Extracellular Overexpression of Chitosanase from Bacillus sp. TS in Escherichia coli

Abstract

The chitosanase gene from a Bacillus sp. strain isolated from soil in East China was cloned and expressed in Escherichia coli. The gene had 1224 nucleotides and encoded a mature protein of 407 amino acid residues. The optimum pH and temperature of the purified recombinant chitosanase were 5.0 and 60 °C, respectively, and the enzyme was stable below 40 °C. The K m, V max, and specific activity of the enzyme were 1.19 mg mL(-1), 674.71 μmol min(-1) at 50 °C, and 555.3 U mg(-1), respectively. Mn(2+) was an activator of the recombinant chitosanase, while Co(2+) was an inhibitor. Hg(2+) and Cu(2+) inhibited the enzyme at 1 mM. The highest level of enzyme activity (186 U mL(-1)) was achieved in culture medium using high cell-density cultivation in a 7-L fermenter. The main products of chitosan hydrolyzed by recombinant chitosanase were (GlcN)3-6. The chitosanases was successfully secreted to the culture media through the widely used SecB-dependent type II pathway in E. coli. The high yield of the extracellular overexpression, relevant thermostability, and effective hydrolysis of commercial grade chitosan showed that this recombinant enzyme had a great potential for industrial applications.

Rational design and structure-based engineering of alkaline pectate lyase from Paenibacillus sp. 0602 to improve thermostability

“…Then, confirmed plasmids were expressed in E. coli BL21 (DE3) to obtain proteins. The proteins with His-tags at the C-terminus were purified by Ni-NTA chelating chromatography and quantified with the Bradford method [ 12 ] using bovine serum albumin as the standard. The degree of purity was assessed by SDS-PAGE on a 12% gel.…”

“…0602 has been overexpressed and characterized in a previous study [ 1 ]. PelN shows excellent characteristics in ramie degumming combined chemical treatment [ 11 , 12 ]. However, PelN is unstable at optimal temperature of 65 °C and lead to lower efficiency substantially [ 1 ].…”

Improved extracellular expression and high-cell-density fed-batch fermentation of chitosanase from Aspergillus Fumigatus in Escherichia coli

“…OU01 and Bacillus sp, respectively, were also expressed in E. coli through secretion under the direction of MSCHITO and PelB signal peptides. However, the extracellular activity were only 67.56 U/mL in shake flask [12] and 186 U/mL in 7-L fermenter [13]. Therefore, the active CSN expressed in E. coli through our optimized method reported the highest activity so far.…”

“…For example, two chitosanasegenes that originated from Microbacterium sp. OU01 and Bacillus sp, respectively, were expressed in E. coli with the extracellular activity of only 67.56 U/mL in shake flask [12] and 186 U/mL in 7-L fermenter [13]. Contrarily, a chitosanase derived from Bacillus subtilis HD145 was expressed in P. pastoris with relative high activity of 9000 U/mg [14].…”

OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system

“…The aim of this study was to investigate the effect of two different signal peptides on recombinant protein expression and N-terminal processing using chitosanase (Csn) from Bacillus subtilis as a model protein. This enzyme is biotechnologically important because it converts chitosan, a recalcitrant waste product from the seafood industry, into value-added chito-oligosaccharides (CHOS), which have been shown to have excellent health and agricultural benefits (Pechsrichuang et al 2013 ; Zhou et al 2015 ). It is a relatively small, 28-kDa extracellular enzyme that is secreted from Bacillus subtilis, a Gram-positive bacteria (Pechsrichuang et al 2013 ).…”