Optimization and kinetic characterization of recombinant 1,3-β-glucanase production in Escherichia coli K-12 strain BL21/pETSD10 - a bioreactor scale study.
UNLABELLED
The Escherichia coli K-12 strain BL21/pETSD10 was used to produce recombinant endocellular 1,3-β-glucanase. This enzyme is responsible for the hydrolysis of the glycosidic bond in specific polysaccharides with tracts of unsubstituted β-1,3-linked glucosyl residues. Conditions for the overproduction were experimentally examined, and the optimal values of the process on a bioreactor scale were found by interpolation of the experimental data. Cell induction was preferred during log-phase with relatively high cell density at OD600 near 1·1 with 0·074 g l(-1) of Isopropyl β-D-1-thiogalactopyranoside (IPTG). The higher concentration of IPTG favors high enzyme production but with an excess of ballast protein. 1,3-β-glucanase production was favoured with moderate culture aeration (0·7-0·9 vvm) and moderate stirring (125-150 rev min(-1) ). The highest specific glucanase activity (252 U g(-1) ) was found during validated experiments carried out at aeration at 135 rev min(-1) and stirring at 0·8 vvm. Due to high-tonnage industrial applications (i.e. to hemicellulose hydrolysis), the enzymatic preparation did not need to be highly purified. After pretreatment (precipitation with ammonium sulphate and dialysis) of the crude preparation, the enzymatic protein was one of the three main proteins in the preparation. The reaction rate with respect to the substrate (CM-curdlan) was described by the first order reaction equation (k = 1·95 l h(-1 ) g(-1) ). Products formed in the reaction are composed of nine glucose units on average. In the reaction conditions, the preparation showed very good stability (t1/2 = 202 h).


SIGNIFICANCE AND IMPACT OF THE STUDY
The results contribute to the knowledge of cultivation parameters of E. coli K-12 strain BL21/pETSD10 on a bioreactor scale to overproduce an enzyme degrading β-1,3-glucans. The optimal values of protein concentration, specific activity and total glucanase activity as a function of aeration and stirring were evaluated by numerical analysis. The obtained values were validated as positive. The protein degrades some bonds in hemicellulose. Thus, the protein could be applied as one of the degrading components for hemicellulose.
['Zaslona|H|H|', 'Trusek-Holownia|A|A|http://orcid.org/0000-0002-3886-7166', 'Radosinski|L|L|', 'Hennig|J|J|']
D019149:Bioreactors D010450:Endopeptidases D048168:Escherichia coli K12 D043326:Glucan 1,3-beta-Glucosidase D006868:Hydrolysis D007544:Isopropyl Thiogalactoside D007700:Kinetics D008969:Molecular Sequence Data D011994:Recombinant Proteins D047071:beta-Glucans
1,3-β-glucanase aeration agitation bioreactor scale enzyme production gluB20-2 gene expression kinetics of sugar polymer hydrolysis process parameters optimization