Introduction

Enzymes are biological catalysts. They increase the rate of chemical reactions taking place within living cells without themselves suffering any overall change. Amylases are among the most important industrial enzymes and are of great significance in present day biotechnology. Two major classes of amylases have been identified namely α-amylases and glucoamylases in addition to this β-amylases, which is generally plant origin has also been reported a few microbial sources. Amylases are characterized by their ability to hydrolyze glucosidic linkages in polysaccharide. Alpha-amylases (endo 1, 4-α–D–glucan glucohydrolase, EC 3.2.1.1) are extracellular and acts on starch components. The α-amylases may be derived from several bacteria, yeasts and fungi. Bacterial amylase, however, is generally preferred over fungal amylase due to its thermostability and  also it minimizes the contamination risk and reduces reaction time. Strains of Bacillus sp., mainly Bacillus subtills, Bacillus licheniformis, Bacillus amyloliquefaciens and Bacillus megaterium are employed. Among these Bacillus subtilis strains are employed for commercial application^1,3 .

Physical entrapment of enzymes in calcium alginate beads has shown to be a relatively easy, rapid and safe technique. Entrapment requires that the immobilized enzyme should be a large enough molecule to be kept inside the gel matrix but the substrate and product should be small enough to pass through the pores of the gel ².

Starch molecules are very large, often reaching a molecular weight of 80 million Daltons. It is expected that starch hydrolysis reaction could occur more effective if enzyme bound to surface. Therefore, a comparison of different methods of immobilization of α -amylase to calcium alginate method would provide useful information on the efficiency of the hydrolysis of starch into smaller sugars.

In this study amylase which was purchased commercially from Himedia was immobilized and compared  with that of the immobilized enzyme synthesized by the  bacteria and comparative study of different immobilization methods was drawn.

Materials and Methods

Immobilization  by Sodium alginate entrapment method

Sodium alginate  purchased from Himedia was prepared by dissolving 3gm sodium alginate in 100ml of boiling water and autoclaved at 121°C for 15minutes.Both alginate slurry and enzyme  equivalent to 0.03gm  were mixed and stirred for 10 minutes to  get a uniform mixture. The slurry was taken  into a sterile syringe and added drop wise into 0.2M CaCl₂  solution from 5cm  height and kept for curing  at 4° C  for  1 hour The cured beads were washed with sterile distilled water  3 to 4 times. When the beads were not being used, they  were preserved in 0.9% sodium chloride  solution in the refrigerator. All operations were carried out aseptically under laminar flow unit ^4,5.

Immobilization of Amylase in Agar-agar

A definite quantity of agar-agar (Hi media, Mumbai, India) was dissolved in 18ml of 0.9% sodium chloride solution to get a final concentration of 2% and sterilized by autoclaving . The enzyme suspension (2ml equivalent to 0.03gm) was added to the molten agar-agar maintained at 40° C shaken well for few seconds (without forming foam  ), poured into a sterile flat bottom 4 inch  diameter petriplates and allowed to solidify. The solidified agar block was cut into equal size cubes(4 mm³) added to sterile 0.1M  phosphate buffer (pH 7.0 ) and kept in the refrigerator for curing. After curing phosphate buffer was decanted and the cubes were washed with sterile distilled water 3to 4 times ⁶.

Immobilization of the  enzyme in Gelatin

5ml of 0.06% of  enzyme suspension  was added to 15ml of  20%  sterile gelatin (Hi media) maintained at 45° C and poured into a sterile petridish. The gel was over layered with 10ml of 5% glutaraldehyde   for hardening at 30°C .The resulting block  was cut into small size cubes and the cubes were washed thoroughly with sterile  distilled water for the complete  removal of excess glutaraldehyde ⁶.

Immobilization of the  enzyme in Polyacrylamide

A  cell suspension was prepared by adding 0.03g cells to 10ml  chilled sterile distilled water. To  another  10ml of 0.2M sterile phosphate buffer(pH 7.0), the following chemicals  were added: 2.85g  acrylamide,0.15g bisacrylamide ,10mg ammonium persulphate   and 1ml TEMED (NNN¹ N¹tetra methyl ethylene diamine). The cell suspension and the above phosphate buffer mixture well and poured into sterile flat bottom 10cm- diameter petriplates. After polymerization the acryl amide gel was cut into equal size cubes(4mm³) transferred  to 0.2M phosphate buffer(pH 7.0) and kept  in the  refrigerater for 1 hour for curing. The cubes were washed 3 to 4 times with sterile distilled wate  and stored  in sterile distilled water at 4  C until use ⁷.

Production of amylase

Bacillus subtilis  was isolated from  the  soil and  identified by Berger’s manual of systematic bacteriology. The organism was grown  in 250ml of Erlenmeyer’s flask,  each  containing 50ml of starch nutrient  broth medium that has the following  composition(g/L) : beef extract ,3.0.,peptone 10.0.,NaCl ,5.0., Starch,10.0.,  distilled water  1000ml. The pH  was adjusted to 7.0 . The flasks  were inoculated with spore suspension  obtained from nutrient agar and incubated at 40°C  with  shaking at 150rev/min at 48hr. The filtrates was then separated by  centrifugation  at 10,000/10min.The reaction mixture  was estimated by taking 0.4ml  of 1%soluble  starch and 0.5ml of each diluted culture supernatant and 1% phosphate buffer . The mixture was incubated at 50°C for 20min and incubated at 10,000rpm and assayed  for reducing sugar⁸ .

Assay of amylase activity

Amylases acts on starch to produce reducing sugars which upon reaction with DNS reagent form orange to reddish colored complex  having maximum absorbance at 540nm. To the above immobilized enzyme 1ml of 1% starch solution and 1ml of distilled water was added and incubated. After incubation 1ml of  2N NaOH  to stop the enzyme reaction . 1ml of DNS  reagent was  added and  placed in the boiling water bath for 10min and OD was measured at 540nm  against blank in colorimeter. 1 units of amylase activity is expressed as the amount of enzyme which produces 1m mole per min of reducing  sugar  with glucose as standard.

Standard graph of maltose was drawn and with the  help of  standard graph,  enzyme activity was calculated. The activity of enzyme  immobilized by various methods was  calculated and further compared  with the activity of the enzyme produced by Bacillus subtilis.

Results and Discussion

Among the immobilization methods activity of enzyme entrapped by sodium alginate was more than that of remaining entrapment methods(2.4 IU/ml). Sodium alginate method is having an advantage that it is non-ionic. The consequence is that the properties of the enzymes are only minimally modified in the presence of the gel matrix. At the same time, the diffusion of the charged substrate and products is not affected, gelation of calcium alginate does not depend on the formation of more permanent covalent bonds between polymer chains. Rather, polymer molecules are cross-linked by calcium ions.

Immobilization in gelatin is having a next higher value after sodium alginate entrapment(1.6 IU/ml) . The main attraction of using gelatin as the immobilization media is that the gel formation process requires only simple equipment and that the reagents are relatively inexpensive and nontoxic. The retention of enzymatic activities for immobilization with a gelatin gel is typically 25-50% of the original free enzyme. Gelatin gel has the advantage that the mass transfer resistance is relatively low compared to other entrapment methods, but the rate of enzyme loss due to leakage is high.

The activity of the enzyme immobilized in polyacrylamide and agar agar were found to be less than remaining methods.Among these two methods polyacrylamide gave high titer value than agar agar. The results are tabulated in Table.1.

Table 1: Standard graph of Maltose

Table 2: Activity of Amylase immobilized by different methods.

Figure 1: Standard Graph of Maltose.   Click here to View figure

Figure 2: Comparative Study of various Entrapment  techniques.   Click here to View figure

Unlike the adsorption and covalent bonding methods, most polymerization reactions that cause cross-linking and gel formation in entrapment methods do not directly involve the formation of bonds between the support material and the enzyme molecules. There are reports that these bonds change the conformation of the enzyme protein and modify the enzyme properties. Since the enzyme molecules do not themselves participate in the polymerization reaction in the entrapment methods, the same entrapment techniques can be successfully applied to a wide range of enzymes with only minor modifications between different enzymes.

References

1. Mohamed A., Abdel-Naby and Amal M. Hashem .,  MicrobioL Res., 153, 1-000 (1998).
2. Emne’us J. and Gorden L.Anal.Chim.Acta., 234,97-106(1990).
3. Mifflin T.E. and Bruns D.E., Clin Chem , 615-620 (1987).
4. Roy I., Sastry, M. S. R., Johri, B. N. and Gupta, M. N., Enzyme Microb. Tehnol.,27,53-65(2000).
5. Johnsen A.and Flink JM.,Enzy.Microb. Technol.,8,737-748(1986).
6. Veelken M., Pape H., J.Appl Microbiol Biotechnol., 15,206-210(1982).
7. rabhakaran G,Pugulvendhan R.,1(4),189-194(2009).
8. Bajpai P and P.Bajpai .,Biotechnol.Bioengg.,33,72-78 (1989).