Chimeric gene construct coding for bi-functional enzyme endowed with endoglucanase and phytase activities
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S H O R T CO M MU N I C A T I O N
Chimeric gene construct coding for bi-functional enzyme endowed with endoglucanase and phytase activities V. Arunodai Reddy · K. Venu · D. E. C. S. Rao · K. V. Rao · V. D. Reddy
Received: 17 March 2008 / Revised: 29 September 2008 / Accepted: 8 October 2008 / Published online: 6 November 2008 © Springer-Verlag 2008
Abstract Phytase and endoglucanase enzymes are being widely used as feed additives in poultry industry. In our earlier studies, the Bacillus phytase, when expressed in Escherichia coli, was found in inclusion bodies, whereas endoglucanase was found in active soluble form. Herein, we report the development of a chimeric gene construct coding for »73 kDa fusion protein and its over-expression in E. coli in soluble form. The novel enzyme exhibited both endoglucanase and phytase activities across broad pH (4.0– 8.0) and temperature (25–75°C) ranges. As such, the bifunctional enzyme seems promising and might serve as a potential feed additive for enhanced nutrition uptake in monogastric animals. Keywords Escherichia coli · Endoglucanase · Fusion protein · Over-expression · Phytase activity
Introduction Phytate is the main storage form of phosphorus in the grains of cereals, legumes and oilseeds (Harland and Morris 1995). Monogastric animals like poultry, swine and Wsh cannot assimilate this form of phosphorus (Baruah et al. 2005). FortiWcation of animal feed with inorganic phos-
Communicated by Jorge Membrillo-Hernández. V. A. Reddy · K. Venu · D. E. C. S. Rao · K. V. Rao · V. D. Reddy (&) Centre for Plant Molecular Biology, Osmania University, Hyderabad 500 007, India e-mail: [email protected] V. A. Reddy BITS-Pilani, Zuarinagar, Goa 403 726, India
phate not only adds to the cost but also results in environmental pollution due to the undigested phytate remnant in the excreta. Phytase catalyzes the hydrolysis of phytate into inorganic phosphate and myo-inositol. Supplementation of animal feed with phytase improves the uptake of phytate phosphorus and essential elements besides reducing environmental pollution. Fungal phytases are currently being used worldwide as feed additives (Kim et al. 1999). Thermo-tolerant phytases from Bacillus might serve as an alternative source of the enzyme. However, commercialization of Bacillus phytase met with little success owing to its low production eYciency (Kerovuo and Tynkkynen 2000). Escherichia coli has been widely used as an expression host for the production of recombinant proteins for industrial purpose, owing to its simplicity, economic viability, rapid high-density cultivation, well characterized genetics, and availability of large number of compatible expression systems (Sorensen and Mortensen 2005; Sawabe et al. 2007; Kamarthapu et al. 2008). In our earlier studies, expression of Bacillus phytase (GenBank Accession No. EF 536824) in E. coli BL21 (DE3) resulted in the formation of inclusion bodies leading to expensive and time consuming downstream processing for recovery of active enzyme (Rao et al. 2008). Another hydrolyti
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