Thermostable and Alkaline xylanasefrom an alkaliphilic ActinomyceteBy: Amare SirawE-mail: firstname.lastname@example.org JULY, 2006
IntroductionIntroduction xylan is the major constituent of hemicellulose is a hetero-polysaccharide usually having a branching chain it composed of a β-1,4 xylopyranoside backbone It can be hydrolyzed to its component sugars using mineralacids or enzymes
Hydrolysis of xylan to its constituent sugars is brought aboutby different enzymes which act cooperatively (Figure 1)Intro…cont’dIntro…cont’d Endo xylanase :randomly cleave xylan back bone ∝-L-Arabinosidase :hydrolysis L-arabinose side ∝-Glucuronidase : hydrolysis glucronic acid Acetyl esterase :deacetylation of acetylated xylan β-Xylosidase: hydrolysis xylooligosaccharides in to xylose
Intro… Cont’dIntro… Cont’d Pulp and paper industrybleaching of kraft pulp – reduction of toxic chemicals needed for ligninextraction Animal diet– hydrolysis of highly viscous arabinoxylans – improvement ofdigestibity of fodders based on cereals Bakery– improvement of dough handling, bread volume and texture bydecreasing polymerization of arabinoxylans in flour – change in thequality of baked products
Intro…cont’dIntro…cont’d Solid- State Fermentation :fermentation involving solids inabsence (or near absence) of free water less effluent generation, requirement for simple fermentationequipments, lower capital investment and lower operating cost uses agro-industrial residues, which have cheaper cost, as asubstrate sources. Why searching of alkaline xylanase is needed to day? In recent years a great deal of attention is given on alkalineactive xylanases
Intro… cont’dIntro… cont’d Most xylanase studied to date are optimally active at, or near,mesophilic temperatures (approximately 40-60 0C) andneutral pHs (Belancie et al., 1995; Khandeparkar and Bhosle,2006). only few xylanase with optimum temperature for activityexceeding 70 0C and above pH 9 have been reported (AmareGessesse, 1998; Gashaw Mamo et al., 2006; Khandeparkar andBhosle, 2006). Most xylanases, which are active and stable at alkaline pH andhigher temperature, are obtained from xylanolytic microoganismsfrom extreme environments
ObjectivesObjectives To isolate xylanase producing alkaliphilicTo isolate xylanase producing alkaliphilicactinomycetes from Lakeactinomycetes from Lake Abjata and characterize theand characterize thecrude enzyme to determine potential application.crude enzyme to determine potential application. production of xylanase of actinomycete sp AS-19 inproduction of xylanase of actinomycete sp AS-19 insolid-state fermentation using agro wastes assolid-state fermentation using agro wastes assubstrate.substrate.
Materials and MethodsMaterials and Methods Water and sediment samples were collected from Lake Abjata Isolation and enumeration of actinomycetes were performedusing Starch-Casein Agar, The plates were incubated at 30 and 37 oC for 5- 7 days All isolates were identified as actinomycetes based onmorphological characteristics following Williams and Cross(1971) and Bergey’s Manual of Systematic Bacteriology(Micheal, 1986).
Materials ….cont’dMaterials ….cont’d Screening for xylanase production All pure isolates of actinomycetes were inoculated for 5 -7 days of incubation the plates were flooded with 0.1 aqueous Congo red Solutionand rinsed with 1M NaCl (Teather and Woo, 1982). all isolates that were positive for xylanase production on xylanagar plates were grown in liquid culture on rotary shaker (120rpm at 37 0C)
Materials … cont’dMaterials … cont’d Effect of moisture level: tested by varying the wheatbran to moisture ratio in the range of 1:0.5 to1:4(w/w). Effect of carbon sources :evaluated by supplementing5 % (w/w) of different carbon with wheat bran Effect of incubation temperature: studied by incubating at differenttemperature (Ambient Temperature, 30, 37 oC) for 72 h
Materials…cont’dMaterials…cont’d Effect of metal ions: tested by incubating the crude enzyme inthepresence of 1 m M solution of different metal ions Xylanase assay : Enzyme activity was assayed following thedinitro salicylic acid (DNS) method (Miller, 1959One unit of xylanase activity was defined as the amount ofenzyme that released 1µ mol of reducing sugar equivalent toxylose per minute
Materials…cont’dMaterials…cont’dEffect of pH and temperature on xylanase activity: The effect of pH on xylanase activity was determined at variouspHs (4- 10). To test the pH stability, enzyme was incubated for 1h at variouspHs( 4- 10). Residual enzyme activity was assayed at pH 9following standard assay condition The effect of temperature on xylanase activity was assessed byincubating under standard assay conditions the reactionmixtures at different temperatures in the range of 35 to 90 oC.
Materials…cont’dMaterials…cont’d Thermostability incubating the enzyme sample for 1 h at varioustemperatures between 40 to 90 oC in 50 m M glycine -NaOHbuffer (pH 9).then, Residual enzyme activity was assayed atpH 9 following standard assay condition Enzyme extractionThe enzyme from each flask was extracted using 100 ml distilledwater. The extract was centrifuged and the clearsupernatant was used as the enzyme source.
Results and DiscussionsResults and Discussions A total of 77 aerobic alkaliphile actinomycetes (slide 1) Twenty-seven strains (35%) formed detectable clear zone on solidmedia (Table 3). A total of 11 isolates produced appreciable xylanase activity in liquidculture, out of which 6 produced xylanse activity greater than 1U. great majority of xylanase producing alkaliphilic strains known so farbelong to genus Bacillus (Subramniyan and Prema, 2000), thisstudy showed that alkaliphilic actinomycetes might also be importantsource of alkaline active xylanase
Results…cont’dResults…cont’d The enzyme was optimally active between 70-80 0C (Fig 2). At 850C it retained about 90% of the optimum temperature while at 900C it retained 67%.
Results…cont’dResults…cont’d The enzyme was stable at temperatures of 50 to 80 0C. At 90 0C,the enzyme retained 47.5% of it original activity (Fig 3a) The enzyme showed good stability at both temperature and pHvalues . After 4h incubation at 75 0C it retained 77.7 and 58.6% ofits original activity at pH 9 and 10, respectively. After 4h at 80 0Cit retained 54 and 58.7% of its original activity at pH 9 and 10respectively (Fig 3b).
Results…cont’dResults…cont’d The xylanase showed to be active in a wide range of pH. Theoptimum pH for activity was at pH 8.5 to 10, and over 70% of thepeak activity was displayed between pH 7 and 10(Fig 4a). The xylanase was stable at pH 8 to 10 (Fig 4b).At pH 7, 56% ofthe original activity was retained.
Results… cont’dResults… cont’d In the kraft process of pulp production, the pulp prior to the normalbleaching operation has an alkaline pH and high temperature(Roncern et al., 2005) Most xylanase known to date are optimally active at temperaturebelow 50 0C and act in acidic or neutral pH (Blanco et al., 1995). The use of alkaline active xylanases allows direct enzymatictreatment of the alkaline pulp and avoids the cost incurring andtime consuming steps of pH re-adjustment So far only few xylanase with optimum temperature for activityexceeding 70 0C and above pH 9 have been reported (AmareGessesse, 1998; Gashaw Mamo et al., 2006; Khandeparkar andBhosle, 2006)
Results…cont’dResults…cont’d Among those metal ions, HgCl2 and ZnSO4 showed stronginhibition while FeSO4, MgSO4 and CaCl2 resulted in partialinhibition (Table 4). A lot of impurities like metal ions, which can potentially inhibit theactivity of xylanase, exist in industrial wastes Thus, in view of processing impure pulp and other environmentalapplication, the resistance of AS-19 xylanase to different metalions could be attractive
Results…cont’dResults…cont’d The rate of substrate degradation by xylanase was rapidbetween 10 and 30 min and 20- 50 min was for birch wood andoat spelt xylan and attained its stationary phase after 30 min and50 min, respectively (Fig 5) This might suggest that AS-19 xylanase do have high bindingaffinity to birch wood xylan than oat spelt xylan.
Results…cont’dResults…cont’d under SSF, in addition to xylanase the organism producedcellulase, and amylase but protease activity was not observed (Table 5). The absence of protease is advantageous The presence of amylase together with xylanase could be seenas advantageous The presence of cellulase do have both advantage anddisadvantage
Results…cont’dResults…cont’d Maximum activity was detected at 30 0C . The amount of enzymeproduced at Ambient Temperature was 95.8 % of that producedat 30 0C ( Table 6). Maximum production at lower temperatures → can reduce therate of evaporation during incubation Enzyme production is possible with out incubation instrumentand this in turn could reduce the cost of enzyme production
Results…cont’dResults…cont’d The highest xylanase production was observed in wheat bran–to-moisture ratio of 1:1.5. With increasing moisture level, enzymeproductivity was decreased (Fig 7). Enzyme production was decreasing with increasing moisturelevel of the substrate, which could be attributed to a reduction inthe degree of aeration with increasing moisture level.
Results…cont’dResults…cont’d Among the six different sugars tested, birch wood xylan wasfound to increase the enzyme activity slightly (110%), where asariabinose, glucose, lactose and xylose were found to decreasethe enzyme activity (Table 7). might be due to catabolite repression of the xylanase production wheat bran supplied enough nutrients without any need foraddition of expensive supplements.
Results…cont’dResults…cont’d Maximum xylanase production was observed when wheat branwas used as a substrate (Table 8 ). Enzyme production on sugarcan bagass showed 83.5% of production on wheat bran. about 30-40% of the production cost taken by growth substrate(Kulkarni et al., 1999).
ConclusionConclusion Actinomycete sp AS-19 produces an alkaline xylanase, havinglow level cellulolytic activity. The enzyme is active and stable upto 80 oC in alkaline solution (pH 9.0). The enzyme is also optimally active and stable in a broad pHrange (8.5-10). These characteristics are important in enzyme-assisted kraft pulpbleaching in paper and pulp processing because theseconditions of temperature and pH are similar to those of pulpproduced in paper industry
Conclution…cont’dConclution…cont’d Strain AS-19 produced relatively its maximum xylanase in wheatbran that used as substrate source. The strain grows in high pH value this can reduce the level ofcontamination, as few organisms are capable of growing underalkaline growth condition.
Results…cont’dResults…cont’d Maximum enzyme production was observed from 24 h up to 72h(138U/g). Further incubation after this time showed a gradualdecline in the xylanase production (Fig 6). allow appreciable reduction in the production cost of the enzymeand products can be found in short period of time
Figure 1 Structure of xylan and enzyme cleavage sitesSlide 3