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

✍ Scribed by Tsukagoshi, N. ;Ihara, H. ;Yamagata, H. ;Udaka, S.

• Publisher: Springer
• Year: 1984
• Tongue: English
• Weight: 584 KB
• Volume: 193
• Category: Article
• ISSN: 0026-8925
• DOI: 10.1007/bf00327414

✦ Synopsis

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.