Journal of Scientific & Industrial Research

Vol. 6 1 , May 2002, pp 36 1 -365

Enzymatic Preparation of L-Glucose, L-Galactose and L-Xylose

Using Galactose oxidase Immobilised on Crab-shell Particles

K K Yadav, S K Vernwal, Z Afaq and K D S Yadav*

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

Received: 27 September 2000; accepted: I I February 2002

Galactose oxidase has been immobilised on a solid support developed by modification and activation of surfaces of crab-shell particles. The conversion of D-sorbitol to L-glucose, galactitol to L-galactose and xylitol to L-xylose using the immobilised galactose oxidase have been demonstrated. The performance of the immobilised galactose oxidase for the conversion of D-sorbitol to L-glucose in a mini batch reactor has been studied. The advantages of this method for preparing L-sugars have been discussed.

Introduction

L-sugars are phannaceutically important compounds being safe non-caloric sweeteners and precursors of many natural products ' -3 . The reported4-6 routes of their synthesis are tedious with poor yields? There is a need to develop convenient methods for the syntheis of L-sugars. Root et al8 have reported that galactose oxidase [EC 1 . 1 .3.9] oxidises xylitol to L-xylose, D-sorbitol to L-glucose and galactitol to L-galactose, stereospecifically and no other products are fonned. However, use of galactose oxidase in solutions for the above conversions is expensive. In solution, conversion needs to be done in batches and after each batch, the enzyme has to be denatured, thus, increasing the cost of the final product. Moreover, the separation of the denatured enzyme from the final product also increases the cost of production . The solution of these problems is to immobilise the enzyme on a solid support and use it in a reactor. Thus, enzyme could be used repeatedly and there will be no problem of separation of the enzyme from the final product. The present communication reports an immobilised preparation of galactose oxidase, prepared by chemical modification and activation of surfaces of crab-shell particles. The conversion of D-sorbitol to L-glucose, galactitol to L­galactose and xyl itol to L-xylose USIng the immobilised enzyme preparation has been

*Corresponding author

demonstrated. The performance of galactose oxidase immobilised on crab-shell particles for the conversion of D-sorbitol to L-glucose in a mini batch reactor has been tested and relevant data reported.

Materials and Methods

Galactose oxidase and catalase were procured from Sigma Chemical Company, St Louis, USA and 3-methoxybenzyl alcohol was procured from Aldrich Chemical Company, Inc . , Wisconsin, USA which was vacuum disti lled. Glutaraldehyde, 3,5-dinitrosalicylic acid and D-sorbitol were procured from CDH, Delhi . All other chemicals used in the present research work were either from S .d. Fine Chemicals, Mumbai or Qualigens Fine Chemicals, Mumbai and used without any further purification .

Crab-shell particles of mesh size about 40, were used for the preparation of the solid support for immobilisation of galactose oxidase. These were prepared by picking up bigger crab-shell particles from river sand, washing with double distilled water, drying overnight at 50 DC in an oven, crushing in mortar with pestle and removing the fine and coarse particles.

Crab-shel l particles ( 1 g) were treated with 1 0 mL of 4N NaOH and left for 24 h at room temperature. NaOH was removed by washing the particles 1 0-times with double distil led water. The particles were treated with 1 .5 mL of 2.5 per cent

362 J SCI IND RES VOL 61 MAY 2002

aqueous glutaraldehyde solution for 1 .5 h at room temperature. The excess of glutaraldehyde was removed by washing l O-times with double distilled water. 1 50 units lO of galactose oxidase was dissolved in 0.5 mL of O. I M sodium phosphate buffer, pH 7.0 and 0.4 mL of the enzyme solution was mixed with glutaraldehyde activated crab-shell particles. The solution was left at room temperature for 2 h after which it was kept in the refrigerator for 24 h . The enzyme solution was removed by decantation and the immobilised enzyme preparation was washed with 1 M NaCI solution in O. I M sodium phosphate buffer pH 7.0 to remove physically adsorbed enzyme molecules from the surface of the activated matrix . The analysis of the enzyme solutions before and after immobilisation using 3-methoxy benzyl alcohol as the substrate I I indicated that more than 90 per cent of the enzyme activity was retained in the matrix . The immobilised enzyme preparation was washed 1 0-times with O. IM sodium phosphate buffer pH 7.0 and was kept in the refrigerator in the same buffer.

For co-immobilising galactose oxidase and catalase on crab-shell derived matrix, all the operations were the same as used for immobilising galactose oxidase alone with the only difference that the galactose oxidase solution in the case of co­immobilisation also contained catalase ( I mglmL).

The activIty of galactose oxidase was determined " using 0.06M methoxybenzyl alcohol in O. I M sodium phosphate buffer, pH 7.0 at 25 °C. Absorbance changes with time at 3 1 4 nm were monitored using UV NIS spectrophotometer (Hitachi, Japan, model U-2000) . In an assay volume of 1 .0 mL, a M314 = 1 .0 is equivalent to 0.37 � of the product.

For testing conversion of D-sorbitol to L-glu­cose using immobilised galactose oxidase the immobilised enzyme preparation was dried by putting it on a piece of Whatman filter paper and then taken in a 5 mL culture tube, washed with sterilised double distilled water prior to addition . Sorbitol solution (3 mL of 50 mM) in O. IM sodium phosphate buffer, pH 7.0 was added to the culture tube which was closed with the cap. The mixture was left for 5 d at room temperature. The quantity of L-gluclose formed in the solution was analysed using DNS reagentl 2. To I mL of the test solution, I mL of I per cent DNS reagent was added and the resulting mixture was heated in a boiling water bath for 30 min. Further, 3 mL Rochelle salt solution was added to it in warm

condition and absorbance at 572 nm was recorded and concentration of L-glucose was read from the calibration curve prepared using a series of L-glucose solutions of known concentrations. The same procedure was adopted for studying other conversions studies and reported in this paper.

In order to test functioning of the immobilised enzyme for the conversion of D-sorbital to L-glucose in a reactor, a mini continuously stirred batch reactor, as shown in Figure 1 , was designed. The reactor is a double wall glass vessel with a stopper at the top, connected to a circulating water bath (ultra cryostate CB-7oo of Remi make) to maintain it at desired temperature. A magnetic bead was kept inside the reactor and the reactor was kept over a magnetic stirrer model 2 MLH, Remi equipment, Mumbai as shown in Figure I . 20 mL of 1 00 mM sorbitol solution in 1 00 mM sodium phosphate buffer, pH 7 .0 was poured into the reactor and water, maintained at 25 °C, was circulated through the double walls of the reactor to maintain it at 25 DC. Immobilised enzyme preparation was added to the sorbitol solution in the reactor and stirred with the magnetic stirrer. 1 mL of the reactor solution was withdrawn at different intervals and analysed for L-glucose formed using DNS reagentl2.

- -

CIRCUlATING WATER BATH

lJMMOB ILIZED EN ZYME

4.MAGNETIC BEAD

S.MAGNETIC STIRRER

- - - ..

3

5

Figure 1 - Continuous stirred reactor based on galactose oxidase immobilized on crab shell particles

YADAV et al.: PREPARATION OF L-GLUCOSE. L-GALACfOSE & L-XYLOSE 363

Results and Discussion

Crab-shell particles have been used for developing solid support for the immobilisation of galactose oxidase. Crab-shell consists of chitin9

which is composed of repeating ( 1 -7 4) linked 2-

aceto arnido-2-deoxy-p-D-glocose moieties. The structure of chitin is shown in Chart 1 along with its modifications effected for the enzyme immobilisation. It is non-toxic, naturally occurring, not easily degradable and is available almost at no cost. The treatment of crab-shell particles with NaOH solution deacetylates the aceto amido group to give

CRAB SHELL PARTICLES

( I) WASHED WITH DOW

(II) OVERNIGHT TREA WITH 4N NaOH

(III) WASHED WITH DOW

1/

PARTICLES WITH FREE

NH2 GROUPS

TREA THENT WITH

2 . 5% GLUTARALD EHYDE

'If

ACTIVATED MATRIX

TREATMENT WITH ENZYME

'l-

IMMOBILISED PREPARATION

free NH2 on the surface of these particles. These free NH2- groups on the surface of crab-shell particles have been activated by treatment with glutaraldehyde. One end of glutaraldehyde combines with the free NHr group and the other aldehydic group is free for attaching enzyme through it. All these steps are shown in Chart 1 . The analysis of the enzyme solution, used for immobilising the enzyme on activated crab-shell particles, before and after immobilization, for the total number of enzyme units present in the solution showed that more than 90 per cent of the activity of enzyme was immobilised on the surface of crab-shell particles.

� CH OH HO O �

o HO

/ 0 CH OH A NH 2

CHITOSAN

1 -N=CH- (CH ) -CHO 2 3

-N=CH- (CH ) -CH=N-E 2 3

Chart 1 - Enzyme immobilization

364 J SCI IND RES VOL 6 1 MAY 2002

The i mmobi l ised preparation of galactose oxidase ach ieved by the above method was used for the conversion of D-sorbitol to L-glucose, galactitol to L-galactose and xylitol to L-xylose. The conversion yields are given in Table 1 . The yields using only the immobilized galactose oxidase are 6.2, 5 .0, and 7.0 per cent, respectively. Though the conversion yields are low but when compared with the reported synthetic routes4-6, for the synthesis of L­glucose and L-galactose, these are not discouraging. In one route, for the synthesis of L-hexoses, the reported4 yields were 3 per cent for L-glucose and L­galactose and the synthetic route is tedious in comparison to the enzymatic method. In another synthetic route5-6, the yield was 40-60 per cent but the route also is not convenient and the obtained product was a racemic mixture. Thus, taking al l factors into account that matter in a process development for the such conversions, the achieved conversion yields are not without merit. Moreover, there are possibi l i ties for enhancing the conversion yields further.

H202 is formed concomitantly with the formation of L-sugars during the course of the enzymatic reaction, as shown below:

D-Sorbitol + D2 Galaclose-oxidase ) L-glucose + HzOz

H202 is known to inhibit the enzyme activity 'J

and i ts accumulation in the reaction medium is one possible reason for low conversion yields obtained. If this is the case, the decomposition of HzOz as it forms during the reaction, should increase the conversion yield of L- sugars. Catalase l4 [E.C. 1 . 1 1 . 1 .6] is known to decompose HZ02 by fol lowing the reaction:

If catalase is co-immobi l ised with galactose oxidase and then used for the conversions carried out

Table I -Percentage conversion yields of L-sugars from polyols using galactose oxidase

Conversion

Sorbitol to L-Glucose

Dulcitol to L-Galactose

Xylitol to L-Xyloseo

Crab-Shell

Galactose oxidase alone

6.2

5.0

7.8

With catalase

1 2.0

1 6.5

1 3 .8

and mentioned above the conversion yields should Increase. The results given in Table I confirm this point.

There is sti l l scope for improving the conversion yields. L-sugars are the end-products of the above enzyme catalysed reaction. The end­products of an enzymatic reaction generally inhibits the activity of the enzyme catalysing their formation. Thus, with accumulation of L-sugars in the reaction medium, lowering of conversion yields is expected. If the immobi l ised enzyme is used in a column reactor in which the substrate solution is circulated then the end-products are also removed away from the site of their production. If another column is connected in series that removes L-sugars continuously, as they are formed, the end-product inhibition could be effectively tackled.

The optimal conditions for the conversion are 1 00 mM D-sorbitol, 25 °C and 7.0 pH. It takes 5 d to reach the maximum conversion and the immobi l ised preparation can be used for at least 6 cycles. The native galactose oxidase is a mixture of oxidised and reduced forms 1 5, and only the former is active. It has been observed that repeated use of immobi l i sed galactose oxidase converts it to reduced and inactive form which can be reactivated by treatment with potassium ferricyanide solution 1 5 . The reacti vated immobil ised enzyme preparation again could be used for 5-6 cycles of the conversions.

The results of the studies on the performance of galactose oxidase co-immobi l i sed with catalase on crab-shell in a mini batch reactorS for the conversion of D-sorbitol to L-glucose are shown in Figure 2 in which absorbance at 572 nm, which is proportional to the concentration of L-glucose, has been plotted against time for which conversion has taken p lace . It is evident from the figure that t ime required for the maximum conversion to take place is nearly 5 d. The final conversion yield calculated using a calibration curve was 1 0 per cent. It clearly shows the feasibi l ity of the above conversion in a mini batch reactor based on i mmobil ised preparation of galactose oxidase . Though the feasibi l i ty of using galactose oxidase immobil ised on crab-shell particles in a continuously sti rred reactor has been tested and reported in this paper, both the matrix for i mmobi l ization, and so also the reactor design can be varied to make the process more advantageous.

-�

YADAV et al. : PREPARATION O F L-GLUCOSE, L-GALACTOSE & L-XYLOSE 365

1 U

9

8

7

� 6 -CII II) 0 5 0 ::l a 4 I ...J

3

2

O +---------�--------�------� o 50 1 00 1 5C

Time(Hrs) Figure 2 - Conversion of L-Glucose ( in percentage) from

Sorbitol Solution

Non avai labi l ity of the enzyme In enough quantity is generally quoted as a l imiting factor in the development of an enzyme catalysed based conversion. Galactose oxidase is secreted by a various fungal genera l 6. With the developments in molecular biology, i t is not impossible to clone and overexpress the gene of galactose oxidase from one of the fungal strains into suitable vector for the production of the enzyme In desired quantity. Moreover, the enzyme from Fusarium graminearum (NRRL 2903) has been extensively studied 1 7. 18 and all relevant information on the properties of this enzyme is avai lable. The purification procedure of the enzyme is s imple" and the enzyme is stable at 25 °C even in 6M urea. Thus the enzyme is a sui tab le reagent for the reported conversions.

Acknowledgements

The financial supports of Department of B iotechnology, Government of India, New Delh i (Project No. BTfTF/06/07/9 1 ) and University Grants

Commission, New Delhi [Project No. F- 1 2-76/97 (SR-I)] are thankfu l ly acknowledged .

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