Nucleotide Sequence of the Gene for an Alkaline Endoglucanase from an Alkalophilic<i>Bacillus</i>and Its Expression in<i>Escherichia coli</i>and<i>Bacillus subtilis</i>

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  • Nucleotide Sequence of the Gene for an Alkaline Endoglucanase from an Alkalophilic Bacillus and Its Expression in Escherichia coli and Bacillus subtilis.

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The gene for an alkaline endoglucanase from the alkalophilic Bacillus sp. KSM-64 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. The nucleotide sequence of a 4.1-kb region of the HindIII insert had two open reading frames, ORF-1 and ORF-2. The protein deduced from ORF-1 was composed of 244 amino acids with an Mr of 27, 865. Subcloning analysis proved that the alkaline endoglucanase was encoded by ORF-2 (822 amino acids with an Mr of 91, 040). Upstream from ORF-2, there were three consensus like sequences of the sigma A-type promoter of Bacillus subtilis, a putative Shine-Dalgarno sequence (AGGAGGT), and a catabolite repression operator-like seauence (TGTAAGCGGTTAACC). The HindIII insert was subcloned into a shuttle vector, pHY300PLK, and the encoded alkaline endoglucanase gene was highly expressed both in E.coli and B.subtilis. One of the three promoter-like sequences in ORF-2 could be suitable for high levels of enzyme expresson in both host organisms.

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