Indian Journal of Biochemistry & Biophysics Vol. 38, June 200 I, pp. 180- 185

Purification and characterization of an invertase produced by Aspergillus ochraceus TS

Kajari Ghosh, Alok Dhar and Timir B Samanla * Departmcnt of Microbiology, Bose Institute, P- 11I 2, CIT Scheme VII -M, Calcutta 700 054, India

Received 10 Febrt/w)' 2000; revised 7 Jllly 2000; accepTed 29 AugusT 2000

Purification and characteri za ti on of an extracellular invertase produced by Aspergilllls ochmcells TS are reportcd. Thc cnzyme was purified (42-fold) from culture filtrate by sal t precipitation, ion-exchange and gcl filtration. Sodiu m dodccy l sulphate polyacrylamide gel electrophoresis (S DS-PAGE) of the purified enzymc showed a single band of molecu lar mass 66 kDa. The molecular mass of the native enzy me was found to bc 130 kDa by gel filtration. The purity of the pmtein was also checked agai nst its anti scrum raiscd in rabbits by two-d imensional immunod iffusion in agarose gel and Wcstern blot that sholVcd a si ngle band. It is a glycoprotein wi th mannosc as its carbohydrate residuc. Thc cnzyme showed high affinity for sucrose wi th a Km of 3.5 mM. The amino acid ana lys is revca lcd a hi gh proportion of ac idic residues but it had a low contcnt of cysteine, hi stidine and arginine comparable to other fungal in vertases.

Interest in the sucrose hydrolytic enzyme also known as ~-D-fructofuranoside fructohydrolase (EC 3.2. 1.26), has appreciably increased in recent times due to its various biotechnological applications. The enzyme is believed to split the terminal ~-(2-1) fructoside linkages of small er oligosaccharides such as sucrose, raffinose and stachyose.

M· . . b . I ? f ·3-8 d 9 Icroorganl sms VIZ. actena '-, ungl an yeasts are reportea to produce both intracellular and ex tracellular invertases . The invert sugar produced by yeasts gets further metaboli zed to ethanoL The conversion of monosaccharides (invert sugar) to ethanol by yeasts is very difficult to controL In contrast, the extracellular in vertase produced by fungi is free from contaminat ion with other enzymes and it prevents conversion of invert sugar to ethanoL The funga l invertases are more advantageous than those produced by yeast for preparation of high fructose syrup. While the yeast invertases are well defined, the invertase produced by fungi is least studied . The invertase produced by Aspergillus ochraceus TS (A . ochraceus TS) was purified with a view to develop it as a model to investigate the biosynthesis and secretion of this enzyme in fungi.

Materials and Methods Materials

Raffinose and standard marker proteins were

*Author for correspondence Phone: 33-337-94 16/92 I 9/9544; Fax: 33-334-3886 E.mail : timir@boseinSLerneLin

obtained from Sigma, USA while ac rylamide and sucrose were suppl ied by Spectrochem, India. DE-52 cell ulose, Sepharose 48 and Sepharose 68 were obtained from Whatman, USA and Pharmacia, Sweden respecti vel y.

Methods A. ochraceus TS, a mesophilic fungus iso lated by

Samanta et al. 1O, was grow n in sterile liquid medium

[composition %, w/v : sucrose (1) , cornsteep liquor (0. 1), K2HPO~ (0.05) pH 7.0] on a rotary shaker at 30°C for 48 hr.

In vertase assay Invertase was assayed at 40°C using sucrose as

substrate in 50 mM acetate buffer, pH 5.0 following the method of Millerll. The reaction mixture containing sucrose ( I % w/v) and enzyme (20 mg protein) taken in acetate buffer (0.05 M, pH 5.0) was incubated at 40°C for 30 min. The reaction was terminated by adding dinitrosalicylic ac id reagent (1 ml). It was then heated on boiling water bath for 10 min and then cooled to room temperature. Absorbance of the colour developed was measured at 540 nm. One unit of enzyme act ivity is defined as the amount of protein which produces one )..l mole of glucose per min .

Protein estimation Protein was estimated by the method of Lowry'2.

..

GHOSH el of: PURIFICATION OF INVERTASE PRODUCED BY ASPERGILLUS OCHRACEUSTS 181

Purification of in vertase Unless otherwise stated, all procedures during

purification of invertase were done at 4°C. The culture filtrate (48 hr old) was centrifuged at 22,000 g for 20 min . The supernatant was dialysed against acetate buffer (0.05 M, pH 5.0) overnight.

Fractionation with (NH4h S04 Solid (NH4hS04 was added to the dialysate to

attain 60-80% saturation. The mixture was stirred for 30 min and the precipitated protein was centrifuged at 22,000 g for 20 min. The pellet (22,000 g) was suspended in acetate buffer (0.05 M, pH 5.0) and was dialysed against the same buffer (0.01 M, pH 5.0). The dialysate was treated with acetone (I :5, v/v) and again centrifuged at 22,000 g.

Chromatography on DE-52 cellulose The protein (250 mg) obtained by solvent

precipitation was applied to a DE-52 cellulose column (35 x 1.6 cm) equilibrated with acetate buffer (0.05 M, pH 5.0). It was eluted with a gradient of NaCI (0-0.5 M) in same buffer. Fractions (2.0 ml each) were collected and assayed for invertase activity.

Gel filtration on Sepharose 4B column The pooled fractions (\42-160) eluted from the

ion-exchange column were lyophilized. The concentrated protein (24.5 mg) after dialysis (acetate buffer 0.05 M, pH 5.0) was applied to a Sepharose 4B column (60 x 0.8 cm), which was equilibrated with acetate buffer (0.05 M, pH 5.0). The enzyme was eluted at a flow rate of7.5 ml Ihr.

Determination of molecular weight The molecular weight of the enzyme was

determined by gel filtration on Sepharose 6B column. Standard proteins, alcohol dehydrogenase (M, ISO kDa), phosphorylase b (M, 92 kDa) and carbonic anhydrase (M, 29 kDa) were used to calibrate the column. Fractions (0.5 ml) were collected and assayed. A standard curve of peak elution volume (Ye) against M, was constructed.

SDS-PAGE of the purified invertase was done by the method of Laemmli 13. After electrophoresis, the gels were stained with Schiff's reagent l4 and silver nitrate lS separately.

Determination of carbohydrate content The carbohydrate content of the enzyme was

determined, by phenol-H2S04 method l6. The purified protein (1-2 mg) taken in sodium citrate (0.1 M, pH 5.5, I ml) was incubated with endoglycosidase (0.5

unit) at 37°C for varying time intervals. The reaction mixture was precipitated with an equal volume of cold TCA (12.5% w/v). The supernatant was assayed for total neutral sugar by phenol-sulphuric ac id method with mannose as standard. The aliquots taken from endoglycosidase treated reaction mixture were resolved on silica gel G plate (TLC) using chloroform : acetic acid: water (60 : 70 : 10) as solvent system. The spot was developed by spraying the plate with a mixture of p-aminophenol, phosphoric acid and distilled alcohol followed by baking at !OO°C for 30 min. The spots developed were compared with authentic samples of glucose, fructose, galactose and man nose.

Determination of pI of purified protein The purified protein (2 mg) was applied to a

chromatofocussing column (PBE 94, Pharmacia, Sweden) (10 x 0.8 cm) equilibrated with imidazole­HCL buffer (0.025 M, pH 7.4). After initial wash with the buffer (20 ml), the protein was eluted with polybuffer (polybuffer 74 diluted I :8, pH 4.0) at a flow rate of 30 ml/hr at 30°C. Polybuffer 74, a unique buffering substance available from Pharmacia, Sweden for use with poly buffer exchangers for chromatofocussing,was used after dilution (1 :8) as elution buffer for the purified invertase in a gradient of pH 7-4. Fractions (1 ml) were assayed using sucrose as substrate. The pH of the fractions was measured throughout the elution.

Amino acid analysis of the purified invertase The purified protein was hydrolysed for 1 h at

150°C with 6N HC\. The hydrolysate was derivatised with phenylisothiocynate (PITC). PITC derivatives were analysed on a reverse phase column by high pressure liquid chromatography 17 (HPLC) , (PICO­TAG System Operator Manual, Part Number 88140).

Antibody preparation Samples containing 100 mg of pure protein were

injected into a rabbit every 15 days. After the fourth injection, the rabbit was bled out and the serum was checked for immunoprecipitation with homologous antigen. For this 50 ml sample of the serum was tested by two-dimensional diffusion in agarose gels in barbitol acetate buffer pH 8.2 by the procedure recommended by Ouchterlonyl 8.

Immunoblotting Immunochemical detection of the antigen on

nitrocellulose paper was carried out by the method of

182 INDIAN J. BIOCI-IEM. BIOPI-IYS., VOL. 38, JUN E 200 1

Towbin ef.Crll9

. with minor modifications20. Immuno­

blotting was done in three major steps i.e. SDS-PAGE of the test sample, electroblott ing and immuno­staining.

EI ect roblott ing The electrophoresed gel was equilibrated with the

blotting buffer [25 mM Tris, 192 mM glycine and 20% (v/v) methanol pH 8.3] for about 10 min. Next the gel was transferred to nitrocellulose paper by a transblot apparatus . The nitrocellulose strips were then washed with buffer (10 mM Tris HCI, pH 7.4) prior to immunostaining. The blotted strips contain ing marker proteins were stained wi th amidoblack (0.1% v/v) .

IlIlInullostain ing Th~b lotted str ips were treated wi th a solution of

BSA (3 %) in Tris-HCI buffer (10 mM, pH 7.4) over­night at 4°C to block the unbound sites. Strips were then washed thrice with PBS (0.15 M, pH 7.4) con­taining Tween 20 (0.05% v/v) followed by incubation for 2 hr at room temperature with appropriate di lu­tions of antiserum in PBS pH 7.4 containing 0.5% BSA. Then these strips were treated with peroxidase labelled goat anti -rabbit IgG (di luted to 1: 10,000 with PBS pH 7.4 containing 0.5% BSA) for 1 hI' at room temperature. Fina lly, the blotted strips were washed and developed by treatment with the substrate solu­tion wh ich contained diaminobenzidine hydrochloride (1 mg/ml) in Tris HCI buffer (10 mM, pH 7.4) con­taining 200 mM NaCI and hydrogen peroxide (30% V/V)1 9.20.

Results and Discussion Purification

Results of the purification of invertase produced by A. ochraceus TS are presented in Table I. About 10-fold increase in specific activity of the enzyme was achieved by DE-52 cellulose chromatography. Out of two peaks, the pooled fract ions (fraction 142-160) of

peak B were further purified 42-fold by gel filtration through a Sepharose 4B column . Peak B showed higher invertase activity than peak A and it was separated for further purification. The purified enzyme showed a single band on SDS-PAGE (Fig. 1). The purity of the enzyme was also checked with polyclonal antibody. It showed immunoprecipitation bands when purified invertase was used as antigen (Fig.2). Moreover, it exhibited a single band in Western blot (Fig. 3).

Physical characteristics The molecular mass of the native protein was

found to be l30 kDa by gel fi ltration (Fig.4). Invertase appeared to be a homodimcr because it showed a si ngle band of - Mr 66 kDa on SDS-PAGE. Thus, the invertase produced by A. orchraceus TS

a b c d kDa

-.. -66

-45 -29

- 14

Fig. I - SDS-PAGE of purifi ed extracellular invertase produced by A. ochracells TS. [Lane a, band d : 2-4 I1lg of purified pro­teins. Lane e : Marker proteins BSA (66 kDa), ovalbumi n (45 kDa), carbonic anhydrase (29 kDa), lysozyme (1 4 kDa)]

Table I - Purification of invertase produced by Aspergillus ochracells TS

Steps Protein Total activity Sp. act ivity Purification fold Yield

(l1lg) (U) (U/l1lg) (%)

Culture filtrate 750 4650 6.2 100

(NI-I4)2S04 saturation (60-80%) 300 2130 7.1 1.1 5 45.80

Acetone prec ipitation 250 2050 8.2 1.32 44.08

DE-52 24.5 1574 64.25 10.36 33.9

Sepharose 4B 5 1302 260.4 42 28

GHOSH el al: PURIFICATION OF INVERTASE PRODUCED BY ASPERGILLUS OCHRACEUSTS 183

(a) (b) Fig.2 - Reaction bctwcen (a) : Serial dilution or puriried protein and serum raised agai nst extracellular invertase rrom A. oehraeells TS. (b) : Serial dilution or sera and purified extracellular invertase rrom A. ochraceus TS in agarose gel

Fig.3 - Western blot profile of purified invertase. rLane (n), Experimental; Lane (b), Control (Pre-immune serum)]

seems to be larger in size than that obtained from Aspergillus aWQl1Iori and Aspergillus ficcum4

. The pi of the purified enzyme was 4.4. The invertase showed specificity towards sucrose and raffinose because it did not hydrolyse ma ltose, cellobiose and lactose indicati ng its specificity towards a ~ (2- 1) glycosidic linkage (Table 2). It is a g lycoprotein with mannose as its carbohydrate residue. In fact, 30% of the total

mass of the enzyme comprised of neutral sugar.

160 150 140

120

.., 0

)(

~ 100

~ ... ..

80 3 u .. '0 60 ~

40 C

20

0 2 2.5 3 3.5 4

V./Vo

Fig.4 - Molecular weight determination or purified in vertase by Sepharose 6B column. fA, alcohol dehydrogenase (150 kDa); B, bovine serum albumin (66 kDa); C, carbonic anhydrase (29 kDa); . , invertase produced by A. oehmeells TS]

Table 2 - Substrate specificity of inve rtase produced by A. oci1rncclIs TS

Substrate

Sucrose Maltose Lactose Cellobiose Raffinose Stachyose Inulin

Enzyme activity

!.l mole/min/mg protein

260.00 o o o

250.02 26.0 2.6

184 INDIAN J. BIOCHEM . BIOPHYS., VOL. 38, JUNE 2001

Table 3 - Physicochcmical properties of in vertase produced by A. oehmells TS

Property

Mol. mass (k Da) (By gel filtration Sephadex 6B)

Content of ca rbohyd rate,

% pl

pH opti mum

pH stab ility

Temperature optimum

Hea t stability

Inhibitors

1.0

C 'e -0 E

8

6

~

3- 2 I~ 0 .8

> 0 c

E

0 0.6 E 2-< 0.4

} l/V m a x

-0.4 - 0·2 0 0.2 ~

-(l /K m )

0

Value

130

30

4.4

5.0

4 - 8

40° C

30 - 55°C

Hg2., Cd-acetate, p-ch I oromercll ri benzoa te, N-ethy Ima le i m ide

----' 20 .40 60

Sucrose (mM )

0·4 0.6 0.8 1.0

,j~ucros~ (mM)l

Fig.S - Lineweaver - Burk plot of purified invertase produced by A. oehmcells TS

Invertase produced by S. pombi l and N. crossa22 are reported to contain galactose and hexosamine as carbohydrate moiety respecti vely . I n the present study, the purified enzyme did not exhibit fructosyl transferase activity since incubation of the enzyme with sucrose or raffinose did not reveal the presence of fructose dimer on thin layer chromatographic plates (data not shown) .

In vertase produced by A. ochraceus TS was fou nd to be thermotolerant. Its optimal temperature in vitro was 40°C. It did not lose much activity even when incubated at 55°C fo r 30 min contrary to earl ier reports (Table 3)1.9. It had a broad pH range, which varied from 4-8. The optimal pH was 5.0 (Table 3). The purified enzyme was insensitive to the metals like

Table 4 - Amino acid composition of invertase produced by Aspergilills mvalllori and Aspergilills

oehmcells TS

Amino acid Composition (%)

A. IIlvalnori A. ocilracells TS

Asp 11.11 9.08

Thr 8.50 1.34

Ser 9.50 3.54

GIll 8.50 16.74

Pro 5 2.71

Gly 10.5 15.69

Ala 9 9.17

Cys 0.5 0.33

Va l 5.5 8.63

Met 1 ND

TIe 3.5 3.92

Leu 7 8.38

Tyr 5.5 2.16

Phe 5 2.21

Lys 4 12.32

His 2.5 2.34

Arg 2.5 1.40

ND : Not d etected

Mg2+, Zn2+, Ca2+ but it was activated (47%) by Mn2+. The enzyme was totally inhibited by HgCI2 (lmM). Sulphydryl reagents VIZ., Cd-acetate, p-chloro­mercuribenzoate (PCMB) and N-ethylmaleimide (NEM) each at 10 mM concentration caused 50, 90 and 100% inhibition respectively indicating the presence of -SH group at its active site (Table 3). Kinetic characterist ics

Kill and Vlllax of the purified enzyme calculated from Line weaver-Burk plot for sucrose and raffinose were 3.5 and 4.5 mM and 7.1 and 10.0 mmoles per min respectively (Fig.S) . The amino acid analysis of the enzyme revealed the presence of aspartic acid , glutamic acid, alanine, glycine and valine in hi gh proportion. The levels of cysteine, hi stidine and arginine were qu ite low but it had a higher level of lysine than the invertase produced by S. cerevisiae. Invertase produced by A. awamoris had been reported to have low level of cysteine, histidine and arginine (Table 4).

GHOSH el a/ : PURIFICATION OF INV ERTASE PRODUCED BY ASPERGILLUS OCHRACEUS TS 185

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