FEMS Microbiology Letters 13.5 (1996) 287-293

Purification and regulation of the synthesis of a from Aspergillus nidulans

Sudeep Kumar, Daniel Ran-h *

P_xylosidase

Departmemo de Biotecnologpiu de Alimentos, Institute de Agroquimica y Tecnologia de Alimentos, Consejo Superior de Irwestigaciones Cientifcas, Apartado de Correos 73, 46100 Burjassot, Valencia, Spain

Received 3 November 1995; revised 17 November 1995; accepted 17 November 1995

Abstract

PXylosidase (EC 3.2.1.37) has been purified from Aspergillus nidulans mycelium grown on oat-spelt xylan as sole carbon source. Its pH optimum for activity was found to be 5.0 and the optimum temperature was 50°C. Its molecular mass

was estimated by gel filtration to be 180000. Using p-nitrophenyl-P-D-xylopyranoside as substrate, the K, and V,,,,, values have been found to be 1 .l mM and 25.6 wrnol min- ’ (mg protein)- ‘, respectivdy. Enzyme activity was inhibited by Hg’+, Ag2+, and CL?+ at a concentration of 1 X lop3 M. The synthesis of /3-xylosidase in A. nidulans is strongly induced by arabinose and xylose and is subject to carbon catabolite repression mediated by the creA gene product.

Keywrds: Aspergillus nidulans; P-Xylosidase; Purification: Carbon catabolite repression: CREA

1. Introduction

Xylan is the major constituent of wood and agri- cultural residues. In nature, the complete degradation of xylan requires the synergistic action of several

enzymes, mainly endo /3- l+xylanases (EC 3.2.1.8) which cleave the p-1,4 glycosidic bond between two different xylose residues to produce xylooligosaccha- rides, and Pxylosidase (EC 3.2.1.37) which cleaves

xylooligosaccharides to yield xylose [l]. The hydrol- ysis of xylan is of great interest for various biotech- nological applications [2].

Filamentous fungi are well known producers of xylan degrading enzymes. Several species of the

* Corresponding author. Tel: + 34 (6) 390 0022; Fax: + 34 (6)

363 6301: E-mail: dramon_iata.csic.es.

genus Aspergillus are efficient producers of such

enzymes and of these A. nidulans has become a model system for studying the mechanisms of con-

trol of gene expression in filamentous fungi [3]. Recently the xylanolytic complex of A. nidtlilans has been identified [4]. In the presence of xylan as sole carbon source A. nidulans secretes at least three

endo-l&3-xylanases with molecular masas of 22, 24, and 34 kDa, named respectively X,, , X 24 and X,. The three proteins have been purified and char-

acterised [5-71. X,, is a nc4&al xylanrtse while the other two xylanases = acidic pnMcins. Thd synthe- sis of the xylanolytic complex of A. n&lans is induced by xylan and xylooIigosaccharitW such as xylobiose, xylotriose and xylotetraose and tiepressed by glucose. This carbon catabolite repressi~ is ap- parently mediated by the CREA protein [8] and only affects the X,, and X,, xylanases [9].

0378-1097/96/$12.00 0 1996 Federation of European Microbiological Societies. All rights reserved

SSDI 0378-1097(95)00468-E

288 S. Kumar, D. Ram& / FEh4S Microbiology Letters 135 (19961 287-293

Previously, an extracellular /I-xylosidase was pu- rified from the culture supematants of a natural

isolate of Emericellu niduluns [lo]. In this paper we report the purification and biochemical characteriza-

tion of a mycelial bound P-xylosidase from A.

niduluns exhibiting different properties to the en-

zyme isolated from E. niduluns as well as the induc-

tion and carbon catabolite repression of the synthesis of the A. niduluns enzyme.

fractions were estimated by measuring absorbance at 280 nm.

2.5. Purification

2. Materials and methods

2.1. Strains

AspergiZZus niduluns CECT2544 was obtained from the Spanish Type Culture Collection and main-

tained on complete agar medium [ 1 I]. The strain

creAd30 [12] was a generous gift of Prof. H.N. Arst.

2.2. Culture conditions

Culture conditions for enzyme purification were

followed as described earlier [7]. The regulation of P-xylosidase production was investigated in replace- ment cultures as described previously [9]. At differ- ent intervals, 5 ml culure aliquots were withdrawn,

filtered and the mycelium used for analysing p- xylosidase activity.

2.3. Enzyme assays

Reaction mixtures containing 250 ~1 of 2 mM

p-nitrophenyl-P-r+xylopyranoside (pNpX) in water, 250 ~1 of suitably diluted enzyme or mycelia in sodium acetate buffer (50 mM pH 5.0) were incu- bated at 50°C for 30 min. The reaction was stopped by adding 1 ml of 2 M sodium carbonate solution and the release of p-nitrophenol was measured at 400 nm. One unit of P-xylosidase activity was de- fined as the amount of enzyme which liberates 1 pmol of p-nitrophenol/min/ml or g of mycelia under the conditions described above.

Mycelium from a xylan grown culture was ex-

tracted twice with saline phosphate buffer containing 0.05% (w/v) Triton X100. The mycelial extract was concentrated in a Minitan Ultrafiltration system (Mil-

lipore Corp., Beldford, MA) using a 10000 molecu-

lar mass cut-off polysulfone filter. The concentrate was loaded onto a 2.5 X 30 cm Q-Sepharose column equilibrated with 50 mM sodium acetate buffer, pH

3.5. Unbound proteins were eluted with equilibrating buffer and bound proteins were eluted with a linear gradient up to 0.5 M NaCl in sodium acetate buffer. The flow rate was 30 ml/h and 5 ml fractions were

collected. Fractions showing @xylosidase activity were pooled, dialysed, concentrated and then applied

to a Mono-Q column (Pharmacia, Uppsala, Sweden) equilibrated with 10 mM piperazine buffer, pH 4.5. Fractions showing P-xylosidase activity were eluted

with a linear gradient up to 0.5 M NaCl. The flow rate was 30 ml/h and 2 ml fractions were collected. Finally the Pxylosidase activity recovered was ap-

plied to a Superdex 200 HR lo/30 molecular gel filtration prepacked column (Pharmacia, Uppsala,

Sweden) equilibrated with 10 mM piperazine buffer, pH 5.5. The flow rate was 30 ml/h and 2 ml

fractions were collected. The molecular mass of the protein was estimated by the gel filtration chro- matography described above using molecular mass standards.

2.6. Electrophoresis and isoelectric focussing

SDS polyacrylamide gel electrophoresis was per- formed according to Smith [14] using an acrylamide concentration of 10% (w/v). The gel was stained with silver as described by Merril et al. [ 151. The low molecular mass calibration mixture from Pharmacia was used as standard. Analytical isoelectric fo- cussing was performed as described previously [7].

2.4. Protein estimation 2.7. Determination of kinetic constants

Protein content was estimated by the method of Bradford [ 131 using bovine serum albumin (fraction V) as standard. Protein content in chromatography

The Michaelis-Menten constants were determined by non-linear regression, using pNpX at concentra- tions ranging from 0.2 to 3.0 mM.

S. Kumar, D. Ram& / FEMS Microbiology Letters 135 f IYY61 287-293 289

2.8. Temperature and pH relationships

The optimum temperature for P-xylosidase activ- ity was determined by incubating the enzyme prepa- ration with pNpX at different temperatures ranging

from 30 to 60°C. Thermal stability was assessed by

incubating the enzyme preparation at 50, 55 and 60°C and assaying activity at various time points using the standard assay.

The pH optimum for P-xylosidase activity was determined by incubating the enzyme preparation with pNpX in Teorell and Stenhagen universal buffer

[ 161 at various pH values between 3.5 and 9.0 at 50°C. pH stability was assessed by incubating the

enzyme at different pH at 25°C and measuring activ- ity at various times using the standard protocol.

2.9. l$hect.s of carious reagents

An appropriate amount of enzyme was mixed

with 50 mM sodium acetate buffer (pH 5.5) contain- ing 1 mM of various reagents. The enzyme activity was measured as described earlier.

2.10. Hydrolysis experiments

End products of the hydrolysis of xylobiose, xy- lotriose and xylotetraose (Megazyme, Sidney, Aus- tralia) were determined by HPLC on a Sugar Pack

column (Waters Associates, Milford, MA) equili-

brated and eluted with Milli-Q water. Peaks were detected by differential refractometry and identified

by comparing elution times to those of appropriate standards.

2. Il. Antibodies and Western blotting

Antibodies against the /3-xylosidase of A. nidu-

lam were raised in rabbit. Cross-reactivity between

Table I Purification of P-xylosidase from A. nidulans

Step

Crude extract

Q-Sepharose

Mono-Q

Superdex-200

Total activity Total protein Specific activity

(U/ml) (mg) KJ/mg)

44.2 36.5 1.2

22.3 2.9 7.6 20.0 0.6 34.1 12.0 0.1 107.1

the antisera and the enzyme was tested by western

blotting as described previously [7]. @Xylosidase was extracted from the mycelia of the xylan grown cultures of A. niger, A. terreus and A. nidulans

with saline phosphate buffer containing 0.05% (w/v>

Triton X- 100.

3. Results and discussion

3. I. Pur$cation

Unlike the situation reported in Emericella nidu-

lans [lo], no extracellular /3-xylosidase activity was detected when A. nidulans was grown on xylan as

sole carbon source. All P-xylosidase activity was found to be mycelium bound. The details of the

purification of /3-xylosidase, following the protocol described in Materials and methods, are presented in

Table 1. Most of the protein content in the crude extract was eluted from Q-sepharose with the equili-

brating buffer. P-Xylosidase activity was eluted at 180-200 mM NaCl (Fig. 1). Fractions showing p-

xylosidase activity were concentrated, dialysed and

loaded onto a Mono-Q ion exchange column. p- Xylosidase activity was eluted by 200 mM NaCl. P-Xylosidase was further purified using a Superdex- 200 gel filtration column. The biochemical character- ization and substrate hydrolysis pattern studies re- ported below were performed with this purified /3-

xylosidase preparation, stored at - 70°C.

3.2. Biochemical characterization

Purified P-xylosidase showed a single sllver- stained protein band on SDS-PAGE. corresponding

to a molecular mass of 85000 (result not shown). Gel filtration column chromatography indicated that the molecular mass of native P-xylosidase was

Recovery

(%)

loo

50

45.2

27.2

Fold purification

I 6.3

28.4

89.2

290 S. Kumur, D. Ramo’n/ FEMS Microbiology Letters 13.5 (19%) 287-293

- 0.00 0 20 40 60 80 100

Fraction Number

Fig. 1. Elution pattern of S-xylosidase by Q-Sepharose column

chromatography. Experimental details are described in the text.

Solid line, absorbance at 280 nm; (A ) Pxylosidase activity.

180 000 suggesting that the native enzyme may be dimeric in nature. The estimated molecular mass of

the A. niduluns &xylosidase is similar to those of

P-xylosidase reported from other fungi [ 10,17,18]. The dimeric nature of Pxylosidase has also been

reported in E. niduluns [lo], Aspergillus niger [19], Penicillium wortmanni [20] and Chaetomium trilate-

tute [21].

Analytical IEF data showed the P-xylosidase to be an acidic protein with an isoelectric point of approximately 3.4. Similar isoelectric points have

been observed for the P-xylosidase of E. niduluns

[ 101 and P-xylosidase II of Aspergillus puluerulen-

tus [lS], although the p1 of A. niger P-xylosidase has been reported to be 4.9 [19].

The pH optimum of the purified P-xylosidase was found to be 5.0. This is the same as that

observed for E. niduluns [lo] and P-xylosidase II of A. puluerulentus [ 181 but different to that of A. niger

which has been reported to be 4.0 [19,22]. The

enzyme is stable over the pH range 4.0 to 6.0 for up to 8 h at room temperature. Stability of @xylosidase was also reported in A. niger [19,22].

The optimum temperature for the purified P_ xylosidase activity at pH 5.0 was found to be 50°C. This was rather lower than the optimum temperature for the A. niger /3-xylosidase [ 191 but very similar to that of E. niduluns [lo]. The enzyme was quite unstable above 45°C. At lower temperatures (40- 45°C) the enzyme was stable up to 8 h, while at the

optimum temperature (50°C) the enzyme became inactivated within 4 to 6 h. In an experiment to

examine thermostability it was observed that over a period of 30 min the enzyme lost all its activity at

60°C but at 55°C it retained 50% of its original activity.

3.3. Kinetic parameters

The Michaelis constants determined on pNpX were different to those found earlier in E. nidulans

[lo]. The calculated K, and V,,, values of p- xylosidase were found to be 1.1 mM and 25.6

~mol/min/mg of protein, respectively. The lower K, value of Pxylosidase from A. nidulans shows

that it has an higher substrate affinity than that of E.

niduluns [ 1 O] but less than that of A. niger [ 191.

3.4. Effects of various reagents on P-qlosidase

actiuity

The effects of various metallic ions and reagents on the activity of purified &xylosidase were investi- gated. As shown in Table 2, activity was dramati- cally inhibited by Cu*+, Ag*+ and Hg*+ and also

some inhibition was observed in the presence of

Zn*+ and Fe*+. Some inhibition was also observed in the presence of SDS. The enzymatic activity was

Table 2

Effects of various reagents (at 1 mM) on the enzyme activity of

purified Pxylosidase of A. nidulans

Reagents Relative activity (o/o)

Control 100

CoCl 2 108& 4

AgW I+ 1 CUCI, 9* 1 ZnCl 2 50+ I

MgCl2 114+ 7

I&Cl, 7* 1 CaClz 111+ 8

CdCI? 96k 5

FeCl, 47* 3

EDTA 126k 7

Cysteine 130* 9

DTT 125+ 9

SDS 71* 10

The results presented are the average (+ standard deviation) of

three different experiments.

4. Kumar, D. Ram& / FEMS Microbiology Leiters 135 (1996) 287-293 291

0 hours 16 hours

r

x1

II

slightly stimulated in the presence of Co*+, Mg’+ and Ca*+. In the presence of DTT, EDTA and

cysteine /3-xylosidase activity was incresed by ap- proximately 30%. In comparison to the enzyme from

E. nidulans [lo], the A. nidulans P-xylosidase shows some resistance towards SDS but high sensitivity to

cu’+.

The effects of D and L forms of xylose were also studied at two different concentrations (25 and 100

mM1. /?-Xylosidase activity was not affected by L-xylose even at 100 mM concentration but inhibi- tion was very clear in the presence of D-xylose at 25

mM (44% inhibition).

3.5. Hydrolysis patterns

The hydrolysis patterns of xylobiose, xylotriose

and xylotetraose with the purified P-xylosidase are

shown in Fig. 2. With xylobiose as substrate, the enzyme effected 90% degradation to xylose within 6

to 9 h while with xylotriose hydrolysis took longer

and the enzyme only effected 70 to 80% degradation after 16 h. When xylotetraose was used as a substrate the hydrolysis was much slower and only 20 to 30% of the substrate was converted to xylose after 16 h. This result indicates that the best substrates for p-

xylosidase are short oligomers of xylose and that as chain length increases the hydrolysis becomes pro- gressively less efficient. No hydrolysis products were

identified with oat, spelt or birch wood xylans (re-

sults not shown). Similar results have been obtained with the /3-xylosidases from Trichoderma r:iride [ 171

and E. nidulans [lo]. In all these cases, the rate of

hydrolysis of xylooligosaccharides decreased with increasing chain length.

3.6. Western blotting

Antibody raised against the purified P-xylosidase from A. nidulans was used to try to detect similar

Fig. 2. Time course of enzymatic hydrolysis of (A) xylobiose, (B)

xylotriose and (C) xylotetraose. Different substrates (1.5 mg/ml

final concentration) were incubated with 100 ~1 of purified

P-xylosidase in 50 mM sodium acetate buffer, pH 5.0 at 50°C. At

0 h and 16 h 100 ~1 aliquots were withdrawn and used for

analysing the end products. Peaks denote: Xl, xylose; X2, xylo-

biose; X3, xylotriose; and X4, xylotetraose.

292 S. Kumar, D. Ram& / FEMS Microbiology Letters 135 (1996) 287-293

Pxylosidases in other Aspergillus species. Enzy-

matic assays had shown the presence of pxylosi- dase activities in A. niger and A. terreus, however

no @-xylosidase bands were detected in A. niger

and A. terreus by western blotting using the A.

niduluns antibody (results not shown). This indicates

that the P-xylosidases present in the other two species of Aspergihs are immunologically different to the

P-xylosidase purified from A. niduhns.

3.7. Induction and carbon catabolite repression of

P-xylosidase

Using culture replacement, the influences of dif-

ferent carbon compounds on P-xylosidase produc-

‘#Ad-type

0

I 1 I

6 12 16 24

Time ( h)

0 6 12 18 24

Time (h)

Fig. 3. Time course of Pxylosidase production during incubation

of washed, fructose grown mycelia of the A. nidufans wild-type strain and creAd30 mutant incubated in minimal medium contain-

ing 1% (w/v) xylan (A) or 1% (w/v) xylan plus 1% (w/v)

glucose ( W 1.

tion were followed in the A. nidulans wild-type

strain. The enzymatic activity was found to be asso- ciated with the mycelium and no activity was de-

tected in culture supematants. The greatest levels of P-xylosidase production were observed in the pres-

ence of arabinose (0.35 U/mg of mycelium) and

xylose (0.31 U/mg of mycelium). The inducing abilities of xylan (0.22 U/mg of mycelium) and

arabitol (0.17 U/mg of mycelium) were less than those of arabinose and xylose. Xylitol acts as non-in-

ducing carbon source.

In order to study carbon catabolite repression, transfer experiments were carried out with both wild-type and the creAd30 mutant strain of A. nidu-

lam. Cultures were transferred to xylan or xylan plus

glucose. As seen in Fig. 3, the presence of glucose represses the synthesis of P-xylosidase in the wild-

type strain but in the case of the creAd30 mutant this repression was partially overcome and the synthesis of &xylosidase started at around 6 h after transfer.

This result indicates that the synthesis of p-xylosi- dase in A. niduluns is carbon catabolite repressible, mediated, at least in part, by CREA. The carbon

catabolite repression effect is similar to that observed on xylanase synthesis in A. nidulans 191.

The P-xylosidase purified from A. niduluns in the present investigation appears to be different to

that previously isolated from E. niduhs [ 101 not only in its cellular location but also in its kinetic

characteristics.

Acknowledgements

This work has been supported by grants from D.G. XII of the European Commission (BIOTECH BI02-CT93-0174) and the Comisi6n Interministerial de Ciencia y Tecnologia of the Spanish Government (ALI93-0809). S.K. is the recipient of a Fellowship

of the Ministerio de Education y Ciencia. The A. niduluns creAd30 strain was provided by Dr. H.N. Arst. Thanks are due to Dr A.P. MacCabe for critical reading of the manuscript.

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