Two β-glucanases from bacterium Cellulomonas flavigena: expression in Pichia pastoris, properties, biotechnological potential

Abstract

In the genome of Cellulomonas flavigena, two genes that potentially encode endoglucanases — Cfla_2912 and Cfla_2913 were identified. We cloned the genes and created Pichia pastoris-based recombinant producers of two proteins that were expressed from the AOX1 promoter. Each of the endoglucanase molecules contains a GH6 catalytic domain, CBM2 carbohydrate-binding module, and TAT signal peptide. The fermentation of the producers was carried out in a 10 L fermenter; Cfla_2912 and Cfla_2913 were purified using affinity chromatography. The yield comprised 10.3 mg/ml (430 U/ml) for Cfla_2913 and 9 mg/ml (370 U/ml) for Cfla_2912. Cfla_2912 and Cfla_2913 were found to have a high activity against barley β-glucan and lichenan, a weak activity against carboxymethyl cellulose (CMC), phosphoric-acid treated cellulose, and no activity against laminarin, xylan, soluble starch, microcrystalline cellulose, cellobiose, and cellotriose. Thus, the proteins exhibited β-glucanase activity. Both proteins had a neutral pH optimum of about 7.0 and were more stable at neutral and slightly alkaline pH ranging from 7.0 to 9.0. Cfla_2912 and Cfla_2913 showed a moderate thermal stability. The products of barley β-glucan hydrolysis by Cfla_2912 and Cfla_2913 were trisaccharide, tetrasaccharide, and cellobiose. Cfla_2912 and Cfla_2913 efficiently hydrolyzed cereal polysaccharides, which indicate that they may have biotechnological potential.

Keywords:

• Cellulomonas flavigena
• enzyme production in bioreactor
• β-glucanase
• Pichia pastoris

Author contributions

Lisov A.V. and Leontievsky A.A. conceived and designed research, analyzed data, and wrote the manuscript. Lisova Z.A. and Belova O.V. conducted a study of the properties of the proteins. Nagel A.S., Shadrin A.M., Andreeva-Kovalevskaya Z.I., Nagornykh M.O., and Zakharova M.V. carried out the development of a recombinant producer, protein synthesis in a bioreactor, and protein purification. All authors read and approved the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.