ISOLATION FROM SOIL OF BACTERIA PRODUCING EXTRACELLULAR ALPHA GALACTOSIDASE

: Extracellular alpha galactosidase (alpha galactnsidasc galactol~ydl-olase EC 3.2.1.22) producing bacteria wcre isolated fiom soil hy the enrichment culture techniyue using raffinose as the induce].. Six hacterial species were isolated hy this method hased on their morphological characteristics. Their raffinose ut.ilization rate varied fi-om 11 mgAl to 27 mgh. Enzyme acivity present in the supernatant varied from 2-11 mU/ml. Two of the isolated species did not show ally alpha galactosidase activily. Three hacterial species having 11ig11 alpha galactosidase activity were identified as K l c b s i ~ l b u ~ ~ ~ ~ . u ~ ~ r ~ ~ . o ~ ~ . i i ~ e ~ Cifrobacter fieu~rtlii. andEsi;Ir.crichiix roli hy their colony morpholo~y and biochemical tests. They were cult,ivated at different pH values with peptone and ammonium sulpllate as the nitrogen source. From t,l~ose studies, it was shown that the highest extracellular alpl~n galact,osidase activity of 14.7 mUIm1 could he obtained h.om Citmboclr!r frer1.17,rlii after 1811 of cultivation in a culture medium with an i.nitial pH of 8 containing peptone as t l ~ e nitrogen source. Extracellulai~ enzyme production was increased up to 1SmUIn1l hy the cullivation of Citrobur:ter f'rrlrlrdii in pH8 pllosphate Bufl'er li)~. 3611 with peptone as the nitrogen source.

Alpha galactosjdase has been reported to occur widely in microorganjsms, plants and in animals.' This enzyme is required for the degradation of galactomannans and the reserve oljgosaccharides during seed germination." It has been isolated from several bacterial species e.g. Bacillr~s subtilis,:: Baci1lrr.s .~tearoth.ernzophilz~,.~,~ K1eb.siella sp. No. PG-2" and also from many species of fungi e.g. Mortierelln udn.aceo," Mo~~ascu..s pilos~s,~ Trich.odern~a reesei Rut C-30." Industrial applications of alpha galactosidase are in beet sugar indust,ry, soy milk processing, pulp and paper industry and manufacture of gelling agents. Attempts have been made in medicine to use al.pha galactosidase in the treatment, of Fabry's disease.' In industrial applications, fungi and bacteria are used as the enzyme source as they can be easily grown and it is usually not difficult to scale up the production process. There is no seasonal variation in the enzyme production with microorganisms. The majority of enzymes that have so far been used in industry are extracellular enzymes produced by microorganisms. With microbial enzymes, it is often possible to increase the yield by changes in the growth conditions, addition of inducers, or strain selection including increasing the number of gene copies by genetic engineering.
In this paper we report the isolation of extracellular alpha galactosidase producing bacteria from soil by using raffinose as the inducer and the optimization of alpha galactosidase production.

METHODS AND MATERIALS
Materials: Analytical grade Serva Fein Biochemicals and Sigma Chemicals were used. Absorbance measurements were carried out using a Shinladzu UV 120-02 spectrophotometer. A bench centrifuge (MSE) and a high speed (Becl~man model 52-21) centrifuge were used for centrifugation. Fisherbrand Hydrus 300 pH meter was used for pH measurements. Sterilization of culture media was done by autoclaving them at 121°C for 15 minutes. Isolatior~. of bacteria producin.g extracellula,r alplza galactosidase: Soil samples were collected from topsoil from humus rich places. Soil samples were pooled and approximately 21x11 of soil sample was incubated in a culture medium containing 1%; raffinose, 3% peptone, 0.05% yeast extract and 10% (vlv) salt solution (1% IGHPO,, 0.9% MgCI,.GH,O, 0.1% CaCl,, 1% NaC1). pH of the culture medium was adjusted to 7.0 with 0.1% NaOH. Culture medium (25ml) in 100ml conical flasks were incubated at room temperature in an orbital shaker at 115 r.p.m. for four days. After four days, lml of each culture was transferred to a fresh culture medium of the same composition and incubated for two days under the same conditions. The process was repeated twice. Afterwards a loopful of culture was transferred on to an agar plate containing the same culture medium solidified with 3% agar and streak plate isolation procedure was performed. Six bacterial species were isolated and inoculated into fresh culture media separately. After 24hrs cultivation the following parameters were tested with each culture.
(i) Optical Density at 510nm (i.i) Wet weight (iii) Rafinose Utilization Rate (jv) Enzyme Activity of the supernatant at different pH values.
Deterni,il~ation, of wet weigh.t: The weight of pellets obtained by centrifugation of 10ml of each bacterial culture at speed 5 for 15 minutes using a MSE bench centrifuge was determined.
Detcrnrinatiori of Rafinmse Utilizatioz~ Rate: Before inoculating sterilized. enrichment culture niediun~ in different flasks with six bacterial species, 2ml of culture niedium from each flask was taken out for raffinose estimation. After 24hrs of incubation 21nl of the supernatant obtained after centrifugation of 10ml of each culture broth was also subjected to rdfinose estimation. 11111 of the culture medium without raffinose was used to prepare the blanlc sol~ztion. 11111 of the culture supernatant to be tested for raffinose estimation was nlixecl witb linl of 0.02 M thio-barbituric acid. lml of concentrated HCI was added to each mixture and kept in a boiling waterbath for 6 minutes. After cooling, the absorbance was measured at 432.5nni.
Raffinose utilization rate mgh for each bacterial culture was calculated using the difference obtained. for the raffinose contents of each culture medium.
Three selected bacterial species were cultured at 30"C, 45°C and 60°C and the raffinose utilization rates were measured with each culture.
Idcntijicao~. of' tlbe isolated bacterial cultures: Three bacterial species which showed high extracellular alpha galactosidase production were identified by their morphological and biochemical characteristics as described by Barrow and Felthanl1 and LennetteL2 as Esch.erich.ia coli, Klcbsiellapneu.monine and Citrohacter fieurrdii. No tests were done to determine whether the bacteria were pathogenic, as they were is01 ated from soil.
Optin~izntion ofenzyrne prodr~ction. lty Bacteria: The three bacterial species which were identified as Esch.erichia coli, Kle bsiella pneum,oniae and Citrohacter freu.ndii were cultivated in a n~edi~ltn with different nitrogen sources (ammonium sulphate and peptone) and with different initial pH values 5, 7,S. A small decrease in initial pH value was observed after autoclaving.   For each bacterial species under each culture condition the following parameters were estimated at 6h intervals.
(i) Optical density at 510nm (ii) Wet weight (iii) pH of the supernatant (iv) Enzyme activity of the supernatant at pH 7. (v) Enzyme activity of the cells at pH 7.
Culture medium was the same as used jri the isolatjon of bacteria. Culture volunie was 500ml (culture flasks 2L) and cultures were inoculated with 50ml of bacteria culture medium cultivated for 24 h. Cultures were incubated in an orbital shalrer at a speed of 115 rpm at room temperature. 20m1 samples were removed at 6 h intervals aseptically using a laminar flow. 3(%arnmoni~zm sulphate was used Instead of 3' 3) peptone when ammonium sulphate was used as the nitrogen source.
Elzzyrne activity ofthc cells: Enzyme activity of the cells was determined by washing the pellet obtained from wet weight determination in 0.15M McIlvaine buffer (pH7). The pellet was suspended in final volume of 2ml ~zsing 0.15M McIlvaine buffer (pH71 and 0. lml if this suspension was incubated with 0.4ml of 0.15M McIlvairie buffer (pH71 and 0.5ni1 10 mM p-nitrphenyl-alpha-D-galactpyranosjde solutjon for 30 minutes at 29°C. The reaction was terminated by adding 5ml of 0.1N Na,CO, solution. Absorbance was measured at 405nn1, using a Shimadzu UV 120-02 spectrophotometer.
C. freundii was also grown on a culture medium containing pH 8 phosphate buffer and the above parameters were estimated at 611 intervals.

RESULTS
Six bacterial species were isolated from soil samples after cultivation for an initial period of four days and cultivation in fresh culture medium for two days three times. The bacterial species were labelled as S,M,H,G,F and W. Detailed studies indicated that supernatant of species H and J have no enzyme activity (Table 1).
They also do not utilize raffnose. Other four bacterial species have extracellular enzyme activity. Their rafinose utilization rate vaned from 11-28mgh (Table 1). In all cases, the enzymes showed high activity at neutral pH. The lowest wet weight and highest activity of llmU/inl were observed in Citrohacter freuPl7,dii.
The three species which showed high production of extracellular enzyme were identified as E. coli, Citrnhacter freun.dii an.d Klebsiella p1rcumon.iae (Table 2).
According to the results obtained from three bacterial species grown in the culture medium containing ammonium sulphate, Citrobacter freulrdii gave the highest enzyme production of 2.5 mU/ml after 12h of cultivation when compared with highest activity of 1.2 mU/ml for E.coli and Klebsiella pn,er~nton,iae at 6h and 12h of cultivation respectively (Table 3); Inorganic nitrogen has led to poor cell growth and low alpha galactosidase production when grown in culture medium with peptone as the nitrogen source. With different initial pH, again higher enzyme production of 14.7 mU/ml was observed with C. freun.dii after 18h of cultivation at pH 8.0 (Figure 1). The enzyme production could be further increased up to 19mU/ml after 36h when grown in pH 8 phosphate buffer ( Figure 2). As shown in the graph, the pH of the medium does not change significantly with time as observed under other culture conditions.

DISCUSSION
In this study, initial pH of the medium was adjusted to 7 because most bacteria grow well at. neutral pH values and also earlier reports showed that bacterial alpha galactosidases are more stable at neutral pH values.I3 Raffinose was used as the inducing sugar in the medium. The results show that C. fi-eun.dii grown in pH 8 phosphate buffer culture medium containing peptone as the nitrogen source is suitable for the production of extracellular alpha galactosidase. In future studies the alpha galactosidase from C. freundii will be purified and characterized to determine its suitability as an industrial enzyme for removal of raffinose sugars from food products.