Study mold strains
Wild-type strains of Aspergillus candidus UCCM 00117 (Asp-C) and Aspergillus sydowii UCCM 00124 (Asp-S) were obtained from University of Calabar Collection of Microorganisms (UCCM) (www.wfcc.info/ccinfo/collection/by_id/652) and reactivated in Czapek-Dox agar (CDA) medium (Sigma-Aldrich, USA) for 96 h at 30 ºC. The strains were grown in minimal media containing 1% (w/v) xylan at 30 ºC and on PDA-NaCl medium containing 25% (w/v) NaCl at 55 ºC. The minimal medium contained 0.35% NaNO2, 0.15% K2HPO4, 0.05% MgSO4.7H2O, 0.05% KCl, 0.001% FeSO4.7H2O supplemented with 1% (w/v) xylan (Sigma Aldrich, Germany) (Brandt et al. 2020). Xylanase production with no-growth on PDA-25% NaCl medium was characteristic of Asp-S while non-xylanase production but luxuriant growth on PDA-25% NaCl was characteristic of Asp-C.
Preparation of protoplasts from wild Aspergillus strains
Effects of mycelial age (36, 72 and 108 h), pH (4, 6 and 8) and incubation time (2, 4 and 6 h) on protoplast isolation were investigated using Box-Benkhen design (BBD) of a response surface methodology (RSM) in Design Expert version 12 (StatEase, Minnesota, USA) (Farjaminezhad and Garoosi 2021). The optimized factor levels were used for protoplast isolation in a medium composed (g.L−1) of glucose 1; KH2PO4 0.1; KCl 0.5; MgSO4.7H2O 0.05; casamino acid 0.1, at optimized pH. Flasks were inoculated, upon cooling, with 1 mL of 3.5 × 106 sfu.mL−1 of each strain and incubated, with agitation, for an optimized period of time (h). Germlings of optimized age were harvested by centrifugation at 2655.25 × g for 20 min and washed twice in 5 mL 25 mM Tris–HCl buffer at pH 7.5. Pellets were re-suspended in a buffer mixture (5 mL) of 25 mM each of Tris–HCl at pH 7.5 and CaCl2, and 1.2 mM sorbitol (Klinsupa et al. 2016). Thereafter, the obtained suspensions were suspended in lytic enzyme mixture comprising glucanase (1200 U), cellulase (850 U), protease (875 U) and chitinase (180 U) and incubated for an optimized period at 30 ºC with slow shaking (50 rpm). Protoplasts were separated from mycelia using sintered glass filter and washed again in the osmotically-stabilized buffer mixture to rid the preparation of lytic enzymes.
Protoplast fusion
Protoplast suspensions of both strains (1.5 mL, 8.5 × 106 cells.mL−1) were centrifuged at 663.81 × g for 15 min. Pellets were re-suspended in 1 mL 40% (w.v−1) 6000 kDa polyethylene glycol in 10 mM CaCl2 mixed with 0.05 M glycine at pH 7.5, thereafter incubated at 30 ºC for 30 min. Suspension was diluted in 6 mL 0.8 M sorbitol-containing MM before centrifuging at 2150.75 × g for 15 min. Pellets of fusants were washed twice in sorbitol solution (8 mL of 0.8 M) and re-suspended in 5 mL of same solution.
Protoplasts were regenerated on yeast malt potato dextrose agar (YMPDA) with 0.8 M of sorbitol or sucrose as osmotic agents. Fifty microliters (50 µL) of protoplast suspension (3.5 × 103 sfu.mL−1) was plated on the surface of sterile regenerating medium containing osmotic stabilizers. Plates were incubated for 72–96 h at 30 ºC. Regenerated colonies were characterized by macro-morphology on CDA for differences in mycelial growth pattern and by l-ASNase activity, combined potentials of xylanase production at 30 ºC and growth on PDA-25% NaCl at 55 ºC. Fusants with prospects were preserved for further studies on CDA plates in a refrigerator and the most promising was maintained in sterile soil and deposited at the UCCM and assigned the collection number UCCM 00130F06.
Comparative fermentative production of L-ASNase by protoplast fusant and cost analysis
l-ASNase production by submerged and solid-state fermentation types were compared in shake flasks. Submerged fermentation (SmF) medium was as detailed in Ekpenyong et al. (2021a). Solid-state fermentation (SSF) utilized sugarcane bagasse (2 g dry mass) as support material in 100 mL Erlenmeyer flasks and 10 mL solution mixture of 10% test spore suspension and 90% of fermentation medium with same composition as SmF. Inoculated flasks for SmF were incubated at 30ºC on orbital shaker (100 rpm) for 96 h while SSF flasks were incubated in a chamber at 30 ºC and 80% humidity (Cachumba et al. 2019). The better fermentation type, assessed by l-ASNase activity, was then operated by batch, fed-batch or continuous mode in 5 L bioreactors (BioStat(R) CPlus, Sartorius Stedim Biotech, Germany) with 3.5 L working volume for l-ASNase production by the fusant.
In the batch mode, the optimized medium reported in Ekpenyong et al. (2021a) was used. Sterilization was conducted in situ and inoculation with 3% (v.v−1) spore suspension. The bioreactor was operated as described in Asitok et al. (2022a) and determinations of total protein (Bradford 1976), biomass concentration (Rodrigues et al. 2006), l-ASNase activity (Imada et al. 1973) and residual carbohydrate (Miller 1959) were made at 6 h interval for 96 h. Briefly, l-ASNase assay protocol by Imada et al. (1973) involved dissolution of 0.04 M of l-asparagine (0.5 mL) in 0.5 M (0.5 mL) Tris–HCl buffer (pH 7.2) and adding the mixture to 0.5 mL of cell-free fermentation broth of fusant mold and making up total reaction volume to 2 mL with sterile distilled water. The preparation was incubated in a water bath at 37 ºC for 30 min and reaction stopped by adding 0.5 mL of 1.5 M trichloroacetic acid. One unit of enzyme activity was defined as the amount of enzyme that liberated one micromole of ammonia from substrate in 1 min. Means of triplicate determinations were subjected to Pearson’s bivariate correlation and subsequently employed for logistic and/or modified Gompertz model fitting (Ekpenyong et al. 2021b).
In the fed-batch mode, fermentation started batch-wise for 24 h with same medium used during batch mode except that the starting molasses concentration was reduced to 30 g.L−1. Medium pH was adjusted to 5.8, temperature to 50ºC, dissolved oxygen to 45% using the agitation cascade of the bioreactor at 100–600 rpm and 1.5 vvm aeration. Feeding solution was composed of 50 g.L−1 molasses, 44 g.L−1 asparagine and 0.5 g.L−1 MnCl2 using a peristaltic pump. Feed rate of the solution was calculated using equation below;
$$F=\frac{\mu {X}_{0}{V}_{0}{e}^{\mu t}}{{Y}_{X/S}{S}_{0}}$$
(1)
where F (h−1) is feed rate, X0 (g.L−1) is mold biomass concentration at end of batch operation, V0 is volume (L) of spent medium at the end of batch operation and S0 (g.L−1) is total substrate concentration, µ (h−1) is the specific growth rate, X/S (g.g−1) is biomass yield on substrate determined from the batch part of the fed-batch fermentation using Eqs. 2 and 3 below;
$$\mu =\frac{1}{X}\frac{dx}{dt}$$
(2)
$${Y}_{X/S}=\frac{{X}_{m}-{X}_{0}}{{S}_{0}-{S}_{m}}$$
(3)
where Xm is maximum fungal dry cell weight (DCW) (g.L−1) at time t, X0 is DCW (g.L−1) at t = 0, S0 is initial substrate concentration (g.L−1) at t = 0 and Sm is final substrate concentration (g.L−1) at time t. Kinetics and modeling studies were conducted as in the batch mode.
In the continuous mode, fermentation was also started batch-wise but for 12 h, and then shifted to continuous mode using two simultaneously operated motor pumps to keep feed-rate and product-withdrawal at constant rate to maintain constant volume in the fermentor. To maintain constant reaction volume of 3.5 L, 2.5 L feed was injected into the bioreactor as 2.5 L was withdrawn by means of the motor pumps for 7 continuous runs. An initial optimization of dilution rates was conducted and the best selected on the basis of L-ASNase activity. Analysis was as described for batch and fed-batch modes. Performances of models were evaluated by adjusted goodness-of-fit, r2, root mean squared error (rmse) and mean absolute error (mse) using the equations detailed in Asitok et al. (2022a).
By considering the costs incurred from purchase of chemicals required to formulate media for fermentation by batch, fed-batch and continuous fermentation modes, and chemicals for post-fermentation analysis; cost of consumable materials as well as labour, the cost incurred from production of l-ASNase by fusant under all three fermentation modes was calculated (Yang and Sha 2022). Energy, equipment and rent costs were omitted since they are known to vary significantly among real-time laboratory arrangements.
RSM and sensitivity analysis of Fusant-06 L-ASNase purification conditions
By combining Plackett–Burman design (PBD) and response surface methodology (RSM), different molecular weights, concentrations of polyethylene glycol (PEG) and salts, pH and temperature, were screened for significant influences on l-ASNase recovery and purification by aqueous two-phase system (ATPS). Levels of selected significant variables were determined as follows: X1 = (Molecular weight—4500)/1500.0; X2 = (PEG concentration—15.8)/5.0; X3 = (Citrate concentration—20.8)/5.0; X4 = (NaCl concentration—12.5)/3.0 and X5 = (pH—7.8)/1.0.
In a typical RSM procedure, a sensitivity analysis is conducted to quantify the relationship between the input and output uncertainties in an attempt to determine how sensitive the response model is to fluctuations in the parameters and data on which it is built. This analysis evaluates the robustness of model assumptions and operates how a given model responds to variations in its assumptions (Zhan et al. 2013). In short, it complements design of experiment by revealing non-linear effects of variables.
In the present study, we adopted Sobol method of global sensitivity analysis to evaluate the first-order, second-order and total sensitivity indices of the RSM models using SobolGSA version 2.0 (Imperial College, London). As a continuation, the RSM model was used to generate new parameters using Sobol’ sequence sampling method and the new samples from RSM were run by Sobol method to obtain the sensitivity indices of input parameters. The variance-based method was selected for the Sobol’ sensitivity analysis as it adopts a variance ratio which estimates the importance of each input factor on the response variable. The total variance of the response model is given by the expression below;
$$V\left(Y\right)=\sum_{i=1}^{n}{V}_{i}+\sum_{i\le j\le n}^{n}{V}_{ij}+\dots +\sum_{i\le \dots n}^{n}{V}_{1\dots n}$$
(4)
where V is the total variance of the model output Y, n is the number of input factors, Vi = first-order effect or partial variance of model output due to xi, \({{X}_{i}(V}_{i}=V[E\left(Y|{X}_{i}\right)])\) and Vij the second-order effect or the partial variance of model output due to interaction between xi and xj, \({{V}_{ij}(V}_{ij}=V\left[E\left(Y|{X}_{i},{X}_{j}\right)\right]-{V}_{i}-{V}_{j})\).
The first-order sensitivity index, Si was therefore given as a ratio of the first-order effect to the total variance as follows;
$${S}_{i}=\frac{{V}_{i}}{V\left(Y\right)}=V[E\left(Y|{X}_{i}\right)])/V(Y)$$
(5)
The second-order sensitivity index, Sij was given as a ratio of the second-order effect to the total variance as follows;
$${S}_{ij}=\frac{{V}_{ij}}{V\left(Y\right)}=V\left[E\left(Y|{X}_{i},{X}_{j}\right)\right]-{V}_{i}-{V}_{j})/V(Y)$$
(6)
while the total sensitivity index, STi was given by
$${S}_{Ti}=\frac{E\left(V\left(Y|{X}_{\sim i}\right)\right)}{V\left(Y\right)}$$
(7)
where ~ i denotes all input variables except i input.
Selected significant factors were employed at their optimum levels for ATPS purification as described by Nascimento et al. (2020). Further purification of the ATPS-extracted protein was performed by molecular exclusion chromatography on Sephadex G-100 of diameter 100 cm by 1.5 cm, using 50 mM phosphate buffer (pH 7.0) and 100 mM KCl as mobile phase at a flow rate of 0.2 mLmin−1. Protein standards ranged from 12.4 to 115 kDa (Fisher Scientific Products, India) and comprised cytochrome C (12.4 kDa), soybean trypsin inhibitor (29.0 kDa), ovalalbumin (44.3 kDa), serum albumin (66.2 kDa), phosphorylase b (97.2 kDa) and β-galactosidase (115 kDa).
Molecular weight, amino acid profile, substrate specificity, inhibitor, organic solvents, metal ions, temperature and, pH activity and stability characterizations of l-ASNase
Purification level and molecular weight of protein were confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) with 12% (wv−1) gel using the method of Laemmli (1970).
The sequence of amino acids in l-ASNase from Fusant-06 was determined by Agilent amino acid analyzer (Asitok et al. 2022b). Sequences obtained were compared with NCBI sequences for Aspergillus l-ASNases and with those of the wild Aspergillus strains.
Substrate specificity of l-ASNase was evaluated by incorporating l-asparagine, urea, acrylamide or l-glutamine as enzyme substrates in assay mixture (Imada et al. 1973). l-ASNase activities were expressed relative to activity in the presence of l-asparagine as control.
Effect of enzyme inhibitors reflecting the four classes of protease were evaluated by pre-incubation of fusant l-ASNase with 5 mM and 10 mM of pepstatin A, phenyl-methyl-sulfonyl fluoride (PMSF), di-iso-propyl-fluorophosphate (DPFP), ethylene diamine tetraacetic acid (EDTA), p-chloro-mercuric benzoate (pCMB), dithiothreitol (DTT), iodoacetamide (IAM), and 5% (vv−1) β-mercaptoethanol (β-MEOH) for 15 min. Thereafter, residual activity was determined using assay mixture without inhibitor as control.
Organic solvents, selected based on their octanol–water partition coefficient, log P to include toluene, acetonitrile, acetone, cyclohexane, glycerol, ethanol, methanol, n-hexane, chloroform, and 2-propanol at 50% (vv−1) was studied for their effect on fusant l-ASNase activity at 50 ºC and 200 rpm agitation for 1 h. Thereafter, residual l-ASNase activities were determined using assay mixture without solvent as control.
Influence of metal ions on fusant l-ASNase was evaluated by pre-incubation with 5 mM of cations including K+, Na+, Ca2+, Mg2+, Zn2+, Mn2+, Fe2+, Ni2+, Cu2+, Ba2+, Co2+, Cr3+, Fe3+ and Mo5+ at 50ºC, pH 7.0 for 15 min before measuring relative activity. Assay mixture without metal served as control.
Effects of temperature (20-70ºC), pH (3–10) and NaCl concentration (5–35%) on enzyme activity were evaluated as described in Ekpenyong et al. (2021a).
Michaelis–Menten kinetics of Fusant-06 L-ASNase
The Michaelis–Menten constant Km, maximum velocity, Vmax, catalytic rate, Kcat and catalytic efficiency, Kcat/Km of Fusant-06 l-ASNase were determined by measuring velocity of reaction at varying concentrations of l-asparagine ranging from 0.005–1.28 mM with 5 µg/mL (0.043 µM) l-ASNase and plotting the Lineweaver–Burk relationship between substrate concentration (1/[S]) and reaction velocity (1/V). Catalytic rate was determined by dividing maximum reaction velocity, Vmax by molar concentration of l-ASNase, [E0] while the catalytic efficiency was calculated as a ratio of catalytic rate to Km.
Potential applications of L-ASNase in health-care and the industry
The MTT-based cytotoxicity assay protocol earlier described (Ekpenyong et al. 2021c) was adopted for in-vitro cytotoxicity of purified l-ASNases against human myeloid leukemia (HL-60), hepatocellular carcinoma (HepG-2) and human breast carcinoma (MCF-7) cell lines. The non-tumor human embryonic cell line (HEK 283 T) was included for selective toxicity evaluation. Test concentrations of all l-ASNases ranged from 0.5 to 1200 µg.mL−1. Percent cell viability was determined from triplicate determinations at 570 nm. Data was analyzed by non-linear regression in GraphPad Prism 8 and IC50 and selectivity index determined.
For acrylamide reduction potential in food industry, two hundred grams (200 g) of strips of freshly peeled sweet potato (Ipomoea batatas), Irish potato (Solanum tuberosum) and yam (Dioscorea esculenta) were oven-dried for 10 min at 85 ºC and afterwards immersed in 100 U of l-ASNase dissolved in double distilled water. A control preparation was set up without prior exposure to test enzyme and activity determined using Nessler’s reagent (Imada et al. 1973). The preparation was fried in 100 mL of sunflower oil until crunchy strips (chips) were obtained. The strips were ground in a mortar and soaked in 50 mL 90% (vv−1) ethanol to extract acrylamide. The ethanol extract was concentrated by evaporation in vaccuo and amount of acrylamide (CH2 = CHCONH2) determined again with Nessler’s reagent.
Antioxidant potential was investigated, in terms of free-radical scavenging potential of the enzyme, using the protocol described by Mihooliya et al. (2020) with ascorbic acid (Sigma-Aldrich, USA) as standard. The change in colour of 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) from deep violet to pale yellow was monitored spectrophotometrically at a wavelength of 517 nm for 3 h. The scavenging effect was calculated using the expression in Eq. 4 (Syame et al. 2022).
$$SE \left(\%\right)=100-\left[\left(\frac{\left({A}_{0}-{A}_{1}\right)}{{A}_{0}}\right)\times 100\right]$$
(8)
Additionally, the scavenging activity of Fusant-06 L-ASNase was also tested against the dark blue 2,2ʹ-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) radical cation (ABTS.+) as reported by Liu et al. (2016). Briefly, a solution of decreasing concentrations (µg/mL) of ABTS cation, with an absorbance of 0.705 ± 0.04 at 734 nm, was mixed with ABTS diluent and gently shaken. Subsequently, 10 µL of purified l-ASNase was added to the reaction mixture and left for 6 min in the dark at 28 ± 2ºC (room temperature) and absorbance drop measured at a wavelength of 734 nm using a microplate reader (Thermo Scientific, USA). The scavenging rate (%) was used to evaluate the ABTS scavenging capacity of l-ASNase as described by Liu et al. (2016).