TWO-STAGE DESIGN OF EXPERIMENTS APPROACH FOR PREDICTION OF RELIABILITY OF OPTOCOUPLERS

ADITHYA THADURI, A. K. VERMA, GOPIKA VINOD, M. G. RAJESH, UDAY KUMAR
2012 International Journal of Reliability, Quality and Safety Engineering (IJRQSE)  
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
more » ... rom 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 OD 600 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: OD 600 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. Bioengineering 2019, 6, 61 2 of 23 recently reviewed [8, 9] . Enhanced production of recombinant β-glucosidase, following DoE, has been detailed for recombinant β-glucosidases from a variety of sources, such as Pichia pastoris [3], A. niger HN-2 [10] and A. niger [11] . However, to date, no attempt has been made to enhance the production of β-glucosidase form Streptomyces sp.; a species known to be an effective source of β-glucosidase genes [12, 13] , with limited sequence conservation compared to P. pastoris (18% conservation) and A. niger (26% conservation). The aim of this study was to optimise the production of S. griseus recombinant β-glucosidase in E.coli BL21 (DE3) by using multiple screening designs to validate the variable selected for optimisation via a response surface methodology. The findings of this study are explored in light of the existing literature and recommendations are offered for future applications of Design of Experiments to enhance recombinant protein production. Materials and Methods Chemical and Materials The recombinant pGEX-4T-1 vector containing S. griseus β-glucosidase gene (GST-tagged) in E. coli BL21 (DE3) was developed in a previous study [14] . Ampicillin, glycerol, Isopropyl-β-D-thiogalactopyranoside (IPTG), LB broth, p-nitrophenyl-β-D-glucopyranoside (pNPG), p-nitrophenol (pNP), cellobiose, fructose sucrose, tryptone, yeast extract, beef extract, CaCl 2 , DTT, KOH, MgCl 2 , (NH 4 ) 2 S 4 , ZnSO 4 , Triton X-100, M PMSF, Lysozyme, Bradford reagent, and Glutathione Sepharose 4B resin were purchased from Sigma Aldrich (Ireland). Production of S. griseus Recombinant β-glucosidase Preparation of Microbiological Media The recombinant pGEX-4T-1 vector containing S. griseus β-glucosidase gene (GST-tagged) in E. coli BL21 (DE3) was inoculated into 5 mL of Luria-Bertani (LB) medium containing ampicillin at a final concentration of 50 µg·mL −1 , and incubated at 37 • C for overnight at 220 rpm. The overnight culture was used to inoculate 10% v/v of fresh medium for small-scale (10 mL) expression studies used during the screening and optimisation processes. Carbon and Nitrogen Sources for S. griseus Recombinant β-glucosidase Expression Identification of good carbon and nitrogen sources for S. griseus recombinant β-glucosidase expression was initially performed. Four carbon sources (glucose, fructose, galactose, and glycerol) and six nitrogen sources (yeast extract, tryptone, beef extract, KNO 3 , NH 4 Cl, and (NH 4 ) 2 SO 4 ) were selected based on their noted effect on recombinant expression in E-coli in general [15, 16] , and on β-glucosidase in particular [17, 18] . The effect of carbon and nitrogen sources was investigated in basal medium supplemented with 0.5% (w/v) of the different carbon sources and 1% (w/v) of the different nitrogen sources [19] . In each case, cells were grown until OD 600 nm reached 0.5, and then induced with 1mM IPTG for 6 h with subsequent culturing at 37 • C, 220 rpm. Effect of Culture Aeration on S. griseus Recombinant β-glucosidase Expression Subsequently, the influence of culture aeration, via baffled culture flasks, on the protein expression was also investigated by cultivating cells with medium supplemented with the selected carbon and nitrogen sources, 0.5% w/v fructose, 1% w/v yeast extract and tryptone (see Section 2.2.2), in baffled and non-baffled flasks, at 37 • C, 220 rpm for 6 h post 1 mM IPTG induction. Screening of Most Significant Medium Components and Induction Condition Using Multiple Screening Designs Fractional Factorial Design (FFD), Plackett-Burman Design (BBD), and Definitive Screening Design (DSD) were employed to comparatively screen for the most significant medium components
doi:10.1142/s0218539312500076 fatcat:dfefr26yjjetvlfpfejf6vu2gi