Occurrence of two forms of extracellular endoinulinase from Aspergillus niger mutant 817

JOURNAL OF FERMENTATION AND BIOENGINEERING

Vol. 78, No. 2, 134-139. 1994

Occurrence of Two Forms of Extracellular Endoinulinase from Aspergillus niger Mutant 817 TOYOHIKO NAKAMURA, YUHI NAGATOMO, SHIGEYUKI HAMADA, YOSHIHIKO NISHINO, AND KAZUYOSHI OHTA*

Department of Biological Resource Sciences, Faculty of Agriculture, Miyazaki University, 1-1 Gakuen Kibanadai Nishi, Miyazaki 889-21, Japan Received 31 January 1994/Accepted 17 May 1994

Aspergillus niger mutant 817 was generated from A. niger 12, a strain that produces high levels of inulinases irrespective of the carbon source, by T-irradiation with 6°Co. A. niger 817 showed 4-fold higher inulinase activity than the wild-type strain 12 in the filtrate of a submerged culture with fructose. An extremely high ratio of inulinase to invertase activity (I/S ratio) of 880 was found in the culture filtrate. Two inulinases, P-IA and P-IB, were purified from the culture filtrate of A. niger 817 grown on sucrose by DEAE-Cellulofine A-500 and Q-Sepharose HP chromatographies. The enzymes were homogeneous as judged by SDS-polyacrylamide gel electrophoresis with apparent molecular weights of 70,000 for P-IA and 68,000 for P-IB. The specific activities were 350 U / m g for P-IA and 340 U/mg for P-IB, and about 3.5-fold higher than that reported for the wild-type endoinulinase P-III. The enzymes were active only toward inulin, and lacked activity toward sucrose, raft]nose or levan. The main products of inulin hydrolysis were inulotriose and -tetraose. Thus, both inulinases exhibited the endo-type mode of action on inulin. The apparent Km values for inulin were 0.48 mM for P-IA and 0.50 mM for P-IB at 40°C and pH 5.0, and lower than the 1.25 mM reported for the wildtype endoinulinase P-III.

Inulin occurs as a reserve carbohydrate in the roots and tubers of plants like Jerusalem artichoke, chicory, or dahlia. It is a polyfructan, consisting of linear /3-2, 1-1inked polyfructose chains terminated by a glucose residue attached through a sucrose type linkage (1). Such inulin sources have recently received attention as a potential feedstock for both fuel ethanol (2, 3) and fructose syrup production (4, 5). Most microbial inulinases are inducible and exo-acting enzymes, which split off terminal fructose units successively from the nonreducing end of the inulin molecule (1). These exoinulinases (/3-D-fructan fructohydrolase, EC 3.2.1.80) hydrolyzed sucrose and the fructose portion of raffinose in addition to inulin. Nakamura et al. (6) previously isolated a novel strain, Aspergillus niger 12, which produced high levels of extracellular inulinases irrespective of the carbon source. A. niger 12 secreted both exo- and endoinulinases (2,1-~-D-fructan fructanohydrolase, EC 3.2.1.7) into the culture medium (7-9). The purified endoinulinase P-Ill hydrolyzed the internal linkage of inulin to yield inulotriose, -tetraose, and -pentaose as the main products, and lacked invertase activity. Since the first report of the microbial endoinulinase P-III from A. niger 12 (7), a few fungal endoinulinases have been found in Aspergillus ficuum (5, 10), Penicillium purpurogenum (11), and Chrysosporium pannorum (12). In this article, A. niger 12 was subjected to mutational experiments by N-irradiation with 6°Co to increase the endoinulinase activity and reduce the exoinulinase activity. A mutant, strain 817, that gave 4-fold higher inulinase activity and lower invertase activity than the wild-type strain 12 in the filtrate of a submerged culture was selected. In our previous paper (2), a submerged cul-

ture of the selected mutant, A. niger 817, was successfully applied as the inulinase source in the production of high concentrations ( > 20%, v/v) of ethanol from pure inulin in combination with Saccharomyces cerevisiae. This article also describes the purification and some properties of two forms of the extracellular endoinulinase produced from A. niger 817. MATERIALS AND METHODS Induction and isolation of mutants A. niger 12 (6) was grown as the parent strain on agar slants (pH 5.5) containing 1.0% inulin, 0.5% peptone, 0.3% (NH4)2HPO4, 0.05% KC1, 0.05% MgSO4.7H20, 0.001% FeSO4.7HzO, and 2.0% agar at 30°C for 7d. The spores were collected and suspended in sterile 0.05 M phosphate buffer (pH 7.0) to give l06 spores/ml. A 10-ml portion of spore suspension was exposed to ~'-ray irradiation with 6°Co over a range of 4× l04 to 8 × l04 rads. The suspension was serially diluted with sterile 0.05 M phosphate buffer (pH 7.0), spread on agar plates of the same composition, and incubated at 30°C for 7 d. Colonies that developed quickly and were distinguishable morphologically from the wild-type were transferred to the above agar slants and further incubated at 30°C for 7 d. Several transfers were done to assess the stability of the mutants. To determine their ability to produce inulinase, the stable mutants were grown in submerged cultures at 30°C for 5 d, using a medium (pH 4.5) containing 3.0% fructose, 2.0% corn steep liquor, 1.2% NH4H2PO4, 0.07% KC1, 0.05% MgSO4-7H20, and 0.001% FeSO4. 7HzO, as previously described (7). The culture filtrates were assayed for inulinase and invertase activities. Culture conditions for inulinase production by A. niger 817 As described in Results below, A. niger mutant 817 was selected as the best inulinase-producing

* Corresponding author. 134

VoL 78, 1994 strain. Although inulin and fructose allow better inulinase production by A. niger 12 than other carbon sources (6), sucrose offers an inexpensive and readily available substrate for inulinase production in volumes of several liters. For this reason, A. niger 817 was grown in a submerged culture at 30°C for 5 d using a medium containing 3% sucrose as the carbon source and 0.5% sucrose fatty acid ester as a surfactant to increase the secretion of inulinases, as previously described (2). Assays of inulinase and invertase activities Inulinase activity [I] is commonly compared with the invertase activity IS] displayed by the same enzyme preparation, and the I/S ratio is used to characterize inulinases (1). The reaction mixture consisting of 0.5 ml of 0.5O/oo (w/v) inulin or sucrose dissolved in deionized water and 0.5ml of suitably diluted enzyme solution in 0.1M acetate buffer (pH 5.0) was incubated at 40°C for 30 min as described by Nakamura and Hoashi (13). The extracellular inulinase and invertase activities in culture filtrates were assayed by measuring reducing sugars released from inulin and sucrose, respectively. Reducing sugars were determined by the dinitrosalicylic acid method (14). One unit of inulinase activity was defined as the amount of enzyme that liberated 1.0 ffmol of fructose equivalent from inulin per min. One unit of invertase activity was defined as the amount of enzyme that hydrolyzed 1.0 ffmol of sucrose per minute. The specific activities were defined as U/mg of protein. Purification of inulinases The 5-day-old submerged culture of A. niger 817 was clarified by filtration; the culture filtrate was used as an enzyme source for purification experiments. All operations were carried out at 4°C. Any insoluble material in the dialyzed enzyme solution was removed by centrifugation at 13,000xg for 20min. Step 1. Concentration o f culture filtrate The pH of the culture filtrate (17,300 ml) was adjusted to 5.0 by 1 N HC1 or 1 N NaOH. The crude enzyme was concentrated to one-tenth of its original volume in a dialysis bag surrounded by a thick layer of dry polyethylene glycol (6000). The concentrate was dialyzed for 2 d against several changes of distilled water. The dialyzed sample was further concentrated to a final volume of 70.0 ml by ultrafiltration through a 3 x 103 molecular-weight cut-off membrane (Diaflo YM3; Amicon Inc., Beverly, MA, USA) in a stirred cell. Step 2. DEAE-Cellulofine A-500 column The concentrated enzyme was loaded onto a DEAE-Cellulofine A-500 (Seikagaku Kogyo, Tokyo) column (2.0x90cm) previously equilibrated with 0.02 M acetate buffer (pH 6.0). The column was washed overnight with the same buffer. The adsorbed enzyme was eluted at a flow rate of 1.0ml/min in a step gradient with 0.1 M, 0.2M and 0.3 M NaC1 in the same buffer (Fig. 1A). Fractions of 15 ml were collected and assayed for enzyme activities and A280. The elution profile showed only one main peak (P-I) with inulinase activity at a concentration of 0.2 M NaCI. The active fractions 78 to 91 were pooled, dialyzed against distilled water, and concentrated under reduced pressure with a collodion bag (Sartorius AG, G6ttingen, Germany) to 67.0 ml. Step 3. Q-Sepharose H P column A portion of the enzyme solution was loaded onto a prepacked anionexchange column of Q-Sepharose HP (1.6 x 10 cm) with a Fast Protein Liquid Chromatography System (Pharmacia LKB, Uppsala, Sweden). The adsorbed enzyme was eluted at a flow rate of 1.0 ml/min with a linear gradient

ENDOINULINASESFROM A. NIGER

135

of 0 to 0.4M NaC1 in 0.02 M acetate buffer (pH 6.0). Fractions of 10 ml were collected. The invertase activity displayed by an exoinulinase appeared first as a minor peak, and the inulinase activity was further separated into two peaks (Fig. 1B). Two peaks of the active fractions 17 to 18 and 20 to 22, were designated as P-IA and P-IB, respectively. To remove contaminating proteins, pooled fractions from either P-IA or P-IB were concentrated as described above, and independently rechromatographed under the same conditions. Both P-IA and PIB showed good correspondence between the protein peaks and inulinase activities (Fig. 1C). Effects of pH and temperature on activity and stability of inulinases The effect of pH was determined over the pH ranges 3.5 to 6.5 (0.1 M acetate buffer), 6.8 to 7.7 (0.1 M phosphate buffer), 8.0 to 8.9 (0.1 M Tris-HC1 buffer), and 9.0 to 10.0 (0.1 M glycine-NaOH buffer). The assays of inulinase activity were carried out in a reaction mixture containing 0.5 ml buffer of the desired pH, 0.25ml 1% inulin solution, and 0.25ml enzyme solution. To determine the pH stability, the enzyme solution was pre-incubated at various pH values at 30°C for 24h, and the residual activity was assayed under standard conditions. The effect of temperature on inulinase activities was determined under standard conditions except that the reaction mixture was incubated at temperatures from 30 to 70°C. To determine the thermal stability, the enzyme solution was treated at temperatures from 30 to 80°C for 30 min, and the residual activity was assayed under standard conditions. Effects of enzyme inhibitors and metal ions on activity of inulinases All metal solutions were prepared as chloride salts except Ag ÷, which was employed as its nitrate. The enzyme solution (0.25 ml) previously dialyzed against deionized water was pre-incubated with an equal volume of 0.1 M acetate buffer (pH5.0) containing 4.0mM of enzyme inhibitor or metal ion at 30°C for 1 h. Then 0.5 ml of a 0.5% inulin solution was added, and the reaction mixture was incubated at 40°C for 30min. Inulinase activity was thus assayed in the presence of each compound at a final concentration of 1.0 raM. The inulinase activity without any added compound was assigned a value of 100. Determination of kinetic parameters The initial velocities of inulin hydrolysis by 0.11 U/ml of purified enzyme were determined at various substrate concentrations ranging from 0.125 to 5.0mM in 0.02M acetate buffer (pH 5.0) at 40°C. The average molecular weight of inulin was assumed to be 6300 from the average degree of polymerization described below. MichaelisMenten constants (Km) and maximal velocities (l/m~x) were estimated by the method of Lineweaver-Burk (15). Analytical procedures Protein content was determined either by the method of Lowry et al. (16) or by A280, using bovine serum albumin as the standard. SDS-polyacrylamide gel electrophoresis (PAGE) was carried out in 8% (w/v) polyacrylamide gel slabs by using 25mM Tris-glycine buffer (pH8.3) containing 0.1% (w/v) SDS as described by Laemmli (17). The standard proteins (molecular weight in parentheses; Boehringer Mannheim GmbH, Mannheim, Germany) for calibration were phosphorylase b (97,400), glutamate dehydrogenase (55,400), and lactate dehydrogenase (36,500). Protein bands were detected by staining the gels with 0.1% (w/v) Coomassie brilliant blue R-250 so-

136

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FIG. 1. Column chromatography profiles of inulinases from A. niger 817. (A) DEAE-Cellulofine A-500 column chromatography of crude enzyme. Protein (8880 mg) was loaded onto a column equilibrated with 0.02 M acetate buffer (pH 6.0) and eluted in a step gradient with 0.1 M, 0.2 M and 0.3 M NaC1 in the same buffer. (B) Fast Protein Liquid Chromatography on a column of Q-Sepharose HP. (C) Rechromatography of inulinases P-IA (I) and P-IB (II) on a column of Q-Sepharose HP. Experimental conditions are given in Materials and Methods. Symbols: o , protein; ©, inulinase activity; ~, invertase activity. Line: .... , NaCI concentration. lution. High-performance thin-layer chromatography ( H P T L C ) o f the products o f inulin hydrolysis was carried out on H P T L C pre-coated silica gel 60 plates (Merck A G , Darmstadt, Germany). The plates were developed three times at r o o m temperature with a solvent system o f c h l o r o f o r m , acetic acid, and water (3 : 10 : 1, v / v / v ) . The sugar spots were visualized by spraying the plates with aniline-diphenylamine-phosphoric acid rea-

gent (18) and heating at 120°C for 5 min. Chemicals Inulin derived from dahlia tubers was obtained from Sigma Chemical Co. (St. Louis, MO, USA). The average degree o f polymerization was estimated to be 38.8 from the ratio o f glucose to fructose in an acid-hydrolyzate o f the pure inulin. Fructose, glucose, sucrose, raffinose, and levan were purchased from W a k o Pure Chemical Industries (Osaka). Inulo- and fructooligosaccharides (F2, F3, GF2, GF3, and GF4) were sup-

ENDOINULINASES FROM A . N I G E R

VoL. 78, 1994 TABLE 1. Strain

137

Comparison of enzyme activities of A . niger mutant 817 and wild-type 12a

Inulinase

Extracellular protein (mg/ml)

Invertase

Activity (U/ml)

Spec. act. (U/rag)

Activity (mU/ml)

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plied by Meiji Seika Co. Ltd. (Tokyo). RESULTS Selection of mutants Fifty-five mutants were isolated and the considerable variations in inulinase and invertase activities were observed among the mutants when grown in submerged cultures with fructose as the carbon source. A mutant designated 817 was selected on the basis of its high values for both inulinase activity and I/S ratio (Table 1). A. niger mutant 817 and wild-type 12 produced roughly the same amounts of extracellular proteins. However, A. niger 817 showed 4-fold higher inulinase activity (17.5U/ml) in the culture filtrate than the wild-type (4.6 U/ml). Conversely, A. niger 817 showed reduced invertase activity (20 mU/ml) as compared to the 87 mU/ml of the wild-type. The resulting I/S ratio of the mutant culture was 880, which is 16-fold that of the wild-type culture. The extremely high I/S ratio suggested that A. niger 817 produced mostly endoinulinases in the culture filtrate. Continual propagation during five serial transfers of mutant 817 on agar slants did not result in any loss of the enhanced inulinase activity in the submerged culture, indicating the genetic stability of the new trait. Unlike the wild-type, conidia formation by A. niger 817 was scant when grown on agar plates at 30°C for 5 d. The selected mutant A. niger 817 was employed for further study. Inulinase production by A. niger 817 in sucrose-containing medium The time courses of growth and inulinase production by A. niger 817 were previously studied in a submerged culture with sucrose as the carbon source and sucrose fatty acid ester as a surfactant at 30°C (2). In this study, A. niger 817 was grown for 5 d and the culture filtrate showed 40.0 U/ml inulinase activity and 5.0U/ml invertase activity. The I/S ratio, which was 880 in the fructose-containing medium, dropped to 7.9 in the sucrose-containing medium due to the induction of invertase activity by sucrose. The increased inulinase activity in the sucrose medium as compared to that in the fructose medium was ascribed to the stimulatory effect of sucrose fatty acid ester on microbial enzyme production, as described by Reese TABLE 2. Step Culture filtrate Dialysis and ultrafiltration DEAE-Cellulofine A-500 P-I Q-Sepharose HP P-IA P-IB

and Maguire (19). Purification of mutant inulinases Chromatography of the crude enzyme on a DEAE-Cellulofine A-500 column revealed only one main peak (P-I) with inulinase activity, which had a high I/S ratio of 72. The inulinase activity of P-I was further separated into inulinases P-IA and P-IB on a Q-Sepharose HP column, each of which was rechromatographed to render it free from invertase activity. Table 2 summarizes the purification steps. The inulinases P-IA and P-IB were purified 177- and 170-fold over the culture filtrate, with the yields of 18.2 and 18.9~, respectively. The purified inulinases had specific activities of 350U/mg for P-IA and 340 U/rag for P-IB. Criteria of enzyme purity and molecular weights Inulinases P-IA and P-IB showed single Coomassiestained protein bands with mobilities corresponding to molecular weights of 70,000 and 68,000, respectively, in SDS-PAGE (Fig. 2). Effects of pH and temperature on activity and stability of inulinases Inulinases P-IA and P-IB showed maximum activity at the same pH of 5.3. Inulinase activities were stable over a pH range from 5.0 to 7.0 for P-IA and a broad pH range from 3.5 to 9.0 for P-IB. The inulinases showed maximum activity at 50°C for P-IA and between 50 and 55°C for P-IB. The two enzymes gave nearly identical stability curves at temperatures from 30 to 80°C. Both enzymes were stable up to 50°C with no loss of activity, and retained 86 and 82%, respectively, of the original activity after heating at 60°C. Complete inactivation was observed when the enzymes were treated at 80°C. Effects of enzyme inhibitors and metal ions on activity of inulinases The effects of various potential enzyme inhibitors and metal ions on the activity of inulinases PIA and P-IB were examined. Mn 2+ appeared to stimulate the activity of the inulinases. N-Bromosuccinimide, Ag ~ or Hg 2÷ caused 100% inactivation of inulinase activity, and an appreciable loss of activity was observed with p-chloromercuribenzoic acid, EDTA or Fe 3+. Inhibition of activity by thiol-group-blocking agents such as p-chloromercuribenzoic acid and HgC12 suggested the possible involvement of sulfhydryl groups in the active

Purification of inulinases P-IA and P-IB from A . niger 817 Total activity (U)

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FIG. 2. Sodium dodecyl sulfate-polyacrylamidegel electrophoresis of the purified inulinases P-IA and P-IB from A. niger 817. The enzymes were subjected to 8.0% SDS-PAGE at pH 8.3. Protein was visualized by Coomassie brilliant blue R-250 staining. Lane 1, Standard proteins (from top): phosphorylase b (97,400), glutamate dehydrogenase (55,400), and lactate dehydrogenase (36,500); lane 2, P-IA; lane 3, P-lB. The molecular weight of each standard is indicated beside the corresponding band. site o f the enzymes, as described for inulinases from Penicillium sp. 1 and A. niger 12 (9). The loss o f activity by the oxidation of inulinases P - I A and P-IB by Nbromosuccinimide suggested that t r y p t o p h a n was also involved in the active site o f the enzymes. Substrate specificity of inulinases The actions o f the inulinases were tested on a variety o f fructose-containing oligo- and p o l y s a c c h a r i d e s - - i n u l i n , sucrose, 1kestose (GF2), nystose (GF3), raffinose, and levan. The reaction mixture, consisting o f 0.5 ml o f each substrate (0.5%) and an equal volume o f the enzyme solution ( 0 . 1 6 U / m l ) , was incubated at 40°C for 3 0 m i n . Under these conditions, both inulinases P - I A and P-IB were active t o w a r d inulin, but did not release any detectable reducing sugars from raffinose and levan when the incub a t i o n time was increased to 3 h, nor from sucrose, 1kestose, and nystose even after 5 0 h o f incubation. Thus, the inulinases were specific t o w a r d 2,1-~-o-fructosidic linkages o f inulin and did not hydrolyze the small fructo-oligosaccharides 1-kestose and nystose. Time courses of inulin hydrolysis by inulinases and reaction products Figure 3A shows the time courses o f inulin hydrolysis by inulinases P - I A and P-IB. The extents o f inulin hydrolysis (%) were calculated as (/~mol o f reducing sugars released//~mol o f fructose equivalent o f i n u l i n ) × 100. P - I A and P-IB displayed fast hydrolysis o f inulin during the first 6 h o f incubation, and then gradually hydrolyzed inulin to the extents o f 53 and 51% after 12 h, respectively. No further appreciable increase in reducing sugars released by the inulinases from inulin was observed due to the lack o f invertase activity. During the course o f inulin hydrolysis by inulinase PI A (Fig. 3A), aliquots (2/L1) o f the reaction mixture were periodically withdrawn and analyzed for the hydrolysis products by thin-layer c h r o m a t o g r a p h y in order to characterize the mode o f action o f the enzyme (Fig. 3B). Ran-

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FIG. 3. Action pattern of inulinases P-IA and P-IB from A. niger 817 on inulin. (A) Time courses of inulin hydrolysis with inulinases P-IA (©) or P-IB ( • ) . The reaction mixture containing 5 ml of 4.0°//ooinulin in deionized water and 5 ml of P-IA (0.25 U/ml) or P-IB (0.21 U/ml) in 0.I M acetate buffer (pH 5.0) was incubated at 40°C. The reducing sugars produced were determined as described in Materials and Methods. (B) Thin-layer chromatogram of inulin hydrolyzate with inulinase P-IA. The reaction mixtures of inulin hydrolysis by inulinase P-IA in Fig. 3A were subjected to thin-layer chromatography. Inulo- and fructo-oligosaccharides were used as standards (S). d o m attack by inulinase P - I A on inulin yielded a series o f oligosaccharides with a degree o f polymerization o f 3 and above during the first 6 h o f incubation. Inulotriose (F3) and -tetraose (F4) were liberated as the main p r o d ucts after 9 to 12 h o f incubation. Only after 48 h o f digestion did small amounts o f fructose, sucrose, and inulobiose begin to a p p e a r in the reaction mixture, when inulinase P - I A further but weakly hydrolyzed F 4 to F 3 and F, GF4 to F3 and G F , and F5 to F3 and F2. Similar c h r o m a t o g r a p h i c patterns were observed for the inulinase P-IB (data not shown). Kinetic parameters of inulinases The affinity o f the purified enzymes for inulin was examined with a Lineweaver-Burk plot. Both enzymes showed MichaelisMenten-type kinetics with inulin as the substrate at 40°C and p H 5.0. Inulinases P - I A and P-IB exhibited apparent Km values o f 0.48 and 0 . 5 0 m M , respectively. The Vm~x values were 109 and 139 p m o l / m i n / m g o f protein for P - I A and P-IB, respectively. DISCUSSION In previous papers (7, 9), an extracellular endoinulinase P - I I I was purified from A. niger 12 and charac-

ENDOINULINASES FROM A. NIGER

VoL 78, 1994 terized as a 6.6-kDa enzyme with a specific activity o f 101 U / m g . The endoinulinase P - I l l was a glycoprotein with 6.7% c a r b o h y d r a t e (9). A . niger m u t a n t 817 induced in this study showed 4-fold higher inulinase activity in the culture filtrate t h a n the wild-type strain 12. Due to the c o n c o m i t a n t reduction in invertase activity, the I / S ratio o f 880 was significantly higher than that in the wild-type culture and the values reported for other endoinulinase-producing fungi (11, 12). While the wildtype A . niger 12 apparently secreted a single endoinulinase P - I l l , as revealed by the elution profile (7), two forms o f the extracellular inulinase, P - I A and P-IB, were purified from A. niger 817. Inulinases P - I A and PIB were h o m o g e n e o u s as j u d g e d by S D S - P A G E , and had estimated molecular weights o f 70,000 and 68,000, respectively, which were similar to the value reported for the wild-type endoinulinase P - I l l (9). However, inulinases P - I A and P-IB differed from the endoinulinase P - I l l in their a p p r o x i m a t e l y 3.5-fold higher specific activities (350 U / m g for P - I A and 340 U / m g for P-IB), which m a y explain the enhanced specific activity o f inulinase in the culture filtrate o f A . niger 817 (Table 1). In addition, the Km values o f 0.48 and 0.50 m M for inulinases P - I A and P-IB were lower than the reported value o f 1.25 m M for endoinulinase P - I l l (7). A l t h o u g h treatment o f endoinulinase P - I l l at 50°C for 30 min resulted in a 22% loss o f the activity (7), inulinases P - I A and P-IB were stable with no loss o f activity after treatment at 50°C. The improved properties o f inulinases P - I A and P-IB, including specific activity, affinity for inulin and thermal stability, might be ascribed to the alteration o f the molecular structure o f the wild-type endoinulinase P - I l l by the mutation. In c o m m o n with the wild-type endoinulinase P - I l l (7), inulinases P - I A and P-IB lacked activity t o w a r d sucrose and raflinose, and hydrolyzed inulin by r a n d o m attack to inulotriose and higher oligomers, suggesting that P - I A and P-IB are endo-acting enzymes and that at least 4 fructose units are required for p r o p e r binding to the enzymes. The two enzymes resembled one another in the properties investigated except for small differences in their electrophoretic mobilities. The occurrence o f the two forms o f endoinulinase in A . niger 817 m a y be attributed to a variety o f post-translational modification steps during cultivation, as described for multiple forms o f Aspergillus awamori glucoamylase (20). This could be caused by glycosylation, limited proteolysis, or both. Structural characterization needs to be conducted to clarify the relationship between the two enzymes. The present work shows that A. niger m u t a n t 817 produced two forms o f highly active endoinulinase extracellularly in a submerged culture with sucrose as the carbon source. The enhanced inulinase activity with high I / S ratio exhibited by A . niger 817 m a y be industrially advantageous for the large-scale saccharification o f inulin in the p r o d u c t i o n o f ethanol and fructose syrups from inulin-containing agricultural crops.

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