Boosting extracellular dextransucrase production by Weissella confusa by combining a statistical and randomized mutational approach during upstream fermentation processing

Figures & data

Table 1. Experimental ranges and coded levels of independent variables for dextransucrase production.

			Levels
Variables	Code	Unit	−1	1
Fermentation time	A	hours	8	24
Temperature	B	°C	20	30
Sucrose	C	gL^−1	10.0	30.0
pH	D	–	5.5	7.5
Yeast extract	E	gL^−1	2.5	10.0
Peptone	F	gL^−1	1.0	5.0
Dipotassium hydrogen phosphate	G	gL^−1	10.0	20.0
Sodium chloride	H	gL^−1	0.005	0.05
Calcium chloride	J	gL^−1	0.01	0.1

Figure 1. Dextransucrase productivity by wild and mutant strains of Weissella confusa KIBGE-IB38. Tukey’s test was performed. # represents a significant difference as compared to the wild with P < 0.001.

Figure 2. Pareto chart illustrating the effect of independent variables on dextransucrase production (the blue colour represents a negative effect and the orange colour represents a positive effect).

Table 2. Plackett-Burman experimental design of independent variables with the experimental and predicted values for dextransucrase production.

		Coded levels of variables											Dextransucrase (DSU ml^−1)
Std.	Run	A	B	C	D	E	F	G	H	J	Dummy 1	Dummy 2	Experimental	Predicted	Residuals
2	1	1	−1	1	−1	1	1	−1	1	−1	−1	1	35.90	35.40	0.50
13	2	0	0	0	0	0	0	0	0	0	0	0	30.00	30.00	0.000
6	3	−1	−1	1	1	−1	1	−1	−1	1	1	1	44.87	43.52	1.34
8	4	−1	1	−1	−1	1	1	1	−1	1	−1	1	38.20	37.70	0.50
1	5	1	−1	−1	1	1	1	1	−1	−1	1	−1	38.90	39.40	−0.50
7	6	1	1	1	−1	−1	−1	1	−1	−1	1	1	51.20	49.86	1.34
10	7	−1	1	−1	1	1	−1	−1	1	−1	1	1	30.50	29.16	1.34
3	8	−1	1	1	1	−1	1	1	1	−1	−1	−1	64.20	65.55	−1.35
12	9	−1	−1	−1	−1	−1	−1	−1	−1	−1	−1	−1	15.70	17.04	−1.34
5	10	1	1	−1	−1	−1	1	−1	1	1	1	−1	43.80	44.30	−0.50
9	11	−1	−1	1	−1	1	−1	1	1	1	1	−1	10.40	10.90	−0.50
4	12	1	1	1	1	1	−1	−1	−1	1	−1	−1	38.60	39.95	−1.35
11	13	1	−1	−1	1	−1	−1	1	1	1	−1	1	22.00	21.50	0.50

A: Fermentation time, B: Temperature, C: Sucrose, D: pH, E: Yeast extract, F: Peptone, G: Dipotassium hydrogen phosphate, H: Sodium chloride, J: Calcium chloride.

−1 and 1 signs show the minimum and maximum value of the input parameters range, respectively

Table 3. ANOVA of Plackett-Burman design for dextransucrase production.

Sources	Coefficient Estimate	Degree of freedom	Sum of Squares	Mean Square	F-value	P-value Prob > F
Model/Constant	35.71	9	2466.73	274.08	17.23	0.0195*
Fermentation time	2.21	1	58.65	58.65	3.69	0.1506
Temperature	8.23	1	812.30	812.30	51.07	0.0056*
Sucrose	4.67	1	261.99	261.99	16.47	0.0270*
pH	3.66	1	160.38	160.38	10.08	0.0503
Yeast extract	−4.11	1	202.29	202.29	12.72	0.0376*
Peptone	8.12	1	791.70	791.70	49.78	0.0059*
Dipotassium hydrogen phosphate	1.29	1	20.10	20.10	1.26	0.3428
Sodium chloride	−1.72	1	35.60	35.60	2.24	0.2315
Calcium chloride	−3.21	1	123.71	123.71	7.78	0.0685
Residuals	-	3	47.71	15.90
Cor Total	-	12	2514.45
Std. Dev.	3.99		PRESS	541.52	R-squared	0.9810
Mean	35.71		Adq. Prec.	15.623	Adjusted R^2	0.9241
C.V. %	11.17				Predicted R^2	0.7846

 ^* Significant values.

Table 4. Central composite experimental design of independent variables including the coded levels, actual values, predicted values and residuals for dextransucrase production.

			Variables			Dextransucrase DSUml^−1
Std.	Run	Type	X1	X2	X3	Experimental	Predicted	Residuals
3	1	Factorial	−1	1	−1	74.22	73.90	0.32
17	2	Center	0	0	0	83.90	83.61	0.29
6	3	Factorial	1	−1	1	96.90	96.84	0.06
2	4	Factorial	1	−1	−1	50.50	50.59	−0.09
13	5	Axial	0	0	−1.68	56.59	56.71	−0.12
18	6	Centre	0	0	0	83.98	83.61	0.37
5	7	Factorial	−1	−1	1	72.59	72.56	0.03
4	8	Factorial	1	1	−1	70.59	70.25	0.34
7	9	Factorial	−1	1	1	64.15	63.68	0.47
11	10	Axial	0	−1.68	0	69.89	69.68	0.21
1	11	Factorial	−1	−1	−1	55.59	55.65	−0.06
10	12	Axial	1.68	0	0	79.22	79.48	−0.26
19	13	Centre	0	0	0	83.49	83.61	−0.12
15	14	Centre	0	0	0	83.53	83.61	−0.08
20	15	Centre	0	0	0	83.06	83.61	−0.55
9	16	Axial	−1.68	0	0	61.86	62.13	−0.27
8	17	Factorial	1	1	1	89.79	89.36	0.43
16	18	Centre	0	0	0	83.80	83.61	0.19
12	19	Axial	0	1.68	0	77.99	78.74	−0.75
14	20	Axial	0	0	1.68	86.59	87.00	−0.41
				Level
Variables		Code	Units	1	−1	0	1.68	−1.68
Temperature		X1	°C	30	20	25	16.59	33.40
Sucrose		X2	gL^−1	30	10	20	3.18	36.81
Peptone		X3	gL^−1	5.0	1.0	3	−0.36	6.36

Table 5. ANOVA of central composite model for dextransucrase production.

Source	Coefficient Estimate	Degree of Freedom	Sum of Squares	Mean Square	F-value	P-value Prob > F
Model/Constant	83.61	9	2959.27	328.81	1527.10	<0.0001*
X1-Temperature	5.16	1	363.17	363.17	1686.71	<0.0001*
X2-Sucrose	2.69	1	99.12	99.12	460.36	<0.0001*
X3-Peptone	9.01	1	1107.50	1107.50	5143.65	<0.0001*
X1X2	0.35	1	0.97	0.97	4.52	0.0594
X1X3	7.33	1	430.27	430.27	1998.34	<0.0001*
X2X3	−6.78	1	368.15	368.15	1709.84	<0.0001*
X1^2	−4.53	1	295.38	295.38	1371.84	<0.0001*
X2^2	−3.33	1	159.34	159.34	740.06	<0.0001*
X3^2	−4.16	1	248.92	248.92	1156.09	<0.0001*
Residuals		10	2.15	0.22
Lack-of-Fit		5	1.57	0.31	2.72	0.1481
Pure Error		5	0.58	0.12
Cor Total		19	2961.42
Std. Dev.	0.46	Adq. Prec.	140.94	R-squared	0.9993
Mean	75.41	PRESS	12.76	Adjusted R^2	0.9986
C.V. %	0.62			Predicted R^2	0.9957

*Significant values.

Figure 3. Diagnostic Plots for dextransucrase production. (A) Normal probability plot of residuals (B) Residuals vs. Predicted values (C) Residuals vs. Experimental runs (D) Actual vs. Predicted values.

Figure 4. Contour and three-dimensional response surface plots showing the effect of independent variables on dextransucrase production. (A and B) temperature vs. sucrose (C and D) temperature vs. peptone (E and F) sucrose vs. peptone.

Figure 5. SEM analysis of dextran produced by the biocatalysis of dextransucrase.

Figure 6. FTIR spectrum of dextran produced by the biocatalysis of dextransucrase.