The banana pseudo stem has efficient carbon source to be used as substrate for solid state fermentation. The xylano-pectinase activities produced from the fermentation of banana pseudo stem waste were analyzed and shown in Fig. 1.Enzyme production from banana pseudo stem waste. The maximum pectinase and xylanase activity was observed at 12–16 hours. The highest pectinase and xylanase activities obtained using banana pseudo stem as carbon source were 124.62 U/ml and 173.81 U/ml respectively. From the result, it is inferred that banana pseudotem has the ability to provide essential nutrient for the growth of bacteria. The higher enzyme activities are due to the higher carbon source which is essential for the growth of the bacteria (Rahul et al., 2017).
Kinetic Studies on Crude Enzymes
It is essential to understand the kinetic behavior of crude enzyme extracts produced from banana pseudo stem waste before scaling up the bioprocess. Thermal deactivation is a major problem in enzymatic degumming of banana fibers. At the initial stage of kinetic process, the enzyme activities were identified as 124.62 U/ml and 173.81 U/ml for pectinase and xylanase respectively. Deactivation of the enzymes are measured as the ratio of enzyme activity at specific time (E) to enzyme activity at initial time (Eo). The temperature was varied from 30oC to 70oC to identify the thermal deactivation of xylanase and pectinase enzyme produced from solid state fermentation of banana pseudo stem waste. The thermal deactivation is expressed as exponential decay
E = Eo x e− kdt
E is the enzyme activity at specific time, Eo is the initial enzyme activity, kd is the deactivation rate constant. It was found that kd values for crude xylano-pectinase in the range of 40-50oC was higher compared to other temperatures. The kd values for pectinase and xylanase enzyme was illustrated in the following Table 1.
Table 1
Deactivation rate constant for pectinase and xylanase
oC
|
K
|
Kd (hour-1) for pectinase
|
Kd (hour-1)
for xylanase
|
30
|
303
|
0.0012
|
0.0135
|
35
|
308
|
0.0024
|
0.0170
|
40
|
313
|
0.0081
|
0.0208
|
45
|
318
|
0.0095
|
0.0286
|
50
|
323
|
0.0098
|
0.02741
|
55
|
328
|
0.0072
|
0.01554
|
60
|
333
|
0.0019
|
0.0100
|
65
|
338
|
0.0017
|
0.0087
|
70
|
343
|
0.0016
|
0.0060
|
The half-life (t1/2) is the time when the enzyme activity decreases to half of its original value. The determination of half-life period is essential for the economic feasibility of banana pseudo stem waste reutilization. The longer shelf-life corresponds to the higher thermostability. The half-life can be calculated by replacing E with Eo/2. Table 2 illustrates the half-life period of pectinase and xylanase enzyme. The mathematical equation for half-life determination is given as
Table 2
Determination of Half-life
oC
|
K
|
t1/2 for pectinase
(hours)
|
t1/2 for xylanase
(hours)
|
30
|
303
|
45.22
|
51.33
|
35
|
308
|
38.19
|
40.764
|
40
|
313
|
28.64
|
33.317
|
45
|
318
|
12.89
|
37.258
|
50
|
323
|
12.67
|
39.8041
|
55
|
328
|
10.82
|
44.594
|
60
|
333
|
33.81
|
69.3
|
65
|
338
|
35.73
|
79.665
|
70
|
343
|
36.67
|
115.5
|
t1/2 = ln (2)/ Kd
The activation energy can be calculated from the Arrhenius equation. The Arrhenius equation can be written as
Kd = Ae− Ead/RT
Ead is the activation energy, R is the universal gas constant and T is the absolute temperature. The activation energy for xylanase and pectinase enzyme when the temperature was in the range of 30 to 50oC is -10.314 KJ/mol and − 23.52KJ/mol respectively. The activation energy increases with increase in temperature. When the temperature was in the range of 55 to 70oC, the activation energy was increased to 59.668 KJ/mol for xylanase enzyme and 47.36KJ/mol for pectinase enzyme.
The Eyring absolute rate equation can also be used to predict the thermodynamic data of pectinase and xylanase enzyme.
Kd = (kBT/h)e(−∆H/RT)e (∆S/R)
∆H = Ead -RT
In this equation, where
h is the planck constant of 6.63x10− 34 J s,
kB is the Boltzman constant of 1.38 x 10–23 J/K
T is the absolute temperature
∆H is enthalpy of activation in KJ/mol
∆S is entropy of activation in J mol-1K−1
∆G is the free energy of activation
The enthalpy of activation can be calculated from the activation energies for denaturation, Further, free energy of activation can be determined from the following equation
∆S = (∆H-∆G)/T
The thermodynamic parameters of pectinase and xylanse enzyme was calculated and illustrated in the Table 3.
Table 3
Thermodynamic study of crude enzymes
Temperature
(oC)
|
Pectinase
|
Xylanase
|
∆H (kJ/mol)
|
∆G
(kJ/mol)
|
∆S
(J mol− 1K− 1)
|
∆H
(kJ/mol)
|
∆G
(kJ/mol)
|
∆S
(J mol− 1K− 1)
|
30
|
10.812
|
56.67
|
-242.72
|
13.347
|
67.46
|
-266.689
|
35
|
10.729
|
61.92
|
-263.38
|
13.56
|
71.92
|
-277.532
|
40
|
11.591
|
74.34
|
-286.72
|
13.508
|
91.01
|
-333.923
|
45
|
11.599
|
44.89
|
-269.119
|
13.458
|
54.632
|
-214.119
|
50
|
11.452
|
48.19
|
-212.388
|
13.409
|
68.19
|
-252.628
|
55
|
45.961
|
40.38
|
-108.62
|
56.62
|
60.43
|
-11.615
|
60
|
45.890
|
40.57
|
15.39
|
56.668
|
67.665
|
33.024
|
65
|
45.182
|
40.81
|
16.311
|
56.71
|
67.108
|
30.763
|
70
|
45.52
|
40.114
|
16.99
|
56.753
|
67.154
|
30.3
|
Thermodynamics data states that when ∆H is positive, ∆S is negative, the process is non-spontaneous at any temperature and the reverse process is spontaneous. Therefore, deactivation rate of xylanase and pectinase could be reversible between 30 to 55oC. Further, on increasing temperature above 55oC, both ∆S and ∆H are found to be positive for both enzymes which indicates that deactivation of enzyme is spontaneous at higher temperature from 60 to 75oC. Therefore, enzymes produced from banana pseudo stem waste are thermally stable.
SEM analysis of fibers
Scanning Electron Microscopy generates signal diversification at the outer layer of the solid specimen by high energy electrons with a focused beam. Pectin is found to be mostly entrapped in the mesial lamella of the cortical tissue. The SEM micrographs of the sample before and after treatment were analyzed. The majority of gummy substance removal can be visualized in the degummed banana fiber by 5–6 hours except other hemicellulosic substances. From the image, it is obvious that the degradation of pectin substance and removal of hemicellulosic substances in degummed banana fibers (Cheng et al., 2019). However, the minimum amount of hemicellulose substance may remain in the dislocation region of the degummed banana fibers (Mao et al., 2019). Figure 3 shows the SEM image of control and treated banana fiber.