Construction and Expression of a Novel Paenibacillus polymyxa GS01 Bifunctional xyn43A-lin16A Gene through Overlap Extension PCR

Abstract

A shotgun method was adopted to clone the beta-xylanase and lichenase genes from a genomic library of a Paenibacillus polymyxa GS01 genome library. Also, a fusion enzyme, Xyn3A-Lin16A, was designed by overlap extension polymerase chain reaction (PCR). The cloned Xyn3A and Lin16A proteins were successfully expressed and exhibited both xylanase and lichenase activities. The xyn43A and lin16A gene amplicons were 1,917 bp and 714 bp in size and encoded proteins of 635 and 238 amino acids, respectively. The Xyn43A and Lin16A gene products showed predicted molecular masses of 65 and 24 kDa with respective calculated pis of 5.97 and 5.77, respectively. Furthermore, the fusion enzyme gene, Xyn43A-Lin16A, was 4,466 bp in length and encoded a protein of 847 amino acids, with apparent molecular mass of 89 kDa and a calculated pI of 5.93. This fusion enzyme showed optimum activity at pH 6.0-7.0 and 50 degrees C. Thus, the xyn43A and lin16A genes from P. polymyxa GS01were able to exist in tandem, and recombinant DNA technologies can be used to improve enzyme productivity. Therefore, the development of functional fusion enzymes (xylanase-lichenase) using recombinant DNA technologies may lead to further improvements and their successful enzyme engineering in industrial application.