INTRODUCTION:

Enzymes are used in a variety of ways in the biochemical industry. Enzymes are the focal point of metabolic and biochemical processes. As a result, they are studied extensively by biologists and process designers/engineers, chemical engineers, pharmacists, manufacturing experts, and other scientists. Enzymes were used in various processes in the past, such as the manufacture of wine and bread. Xylanase is one of them which are widely used in various purpose. Xylanase is a naturally occurring enzyme present in bacteria and fungi that can help humans digest carbohydrates. Plant cell walls contain three major polymeric constituents: cellulose (insoluble fibers of - 1,4-glucan), hemicellulose (non-cellulosic polysaccharides such as glucans, mannans, and xylans), and legnin (a complex polyphenolic structure).

Inwood, xylan is the most abundant hemicellulose. In terrestrial plants, xylan has a backbone of glycosidically 1,4-linked xylopyranose units, whereas, in marine algae, it has a 1,3-linked backbone¹. Xylanase's uses are Food Industry, Bread Quality Improvement, Degumming, Agro waste treatment, Food industry, pharmaceutical industries, etc. Wheat bran is one of the cheapest and readily available in West Bengal, India, for this enzyme production.

MATERIALS AND METHODS:

Production of xylanases under SSF and SMF:

Xylanases are produced by solid-state or submerged fermentation (SMF) techniques. The enzyme productivity via solid-state fermentation (SSF) The rate of submerged fermentation is usually much higher. Because of the process's economic and engineering advantages, solid-state fermentation (SSF) techniques are increasingly being used to generate a wide variety of enzymes, like xylanases from fungal sources².

SSF processes have many advantages over SMF processes, including lower fermentation cultivation costs, lower risk of contamination, improved enzyme stability, mimicking the fungus' natural habit, producing enzymes with higher specific activities, generating a protein-enriched byproduct, and easier downstream processing of the enzymes produced³.

Fungi grow best in SSF conditions because they can grow at low water activities, unlike most bacteria and yeast, which will not proliferate under these conditions⁴.

Submerged fermentation allows for more precise monitoring of fermentation conditions. For the development of xylanase, submerged aerobic microorganism fermentation is a well-known and widely used process⁵.

When more purified enzymes are needed, SMF is preferred. In SSF preparations using complex substrates, however, synergistic effects from a battery of xylan-degrading enzymes can easily be detected. The latter, on the other hand, is commonly sought in animal feed applications⁶.

Xylanases have been categorized into the GH10 or GH11 families, but research on xylanases in families 5, 7, 8, and 43 is still in its early stages. The conversion of xylan to valuable goods is part of our efforts to improve the overall economics of lignocellulosic biomass processing and develop new renewable energy production methods. Xylanase enzymes are among those with a wide variety of significant industrial applications. As a result, new methods for producing these enzymes that are simpler and less expensive will be created in the future to meet the needs of different industries. In this context, the use of lingo cellulosic agricultural waste for enzyme production through submerged or solid-state fermentation and molecular techniques being tested to improve the enzyme's characteristics and increase its expression rates has been very appealing. Furthermore, since the native enzyme does not fulfill all of the process criteria, bioprospecting for new genes, logical engineering, and directed evolution of existing genes can all be used to improve these enzymes.

Microorganism:

The microorganism was isolated from soil and identified as Aspergillus spp. was used for experiments.

Composition of inoculums medium:

The inoculum medium's composition was glucose 50g/l, KH₂PO₄ 1.0g/l, KCl 0.5g/l, NaNO₃ 2.0g/l, MgSO₄. 7 H₂O 0.5g/l, FeSO₄. 7 H₂O 0.001g/l, Agar 30g/l. pH 4.0-4.2. The calculated amount of ingredients was dissolved in distilled water except agar. Then the pH of the mixture was adjusted using 0.1N HCL and NaOH. After pH adjustment, the agar was added and dissolved in a hot water bath. The medium without agar was distributed in 250ml Erlenmeyer flasks. The flasks were cotton plugged and covered by brown paper. The Slants were prepared by transferring 5 ml of hot medium containing agar in test tubes, and then Erlenmeyer flasks containing 50ml medium and test tubes for slants were sterilized at 121⁰C for 15 minutes.

Preparation of Fermentation Medium:

Wheat Bran was taken as a fermentation medium for the production of xylanase. 25gm of wheat bran was taken in 500ml Erlenmeyer flask. 20ml distilled water was added to it and mixed well. After that, the flask was cotton plugged and covered by brown paper. Then the flask was sterilized in the autoclave.

Fermentation:

The spore of Aspergillus sp. was scraped by a sterile needle and transfer to 10ml sterile distilled water. Then 1.25ml of spore suspension was added to a sterile wheat bran flask in aseptic condition. Then the flask was incubated at 27^oC - 30^oC for three days (72 hrs.).

Extraction of Enzyme:

After completing the fermentation process, 100ml distilled water was added to the fermentation flask and mixed well. Then the flask was put into the shaker for 1 hour. After that, the liquid portion was collected as filtrate after filtration by filter paper. Then the extracted liquid was centrifuged at 5000 rpm for 15 minutes. Then the supernatant was collected as the source of crude xylanase.

Xylanase Assay:

The enzyme activity was determined using xylose as a substrate. 0.5gm xylose was dissolved in 50ml of 0.2 M NaOH in a 100ml volumetric flask. Then pH was maintained at 4.8-5.2 by glacial acetate acid, and then the volume was made up to 100ml. For enzyme assay, 1 ml crude enzyme solution and 1ml substrate were taken in a test tube and incubated at 60^oC in a water bath for 5 mins. Then 3ml. of DNSA was added to it, and then this was transferred immediately to boiling water bath for 10 mins. After that, the test tube was put into cold water for cooling. Then the optical density was measured at 540 nm in a spectrophotometer.

Enzyme activity = (Dilution factor X 2 X 100 X Sugar concentration)/(Incubation time x MW of xylose)

M.W of xylose = 150 and Time of incubation = 5 mins.

Standard curve for xylanase assay:

The final solution's optical density was measured, and these were plotted against the known sugar solution. A straight line was obtained, and the equation of that straight line is y = 1.2193x + 0.0403. From the measured optical density of the solution (y), we can calculate the unknown sugar concentration (x) produced by an enzymatic reaction. From the calculated sugar concentration, enzyme activity was measured by applying the above equation.

DNA Preparation:

75g Na-K tartrate dissolve in 250ml distilled water, 4g NaOH was dissolved in 50ml distilled water, and 3.5g DNSA was taken as a reagent. In a 500ml volumetric flask, 2.5g DNSA was taken. Then a certain amount of Na-K tartrate was added. Next, NaOH was added, and then the remaining tartrate was added, and volume was made up to 500ml.

Protein Assay: (Folin Method):

For protein assay following reagents were taken for the folin method for assay of protein.

A. 2% Na₂CO₃ dissolve in 0.1 N NaOH.

B. 0.5% CuSO₄ and 1% Na-K tartrate.

C. 1ml CuSO₄ and 1ml of Na-K tartrate mixed with 48 ml. of 2% Na₂CO₃ in 0.1 NaOH.

D. Folin reagent, diluted with water 1:1.

0.4ml of the sample containing 10-100mg of protein was mixed with 2ml of reagent C. The solution allowed to stand for 10 mins. 0.2ml reagent D pipette out rapidly into the mixture with thorough mixing and measured OD value at 750nm, after 30 minutes.

Standard curve for protein Assay:

The optical density of the reaction mixture was measured and plotted against standard BSA concentration. A straight line was obtained from this graph, and the equation is y = 0.777x + .047. From measured optical density, we can calculate the unknown protein concentration.

Enzyme Purification:

The crude enzyme was purified firstly by salting out method using anhydrous (NH₄)₂SO₄.20 ml crude enzyme was taken in 50ml beaker. Then 30%, 40%, 50%, 60%, 70% and 80% saturated (NH₄)₂SO₄. Were dissolved into each beaker and marked. Then these beakers were put overnight under refrigerated conditions for precipitation of protein. After that, these were taken out and were centrifuged at 5000rpm for 15 minutes. The supernatant was discarded, and the precipitate was dissolved in 5ml 0.1 M (pH – 5.2) acetate. After that, enzyme assay and protein assay were done for each sample. Then the enzyme was purified by dialysis after dissolving the precipitation in the buffer.

Preparation of 0.1 M; pH 5.2 acetate buffer:

Reagents:

A. 1.5ml glacial acetic acid was taken, which was made up to 100ml with distilled water.

B. 0.64g Na-acetate or 2.72g Na-acetate trihydrate was dissolved in 100ml distilled water.

In a 100ml volumetric flask, 32.2ml of reagent A was used to make a 100ml 1M pH 5.2 acetate buffer. Then 14.8ml of glacial acetic acid was added to it. Then volume was made up by distilled water.

Storage:

The partially purified enzyme was stored by lyophilization. Then the lyophilized enzyme was collected and stored in a vialed and kept in the deep freeze for further use.

RESULTS AND DISCUSSION:

Fermentation was performed in 250ml Erlenmeyer flasks with 25gm wheat bran 265ml of the crude enzyme was produced. Enzyme and protein assay was done for these enzymes of 50-time dilution and ½ diluted protein.

Activity of the enzyme was measured and it was 60.493 IU/ml and protein assay 1.549mg/ml. Then in purification step 30%, 40%, 50%, 60%, 70%, 80%, (NH₄)₂SO₄ done, and measured enzyme activity were 22.986 IU/ml; 49.986 IU/ml, 60.54 IU/ml, 61.41 IU/ml, 63.59IU/ml, 62.626IU/ml respectively. Measured protein assay were 1.145mg/ml, 1.159mg/ml, 1.364mg/ml, 1.310mg/ml, 1.516mg/ml, 1.564mg/ml.

Also enzyme and protein assay were done for supernatant of 30%, 40%, 50%, 60%, 70%, 80%, (NH₄)₂SO₄ fractionate. Measured enzyme activity of these supernatant were 52.986IU/ml, 52.933IU/ml, 26.799IU/ml, 16.373IU/ml, 9.69IU/ml, 9.85IU/ml, measured protein assay of their enzyme were 1.327 mg/ml, 1.350mg/ml, 1.3mg/ml, 1.242mg/ml, 1.255 mg/ml, 1.304mg/ml.

Table 1: Enzyme and Protein Assay of different fractionation of precipitation

	Crude	30%	40%	50%	60%	70%	80%
Enzyme OD	0.595/0.592	0.241/0.260	0.505/0.490	0.598/0.590	0.613/0.600	0.620/0.600	0.620/0.606
Protein OD	0.646/0.652	0.253/0.239	0.494/0.501	0.573/0.581	0.563/0.549	0.635/0.639	0.657/0.653
Enzyme Activity (IU/ml)	60.493	23.986	49.986	60.54	61.41	63.59	62.626
Protein Assay (mg/ml)	1.549	1.145	1.159	1.364	1.310	1.516	1.564
Specific Activity (IU/mg)	39.052	20.07	43.128	44.384	47.259	42.18	40.04
Purification Found	1.0	0.5139	1.104	1.1365	1.210	1.080	1.0252
Total IU	1209.86	229.86	499.86	605.40	619.10	635.90	626.26
Total Protein	30.98	11.45	11.59	13.64	13.10	15.16	15.64
Yield	100.0	18.998	41.315	50.03	51.17	52.559	51.76

Table 2: Enzyme and Protein Assay of different fractionation of supernatant

	Crude	30%	40%	50%	60%	70%	80%
Enzyme OD	0.493/0.557	0.521/0.530	0.467/0.460	0.285/0.286	0.194/0.186	0.129/0.129	0.135/0.126
Protein OD	0.576/0.569	0.562/.0563	0.576/0.567	0.564/0.553	0.528/0.531	0.536/0.533	0.557/0.550
Enzyme Activity (IU/ml)	52.999	52.986	52.933	26.799	16.373	9.69	9.85
Protein Assay (mg/ml)	1.353	1.327	1.350	1.31	1.242	1.255	1.304
Specific Activity (IU/mg)	39.171	39.92	39.20	20.457	13.183	7.721	3.719
Total IU	1059.98	1059.72	1058.66	535.98	327.46	193.8	197.0
Total Protein	27.06	26.54	27.0	26.20	24.84	25.1	26.08
Yield	100	99.97	99.87	50.56	30.89	18.28	18.58

The above data found that 70% saturated (NH₄)₂SO₄ fractionation was taken for further study of purification of the enzyme.

Table 3: Enzyme and Protein Assay of Xylanase:

	Blank	Crude Enzyme	70% saturated (NH₄)₂SO₄. Fractionation ate + 20 ml buffer
Enzyme OD	0.066/0.069	0.600/0.598	0.071/0.071 (Blank)	0.626/0.624
Protein OD		0.655/0.660		0.668/0.663
Enzyme Activity (IU/ml)		53.71		56.174
Protein Assay (mg/ml)		1.571		1.592
Specific Activity (IU/mg)		34.188		35.285
Total IU		140x53.71=7519.4		20x56.174=1123.48
Total Protein		140x1.571=219.94		20x1.592=31.84

After lyophilization, 0.067 g of purified enzymes were produced, and it was placed in clean sterilized ampoules. Then it was stored in desiccators.

Production of Xylanase In the second batch:

Fermentation was done in a 250ml Erlenmeyer flask with 25g wheat Bran 173ml of the crude enzyme produced. Enzyme and Protein assay was done for these enzymes of 50 times diluted and ½ diluted protein. The enzyme activity was measured, and it was 53.71 IU/ml and protein assay 1.571mg/ml. Then purification was done of 140ml crude enzyme by 70% saturated (NH₄)₂SO₄. Fractionation and precipitate were dissolved in 20 ml buffer. After that activity of the enzyme was measured, and protein assay was also done. The enzyme activity was 56.174 IU/ml, and the protein assay was 1.592 mg/ml.

Application in Pharmaceutical and Food Industries:

Since turbidity is caused by both pectic substances and other materials suspended in a stable colloidal structure, xylanase and amylase, pectinase, and carboxymethyl cellulose may be used to clarify and extract juices⁷.

Xylanase is also improving plant oils coffee extraction, extraction, and starch extraction⁸.

The xylose generated by xylan depolymerization can also be converted to xylitol, a valuable sweetener for pharmaceutical and food industries⁹.

Xylanase can improve bread quality in the bakery industry by raising the specific volume of bread. This trait was improved when combined with amylases, as seen with the introduction of Aspergillus niger. Since enzymes are most active at temperatures below 35°C, which is widely used for dough preparation, they may be suitable for the bakery industry. These enzymes may also be more efficient baking additives than currently available commercial mesophilic enzymes, which are most active at higher temperatures¹⁰.

Agricultural silage and grain feed can benefit from the addition of xylanase to improve nutritional value. Intestinal viscosity was related to decreased weight gain and feed conversion efficiency in rye-fed broiler chicks when this enzyme was used in poultry diets.

Intestinal viscosity was reduced when xylanase from Trichoderma longibrachiatum was added to a rye-based diet for broiler chickens, resulting in improved weight gain and feed conversion efficiency.

Xylanases can be used as a pre-treatment for arabinoxylan-containing substrates in cereals since arabinoxylans are partly water-soluble and create a viscous aqueous solution. This high viscosity of cereal grain water extract could delay filtration or cause haze formation in beer, which could cause brewing issues. Furthermore, it is unfavorable in cereal grains for animal feed¹¹.

a. Xylanase is increasing the production of juice from fruits and vegetables. It also improves the filter performance of juices, which reduces the viscosity of the fruit juice.

b. Hemicellulose and Cellulose scarification in biomass gives sugar-rich liquid, producing various value-added products like furfural, ethanol, and various functional biopolymers.

c. Treatment xylanase increases the nutritive sugar content in the animal feed. It thus is helpful for digestion in cows and other ruminants, useful in the production of beer, as it improves the extraction of more fermentable sugar from wheat and extraction of more fermentable sugar from barley.

Bread Quality Improvement:

a. Xylanase enzymes play a crucial role in the bakery industry and use in bread dough making.

b. Xylanase improves dough machinability, dough stability, oven rotation, loaf volume, crumb structure, and shelf life when used in optimum amount¹².

c. With an improvement in the essential volume of bread, xylanase increases bread consistency. Combining amylase with xylanase will improve this even further.

d. Used as additives in the baking industry because they improve the gluten network's elasticity in bakery dough. Dough handling and stability benefit from increased elasticity—the addition of xylanase to wheat flour results in a loaf that is approximately 10% more voluminous.

e. Xylanase improves crumb softness after storage, but not all xylanases help the baking industry due to substrate specificities, action patterns, inhibitor interactions, and kinetics.

Food Industry:

a. Xylanase with pectinase and cellulase is used for clarifying must and juices, for liquefying fruits and vegetables–L-arabinose-furanosidase and-D- glucopyranosidase have been employed for aromatizing musts wines and fruit juices.

Seed germination:

a. The reserve food is converted to the assailable end product by xylanases from the germinating plant seed. Cell elongation and fruit softening are thought to be aided by xylanase^13,14.

Degumming:

a. Degumming of bast fibers such as flax, jute, hemp, and ramie can be accomplished using a xylanase system combined with a pectinolytic enzyme system.

CONCLUSION:

Microbial xylanases have much potential in the industrial world. The xylanase enzyme should be promoted in the food processing, pulp, and biopharmaceutical industries to replace the chemical used during processing. In conjunction with another enzyme, the xylanase enzyme will produce better results for long-term industrial processes. In this work, wheat bran is used to produce xylanase as a raw material that is cheap and readily available in the Indian market. Solid-state fermentation is done with 80% moisture content for the production of this extracellular enzyme. Solid-state fermentation is done because minimum control is necessary. So, the production cost is less. After fermentation, purification is done in 70% saturated ammonium sulfate salt fractionation because the best result is found in this condition. After production and purification of xylanase, which is stored in desiccators cause, the enzyme is hygroscopic.

ACKNOWLEDGEMENT:

The authors want to acknowledge Jadavpur University, Kolkata-700032, India, for providing samples and the necessary instrumental facilities to complete the work.

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

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14. Burugu A. Addanki M. Surepalli S. Chanda C. Optimization of xylanase production from penicillium funiculosum using agricultural (Corn cob) waste. Research J. Pharm. and Tech 2020; 13(9):4111-4114. doi.org/10.5958/0974-360X.2020.00726.X

Received on 20.04.2021 Modified on 22.05.2021

Research J. Pharm. and Tech. 2022; 15(6):2616-2620.

DOI: 10.52711/0974-360X.2022.00437