About Author:
E.Ashwini
Microbiologist In Institute Of Health Systems, Hyderabad
M.Sc Microbiology From Osmania University.
ABSTRACT
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 eithter 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 degree C and 60 degree 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
STRUCTURE OF XYLAN:
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 3.2.1.8). 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 feed stuff.
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
In 1986, dioxin was found in the milk consumed by students at a Montreal elementary school. Tests revealed that the source of this carcinogenic compound was the milk cartons, manufactured using chlorine-bleached pulp. Since then, regulatory and customer concerns have induced the Pulp & Paper industry to make remarkable improvements in their bleaching process. Today, further progress has been achieved through biotechnology. Back in 1986, researchers were quick to identify and promote natural xylanase enzymes as a biochemical approach to reduce the use of chlorine bleach. It was cost effective and easy to use, but the temperatures and pH levels tolerated by these enzymes were below those normally encountered in most pulp mills. Ottawa's Iogen Corporation recognized the business potential and approached the National Research Council of Canada's Institute for Biological Sciences (NRC-IBS) to assist them in their goal to produce a commercial enzyme with an enhanced operating range. By 1993, several research groups had concluded that improvement of this target xylanase was not possible." NRC-IBS' expertise in protein engineering eventually led to the design and development of enzymes that can function at a higher temperature and pH level than the natural enzymes.
The first modified xylanases on a commercial scale was produced three years from the onset of collaboration with NRC-IBS (Iogen 2000), with the first mill trial and subsequent sale of the modified enzyme product in the following year. By 2002, these enzymes were processing over 4.5 million tons of pulp annually in Canadian and US mills, with a cumulative total passing the 10-million ton mark in the spring of 2002. Iogen has now proceeded, in partnership with NRC-IBS, to the development of fourth generation products. The use of these modified xylanases has enabled pulp mills to take a significant step toward the elimination of chlorine bleach. This in turn has decreased toxic organochlorine discharges into Canadian waterways by several hundred tons. The saving in bleaching chemicals also creates a net reduction of $0.5M/ mill/year in the overall production cost. In addition, the mills are now able to offer chlorine-free pulp, which has helped them to hold existing customers as well as expand into new markets. "When most people think of biotechnology, they tend to think of agricultural or medical applications, Industrial bioproducts, however, are also important. They can protect our environment, maintain our quality of life and increase the competitive edge for Canadian manufacturing industries."(Dr. Sung, 2002).
The pulp and paper industry processes huge quantities of lignocellulosic biomass every year. The technology for pulp manufacture is highly diverse, and numerous opportunities exist for the application of microbial enzymes. Historically, enzymes have found some uses in the paper industry, but these have been mainly confined to areas such as modifications of raw starch. However, wide ranges of applications in the pulp and paper industry have now been identified. The use of enzymes in the pulp and paper industry has grown rapidly since the mid 1980s. While many applications of enzymes in the pulp and paper industry are still in the research and development stage, several applications have found their way into the mills in an unprecedented short period of time. Currently the most important application of enzymes is in the prebleaching of Kraft pulp. Xylanase enzymes have been found to be most effective for that purpose. Xylanase prebleaching technology is now in use at several mills worldwide. This technology has been successfully transferred to full industrial scale in just a few years. The enzymatic pitch control method using lipase was put into practice in a large-scale papermaking process as a routine operation in the early 1990s and was the first case in the world in which an enzyme was successfully applied in the actual papermaking process. Improvement of pulp drainage with enzymes is practiced routinely at mill scale. Enzymatic deinking has also been successfully applied during mill trials and can be expected to expand in application as increasing amounts of newsprint must be deinked and recycled. The University of Georgia has recently opened a pilot plant for deinking of recycled paper. Pulp bleaching with a laccase mediator system has reached pilot plant stage and is expected to be commercialized soon. Enzymatic debarking, enzymatic beating, and reduction of vessel picking with enzymes are still in the R&D stage but hold great promise for reducing energy. Other enzymatic applications, i.e., removal of shives and slime, retting of flax fibers, and selective removal of xylan, are also expected to have a profound impact on the future technology of the pulp and papermaking process.
Xylanases was produced, purified and characterized from a hyperxylanolytic mutant of Aspergillus ochraceus (11) (S.R.Biswas, et.al 1990).
Xylanase production by Aspergillus awamori and the development of a medium and optimization of the fermentation parameters for the production of extra cellular xylanase
and β – xylosidase was reported while maintaining low protease production (12) (David.C.Smith, and Thomas.M.Wood 1991).
A variety of materials have been used for induction of xylanases: pure xylan (13, 14, 15, 16, 17) and xylan-richnatural substrates, such as sawdust (18), corn cob (19, 20, 17), wheat bran (21), sugar beet pulp (22), and sugarcanebagasse (23), paper of inferior quality was an excellentcarbon source and inducer for xylanase in Thermoascus aurantiacus(24), Humicola lanuginosa (25), and Paecilomyces varioti(26). In Melanocarpus albomyces (27) and Thermomyces lanuginosus(17), xylose, the pentosan unit of xylan, could also inducexylanase. Xylanases are often coinduced with cellulases by purecellulose, as in T. aurantiacus (28), Chaetomium thermophilevar. coprophile (29), and H. insolens. In M. albomyces(27), and T. lanuginosus (20,30), xylanase, but littleor no cellulase, was produced. Crude culture filtrates of thesefungi can therefore be used for biobleaching of paper pulp. Themajority of xylan-degrading enzymes from thermophilic fungi areendoxylanases.
Malbranchea pulchella var. sulfurea also produced an extra cellular xylosidase (31), but in H. grisea var. thermoidea (32) and Talaromyces emersonii (33), the xylosidase was periplasmic.Interestingly, the best xylanase-producing strains of T. lanuginosussecreted small amounts of xylan-debranching enzymes and did not produce -mannan- and arabinan-degrading enzymes, whereas thelow-xylanase-producing strains exhibited a higher degree of xylanutilization and also the ability to produce a mannan-degradingenzyme system. This suggests that utilization of xylanis facilitated by the removal of other polysaccharides that aretightly bound or cross-linked toxylan.
3. MATERIALS AND METHODS
Serial dilution
1) Collected samples are taken
2) Labelled test tube containing 9ml of sterile saline water and sterile Petri dishes were taken and labelled accordingly (10-2 ,10-3…10-9) with a marking pencil.
3) Add 1 ml of sample and prepare it to 9ml sterile saline water to make 1:10(10-1)
4) Vigorously shake the dilution to obtain uniform suspension of microorganisms.
5) Transfer 1ml of suspension from the 1st test tube into 2nd test tube with a sterile pipette under aseptic conditions to make 1:100(10-2) dilution and shake it well.
6) Prepare another dilution 1:1000 by pipetting 1ml suspension from 1:100 dilution tubes into 3rd test tube.
7) Make further dilutions 10-4 to 10-9, by pipetting 1ml suspension into remaining test tubes.
Culturing in Petri dishes:
8) Nutrient media for the organisms which grow was prepared as per composition an sterile in autoclave at 121 degree C, 15 lbs pressure for 15 mins.
9) Transfer the sterilized media into Petri dishes and allowed it to solidify
10) Transferred 0.1ml of aliquots from 10-3, 10-5, and 10-7 to different Petri dishes and three control Petri dishes were also maintained.
11) Incubate all the plates at 37degree C for 24 hrs and plates were observed for the development of colony and gram staining was performed for the observed colony.
12) The pure cultures obtained (of the bacterial species) were cultured in NAM medium by incubating at 37°cfor 24 to 48 hrs in the form of agar plates, slants and also cultured in Nutrient broth.
Streak Plate Technique:.
Procedure.
1) Prepare Petri dishes containing nutrient agar growth medium solidified.
2) Flames sterilize a wire loop. Using aseptic technique, use the sterile loop to make parallel streaks of the suspension on the agar. (Note: there should be 16 streaks, 4 sets of 4, and whole surface of plate should be used)
3) Cover the plate. Invert and incubate under low light at constant temperature.
4) Select colonies free from these organisms. For further isolation, remove a sample using a sterilized wire loop and place in a drop of sterile culture medium on a glass slide. Check microscopically that the desired species has been isolated.
5) Repeat the streaking procedure with the cells from single colony and again allow colonies to develop. This second streaking reduces the possibility of bacterial contamination and of colonies containing more than one species.
6) Select colonies that are free of other organisms for further isolation. Remove a sample using a sterilized wire loop and place on a drop of sterile culture medium on a glass slide. Check microscopically that performing gram and negative staining has isolated the desired species.
7) Transfer selected colonies to liquid or agar medium.
GRAM STAINING
Principle
The Gram stain, a differential stain was developed by Dr. Hans Christian Gram, a Danish physician, in 1884 that is why Gram staining. It is a very useful stain for Identifying and classifying bacteria into two major groups: the gram-positive and gram-negative. In this process the fixed bacterial smear is subjected to four different reagents in the order listed: crystal violet (primary stain), iodine solution (mordant), alcohol (decolorizing agent) and safranin (counter stain). The bacteria which retains the primary stain (appear dark blue or violet) (i.e. not decolorized when stained with Gram’s Method) are called gram-positive, whereas those that lose the crystal violet and counter stained by safranin (appears red) are referred to as gram-negative.
The differences in staining response to the Gram stain can be related to chemical and physical differences in their cell walls. The gram-negative bacterial cell wall is thin, complex, multilayered structure and contains relatively a high lipid contents, in addition to protein and mucopeptides. The higher amounts of protein is readily dissolved by alcohol, resulting in the formation of large pores in the cell wall which do not close appreciably on dehydration of cell-wall proteins, thus facilitating the leakage of crystal violet-iodine (CV-I) complex and resulting in the depolarization of the bacterium which later takes the counter stain and appears red. In contrast the gram-positive cell walls are thick and chemically simple, composed mainly of protein and cross-linked mucopeptides. When tested with alcohol, it causes dehydration and closure of cell wall pores, thereby not allowing the loss of complex and cells remain purple.
Materials
* 24-hrs (or less old) culture .
* Gram staining reagents:
• Crystal violet
• Gram’s iodine solution
• 95% ethyl alcohol
• Safranin
* Slide rack
* Wash bottle of distilled water
* Droppers
* Inoculating loop
* Glass slides
* Blotting paper
* Lens paper
* Bunsen burner/ spirit lamp
* Microscope
Methodology
* Make thin smear of culture on a clean glass slide.
* Let the smear air-dry.
* Heat fixes the smear.
* Hold the smear using slide rack.
* Flood the smear with crystal violet for 30 seconds.
* Wash the slide with the distilled water for a few seconds, using wash bottle.
* Cover the smear with Gram’s iodine solution for 60 seconds.
* Wash off the iodine solution with 95% ethyl alcohol. Add ethyl alcohol drop by drop, until no more color flows from the smear
* Wash the slide with distilled water and drain.
* Apply safranin to smear for 30 seconds (counter staining)
* Wash with distilled water and blot dry with blotting paper.
* Let the stained slide air-dry.
* Observe microscopically using oil-immersion.
NEGATIVE STAIN TEST
PRINCIPLE
The negative stain uses the dye nigrosin, which is an acidic dye. By giving up a protein (as an acid) the chromophore of the dye becomes negatively charged. Because the cell wall is also negatively charged only the background around the cells will become stained, leaving the cells unstained.
MATEREALS
* Slides
* Nigrosin dye
* Inoculation loop
* Sample
PROCEDURE
1. Place a very small drop (more than a loop full--less than a free falling drop from the dropper) of nigrosin near one end of a well-cleaned and flamed slide.
2. Remove a small amount of the culture from the slant with an inoculating loop and disperse it in the drop of stain without spreading the drop.
3. Use another clean slide to spread the drop of stain containing the organism using the following technique.
4. Rest one end of the clean slide on the center of the slide with the stain. Tilt the clean slide toward the drop forming an acute angle and draw that slide toward the drop until it touches the drop and causes it to spread along the edge of the spreader slide. Maintaining a small acute angle between the slides, push the spreader slide toward the clean end of the slide being stained dragging the drop behind the spreader slide and producing a broad, even thin smear.
5. Allow the smear to dry without heating.
6. Focus a thin area under oil immersion and observe the unstained cells surrounded by the gray stain.
FERMENTATION OF CARBOHYDRATES
Principle
Fermentation degradation of various carbohydrates such as glucose (a monosaccharide), sucrose (disaccharide), cellulose (polysaccharide) by microbes, under anaerobic condition is carried out in a fermentation tube. A fermentation tube is a culture tube that contains a Durham tube (i.e. a small tube placed in an inverted position in the culture tube) for the detection of gas production, as an end product of metabolism. The fermentation broth contains ingredients of nutrient broth, a specific carbohydrate (glucose, lactose, maltose, sucrose, or mannitol) and a PH indicator (phenol red), which is red at a neutral PH (7) and turns yellow at or below a PH of 6.8 due to the production of a an organic acid.
Materials
1. culture.
2. Sterile fermentation tubes along with Durham’s tubes of:
Glucose broth (3)
Sucrose broth (3)
Lactose broth (3)
3. Inoculating loop
Methodology
Preparation of fermentation medium whose constituents are as follows:
Tryptone /peptone- 1.0 g
Carbohydrate*- 0.5 g
Sodium chloride- 1.5 g
Phenol red- 0.0018 g
Distilled water- 100.0 ml
(* A specific carbohydrate--- such as glucose, sucrose and lactose is added)
1) Broth taken into fermentation tubes and Durham’s tubes are inserted into the fermentation tube such a way that the broth enters into the Durham’s tube also. Autoclave at 121oC for 15 minutes.
2) After autoclaving cool them to room temperature.
3) Inoculate the culture Incubate all the 6 inoculated and 3 uninoculated tubes at 35oC for 24-48 hours.
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