Document Type

Article

Rights

This item is available under a Creative Commons License for non-commercial use only

Disciplines

1. NATURAL SCIENCES, 1.2 COMPUTER AND INFORMATION SCIENCE, 1.4 CHEMICAL SCIENCES, 1.6 BIOLOGICAL SCIENCES

Publication Details

Uhoraningoga, A., Kinsella, G. K., Frias, J. M., Henehan, G. T., & Ryan, B. J. (2019). The Statistical Optimisation of Recombinant β-glucosidase Production through a Two-Stage, Multi-Model, Design of Experiments Approach. Bioengineering, 6(3), 61.

Abstract

β-glucosidases are a class of enzyme that are widely distributed in the living world, with examples noted in plants, fungi, animals and bacteria. They offer both hydrolysis and synthesis capacity for a wide range of biotechnological processes. However, the availability of native, or the production of recombinant β-glucosidases, is currently a bottleneck in the widespread industrial application of this enzyme. In this present work, the production of recombinant β-glucosidase from Streptomyces griseus was optimised using a Design of Experiments strategy, comprising a two-stage, multi-model design. Three screening models were comparatively employed: Fractional Factorial, Plackett-Burman and Definitive Screening Design. Four variables (temperature, incubation time, tryptone, and OD600 nm) were experimentally identified as having statistically significant effects on the production of S.griseus recombinant β-glucosidase in E. coli BL21 (DE3). The four most influential variables were subsequently used to optimise recombinant β-glucosidase production, employing Central Composite Design under Response Surface Methodology. Optimal levels were identified as: OD600 nm, 0.55; temperature, 26 °C; incubation time, 12 h; and tryptone, 15 g/L. This yielded a 2.62-fold increase in recombinant β-glucosidase production, in comparison to the pre-optimised process. Affinity chromatography resulted in homogeneous, purified β-glucosidase that was characterised in terms of pH stability, metal ion compatibility and kinetic rates for p-nitrophenyl-β-D-glucopyranoside (pNPG) and cellobiose catalysis

DOI

https://doi.org/10.3390/bioengineering6030061

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