Title of Invention

"A PROCESS FOR THE PREPARATION OF XYLANASE FROM RAGI MALT"

Abstract

The present invention relates to a process for obtaining xylanase from ragi malt wherein several parameters were examined i.e extraction buffer, ionic strength of extractant, number of extractions, detergent, phenolic adsorbent and defined metal ion concentration to yiled high xylanase activity. In this invention Larchwood xylan is used as the substrate and xylanase activity is estimated using dinitro salicylic acid (DNS) method. Process involves steps of adding ionic detergent, phenolic adsorbent and metal ion to dispersed solution of ragi malt, stirring, centrifuging and dialyzing to obtain xylanase.

Full Text

The present invention relates to a process for the preparation of xylanase from ragi malt. The invention is aimed at obtaining xylanase from ragi malt which yielded high activity. Xylanases (E.C.3.2.1.8) are hydrolases which depolymerize the xylan backbone of heteroxylans producing xylooligosaccharides varying in their degree of polymerization (D.P.2-10). Xylan is the major component of the plant cell walls especially hemicelluloses. Xylans differ structurally in their composition with respect to arabinose: xylose ratio, glucuronic acid and percentage of acetic and ferulic acids. Thus the enzymatic degradation of arabinoxylans to their monomers, i.e. xylose and arabinose is a complex process involving a battery of enzymes. Endoxylanase and p-xylosidase are the most important enzymes involved in xylan hydrolysis. Side chain cleaving enzymes such as ot-arabinofuranosidase, a-glucuronidase and acetyl xylan esterase play important roles in the removal of side substituents of heteroxylans. Subtituent groups in xylan are limiting factors in achieving the efficient hydrolysis of the substrate. The interest in xylanases has increased greatly in the past decade due to their potential biotechnological applications, especially in the paper industry. Due to their growing usage intensive investigations have been undertaken mainly with xylanolytic enzymes derived from mesophilic fungi and bacteria. However due to the environmental concern, disposal problems and safety aspects coming into forefront plant enzymes are being prefered over microbial enzymes due to the simple cost effective procedures for the preparation of starting material i.e. cereal malts. Xylanases from cereal malts are prefered due to their properties which are intermediate between fungal and bacterial xylanases. So there is ample scope for utilizing the cereal malts as a substituent for widely used microorganisms for obtaining xylan degrading enzymes.Ragi malt is used in infant, geriatric and health foods. It is having more xylanase activity compared to arabinase and 1,3/1,4 P-Dglucanase. Reference may be made to Cleemput G., Hessing M., Van Oort, M., Deconynck, M. and Delcour, J.A. Plant Physiol.1997, 113, 377-386 wherein xylanase was extracted from wheat flour and reported low activity, whereas, in the present invention xylanase was extracted from ragi malt to get high activity. References may be made to Debyser, W., Derdelinckx and Delcour. J.A. J. Cereal Sci., 1997,26, 67-74; Sungurtas, J., Swantston, J.S., Davies,H.V. and Mcdougall,GJ. J.Cereal Sci., 2004, 39, 273-281 wherein they have reported low activity and used Azurine crosslinked (AZCL)- xylan which is a costly substrate, whereas, in the present invention Larchwood xylan is used as the substrate for the xylanase assay which is cost effective compared to AZCLxylan and yielded high activity. Reference may be made to Liu, W., Zhu, W., Lu,Y., KongJ. and Ma,G. Proc. Biochem., 1998, 38, 331-336 wherein they have isolated xylanase from Trichosporon cutaneum SL409 employing enrichment culture, which is costly, cumbersome, time consuming and involves the usage of more quantity of chemicals and reported low yield. Reference may be made to Sengupta et al, United States Patent, 6,569,646, May 27, 2003, Wherein they have isolated xylanase from culture broth of Termitomyces clypeatus having accession no. 11CB-411 and consumed costly media ingredients such as micronutrients, soya- peptone, ammonium nitrate and bacto peptone. Reference may be made to Ratanakhanokchai, K., Kyu, K.L., and Tanticharoen, M., 1999, 65, 694-697, wherein xylanase was isolated from the culture supernatant of Bacillus sp. Strain K-l, which involves the usage of costly culture media, viz, Berg's mineral salt media and Tris buffer whereas, in the present invention acetate buffer is used which is cost effective compared to Tris buffer and yielded high xylanase activity. The main object of the present invention is to provide a process for the preparation of xylanase from ragi malt which obviates the drawbacks as mentioned above. Another object of the present invention is to use ragi, which is having at present low economic value compared to wheat, barley and microorganisms. Still another object of the present invention is to have maximum xylanase activity by using acetate buffer which is cost effective compared to tris, MeS and phosphate buffers. Yet another object of the present invention is to use Larchwood xylan which is a cost effective substrate for xylanase assay. Accordingly the present invention provides a process for the preparation of xylanase from ragi malt, which comprises the steps of: a) extracting ragi malt in acetate buffer having pH in a range of 4.5-6.0 and ionic strength ranging from 0.05-0.2 M in order to obtain ragi malt extract; b) adding reduced glutathione, ionic detergent, phenolic adsorbent and metal ion to extract solution obtained in step (a) in order to obtain dispersed solution of ragi malt extract; c) stirring the dispersed solution of ragi malt extract obtained in step (b) fo a period in the range of 55-65 min consecutively for two times at a temperature in the range of 4- 6 degree C; d) centrifuging, the resultant extract obtained in step (c), at an angular speed in the range of 6000-7500 rpm for a period of 10- 20 min. at a temperature ranging from 4- 6 degree C in order to obtain the supernatant containing xylanase; AND e ) dialysing the supernatant containing xylanase obtained in step (d) against the extraction buffer at a temperature in the range of 4-6 degree C in order to to obtain the said xylanase. In one of the embodiments the reduced glutathione used in step (b) is at the concentration in the range of 2.5 to 10 mM. Another embodiment provides a process wherein the ionic detergent used in step (b) is preferably Triton X-100 at the concentration in the range of 0.25 to 1 percent (v/v). Yet another embodiment provides a process wherein the phenolic adsorbent used in step (b) is preferably polyvinyl polypyrrolidine (PVPP) at the concentration in the range of 0.25 to 1 percent (v/v). Yet another embodiment provides a process, wherein the metal ion used in step (b) is preferably calcium chloride at the concentration in the range of 5 to 20 mM (milli mole). Yet another embodiment provides a process, wherein the activity of xylanase obtained is in the range of 1.0-1.55 micro moles per minute estimated using Dinitro Salicylic acid (DNS) method. In yet another embodiment of the present invention ragi malt is extracted twice to get the maximum activity. In still another embodiment of the present invention ragi malt extract is dialysed to remove salt and free sugars. Process in detail: In the present invention ragi malt was dispersed in acetate buffer (pH 4.5- 6.0) of varying ionic strength (0.05-0.2 M) consisting of reduced glutathione (2.5- 10 mM), polyvinyl polypyrrolidine (0.25-1.0%), Triton X-100 (0.25- 1.0 %), calcium chloride (5- 20 mM ) and magnesium chloride. (5- 20 mM). Extraction was carried out consecutively twice using a magnetic stirrer at 4-6 degree C for 55-60 min. and centrifuged at 6000-7500 rpm at 4-6 degree C for 10-20 min. to get the supernatant The supernatants of extraction 1& 11 were pooled and dialysed against the acetate buffer overnight at 4-6 degree C. Dialysed supernatant was designated as the enzyme extract and assayed for xylanase activity. The enzyme extract was incubated with Larchwood xylan at 50°C for 1 h and xylanase activity was expressed as the reducing sugar liberated and estimated using dinitro salicylic acid (DNS) method. Novelty of the present invention: 1) Use of ragi malt as a source of xylanase. 2) Use of acetate buffer in combination with reducing agent, phenolic adsorbent, detergent and metal ion in a defined concentration to get high xylanase activity. The following examples are given by way of illustration of the present invention and therefore should not be considered to limit the scope of the present invention. EXAMPLE- 1 Effect of the extractant on the isolation of maximum xylanase activity Ig of ragi malt was dispersed in beaker containing 3 ml of 0.1 M acetate buffer (pH 6.0) and stirred on magnetic stirrer using magnetic bar for 1 h at 4 degree C, followed by centrifugation at 7,500 rpm at 4 degree C for 10 min. Supernatant was collected and dialysed overnight against the respective extraction buffer at 4 degree C. Xylanase activity in the supernatant was determined by DNS (Dinitro salicylic acid) method using Larchwood xylan as the substrate and was highest in acetate buffer extraction, pH 6.0 (1.33 umol min"1) compared to the rest of the extractions employed and their values are as follows: pH Accordingly ragi malt acetate buffer extraction at pH 6.0 was chosen for further standardization to isolate the maximum xylanase activity. EXAMPLE- 2 Effect of the ionic strength of extractant on the isolation of maximum xylanase activity 1 g of ragi malt was dispersed in beaker containing 3 ml of 0.1 M acetate buffer (pH 6.0) of ionic strength (0.1 M ), stirred on magnetic stirrer using magnetic bar for 1 h at 4 degree C, followed by centrifugation at 7,500 rpm at 4 degree C for 10 min. Supernatant was was collected and dialysed overnight against acetate buffer (pH 6.0 ) of respective ionic strength at 4 degree C. Xylanase activity in the supernatant was determined by DNS (Dinitro salicylic acid) method using Larchwood xylan as the substrate and was maximum in 0.1 M acetate buffer (1.18 umol min"1) compared to the rest of the extractions and their values are as follows: Ionic strength (Table Removed) EXAMPLE- 3 Effect of the number of extractions on the isolation of maximum xylanase activity 1 g of ragi malt was dispersed in a beaker containing 3 ml of acetate buffer, pH 6.0 and stirred on a magnetic stirrer using a magnetic bar for 1 h at 4 degree C, followed by centrifugation at 7,500 rpm at 4 degree C for 10 min. The extraction was carried out consecutively for two times keeping all the conditions constant as mentioned above. The supernatants obtained from first, second and third extractions were collected separately and dialysed overnight against 0.1 M acetate buffer, pH 6.0 at 4 degree C. The xylanase activity was determined by DNS (Dinitro salicylic acid) method using Larchwood xylan as the substrate. More than 90% of the activity was obtained in the first two extractions .(1.29|iimolniin"1) and the result is as mentioned below: No: of extractions (Table Removed) EXAMPLE- 4 Effect of reduced glutathione on the isolation of maximum xylanase activity 1 g of ragi malt was dispersed in beakers containing 3 ml of 0.1 M acetate buffer (pH 6.0) consisting of reduced glutathione (5.0 mM) stirred on magneic stirrer using magnetic bar for 1 h at 4 degree C, followed by centrifugation at 7,500 rpm at 4 degree C for 10 min. Supernatant was collected and dialysed overnight against 0.1 M acetate buffer (pH 6.0 ) at 4 degree C. Xylanase activity as determined by DNS (Dinitro salicylic acid) method using Larchwood xylan as the substrate was found to be enhanced by the addition of reduced glutathione compared to the control (1.1 umolmin"1) and was high with 5.0 mM reduced glutathione (1.41 umolmin'1) Xylanase activity was found to be varied with different concentrations of reduced glutathione and the values are mentioned below: Reduced glutathione (mM) (Table Removed) EXAMPLE- 5 Effect of phenolic adsorbent on the isolation of maximum xylanase activity 1 g of ragi malt was dispersed in beaker containing 3 ml of 0.1 M acetate buffer (pH 6.0) consisting of polyvinyl polypyrrolidine (PVPP,0.5 %) stirred on magnetic stirrer using magnetic bar for 1 h at 4 degree C, followed by centrifugation at 7,500 rpm at 4 degree C for 10 min. Supernatant was collected and dialysed overnight against 0.1 M acetate buffer (pH 6.0) at 4 degree C. Xylanase activity as determned by DNS (Dinitro salicylic acid) method using Larchwood xylan as the substrate was found to be enhanced by the addition of PVPP compared to the control (1.1 (iimolmin"1) and was high with 0.5% (1.38 umolmin"1) PVPP incorporated extract and the values obtained with different concentrations of PVPP are as follows: PVPP (% ) 0.25 0.5 0.75 1.0 Activity (umolmin"1) 1.12 1.38 1.17 1.14 EXAMPLE- 6 Effect of detergent on the isolation of maximum xylanase activity 1 g of ragi malt was dispersed in beakers containing 3 ml of 0.1 M acetate buffer (pH 6.0) into which Triton-X 100 (1.0%) was added and stirred on magnetic stirrer using magnetic bar for 1 h at 4 degree C, followed by centrifugation at 7,500 rpm at 4 degree C for 10 min. Supernatant was collected and dialysed overnight against 0.1 M acetate buffer (pH 6.0) at 4 degree C. Xylanase activity was determined by DNS (Dinitro salicylic acid) method using Larchwood xylan as the substrate and was found to be enhanced by the addition of Triton X- 100 compared to the control (1.1 umolmin"1). Activity was high with 1.0% (1.33 (umolmin"1) Triton X-100 and the values are as mentioned below: Triton-X 1 00 (%) 0.25 0.5 0.75 1.0 Activity (umolmin"1) 1.23 1.23 1.23 1.33 EXAMPLE- 7 Effect of metal ions on the isolation of maximum xylanase activity 1 g of ragi malt was dispersed in beakers containing 3 ml of 0.1 M acetate buffer (pH 6.0) into which metal ions, calcium chloride (CaCl2, 20 mM ) and magnesium chloride (MgCl2, 10 mM ) were added individually and stirred separately on magnetic stirrers using magnetic bars for 1 h at 4 degree C, followed by centrifugation at 7,500 rpm at 4 degree C for 10 min. Supernatants were collected and dialysed overnight against 0.1 M acetate buffer (pH 6.0) at 4 degree C. Xylanase activity was determined by DNS method using Larchwood xylan as the substrate and was high in the extract with 20 mM CaCl2 (1.29 umolmin"1). Xylanase activity in the extracts with different concentrations of CaCl2 and MgCl2is as follows: Cone, of metal ions Activity (umolmin"). (Table Removed) EXAMPLE- 8 Best conditions chosen for the isolation of maximum xylanase activity from ragi malt 1 g of ragi malt was dispersed in beaker containing 3 ml of 0.1 M acetate buffer (pH 6.0) consisting of reduced glutathione (5.0 mM ), polyvinyl polypyrrolidine, PVPP ( 0.5 % ) , Triton X-100 ( 1.0 % ) and calcium chloride ,CaCl2 ( 20 mM ), stirred on a magnetic stirrer using magnetic bar for 1 h at 4 degree C, followed by centrifugation at 7,500 rpm at 4 degree C for 10 min. Supernatant was collected and dialysed overnight against 0.1M acetate buffer (pH 6.0) at 4 degree C. Xylanase activity was determined by DNS method using Larchwood xylan as the substrate (1.55 jimolmin"1). The main advantages of the present invention are: 1. Obtaining xylanase from ragi malt which yielded high activity. 2. Easy availability of ragi which is used as a source to get xylanase. 3. Ragi, having low economic value at present is used to obtain high activity xylanasae. 2. The process as claimed 1, wherein reduced glutathione used in step (b) is at the concentration in the range of 2.5 to 10 mM. 3. The process as claimed in claims 1 and 2, wherein the ionic detergent used in step (b) is preferably Triton X-100 at the concentration in the range of 0.25 to 1 percent (v/v). 4. The process as claimed in claims 1-3, wherein the phenolic adsorbent used in step (b) is preferably polyvinyl polypyrrolidine (PVPP) at the concentration in the range of 0.25 to 1 percent (v/v). 5. The process as claimed in claims 1-4, wherein the metal ion used in step (b) is preferably calcium chloride at the concentration in the range of 5 to .20 mM ( milli mole). 6. The process as claimed in claims 1-5, wherein the activity of xylanase obtained is in the range of 1.0 -1.55 micro moles per minute estimated using Dinitro Salicylic acid (DNS) method.

Documents:

326-DEL-2006-Abstract-(27-03-2012).pdf

326-del-2006-abstract.pdf

326-DEL-2006-Claims-(27-03-2012).pdf

326-del-2006-correspondece-others.pdf

326-DEL-2006-Correspondence Others-(27-03-2012).pdf

326-del-2006-correspondence-others 1.pdf

326-del-2006-description (complete).pdf

326-del-2006-description (provisional).pdf

326-del-2006-drawings.pdf

326-DEL-2006-Form-1-(27-03-2012).pdf

326-del-2006-form-1.pdf

326-del-2006-form-18.pdf

326-del-2006-form-2.pdf

326-DEL-2006-Form-3-(27-03-2012).pdf

326-del-2006-form-3.pdf

326-del-2006-form-5.pdf