Cloning, expression and characterization of a pectate lyase from Paenibacillus sp. 0602 in recombinant Escherichia coli
- Equal contributors
1 National Engineering Laboratory for Industrial Enzymes and Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
2 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
3 Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville Campus, 381 Royal Parade, Parkville, Victoria 3052, Australia
4 Department of Biochemistry and Molecular Biology and ARC Centre of Excellence in Structural and Functional Microbial Genomics, Faculty of Medicine, Monash University, Clayton, Victoria 3800, Australia
BMC Biotechnology 2014, 14:18 doi:10.1186/1472-6750-14-18Published: 10 March 2014
Biotechnological applications of microbial pectate lyases (Pels) in plant fiber processing are considered as environmentally friendly. As such, they become promising substitutes for conventional chemical degumming process. Since applications of Pels in various fields are widening, it is necessary to explore new pectolytic microorganisms and enzymes for efficient and effective usage. Here, we describe the cloning, expression, characterization and application of the recombinant Pel protein from a pectolytic bacterium of the genus Paenibacillus in Escherichia coli.
A Pel gene (pelN) was cloned using degenerate PCR and inverse PCR from the chromosomal DNA of Paenibacillus sp. 0602. The open reading frame of pelN encodes a 30 amino acid signal peptide and a 445 amino acid mature protein belonging to the polysaccharide lyase family 1. The maximum Pel activity produced by E. coli in shake flasks reached 2,467.4 U mL−1, and the purified recombinant enzyme exhibits a specific activity of 2,060 U mg−1 on polygalacturonic acid (PGA). The maximum activity was observed in a buffer with 5 mM Ca2+ at pH 9.8 and 65°C. PelN displays a half-life of around 9 h and 42 h at 50°C and 45°C, respectively. The biochemical treatment achieved the maximal reduction of percentage weight (30.5%) of the ramie bast fiber.
This work represents the first study that describes the extracellular expression of a Pel gene from Paenibacillus species in E. coli. The high yield of the extracellular overexpression, relevant thermostability and efficient degumming using combined treatments indicate its strong potential for large-scale industrial production.