Archiv ffir Mikrobiologie 56, 300--304 (1967)

Induction of Sorbitol Dehydrogenase by Sorbitol in Aspergillus niger

B. M. DEsAI, V. V. MoDI and V. K. S ~

Department of Microbiology, Faculty of Science, M.S. University of Baroda

Received January 11, 1967

There are m a n y reports on the induc t ion of enzymes responsible for the metabol i sm of polyols in bacteria, ei ther direct ly or through the i r phosphory la ted in termedia tes (S~ow, 1956; MAGASA~rK, 1953; S~BEK and RANDL~R, 1959). However, there is negligible da ta on the polyol metabol i sm by f i lamentous fungi (YAMADA et al., 1959). I n this communi- cat ion we wish to repor t our studies on sorbitol metabol i sm and re la ted enzymes in Aspergillus niger. We have observed t h a t sorbi tol dehydro- genase is present a t high level in Aspergillus niger during growth on sorbi- tol. Sorbitol dehydrogenase and fructokinase are induced in the presence of sorbitol and glucokinase is repressed.

Materials and Methods

A strain of Aspergillus niger (NCIM 611) was obtained from National Collection of Industrial Microorganisms, National Chemical Laboratories, Poona, India. This strain was grown on Currie's medium (CuRRIE, 1917) for the control experiments. Sorbitol adapted strain was obtained by cultivating the parent strain on a modified medium prepared by substituting increasing concentrations of sorbitol in place of sucrose in Currie's medium, as discussed below.

The parent strain was first allowed to grow and sporulate on medium containing 1~ sorbitol. The spores from these flasks were successively transferred to media containing 2, 3, 4, 5 and 60/0 sorbitol.

After 6 to 7 transfers on 60/0 sorbitol medium the resulting strain was transferred onto sorbitol agar medium containing 6.0g-~ sorbitol; 0.075g-~ KH~PO4; 0.02 g-~ MgS04; 71-120; 0.02 g-~ NH4~O ~ and 2.0 g-~ agar. This strain was maintained on this solid medium for regular use and stock culture was prepared in soft according to the method described by FOSteR (1949).

The strain obtained through sequential transfers as explained above will be hereafter referred to as sorbitol adapted strain.

For induction experiments on Aspergillus niger the parent strain was grown on Currie's medium for 48 hrs on a rotary shaker and pellets harvested on a Buehner funnel, washed with sterile distilled water and resuspended on carbon free salt medium for 12 hrs. After this period the pellets were again washed on Buchner

Sorhitol Dehydrogenase by Sorbitol in Aspergillus niger 301

funnel and resuspended on 60/0 sorbitol medium. After 16 hours' holding on this medium the pellets were washed on Buehner funnel and blotted.

Weighed pellets were ground in previously chilled mortar and pestle with half their weight of alumina (Alcoa-301) and three volumes of 0.02 ~ potassium phos- phat~ buffer pH 7.2. The homogenate was centrifuged a~ 20,000 • g for 10-- 12 rain at 0--5~ in Sorvell super speed centrifuge. The supernatent was filtered through cheese cloth and stored at 0--4~ until used.

Sorbitol dehydrogenase was assayed by the method of Ulrich Gcrlaeh as described in BERO~ErER (1963). The enzyme was measured by following the rate of NADH~ oxidation at 340 m~ in 3 ml reaction mixture containing 0.5 mg NADH~ 100 ~zm potassium phosphate buffer pH 8.6, and 0.1 ml of cell free extract. Fructose 1.2 m~ was added to initiate the reaction.

One unit of enzyme activity is defined as that amoun~ which under the assay conditions, oxidizes, 1 v.m of NADH~ in 1 min at 30 ~ C. The units are computed in tables per hour per total weight of pellets in a flask. Specific activity is measured as units per mg protein.

Protein was determined by the method of Warburg and Christian as given in COLOWICX and K~LAz~ (1957).

Experiments on fructokinase and glucokinase were carried out on the parent as well as the sorbitol adapted strain. The cultures were grown on sucrose and sorbitol respectively and on the third and the fourth days of growth at 30~ the mats were harvested and ground with half their weights of alumina and three volumes of 0.02 ~ tris buffer pH 7.0. The homogenate was centrifuged at 900 • g for 5 rain and the supernatant used for enzyme assay. The kinases were determined by the method as described by HEgs (1955). One ml of crude extract was incubated at 30~ with 9 ~m fructose or 200 tzm glucose, 20 vm ATP, 5 ~zm magnesium acetate, 1.2 m potassium acetate and 50 ~zm of sodium fluoride. Final volume was made up to 3 ml with distilled water, and the tubes incubated for 15 min. The hexokinase activity was determined by sugar disappearence. Glucose was determined by the method of SoMoGYI (1952) and fructose by the method of Ron (1949). The unit of hcxokinase activity was defined as the amount of enzyme required to phosphorylate one micro- mole of hexose in 15 rain at 30~

Results and Discussion

The induc t ion of sorbi tol dehydrogenase fo rmat ion by sorbi tol was

measured in two ways.

I n the first exper iment the induct ion of sorbi tol dehydrogenase for-

m a t i o n was measured in the sorbitol adap ted s train obta ined af ter

several sequent ia l t ransfers o f the pa ren t cul ture in a sorbito] conta ining medium, and compared wi th the enzyme con ten t of the s train grown on

sucrose medium. The resul ts are shown in Fig. 1. I t m a y be seen t h a t the

increase in enzyme ac t iv i ty is observed t i l l th i rd day in sorbi tol medium, where as the basal enzyme in sucrose med ium decreases. The resul ts l is ted in Table 1, indicate t h a t in the paren t s t rain grown on med ium

containing sorbitol, the induct ion of sorbitol dehydrogenase format ion

resul ted in 14 fold rize in the ac t iv i ty .

302 ]3. M. D~s~, V. V. l~oI)i and V. K. SHoat:

The results of the estimations of glucokinase and fructokinase levels in the parent and sorbitol adapted strain (Table 2) suggest that sorbitol represses glucokinase by 5--6 times and induces fructokinase by 10--15 folds.

Thus sorbit01 dehydrogenase and fructokinase are simultaneously induced and glucokinase is repressed in the sorbitol strain.

2 000

/ 600

800

%

goo

Sorbilol medium I Sucrose ,

J ~ 5 8 1 2 Days of /zcuSg/]on

Fig. 1. Sorbitol dehydrogenase activity of sorbitol adapted strain grown on sucrose and sorbi~ol media

Table 1. Sorbitol dehydrogenase activity in Aspergillus niger, parent strain

Sucrose medium Sorbitol medimn

Holding period 4 days 16 hours Specific activity 4.04 56.5

Efforts were made to identify the first product of sorbitol metabolism in the mold. Many unsuccessful attempts were made to isolate the first metabolic product from the parent strain. Finally it was shown to be fructose by the following method. The parent strain was grown on sucrose for 48 hrs and transferred to carbon free salt medium for 12 hrs. The mats were then transferred to (t) 6~ sorbitol medium and (2) 60/0 sorbitol solution, in distilled water. After 4 - -6 hrs incubation at 30~ the mats were removed and the fermented solutions were tested for reducing sugar with Benedict's solution. The flasks containing 6~ sorbitol medium did not show any reducing sugar. But the flask containing only sorbitol contained reducing sugar. The solutions were then concentrated at

Sorbitol Dehydrogenase by Sorbitol in Aspergillus niger 303

4 5 - - 5 0 ~ and the concen t ra tes t e s t ed for presence of f ructose b y resorci- nol tes t , osazones and p a p e r ch roma tog raphy . .A l l the th ree tes t s showed the presence of f ructose and no o the r sugar . These f indings suggest t h a t f ructose is fo rmed from sorbi tol .

Table 2. Glucokinase and/ruetoklnase activities o/ two strains o/Aspergillus niger on 3rd and 4th days o/incubation

Paren~ strain Sorbitol adapted strain Units/1 ml ext. Units/1 ml ext.

Glucokinase

3rd day 77 14.2 4th day 181 35.0

Fruetokinase

3rd day 00.069 01.29 4th day 00.1604 02.51

The resul ts suggest t h a t in Aspergillus niger, free f ructose is the first p roduc t fo rmed b y the enzyme sorbi to l dehydrogenase , t hus causing the need for the induc t ion of f rue tokinase a long wi th sorb i to l dehydrogenaso for the u t i l i za t ion of sorbi tol .

Summary Evidence is p resen ted for the s imul taneous induc t ion of sorb i to l

dehydrogenase along wi th f ruc tokinase and repress ion of g lucokinase b y sorb i to l in Aspergillus niger. Fruc tose is the first p roduc t of sorb i to l ca tabol i sm.

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304 B.M. D~SAI et al. : Sorbitol Dehydrogenase by Sorbitol in Ass niger

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Dr. V. V. MoDI Department of l~/Iicrobiology Faculty of Science M. S. University of Baroda Baroda (India)