Optimization of intracellular L-Asparaginase production by Streptomyces paulus CA01 isolated from wheat bran using the response surface methodology

Figures & data

Table 1. Matrix of experimental design.

StdOrder	RunOrder	PtType	Blocks	Coded factor			L-ASP (IU/mL)
				T	SC	GC
15	1	0	1	0	0	0	7.5
14	2	0	1	0	0	0	6.98
9	3	2	1	0	−1	−1	2.14
4	4	2	1	1	1	0	3.94
10	5	2	1	0	1	−1	4.25
6	6	2	1	1	0	−1	1
11	7	2	1	0	−1	1	4.11
8	8	2	1	1	0	1	3.11
13	9	0	1	0	0	0	6.53
7	10	2	1	−1	0	1	3.61
3	11	2	1	−1	1	0	3.66
12	12	2	1	0	1	1	8.01
5	13	2	1	−1	0	−1	1.64
2	14	2	1	1	−1	0	0.46
1	15	2	1	−1	−1	0	2.02

T, temperature of cultivation (–1: 25 °C, 0: 27.5 °C, 1: 30 °C); SC, substrate concentration (–1: 0.75, 0: 1, 1: 1.25%); and GC, glucose concentration (–1: 0.1%, 0: 0.2%, 1: 0.3%).

Figure 1. Morphology of CA01 strain observed by CETI optical microscope equipped with a camera at G100. (A) Micromorphology (B) Macromorphology.

Table 2. Biochemical, physiological and morphological characteristics of the isolated CA01 strain [9].

Studied characteristics	Result for strain CA01
Sugar and products of degradation	Glucose; lactose; citrate and mannitol
Mode of sugar degradation	Aerobic
Metabolic enzymes production	Tryptophane Deaminase (TDA); nitrate reductase (NR); catalase.
Mobility	Absence
Polymers degradation	Casein
Growth temperature	25 °C at 37 °C.
Growth pH	From 6 to 9.
Tolerance of NaCl	From 5 to 20%.
Aerial mycelium / Substrate mycelium	White / Greyish.
Spores	Powdery – greyish.
Gram staining	Gram+.

Figure 2. Phylogenetic tree of molecular identification after sequencing of 16s rDNAgene using Sanger method of sequencing and alignment by CHROMAS Pro software. QRY_Contig_CA01-16S-Complet_2^ND_ENVOI: our studied strain.

Figure 3. Illustration of the L-Asparaginase activity of intracellular protein fraction of Streptomyces paulus CA01 strain, demonstrated by the diffusion on wells approach. C: Negative control: sterile ADS medium; I: intracellular protein fraction of Streptomyces paulus CA01strain.

Table 3. Coded coefficients for transformed response.

Term	Effect	Coefficient	SE Coefficient	T-value	P-value	VIF
Constant		3.7414	0.0753	49.68	0.000
T	−0.3812	−0.1906	0.0461	−4.13	0.009	1.00
SC	1.3068	0.6534	0.0461	14.17	0.000	1.00
SG	1.1281	0.5640	0.0461	12.23	0.000	1.00
T^2	−2.9811	−1.4906	0.0679	−21.96	0.000	1.01
SC^2	−0.9288	−0.4644	0.0679	−6.84	0.001	1.01
GC^2	−1.0292	−0.5146	0.0679	−7.58	0.001	1.01
T × SC	0.5719	0.2859	0.0652	4.38	0.007	1.00
T × GC	0.0845	0.0422	0.0652	0.65	0.546	1.00
SC × GC	0.2498	0.1249	0.0652	1.92	0.114	1.00

Note: S = 0.130433; R² = 99.47%; adjusted R² = 98.5%; predicted R² = 96.79%.

Term: the different terms of the model: T: temperature of cultivation; SC: substrate concentration; GC: glucose concentration; Effect: the effects of the different terms of the model, on the response Y; SE Coefficient, Standard Error Coefficient; VIF: measures the magnitude of the variance of a regression coefficient.

Table 4. Analysis of variance for transformed response.

Source	DF	Adj. SS	Adj. MS	F-value	P-value
Model	9	15.8306	1.75896	103.39	0.000
Linear
T	1	0.2906	0.29064	17.08	0.009
SC	1	3.4154	3.41543	200.76	0.000
GC	1	2.5452	2.54520	149.60	0.000
Square
T2	1	8.2036	8.20360	482.20	0.000
SC2	1	0.7963	0.79620	46.80	0.001
GC2	1	0.9777	0.97774	57.47	0.001
2-Way interaction
T × SC	1	0.3270	0.32704	19.22	0.007
T × GC	1	0.0071	0.00714	0.42	0.546
SC × GC	1	0.0624	0.06241	3.67	0.114
Error
Lack of Fit	3	0.0232	0.00774	0.25	0.857
Pure Error	2	0.0618	0.03092
Total	14	15.9157

Note: DF, the degrees of freedom of the different terms of the model; Adj SS (Adjusted sum of squares): The sums of the squares adjusted to the terms of the model; Adj MS (Adjusted means squares): The squares adjusted averages of the terms of the model.

Figure 4. Surface plot illustration of the effect of interaction of the selected factors (T, temperature; SC, substrate concentration; GC, glucose concentration) on the L-asparaginase production by Streptomyces paulus CA01 using Minitab 17 software.

Figure 5. Optimization of the intracellular L-Asparaginase production by the Streptomyces paulus CA01 strain.

T: temperature; SC: Substrate concentration; GC: Glucose concentration.

Note: The equation of the coded values of the model is: (3) $$Activit{y}_{L-Asparaginase}=2,6453\text{-}0,1360T+0,4000SC+0,3395GC\text{-}0,8777T^2\text{-}0,2682S{C}^2\text{-}0,2816G{C}^2+0,2037T\text{*}SC$$(3)

Figure 6. Normal plot for the experimental response.

Chergui A, Kecha M, Tighrine A, et al. Characterization and optimization of extracellular L-Asparaginase production by selected actinomycete strain isolated from an algerian wheat bran. Cell Mol Biol. 2018;64(14):53–60. doi:10.14715/cmb/2018.64.14.9.

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Data availability statement

All data generated or analyzed during the current study are available from the corresponding author (M. S. B) on reasonable request.