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Purification and properties of a thermostable [alpha]-amylase by acremonium sporosulcatum.

Introduction

The Starch degrading amylolytic enzymes have a great commercial value in biotechnological applications ranging from food, fermentation, textile to paper industries (Lin and Hsu, 1977; Pandey et al., 2000). Although Amylases can be derived from several sources such as plants, animals and microbes, the microbial amylases meet industrial demands; literature contains a large number of such available commercially and they have almost completely replaced chemical hydrolysis of starch in starch processing industries (Pandey et al., 2000). The major advantage of using microorganisms for production capacity is their amino ability, microbial amipulation to obtain the enzyme production enzymes of desired characters (Lonsane and Ramesh 1990). [alpha]-Amylase has been derived from several fungi, yeasts, bacteria and actinomycetes. However, enzymes from fungal and bacterial source have dominated applications in industrial sectors (Pandey et al., 2000). Fungal sources are confined to terrestrial isolates mostly to species of Aspergillus and, Pencillium (Haska and Ohta, 1944; pandey et al., 2000).

[alpha]-Amylase family

The [alpha]-amylase, i.e. the clan GH-H of glycoside hydrolyses, is the largest family of glycoside hydrolases, transferases and isomerases comprising nearly 30 different enzyme specificities. (Henrissat B. 1991). A large variety of enzymes are able to act on starch.

Amylases are a group of enzymes that break down starch or glycogen. Microorganisms synthesize and release amylases extracellularly. Amylases are classified on the break down basis of characters of starch molecule. [alpha]-Amylases (EC 3.2.1.1), hydrolysis the internal [alpha]-1,4 linkage in starch in a random fashion leading to the formation of soluble maltodextrins, maltose, and glucose. Most of the [alpha]-Amylase are metalloenzymes, which require calcium ions ([Ca.sup.2+]) for their activity, structural integrity, and stability. They belong to family 13(GH-13) of the glycoside hydrolase group of enzymes (Bordbar, et al, 2005)

These are endoenzymes that split the substrate in the interior of the molecules and are classified according in their action and properties. For example amylase that produce sugars are termed " saccharogenic" and those the liquefy starch without producing free sugars are known as "starch liquefying". [beta]-amylases ([alpha]-1-4- glucan maltohydrolase, EC 3.2.1.2) are exoacting enzymes that cleave non-reducing chain ends of amylase, amylopectin and glycosidic linkages in amylopectin and results in incomplete degradation of the molecule yielding maltose and [beta]- limit dextrins.

Amyloglucosidase (exo-1-4-[alpha]-D-glucan glucano hydrolase,(EC 3.2.1.3) hydrolyses single glucose unit from non-reducing ends of amylase and amylopectin in stepwise manner. Unlike [alpha]- amylase, most glucoamylases are also able to hydrolyse 16-[alpha]-linkages at the branching points of amylopectin although at a slower rate than 14-linkages. Thus glucose, maltose and limit dextrins are the end products of glucoamylase action.

Extracellular [alpha]-amylase producing fungi

Studies on fungal amylase especially in the developing countries have concentrated mainly on Rhizopus spp and Aspergillus spp, probably because of the ubiquitous nature and non-fastidious nutritional requirements of theses organisms (Abe et al., 1988). It has been reported that while a strain of Aspergillus niger produced 19 types of enzymes, [alpha]- amylase was being produced by as many as 28 microbial cultures (Pandey et al., 1999). Thus, the selection of a suitable strain for the required purpose depends upon a number of factors, in particular upon the nature of the substrate and environmental conditions.

Optimization of growth conditions is important for best growth of fungi. The growth requirements for fungi may vary from stain to stain, although cultures of the same species and genera tend to grow best on similar media. Similarly growth responses of fungi also vary though they are grown on same conditions (Smith and Onions, 1994). Fungi grow over a wide range of pH condition and must thus be able to tailor gene expression to the particular pH of their growth environment. Filamentous fungi vary in pH requirements. Most commonly fungi grow well over the same range pH 3 to 8. although some can grow at pH 2 and below e. g. Monilella acetobutans, Aspergillus niger. Penicillium funictlosum (smith & Onions, 1994). Many reports were available in literature on the capability of fungal enzymes, which hydrolyze starch. Production of [alpha]- amylase by different fungal stains and then enzyme activities are presented table 1.

Materials and methods

Microorganism and culture maintenance

A fungal strain of Acremonium sporosulcatum (IMI 393096, Kew, UK and NCIM 1319, Pune,. India) was used in this study. Culture was maintained by using Potato dextrose agar media under 30[degrees]C for seven days and it was stored at 4[degrees]C. These sub cultured fortnightly

Enzyme production

Acremonium sporosulcatum was grown on the PDA broth at 30[degrees]C for 48hrs for inoculum preparation. 1ml of the culture was transferred to Production medium it was composed of Maize starch 5.0 gm/lit, Yeast extract 1.0, [K.sub.2] HP[O.sub.4] 0.5,MgS[O.sub.4]. 7[H.sub.2]O 0.1, pH 6.8-7.0 and 1ml of Microelement Solution (Fe [(N[H.sub.4]).sub.2] (S[0.sub.4]).sub.3] 1.0, ZnSO4 1.0, MnS[O.sub.4] 0. 5, CuS[O.sub.4] 0. 08, CoS[O.sub.4] 0. 1, [H.sub.3] B[O.sub.4] 0.1,pH 6.8-7.0 gms/100ml). The Production was carried out in a 250 ml Erlenmeyer flask inoculated with 2ml of broth culture and incubated at 30[degrees]C at 240rpm. The sample was with drawn after 48hrs of fermentation. The enzyme production and extraction process were carried out by according to Baysal (Baysal et al 2003a, 2003b) Extract was pooled and centrifuged at 10,000 x g for 15min clear supernatant solution was used as crude enzyme source for purification.

[alpha]-Amylase purification

The amylase was purified by ammonium sulfate 30% to gradual increase up to 70% saturation, ion-exchage chromatography (DEAE cellulose-50) and gel filtration (Sephadex G-100). After ammonium sulfate saturation, protein precipitate was centrifuged at 10,000 x g for 15 min at 4[degrees]C. The pellet was then dissolved in 0.1M Phosphate buffer (pH 6.9) and dialyzed against the same buffer overnight. The undissolved material in the dialysate was removed by centrifuged at 15,000 x g for 10mim. The supernatant collected and concentrated in a lyophilizer and applied on a DEAE cellulose column (5X20 cm glass column, flow rate 20ml/1hrs) equilibrated with 0.01M phosphate buffer (pH 6.9). The protein was then eluted with a linear gradient of NaCl (0.1 to 1 M) in the same buffer. The active fractions were collected, dialyazed and applied on a Sephadex G-100 column (2.5x10 cm, flow rate 5ml/1h) equilibrated with 0.05 M Tris-HCl buffer (pH 7.0) the active fractions were collected and lyophilized.

Gel electrophoresis

The purity was checked by SDS-PAGE. Polyacrylamide gels were prepared by the method of Laemmli (1970). Gel was stained with coomasie Brillant Blue R-250. the SDS-PAGE molecular weight markers) sigma were Bovine serum albumin (66,000) albumin egg (45,000) and carbonic anhydrase (29,000).

Protein determination

Protein was estimated according to the method of Lowery taking crystalline bovine serum albumin as the standard (Lowry et al., 1951)

Assay of alpha amylase activity

[alpha]-Amylase activity was assayed by the method Bernfeld (1955) Using soluble substrate (1% w/v) in 0.2M acetate buffer (pH 4.5) and 100 [degrees]l of enzyme solution was incubated for 20 min at 37[degrees]C. The reaction was stopped by adding .04 ml of 3, 5-dinitrosalicylic acid solution, followed by heating in a boiling water bath for 5min and cooling to room temperature, then 8ml of deionized water was added. Absorbance of each solution contain the brown reduction product was measured at 540nm by UV-Visible spectrophotometer.

All the experiments performed independently in triplicate and the results are presented as the mean of three.

The effect of Effect of trace elements on enzyme activity

The determine the effect of trace elements of [alpha]-amylase activity, enzyme was preincubated with final concentration 5mM for different trace elements such as Fe[(N[H.sub.3]).sub.2]] [So.sub.4], Zn[So.sub.4], Mn[So.sub.4], Co[So.sub.4], Co[So.sub.4], and [H.sub.3] [Bo.sub.4]. 0.1ml of trace elements solution was added to each flask containing 50ml sterile fermentation medium in 250ml Erlenmeyer flasks. Simultaneously the individual components of the trace elements solution were separately added to each flask containing 50ml fermentation medium. Triplicates were used for each element. Fermentation was carried out as before at the end of which enzyme activities and final pH of each test were noted. Addition of complete trace element solution has contributory role in enhancing enzyme activity. In comparison with other Fe[(N[H.sub.3]).sub.2]S[O.sub.3] is found essential for the enzyme synthesis

Substrate specificity

The substrate specificity of the enzyme from both the crude extract and post dialysis solution (ammonium salt precipitation) upon 3 different substrates: soluble starch, glycogen and dextrin. The enzyme activity typically behaves on different substrates. The variation between the activity of the crude extract and partially purified enzyme acting on starch can be explained in two ways. First the solutions used as enzyme source are not equal regarding their enzyme activity/ml. Second the crude extract offers conditions much closer to those found in vivo (different types of ions, different biological molecules) that could stabilize the enzyme.

Results and Discussion

Effect of substrate concentration

Submerged fermentation was carried out with substrates like maize starch, potato starch, rice starch, sweet potato starch, tapioca starch and maize cob powder for the production of alpha amylase with the initial pH 5.8 to 6.9 at 30[degrees]C using Acremonium sporosulcatum. Among all substrates maize starch (13.59 Units/ml) gave the highest and tapioca starch was lowest yields of alpha amylase activity (4.66 Units/ml). Potato starch was given second highest alpha amylase activity. There is a decrease in the activity of Rice starch sweet potato starch this may be due to the fermentation of other metabolites during fermentation and also because of increase in viscosity of the broth. The temperature, pH and rpm were maintained at 30[degrees]C receptivity, the substrate concentration was maintained at 1% w/v for optimization of operating variables in the further investigation.

Influence of nitrogen source

Studies on influence of inorganic and organic nitrogen source such as sodium nitrate, Ammonium nitrate, aluminum ammonium sulfate, potassium nitrate, ammonium sulfate and Urea, Yeast extract, Soya been meal, peptone and beef extract at 1% (w/v) concentration to the fermentation medium showed a mixed trend on enzyme production. Of all the inorganic nitrogen sources sodium nitrate was found to be best for the production and ammonium nitrate was found next best source for the production. Lowest activities of the enzyme were observed in the presence of potassium nitrate and aluminum ammonium sulfate. In the presence of aluminum ammonium sulfate the mycelium poorly developed with large vacuoles. Organic source of nitrogen urea was found almost unsuitable for the synthesis of the enzyme although biomass developed well yeast extract (9.71 Units/ml) and Soya bean meal (8.5 units/ml) was respectively found suitable organic sources of nitrogen for the mold to elaborate the enzyme.

Partial purification of alpha amylase

Partial purification and different fractions of enzyme showed different degree of enzyme activates (Table 2). The fraction with the maximum enzyme yield was up to 70%, Hence, further charactering of the enzyme was done with this fraction..

Characterization of partially purified alpha amylase

Maize starch is suitable for the best for purification steps for protein and [alpha]-amylase activity assay. The ammonium sulfate appears to interfere with the determinations and we had to give up the samples with high sulfate concentrations: the supernatant after the precipitation with 35% ammonium sulfate and the supernatant with 70% ammonium sulfate. The acetone-washing step was necessary. The total protein decreases from 12.5mg to 5mg and the purification factor increases. The evolution of the purification factor and yield along the purification steps are shown in the table 2.

Substrate specificity

Studies on substrate specificity of the enzyme from both the crude extract and post dialysis solution upon three different substrates: Soluble starch, glycogen and dextrin the result was showed in graph1. The enzyme activity typically behaves on different substrates. The variation between the activity of the crude extract and partially purified enzyme acting on starch can be explained in two ways. First the solutions used as enzyme source are not equal regarding their enzyme activity/ml. Second the crude extract offers conditions much closer to those found in vivo (different types of ions, different biological molecules) that could stabilize the enzyme.

[GRAPHIC 1 OMITTED]

Influence of substrate concentration

Starch degrading rates upon starch concentration is presented (graph 2). The data shows that the enzyme does not behave typically according to a Michaelis-Menten Kinetic but more like as substrate-inhibited enzyme. This behavior has two explanations one is for the hydrolase's the water may be considered on of the substrates, the increasing of the starch concentration leading this to the diminishing water concentration the other explanation is the starch degrading mechanisms

[GRAPHIC 2 OMITTED]

Thermo stability and pH stability

The enzyme remained stable at a temperature range 20[degrees]C to 70[degrees]C. above which the stability a rapidly declined. The maximum activity was displayed at 50[degrees]C graph 3. And the effect of pH on amylases activity was characterized by stability from pH 3.0 to 8.0 after 72hrs. of incubation at 30[degrees]C the optimum pH activities were 6.8 to 8.3 (Graph 4).

[GRAPHIC 3 OMITTED]

[GRAPHIC 4 OMITTED]

Influence of inorganic ions

The purification process in the presence of ammonium sulfate at high concentration the enzyme activity decreases dramatically Graph 5.

[GRAPHIC 5 OMITTED]

Effect of calcium ion

The calcium ions have a specific importance for the alpha amylase, because it has a thermo stabilizing role. Without it, the enzyme activity disappears at 70[degrees]C because of thermo denaturation of the enzyme. Therefore the effect of calcium ions on [alpha]-amylase was studied in some detail this ion has an activating effect on this enzyme. The results are presented in the following graph (Graph 6)

[GRAPHIC 6 OMITTED]

In the presence of the Ca[Cl.sub.2] 0.001M the amylase activity increases to about 96% of the control value. Further increase in the salt concentration lead to a loss of enzyme activity. At 0.005 M Ca[Cl.sub.2] the activity decreased to 3.04 [micro]mol/ml. and at 0.1M becomes 0.52 [micro]mol/ml although the activation effect decreased. It maintains above the control level values. In order to explain this effect we have to consider both the [Ca2.sup.+] and [Cl.sup.-] ions.

Conclusion

Pure colonies of Acremonium sporosulcatum have been isolated by natural selection and among which relatively fast growing strains with ability to hydrolyze starch was selected. The strain no 325 were selected for the fermentative products thermostable of [alpha]- amylase under submerged cultivation. It has wide range of temperature tolerance form 4[degrees]C to 37[degrees]C with an optimum temperature of 30[degrees]C for growth, reproduction and elaboration of industrial enzymes of the present study. Similarly it grows and produces the enzyme form pH 3-9. The optimum pH for [alpha]- amylase.

Fermentations were carried out as biphasic system--i.e. the seed stage and fermentation stage. Several of the naturally occurring starchy substances like potato starch, maize starch, tapioca starch etc are studied out of which maize starch at a concentration of 7.5 % was founded optimum for the synthesis of the enzyme. The fungus needs both inorganic and organic nitrogen source like sodium nitrate and an organic nitrogen source like yeast extract are found essential for the synthesis of [alpha]-amylase, peptone and ammonium sulfate are essential for the synthesis of pectinase and gelatin, ammonium nitrate are found essential nitrogen source for alkaline protease. Aeration, inoculum concentrations, effect of micronutrients is also investigated.

The enzyme is partially purified by ammonium sulfate precipitation method and it is found to be stable upto 70oC after dialysis. The total protein decreases from 12.5mg to 5mg and the purification factor increase. The molecular weight of [alpha]-amylase was 58kDa(plate1) on SDS-PAGE. These properties indicate the possibilities for use of the amylase in the starch processing industry. This enzyme can be exploited commercially.

[ILLUSTRATION OMITTED]

Plate 1 : SDS-PAGE of samples taken in the purified [alpha]-amylase. Lane 1-Crude Extract, Lane 2-Ammonium sulfate preipitation, Lane 3-After dialysis

References

[1] Abe, J., F.W. Bergman, K. Obeta and S. Hizukuri. 1988. Production of the raw starch degrading amylase of Aspergillus sp. K-27. Appl. Microbiol. Biotechnol. 27: 447-450.

[2] Baysal Z., Uyar F., Aytekin C. (2003a). Production of a-amylase by thermotolerant Bacillus subtilis in the presence of some carbon, nitrogen containing compounds and surfactants. Ann. Microbiol., 53: 325-330.

[3] Baysal Z., Uyar F., Aytekin C. (2003b). Solid state fermentation for production of a-amylase by thermotolerant B. subtilis from hot-spring water. Process Biochem. (In press).

[4] Bernfeld P. (1955). Amylases, a and [beta]. In: Methods in Enzymology, Vol. 1, Academic Press, New York, pp. 149-154.

[5] Bordbar, K. Omidiyan, R. Hosseinzadeh, Study on in teraction of a -amylase from Bacillus subtilis with cetyl tri methylammonium bromide, Colloids Surf. B: Biointerfaces, 40 (2005) 67-71.

[6] Haska, R. Ohta, Y (1994). Starch/Starke 46: 480-485.

[7] Henrissat, B. 1991. A classification of glycosyl hydrolases based on amino acid sequence similarities, Biochemical Journal. 280: 309-316.

[8] Kundu and S. Das 1969. Production of amylase in liquid culture b y a strain of Aspergillus oryzae. Applied Microbiology April p 598-603.

[9] Laemmli U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685.

[10] Lin L.L, Hsu W. H (1977). A gene en coding for [alpha]-Amylase from thermophilic Bacillus sp., strain TS-23 and it expression in Escherichia coli.J.Appl. Microbial 82: 325-334.

[11] Lonsane, B. K, Ramesh M. V (1990). Production of bacterial thermostable [alpha]-Amylase by solid-state fermentation: a potential tool for achieving economy in enzyme production and starch hydrolysis in advances in J. Applied Microbiol 35: 1-56.

[12] Lowry O.H., Rosebrough N.J., Farr A.L. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275.

[13] Mitidieri, S., A. H Souza Martinelli, A. Schrank, and M. H Vainstein 2006. Enzymatic detergent formulation containing amylase from Aspergillus niger: a comparative study with commercial detergent formulations. Bioresour Technol, 97(10): 1217-24

[14] Pandey A, Nigam P, Soccol C R, Soccol V T, Singh D and Mohan R (2000). Advances in microbial amylases. Biotechnol. Appl. Biochem. 31: 135-152.

[15] Pandey, A., C.R. Soccol, P. Selvakumar and P. Nigam. 1999. Solid state fermentation for the production of industrial enzymes. Current Science, NewDelhi-INDIA, 77(1): 149-162

[16] Pisano, M. A., W. S. Oleniacz, R. T. Mason, A. I. Fleischman, S. E. Vaccaro and G. R. Catalano 1962. Enzyme production by species of Cephalosporium. VIII International Congress for Microbiology, Montreal.

[17] Shinsaku Hayashida and Juji Teramoto 1986. Production and Characteristics of Raw starch Digesting [alpha]-amylase from a protease-negative Aspergillus ficum mutant

[18] Smith, D. and A.H.S. Onions. 1994. The preservation and maintenance of Living Fungi. Second edition. IMI Technical Handbooks No. 2, pp 122. Wallingford, UK: CAB INTERNATIONAL.

[19] Steen, A. Susanne H. E., J. Orgen Olsen and Bo Jensen 1998. Increased production of [alpha]-amylase from thermomyces lanuginosus by the addition of Tween 80. Enzyme Microbial Technology. 23: 249-252

[20] Tony M. Silva, Derlene A. A, Ana F.A. C, Roberto D. S Mauricio, B. and Eleni Gomes 2005. Production of Saccharogenic and Dextrinogenic amylase Rhizomucor pusillus A 13.36. the Journal of Microbiology. p 516-568.

Vasant. K. Valaparla

Centre for Biotechnology, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur- 522 510, A.P., India. E-mail: vasanth_msv@yahoo.com
Table 1: Production of [alpha]-amylase by different fungi

Microorganism                       substrate         amylase activity

Aspergillus niger,                  corn gluten       18
Aspergillus oryzae                  soybean meal      10.80
Aspergillus awamori                 raw starch        10
Cephalosporium longisporum          casine            14
Thermomyces lanuginosus             dextran           19
Rhizomucorpusillus                  potato starch     13
Acremonium sporosulcatum sp.nov*    maize starch*     13.59*

Microorganism                       references

Aspergillus niger,                  Mitidieri, S et al 2006
Aspergillus oryzae                  Kundu and S. Das 1969
Aspergillus awamori                 Shinsaku, H and Juji, T 1986
Cephalosporium longisporum          Pisano M. A et al 1962
Thermomyces lanuginosus             Steen. A. et al 1998
Rhizomucorpusillus                  Tony M. Silva et al 2005
Acremonium sporosulcatum sp.nov*    present strain

Table 2 Purification steps of [alpha]-amylase by Acremonium
sporosulcatum using maize starch

Step             Volume     Total      Total    Specific   Yield
                  (ml)    activity.   protein   activity     %
                          Units//ml    (mg)      (U/mg)

Crude extract     100        936        34       27.52      100

Ammonium           25        475        13       36.53     50.74
sulphate
Precipitation

After dialysis     20       442.8       08       55.35     45.72

Step             Purification
                  factor or
                     fold

Crude extract         1

Ammonium             1.31
sulphate
Precipitation

After dialysis       2.01
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Author:Valaparla, Vasant. K.
Publication:International Journal of Biotechnology & Biochemistry
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Date:Jan 1, 2010
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