase produced in E. coli closely resembled those of the donor strain α-amylase and the temperature range for the maximal activity was from 65° C to 80° C. Nearly 100% of the activity remained after heating at 80° C for 15 min. The α-amylase was shown to be accumulated in the periplasmic space. It was purified to a nearly homogenous protein with a molecular weight of 61,000, which was very similar in size to that produced by B. stearothermophilus DY-5.