Partial Puri fi cation , Stability Analysis , and Preservation of Xylanase from Xylanolytic Alkalophylic Bacteria

Abtsract A xylanase, which produces xylose from oat spelt xylans, was isolated from the culture medium of xylanolytic alkalophylic bacteria mutant. The enzyme was purifi ed by ammonium sulphate with level 30, 40, 50, 60, 70, 80, and 90%. The purify of the fi nal preparation was demonstrated by sodium dodecyl sulphatepolyacrylamide gel electrophoresis. The molecular masses of the purifi ed xylanase were 137.61 and 165.34 kDa. Result of ammonium sulphate saturation with the highest activity was used as standart for saturation for enzyme production and preservation, using corn, tapioca, soy bean meal and gaplek fl our as carriers. Addition of 60% ammonium sulphate showed the highest xylanase activity (62.03 U/g), and produced 89.40% enzyme recovery. Tapioca, as a carrier, produced the highest xylanase activity.


Introduction
The plant cell wall is a complex composite of structural polysaccharides that represents the most abundant source of organic molecules in the biosphere.The annual recycling of 1011 tons of plant structural polysaccharides is an important biological process that is integral to the carbon cycle (Taylor et al., 2006).The catabolic breakdown of hemicellulose thus represents a critical step in the recycling of carbon in nature and has been targeted as a subject of intense research with respect to renewable energy resources.β-1,4-linked xylopyranose is the principal component of plant cell wall hemicellulose, which represents the second largest reservoir of fixed carbon in the biosphere (Dodd et al., 2009).Hemicelluloses are a complex heteropolymer made up of glucoronoxylans, arabinoxylans, glucomannans, arabinogalactans and galactomannans (Singh et al.,2007).Xylan is the most abundant renewable polysaccharide after cellulose (Gupta and Kar, 2009) and as a major component of hemicellulose contributing 15-30% of the total dry weight in angiosperm an 7-12% in gymnosperm.The backbone of xylan consists of β-1,4-xylopyranosyl residues (Singh et al., 2007), depending on the plant source, can be variably substituted by side chains of arabinosyl, glucuronosyl, ethylglucuronosyl, acetyl, feruloyl and p-coumaroyl residues (Pastor et al., 2007).Xylanases are glycosidase, which hydrolyze the endo-β-1,4-xylopyranosyl linkages and degrade xylan into xylooligosaccharides.They are the key enzymes for xylan degradation and differ in their specifi cities toward the xylan polymer (Pastor et al., 2007).Due to its heterogenecity and complexity, the complete hydolysis of xylan requires a large variety of cooperatively acting enzymes.Endo-  (Singh et al, 2007;Dodd and Cann, 2009).
There is an increasing trend towards using enzymes for catalyzing biotransformations (Dalal et al., 2007).Due to their biotechnology application in various industrial processes, xylanase from microorganism have attracted increasing attention in the last decade.Their applications include biopulping wood and pulp bleaching, wastewater treatment, treating animal feed to incease digestibility, processing food to increase clarifi cation, pretreatment of forage crop converting lignocellulosic substances into feedtocks and fuels, improve cell wall maceration for the production of plant prototypalsts.Purifi cation has become a necessity as it leads to reduction in bulk, concentration enrichment, removal of specifi c impurities (e.g.toxins from therapeutic products), prevention of catalysis other than the type desired (as with enzymes), prevention of catalyst poisoning (as with enzymes), recommended product specification (e.g.pharmacopocia requirement), enhancement of protein stability and reduction of protein degradation (e.g. by proteolysis).Most of the purification methods, which are used in laboratory research, can be scaled up to industrial processes.Such methods are filtration, centrifugation, microfiltration, ultrafiltration, diafiltration, precipitation, ion-exchange chromatography and gel fi ltration.Ammonium sulphate precipitation is an effi cient method for removal of lower molecular mass xylanases (Kuhar et al., 2007) Selected strain from the xylanolytic alkalophylic mutan has been shown to be producer of an active xylan degradation enzyme.Previously, we have isolated the xylanolytic alkalophylic bacteria from the crabs and mutation them by ethyl methanesulfonat.The present article reports the purifi cation and preservation of xylanase obtained from liquid state culture of xylanolytic mutan when grown on medium containing xylans from oat spelt and xylose as substrates.

Xylanase assay
Xylanase activity was assayed by measuring the amount of reducing sugar liberated from enzymatic hydrolysis of soluble oat spelt xylan.Briefl y, assays containing 0.4 ml of 50 mM sodium acetate buffer (pH 6.0) with 0.2 ml of 4% soluble oat spelt xylan, and 0.2 ml of enzyme preparation were incubated at 50°C for 20 min (Ruiz -Arribas et al., 1995), after which the amount of reducing sugar was measured by the Nelson-Somogyi method with D-xylose as the standard (Plummer, 1978).Substrat and enzyme controls were always used.All assays were performed in duplicate.One unit of activity is defi ned as the amount of xylanase needed to liberate 1 μmol of D-xylose per min under these assay condition (Ruiz-Arribas et al., 1995).

Protein measurement
The protein concentration of the enzyme preparation was measured by Lowry method (Plummer, 1978), with bovine serum albumin as the standard.

Enzyme purifi cation
Xylanase was purified from 1.5 l of a culture supernatant.The cell-free culture supernatant, as a crude enzyme, was extracted by centrifugation (14,000 x g for 20 min, 5°C), and devided into 7 tubes (each 50 ml).Enzyme was partially purified by ammonium sulphate (30-90% saturation) precipitation.After incubating overnight at 4°C, the precipitate was discarded by centrifugation (3,000 x g for 20 min, 5°C), and followed by dialysis (dialysis tubing 22 kDamolecular weight-cutoff membranes) that performed against 10 mM sodium acetate buffer (pH 6.0).The dialysat was determined xylanase activity.The level of ammonium sulphate that gave the highest activity was carried out to enzyme production, and then it was measured the molecular mass and preserved with various carriers.

Molecular mass estimation
The molecular mass of xylanase was estimated by 12% SDS-PAGE.Proteins were stained with Coomassie brilliant blue, and PageRuler Unstained Protein Ladder was used as molecular mass marker.

pH and thermal stability analysis
For pH stability determination, partially purifi ed enzymes were incubated in buffers of varying pH (sodium acetate buffer for pH 3.0-6.0,sodium phosphate buffer for pH 6.5-7.5) at room temperature and for 1h.For thermal stability characterization, various temperature (30-60°C) were used at sodium acetate buffer ph 6 for 1h.The residual xylanase activity was assayed under standard conditions.

Enzyme preservation
Xylanase was preserved with various carriers (tapioca, corn, gaplek and soy bean meal), and freeze-dried.Each treatments were determined for xylanase activity.

Statistical methods
Treatments were arranged in a one way design, with the main factors being kinds of carrier (tapioca, corn, gaplek and soy bean).The data in the main study were analyzed as a one way arrangement.The differences of mean value were analyzed by Duncan's new multiple range test (Rosner, 1990).

Enzyme purifi cation
The xylanase was tested for the effect of level of ammonium sulphate on the activity (Figure 1).Precipitation with 60% of ammonium sulphate was optimum level that result the highest activity (62.03U/g).After the enzyme was precipitated with 50% of ammonium sulphate, 38% of the activity was retained.Thirty seven percent of the activity remained after the enzyme was precipitated with 70% of ammonium sulphate.Rapid inactivation of enzyme activity was observed bellow and above those level.The xylanase was purified with 60% of ammonium sulphate to apparent homogenity.Two protein bands were seen after Coomasie brilliant blue staining, and  2).Specifi c activity (62.03U/g) of the purifi ed protein was higher than that crude enzyme (19.66U/g), as presented in Table 1.It increased 3.15-fold during this step, and resulting 89.40% of enzyme recovery.This purifi cation procedure led to the recovery of 0.045 g of xylanase protein per liter of culture with a specifi c xylanase activity of 62.03 U/g of protein.

Stability analysis of enzyme
Analysis of the effect of pH and temperature on the hydrolytic activity of xylanase on oat spelt xylan showed similar pH and temperature profi les (Figure 3 and  4).Analysis of the infl uence of pH on enzyme stability showed that while the enzyme retained more than 80% of its initial activity after incubation at room temperature for 1h in buffers ranging from pH 3.0 to 7.5, the enzyme was less stable under these conditions, losing more than 50% of its initial activity in buffers at pH lower than 3.5 or higher than 7.0.Thermostability assays showed that the enzyme remained highly stable at 35 to 40°C after 1h of incubation at pH 6.0, while it lost 30% of its initial activity after incubation more than 40°C.

Enzyme preservation
The purified enzyme was tested for the effect of kind of carriers on the activity.The activity of this enzyme was highest   with tapioca as carrier (60.30U/g) (P<0.01), as presented in Table 2. Corn, gaplek and soy bean meal as carriers gave less xylanase activity, those were lower 22.39, 41.81, and 53.07%respectively than that tapioca.

Discussion
The solubility of a globular protein in an aqueous solvent is influenced by four main factors: salt concentration, pH, the organic content of the solvent and the temperature (Palmer, 1991).Ammonium sulphate precipitation is an effi cient method of lower mass xylanases.The material needed for the method is cheap, and finds much use in industrial purifi cations.Ammonium sulphate precipitation led to several fold purifi cation such as 1.2 to 11-fold (Kuhar et al., 2007).Partially purifi ed xylanase in the culture supernatant of Streptomyces galbus NR by salting out at 40-60 and 60-80% ammonium sulphate saturation which led to the purifi cation of 9.63-fold and 68.80% recovery (Kansoh and Nagieb, 2004).In this study, 60% ammonium sulphate saturation led to the purifi cation 3.15-fold and 89.40% recovery.In the enzyme purifi cation steps, addition of a small amount of neutral salt to a solution increased the solubility of a protein.The added ions changes ionization of amino acid side chains and can also interfere with interactions between protein molecules, the overall effect being to increase interactions between solute and solvent.At very high salt concentration, the abundance of interactions between the added ions and water decreases the possibilities for proteinwater interactions, often resulting in the protein being precipitated from solution (Palmer, 1991).
After the enzyme has beed recovered and concentrated, it is usually formulated in order to meet the product stability specifi cations suited to the application and handling practices employed by the end-user.Food-grade ingredients are required for food and feed applications.For solid products, the enzyme concentrate or formulated product, if suitable, can be granulated or dried in a manner that provides a solid product that meets health and safety requirements, such as low dusting (Clarkson et al., 2001).Most feed enzymes are supplied as liquid formulations at the end-user level, as these are convenient to use.It is mostly dry enzyme premixes that are being sold to the feed manufacturer.However, solid formulations can provide some signifi cant advantages, such as enhanced stability, delayed or controlled release and protection against deactivation during harsh applications (Kuhar et al., 2007).
It could be concluded that the molecular masses of the xylanase were 137.61 and 165.34 kDa.Addition of 60% ammonium sulphate showed the highest xylanase activity (62.03U/g) and 89.40% enzyme recovery.Tapioca, as a carrier, had the highest xylanase activity.

Figure 1 .
Figure 1.The effect of ammonium sulphate precipitation on enzyme activity (U/g)

Figure 2 .
Figure 2. SDS-PAGE analysis of xylanase from alkalophylic xylanolytic bacteria.Gels were loaded with molecular mass marker (lane 1), and protein after precipitation with 60% of ammonium sulphate (lane 2)

Figure 3 .
Figure 3. Infl uence of the pH on the activity of partially purified xylanase.For the pH profile, the enzyme activity was measured at room temperature in 50 mM sodium phosphate and sodium actetate buffer adjusted to the correct pH.Values are the means of results of duplicate experiments.Each point represents the mean of standard deviation (SD) (indicated by error bar) for two different enzymes.

Figure 4 .
Figure 4. Infl uence of the temperature on the activity of partially purifi ed xylanase.For the temperature profi le, the enzyme activity was measured in 50 mM sodium actetate buffer (pH 6.0) at different temperatures.Values are the means of results of duplicate experiments.Each point represents the mean of standard deviation (SD) (indicated by error bar) for two different enzymes.

Table 1 .
Purifi cation chart of xylanase from xylanolytic alkalophylic bacteria had mass of 137.61 and 165.34 kDa on SDS-PAGE (Figure

Table 2 .
The effect of various carriers on xylanase activity a,b signifi cantly different (P<0.01)