The Statistical Optimisation of Recombinant β-glucosidase Production through a Two-Stage, Multi-Model, Design of Experiments Approach

• Published in: Bioengineering (Basel) 2019-07, Vol.6 (3), p.61
• Main Authors: Uhoraningoga, Albert, Kinsella, Gemma K, Frias, Jesus M, Henehan, Gary T, Ryan, Barry J
• Format: Article
• Language: English
• Subjects: Affinity chromatography; Bioengineering; Carbon; Catalysis; Cellobiase; Cellobiose; Definitive Screening Design; Design of experiments; Design optimization; E coli; Enzymes; Fractional Factorial design Plackett-Burman Design; Glucosidase; Glycerol; Industrial applications; Metal ions; Nitrogen; p-Nitrophenyl-b-D-glucopyranoside; Protein expression; Proteins; recombinant β-glucosidase; Response Surface Methodology; Screening; Statistical analysis; Streptomyces griseus; Variables; β-Glucosidase
• ISSN: 2306-5354; 2306-5354
• DOI: 10.3390/bioengineering6030061

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 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 OD ) were experimentally identified as having statistically significant effects on the production of recombinant β-glucosidase in 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: OD , 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 -nitrophenyl-β-D-glucopyranoside ( NPG) and cellobiose catalysis.