biotropia book final.indd 119 USING STREPTOMYCES XYLANASE TO PRODUCE XYLOOLIGOSACHARIDE FROM CORNCOB ANJA MERYANDINI1*, TITI CANDRA SUNARTI2, APRILIA NAOMI1 and FERY MUTIA2 1. Department of Biology, Faculty of Mathematics and Natural Sciences, Bogor Agriculture University, Bogor, Indonesia 2. Department of Agrotechno Industry, Faculty of Agricultural Technology, Bogor Agriculture University, Bogor, Indonesia ABSTRACT Streptomyces 234P-16 and SKK1-8 are xylanase-producing bacteria. Corncob xylan were extracted using acidifi ed method. Crude enzymes (produced by centrifuging the culture) were used to hydrolyze xylan from 2 varieties of corncob. Crude extract activity was measured by using DNS (Dinitrosalisilic Acid) method. Xylanase from strain 234P-16 has the highest activity if cultivated in 1% Hawaii xylan, whereas strain SKK1-8 on 1.5% Bisma xylan. SKK1-8 xylanase can hydrolize corncob xylan (1% Hawaii or 1.5% Bisma xylan) within 4 hours and produce xylooligosacharide with polymerization degree of 4.76 and 6.37, respectively. Key words: Xylanase, Xylooligosacharide, Streptomyces. INTRODUCTION Besides rice, corn is the major carbohydrate sources in Indonesia especially for feed and industrial raw materials. Most of corn is used as raw material for food and non- food industries. But these applications are limited to the corn kernel, and other parts of corn plant such as corncob was not utilized yet. About 30% of the corn is corncob; the rests are corn stover and kernels. Data from Badan Pusat Statistik (Indonesian Bureau of Statistics) showed that maize production is increasing from 9.82 million ton in 2002 to 11.35 million ton in 2004. Th is increase is also followed by the increase of corncob as an agricultural waste. Corncob consists of protein, fat, nitrogen free extract and also hemicellulose and cellulose (Johnson 1991). Th e content of xylan (major part of Hemicellulose) in corncob could go up to 40-g/100 g, the highest among all of the agricultural waste (Yang et al. 2005). Most xylan occurs as heteropolysaccharide, containing diff erent substituent groups in the backbone chain and in the side chain (Beg et al. 2001). Due to the corncob nutritional content, it can convert into a commercial product or be used as a medium to BIOTROPIA VOL. 15 NO. 2, 2008 : 119 - 128 *Corresponding author: ameryandini@yahoo.com 120 cultivate microorganisms. In our previous research we have identifi ed xylanase-producing Streptomyces (234P-16 and SKK1-8). Xylanase from 234P-26 has an optimum condition at pH 5 and 90 oC whereas from SKK 1-8 the optimum condition is at pH 6 and 50 oC (Meryandini 2005). In this research we will use this Streptomyces xylanase to produce xylooligosaccharide from corncob xylan. MATERIALS AND METHODS Materials In this research we use 2 local varieties of corncobs (Bisma and Hawaii) and 2 acid-xylanases from Indonesian Streptomyces: Streptomyces 234P-16 from Padang and Streptomyces SKK1-8 from Sukabumi. Delignifi cation Delignifi cation of corncobs methods had been investigated previously (Widyani 2002). About 1000 g of corncob grits is immersed in 10 liters of 1% NaOCl solution for 5 hours at room temperature (28 oC). After 5 hours, the sample is decanted and rinsed by distilled water for several times and fi ltered, the solid part i.e. delignifi ed samples are then dried by oven drying at 50 oC for 48 hours. Th e chemical composition of this sample will be examined as cellulose, hemicellulose, and lignin. Xylan extraction & purifi cation Th e method for separation of xylan using acidifi ed method from delignifi ed samples has been investigated by Anggraini (2003). Delignifi ed corncob grits are immersed in 15% NaOH solution for 24 hours at room temperature (28 oC). Th is step liberated the xylan into soluble fraction and neutralized by 6 N HCl solutions (pH 4.5-5.0). Th e supernatant is centrifuged on 4000 rpm for 30 minutes to obtain residue such as xylan. Purifi cation is conducted by re-dissolving the crude xylan into 4% NaOH and then fi ltered. Filtrate was acidifi ed using 6 N HCl (pH 4.5-5.0), and then centrifuged on 4000 rpm for 30 minutes. Precipitates were dissolved in 95% ethanol and centrifuged, and then dehydrated using 50 o C drying oven. Xylanase production Th e Streptomyces isolates are cultured on YM (Yeast- Malt) agar and then on oat- spelt xylan agar medium (Yeast extract 0.2%, sucrose 10%, K2HPO4 1.5%, MgSO4 .7H2O 0.025%, NaCl 0.23%, Na2HPO4 .2H2O 5 %, oat spelt xylan 0.5%) for 4 days at room temperature. Two cookbores of Streptomyces are cultured in 100 ml liquid xylan media and incubated at room temperature using a shaking incubator with an agitation speed of 240 rpm. Every day the culture should be centrifuged and the supernatans collected for enzyme assay. BIOTROPIA VOL. 15 NO. 2, 2008 121 Xylanase production on several substrates Two cookbores of Streptomyces are cultured in 100 ml liquid xylan media (oat- spelt and several concentration of 2 local corncob xylan) in a 500 ml Erlenmeyer and incubated on a shaker for 10 to 12 days at room temperature. Duration of xylan hydrolysis by crude xylanase Streptomyces xylanase produced in 1% of corncob xylan was used to hydrolyze 1% of corncob xylan for 4 hours at optimum pH and temperature condition. Degree of hydrolysis and degree of polymerization will be monitored every hour and the reducing sugars analyzed by Dinitroasalicilycacid Method, and total sugar by Phenol-H2SO4 method. Assay of xylanase activity Th e culture was centrifuged 5 minutes at 10.000 x g to obtain the xylanase crude extract. Xylanase activity was measured with oat-spelt xylan as a substrate. Enzyme solu- tion (100 μl) was added to 1 ml substrate solution which contain 0.5% xylan and the mixture was incubated at optimum temperature for each enzyme (90 oC for 234P-26 and 50 oC for SKK 1-8) for 30 minutes. Crude extract activity was measured by using DNS (Dinitrosalisilic Acid) method by Miller (1959) with xylosa as the standard. Th e reducing sugar of the references samples (substrate solution incubated with- out enzyme and diluted enzyme solution in buff er) were deduced from the values of the test samples. Th e reducing-sugar was detected by spectrophotometer (λ = 540 nm). One unit xylanase activity was defi ned as the amount of enzyme which produces 1 μmol xylosa per minute. RESULTS AND DISCUSSIONS Xylan extraction Th e chemical composition of corncob is described in Table 1, and the delignifi cation eff ect of corncobs and recovery of cellulose fraction to the fi ber components are shown in Table 2. Table 1. Chemical composition of corncobs Constituent Bisma Variety Hawaii Variety Ash (% db) 1.62 1.67 Lipid (% db) 3.02 4.68 Crude Protein (%db) 2.41 4.82 Crude Fiber (% db) 38.07 40.65 Carbohydrate (% db, by difference) 51.93 44.14 Streptomyces xylanase to produce xylooligosacharide - A. Meryandini et al. 122 Table 2. Composition of fibers before and after delignification. Constituent Before delignifi cation (%) After delignifi cation (%) Bisma Variety Cellulose 65.96 44.36 Hemicelulose 10.82 30.38 Lignin 23.74 19.21 Hawaii Variety Cellulose 60.04 41.88 Hemicelulose 18.11 30.18 Lignin 16.14 15.12 Elimination of lignin should be conducted since lignin will reduce the eff ectiveness of the utilization of xylan as carbon source for microbial growth and will infl uence the production of enzyme (Agustine 2005). Post-delignifi cation, the product composition was analyzed and small amount of lignin liberated. Th e initial lignin of Bisma Variety (23.74%) and Hawaii Variety (16.14%) were reduced to 19.27% (Bisma Variety) and 15.2% (Hawaii Variety), respectively. Delignifi cation cannot eliminate lignin from lignocellulosic materials completely. Cellulose microfi bril was integrated in hydrophobic matrix covered by lignin, and the lignin linked with cellulose and hemicellulose in covalent linkages (Agustine 2005). According to Fengel and Wegener (1995), lignin, cellulose and hemicellulose cannot separate completely even by special separation and purifi cation. Lignin and cellulose can be detected in purifi ed cellulose and lignin. Delignifi cation was conducted using 1% NaOCl as strong oxidant (Agustine 2005). Anggraini (2003) and Widyani (2002) stated that hypochlorite ion from NaOCl can cleave the carbon linkage on lignin structure, and delignifi cation caused the opening of the linkages between lignin and other polysaccharides and infl uenced the increasing use of xylan by bacteria. Xylan extraction was conducted by submerging the delignifi ed materials into 15% NaOH. Anggraini (2003) and Widyani (2002) concluded that hemicellulose can be dissolved in alkaline solution, such as 15% NaOH can produce brighter and clear powder, relatively clean from impurities, and easily dissolved in water and produced high yield. Acid solution 6 N HCl was added to neutralize up to pH 4.5-5.0 to precipitate again the xylan. Th e yield of xylan recovery was 9.26% from Bisma Variety, and 9.94% from Hawaii Variety. Based on research results of Widyani (2002) and Anggraini (2003) 7.64- 12.94 % and 7.31-11.45 %, respectively, of xylan can be produced from corncob using the same acidifi ed methods. Production of xylanase Th e daily production curve of xylanase Streptomyces 234P-16 and SKK 1-8 assayed at pH 7.2 and 37 oC is shown in Figure 1. Th e highest xylanase production was reached on day-10 with the activity of 0.625 Unit/ml for SKK1-8 and on day-5 with the activity of 0.27 U/ml for 234P-16. Th e optimum time of xylanase production was then used as the standard harvest time for the next xylanase production. BIOTROPIA VOL. 15 NO. 2, 2008 123 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 4 5 6 7 8 9 10 11 12 Days A ct iv ity ( U /m l) 234P-16 SKK1-8 Figure 1. Production curve of Streptomyces. 234P-16 and SKK 1-8 xylanase assayed on 37 oC and pH 7.2. Xylanase activity on several concentrations of corncob xylan 0 20 40 60 80 100 120 140 160 Oat spelt Hawai Bisma Substrate R el at iv e A ct iv ity (% ) 234P-16 SKK1-8 Figure 2. Relative activity of Streptomyces xylanases on 0.5% xylan from oat spelt, Bisma dan Hawaii corncob Figure 2 shows diff erences in xylanase activity on several 0.5% substrates. Xylanase from strain 234P-16 showed the highest activity on Hawaii xylan, while xylanase from SKK1-8 on oat spelt. Xylanase 234P-16 showed the lowest activity on Bisma xylan, whereas xylanase 45I-3 on Hawaii xylan. Hendarwin (2005) also reported activity diff erences in various substrates. Xylanase activity in several concentrations of corn xylan and 0.5% oat spelt xylan Streptomyces was inoculated in 0.5%, 1% and 1.5% Bisma or Hawaii xylan medium and compared with 0.5% oat spelt xylan. Figure 3 shows the Streptomyces 234P-16 xylanase activity on several concentrations of Hawaii xylan (Figure 3A) and several concentrations of Bisma xylan (Figure 3B ). Xylanase from strain 234P-16 has the highest activity if cultivated in 1 % Bisma xylan (0.57 U/ml) or in 1% Hawaii xylan (0.947 U/ml) Streptomyces xylanase to produce xylooligosacharide - A. Meryandini et al. 124 A. Activity of isolate 234P-16 xylanase 0 0,2 0,4 0,6 0,8 1 4 5 6 7 8 9 10 Days A ct iv it iy ( U /m l) Oat sp elt 0,5% Hawai 0,5% Hawai 1% Hawai 1,5% B. Activity of isolate 234P-16 xylanase 0 0,2 0,4 0,6 4 5 6 7 8 9 10 Days A ct iv ity (U /m l) Oat sp elt 0,5% Bisma 0,5% Bisma 1% Bisma 1,5% Figure 3. Xylanase activities from Streptomyces 234P-16 on (A) Hawaii corncob ylan and (B) Bisma corncob xylan compared with oat spelt xylan Figure 4 shows the Streptomyces SKK1-8 xylanase activity on several concentrations of Hawaii xylan (Figure 4 A) and several concentrations of Bisma xylan (Figure 4B). Xylanase from strain SKK1-8 has the highest activity if cultivated in 1.5 % Bisma xylan (49 U/ml) or in 1% Hawaii xylan (5.75 U/ml). A. Xylanase activity from isolate SKK1-8 0 2 4 6 8 4 5 6 7 8 9 10 11 12 Days A ct iv it y (U /m l) Oatspelt 0,5% Hawai 0,5% Hawai 1% Hawai 1,5% BIOTROPIA VOL. 15 NO. 2, 2008 125 B. Xylanase activity from isolate SKK1-8 0 10 20 30 40 50 60 4 5 6 7 8 9 10 11 12 Days A ct iv it y (U /m l) oat spelt 0,5% Bisma 0,5% Bisma 1% Bisma 1,5% Figure 4. Xylanase activities from Streptomyces SKK1-8 on (A) Hawaii corncob xylan and (B) Bisma corncob xylan compared with oat spelt xylan Th e production of xylanase in 0.5% oat spelt xylan was compared with the production in several concentrations of corncob xylan. Xylanase from strain 234P-16 has the highest activity if cultivated in 1% Hawaii xylan, whereas strain SKK1-8 on 1.5% Bisma xylan (Figure 3 and 4 ). Th e production of xylanase is usually induced in medium containing pure xylan or xylan-rich residues. Several reports showed that xylanase can be induced with lignocellulose material such as wheat bran, rice straw, corncob and sugarcane bagase (Beg et al. 2001). Corncob xylan is a rich carbon source with several kinds of carbon content as according to Fengel and Wegener (1995) lignin, cellulose and hemicellulose cannot be separated perfectly although the usage of a special separation and purifi cation methods and this content gives an eff ect on xylanase production. All carbon sources will give an eff ect on enzym production as reported by Ambarawati (2005) that medium containing mannan can induce xylanase from strain 45I-3 and vice versa. Th e optimum concentration to induce xylanase was 1% for Hawaii xylan and for Bisma xylan. Th is greater result on Hawaii xylan could be due to the higher concentration of Hemicellulose in Hawaii xylan. Hydrolysis of xylan Sugar composition was monitored by the increasing of reducing sugar and the decreasing degree of polymerization and the result is shown in Table 3. Table 3 shows the decreasing of DP during hydrolysis, while the reducing sugar continued to increase. Smaller number of DP shows that polysaccharide was depolymerized into short-chains compounds. Li et al. (2000) also reported that each microorganism will liberate specifi c xylanase, and showed diff erent activity on the same substrate. High diversity of xylanase may be caused by many diff erent structures of xylan in the nature. Isolates SKK1-8, but not 234P-16, can be used for XOS production using corncob xylan within 4 hours hydrolysis time. Streptomyces xylanase to produce xylooligosacharide - A. Meryandini et al. 126 Chen et al. (1997) stated that xylo-oligosaccharide (XOS) composed of 2-5 units xylose. Vazque et al. (2000) reported that XOS for food application should be 2-4 unit monomers, especially DP 2 xylobiose. Table 3. Reducing sugar, total sugar and the degree of polymerization Isolate Substrate Time (hours) Sugar production (mg/ml) Total sugar (mg/ml) Degree of Polymerization 234P-16 1 % Hawaii xylan 0 2.05 184.79 90.14 1 2.91 184.79 63.50 2 3.4 184.79 54.35 3 4.3 184.79 43.18 4 4.8 184.79 38.50 1% Bisma xylan 0 1.53 151.47 99 1 2.06 151.47 73.53 2 2.39 151.47 63.38 3 3.13 151.47 48.39 4 3.39 151.47 44.68 SKK1-8 1 % Hawaii xylan 0 12.24 184.56 15.08 1 27.43 184.56 6.73 2 36.29 184.56 5.09 3 34.84 184.56 5.30 4 38.70 184.56 4.76 1.5% Bisma xylan 0 1.36 182.61 134.27 1 2.45 182.61 74.53 2 26.84 182.61 6.80 3 24.72 182.61 7.39 4 28.65 182.61 6.37 CONCLUSIONS Xylanase from strain 234P-16 has the highest activity if cultivated in 1% Hawaii xylan, whereas strain SKK1-8 on 1.5% Bisma xylan. Using 1.5% of Bisma xylan, xylanase from SKK1-8 can produce xylooligosacharide with 6.37 degree of polymerization whereas using 1 % of Hawaii xylan with 4.76 degree of polymerization within 4 hours of hydrolysis time. Xylanase from strain 234P-16 is not suitable for producing xylooligosacharides. Xylanase from strain 234P-16 has the highest activity if cultivated in 1% Hawaii xylan, whereas strain SKK1-8 on 1.5% Bisma xylan. Using 1.5% of Bisma xylan, xylanase from SKK1-8 can produce xylooligosacharide with 6.37 degree of polymerization whereas using 1 % of Hawaii xylan with 4.76 degree of polymerization within 4 hours of hydrolysis time. Xylanase from strain 234P-16 is not suitable for producing xylooligosacharides. ACKNOWLEDGMENTS Th is research was funded by DIP Pusat Studi Regional Penelitian Biologi Tropika (SEAMEO BIOTROP) with contract agreement no 044.1/PSRP/SP-PEN/IV/2006 on 6th April 2006 to Anja Meryandini. We thank Dr. Ir. Yulin Lestari for the isolate. 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