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

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 alpha-amylase. In attempts to reduce the size of the insert, the alpha-amylase gene was shown to be contained in a 3.1 Kb HindIII - BamHI fragment of the donor strain DNA. The alpha-amylase gene was stably maintained and expressed efficiently in E. coli. The enzymic properties of alpha-amylase produced in E. coli closely resembled those of the donor strain alpha-amylase and the temperature range for the maximal activity was from 65 degrees C to 80 degrees C. Nearly 100% of the activity remained after heating at 80 degrees C for 15 min. The alpha-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.