Display of Bombyx mori Alcohol Dehydrogenases on the Bacillus subtilis Spore Surface to Enhance Enzymatic Activity under Adverse Conditions

Alcohol dehydrogenases (ADHs) are oxidoreductases catalyzing the reversible oxidation of alcohols to corresponding aldehydes or ketones accompanied by nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as coenzyme. ADHs attract major scientific and industrial interest for the evolutionary perspectives, afforded by their wide occurrence in nature, and for their use in industrial synthesis. However, the low activity of ADHs under extremes of pH and temperature often limits their application. To obtain ADH with high activity, in this study, we used Bombyx mori alcohol dehydrogenases (BmADH) as foreign gene and constructed a recombinant integrative plasmid pJS700-BmADH. This pJS700-BmADH was transformed into Bacillus subtilis by double cross-over and produced an amylase inactivated mutant. The fusion protein containing BmADH was expressed on the spore surface and recognized by BmADH-specific antibody. We also assayed the alcohol dehydrogenase activity of the fusion protein together with the native BmADH at different pH and temperature levels, which indicated the recombinant enzyme exhibits activity over wider ranges of temperature and pH than its native form, perhaps due to the resistance properties of B. subtilis spores against adverse conditions

Proteomic profiling of liver from Elaphe taeniura, a common snake in eastern and southeastern Asia

Snake liver has been implicated in the adaptation of snakes to a variety of habitats. However, to date, there has been no systematic analysis of snake liver proteins. In this study, we undertook a proteomic analysis of liver from the colubrid snake Elaphe taeniura using a combination of two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flightmass spectrometry (MALDI-TOF MS). We also constructed a local protein sequence database based on transcriptome sequencing to facilitate protein identification. Of the 268 protein spots revealed by 2-DE 109 gave positive MS signals, 84 of which were identified by searching the NCBInr, Swiss-Prot and local databases. The other 25 protein spots could not be identified, possibly because their transcripts were not be stable enough to be detected by transcriptome sequencing. GO analysis showed that most proteins may be involved in binding, catalysis, cellular processes and metabolic processes. Forty-two of the liver proteins identified were found in other reptiles and in amphibians. The findings of this study provide a good reference map of snake liver proteins that will be useful in molecular investigations of snake physiology and adaptation

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

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

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

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

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