Biotechnol Ind J, Volume: 13( 4)
The Production of Xylanase Enzyme (E.C. Number=22.214.171.124) Using Solid Substrate Fermentation
- Nitin Kumar Singh, IMS Engineering College , Ghaziabad, India, Tel: 01332 285 311; E-mail: [email protected]
Received: June 04, 2017; Accepted: July 21, 2017; Published: July 24, 2017
Citation: Nitin KS, Vivek KT, Santosh KM. The Production of Xylanase Enzyme (E.C. Number = 126.96.36.199) Using Solid Substrate Fermentation. Biotechnol Ind J. 2017;13(4):145.
The production of Xylanase enzyme (E.C. number=188.8.131.52) was studied using solid substrate fermentation. Various solid substrates were selected as a solid substrate for Xylanase production using SSF. Optimum xylanase activity was observed when Pea peel has been used as solid substrate. The production of the xylanase is carried out some other solid agricultural waste because. Agro industrial waste are a good source of nutrition for the growth of the microorganisms as they are rich in carbon source and agro-industrial wastes such as wheat bran, sugarcane bagasse, corn cob, rice bran and wheat straw are abundantly available and cheapest natural carbon sources. The present study is an attempt for process optimization for xylanase production using agro industrial waste as a sole carbon source. The production of the enzyme xylanase has industrial use and it can be used for many commercial purposes including chlorine free bleaching of the wood pulp prior to papermaking. Xylanases can also be used as food additives in poultry, in wheat flour for improving the handling of dough and increasing the quality of baked products, it is also used for the extraction of coffee, extraction of plant oils and starch. Different physical and chemical parameters which affects the production of xylanase has been optimized by batch experiment as well as using statistical tool i.e., Design-Expert. Experimental outcomes showed that agro-industrial residue having excellent potential for the production of industrial important enzyme i.e., Xylnase.
Agro-industrial; Plant oils; Xylanase enzyme; Fermentation
The Global market for industrially useful enzymes was estimated to be about $4.2 billion in year 2014 and is expected to develop at a compound annual growth rate (CAGR) of approximately 7% over the period from 2015 to 2020 to reach nearly $6.2 billion in the year 2015 at Industrial Enzyme Market [1-9]. Within the grain-processing proteins sector alone which accounts for the widest sector where enzymes are used the growth is significant. Presently the technical industries, dominated by the detergent, textile, starch and fuel alcohol industries, account for the bulk of the overall enzymes used in the industrial sector, with the feed and food enzymes along totaling solely regarding thirty fifth of total. Enzymes are a group of protein which increases the rate of the biological reaction. Xylanase is the class of enzymes which convert the polysaccharide beta-1,4-xylan into xylose, it breaks hemicellulose, which is the major components of plant cell walls. It plays a major role for the degradation of plant cells hemicelluloses into the usable nutrients. Xylan is found in massive quantities in softwoods from the gymnosperms i.e., 7% to 10% and hardwoods from the angiosperms i.e., 15% to 30% of the plasma membrane content and similarly as in annual plants about 30%. It is mainly present in the secondary cell wall of plants, but it is also found in primary cell wall, mainly in monocots. The ecological niches of those micro-organisms which having capability to produce xylanase are numerous and widespread and generally embrace environments wherever stuff accumulate and deteriorate, similarly as within the tum of ruminants [10-14]. Xylanases are produced by bacteria, fungi, snails, marine algae, protozoans, yeast, seeds, insect, crustaceans, etc., (mammals do not produce xylanases). The main source for industrially important xylanases is the filamentous fungi. Solid state fermentation (SSF) process is the preferable biochemical process for the production of xylanases using various agro industrial waste although various methods use submerged fermentation for the enzyme. The process of the SSF is more economical and it is easy to isolate the enzyme in a SSF. Use of SSF provide various advantages to the system the low wetness content of the substrates eliminates the borderline contamination, lowering the operational value of the reactors, it is also economical [15-21]. For SSF the product separation is simple and fewer cumbersome. One of the major advantages of using SSF is that there is low waste water output and also no issues of foaming. Xylanases can be of fungal origin or the bacterial origin and based on the climatic conditions where the enzyme is originated it can be Extremophilic xylanases, Thermophilic xylanase, Psychrophilic xylanase or alkaliphililic and acidophilic xylanase. Xylanase is an industrially important enzyme which is used in many industrial processes. The major current application of xylanases is within the pulp and paper industries for economical biobleaching. Xylanases would even be needed for detergent applications, xylanase would be helpful in animal feeds if accessorial to the feeds, they'd be most fitted to use within the baking business as dough preparation. The use of Xylanase can be seen in production, cut back haze within the final product, to extend wort filterability. In occasional extraction and within the preparation of soluble occasional. It is also used in the production of pharmacologically active polysaccharides to be used as, within the proto-plastation of plant cells, antimicrobial agents or antioxidants, and within the laundry of exactitude devices and semiconductors, within the production of alkyl radical glycosides to be used as surfactants. Further, this study can be useful in reducing the cost of enzyme. The future prospects of this enzyme lie in its use as Biofuel and replacement of chlorine from the industries which is used in beaching purposes. The objective of the research work was to isolate and screen the microorganism producing xylanase and its process optimization using a statistical tool i.e., Response Surface Methodology. The study was done using the agricultural waste products.
Materials and Methods
Culturing and sub culturing of microorganisms- Source microorganism culture was provided by IMSEC culture bank. This culture was grown on Potato Dextrose Broth. Culture was revived after two or three weeks regularly. Maintenance of culture and inoculum preparation- Once the reviewed strain was inoculated in the Petri plates containing PDA media. Culture plates are covered with parafilm and incubated at 28°C in BOD incubator [22-25]. YEPD broth was prepared by using yeast, peptone, dextrose and distilled water. This broth was incubated at 30°C for 4 days for the growth and kept in refrigerator for the maintenance of culture [26-32]. For inoculum development prepare a broth of Aspergillus nidulens and inoculate 500 μl from it to all the autoclaved flask keep it in incubator at 30°C for 5 days to observe growth Screening and selection of agro waste for the Xylanase production- Various agro industrial waste were taken, used and screened for their ability to produce Xylanase. After the grinding the substrate was autoclaved and then seeded with the microorganism, after 72hrs of incubation the growth of fungus was optimum and further enzyme was extracted and assayed. Out of the various solid Substrates used this has been observed that growth of the microorganism was maximum in Pea peels, therefore pea peels were selected for the further research work for Xylanse production.
The amylase assay was performed according to the protocol. One unit of enzyme activity is defined as the quantity of enzyme that caused 0.01% reduction of color intensity of xylan solution at 50 1C in 1 min per ml.
Optimization of process parameters
Optimization of various process parameters were carried out based on the growth of the micro-organism and how that particular parameter is affecting the activity of the enzyme. Various previous studies done for the production of the enzyme estimated that the growth of the enzyme is optimal at the temperature of around 30°C to 35°C, experiments were set accordingly keeping in mind the optimal temperature range and since the presence of the moisture content provides a better environment for the growth of the fungus, this parameter needs to be optimized to get the maximum enzyme activity [33-35]. Since carbon source is required to meet the energy requirements of the cell, so an additional carbon source other than the raw substrate is provided in order to meet the requirements of the microorganism during the growth phase. Nitrogen is also an important macronutrient which is used by the microorganism for DNA synthesis and other work such as synthesis of the amino acids. So, on providing an optimal amount of the carbon and nitrogen source the growth of the microorganism can be optimized.
Optimization by response surface methodology: Response surface methodology i.e., RSM is a collection of statistical and mathematical techniques for empirical model building. By careful design of experiments, the objective is to optimize a response which is influenced by several independent variables (input variables). An experiment is a series of tests, called runs, in which changes are made in the input variables in order to identify the reasons for changes in the output response [36-39]. This was done using Design Expert tool by which we can use the RSM effectively. Different sets of experimental runs were provided by this tool by using different variables which were used to set up the experiment. The variables taken here are the temperature, moisture content, carbon source and nitrogen source. Different runs of the experiment were done and the results were analysed for the different runs of the experiment.
Result and Discussion
During the present research work, this has been observed that different solid agro industrial substrate can be used for the production of xylanse and product formation is directly associated with the growth of fungi. Further on the basis of initial screening that Pea Peals having excellent potential for the production of xylanase as solid substrate the Xylanase production process was optimized. The results indicate that Pea peels when used as solid substrate the maximum absorbance was observed at 540 nm wavelength with 100% initial moisture content used. It has been also found that initial moisture content plays significant role in the growth of microorganism and subsequently in the production of Xylanase [40-45]. Temperature also play another critical role in the growth of fungi and product formation Result of experiment showed that the growth of the microorganism was maximum at 30°C. Experimental outcome indicates that additional Carbon and Nitrogen sources enhance the growth of A. nidulans and resulting in the enhanced production of xylanase. It was observed that during the experiments Dextrose was preferred carbon source which promotes the growth of fungus. On the other hand 0.1M urea when used as supplementary nitrogen source in the solid substrate enhances the maximum growth of the microorganisms After the initial results of individual experiment the effect of all the four factors we have used DESIGN EXPERT tool to see the combined effects of the factors on the growth of the microorganism. Eight different experiments were performed in different flasks according to runs provided by RSM tool of Design expert and the further enzyme activity has been recorded.
The effect of different physical and chemical parameters on the Xylanase production were observed. During this research work the major parameters that affects the growth of the filamentous fungi and production of xylanase were moisture content, temperature, Carbon source and nitrogen source. Experimental outcome of the various physical and chemical parameters has been given below:
For optimization of the moisture content three flasks with solid substrate including desired moisture content and other supplementary nutrients were incubated at 30°C for 72 hrs. The crude enzyme was extracted and enzyme activity was observed. Results reveals that when moisture content was used 100% w/v the enzyme production was maximum further this has also been observed that when moisture content is increased it decreases the porosity of the solid substrate that prevents the growth of the filamentous fungi. However, lower moisture content i.e., 80% seems to unavailability of desired water content for the growth of filamentous fungus resulting in the decrease of enzyme activity. Similar type of study was performed by Ajay Pal  by using soybean cake with variable moisture content as solid substrate the optimum growth of microorganism was observed with 70% moisture content. Result of moisture content showed that moisture content is a critical factor that affects the growth of microorganism. The variable moisture content is needed by different solid substrate due to variable properties of solid substrate i.e., porosity, particle size and surface area etc (Figure 1).
For optimizing the temperature for the growth of the microorganisms on the solid substrate, the solid substrate was three different flasks and the 100% moisture content was provided, the flasks were incubated at three different temperatures i.e., 25°C, 35°C and 30°C. After inoculating the flasks with A. nidulens maximum enzyme activity was observed at 30°C when pea peels were taken as solid substrate. Sanghi et al.  performed similar type study in which effect of temperature on xylanase production was optimized using wheat bran as asolid substrate and maximum enzyme activity was at 30°C when pea peels were taken as solid substrate (Figure 2)
Xylanase research suggests that supplementary carbon source promotes the growth of microorganism when used in desired concentration. For the optimization of the role of additional carbon content, initially four carbon sources i.e., Xylose, Dextrose, Maltose and Fructose were used in solid substrate. Experiments were set up in four different flasks in which the substrates were provided with these additional carbon sources. Result of these experiment showed that when Dextrose were used in 1.5% w/v 5 ml in each flask was used there is maximum growth of fungi. Jatinder et al.  investigated the effect of carbon source and it was found that using RSM the most suitable additional carbon source was Dextrose, in our study we found that with Dextrose as additional carbon source the enzyme activity was maximum (Figure 3).
For the optimization of the additional Nitrogen source which is to be provided to the substrate in order to support the growth of the microorganisms, four different flasks with different nitrogen sources and their different concentrations were incubated at 30°C. A similar study for the optimization of the additional nitrogen source was done by Yang et al. in the year 2005. In our study, it was found that 0.1 M urea was best suited for the growth of the microorganism and the enzyme activity was maximum at 0.1 M urea concentration (Figure 4).
Result of optimization of process parameters using design expert: Design Expert tool is the statistical way to know the effect of one variable on the other variable. After studying the effect of various parameters individually, there was a need to study that how the presence of one variable affects the other variable when both the parameters are used simultaneously in the growth media. The experimental design using Response Surface Methodology was used to estimate the coefficients in a mathematical model, to predict the response and to check the applicability of the model. These four independent variables were studied at different levels and their minimum and maximum values are listed. As evident from table below, run orders with different combinations of Temperature, Moisture, Nitrogen and Carbon source levels enhanced Xylanase activity. Similar result was also obtained by Liu et al.  where Plackett–Burman design with response surface methodology was proved better for optimization for the production of the enzyme. Cotarlet and Bahrim  also mentioned the importance of statistical designs over “one-variable-at-a-time” conventional approach for optimization. Relevance of statistical approach over conventional methods was also mentioned in literature reports. Significance of statistical designs for production of enzymes was also in agreement with the results of Kammoun et al.  were the yield of the enzyme was increased. For the analysis by using the statistical tool eight different experiments were set according to the runs provided by Design Expert using RSM, these experiments were set and the enzyme assay was done. The table below shows the runs and the results of the assays. The contour plots were obtained which shows the dependency of one variable on the other variable.
Different graph patterns were obtained which show the dependency of one variable on the other variable, like the dependency of Enzyme activity on Moisture and Temperature, Moisture and additional Carbon source, additional Nitrogen source and Temperature on Enzyme activity, additional Carbon source and Temperature on Enzyme activity (Table 1). The graph plots show how the presence of one variable affects the utilization of the other variable for the growth of the microorganism (Figure 5-8).
Table 1: Runs of experiments by design expert.
During the present research work it has been observed that Aspergillus niger have excellent potential for the production of enzyme Xylanase when Pea peels were used as solid substrate. Simultaneously this has been found that Orange peels, Bagasse, Pineapple peels, Mousseme peels also have good potential as a solid substrate for Xylanase production. Good potential to utilize agricultural waste for the production of Xylanase, traditionally many agro waste have been used for the production of this enzyme. It has been found that Pea peels are a good substrate for Xylanase production. Moisture content also plays a major role in enzyme activity. Apart from Moisture content other factors like temperature, additional carbon and nitrogen content were also optimized.
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