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Inulinase from Chrysosporium pannorum
[J. Ferment. Technol., Vol. 66, No. 5, 553-558. 1988]
Inulinase from Chrysosporium pannorum RONG XIAO, MASATOSHITANIDA, a n d SHOICHI TAKAO*
Departmentof AgriculturalChemist~,Facultyof Agriculture, Hokkaido University,Sapporo 060, Japan A mold, ChrysosporiumpannorumAHU 9700, isolated from soil, was found to produce a very active inulin-hydrolyzing enzyme. The inulinase activity of this strain was induced by inulin, but not by sucrose, glucose, or fructose. The highest inulinase activity, 115 units/ml, was obtained under the optimum conditions. A crude enzyme preparation was active against inulin, levan, sucrose, and raffinose, but not melezitose. An inulin suspension of 10% was hydrolyzed completely at pH 5.6, 50°C, in 8 h, and inulo-oligosaccharides were detected in the initial stage of enzymatic reaction. Jerusalem artichoke extract was also rapidly hydrolyzed into fructose and small amounts of glucose. These results indicate that the G. pannorum crude enzyme contained exoand endo-inulinases. The inulinase ofC. pannorumAHU 9700 might be used industrially to prepare high fructose syrup from inulin-containingagricultural crops.
I n u l i n is usually f o u n d as a reserve carboh y d r a t e i n the roots a n d tubers of various plants like the J e r u s a l e m artichoke, d a n d e l i o n , chicory, d a h l i a , a n d several other m e m b e r s of the f a m i l y Compositae. 1) I n u l i n is a fl2,1-1inked fructose p o l y m e r t e r m i n a t e d b y a sucrose u n i t residue. R e c e n t l y , it has received a t t e n t i o n as a substrate for h i g h fructose syrup production~) a n d for e t h a n o l fermentation.S-s) I n u l i n c a n be hydrolyzed b y acid, p H 1 to 2 at 80-100°C, b u t fructose is easily d e g r a d e d at low pHs a n d the process gives rise to color a n d b y - p r o d u c t f o r m a t i o n such as difructose a n h y d r i d e . 6) O n the other h a n d , e n z y m a t i c hydrolysis has no such problems t h o u g h it takes longer for complete hydrolysis. Several inulinases (2,1-fl-D-fructan fruct a n o h y d r o l a s e E C 3.2.1.7) have b e e n described i n the l i t e r a t u r e as o r i g i n a t i n g from p l a n t m a t e r i a l such as i n u l i n - c o n t a i n i n g roots a n d tubers 7,~) or from m i c r o o r g a n isms.2) The m i c r o b i a l inulinases were derived from fungi, 9-1x) yeasts, a~-14) a n d bacteria, x5-17) Yeasts such as Kluyveromyces
myces cantarellii, a n d fungi, Penicillium a n d Aspergillus species are c o m m o n i n u l i n a s e producers. W e tried to isolate active i n u l i n a s e producers a n d f o u n d one mold w h i c h was identified as Chrysosporium pannorum. I n this report, we describe the i n u l i n a s e activity of C. pannorum a n d some properties of the c r u d e enzyme. M a t e r i a l s and M e t h o d s Microorganism C. pannorum AHU 9700 was used. The strain was isolated from soil using a modification of the medium of MandelsTM containing inulin as the sole carbon source and was maintained on potato dextrose agar slants. Cultivation The strain was grown in 100 ml of liquid medium in 500-ml shaking flasks for 5 d at 27°C. The basal medium contained 1.0% inulin, 1.5% corn steep liquor, 1.2% NH4HzPO4, 0.05% MgSO4'7H20, 0.05% KC1, and 0.001% FeSO4.7H20. The initial pH was 5.0. The medium was autoclaved at 15psi for 10rain without inulin. Inulin was sterilized with ethylene oxide and then added to the autoclaved medium.
fragilis, K. marxianus, Candida kefyr, Debaryo-
E x t r a c t i o n o f juice f r o m J e r u s a l e m a r t i c h o k e tubers The tubcrs were washed thoroughly to
* Corresponding author
remove any remaining soil, then cut into small slices. The sliced tubers were placed in an Erlenmeyer flask
XIAO, TANIDA, and TAKAO
554
for 1 h with an equal weight of tap water and the sugars were extracted at 70°C. The slurry was filtered through a cloth to collect the extract TM and then decolorlzed by an active carbon column. The juice obtained was stored at --20°C for further use. Crude exlzyme preparation About 3 m l of culture broth were withdrawn at regular intervals, and centrifilged at 3000 rpm for 20 rain at 4°C to remove mycelia. The supernatants were used as the crude enzyme throughout the experiments for the enzyme assays. Enzyme assays Inulinase activity The reaction mixture contained 0.1 ml of 1.0% inulin in 0.1 M sodium acetate buffer (pH 5.6) and 0.1 ml of diluted crude enzyme. The mixture was incubated at 50°C for 30 rain. The amount of reducing sugar was measured by the 3, 5dinitrosalicylic acid method. TM One unit of inulinase activity was defined as the amount of enzyme liberating one micromole of fructose per rain. Invertase activity The reaction mixture contained 0.1 ml of 1.0% sucrose in 0.1 M sodium acetate buffer (pH 5.6) and 0.1 ml of diluted crude enzyme. The reaction conditions were the same as those described for the assay ofinulinase activity. One unit ofinvertase activity was defined as the amount of enzyme which catalyzed the hydrolysis of one micromole of sucrose per rain. A n a l y s i s of h y d r o l y s i s p r o d u c t s The hydrolysis products of inulin and Jerusalem artichoke extract by crude enzyme were analyzed by high performance liquid chromatography (Hitachi, model 635) with a column of Shodex NH pak J-411 (Showa-Denko, K.K.) and R I detector (ERC-7510, Erma Optical Works, l,td.). Results
and Discussion
Isolation of inulinase-producing fungi In the screening procedure, some fungi were selected for their promising inulinase producing capacity. After comparison of
inulinase activity and the ratio of the activity
on inulin versus sucrose (I/S ratio), one s t r a i n t h a t w a s i d e n t i f i e d as C. pannorum
Czapek's sohttion agar at room temperature, floccose, granular or finely powdery but also sometimes smooth or with scattered tufts of aerial mycelium; the color was from white to yellow or gray; reverse normally yellow with the pigment diffusing into the agar. Conidiophores arising from aerial hyphae, erect or standing out from mycelial tufts in all directions, to 50/~m long, bearing short chains of 2-4 alternate arthroconidia; arthroconidia cuneiform, pyriform to clavate, with a broadly truncate base when terminal but otherwise with both ends truncate; smooth to distinctly roughened, hyaline to greyish, mostly 2-5 × 2-4/~m. Culture
Carbon sources hmliu Starch Cellulose Xylan Pectin
Xylose Cellobiose
Hokkaido,
Colonies usually slow-growing, reaching 1 . 0 - 2 . 0 c m diameter in two weeks on
pro-
Table 1. Effects of various carbon sources on enzyme production.
Habitat: 1985.
Ishikari,
for inulinase
Effects of carbon sources The various kinds of carbon sources shown in Table 1 w e r e a d d e d to t h e b a s a l m e d i u m , a n d t h e i r effects o n t h e i n u l i n a s e p r o d u c t i o n w e r e investigated. A s s h o w n i n T a b l e 1, t h e i n u l i n a s e p r o duction was found only in the culture medium containing inulin, but not in the media containing sucrose, glucose, fructose, or others. C. pannorum i n u l i n a s e a p p e a r e d to b e a n i n d u c i b l e e n z y m e . Effects of organic nutrients T h e effects o f
Raffinose Sucrose Maltose Glucose Fructose
soil,
conditions
duction
A H U 9 7 0 0 w a s s e l e c t e d as t h e b e s t i n u l i n a s e producer, and studied further. Description of C. P a n n o r u m A H U 9700 from
[J. Ferment. Technol.,
Inulinase (I) (units/ml) 16.99 0.01 0. 01 O. 02 0. 03 0. 32 0. 20 0. 01 0. 06 0. 03 0.01 0.01
Each carbon source was 1%. at 27°C.
Invertase (S) (units/ml) 11.58 0.01 0. 00 O. O0 0. 04 0. 44 0. 21 0. 00 0. 01 0. 04 0. 00 0.00
I/S 1.47 1.00 --0. 75 0. 73 0. 95 -6. 00 0. 75 ---
Cultivation was for 5 d
Vol. 66, 1988]
555
Inulinase from C. pannorum
Table 2. Effects of organic nutrients on enzyme production. Nutrients
Concentration (%)
Inulinase (unlts]ml)
Invertase (units/ml)
I/S
Meat extract
0. 2 0. 5 1.0
0. 45 12.04 24. 91
0. 63 12.45 20. 33
0. 71 0. 97 1.23
Polypepton
0. 2 0. 5 1.0
5. 82 15.37 16.05
6. 14 10. 76 11.14
0. 95 1.43 1.44
Yeast extract
0. 2 0.5 1.0
7.61 13.71 12.32
7.47 11.90 11.28
1.02 1.15 1.09
Corn steep liquor
0. 5 1.0 1.5
0. 81 12. 59 18. 55
0. 96 12. 67 14. 22
0. 84 0. 99 1.30
--
0. 00
0. 00
--
None Cultivation was for 5 d at 27°C.
organic nutrients on inulinase production were tested. It has been reported that Polypepton a n d corn steep liquor were better nitrogen sources t h a n yeast extract for the p r o d u c t i o n o f inulinase b y Penicillium sp. 9) and b y Aspergillus niger-12 strain. TM I n our investigation, however, 1.0% m e a t extract (Wako) gave the highest inulinase production (Table 2). Inulinase was also p r o d u c e d by a d d i n g Polypepton (Daigo), yeast extract (Oriental Yeast), or corn steep liquor, but the level o f inulinase activity was less than in meatextract-containing m e d i u m . Effects of inorganic nitrogen sources A m o n g inorganic nitrogen sources, the addition of 0.02 M (NH4)2SO4, (NH4)2HPO4, NH4C1, or N H 4 N O s gave a higher inulinase yield (20 to 22units/ml) t h a n N H 4 H 2 P O 4 or N a N O ~ (15 to 19units/ml). Increases in concentration to 0.1 M o f each o f the nitrogen sources h a d no stimulative effect. Effects of initial p H T h e initial p H o f culture m e d i u m was adjusted to different values with 1 N hydrochloric acid or sodium hydroxide solution. Inulinase production was not significantly different from p H 4.5 to 5.5 (15 to 21 units/ml).
Effects of culture temperature T h e effects o f temperature on C. pannorum A H U 9700 were investigated. It is reported that the o p t i m a l temperature for inulinase production b y Candida kefyr14) was 27 to 30°C, and higher temperatures lowered inulinase production. T h e inulinase production by this strain at 22, 27, a n d 32°C was 47, 22, a n d 0 units/ml, respectively. Effects of inulin and meat extract concentration This experiment was done with the m e d i u m (shown in T a b l e 3), of various concentrations of inulin and m e a t extract at 22°C. T h e c o m b i n a t i o n of 5.0% inulin and 4.0% m e a t extract gave the most units of inulinase, 115 units/ml. A m a x i m u m inulinase activity (212units/ml) has been reported for K. marxianus U C D (FST) 55-82, TM but the I/S ratio was 0.28. A n I/S value of 1.18 was found for A. niger, TM while only 20.1 units/ml o f inulinase was obtained. C o m p a r e d with these inulinase-producing strains, C. pannorum A H U 9700 showed several advantages for enzyme production. T h e inulinase productivity (115 units/ml) was higher than the productivities o f Penicillium sp. (2.1 units] ml), 9) A. niger strain (20.1 units/ml),n) and K. fragilis A T C C 12424 (100units/ml),l~)
556
XIAO, TANIDA,
Table 3. Meat extract (%)
and TAKAO
[J. Ferment. Technol.,
Effects of inulin and meat extract on inulinase production. .
0.2 0.5 1.0 1.5 2.0 3.0 4. 0 5. 0
.
Inulinase activity (units/ml) . . . . . . . . . . . . . . . . . . . . ! nulin ( °/o)_ _ 1.0 2.0 3.0 4.0
0.0 0.0 42.8 b) 32.3 a) 31.9 a) ----
. 0.0 0.2 51.7 a) 60. 9 b) 56. 7b) --
--
.
. -0.1 0. 2 62. 8 d) 61.2 b) 50. 98> --
5.0
. --0. 1 7.8 91.4 b) 48. 8 b) 42. 8b)
---I. 1 109. 0 c) 115.0 b) 68. 5 b)
The medium consisted of (NH4),SO4 0.13%, MgSO4'7H~O 0.05%, FeSO,.7H20 0.001% and Tween 60 0.5%, pH 5.0. Cultivation was for 6 d (a), 8 d (b), 10 d (e), and 12 d (d) at 22°C. a n d t h e I / S v a l u e w a s also h i g h e r t h a n these i n u l i n a s e - p r o d u c i n g strains. Properties of C. p a n n o r u m crude intdinase preparation
Effects of p H on inulinase activity and stability T h e o p t i m u m p H for t h e r e a c t i o n a n d p H s t a b i l i t y o f t h e e n z y m e w e r e f o u n d (Fig. 1).
Effects of temperature on inulinase activity and stability T h e effects o f t e m p e r a t u r e o n t h e a c t i v i t y w e r e e x a m i n e d at p H 5.6. As s h o w n i n F i g . 2, t h e o p t i m u m t e m p e r a t u r e was 50°C. T h i s t e m p e r a t u r e is r e l a t i v e l y high and advantageous in the view of cont r o l l i n g m i c r o b i a l c o n t a m i n a t i o n o f t h e reaction mixture. The higher temperature also p e r m i t s t h e use o f h i g h e r c o n c e n t r a t i o n s o f i n u l i n as a s u b s t r a t e b e c a u s e o f its solubility. T h e e n z y m e was s t a b l e u p to 5 0 ° C for 10 m i n o f t r e a t m e n t .
1 O0
;~" 75
50 "3
0
T h e o p t i m u m p H was 5 . 5 - 5 . 6 . This value is n e a r l y t h e s a m e as t h a t for A. niger-121°) a n d K. fragilis TM i n u l i n a s e s . The enzyme a c t i v i t y was s t a b l e b e t w e e n p H 4 . 5 - 7 . 0 w h e n tested i n t h e r a n g e o f p H 3.2 to 8.0 at 3 0 ° C for 24 h.
I
I
I
I
I
t
3
4
5
6
7
8
pH Fig. 1. Effects of pI-I on inulinase activity and stability. The reaction was done at 50°C for 30 rain. Buffers used were 0.1 M acetate buffer (pH 3.2-6.0) and phosphate buffer (pH 6.0-8.0). The enzyme was incubated in buffers at 30°C for 24h. O, optimum pH; O, pH stability.
Hydrolysis of inulin and Jerusalem artichoke extract Hydrolysis of inulin b y C. pannomm c r u d e e n z y m e w a s i n v e s t i gated. An inulin suspension which cont a i n e d 0.5, 1.0, o r 1.5 g o f i n u l i n i n 5 m l o f 0.1 M a c e t a t e b u f f e r ( p H 5 . 6 ) w a s m i x e d with 5 ml of a crude enzyme solution (240 u n i t s / m l ) , a n d t h e m i x t u r e s w e r e i n c u b a t e d o n a s h a k e r at 50°C. A f t e r t h e i n d i c a t e d t i m e o f i n c u b a t i o n , 0.5 m l o f t h e reaction mixture was pipetted out and the r e d u c i n g s u g a r l i b e r a t e d was m e a s u r e d as fructose and the product of enzymatic hydrolysis of inulin was analyzed by
Vol. 66, 1988]
Inulinase from G. pannorum
557
I00 E "~ 5O
z 75
+a
>~ 2%
5O
=~ 25
0
I
I
I
I
30
40
50
60
N
70
Temperature (°C) Fig. 2. Effects of temperature on inulinase activity and stability. The reaction was done at various temperatures for 30rain in 0.1 M acetate buffer (pH5.6). The enzyme solution in 0.1 M acetate buffer (pH 5.6) was kept at various temperatures for 10 min. O, optimum temperature; O, temperature stability.
Fig. 3. Hydrolysis of various concentrations of inulin by crude enzyme of C. pannorum A H U 9700. The reaction mixture consisting of 5 ml of the crude enzyme solution (240 units/ml) and 5 ml of inulin suspension (10%, 20% and 30%) in acetate buffer (pI-I 5.6) was incubated at 50°C. After the indicated time of incubation, 0.5 ml of the reaction mixture was pipetted out and reducing sugar liberated was measured as fructose.
t , 5%; O, 10%; A, 15%.
of HPLC analyses before and after hydrolysis. The Jerusalem artichoke extract contained fructose, glucose, and inulo-oligosaccharides. After 2 h incubation, the Jerusalem artichoke
A
high performance liquid chromatography. Figure 3 shows the course of enzymatic hydrolysis of inulin. Inulin in 5%, 10%, and 15% suspensions was completely hydrolyzed within 4 h, 8 h, and 24 h, respectively. The hydrolysis product was fructose and small amounts of glucose though inulooligosaccharides (D.P. 2-7) were observed in the initial stage of the enzymatic reaction (30rain). The result indicates that the C. pannorum enzyme contained exo- and endo-inulinases. We tried to produce high fructose syrup from Jerusalem artichoke tubers by C. pannorum crude enzyme. The reaction mixture consisting of 5 ml of Jerusalem artichoke extract (total sugar was about 10%) and 5 ml of crude enzyme solution (240 units/ml) was incubated on a shaker at 50°C. About 0.5-ml portions of the reaction mixture were pipetted out at regular intervals, and analyzed by HPLC. Figure 4 shows the results
truct0se.~luc0se
I
0
2
4
6
8
lO 12 14 16 18 20
0
4
6
Retention time (min)
Fig. 4. High performance liquid chromatograms of Jerusalem artichoke extract (A) before hydrolysis and (B) after 2 h hydrolysis. The reaction mixture consisting of 5 ml of Jerusalem artichoke extract and 5 ml of crude inulinase (240 units/ml) was incubated at 50°C. After 2 h incubation, 0 . 5 m l of the reaction mixture was pipetted out and analyzed by HPLC. (Column, Shodex N i l pak J--411; eluent, acetonitrile: water 70 : 30; detector, Erma Optical Works, Ltd., model ERC-7510). D.P.: degree of polymerization.
558
XIAO, TANIDA,and TAKAO
extract was completely hydrolyzed to fructose and a small amount of glucose. The result indicates that the inulinase produced by C. pannomm was an industrially attractive enzyme for high fructose syrup preparation from inulin-containing agricultural crops such as Jerusalem artichoke, chicory, and dahlia.
1) 2) 3) 4) 5) 6) 7) 8) 9)
Kaishi, 43, 599 (1969). I0) Nakamura, T., Hoashi, S., Nakatsu, S.: Nippon Nogeikagaku Kaishi, 52, 105 (1978). 11) Derycke, D.G., Vandamme, E.J.: J. Chem. Tech. Biotechnvl., 34, 45 (1984). 12) GrootWasslnk, J.W.D., Fleming, S.: Enzyme Microb. Technvl., 2, 45 (1980). 13) Margaritis, A., Bajpai, P.: Biotechnol. Bioeng., 24, 94 (1982). 14) Negoro, tt., Kito, E.: J. Ferment. Technol., 51, References 103 (1973). Demeulle, S., Guiraud, J.P., Galzy, P.: Z. 15) Uehiyama, T.: Biochim. Biophys. Aeta, 397, 153 Allg. Mikrobiol., 21, 181 (1981). (1975). Vandamme, E.J., Derycke, D.G.: Adv. Appl. 16) Allais, J.J., Kammoun, S., Blanc, P., Girard, C., Microbiol., 29, 139 (1983). Baratti, J.C. : Appl. Environ. :l/licrobiol., 52, Margaritis, A., Bajpai, P.: Appl. Environ. Micro1086 (1986). biol., 45, 723 (1983). 17) Allais, J.J., Lopez, G. H., Kammoun, S., Baratti, Toran, D. L.,Jain, V. K., Allais, J.J., Baratti, J.: J. : Appl. Environ. Microbial Microbiol., 53, Biotechnol. Lett., 7, 527 (1985). 942 (1987). Guiraud, J. P., Gailland, J. M., Galzy, P.: Eur. 18) Mandels, M., Weber, J." Adv. Chem. Set., 95, 391 (1969). d. Appl. Microbiol. Biotechnol., 14, 81 (1982). Fleming, S.E., Grootwassink, J . W . D . : Grit. 19) Margaritis, A., Bajpai, P.: Biotechnol. Bioeng., Rev. Food Sci. Nutr., 12, 1 (1979). 24, 1473 (1982). Edelman, J., Jefford, T.G.: Biochem. J., 93, 20) Miller, G.L.: Anal. Chem., 31, 426 (1959). 148 (1964). 21) Parekh, S., Margaritis, A.: Agric. Biol. Chem., Rutherford, P.P., Deacon, A.C.: Biochem. d., 59, 1085 (1986). 126, 569 (1972). Nakamura, T., Hoashi, S.: Nippon Nogeikagaku (Received March 31, 1988)
Inulinase from Chrysosporium pannorum RONG XIAO, MASATOSHITANIDA, a n d SHOICHI TAKAO*
Departmentof AgriculturalChemist~,Facultyof Agriculture, Hokkaido University,Sapporo 060, Japan A mold, ChrysosporiumpannorumAHU 9700, isolated from soil, was found to produce a very active inulin-hydrolyzing enzyme. The inulinase activity of this strain was induced by inulin, but not by sucrose, glucose, or fructose. The highest inulinase activity, 115 units/ml, was obtained under the optimum conditions. A crude enzyme preparation was active against inulin, levan, sucrose, and raffinose, but not melezitose. An inulin suspension of 10% was hydrolyzed completely at pH 5.6, 50°C, in 8 h, and inulo-oligosaccharides were detected in the initial stage of enzymatic reaction. Jerusalem artichoke extract was also rapidly hydrolyzed into fructose and small amounts of glucose. These results indicate that the G. pannorum crude enzyme contained exoand endo-inulinases. The inulinase ofC. pannorumAHU 9700 might be used industrially to prepare high fructose syrup from inulin-containingagricultural crops.
I n u l i n is usually f o u n d as a reserve carboh y d r a t e i n the roots a n d tubers of various plants like the J e r u s a l e m artichoke, d a n d e l i o n , chicory, d a h l i a , a n d several other m e m b e r s of the f a m i l y Compositae. 1) I n u l i n is a fl2,1-1inked fructose p o l y m e r t e r m i n a t e d b y a sucrose u n i t residue. R e c e n t l y , it has received a t t e n t i o n as a substrate for h i g h fructose syrup production~) a n d for e t h a n o l fermentation.S-s) I n u l i n c a n be hydrolyzed b y acid, p H 1 to 2 at 80-100°C, b u t fructose is easily d e g r a d e d at low pHs a n d the process gives rise to color a n d b y - p r o d u c t f o r m a t i o n such as difructose a n h y d r i d e . 6) O n the other h a n d , e n z y m a t i c hydrolysis has no such problems t h o u g h it takes longer for complete hydrolysis. Several inulinases (2,1-fl-D-fructan fruct a n o h y d r o l a s e E C 3.2.1.7) have b e e n described i n the l i t e r a t u r e as o r i g i n a t i n g from p l a n t m a t e r i a l such as i n u l i n - c o n t a i n i n g roots a n d tubers 7,~) or from m i c r o o r g a n isms.2) The m i c r o b i a l inulinases were derived from fungi, 9-1x) yeasts, a~-14) a n d bacteria, x5-17) Yeasts such as Kluyveromyces
myces cantarellii, a n d fungi, Penicillium a n d Aspergillus species are c o m m o n i n u l i n a s e producers. W e tried to isolate active i n u l i n a s e producers a n d f o u n d one mold w h i c h was identified as Chrysosporium pannorum. I n this report, we describe the i n u l i n a s e activity of C. pannorum a n d some properties of the c r u d e enzyme. M a t e r i a l s and M e t h o d s Microorganism C. pannorum AHU 9700 was used. The strain was isolated from soil using a modification of the medium of MandelsTM containing inulin as the sole carbon source and was maintained on potato dextrose agar slants. Cultivation The strain was grown in 100 ml of liquid medium in 500-ml shaking flasks for 5 d at 27°C. The basal medium contained 1.0% inulin, 1.5% corn steep liquor, 1.2% NH4HzPO4, 0.05% MgSO4'7H20, 0.05% KC1, and 0.001% FeSO4.7H20. The initial pH was 5.0. The medium was autoclaved at 15psi for 10rain without inulin. Inulin was sterilized with ethylene oxide and then added to the autoclaved medium.
fragilis, K. marxianus, Candida kefyr, Debaryo-
E x t r a c t i o n o f juice f r o m J e r u s a l e m a r t i c h o k e tubers The tubcrs were washed thoroughly to
* Corresponding author
remove any remaining soil, then cut into small slices. The sliced tubers were placed in an Erlenmeyer flask
XIAO, TANIDA, and TAKAO
554
for 1 h with an equal weight of tap water and the sugars were extracted at 70°C. The slurry was filtered through a cloth to collect the extract TM and then decolorlzed by an active carbon column. The juice obtained was stored at --20°C for further use. Crude exlzyme preparation About 3 m l of culture broth were withdrawn at regular intervals, and centrifilged at 3000 rpm for 20 rain at 4°C to remove mycelia. The supernatants were used as the crude enzyme throughout the experiments for the enzyme assays. Enzyme assays Inulinase activity The reaction mixture contained 0.1 ml of 1.0% inulin in 0.1 M sodium acetate buffer (pH 5.6) and 0.1 ml of diluted crude enzyme. The mixture was incubated at 50°C for 30 rain. The amount of reducing sugar was measured by the 3, 5dinitrosalicylic acid method. TM One unit of inulinase activity was defined as the amount of enzyme liberating one micromole of fructose per rain. Invertase activity The reaction mixture contained 0.1 ml of 1.0% sucrose in 0.1 M sodium acetate buffer (pH 5.6) and 0.1 ml of diluted crude enzyme. The reaction conditions were the same as those described for the assay ofinulinase activity. One unit ofinvertase activity was defined as the amount of enzyme which catalyzed the hydrolysis of one micromole of sucrose per rain. A n a l y s i s of h y d r o l y s i s p r o d u c t s The hydrolysis products of inulin and Jerusalem artichoke extract by crude enzyme were analyzed by high performance liquid chromatography (Hitachi, model 635) with a column of Shodex NH pak J-411 (Showa-Denko, K.K.) and R I detector (ERC-7510, Erma Optical Works, l,td.). Results
and Discussion
Isolation of inulinase-producing fungi In the screening procedure, some fungi were selected for their promising inulinase producing capacity. After comparison of
inulinase activity and the ratio of the activity
on inulin versus sucrose (I/S ratio), one s t r a i n t h a t w a s i d e n t i f i e d as C. pannorum
Czapek's sohttion agar at room temperature, floccose, granular or finely powdery but also sometimes smooth or with scattered tufts of aerial mycelium; the color was from white to yellow or gray; reverse normally yellow with the pigment diffusing into the agar. Conidiophores arising from aerial hyphae, erect or standing out from mycelial tufts in all directions, to 50/~m long, bearing short chains of 2-4 alternate arthroconidia; arthroconidia cuneiform, pyriform to clavate, with a broadly truncate base when terminal but otherwise with both ends truncate; smooth to distinctly roughened, hyaline to greyish, mostly 2-5 × 2-4/~m. Culture
Carbon sources hmliu Starch Cellulose Xylan Pectin
Xylose Cellobiose
Hokkaido,
Colonies usually slow-growing, reaching 1 . 0 - 2 . 0 c m diameter in two weeks on
pro-
Table 1. Effects of various carbon sources on enzyme production.
Habitat: 1985.
Ishikari,
for inulinase
Effects of carbon sources The various kinds of carbon sources shown in Table 1 w e r e a d d e d to t h e b a s a l m e d i u m , a n d t h e i r effects o n t h e i n u l i n a s e p r o d u c t i o n w e r e investigated. A s s h o w n i n T a b l e 1, t h e i n u l i n a s e p r o duction was found only in the culture medium containing inulin, but not in the media containing sucrose, glucose, fructose, or others. C. pannorum i n u l i n a s e a p p e a r e d to b e a n i n d u c i b l e e n z y m e . Effects of organic nutrients T h e effects o f
Raffinose Sucrose Maltose Glucose Fructose
soil,
conditions
duction
A H U 9 7 0 0 w a s s e l e c t e d as t h e b e s t i n u l i n a s e producer, and studied further. Description of C. P a n n o r u m A H U 9700 from
[J. Ferment. Technol.,
Inulinase (I) (units/ml) 16.99 0.01 0. 01 O. 02 0. 03 0. 32 0. 20 0. 01 0. 06 0. 03 0.01 0.01
Each carbon source was 1%. at 27°C.
Invertase (S) (units/ml) 11.58 0.01 0. 00 O. O0 0. 04 0. 44 0. 21 0. 00 0. 01 0. 04 0. 00 0.00
I/S 1.47 1.00 --0. 75 0. 73 0. 95 -6. 00 0. 75 ---
Cultivation was for 5 d
Vol. 66, 1988]
555
Inulinase from C. pannorum
Table 2. Effects of organic nutrients on enzyme production. Nutrients
Concentration (%)
Inulinase (unlts]ml)
Invertase (units/ml)
I/S
Meat extract
0. 2 0. 5 1.0
0. 45 12.04 24. 91
0. 63 12.45 20. 33
0. 71 0. 97 1.23
Polypepton
0. 2 0. 5 1.0
5. 82 15.37 16.05
6. 14 10. 76 11.14
0. 95 1.43 1.44
Yeast extract
0. 2 0.5 1.0
7.61 13.71 12.32
7.47 11.90 11.28
1.02 1.15 1.09
Corn steep liquor
0. 5 1.0 1.5
0. 81 12. 59 18. 55
0. 96 12. 67 14. 22
0. 84 0. 99 1.30
--
0. 00
0. 00
--
None Cultivation was for 5 d at 27°C.
organic nutrients on inulinase production were tested. It has been reported that Polypepton a n d corn steep liquor were better nitrogen sources t h a n yeast extract for the p r o d u c t i o n o f inulinase b y Penicillium sp. 9) and b y Aspergillus niger-12 strain. TM I n our investigation, however, 1.0% m e a t extract (Wako) gave the highest inulinase production (Table 2). Inulinase was also p r o d u c e d by a d d i n g Polypepton (Daigo), yeast extract (Oriental Yeast), or corn steep liquor, but the level o f inulinase activity was less than in meatextract-containing m e d i u m . Effects of inorganic nitrogen sources A m o n g inorganic nitrogen sources, the addition of 0.02 M (NH4)2SO4, (NH4)2HPO4, NH4C1, or N H 4 N O s gave a higher inulinase yield (20 to 22units/ml) t h a n N H 4 H 2 P O 4 or N a N O ~ (15 to 19units/ml). Increases in concentration to 0.1 M o f each o f the nitrogen sources h a d no stimulative effect. Effects of initial p H T h e initial p H o f culture m e d i u m was adjusted to different values with 1 N hydrochloric acid or sodium hydroxide solution. Inulinase production was not significantly different from p H 4.5 to 5.5 (15 to 21 units/ml).
Effects of culture temperature T h e effects o f temperature on C. pannorum A H U 9700 were investigated. It is reported that the o p t i m a l temperature for inulinase production b y Candida kefyr14) was 27 to 30°C, and higher temperatures lowered inulinase production. T h e inulinase production by this strain at 22, 27, a n d 32°C was 47, 22, a n d 0 units/ml, respectively. Effects of inulin and meat extract concentration This experiment was done with the m e d i u m (shown in T a b l e 3), of various concentrations of inulin and m e a t extract at 22°C. T h e c o m b i n a t i o n of 5.0% inulin and 4.0% m e a t extract gave the most units of inulinase, 115 units/ml. A m a x i m u m inulinase activity (212units/ml) has been reported for K. marxianus U C D (FST) 55-82, TM but the I/S ratio was 0.28. A n I/S value of 1.18 was found for A. niger, TM while only 20.1 units/ml o f inulinase was obtained. C o m p a r e d with these inulinase-producing strains, C. pannorum A H U 9700 showed several advantages for enzyme production. T h e inulinase productivity (115 units/ml) was higher than the productivities o f Penicillium sp. (2.1 units] ml), 9) A. niger strain (20.1 units/ml),n) and K. fragilis A T C C 12424 (100units/ml),l~)
556
XIAO, TANIDA,
Table 3. Meat extract (%)
and TAKAO
[J. Ferment. Technol.,
Effects of inulin and meat extract on inulinase production. .
0.2 0.5 1.0 1.5 2.0 3.0 4. 0 5. 0
.
Inulinase activity (units/ml) . . . . . . . . . . . . . . . . . . . . ! nulin ( °/o)_ _ 1.0 2.0 3.0 4.0
0.0 0.0 42.8 b) 32.3 a) 31.9 a) ----
. 0.0 0.2 51.7 a) 60. 9 b) 56. 7b) --
--
.
. -0.1 0. 2 62. 8 d) 61.2 b) 50. 98> --
5.0
. --0. 1 7.8 91.4 b) 48. 8 b) 42. 8b)
---I. 1 109. 0 c) 115.0 b) 68. 5 b)
The medium consisted of (NH4),SO4 0.13%, MgSO4'7H~O 0.05%, FeSO,.7H20 0.001% and Tween 60 0.5%, pH 5.0. Cultivation was for 6 d (a), 8 d (b), 10 d (e), and 12 d (d) at 22°C. a n d t h e I / S v a l u e w a s also h i g h e r t h a n these i n u l i n a s e - p r o d u c i n g strains. Properties of C. p a n n o r u m crude intdinase preparation
Effects of p H on inulinase activity and stability T h e o p t i m u m p H for t h e r e a c t i o n a n d p H s t a b i l i t y o f t h e e n z y m e w e r e f o u n d (Fig. 1).
Effects of temperature on inulinase activity and stability T h e effects o f t e m p e r a t u r e o n t h e a c t i v i t y w e r e e x a m i n e d at p H 5.6. As s h o w n i n F i g . 2, t h e o p t i m u m t e m p e r a t u r e was 50°C. T h i s t e m p e r a t u r e is r e l a t i v e l y high and advantageous in the view of cont r o l l i n g m i c r o b i a l c o n t a m i n a t i o n o f t h e reaction mixture. The higher temperature also p e r m i t s t h e use o f h i g h e r c o n c e n t r a t i o n s o f i n u l i n as a s u b s t r a t e b e c a u s e o f its solubility. T h e e n z y m e was s t a b l e u p to 5 0 ° C for 10 m i n o f t r e a t m e n t .
1 O0
;~" 75
50 "3
0
T h e o p t i m u m p H was 5 . 5 - 5 . 6 . This value is n e a r l y t h e s a m e as t h a t for A. niger-121°) a n d K. fragilis TM i n u l i n a s e s . The enzyme a c t i v i t y was s t a b l e b e t w e e n p H 4 . 5 - 7 . 0 w h e n tested i n t h e r a n g e o f p H 3.2 to 8.0 at 3 0 ° C for 24 h.
I
I
I
I
I
t
3
4
5
6
7
8
pH Fig. 1. Effects of pI-I on inulinase activity and stability. The reaction was done at 50°C for 30 rain. Buffers used were 0.1 M acetate buffer (pH 3.2-6.0) and phosphate buffer (pH 6.0-8.0). The enzyme was incubated in buffers at 30°C for 24h. O, optimum pH; O, pH stability.
Hydrolysis of inulin and Jerusalem artichoke extract Hydrolysis of inulin b y C. pannomm c r u d e e n z y m e w a s i n v e s t i gated. An inulin suspension which cont a i n e d 0.5, 1.0, o r 1.5 g o f i n u l i n i n 5 m l o f 0.1 M a c e t a t e b u f f e r ( p H 5 . 6 ) w a s m i x e d with 5 ml of a crude enzyme solution (240 u n i t s / m l ) , a n d t h e m i x t u r e s w e r e i n c u b a t e d o n a s h a k e r at 50°C. A f t e r t h e i n d i c a t e d t i m e o f i n c u b a t i o n , 0.5 m l o f t h e reaction mixture was pipetted out and the r e d u c i n g s u g a r l i b e r a t e d was m e a s u r e d as fructose and the product of enzymatic hydrolysis of inulin was analyzed by
Vol. 66, 1988]
Inulinase from G. pannorum
557
I00 E "~ 5O
z 75
+a
>~ 2%
5O
=~ 25
0
I
I
I
I
30
40
50
60
N
70
Temperature (°C) Fig. 2. Effects of temperature on inulinase activity and stability. The reaction was done at various temperatures for 30rain in 0.1 M acetate buffer (pH5.6). The enzyme solution in 0.1 M acetate buffer (pH 5.6) was kept at various temperatures for 10 min. O, optimum temperature; O, temperature stability.
Fig. 3. Hydrolysis of various concentrations of inulin by crude enzyme of C. pannorum A H U 9700. The reaction mixture consisting of 5 ml of the crude enzyme solution (240 units/ml) and 5 ml of inulin suspension (10%, 20% and 30%) in acetate buffer (pI-I 5.6) was incubated at 50°C. After the indicated time of incubation, 0.5 ml of the reaction mixture was pipetted out and reducing sugar liberated was measured as fructose.
t , 5%; O, 10%; A, 15%.
of HPLC analyses before and after hydrolysis. The Jerusalem artichoke extract contained fructose, glucose, and inulo-oligosaccharides. After 2 h incubation, the Jerusalem artichoke
A
high performance liquid chromatography. Figure 3 shows the course of enzymatic hydrolysis of inulin. Inulin in 5%, 10%, and 15% suspensions was completely hydrolyzed within 4 h, 8 h, and 24 h, respectively. The hydrolysis product was fructose and small amounts of glucose though inulooligosaccharides (D.P. 2-7) were observed in the initial stage of the enzymatic reaction (30rain). The result indicates that the C. pannorum enzyme contained exo- and endo-inulinases. We tried to produce high fructose syrup from Jerusalem artichoke tubers by C. pannorum crude enzyme. The reaction mixture consisting of 5 ml of Jerusalem artichoke extract (total sugar was about 10%) and 5 ml of crude enzyme solution (240 units/ml) was incubated on a shaker at 50°C. About 0.5-ml portions of the reaction mixture were pipetted out at regular intervals, and analyzed by HPLC. Figure 4 shows the results
truct0se.~luc0se
I
0
2
4
6
8
lO 12 14 16 18 20
0
4
6
Retention time (min)
Fig. 4. High performance liquid chromatograms of Jerusalem artichoke extract (A) before hydrolysis and (B) after 2 h hydrolysis. The reaction mixture consisting of 5 ml of Jerusalem artichoke extract and 5 ml of crude inulinase (240 units/ml) was incubated at 50°C. After 2 h incubation, 0 . 5 m l of the reaction mixture was pipetted out and analyzed by HPLC. (Column, Shodex N i l pak J--411; eluent, acetonitrile: water 70 : 30; detector, Erma Optical Works, Ltd., model ERC-7510). D.P.: degree of polymerization.
558
XIAO, TANIDA,and TAKAO
extract was completely hydrolyzed to fructose and a small amount of glucose. The result indicates that the inulinase produced by C. pannomm was an industrially attractive enzyme for high fructose syrup preparation from inulin-containing agricultural crops such as Jerusalem artichoke, chicory, and dahlia.
1) 2) 3) 4) 5) 6) 7) 8) 9)
Kaishi, 43, 599 (1969). I0) Nakamura, T., Hoashi, S., Nakatsu, S.: Nippon Nogeikagaku Kaishi, 52, 105 (1978). 11) Derycke, D.G., Vandamme, E.J.: J. Chem. Tech. Biotechnvl., 34, 45 (1984). 12) GrootWasslnk, J.W.D., Fleming, S.: Enzyme Microb. Technvl., 2, 45 (1980). 13) Margaritis, A., Bajpai, P.: Biotechnol. Bioeng., 24, 94 (1982). 14) Negoro, tt., Kito, E.: J. Ferment. Technol., 51, References 103 (1973). Demeulle, S., Guiraud, J.P., Galzy, P.: Z. 15) Uehiyama, T.: Biochim. Biophys. Aeta, 397, 153 Allg. Mikrobiol., 21, 181 (1981). (1975). Vandamme, E.J., Derycke, D.G.: Adv. Appl. 16) Allais, J.J., Kammoun, S., Blanc, P., Girard, C., Microbiol., 29, 139 (1983). Baratti, J.C. : Appl. Environ. :l/licrobiol., 52, Margaritis, A., Bajpai, P.: Appl. Environ. Micro1086 (1986). biol., 45, 723 (1983). 17) Allais, J.J., Lopez, G. H., Kammoun, S., Baratti, Toran, D. L.,Jain, V. K., Allais, J.J., Baratti, J.: J. : Appl. Environ. Microbial Microbiol., 53, Biotechnol. Lett., 7, 527 (1985). 942 (1987). Guiraud, J. P., Gailland, J. M., Galzy, P.: Eur. 18) Mandels, M., Weber, J." Adv. Chem. Set., 95, 391 (1969). d. Appl. Microbiol. Biotechnol., 14, 81 (1982). Fleming, S.E., Grootwassink, J . W . D . : Grit. 19) Margaritis, A., Bajpai, P.: Biotechnol. Bioeng., Rev. Food Sci. Nutr., 12, 1 (1979). 24, 1473 (1982). Edelman, J., Jefford, T.G.: Biochem. J., 93, 20) Miller, G.L.: Anal. Chem., 31, 426 (1959). 148 (1964). 21) Parekh, S., Margaritis, A.: Agric. Biol. Chem., Rutherford, P.P., Deacon, A.C.: Biochem. d., 59, 1085 (1986). 126, 569 (1972). Nakamura, T., Hoashi, S.: Nippon Nogeikagaku (Received March 31, 1988)