Indian Journal of Chemical Technology Vo l. 9. November 2002, pp. 535-539

Articles

Comparative study of catalytic activity of immobilized invertase in sodium alginate gel on sucrose hydrolysis

C N Khobragade* & S G Chandel

Schoo l o f Life Sc ie nces. Swami Ram::mancl Teerth Mara th wada Uni vers ity. Nanded 43 1 606. India

Rc:ccil •c:d 26 July 2001: rc:l'ised recei\•l'(/ 17 Julv 2002; accep!ed 25 Augus / 2002

Invertase is an intracellular hydrolytic enzyme widely used in the inversion of sucrose into glucose and fructose. Both glucose and fructose are the primary compounds used as sweeteners in the food processing industries. In the present investigation invertase was immobilized in sodium alginate gel by entrapment method and the catalytic activity of immobilized enzyme was compared with the activity of native enzyme at variable pH and temperatures. Experimental findings revealed that for native enzyme optimum activity was at pH 4.2 and at temperature 30°C, while for immobilized enzyme it was observed at pH 4.8 and at temperature 50°C. Kinetic parameters K111 and Ymax as well as catalytic elliciency of invertase was also determined for both the native and immobilized enzyme.

In ve rtase is one of the most important hydrolyti c enzyme used as a catalyst in the invers ion of sucrose into g lucose and fruc tose as the sweeteners in food industri es. To avoid the problem associated with the repeated use of so luble invertase, different techniques of immobilization are widely used. Presently both glucose and fructose (invert sugar) are mainly produced by the method of inversion using immobili zed in vertase as it IS efficient and

. I I economt ca process An enzyme in vertase is treated with sucrose as it

has two active sites and completely splits sucrose molecule into g lucose and fructose by acting on a-1 ,4 g lycosidic bond23

The conventional industrial processes involve the incubation of a mixture of soluble enzyme and substrate. After completion of each set of reactions the product formed is recovered by denaturation of enzyme. ln thi s process loss of active sites of enzyme takes place and enzyme can' t be reused. Hence during each set of reactions the use of fresh enzyme is necessary to convert the substrate into product, which is an uneconomical process.

To overcome the problems associated with conventional processes invertase is immobilized on different supporting materials with retention of its catalytic activity, which can be used continuously, and repeatedl/ .6

In the present investigation invertase is immobilized in sodium alginate gel by entrapment

*For correspondence (E-mail : cnkhobragade@ rediffmail.com; Fax: (02462)-29245)

method, which has its own advantages over other techniques. Alginate is a linear polysaccharide ext racted from strai ns of marine brown seaweed and algae. It has a property to make enzy me more thermostable clue to its hydrophili c nature7

. Thi s property of alginate made us to se lect it as a supporting materi al for immobilizat ion. Immob ili z­ation leads to conformat ional changes in enzy me structure, thereby altering the properti es of enzyme. Therefore, it is necessary to invest igate the optimum pH and temperature along with the kinetic constants for both native and immobilized invertase8

·9

.

Experimental Procedure

Sodium alginate L.R. grade (Laboratory Rasayan Mumbai), Invertase, extracted fro m yeast (BDH Laboratory, England), acetic acid, sodium acetate and 3,5 DNSA (Himedia Laboratory Ltd . Mumbai), Sucrose and CaC)z (Qualigens Fine Chemical s, Division of Glaxo India Limited, Mumbai ) were used without further purification and were pure unless stated otherwise.

Preparation of sodium alginate gel (3%)

3 g of pure and dried sodium alginate powder was dissolved in 100 mL of slightly warm distilled water. It was thoroughly mixed on cyclomixer for 5 min to make a transparent gel. The resulting gel was used further for the entrapment of the enzyme 10

. One unit

of enzyme is defined as l f...lg of product formed mL!min of enzyme.

Articles

Preparation of sodium alginate immobilized invertase

3 g of sodium alginate was suspended in 100 mL of warm di stilled water and suspension was stirred for 5 min. After coo ling, to thi s 0.5mL enzyme (0.5%) was added. The beads of uniform size were prepared by dripping the polymer in freshly prepared 0.2 M CaCI2 (250 mL solution). Beads were resuspended in acetate buffer (pH 4.2) and then packed into the chromatography column ( 18x200 mm) about half the length of co lumn . The beads were washed several times with acetate buffer until the unbound enzy me was removed completely. Amount of unbound in vertase was tes ted by taking 5 mL fraction of eluant, I mL of sucrose solution along with 0.5 mL of Beneclicts reagent . The contents of tubes were heated directly on fl ame for 3-5 min along with the proper blank. Reel co lour appeared which indicated that 100% immob ili zation was not achieved. Therefore, washing was continued till no reel colour appeared. After proper washing of the column the amount of invert sugar produced was determined spectrophoto­mctrically from th e standard calibration curve and finally the units of enzy me entrapped in ge l were calculated by subtracting the units of non-entrapped enzy me from the total units of the enzyme ( 100 units).

Comparison of the activity of immobilized and native invertase

The acti vity of immobilized and native invertase was determined by carrying out the hydrolysis of sucrose as a substrate and estimating the amount of in vert sugar (mixture of glucose and fructose) by modified DNS methocl 11

•

To determine the activity of nati ve enzyme 1 mL of enzy me (0.5 %) and I mL of sucrose (0. 1%) was added into the series of test tubes and the reaction mi xture was incubated for 30 min . After completion of incubati on peri od I mL of DNSA was added in all tubes and the contents were thoroughly mixed on cyclomixer. The reaction mixture was warmed in a boiling water bath for 5 min . After addition of 3 mL di still ed water in all tubes the optical density of the contents of test samples were recorded against the proper bl ank at 540 nm on visible spectrophotometer (Shimadzu make). Same procedure was followed to compare activity of immobilized invertase with the acti vity of nati ve enzyme 12

.

EffectofpH The amount of in vert sugar produced was estimated

at different pH by DNS method. The optimum pH for

536

Indian J. Chern. Tednol.. November 2002

native and immobili zed invertase was determined by measuring the activity of native and immobili zed enzyme at different pH. The concentration of sucrose and enzy me was maintained constant and hydrolysis was allowed to proceed for 30 min at appropri ate pH in acetate buffer solution (Tab le I & Fi g. I ).

Effect of temperature The effect of temperature on act ivi ty of nati ve and

immobilized in vertase was determined by carry ing out hydrolys is at different temperatu res ranging from 20-80°C. For thi s system. pH and concen tration of sucrose and enzy me was maintai ned co nstant and reaction was all owed to proceed for 30 min in a se ri es of tubes. This procedure was followed for both th e nati ve and immobili zed enzyme. The activity of enzy me was calculated by estimatin g the amount of invert sugar produced using DNS method (Tabl e 2 & Fig. 2).

E.ff'ect of the concentration of substrate on kinetic parameters

The kinetic constan ts K,11 and Vm.,, were determined for both nati ve and immobil ized invertase. In case of immobilized in vertase Km and Vm., , are not the true kinetic constants, but are referred to as apparent values because of eli ffusional effect o f enzy me from the ae l 1.~

b .

The kineti c constants were determined by using appropri ate concentration of sucrose by keeping the pH and temperature constant. The react ion was allowed to proceed for 30 min and the amount of invert sugar produced was again estimated by DNS method. Similar experiment was also carried out for the nati ve enzyme for comparative study.

In a study of different pH on the activity immobilized invertase the amount of enzyme protein that came down into the filtrate was estimated as 20 units and the units of enzyme that was entrapped in sodium alginate gel was 80 units (total I 00 units). The stability of immobilized enzy me was also more than the native free enzy me.

In the study of effect of different temperature on activity of immobilized invertase the units of enzyme that was entrapped in sodium alginate gel was 9 1 units.

Activity The activity of immobili zed enzy me at optimum

pH and temperature was calculated as 80% & 9 1% respecti vely. The decrease 1n the acti vity or immobilized invertase was due to the conformational

Khobragade & Chandel: Catalytic activity of immobilized invertase in sodium alginate gel on sucrose hydrolysis Articles

changes of enzyme, physical and chemical properties of carrier and the favourable environment caused by the carrier for the formation of product.

Results and Discussion

EffectofpH Effect of different pH on the actiVIty of

immobilized and native free enzyme was studied. It was noticed that change in pH affects the activity of both native/free and immobilized enzyme on hydrolysis of sucrose. For native free enzyme the optimum pH was 4.2 while for immobilized enzyme optimum pH was observed at pH 4.8 (Table 1 & Fig. I). This increase in optimum pH from 4 .2 to 4.8 may be due to change in the H+ ion concentration of the microenvironment of the immobilized invertase in sodium alginate gel. The H+ ion concentration in the entrapped enzyme and sodium alginate gel is very high as compared with the H+ ion concentration around the immob ilized enzymes. Hence, to adjust the same H+ ion concentration in both the environment, there may be diffusion of H+ ions from inside towards the outer environment, thereby shifting optimum pH towards alkaline side. In addition to thi s another reason may be attributed to the sod ium alginate immobilized enzy me which becomes more acidic than the external and the optimum pH to alkaline side.

E.lfect of temperature Temperature variation shows strong effect on

activity of native and immobilized invertase. The optimum temperature for native and immobilized

invertase was observed at 30 and 50°C respectively (Table 2 & Fig. 2). This shift in optimum temperature

from 30 to 50°C was attributed to the hydrophilicity of the alginate gel, the hydrophobicity of the enzyme itse lf and the flexibility of the enzyme 14

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Effect of concentration of substrate For the native enzyme the saturation was observed

at 0.8 mg/mL while for immobilized enzyme it was observed at 0.9 mg/mL (Table 3 & Fig. 3). There is no remarkable difference in the saturation concentration of both the native and immobilized enzyme. The s light difference in the saturation concentration may be due to the availability of active sites for the binding of the substrates. The results revealed that for native enzyme, active sites are available more 111 number as compared to immobilized enzyme.

Table !-Effect of pH on the activity of the native and immobili zed invertase in sod ium alginate gel.

Sr pH Amount of invert sugar produced (mg/mL)

no Native Immob ili zed

I 3.8 6.1 2.9

2 4.0 7.0 3.8

3 4.2 9.2 4.4

4 4.4 8.4 4.8

5 4.6 R.S 5.9

6 4.8 8.2 7. 1

7 5.0 7.6 5.4

Table 2-Effect o f temperature on the activity of the nati ve and immobi li zed in vertase in sodium algina te ge l.

Sr Temp. A moun t of in ve rt sugar produced (mg/mL )

no oc Native Immob ili zed

I 20 8.0 1.6

2 30 8.5 3. 1

3 40 8.0 6.8

4 50 8. 1 7.4

5 60 R.O 5.5

6 70 7.0 2.5

7 80 0.7 0.5

Table 3-Kinetic properties of free and immobili zed in ve rtase

Parameter Free/N ative Immob ili zed In ve rtase ln ve rt ~be

Saturation concentrat ion 0.8 0.9

(mg/mL)

Krn 0.1315 0. 384

\1111.1\ 0.042 0.02 (1

Optimum pH 4.2 4.8

Optimum temperature (°C) 30 50

Catalytic effici ency ( Vmaxl Km) 0.32 0.068

Km & Vmax were determined from Lineweaver-Burk plot.

] e Q.

~:3" 6 ~ E

"'" i .§. 4

0 c ~ 0 E <

2

3.8 4 .0 4 .2

-B-Native.

4.4

pH

---4.6 4.8

Immobilized

Fig. !-Effect of pH on the acti v ity of native & immobili zed invertase

Kinetic constants The Km and Vmox values for native enzyme were

observed as 0.1315 & 0.0425 respectively while that of Km (app) and Vmax (app) for immobilized enzyme

537

Articles

10

g>::i 6 ~ E

""' -~ s 4

0

0

20 25 30 35 40 45 50 55 60 65 70 75 80

Temperature °C

-a- Native. ~ Immobilized

Fig. 2- E!Tect of temperalllre on the acti vity of nati ve & immobili zed in vertase

u 12 g u ,

10 u 0 Q. . 8 [

.~ 6

0 4 c

2 , 0 E .. 0 ., I I I ' I I I I I I

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0 .8 0.9 1.0

Substrate concentration (mg/ml)

-B- Native, ~ Immobilized

Fig. 3- Effcc t of substrate concentrati on of the activity of nati ve & immobili zed in ve rtase

were 0 .384x I 03 g/L and 0 .0263x I 03 g/30 min/80 units

respec tively. The Km and Vmax for invertase were

ca lculated from Lineweaver-Burk plo t. Intercept on

X-axis was considered as -l/K111 while intercept on Y­axis was value of l!Vmax (Table 3, Figs 4 & 5) 16

. The

value of Km for native free enzyme was less as

compared to the immobili zed enzyme and indicates

that ES complex of the native enzyme is more stable

than the immobili zed enzy me, while Vmax value was

more for native enzyme as compared to immobili zed

enzyme thereby yielding more product. The decrease

in Vmax was observed after immobilization. This may

be due to the change in microenvironment of the

enzy me or con formati o nal changes in the enzyme 17•

The increase in Km value of the immobilized enzyme

was due to less stability of ES complex . This is

attributed to the non availability of active sites of

enzy me.

Continuous production of invert sugar in laboratory scale - A column of 18x200 mm was loaded with sodium a lg inate immobilized invertase to give a total bed

538

Indian J. C hem. Techno!.. November 2002

54

51

+i

12

-10 - -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10

_..1_-ISJ

Fig. 4- Detenninatio n of kinetic paramete rs K, , V""" fo r nati ve/ free in vertase (Lineweaver-B urk -p lot )

+i

-1

IC -10 -9 -8 -7 -6 ·5 -4 -3 -2

190

180

170

160

150

145

140

135

130

125

120

110

100

90

80

70

60

50

40

_..1_­ISJ

I ·I

3 4 6 7 8 9 10

Fig . 5- Determinatio n of kinetic parameters Km V""" for immobili zed invertase (Lineweaver-Burk-plot)

volume of 220 mL. Co lumn was equilibrated with acetate buffer (pH 4 .2) . A 0.1 % sucrose solutio n was fed continuously in the column with infl ow and outflow rate of I mL/30 min. The column was stable for 7 days without any loss of the activity of enzyme.

Khobragade & Chandcl: Catalyt ic act ivity of immobilized in vertase in sodi um alginate gel on sucrose hydro lys is Articles

Conclusions Sodium alginate is a very good carrier material for

immobilization of invertase. It is necessary to keep the system always in wet condition to avoid the loss of activity. This immobi li zed system can be extrapolated for continuous production of in vert sugar from sucrose in sy rup manufacturing industries. The system can be recycled and recharged after long use. Production of in vert sugar will be max imum at pH 4.8 and at temperature 50°C.

Acknowledgement The authors are thankful to the Head of School of

Life Sciences fo r providing the necessary facilities during this work.

References I Allen I Laskin. En ;.YIII es & I111111 0bilized Cells in

Biolrchnolog,· (The Benjamin/Cumming Publi shing Co mpany. Inc. Menlo Park. Ca li forni a). 1985. 104.

2 Bows ki L. Sain i R. Ryu D Y & Vieth W R. Biotrchno/ogv & Bioengineering Vol XIII (John Wiley & Sons, Inc. New York ). 1971 , 64 1.

3 Helmut Uhli g. Indus/rial Enzymes & their Applicalions (John Wiley & Sons, Inc. New York). 1998.77.

4 Wiseman Alan. Hcmdbno/..: o( En ;.YIIIe Bimecllllologv (Ell is Hard wood Limited Chichester. West Sussex Po. I ') I El3 Eng land). 1987. 147.

5 Sweigart R V. Indus/rio! Applicalion,· o( lnunobili:etf En;.y111es. State-of-A rt. In Enzyme. edited by Broun G 8 Maneck & Wingard L 13. Vol 4 (Pl enum Press. New York ). 1978. 229.

6 Monsan P & Combes D. Bioll:cluwl !Jioeng. 26 (I ') ~4 ) ]4 7. 7 Wang Nam Sun. En :nnr lnunobili:alion bv Gel Elllmptll l' l/1 .

(Prepared by Dept. of Chem ical Engineering. Un iversity of Maryland. College Park. M D-20742-2 1 II ENCH-4R5 ).

8 Can tarella M. Can tarclla L. Gallifuoco A & Alfani F . .I Bimech. I (1992) 159.

9 Mclo J S & D'Souza S F, IIppi Bioche111 Biolech. Jan -Mar ( 1992) 159.

10 Lee J M & Woodward J. Biolech Bioeng. 25 ( 198] ) 24-11. II Sadasivam S & Manikam A. !Jioclrelllical Mellwds. 2nd

ed ition (New Age In ternati onal Pvr. Ltd .. New Delh i). 1996.6.

12 Mathew J. Alli son C & Lurry Bering. 1 Chenr Educ. 75 (1998) 10.

13 Rani A S, Das M L M. Rajeshwar Rao A & Satyanaraya na S. Indian 1 Che111 Techno/, 5 ( 1998) 397.

14 Das G & Prabhu K A. En: Microb Tech. 12 ( I 090) 625. 15 Gupta M N. Tlr emwswbilitr o( En :_l'llres (Narosa Publi shing

House. New Delhi ) 1993. 162. 16 Copeland R A. En zytnl's (VCH Publi shers. Inc. 3337'h

A venue. New York , I 000 I). 1996. I 00. 17 Goyal L, Singh S & Pundir C S. Indian 1 Clre1n Tecluwl. 7

(2000) 142.

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