Purification and characteristics of two exoinulinases from Chrysosporium pannorum

JOURNALOF FERMENTATIONAND BIOENGINEERING Vol. 67, No. 5, 331-334. 1989

Purification and Characteristics of Two Exoinulinases from Chrysosporium pannorum R O N G X I A O , M A S A T O S H I T A N I D A , § AND S H O I C H I T A K A O *

Department of Agricultural Chemistry, Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan Received 27 December 1988/Accepted 15 February 1989 Two types of exoinulinase (2,1-]l-o-fructan frucianohydrolase EC 3.2.1.7) were purified from Chrysosporium pannorum AHU 9700. The enzymes, F2 and F3, were glyco-proteins having isoelectric points around pH 4.6 and 4.45. The molecular weights were estimated by SDS-polyacrylamide gel electrophoresis to be 84,000 and 70,000, respectively. The enzymes were active on inulin, sucrose, raflinose, stachyose, and fructo-oligosaccharides, but not on melezitose. Levan could be hydrolyzed by exoinulinase F3, but not by exoinulinase F2. The hydrolysis products of inulin by the two enzymes were fructose and small amounts of glucose.

Electrophoresis Polyacrylamide gel disc electrophoresis was performed with 7.5%o polyacrylamide gel according to the method described by Davis (12). lsoelectric focusing For determination of the pl value of the enzymes, gel disc isoelectric focusing (13) was carried out as previously described (11). Determination of molecular weight The molecular weights of purified exoinulinases were determined by SDSpolyacrylamide gel electrophoresis (14). Determination of protein The amount of protein was determined by the method of Lowry et al. (15) using bovine serum albumin as a standard, or by measuring absorbance at 280 nm. Determination of carbohydrate in the enzyme protein Total carbohydrate was determined by the phenol sulfuric acid method (16). Analysis of hydrolysis products The hydrolysis products of inulin and fructo-oligosaccharides by the two exoinulinase were analyzed by high performance liquid chromatography (HPLC) according to the methods described previously (10).

Inulin-degrading enzymes have been purified from Jerusalem artichoke (1), dahlia (2), and dandelion (3). These plant inulinases displayed no invertase activity at all. Several microbial inulinases have also been purified and their properties investigated (4-9). In contrast to the plant inulinases, many of these purified microbial inulinases possess remarkable invertase activity. Most of the microbial inulinases described in the literature are exoenzymes. Their I/S (the activity on inulin versus sucrose) ratio ranged from 0.02 (7) to 1.95 (8). The exoinulinases split terminal fl (2 ~ 1) fructofuranosidic linkages of inulin and smaller oligosaccharides such as sucrose, raffinose, and stachyose. However, they are distinctly different from invertase (fl-D-fractofuranosidase EC 3.2.1.26) which has a high activity on sucrose and an extremely low activity on inulin. Recently, we reported that a mold strain of Chrysosporium pannorum A H U 9700 produced a very active inulinase (10), and described purification and some properties of an endoinulinase from this strain (11). In the latter paper, four peaks of protein having inulinase and invertase activity in different levels were obtained (11). In this paper, we describe the purification and characteristics of two exoinulinases from fraction 2 (F2) and fraction 3 (F3).

RESULTS Purification of enzymes Fraction 2 (F2) of DEAESepharose CL-6B column chromatography of the culture supernatant (Fig. 1) was concentrated and desalted by a Bin-Gel P-6 column (2.6 × 30 cm) equilibrated with 10 mM sodium acetate buffer (pH 4.0), and the protein fraction was applied to a CM-Sepharose CL-6B column (2.0× 12cm) equilibrated with same buffer, and eluted with a linear gradient of NaCI (0-0.2 M, 200 ml-200 ml). Three peaks (F2-a, F2-b, and F2-c) of protein having inulinase activity were obtained, and F2-b containing the largest part of activity was further purified. F2-b fractions were pooled and desalted by Bin-Gel P-6 (2.6× 30cm) equilibrated with 10 mM acetate buffer (pH 5.0) containing 20%o saturation of ammonium sulfate, and applied to a Butyl-Toyopearl M 650 column and eluted with a linear gradient of (NH4)2SO4 (20 tO 00//00,250 ml-250 ml). The result of Butyl-Toyopearl M 650 column chromatography is shown in Fig. 2. The active fractions of no. 35 to 42 were pooled and tentatively designated as inulinase F2.

MATERIALS AND METHODS Microorganisms C. pannorum A H U 9700 was used in this study. The organism was maintained on potato dextrose agar slants. Medium and cultivation The medium for enzyme production and the cultivation conditions were the same as those in the previous paper (11). Preparation of crude enzyme The method of preparation of the crude enzyme was the same as in the previous paper (11). Enzyme assay The activities of inulinase and invertase were assayed according to the methods described previously (11).

* Corresponding author. § Present address: Hokkaido Green-Bin Institute, Naganuma. 331

332

XIAO ET AL.

J. FERMENT. B I O E N G . ,

F1

F2 F3

F4

1°3 112 0102

i .......................

0

1.0

100

7s

-~ g

=E

o >

5O

o.5

o m

0

i

"2,

t~

25

25

50

75 I00 Fraction number(6 ml)

125

150

FIG. 1. DEAE-Sepharose CL-6B column chromatography of crude enzyme from Chrysosporium pannorum. 280 nm; e, activity on inulin; A, activity on sucrose.

Fraction 3 (F3) in Fig. 1 was concentrated and desalted by a Bio-Gel P-6 column (2.6 × 30 cm) equilibrated with 10 mM sodium acetate buffer (pH 4.2). The protein fraction was applied to a CM-Sepharose CL-6B column (2.0 x 12 cm) equilibrated with same buffer and eluted with a linear gradient of NaC1 (0-0.2 M, 300 ml-300 ml). The fractions containing inulinase activity were pooled and concentrated to about 3 ml, and applied on a Sephacryl S-200 column ( 2 . 6 × 9 0 c m ) equilibrated with 0.1 M sodium phosphate buffer, pH 6.0, and elution was made at a flow rate o f 10 ml/h. As shown in Fig. 3, a sharp and symmetrical protein peak having enzyme activity was obtained. The enzyme active fractions of no. 82 to 87 were collected,

Symbols: c , absorbance at

concentrated, and desalted through a Bio-Gel P-6 column equilibrated with 10mM acetate buffer (pH 5.6). This enzyme was designated as inulinase F3. The recovery of the activity of the two enzymes at each step is summarized in Table 1. The I/S values of inulinase F2 and F3 were 0.67 and 0.29, respectively. Homogeneity of purified inulinase F2 and F3 The purified enzymes showed a single protein band on polyacrylamide gel disc electrophoresis (Fig. 4). Molecular weight and carbohydrate content The molecular weights of the enzymes were determined by SDS polyacrylamide gel electrophoresis. The molecular weight

3

0.15

~o ~ 2010I ~

~

E to 4~

~

'7

E c-

v

0

o oo

z

0.I0

30 =

0.04

(D >

,~ 0.02

)

15

oJ

0.05 o

LLA

o <

0

I

20

a

!

I

40 60 80 Fraction number(5 m])

FIG. 2. Chromatography of F2-b on Butyl-Toyopearl M650 column. Symbols: o , absorbance at 280 nm; o, activity on inulin; a, activity on sucrose.

,

m

c~

60 Fraction

~

80

I

1 O0

number (3 ml)

FIG. 3. Chromatography of F3 on Sephacryl S-200 column. Symbols: ~;, absorbance at 280 nm; e, activity on inulin; ±, activity on sucrose.

EXOINULINASE FROM C H R Y S O S P O R 1 U M P A N N O R U M

VOL. 67, 1989 TABLE 1. Step

Protein (rag)

Supernatant (1000 ml)

Ammonium sulfate

Purification of inulinase F2 and F3 Total activity for inulin (U)

Specific activity (U/mg) I/S

Yield (o6)

7.1 4.8

1.23 1.50

100 50.8

for inulin

for sucrose

8.7 7.2

10927 5547

1254 766

333

fractionation

DEAE-Sepharose CL-6B F2 F3 F2 CM-Sepharose CL-6B Butyl-Toyopearl M650 F3 CM-Sepharose CL-6B Sephacryl S-200

11.8 35.7

458.4 813.9

38.7 22.7

51.8 30.1

0.75 0.76

4.2 7.5

6.8 4.2

239.4 174.3

35.2 41.5

48.0 62.3

0.73 0.67

2.2 1.6

12.7 8.2

149.8 86.1

11.8 10.5

34.1 36.2

0.35 0.29

1.4 0.8

I/S=Specific activity for inulin/Specific activity for sucrose.

of inulinase F2 was estimated to be 84,000, and that of inulinase F3 was estimated to be 70,000 (Fig. 5). The carbohydrate contents of inulinase F2 and F3 were about 7.3% and 1.4% respectively, determined by the phenol sulfuric acid method (glucose as a standard). lsoelectric point From the results of analytical isoelectric focusing of the enzymes on an Ampholine polyacrylamide gel, the isoelectric points (pI) of inulinase F2 and F3 were determined to be pH 4.6 and 4.45, respectively. Effects of pH on the activity and stability of the enzymes The pH optimum for the enzyme activity was investigated

'_)

using inulin as a substrate. The reaction mixture adjusted to an appropriate pH with McIlvaine's buffer, was incubated at 50°C for 30min. The optimum pHs of inulinase F2 and F3 were found to be pH 5.0 and 6.0, respectively. Inulinase F2 was stable between pH 5.0 and 7.5, and inulinase F3 was stable between pH 5.0 and 8.5 after standing at 30°C for 24 h at various pHs. Effects of temperature on the activity and stability of the enzymes The temperature optimum of inulinase F2 and F3 was revealed to be around 55°C. Inulinase F2 was stable up to 50°C, and inulinase F3 was stable up to 45°C after keeping for 30 min at various temperatures. Substrate specificity of the inulinase F2 and F3 Relative activities of inulinase F2 and F3 on inulin, levan, sucrose, raffinose, fructo-oligosaccharides, and melezitose were compared. Each substrate was added to the enzyme solution (17 g / m l ) to a final concentration of 0.5% (w/v), and incubated at 50°C for 30 min. The ratios of activities to each substrate of inulinase F2 and inulinase F3 are shown in Table 2. Both enzymes hydrolyzed inulin, sucrose, 1kestose, nystose, and lf-frutofuranosyl nystose, and partly hydrolyzed rafflnose and stachyose. On the other hand, levan (from Acetobacter levanicum, Sigma) could be

20

1

o

alactosidase (116,000) sphorylase (97,400) Inulinase F2~I,,. Inulinase F 3 ~ b u m i n Bovine (66,000)

I0

"T, 3=

Albumin EGG ( 4 5 , 0 0 0 ) ~

%

Carbonic anhydrase (29,000)

W

~6

(+) (A)

(B)

FIG. 4. Disc electrophoresis of the purified inulinase F2 and F3 of C. pannorum AHU 9700. The purified enzyme was subjected to 7.5% polyacrylamide gel disc electrophoresis at pH 8.3. (A), Exoinulinase F2; (B), exoinulinase F3.

1

I

0.5 Relative mobility

I

1.0

FIG. 5. Estimation of the molecular weight of inulinase F2 and F3 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

334

XIAO ET AL. TABLE 2.

J. FERMENT. BIOENG..

Substrate specificity of inulinase F2 and F3

Substrate Inulin Sucrose (GF) l-Kestose (GF,) Nystose (GF.0 l~-fru-nystose (GF4) Raffinose Stachyose Melezitose Levan

Ratio of the activity a (%) F2

F3

66.7 100.0 92.1 83.2 79.3 44.4 30.2 0 0

29.6 100.0 91.5 78.3 67.9 42.3 12.6 0 37.6

The concentration of each substrate was 1.0% in 0.1 M acetate buffer (pH 5.0). The reaction mixture was incubated at 50°C for 30 rain. Activity toward sucrose was taken as 100%.

h y d r o l y z e d b y i n u l i n a s e F3, b u t n o t b y i n u l i n a s e F2. T h e p r o d u c t s o f e n z y m a t i c h y d r o l y s i s o f i n u l i n was a n a l y z e d by HPLC during the incubation. In contrast to the products ( i n u l o t r i o s e , i n u l o t e t r a o s e , a n d i n u l o p e n t a o s e ) o f p r e v i o u s l y r e p o r t e d e n d o i n u l i n a s e (11), o n l y f r u c t o s e a n d s m a l l a m o u n t s o f g l u c o s e were o b t a i n e d in this experiment. A s a c o n s e q u e n c e o f the results, these e n z y m e s were called e x o i n u l i n a s e F 2 a n d F3. DISCUSSION Two exoinulinases (2,1-fl-D-fructan fructanohydrolase E C 3 . 2 . 1 . 7 ) were p u r i f i e d f r o m t h e f u n g i Chrysosporium p a n n o r u m A H U 9700. T h e p u r i f i e d e n z y m e s were active o n s u c r o s e a n d i n u l i n . H o w e v e r , t h e r e were g r e a t d i f f e r e n c e s in t h e i r activities o n t h e s e s u b s t r a t e s as expressed b y t h e I / S v a l u e s a n d t h e i r activities o n levan. Several m i c r o b i a l i n u l i n a s e s h a v e b e e n r e p o r t e d (4-9). M o s t o f t h e m are e x o e n z y m e s , a n d t h e i r I / S values r a n g e d f r o m 0.02 (7) t o 1.95 (8), w i t h t h e I / S v a l u e s o f e n z y m e s f r o m yeast a n d b a c t e r i a u s u a l l y b e i n g l o w e r ( f r o m 0.02 to 0.28 (9)) t h a n t h o s e o f e n z y m e s f r o m f u n g i ( f r o m 0.16 (6) to 1.95). O u r p u r i f i e d e x o i n u l i n a s e s F2 a n d F3 s h o w e d relatively h i g h I / S v a l u e s (0.67 a n d 0.29). O n the o t h e r h a n d , m i c r o b i a l e x o i n u l i n a s e s c a n be divided i n t o a c t i v e a n d i n a c t i v e types a c c o r d i n g t o t h e i r l e v a n h y d r o l y s i s activity. F o r i n s t a n c e , t h e e x o i n u l i n a s e s f r o m Kluyveromyces fragilis (17) a n d Penicillium sp. (8) are active o n levan, b u t t h e e x o i n u l i n a s e s f r o m Aspergillus niger (18) a n d Panaeolus papillonaceus are i n a c t i v e . In this r e p o r t , it was r e v e a l e d t h a t w h e r e a s e x o i n u l i n a s e F3 was active o n l e v a n , e x o i n u l i n a s e F2 was i n a c t i v e . U n t i l n o w , t h e r e h a s b e e n n o r e p o r t o f t w o types o f e x o i n u l i n a s e , active o n l e v a n a n d i n a c t i v e o n l e v a n , b e i n g d i s c o v e r e d f r o m o n e s t r a i n at t h e s a m e time. In o u r i n v e s t i g a t i o n , we o b t a i n e d o n e e n d o i n u l i n a s e (F4) a n d t w o e x o i n u l i n a s e (F2 a n d F3) w i t h different specific activities o n l e v a n f r o m C. p a n n o r u m . W e p r o p o s e to discuss the m e c h a n i s m o f i n u l i n h y d r o l y s i s by t h e s e t h r e e types o f i n u l i n a s e in o u r n e x t p a p e r .

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