Cloning, expression and characterization of a pectate lyase from Paenibacillus sp. 0602 in recombinant Escherichia coli

Abstract

BackgroundBiotechnological 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.ResultsA 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.ConclusionsThis 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.

Improving the thermoactivity and thermostability of pectate lyase from Bacillus pumilus for ramie degumming

“…Therefore, thermostable enzymes that have high catalytic activity under alkaline conditions are desirable for industrial degumming applications. For this purpose, alkaline Pels have been cloned from microbes (Berensmeier et al 2004;Boland et al 2010;Hatada et al 2001;Li et al 2014;Tardy et al 1997;Wang et al 2014;Zhang et al 2013) or from metagenomic DNA obtained from alkaline environment soils . In this study, BpPel from B. pumilus (ATCC 7061) was successfully engineered to improve its catalytic activity and thermostability for potential industrial application for ramie degumming process.…”

“…However, thus far, enzymatic degumming in the textile industry has been hampered by the high cost and partial degumming performance of the currently available enzymes. Microbial or enzymatic degumming processes are commonly combined with a chemical degumming process for maximal effect to meet the industrial requirements (Basu et al 2009;Li et al 2014). Due to the increasing interest and potential markets for enzymatic degumming, alkaline Pels have been obtained from various sources, cloned, and characterized (Berensmeier et al 2004;Kapoor et al 2001;Wang et al 2014;Zhang et al 2013); however, few Pels were found to be economically efficient for industrial applications owing to their low activity or poor stability under the required processing conditions (operation at moderate temperature [40-70°C] and alkaline pH [8][9][10][11]).…”

Directed Evolution and Structural Analysis of Alkaline Pectate Lyase from the Alkaliphilic Bacterium Bacillus sp. Strain N16-5 To Improve Its Thermostability for Efficient Ramie Degumming

“…Therefore, enzymes that are thermostable and active under alkaline conditions are desirable for industrial degumming applications. Because of the increasing interest in and potential market for enzymatic degumming, various alkaline Pels have recently been cloned and isolated from microbes (17,25,26,(28)(29)(30)(31)(32)(33)(34) or …”

“…However, enzymatic degumming has not been widely used in the textile industry because of the high cost and unsatisfactory degumming efficiency of currently available enzymes. Thus, in the textile industry, microbial or enzymatic degumming processes are commonly combined with chemical degumming processes for maximal efficiency (24,25).…”

“…However, enzymatic degumming has not been widely used in the textile industry because of the high cost and unsatisfactory degumming efficiency of currently available enzymes. Thus, in the textile industry, microbial or enzymatic degumming processes are commonly combined with chemical degumming processes for maximal efficiency (24,25).In enzymatic degumming, an alkaline environment at a moderate temperature is critical for effective degumming and improvement of fiber quality because the gumlike materials are soluble under these conditions (26); thus, operation of industrial degumming processes is generally carried out at moderate temperatures (40°C to 70°C) and alkaline pH (pH 8 to 11) (27). Therefore, enzymes that are thermostable and active under alkaline conditions are desirable for industrial degumming applications.…”