One-Factor-at-a-Time (OFAT) Optimization of Variables for Maximum L-Asparaginase Activity

The preferred extraneous carbon source to drive maximum L-asparaginase activity by the study mold was selected by the traditional one-factor-at-a-time (OFAT) approach. The method involved varying the source of carbon and holding every other parameter constant (Ekpenyong et al. 2017a). Glucose (GLU), mannose (MAN) and fructose (FRU) as monosaccharides, sucrose (SUC), maltose (MAL) and lactose (LAC) as disaccharides and agro-industrial waste products including molasses (MOL), cassava processing effluent (CPE) and rice processing effluent (RPE) were the carbon substrates screened. Sugars were purchased from Sigma-Aldrich (Sigma-Aldrich Inc., St. Louis, MO, USA) while effluents were obtained from local industries in Lagos and Calabar, Nigeria and stored at -20 °C until required.

The minimal medium for screening contained (g/L) Na₂HPO₄ 6; KH₂PO₄ 3; MgSO₄·7H₂O 0.2; FeSO₄·7H₂O 0.05, NaCl 0.5; CaCl₂ 0.5; KCl 0.5 (Asitok and Ekpenyong 2019) and was supplemented with 1 mL trace mineral solution composed (g/L) of ZnSO₄·7H₂O 0.005; MnSO₄·4H₂O 0.005; H₃BO₃ 0.005; CuSO₄.5H₂O 0.005; CoCl₂·6H₂O 0.005; MoCl₅ 0.005, BaCl₂ 0.005; CrCl₃·6H₂O 0.005; FeCl₃ 0.005 and NiCl₂·6H₂O 0.005 (Ekpenyong et al. 2017b). The fully-constituted medium was supplemented with 1% (w/v) L-asparagine (Merck) and pH adjusted to 5.8 using 1 N HCl. Medium was dispensed into 250 mL Erlenmeyer flasks at 20% (v/v) and carbon sources (except the effluents) incorporated at 1% (w/v). The effluents were retrieved from the freezer and allowed to thaw under ambient laboratory conditions and subsequently boiled to facilitate removal of un-dissolved solids. Upon cooling, effluent was centrifuged at 10,000 rpm for 20 min and supernatant dispensed as 20% (v/v) into 250 mL Erlenmeyer flasks without adjusting pH (Nitschke et al. 2004). A control flask without extraneous carbon where asparagine served as carbon source was also set up. All arrangements were made in triplicates and flasks sterilized by autoclaving at 121 °C for 15 min.

Upon cooling, one agar plug (6 mm) of reactivated and equilibrated mold culture was aseptically added to each flask. Flasks were plugged with sterile cotton wool and incubated at 30 °C on a rotary shaker agitating at 150 rpm for 72 h. Fermentation broth was first filtered with Whatman No.1 filter paper to remove mycelia and then centrifuged at 8,000 rpm for 15 min. Supernatant was twice filtered with 0.45 µm and 0.22 µm membrane filter (Millipore, USA) and L-asparaginase activity quantified for each flask by the Nesslerization protocol of Imada et al. (1973) using Tris–HCl buffer at pH 7.2. One unit of L-asparaginase activity (U) was defined as the amount of enzyme required to liberate one micromole of ammonia by L-asparagine hydrolysis in one minute under the study conditions. A one-way analysis of variance (one-way ANOVA) in GraphPad Prism 8 software (GraphPad, San Diego, CA, USA) was used to compare performances of the carbon sources and significant mean differences were separated by Tukey HSD test using 95% confidence interval.

Extraneous nitrogen sources were also screened by the OFAT approach. Nitrogen sources included ammonium chloride (NH₄Cl), potassium nitrate (KNO₃), ammonium nitrate (NH₄NO₃), bean processing effluent (BPE), corn steep liquor (CSL), peptone (PEP) and tryptone (TYP). Organic and inorganic nitrogen sources were added at 1% (w/v) to minimal medium supplemented with 1% (w/v) L-asparagine and selected carbon substrate. Effluent substrates were prepared as described in the section for carbon screening and used as also described. A control flask without extraneous nitrogen was also prepared. Experimental set-up, sterilization, inoculation, incubation, harvest, L-asparaginase activity assay and statistical analyses were as described under carbon screening.

To select most suitable inoculum size (spore density) for the fermentation, inoculum sizes were screened by the OFAT approach by inoculating 3% (v/v) spore suspension of different inoculum sizes ranging from 10³ to 10⁹ spore-forming units per milliliter (sfu/mL) into minimal medium containing selected extraneous carbon and nitrogen substrates. Working spore concentrations were prepared by the spectrophotometric method as described in Ekpenyong et al. (2020a) and experimental set up, incubations, harvest, determinations of enzyme activity and statistical analyses were as described under carbon screening section.