Overproduction of an alkali- and thermo-stable xylanase in tobacco chloroplasts and efficient recovery of the enzyme

Overproduction of cellulolytic enzymes through conventional nuclear transformation approaches posed a major challenge as they can potentially degrade the cell wall components and thereby affect transgenic plant growth and development. In this study, we have tested the possibility to over produce an alkali-thermostable xylanase gene from Bacillus sp. Strain NG-27 in tobacco plants through chloroplast expression. Our results showed that the xylanase expression can reach up to 6% of the total soluble protein, a value comparable to high level expression reported for several non-cellulolytic proteins in tobacco chloroplasts. The chloroplast-expressed xylanase retained its activity even when the leaves were dried under sun or at 42degreesC, offering flexibility in the agricultural system in transport and storage. The recombinant enzyme was purified to homogeneity using single step chromatography with more than 85% recovery. Most importantly, transgenic plants were indistinguishable from the control untransformed plants in their morphology, growth and in seed setting. These results open up new avenues for large scale production of several other industrially useful cellulolytic enzymes through chloroplast expression.