Indian Journal of Chemical Technology Vol. 8, July 2001 , pp. 314-318

Immobilisation of a-L-rhamnosidase of Aspergillus terreus on bagasse particles based matrix

Sarita Yadav & K D S Yadav*

Department of Chemistry, D D U Gorakhpur University, Gorakhpur 273 009, India

Received 13 Apri/2000; accepted 27 March 2001

a-L-rhamnosidase from Aspergillus terreus has been immobilised on bagasse particles based matri x. Km, pH and temperature optima of the immobilized enzyme using narigin as the substrate have been found to be 0.24 mM , 4.0 and 55°C respectively . The corresponding values for the same enzyme in the solution phase are 0.16 mM, 4.0 and 55°C respectively. The immobilized preparation of a-L-rhamnosidase hydrolyses naringin present in Citrus sinansis juice indicating its applicability in the debittering of citrus fruit juices.

a- L-rhamnosidase 1 [E.C.3.2.1.40] which catalyses the hydrolysis of naringin to prunin and rhamnose, is a biotechnologically important enzyme which has been used in the debittering of citrus fruit juices2

, in clarification of fruit and vegetable juices3

, in deglycosylation of chloropolysporin4, in manufacture of a-L-rhamnose5

·6 and in quantitative analysis of

rhamnosides7'8 containing terminal a-L-rhamnose.

Keeping these points in view, enzymological studies on a-L-rhamnosidase from different fungal strains

OH OH

Rh-Glu-0 ~ Glu-0 ~~ Rmmno~ 1¥1, ) a-L-Riuimnosida~ 1 ) - + OH 0 H 0

Naringin Prunin ( 4:5, 7-trihydroxyflavanone-7-rhamnoglucoside) ( 4 ',5, 7 -trihydroxyflavanone-7-

glucoside)

have been initiated with the objective of identifying the enzymes suitable for the above applications. Secretion of a-L-rhamnosidase in the culture medium by Aspergillus terreus strain MTCC-3374 has been reported earlier9

. In this communication, immobilisation of a-L-rhamnbosidase from A. terreus on a matrix prepared by activating the bagasse particles is reported. The use of the immobilised preparation in the hydrolysis of naringin present in C. sinansis juice has been demonstrated.

Experimental Procedure Naringin, 1,6-hexanediamine and p-nitrophenyla-

*For correspondence

L-rhamnopyranoside were procured from Sigma Chemical Company, (St. Louis, U.S.A.). All other chemicals used in these studies were either from CDH (New Delhi)or from Loba Chemie (Mumbai) and were used without further purifications. C. synansis juice was extracted in the laboratory by washing, wiping, squeezing through four layers of cheese cloth and was centrifuged to clear it before use.

The enzyme a-L-rhamnosidase used in these studies was prepared by growing the fungal strain Aspergillus terreus which has been isolated in the laboratory and has been deposited at the MTCC centre in the Institute of Microbial Technology, Chandigarh as the fungal strain MTCC-3374.

The methods for maintaining the fungal strain on agar slant and growing it in the liquid culture medium for the extraction of the enzyme have already been reported9

• One mL of spores' suspension (spore density 8x106 spores/mL) from the agar slant was inoculated aseptically into sterilized liquid culture medium (15 mL contained in 100 mL culture flasks). The liquid culture medium contained CaC]z lg, MgS04.7H20 3g, KHzP04 20g, N (CHzCOONa)J 1.5g, MnS04 lg, ZnS04.7HzO O.lg, CuS04.SH20 O.lg, FeS04.7H20 O.lg, H3B03 lOg, sucrose 40g, ammonium tartrate 8g and naringin 0.33g, in one litre of quartz double distilled water. The composition of the agar medium was the same as above except that 20g of agar per litre was added. The culture flasks were incubated in a BOD at 30°C and the fungal strain was allowed to grow under stationary conditions. The maximum activity of

. a-L-rhamnosidase appeared on the third day and fungal mycelia were removed from the culture medium

YADAV & YADAV: IMMOBILISATION OF a-L-RHAMNOSIDASE ON BAGASSE PARTICLES 315

on the same day by filtering the medium through four layers of cheese cloth. The resulting filtrate was passed through Sartorius membrane filter (0.22 !J.m) to remove fine particles and was concentrated 10 times using freeze dryer. The concentrated enzyme sample (contained 0.05 units of enzyme/mL), was dialysed over night against quartz double distilled water at 30°C; volume ratio of culture filtrate to water being 1:1000. The dialysed samples of culture filtrate were used for experimental work using p-nitrophenyl-a-L­rharnnopyranoside as the substrate. The filtered and concentrated enzyme retains its activity for more than 3 months if stored at 4°C.

During the course of enzyme extraction , enzyme activity has been measured by the method reported by Romero et a/. 10 0.5 mL of 3.5 mM p-nitrophenyl-a-L­rhamnopyranoside in 0.1 M potassium hydrogen phthalate/HCl buffer pH 3.5 was added to O.SmL of the same buffer. The reaction mixture was incubated at 57°C and a suitable aliquot of the enzyme sample was added. 50!J.L of the reaction mixture was withdrawn at the interval of every 2 min, diluted to 3mL with O.SM NaOH and their absorbance at 400nm was measured. Molar extinction coefficient value of 21.44 mM-1 cm-1 for p-nitrophenolate has been used and one enzyme unit is defined as the amount of enzyme which liberates 1!J.M of nitrophenolate per minute.

The hydrolysis of naringin by a-L-rhamnosidase has been studied using Davis 11

'12 method. 20 !J.L of the

soluble enzyme or 20 particles of immobilised enzyme were added to 2mL of 0.86 mM naringin solution in 0.2M sodium acetate /acetic acid buffer pH 4.0 maintained at 57°C. 0.1 mL aliquots of the reaction mixture were withdrawn at the intervals of 10 min and were added to 2.5mL of 90% diethylene glycol followed by addition of 0.1 mL of 4N NaOH. The samples were maintained at ambient temperature for 10 min and absorbance at 420 nm were measured. The concentrations of unhydrolysed naringin were determined by calibration curve showing the variation of absorbance at 420 nm against the known concentrations of naringin solutions treated in the way mentioned above.

All spectrophotometric measurements were performed on UV NIS spectrophotometer Hitachi (JAPAN) model U -2000 which was connected to an electronic temperature control unit for maintaining temperature in the cuvettes. The least count of the absorbance measurements was 0.001 absorbance unit.

Bagasse particles based matrix for immobilisation of enzyme has been prepared by the reported method13

. The basic idea involved is the conversion of surface alcoholic groups of saccharides present on the Bagasse particles into aldehydic groups which have been activated by 1 ,6-hexanediamine and glutaraldehyde respectively. Tentative sequence of reactions involved in enzyme immobilisation are shown in Scheme 1.

Dry Bagasse pieces were crushed to the particles in a mortar with pestle. Coarse and very fine particles were removed. 1g of particles of approximate size 0.1cm dia were thoroughly washed with glass double distilled water , treated with 10mL of 0.2M sodium metaperiodate and incubated in dark for 16 h at 25°C. The particles were washed with water , then with 3% sodium thiosulphate and finally with water. These activated particles were treated with 50 mL of 10% 1,6-hexanediarnine and pH was adjusted to - 11. The solution was stirred for 24 h at 25°C. The particles were washed first with 15mL of 10% 0.1M sodium acetate/acetic acid buffer pH 4.8, then with 25mL of 0.1M borate buffer pH 8.5 and finally with distilled water and pH was adjusted to 10-11 and the solution was stirred for 15 h at 4°C. The resulting matrix was

Baggase particles with surface alcoholic groups ~\{~~

L-----__J-+ OH?oo +rrear:ment with Nal04

H ~. HOf!~O 1...--------'-+ ~p

Baggase particles with surface aldehyde groups

f'reaunent with diaiTI..ino hexene H ~ OH ~

Baggase particles with ~H, c; ... N-{CHV.,..NH2

surface amino groups

-+ow·

freatmcnt with NaBH..t H H QH

-+ ~~· :NH-( CH,J.-NH, Baggase panicles with reduce Schiff" s base

OH '

H .. OH

+Treatment with glutaraldehyde ~ H

H I+.~CH·NH-f CHll,s-N=CH-{CH2)3-CHO

Baggase particles with surface aldehyde groups -+ ow-:

+ Treatment with enzyme H qH

Baggase particles with ~-·1· 1 C: · NH-{CH,J,- N -ell - ( CH,), CH•N·E enzyme attached at the ._..., surface mr

_ Where E stands for enzyme H

Scheme 1 - Though the surface carbohydrate on bagasse particles may have complex structures glucose has been considered as an example for simplicity.

316 INDIAN J. CHEM. TECHNOL., JULY 2001

o (a)

0

4.0 0

(b)

•

0.4 0.8 1.2

[S] [mM]

Fig. !-Michaelis- Menten plots using naringin as the substrate. (a) Enzyme in solution phase ( o ); (b) Immobilized enzyme ( • )

"' 0

1.50r----------------,

(b)

X 1.00

·~ c • .E --~ .... <{

<l 0.50

I >

OL----~-----L------~

1 -1 [S] [mM]

Fig. 2-Double reciprocal plots using naringin as the substrate. (a) Enzyme in solution phase ( o ); (b) Immobilized enzyme ( •)

treated with 1.5 mL of 2.5% aqueous glutaraldehyde for 2 h at room temperature. The excess glutaraldehyde was removed by washing with distilled water. The resulting matrix was treated with 1 mL of a-L-rhamnosidase stock and solution was left in the refrigerator at 4 °C overnight. The unreacted enzyme was removed by decanting the solution from the matrix and the matrix was washed with 0.2M sodium acetate/acetic acid buffer pH 4.0 containing 1M NaCl to remove the non-covalently bound enzyme. Analysis of the enzyme activity indicated that more than 70% enzyme activity is covalently bound. The immobilised enzyme was preserved in 0.2M sodium acetate/acetic acid buffer pH 4.0 at 4°C

7.5.-----------------

"' I 0 .,... 5.0 X ~

c: .E --0

"' .... <{

<J 2.5

>

OL-----~~----~----~~----~ 2.0 4.0 6.0 8 .0

pH Fig. 3-Dependences of the enzyme activity on pH. (a) Enzyme in solution phase ( o ); (b) Immobilized enzyme( •)

in the refrigerator. For studies of enzymatic hydrolysis, 20 particles containing immobilised enzyme were withdrawn , dried with filter paper and were added to reaction solution.

Results and Discussions

The variation of steady state velocity of the enzyme-catalysed reaction at the different concentrations of naringin has been presented in Fig.l . Fig. 2 shows the double reciprocal plots corresponding to the data shown in Fig.l. The Km values of the enzyme in the solution phase and in immobilised form have been found to be 0.16 mM and 0.24 mM respectively. Thus the Km value of the immobilised enzyme is higher than the Km value of the enzyme in solution phase indicating that the enzyme has lower affinity for the substrate in the immobilized form in comparison to its substrate affinity in the solution phase. The enzyme after immobilisation might have undergone some conformational change resulting into lower affinity for the substrate.

The variation of steady state velocity of the enzyme-catalysed reaction with the variation of pH is presented in Fig. 3. It is obvious from the curves that the pH optimum of the enzyme in solution phase is 4.0 and it does not change after immobilisation. It is worth noting that a -L-rharnnosidase has been used for debittering of citrus fruit juices, the pH of which lie in the temperature 14 range 3.0-3.8. Thus the pH optimum value 4.0 of this enzyme is suitable for its debittering action in the natural citrus fruit juices.

Y ADA V & Y ADA V: IMMOBILISATION OF a-L-RHAMNOSIDASE ON BAGASSE PARTICLES 317

5.0,...--------------------,

"' I 0 ~

X

c: .E 2.5

o~----~-----~----~ 30 60 90

Temperature [ °C ]

Fig. 4-Dependence of the enzyme activity on temperature. (a) Enzyme in solution phase ( o ); (b) Immobilized enzyme ( •)

o.so..--------------,

~ 0.30 <(

(f)

OL----~3~0----~60~~

Time )min.]

Fig. 5-Enzymatic hydrolysis of naringin present in C. sinansis juice. Enzyme in solution phase in natural C. sinansis juice. ( o ); Denatured enzyme in the juice ( • ); Control with no enzyme in the juice (o ); Immobili zed enzyme in natural C. sinansis juice. (il); Denatured immobilized enzyme in the natural C. sinansis juice (4).Immobilized enzyme in 50% diluted juice. (•).

The variation of steady state velocity of the enzyme catalysed reaction with temperature has been shown in Fig. 4. It is obvious from the curves that the optimum temperature for the enzyme activity is 55°C and it does not change as a result of immobilisation of the enzyme. Though the optimum temperature for the enzyme activity is 55°C, it has appreciable activity in the temperative range 30-50°C and can be used even below 55°C but with the reduced efficiency of the enzymatic conversions.

The enzymatic hydrolysis of naringin present in C. sinansis juice is shown in Fig.5 where absorbance at 420 nm that is proportional to the naringin concentration has been plotted against time. The curve (a) in Fig. 5 is for the juice containing enzyme in solution phase. The curve (b) is for the juice containing denatured enzyme in solution phase where as curve (c) is for the juice with no enzyme. The increased level of absorbance in curve (b) is due to denatured enzyme. It is obvious from line (a) that the enzyme is hydrolyzing naringin present in the

40.-------------------.

u "' ~ 0 u >­.c .g, 20 c:

·~ z 0 •

120

(b)

(a)

240 360

Time [min.]

Fig. 6--Dependence of the percentage of naringin hydrolysed on reaction time in C. sinansis juice. (a) Enzyme in solution phase (o); (b) Immobilized enzyme (•)

C. sinansis juice. Curve (d) in Fig. 5 is for the immobilized enzyme in C. sinansis juice and curve (e) is for the denatured immobilized enzyme in the same juice. The hydrolysis of naringin present in the C. sinansis juice by the immobilized enzyme is obvious from curve (d) in Fig. 5. The background level of absorbance at /..=420 nm in .case of line (d) is lower because the enzyme in the immobilised form is not contributing any absorbance at 420 nm. Whereas in case of curve (a), enzyme being in the solution phase contributes to the background absorbance at 420 nm. Curve (f) is for the immobilised enzyme kept in C. sinansis juice which has been 50% diluted. Thus in all the cases where C. sinansis juice contains active enzyme in solution or in immobilised form, the naringin concentration which is indicated by absorbance at 420 nm is decreasing with time showing the enzymatic hydrolysis of naringin present in C. sinansis juice.

In order to estimate the time required for the enzymatic hydrolysis of naringin present in the C. sinansis juice, percentage naringin hydrolysed has been calculated at different time intervals. The results are plotted in Fig. 6. It is obvious from the figure that nearly 6 h are required and only 35 % of naringin present in the fruit juice is hydrolysed. The longer time required and lower percentage of naringin hydrolysed may be due to the inhibitory actions of some non-identified components present in C. sinansis juice or inhibition by rhamnose produced by the enzymatic hydrolysis of naringin present in the juice. In order to identify the inhibition, percentage naringin hydrolysed by the enzyme in sodium acetate/acetic acid buffer pH 4.0 was determined at different time intervals (Fig. 7). It is clear that the time required for the hydrolysis is only 2 h which is

318 INDIAN 1. CHEM. TECHNOL., JULY 200 I

40.---------------------------------,

!a)

(b)

240 360 Time [min.]

Fig. ?-Dependence of the percentage of naringin hydrolysed on reaction time. (a) Enzyme in solution phase ( o ); (b) Immobilized enzyme (•)

7.5,----------------,,--------------------,

~

' ~ 5.0

:5. c e <f.~ '2 2.5

0 >

2.0 4,0

ConcenlraUon of Inhibitor ( mM]

Fig 8-Inhibition of the enzyamatic activity by rhamnose, glucose and citric acid. (a) Rhamnose (o); (b) Glucose .(M; (c) Citric acid (Q) .

one third of the time required for the full hydrolysis in case of natural fruit juice. It is obvious that some components present in the juice are inhibiting the enzyme activity and increasing the time required for reaching the saturation value of naringin hydrolysis. However the level of the saturation value of naringin hydrolysed in case of naringin in the sodium acetate/acetic acid buffer is also nearly 30% which is not much different from the corresponding value of 35% obtained in case of the natural fruit juice. Since rhamnose is concomitantly released during the hydrolysis of naringin, its accumulation in the reaction medium and inhibition of the enzymatic activity might be solely or partially responsible for the low saturation value of naringin hydrolysis in Citrus

juice as well as the naringin solution in the buffer. In order to estimate the relative inhibition of the enzyme activity by rhamnose , glucose and citrate, the components generally present in citrus fruit juices, the enzyme activities in presence of varying concentration of these inhibitors have been measured (Fig. 8). The results are shown in Fig. 8. It is obvious from these curves that the concentration of rhamnose required to inhibit the activity of the enzyme to half its value is - 0.38 mM where as the corresponding values for glucose and citrate are - 33mM and - 66 mM respectively. Thus rhamnose dominates the inhibitory action of the enzyme.

The studies reported in this communication clearly show that a-L-rhamnosidase produced by Aspergillus terreus can be used for controlled hydrolysis of naringin which imparts bitterness to the citrus fruit juices 10

.

Acknowledgement The financial support of CST, U.P. Lucknow is

thankfully acknowledged.

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