1. 1021 Triveni Enterprises J. Environ. Biol. Vikas Nagar, Lucknow, INDIA33, 1021-1025 (2012) editor@jeb.co.inISSN: 0254- 8704 Full paper available on: www.jeb.co.in CODEN: JEBIDPKinetics of fungal extracellular D-amylase from Fusarium solaniimmobilized in calcium alginate beadsAuthor DetailsDevendra KumarDivision of Post Harvest Management, Central Institute for Subtropical Horticulture, Rehmankhera,Lucknow - 227 107, IndiaM. Muthukumar Division of Crop Improvement and Biotechnology, Central Institute for Subtropical Horticulture,Rehmankhera, Lucknow - 227 107, IndiaNeelima GargDivision of Post Harvest Management, Central Institute for Subtropical Horticulture, Rehmankhera,(Corresponding author)Lucknow - 227 107, Indiae-mail : neelimagargg@rediffmail.com AbstractExtracellular D-amylase mass produced by Fusarium solani using mango kernel as substrate was immobilizedin calcium alginate beads through entrapment technique. Maximum enzyme immobilization efficiency wasPublication Dataachieved in 2 mm size beads formed by 6.5 % (w/v) of sodium alginate in 2% (w/v) calcium chloride. Thecatalytic properties of the immobilized D-amylase were compared with that of free enzyme (soluble). ThePaper received: activity yield of the immobilized enzyme was 81% of the free enzyme. The immobilized enzyme showed30 April 2011 optimum activity at pH 4.5-6.0 and temperature 40 C, in contrast to the free enzyme at 5.5 and 30C,respectively. Thermal stability of the immobilized enzyme was found to be more than the free enzyme overRevised received: a longer time interval. The immobilized enzyme retained activity upto 20% of optimum even after 180 min.28 September 2011 While the free enzyme lost its 80% activity after 60 min and lost total activity down to zero by 120 min. Thekinetic constants, viz., KM (Michaelis constant), Vmax and activation energy were affected by immobilization.Accepted: However, the immobilized D-amylase in calcium alginate beads supports its long term storage which has15 October 2011 immense industrial applications. Key wordsCalcium alginate, Immobilization, D-amylase, Fusarium solani, Mango kernel Introduction cumbersome which hinders its commercial utilization. Immobilizationof the enzymes onto solid supports that are either organic or inorganic Extracellular amylase s produced by several filamentousis a very effective way to increase enzyme stability and operationalfungi have been used in baking, detergent, paper, textile and foodlife time as well as eases its downstream applications (Doaa et al.,industry (Selvakumar et al., 1996; Pandey et al., 2000; Mishra and2009). Entrapping method of immobilization involving gelation intoDadich, 2010). Moreover, mass production of extracellular D-porous gel facilitates immobilized enzyme with high retention of specificamylases were reported by fungi like Aspergillus, Bacillus, activity and stability. Over decades, several matrices have beenTrichoderma and Fusarium (Saville et al., 2004; Kubrak andreported for entrapping, among which alginate in the form of beads,Luschchak, 2008; De Castro et al., 2010) using various substrates.was found to be reasonably safe, simple and cheap offering goodFusarium solani was used to mass produce D-amylase using mangomechanical strength (Le-Tein et al., 2004). Calcium alginate beadskernel, a solid processing waste as substrate. Product recovery are widely used in immobilization of enzymes like D-amylases,and long term storage of the mass produced and purified enzyme is proteases, etc. (Dey et al., 2003; Le-Tein et al., 2004; Ahmed et al., Journal of Environmental Biology November 2012

2. 1022Kumar et al.2008). In this study extra cellular D-amylase from Fusarium solani Kinetics of the immobilized as well as free enzymes waswas immobilized in calcium alginate beads for longer storage stability analyzed using Arrhenius, Lineweaver-Burke and Michaelis-over a range of pH and temperature.Menton plots. The enzyme kinetics experiment was performed by measuring the initial enzyme reaction velocity at different substrate Materials and Methods concentration of starch in 50mM acetate buffer. The Lineweaver- Burke plot was used to establish the Michaelis constant (KM) andProduction and purification of D-amylase: For production of maximum velocity (Vmax) of the enzyme reaction.enzyme using mango kernel as a substrate by Fusarium solani(NAIMCC-F-02956) the process described by Kumar et al. (2010)Results and Discussionwas followed. After fermentation, D-amylase being extracellularprotein was isolated by centrifugation at 12000 rpm and was partially D-amylase, an enzyme of hydrolase family, is producedpurified by ammonium sulphate precipitation and dialyzed using extracellularly by many fungi. In our earlier study, D- amylase wasacetate buffer (50mM, pH 5.0). The specific activity of the enzyme mass produced using submerged fermentation of mango kernel aswas determined as per method described by Sadasivam andsubstrate by Fusarium solani. The crude enzyme was extracted,Manickam (1996). The partially purified enzyme was used forprecipitated by ammonium sulphate upto 70% saturation and dialyzedimmobilization studies.twice in acetate buffer (50mM, pH 5.0) which resulted in purification of 4.083 fold. The specific activity before and after purification wereImmobilization of D-amylase and assay : The partially0.092 and 0.375 U mg-1, respectively. This partially purified enzymepurified D-amylase was mixed with sodium alginate solution inwas immobilized in calcium alginate beads via entrapment technique1:1 ratio by varying the final concentration of the latter between and the enzymatic properties were characterized.3.5-7.5 % (w/v). The D-amylase-alginate mixture was addeddrop-by-drop into calcium chloride 2% (w/v) solution with Alginate, a natural polysaccharide, is a copolymer ofcontinuous shaking at 4C. The beads thus formed were washed alternating sequences of E-D-mannuronic acid and D-L guluronic3-4 times with de-ionized water and finally with 50mM acetateacid residues linked by 1-4 glycosidic bonds (Le-Tien et al., 2004).buffer of pH 5.0. The beads were dried and stored at 4C for Immobilization of D-amylase by entrapment technique into gelfurther studies. matrix under mild conditions using calcium alginate involves ionotropic gelation. When D-amylase-sodium alginate mixture was dropped Assay reaction for the immobilized D-amylase was set up into CaCl2 solution, Na+2 ions of Na-alginate were replaced by theaccording to the protocols described by Dey et al. (2003) andCa+2 ions of CaCl2 forming Ca-alginate beads and ionic cross linkingKumar et al. (2006) with minor modifications. For assay, 200 mgof carboxylate group in uronate block of alginate occurs giving it aof calcium alginate beads was incubated with 400l of 1% (w/v) gel like character (Le-Tien et al., 2004). The D-amylase remainsstarch solution in acetate buffers (50mM, pH5) at 30C for 60 min. entrapped within the gel matrix of calcium alginate beads.Starch was digested by D-amylase and the products formedwere assayed using DNS (Di-nitro salicylic acid) reagent. TheThe immobilization yield is a key parameter whichreaction was stopped by incubating for 10 min at 100C is waterinfluences immobilization efficiency. The effect of sodium alginatebath. Enzyme activity was recorded in Units, wherein, one unit concentration on immobilization yield was assessed to determinewas defined as the amount of D-amylase that produced 1 mole the per cent concentration forming uniform sized beads retainingof reducing sugar under assay condition per gram of bead. Proteinmaximum enzyme activity and greater stability. Different researchersconcentration was estimated by Lowrys method using with bovinehave earlier reported that sodium alginate concentration rangingserum albumin as a standard (Lowry et al.,1951). Immobilizationbetween 26% (w/v) was suitable for the immobilization of D-amylaseefficiency was determined from the difference in enzyme activity in(Konsoula et al., 2005; Kumar et al., 2006; Prabakaran andthe solution before and after the immobilization (Konsoula et al., Pugalvendhan, 2009). The effect of sodium alginate concentration2006). on immobilization efficiency of D-amylase is reflected in Table1. The percent entrapped activity of calcium alginate immobilized D-amylaseOptimization of temperature and pH : The activity of free andwas found maximum at 6.5% (w/v) sodium alginate. Enzyme beadsimmobilized D-amylase was assayed at various temperatures (20 having less than 6.5% (w/v) sodium alginate resulted in lower80 C) at 10C interval by the method of Anwar et al. (2009) and byentrapped activity as well as immobilization efficiency becausefollowing assay conditions as per Dey et al. (2003) with minor leakage of enzyme from beads occurred due to larger pore size ofmodifications. the less tightly crossed linked and fragile Ca-alginate beads. But at sodium alginate concentration of 7.5% (w/v), the entrapped enzyme Free and immobilized enzymes were incubated at 60C activity and immobilization efficiency were found to be low. At higherin 50mM acetate buffer for 3 hr. Sample were taken at differentsodium alginate concentration, decrease in immobilization yield andintervals and activity were measured by method as describedefficiency occur at which might be due to decreased gel porosity,earlier. The change in activity as a function of time was measured ashigh viscosity of enzyme entrapped beads and substrate diffusionthe temperature stability (Zeng et al., 2009). limitation (Ahmed et al., 2008). Reduced pore size reduces leakageJournal of Environmental Biology November 2012 3. Kinetics of immobilized fungal D-amylase 1023though; some initial leakage of the enzyme molecule is certain surface interaction between enzyme and substrate affected by(Zaborsky et al., 1973). alginate beads entrapment. This broader pH profile of the immobilized enzyme than that of the free enzyme indicated that the calciumBead size is another major factor that influences thealginate immobilization method retain the enzyme activity in a widerimmobilization efficiency. The substrate has to diffuse into the beads pH range. This may also be due to diffusion limitations or secondaryfor the enzymatic reaction to take place in the immobilized enzyme interactions between the enzyme and the matrix (Reshmi et al., 2006).system. The rate of hydrolysis is thus affected by the size of the finallattice formed by the bead. In such situations, both the intra-particular The immobilized enzyme was active at a higherdiffusion and the external mass transfer should be taken intotemperature than free enzyme with respect to relative activity. Theconsideration for assessing immobilization efficiency. However, in temperature optimum shifted from 30C (free enzyme) to 40C forthe present study, the external transport has not been consideredthe entrapped enzyme. The relative activity of free D-amylaseon the assumption that greater contribution is from the intra-particle reduced sharply at 50C to 80% of optimum while immobilizedmass transfer. Using an enzyme loading concentration 0.054 U and D- amylase retained 80% of optimum. This result indicated that asbead (diameter 2mm) amount of 33 mg, maximum enzyme activity the temperature increases, relative activity of the free enzyme(0.54 U ml-1) in terms of highest rate of starch hydrolysis wasreduces rapidly compared to immobilized form. Similar result wasobserved the activity yield reduce (81.02%) of the free enzyme. also reported in immobilized D-amylase by Kahraman et al. (2007).Contrarily, the larger bead sizes (diameter 3 mm and above) resulted The temperature optimum of immobilized D- amylase was alsoin lower enzyme activity. Moreover, the immobilization efficiencywider in the range between 30 to 60 C with the relative activitywas drastically influenced by bead size as shown in Table 2. Itevinced that the beads of 2mm diameter offered lesser diffusionTable- 1: Effect of sodium alginate concentration on immobilization efficiencyresistance compared to the larger beads which is in accordance toSodium alginateImmobilizationthe earlier reports (Dey et al., 2003; Ertan et al., 2007). It was (g % ) efficiencya (%)reported that larger alginate bead size resulted in structuraldeformation or denaturation of enzyme during immobilization,3.526.884.557.43eventually altering the catalytic site leading to loss of activity or even5.566.83deactivation of enzyme. Other factor probably related to lower6.590.33reaction rate of immobilization is the steric hindrance of the alginate 7.5 3.38matrix which limits accessibility of the substrate to the enzyme active Values are mean of three replicates ; aImmobilization efficiency % wassite; influenced by bead size. determined from the difference in enzyme activity in the solution before andIn immobilized enzyme system, the temperature and pH after the immobilization .affect relative activity because the behavior of an enzyme moleculegets modified by its immediate microenvironment. Based on theTable- 2: Effect of bead size on immobilization efficiencyrelative activity, the pH optimum of the free enzyme was 5.5. Incontrast, pH optimum of the entrapped enzyme shifted towards acidic Bead sizeImmobilizationby 0.5 units to 5.0. The variation of activity with pH, within a range(mm)efficiencya (%)of 23 units each side of the pI (isoelectric point), is normally a 1.585.16reversible process (Bayramoglu et al., 2004). The conformation of2100the enzyme will be more favorable in the higher pH range so that2.590.46maximum activity is achieved. Extremes of pH will, however, cause3 85.75a time and temperature-dependant, essentially irreversible3.5 45.6denaturation. A change in pH affects the intramolecular hydrogen Values are mean of three replicates ; aImmobilization efficiency % wasbonding leading to a distorted enzyme conformation that reduces itsdetermined from the difference in enzyme activity in the solution before andrelative activity (Reshmi et al., 2006). However, the relative activityafter the immobilizationof immobilized D-amylase was wider in the pH range of 4.5 - 6.0,It was also noted that it was stable upto 77% of the optimum. Similarly, Table- 3: Properties distinguishing immobilized and free D-amylaseimmobilized D-amylase from Bacillus subtilis retaining 77% activitywas earlier reported by Prabakaran and Pulgalvendhan (2009). Kinetics property Free enzyme Immobilized enzyme Immobilization of D-amylase results in the formation of Optimum pH 5.54.5-6.0less polymerized products resulting in an apparent decrease in the Temperature (C)30C 40Cnumber of transglycosylation reactions. It was demonstrated that Thermal stability (min)120 > 180diffusional resistances were in direct relation to the apparentKM (mg ml-1)27.4718.52modification of the enzyme action pattern after immobilization (Siso Vmax (mole min-1 ml-1)5.281.23et al., 1990). Immobilized enzyme was more active also because ofActivation energy, Ea (Kcal mol-1)29.4320.95Journal of Environmental Biology November 2012 4. 1024Kumar et al.Fig. 1: Lineweaver-Burke plot to determine apparent Km for immobilized Fig. 2: Arrhenius plot showing activation energy levels of immobilized andand free enzymes. The plot shows relationship between inverse of substrate free enzyme. Immobilized enzyme resulted in activation energy (20.95concentration and reaction velocity for both immobilization and free enzymeK cal mol-1 ) that has lower affinity towards the substrate as compared to thewhere in the raised slope of immobilization enzyme indicates higher apparent free enzyme with activation energy (29.43 K cal mol-1)Km value reflecting lower substrate affinity over free enzymereduced by only 10-40% of optimum. Entrapped enzyme presentedaccordance with that reported by Saville et al. (2004). Earlier, thelarger activity profile than the free enzyme which was in accordance maximum reaction rate (Vmax) of immobilized and free amylase ofwith earlier report of Le-Tien et al. (2004).10.4 and 25.7 mg starch degraded ml-1 min-1 mg protein, respectively was reported (Tee and Kaletunc., 2009). The KM value was knownThermal stability of the immobilized D-amylase is a most as the criterion for the affinity of enzymes to substrates, and theimportant factor for industrial applications. The immobilized D- lower value of KM represented the higher affinity between enzymesamylase was found to be more thermostable than the free enzyme and substrates (Shuler and Kargi., 2002). The decreasing KM valuesover a long time interval after heat inactivation at 60C. The might be due to conformational changes of enzyme duringimmobilized enzyme also retained activity upto 20% of optimumimmobilization and inability of high molecular substrate (starch) toeven after 180 min. However, as the temperature increases, the diffuse rapidly into the Ca-alginate matrix resulting in reducedstability of free enzyme reduces rapidly compared to immobilized substrate access to the active site of entrapped enzyme (Abdel-form. The free enzyme showed less than 20% activity after 60 min Naby et al., 1998 and Norouzian et al., 2003). The same reasonand reached zero level after120 min. Thus the immobilized D- could be accounted for Vmax of immobilized D-amylase in comparisonamylase presented better thermo stability than the free enzyme with the free enzyme or higher values indicated that the enzymesimilar to earlier reports of immobilized endo-E-gluconase (Busto et converted more substrate to product per unit of time upon saturatedal., 1998). Increased thermal stability has also been reported for a with substrate. At the same time, the diffusion resistance encounterednumber of immobilized enzymes, and the support material is by the product molecules might have caused the product tosupposed to preserve the tertiary structure of the enzyme. The accumulate near the center of the gel to undesirable levels, leadingthermal stability of enzymes might be drastically increased if theyto product inhibition (Le-Tien et al., 2004).are attached to a complementary surface of a relatively rigid supportin a multipoint (Zeng et al., 2009). The activation energy (Ea) of the immobilized and free enzymes were estimated from Arrhenius plot (Fig. 2) by plotting the Kinetics of the immobilized D-amylase was determined at log of the reaction rate against 1/T. The Ea of immobilized and freepH and temperature of 5.0 and 40 C while for free enzyme at 5.5 D-amylase was 20.95 and 29.43 Kcal mol-1, respectively. Similarand 30C. Michaelis-Menten constant (KM) of immobilized D-amylaseresult was found in immobilized D-amylase obtained from Bacilluswas determined by hydrolysis of starch at varying concentrations circulans GRS 313 using the method of immobilization on coconut(0.25-10 mg ml-1) from Lineweaver-Burke plot drawn between the fiber support (Dey, 2002). However, there was slight decline ininverse of starch concentration and reaction (Fig.1). The KM and activity enzyme which might due to reduced conformational flexibilityVmax value for immobilized D-amylase was found 27.47 mg ml-1 and of the immobilized enzyme as a result of the covalent immobilization5.28 5 mole min-1 ml-1, respectively while for free enzyme, 18.52 mg (Table 3).ml-1 and 1.23 mole min-1 ml-1, respectively. KM values of D-amylasewere found to be significantly larger upon immobilization, indicating It may, therefore, be concluded that calcium alginatedecreased affinity of the enzyme for its substrate. Unlike KM values,immobilization of D-amylase from Fusarium solani enhances theVmax was smaller for immobilized amylase and results are inenzyme stability over wider pH and temperature range resulting inJournal of Environmental Biology November 2012 5. Kinetics of immobilized fungal D-amylase1025longer storability of the enzyme.Konsoula, Z. and M. Liakopoulou-Kyriakides: Thermostable D-amylase production by Bacillus subtilis entrapped in calcium alginate gel Acknowledgments capsules. Enz. Microbiol. Technol., 39, 690696 (2006). Kubrak, O. and M. Lushchak: Optimization of conditions for immobilization of alpha amylase from Bacillus sp BKL20 in Ca2+ alginate beads.Authors are thankful to the Director, Central Institute for Ukr. Biokhim Zh., 80, 32- 41(2008).Subtropical Horticulture, Lucknow for his keen interest in the workKumar, R.S.S., R.K.S Vishwanath, S.A Singh and A.G. Appu Rao:and constant support. 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