Kinetic Analysis of Crude Enzyme Extract Produced Via Solid State Fermentation of Banana Pseudo Stem Waste

Banana Pseudo stem waste after each harvest contributes about 70–80 Milli Tons Per hector. The banana pseudo stem will be thrown as waste biomass after each harvest as it is unstable for the upcoming harvest. The biggest challenge in banana cultivation is the utilization of biomass of banana pseusostem waste into valuable products. In this study, Xylano-pectinase enzyme extract was produced from banana pseudo stem waste under solid-state fermentation by Enterobacter cloacae PMC04. 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. Thermodynamics stated that range 40-50 o C were considered to be the optimal temperature for xylano-pectinase enzyme production and subsequent degumming of banana bers. The crude enzyme extract were then used in the degumming of banana bers for textile application. Textile processing of banana ber necessitates the removal of hemicellulose substance which can be achieved by crude xylano-pectinase enzyme. It was found that crude xylano-pectinase was ecient in the removal of hemicellulose substance from the bers. Results obtained from this study demonstrate that the proposed bioprocess could be successfully applied for the degumming of banana bers sustainably.


Introduction
Globally, One third of food produced for human consumption is wasted and contributes to food pollution.
The occurance of food waste can be from the initial agricultural production to the nal household consumption. According to Food and Agriculture Organization (FAO) the estimation of food waste is approximately 1.3 billion tonnes every year all over the world. The anaerobic decomposition of food waste causes methane emissions which is 25 times more potent than carbondioxide at trapping heat (Motelica et al., 2020). Therfore, proper incineration and utilization of waste resource is essential to reduce the food waste in the environment. India leads in banana production with 30 million tonnes per year with major cultivators in the southern states such as Tamilnadu, Maharashtra, Gujarat, Andhra Pradesh, and Karnataka. Waste biomass generated from the banana cultivation incorporates banana pseudo stem, peduncle, and other materials (Balakrishnan et al., 2019). Banana Pseudo stem waste after each harvest contributes about 70-80 Milli Tons Per hector. The pseudo stem, linchpin of banana ber will be hurled as waste after harvesting of banana since it is unstable for further cultivation The biggest challenge in banana cultivation is the utilization of biomass of banana pseusostem waste into valuable products. In this research, banana pseudo stem is utilized as solid substrate for the production of pectinase and xylanase enzyme. The production of industrial enzymes using agricultural waste substrate could improve the global market value of industrial enzyme.
Pectinase is the enzyme used to break down the pectin molecules, polysaccharides found in the cellwall of plants and natural bers. Pectinases catalyzes the pectin degradation process either by depolymeriztion or deserti cation reactions. The ocurance of pectinase can be pectin methyl esterases, pectin acetyl esterases, polygalacturonic acid and rhamnogalacturon (Handa et al., 2016). The microbial source of pectinases are in large demand due to their ease of multiplication, maintenance under controlled condition and cost-effective. Xylanase is a linear class of enzyme employed in the degradation of linear polysaccharide xylan into xylose. The xylan is the major component of hemicellulose which is about 30% of the plant cellwall (Cao et al., 1992).The major role of xylanases is the conversion of industrial agricultural waste into biomass which can be further used as animal feed or biofertilizers.
Solid-state fermentation possesses a series of advantages such as higher enzyme yield, negligence of foam, contaminants, and catabolite repression, ecofriendly in downstream processing, low energy, and medium requirements. The choice of agricultural residual substrate is an indispensable strategy for industrial pro tability with cost-effective biosynthesis (Zeng et al., 2017).
Considering all these aspects, our primary interest is focused on the conversion of banana pseudo stem waste to valuable products. The research highlights the utilization of banana pseudo stem as solid substrate for enzyme production and characterize the kinetic properties of crude enzymes produced.
Therefore, aim of the research is to a) produce xylanase and pectinase by solid state fermentation b)kinetic study of crude xylanase and pectinase extracts c)application of crude enzyme in ber degumming.

Microorganisms:
The microorganism was isolated from the banana waste decomposed soil in and around Tiruchirapalli.
The isolated microorganism was subjected to 16s RNA sequencing and identi ed as Enterobactor cloacae PMC04.

Raw Materials:
The banana pseudo stem was collected from NRCB (National Research centre for Banana). The banana pseudo stem were sterilized at 121 o C for 20 minutes. The sterilized banana pseudo stem waste was used as solid substrate for solid state fermentation.

Solid State Fermentation
Banana pseudo stem (dried) powder 30g was taken in 500ml of Erlenmeyer ask moistened with 70% solution containing (2g/L) each of K 2 HPO 4 and KH 2 PO 4 . The 10ml of xylano-pectinase bacterial culture were inoculated and incubated at 35 o C for 48 hours. After incubation, 1g of the fermented substrate was evacuated and extracted using 10 ml of pH 5, 0.2M citrate buffer. Using the whatman lter, the extracted enzyme was ltered and then centrifuged. The enzyme supernatant was used to analyze the pectinase activity (Pagarra et al., 2019) Xylano-Pectinase Asssay: 1ml of standard solution containing 0.1% pectin, 0.1% xylan was added with 2ml of sodium citrate buffer and 1ml of supernatant. The mixture was subjected to 25min incubation at 45 o C. After incubation, DNS was added as a stopper and subjected to a heating bath for 10min. The addition of 4ml distilled water to attain the nal volume and OD was measured at 570nm. One unit of enzyme activity corresponds to the micromoles of glucose as reducing sugar molecules produced under the reaction condition per minute (Hidayah et al., 2020) Thermal Deactivation of Crude Enzyme Extract Thermal studies were experimented by incubating the crude enzyme extract at speci c temperature in the absence of the substrate. The standard amount of enzyme extracts were taken in test tube and placed in waterbath. The temperature was varied from 30 to 70 o C for 120 minutes.

Banana Fiber Degumming
Mechanically extracted banana pseudo stem bers were subjected to enzymatic degumming. The crude enzyme extracted from solid-state fermentation by the isolate PMC04 was used for banana ber degumming. The banana bers were autoclaved for 20 minutes. The parameters observed for the degumming ber treatment are degumming temperature, degumming time, and enzymatic dosage (Jiang et al., 2017). The degummed ber was analyzed and compared with control bers.

Results And Discussion
The banana pseudo stem has e cient 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 bers. At the initial stage of kinetic process, the enzyme activities were identi ed 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 speci c time (E) to enzyme activity at initial time (E o ). The temperature was varied from 30 o C to 70 o C 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 is the enzyme activity at speci c time, E o is the initial enzyme activity, k d is the deactivation rate constant. It was found that k d values for crude xylano-pectinase in the range of 40-50 o C was higher compared to other temperatures. The k d values for pectinase and xylanase enzyme was illustrated in the following Table 1.  The Eyring absolute rate equation can also be used to predict the thermodynamic data of pectinase and xylanase enzyme. 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 The thermodynamic parameters of pectinase and xylanse enzyme was calculated and illustrated in the Table 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 55 o C. Further, on increasing temperature above 55 o C, 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 75 o C. Therefore, enzymes produced from banana pseudo stem waste are thermally stable.

Enzymatic degumming of banana bers
The mechanically extracted banana bers must undergo hemicellulose removal for textile application. The removal of hemicellulose substance can be done by enzymatic method. In this study, Banana bers are treated with pectinase and xylanase enzymes which resulted in the release of galacturonide. The amount of galacturonide release for 12 hours with time interval of 1 hour was done to screen out the optimum time for ber degumming. The degumming process was analyzed at 45 o C with (15% pectinase, 15% xylanase) enzymatic dosage for 12 hours at an interval of 1 hour. The maximum galacturonide released (0.6mmol/ml) concerning ber degumming time is identi ed to be maximum from 4 to 7 hours which is a relatively very period of treatment ( Figure 3 shows the SEM image of control and treated banana ber.

Conclusion
The banana pseudo stem waste was used as substrate for the production of pectinase and xylanse by Enterobactor cloacae PMC04. The deactivation of crude enzymes could be reversible between 30 and 55 o C. Further, deactivation of crude enzymes were spontaneous between 60-70 o C. The results infer that combination of crude enzymes could be better strategy for degumming of banana bers. The study clearly shows the crude pectinase and xylanase can be successfully produced from banana pseudo stem solid waste under solid state fermentation. The research also highlights an alternative approach of waste management to reduce the food waste problem.

Declarations Author Contributions
Mira chares Subash: Conceptualization, Methodology, Data curation, Writing-Original draft preparation, Writing-Reviewing, and Editing Enzyme production from banana pseudo stem waste.