Isolation And Molecular Characterization Of Xylanase Enzyme From Soil


About Author:
Microbiologist  In  Institute Of Health Systems, Hyderabad
M.Sc Microbiology From Osmania University.

The purpose of this study was to determine the effect of some cultural conditions on the xylanase enzyme production by two Isolated Species from the industrial soiland to investigate its potential to produce xylanase utilizing tomato pomace as a substrate. Xylanase activity was detected using the Dinitrosalicylic acid assay method.
The Alkalophilic bacteria isolated from the industrial soil, secreats extra cellular xylanases when grown in liquid media supplemented with eighter rice bran, grass, corn cob, or sugar baggage as a carbon sources (which were treated with 2N NaoH for removing the cellulose from these substrates). The two bacteria belonging to the species Sporo lactobacilli and Acrobacter respectively shows the high enzyme activity at high temperatures 50?C and 60?C and high enzyme activity was found at pH 8 and pH 9 for two organisms. The extra cellular enzyme has an apparent molecular weight of 66 KD & 67KD for both the organism respectively, as determined by SDS-PAGE. The purified enzyme has two peptides and was conformed by Zymogram analysis. The species sporo lactobacilli show high enzyme activity of 4.7 U/ml and the species Acrobacter shows the enzyme activity of 7.46 U/ml.

Xylan is a major component of the cell walls of monocots and hardwoods, representing up to 35% of the dry weight of plants (1). This polymer is second only to cellulose in natural abundance and represents a major reserve of fixed carbon in the environment. Unlike cellulose, xylan is a complex polymer consisting of a β-D-1, 4-linked xylopyranoside backbone substituted with acetyl, arabinosyl, and glucuronosyl side chains. Hydrolysis of the xylan backbone is catalyzed by endo β-1, 4-xylanases and β-D-xylosidases (2). Endo-β-xylanases act on xylans and xylo-oligosaccharides, producing mainly mixtures of xylo-oligosaccharides. D-Xylosidases hydrolyze xylo-oligosaccharides to D-xylose (2). Many bacterial and fungal species are able to utilize xylans as a carbon source. Interest in the enzymology of xylan hydrolysis has recently increased because of the application of β-xylanases in biobleaching (3, 4) and in the food (5) and animal feed (6) industries. Several microbial sources have been investigated for b-xylanase production. The use of xylanases in the utilization of lignocellulosic materials is under extensive study because of the production of xylose, which as a fermentation feedstock is a raw material for single cell protein, or in the production of xylonic acid, xylitol and ethanol. There is also much interest recently in the use of xylanases in the biobleaching of cellulose pulps, which decreases the demand for chlorines in conventional bleaching in papermaking.

Reference ID: PHARMATUTOR-ART-1182

The compositions and structures of xylans vary according to their sources, but all xylans are composed of a backbone chain consisting of 1,4-D xylose residues. The backbone may be branched and often has side residues of O-acetyl, arabinosyl and methylglucuronosyl substituents (7,8). Endo-β-1,4-xylanase (1,4-b-D-xylan xylanohydrolase) is the main enzyme responsible for the cleavage of the linkages within the xylan backbone (9). β-1, 4-Xylanase and b-xylosidase are the enzymes that are responsible for cleavage of the backbone chains of xylans to xylose and substituted xylooligomers, which are quickly hydrolyzed in the presence of debranching enzymes, such as α-arabinosidase, acetyl xylan esterase, and α-glucuronidase.
The cost of many important fermentation products strongly depends on the cost of carbohydrate raw material. Lignocellulosic biomass conversion offers the potential for less expensive fermentable sugars. Two important research objectives for this potential are: 1) Development of effective and economical biomass pretreatment to increase the yield of the fermentable sugar from biomass hydrolysis and 2) Maximal utilization of the various polymeric sugars available in the heterogeneous lignocellulosic material. Xylanalitytic enzymes are hemicellulase enzymes that catalyze hydrolysis of xylan, usually associated with cellulose and lignin component of plant cell walls. So, xylan-degrading enzymes produced by a bacterial source are able to use the fallowing lignocellulosic substrates as a carbon source.
· Corn meal
· Corncob
· Wheat bran
· Cellulose
·Saw dust and any other vegetable garbage.

Several enzymes are involved in the hydrolysis of xylan polymers of which the most important are the endo- 1,4- β xylanase (EC The majority of microorganisms growing on plant residue in nature usually produce both cellulolytic and xylanolytic enzyme. It is generally accepted that cellulases and xylanases are inductible in microbial cells by fragments of corresponding polysaccharides.

Practical Uses of Xylanases
The earliest U.S. patent for a method of xylanase production was issued in 1979 for an enzyme mixture used as an animal feed additive for dairy cattle. Xylanase has since proven useful in many ways:

1.Biobleaching paper pulp.Paper producers need to retain cellulose while removing the lignin from paper pulp. The classic way to perform this operation is to add chlorine-based bleaches to the pulp that generates organo-chlorine pollutants into the environment. Xylanase breaks the hemicellulose chains that are responsible for the close adherence of lignin to the cellulose network. There is thus a reduced need for bleach to remove the loosened lignin. The use of xylanase leads to a reduction in organo-chlorine pollutants such as dioxin from the paper making process that generates during chlorine based bleach. In addition, chlorine-free bleaching (such as peroxide or ozone bleaching) can achieve brighter results with the addition of xylanase. Because xylanase does not harm cellulose, the strength of the paper product is not adversely affected.

2.Improving animal feed.Adding xylanase stimulates growth rates by improving digestibility, which also improves the quality of the animal litter. For example, chicken feed based on wheat, rye, and many other grains is incompletely digested without added enzymes. These grains tend to be too viscous in the chicken's intestine for complete digestion. Xylanase thins out the gut contents and allows increased nutrient absorption and increased diffusion of pancreatic enzymes in the digesta. It also changes hemicellulose to sugars so that nutrients formerly trapped within the cell walls are released. The chicken gets sufficient energy from less feed. The bran is cleaner because the feed is more thoroughly digested so the chicken waste is drier and less sticky. In addition, chicken eggs are cleaner because the excrement in the laying area is drier. In a sense, the addition of xylanase to animal feed pre-digests that feed.

3.Making bread fluffier and keeping it fresh longer. Added xylanase modifies wheat
 flour arabinoxylans and can result in a loaf with more than 10% greater volume.      Crumb softness after storage is also improved.

4.Aiding in separation of wheat or other cereal gluten from starch.

5.Increasing juice yield from fruits or vegetables. Xylanase aids in the maceration (chewing up) process. In addition, added xylanase can reduce the viscosity of the juice, improving its filterability.

6.Extracting more fermentable sugar from barley for making beer, as well as processing the spent barley for animal feed. In both cases, xylanase has the ability to break hemicellulose down into sugars. In addition, added xylanase can reduce the viscosity of the brewing liquid, improving its filterability.

7.Improving silage (or enhanced fermentative composting). Treatment of forages with xylanase (along with cellulase) results in better quality silage and improves the subsequent rate of plant cell wall digestion by ruminants. There is a considerable amount of sugar sequestered in the xylan of plant biomass. In addition to converting hemicellulose to nutritive sugar that the cow or other ruminant can digest, xylanase also produces compounds that may be a nutritive source for the ruminal micro flora.

8. Xylanases are reported to improve degradability of plant waste material (for instance, agricultural wastes) thereby reducing organic waste disposal in landfill sites.

9. Xylanases are reported to improve the cleaning ability of detergents that are especially effective in cleaning fruit and vegetable soils and grass stains.

10. Xylanase decreases the viscosity of the mash and prevents fouling problems in distilling equipment.

11. Xylanases improve the extraction of oil from oil-rich   plant material such as corn oil from corn embryos.

12. Xylanases improve retting of flax fibers. Retting is the decomposition of the outer stem of the flax plant necessary before the fibers are processed into linen.

Functions of xylanases:
1) Decrease the viscosity of chyme in gastrointestinal tract & stimulate the digestion and absorption of nutrient.                   
2) Release nutrient and improve the availability of feedstuff. 
3) Ameliorate the micro-ecosystem in gastrointestinal tract.

Active Mechanism of xylanases:
1) Break the solid structure of cell wall by means of degrading xylan. 
2) Facilitate the flow ability of endo-digestive enzyme as a result of limiting the viscosity of chyme in gastrointestinal tract. 
3) Strengthen the activity of endo-digestive enzyme and stimulate the digestion and Absorption of nutrients. 
4) Resist against the growth of anaerobe and reduce the incidence of intestinal canal diseases



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