Cloning strategy.

<p>(A) The construction of integration plasmid pJS700-BmADH. The fragments <i>amyE</i> 5′ and <i>amyE</i> 3′ in plasmid are homologous to the upstream and downstream of the amylase gene in <i>B. subtilis</i> 168 (trp-), respectively; <i>Em</i>^r, erythromycin resistant site; <i>CotC</i>, a <i>B. subtilis</i> spore coat protein encoding gene. (B) The schematic integration of CotC-BmADH to <i>amyE</i> locus. Arrows indicate the positions of primer pairs used in the site-directed PCR for confirmation of the correct integration.</p

Alcohol dehydrogenase specific activities at different temperatures.

<p>*indicates that the difference is significant (P<0.05).</p><p>Mean ± SD from three independent experiments are shown.</p

SDS-PAGE analysis of CotC-BmADH and Western blotting.

<p>(A) SDS-PAGE stained by coomassie-blue. (B) CotC-BmADH detected by BmADH specific antibody. Lane 1, <i>B. subtilis</i> 168 (trp-); lane 2, CotC-BmADH strain.</p

Relative remained activity of BmADH and CotC-BmADH after incubation at 37°C for 30 min.

<p>The activity was assayed at pH 9.0, 25°C. *indicates the difference is significant (P<0.05).</p

Identification of the mutant with CotC-BmADH integration at <i>amyE</i> locus.

<p>(A) Analysis of amylase activity. CotC-BmADH mutant strains and <i>B. subtilis</i> 168 (trp-) wide type grew on the starch-containing LB plate before (1) and after (2) being stained by iodine. The integration of CotC-BmADH might disrupt <i>amyE</i> and made the strain amylase deficient, while the while wide type strain showed a big white halo around colony due the secretion of amylase. (B) Site-directed PCR analysis using different primer pairs. Marker, <i>λ</i> DNA digested by <i>Eco</i>T14I; W: <i>B. subtilis</i> 168 (trp-) wide typ; M: CotC-BmADH mutant; primer pairs used in PCR are labeled below agarose gel.</p

Alcohol dehydrogenase specific activities at different pHs.

<p>*indicates that the difference is significant (P<0.05).</p><p>Mean ± SD from three independent experiments are shown.</p