1. INTRODUCTION:

Xylan is the second most abundant polysaccharide present in almost all plant cell walls. It is a polymer of D-xylose, a 5- member ring, linked to pyranosyl groups via β-1, 4 linkages. Breakdown of xylan is of commercial attraction these days because of its striking applications in food and pulp industries, production of biofuels through fermentation and paper industries¹. Xylanases are the key enzymes which hydrolyses the complex polysaccharide xylan to its simple form, xylose. Currently there exists a wide range of xylanases reported till date and are categorized into the glycoside hydrolases 10 and 11 (GH10 and 11) families². This classification is majorly done by considering their structural similarity, catalytic activity and some physiochemical properties such as molecular weight and their pH range³. A wide range of microorganisms have the ability to produce a variety of xylanases including bacteria and fungi.

Among these microorganisms, filamentous fungi such as Aspergillus and penicillium species have a remarkable ability of producing xylanases since they possess an abundant enzyme system required for the production of xylanases. These microorganisms utilize carbon sources from agro wastes such as wheat bran, rice bran, wheat and rice straw, corn cob, oat spelt, sugarcane bagasse etc⁴.

In paper and pulp industries, bleaching is the usual process to remove lignin in order to improve the quality of paper⁵. Recently, scientists have discovered that, xylanases alongside some hemicellulases can be very efficient and cost effective process compared to the traditional bleaching process⁶. Hence production of xylanases has become commercially important. Apart from this, xylanse production attained a huge attention in the field of pharma, food and feed processing industries. Clarification of juices is a major step in food processing industry. Turbidity in juices is mostly because of pectin materials and this can be precipitated by the application of xylanases in combination with pectinases and alpha amylases⁷. Conversion of xylan to its depolymerized product, xylitol, is an artificial sweetener⁸. Xylan hydrolysis liberates a variety of oligomers called xylooligosaccharides (XOs) which possess many advantages such as reducing cholesterol levels, balancing gastrointestinal health by acting as a prebiotic product and maintains the biological availability of calcium⁹. Xylanase production is also important in the processing animal and poultry diet in terms of nutritional value and reducing the complexity and making them easily digestible¹⁰.

Production of xylanases using microorganisms is majorly provisioned through fermentation technology. Two types of fermentation practices are currently being used by industries: one is solid state fermentation and the other is submerged fermentation. Solid state fermentation is the commonly chosen method of producing xylanases over submerged fermentation process because of several reasons, such as low economy and simple downstream processing whereas submerged process limits in terms of aeration, complex purification methods and economy¹¹. Major downstream process include in the purification of xylanases are centrifugation, column chromatography and dialysis. In this current study, we are reporting production and purification of xylanase from penicillium funiculosum and corn cob powder as carbon source using solid state fermentation process.

2. MATERIALS AND METHODS:

2.1. Media and growth Conditions:

P. funiculosum was obtained from the National Chemical Laboratory, Pune. Fungal strains were grown in potato dextrose agar (basal media)flasks (100 ml) at 30°C for 96 h.

Inoculum was then transferred on to reese and mandel medium. Composition of the medium (for 1L) is as follows: KH₂PO₄-3g, (NH₄)₂SO₄-1.5g, MgSO₄-0.3g, urea-0.3g, CaCl₂-0.3g, peptone-2.5g, yeast extract-2g, FeSO₄·7H₂O-0.005g, MnSO₄·H₂O-0.0016g, ZnSO₄·7H₂O-0.0014g, and CoCl₂-0.0012g (pH 5.0). 1% glucose was supplied after sterilizing the media.

The above heat sterilized media was inoculated with P.funiculosum and incubated for 96h at 30°C. Mycelia was collected from the media after incubation by centrifuging at6, 000rpm for 20 min. The pellet was washed, ` and resuspended in 100 ml of 20mM sterile phosphate buffer, pH 4.8 and stored at 4°C and was used as inoculate fermentation medium.

2.2. Enzyme production by solid-state fermentation (SSF):

Corncob was collected from agricultural fields, powdered after proper drying and autoclaved. Fermentation media was prepared by adding yeast extract (0.1% w/v), corn cob powder (5% w/v) in appropriate proportions and sterilized¹². 5% (v/v) of inoculum from reese-mandal medium was added to this medium and kept for growth at 30°C for 14 days. 40% of moisture was maintained within the flask throughout the incubation period. Once growth was observed, media was resuspended in 20mM phosphate buffer pH-4.8 and subjected to centrifugation at 6000rpm for 10min for the separation of mycelium.

2.3. Xylanase purification:

Supernatant was collected and subjected to ammonium sulphate (75%) precipitation¹³. After precipitation, this mixture was subjected to centrifugation at 6000rpm for 10 min. Pellet was resuspended in 10ml of buffer and subjected to dialysis to eliminate salts. Dialysed protein sample was concentrated using sucrose powder.

Cation exchange column chromatography:

In order to collect the specific enzyme, the above concentrated sample was allowed to run through a cation exchange column. CM Sephadex C-50 matrix soaked in distilled water overnight and column (10cm X 2cm) was packed with a bed volume of 15 ml¹⁴. The column was washed with distilled water, equilibrated with 10mM acetate buffer and the sample was loaded on to the column. Flow through was collected about 10ml and a gradient of NaCl was established (0-0.5) and the fractions were collected. Absorbance of these fractions was measured at 280nm and the protein concentrations were estimated. A chromatogram was plotted against the absorbance values with the fraction number (figure2).

2.4. Xylanase activity assay:

Since xylanase reduces polysaccharide xylan to its subsequent sugar xylose, standard graph (Figure1) was prepared using 0–500μg xylose. 900μL of 1% xylan solution was added with 100μL enzyme solution in a test tube. 1.5mL of 3, 5-Dinitrosalicylic acid (DNS) reagent was added and incubated in hot water bath at 50°C for 5 min. Optical density values were measured at 540nm and the reaction in the control tubes were terminated at zero minutes time. One unit of xylanase activity was estimated as the amount of enzyme produced 1µmolof xylan being reduced to xylose per minute under the assay conditions described¹⁵.

3. RESULTS:

3.1. Production of xylanase using solid state fermentation:

P.funiculosum inoculum size was optimized for the adequate amount of xylanase production. From reese-mandal medium, different inoculum sizes ranging 5-20% were chosen to optimize the inoculum volume. Similarly, pH and temperature optimizations of the fermentation media were also done to improve the yield of xylanase production. Sample collected after ammoniumsulphate precipitation and dialysis was taken as a crude protein sample which was having xylanase in the pool.

3.2. Purification of xylanase using ion exchange chromatography:

Further purification was achieved by running the fraction on a cation exchange column chromatography¹⁶. From the chromatogram (Figure2) obtained after purification, few peaks (7, 11and12) were selected and tested for xylanase activity by incubating the fraction with 1% xylan. From the standard graph of xylose (Figure1) amount of xylose liberated was deduced and subsequently converted to its enzyme activity.

Figure 1. Xylose standard curve

Figure 2 purification of xynalase from the crude protein obtained from ammonium sulfate precipitation using ion exchange column chromatography. Fractions 1-10 were flow through and 1 1-20 are charged proteins by 0-0.5 M Nacl gradient. Highlighted peaks were taken for testing xynalase activity.

3.3. Optimization of xylanase production:

3.3.1. Effect of inoculum size on xylanase production:

No effect of inoculum size on xylanase production was observed using solid state fermentation. Increasing the inoculum size from 1% to 10% did not show any effect on xylanase production (Figure 3).

Figure 3. Effect of inoculum size

3.3.2. Effect of initial substrate concentration on xylanase production:

Higher initial substrate concentration of substrate up to 5% showed a substantial increase in the production of xylanases by solid state fermentation (Figure 4).

Figure 4. Effect of substrate concentration

3.3.3. Effect of supplement carbon on xylanase production:

Addition of supplementary carbon source has mild effect onxylanase production. Increasing the supplementary carbon i.e., glucose from 1% to 5% showed mild increase in the production of xylanases (Figure 5).

Figure 5. Effect of glucose as carbon source

3.3.4. Effect of pH on xylanase production:

Maintaining a pH-6 during fermentation was observed to yield better productivity of xylanases (Figure6).

Figure 6. Effect of pH on the production of xylanase

4. DISCUSSION:

Xylanases with improved quality can be a remarkable trend in food, feed and pharma industries also they can be the future biofuel stimulators. Much more-light should be shed on various roles of xylanases in order to meet the increasing demand of the population across the globe. The present study focusses on production of xylanases using agricultural wastes and optimizing the growth conditions for better yield of xylanases. Approximately 668 Units of xylanase enzymeper gram of Corn Cob powder was produced in solid state fermentation. Since the production of xylanases is taking advantage of using agro wastes, it will be helpfulin reducing the soil pollution as well as having a cost-effective production.

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Received on 24.09.2019 Modified on 19.11.2019

Research J. Pharm. and Tech 2020; 13(9):4111-4114.

DOI: 10.5958/0974-360X.2020.00726.X