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

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.

Purification and characterization of a recombinant beta-glucosidase in Escherichia coli

“…These results could be due to the catalytic region of GH3S2 containing a divalent metal ion binding site. Increasing the BGL activity in the presence of Ca 2+ has also been mentioned in previous studies, such as the activity of BGL derived from subtropical soil microorganisms was increased by 131% (Yin et al, 2019), that of Streptomyces griseus was increased by 118% (Uhoraningoga et al, 2019).…”

Application of β-glucosidase Immobilized on Chitosan microspheres in Degradation of Polydatin in Polygonum cuspidatum

“…Β-glucosidase can catalyze the hydrolysis of β-Dglucosidase bond and convert polyglycoside into resveratrol, but the low yield of β-glucosidase limits its use in industry. Through the experimental design of [2] recombinant β-glucosidase, the yield can be further improved [23,24]. Compared with free enzyme, immobilized enzyme has become a research hotspot because of its high stability, reusability, easy separation of products after catalytic reaction and low industrial cost.…”