properties of flavobacterium odoratum ku isoamylase

Properties of Flavobacterium odoraturn KU Isoamylase

S. Hizukuri, T. Kozuma, H. Yoshida, J. Abe, Kagoshima (Japan), K. Takahashi, M. Yamamoto, and N. Nakamura, Shizuoka (Japan)

Isoamylase was purified from the culture filtrate of Flavobacterium odoratum KU to a single protein by starch adsorption and gel-permeation chromatography on a Toyopearl HW-55s column. The molecular weight and isoelectric point of the enzyme were found to be 78000 and 8.7, respectively. The enzyme was stabilized and stimulated by Ca2+. Its optimum temperatures for activity were 50 and 45°C in the presence and absence of CaCl, (lmM), respectively, and the optimum pH was 6.0 in the presence of CaC1, (1mM). The enzyme debranched amylopectin and glycogen completely. The Km and It values of the enzyme for amylopectins and glycogens were similar but were slightly lower than the values for the P-limit dextrins of these polysaccharides. The ko values for the @-limit dextrins of potato amylopectin and rabbit liver glycogen were slightly lower and higher than those of the mother polysaccharides, respectively. Pullulan was practically not hydrolyzed. Flavobacterium isoamylase did not hydrolyze (1~6)-a-~-glucosylcyclodextr~ris (CDs), and it debranched branched CDs having longer (l-4)-a-glucan side chains than maltotriosyl residues, and maltosyl CDs with difficulty. The enzyme practically did not hydrolyze raw starches but it enhanced the raw-starch digestion of glucoamylases synergistically.

Die Eigenschaften von Flavobacteriurn odoraturn KU isoamylase. Aus dem Kulturfiltrat von Flavobacterium odoraturn KU wurde die Isoamylase zu einem Einzelprotein durch Starkeadsorption und Gel- Permeationschromatographie auf einer Toxopearl-HW-55s-Saule ge- reinigt. Das Molekulargewicht und der isoelektrische Punkt des En- zyms wurde mit 78000, bzw. 8,7 festgestellt. Das Enzym wurde stabili- siert und durch Ca2+ stimuliert. Seine optimalen Temperaturen fur die Aktivitat betrugen 50 und 45°C in Gegenwart und Abwesenheit von CaC12 (ImM), und der optimale pH-Wert betrug 6,O in Gegen- wart von CaCI, (1mM). Das Enzym entzweigt Amylopektin und Gly- cogen vollstandig. Die Km- und ko-Werte des Enzyms fur Amylopec- tine und Glykogene waren ahnlich aber wenig geringer als die Werte fur P-Grenzdextrine fur diese Polysaccharid. Die It-Werte fur die 0-Grenzdextrine von Kartoffel-Amylopektin und Kaninchen-Le- berglykogen waren etwas niedriger, aber hoher als diejenigen der an- deren Mutterpolysaccharide. Pullulan wurde praktisch nicht hydroly- siert. Flavobacterium-isoamylase hydrolisierte nicht (1+6)-a-~-Glu- cosyldextrine (CDs), jedoch entzweige verzweigte CDs mit langeren (1,4)-a-Glucan-Seitenketten als Maltotriosylreste und Maltosyl-CDs unter Schwierigkeiten. Das Enzym hydrolysierte praktisch keine Rohstarken, jedoch steigerte es synergistiscb den Rohstarkeabbau durch Glucoamylasen.

1 Introduction Isoamylase (EC 3.2.1.6s) hydrolyzes specifically a-( 1-6)-

linkages in glycogen and starch. The enzyme was first found on yeast cells by Maruo and Kobayashi [l], and then was found in the culture filtrate of Pseudornonas by Harada et al. [2] and Tognoni et al. [3], and some other organismus [4-81 including Navobacterium [9-lo]. The DNA sequences of chromosomal DNA of Pseudomonas enzymes have been reported by several groups [3, 11, 121. The enzyme is useful for the analysis of the fine structures of glycogen [13] and starch [14] and also for the production of maltose and other malto-oligosaccharides from starch. Bacterial pullulanase (EC 3.2.1.41), which also hydrolyzes the a-(l-+6)-linkages of amylopectin but hardly the linkages of glycogen, is also utilized for the same purpose. Since the debranching activity of isoamylase for amylopectin and glycogen is remarkably stronger than that of pullulanase [15], it has considerable merit in the starch industry. However, the optimum pH of the commercially available Pseudornonas isoamylase is more acidic (pH 3.5) than other common amy- lolytic enzymes. Consequently, the enzyme is hardly used to- gether with other enzymes such as p-amylase and glucoamylase in the cases of production of maltose and glucose from starch. Now we report an isoamylase having its optimum pH at 6.0 which was produced by Flavobacterium odoratum KU isolated from soil.

2 Materials and Methods 2.1 Materials

A strain Flavobacterium odoraturn KU was used throughout this study. The cultivation of the bacterium was cultivated as

reported elsewhere [16]. After cultivation, the cells were re- moved by centrifugation and its supernatant was used as a crude enzyme solution.

Soluble waxy rice starch, the substrate o f the enzyme, was prepared by steeping the starch in 0.7N HCl at 3OoC for 30h. Potato amylopectin was prepared by using 1-butanol as an amylose precipitant [17]. Oyster and rabbit liver glycogens were purchased from Sigma Chem. Co. (St. Louis, Mo.) and they were purified by dissolving in water and precipitating with ethanol twice. The /?-limit dextrins of amylopectin and glycogen were prepared as described previously [13]. a-Cy- clodextrin (a-CD), 6-0-a-glucosyl-a-CD (Gl-a-CD), and 6-0-a-glucosyl-p-CD (G1-p-CD) were donated from Ensuiko Sugar Refining Co. (Yokohama), /?-CD and y-CD were the products of Nihon Shokuhin Kako Co. (Shizuoka), and Mer- cian Co. (Tokyo), respectively, and the branched CDs with side chains of various lengths of a-(1+4)-glucan chains were prepared in our laboratory as described elsewhere [IS-201. All the CDs were purified by HPLC of using Hibar LiChrosob RP-18 (25Ox4mm, Merck Darmstadt, Germany) and Asa- hipak GS-320 (500 x 7.6mm, Asahi Kasei, Tokyo) columuns as reported [21]. Crystalline Pseudomonas isoamylase and Kleb- siella pullulanase were obtained from Hayashibara Biochemi- cal Laboratories (Okayama), and Apergillus sp. K-27 glucoamylase [22] and crystalline sweet potato /?-amylase [23] were prepared as described elsewhere. Waxy corn and corn starches were provided by Sanwa Denpun Kogyo Co. Ltd., rice [24], sweet potato [25], lotus [26], water chestnut [27], and nagaimo [28] starch specimens were as described previously. Potato starches was donated from Dr. Yoshioka (Hokkaido Agricul- tural Experimental Station).

Starch/Starke 48 (1996) Nr. 7/8. S . 295-300 0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim. 1996 0038-9056/96/0707-0295$10.00+.25/0 295

2.2 Assay of isoamylase Reducing-sugar method

The enzyme solution (2OOpl), which was appropriately diluted with 50mM acetate buffer, pH 6.0, containing 1mM CaClz and 0.005% bovine serum albumin (BSA),was added to 1.25% potato amylopectin solution (800~1, dissolved in the same buffer solution as in the enzyme solution), and incubated at 45°C for 15min. The enzyme was inactivated by heating the mixture in a boiling water bath for lmin and the reducing power was determined by the Park-Johnson method with modifications [29,30] or by Somogvi’s method [31] with the ex- tended heating period (30min) and the use of Nelson’s reagent [32]. One unit (reducing unit, RU) of activity was defined as the amount of enzyme which increases the reducing power equivalent to lpmole of glucose min-’ under the above conditions. One RU corresponds to 200 iodine colorimetric units (CU) as described below.

Iodine method The method was slightly modified from that described by

Yokobayashi et al. [33]. The appropriately diluted enzyme solution (100~1) in 50mM acetate buffer, pH 6.0, containing 1mM calcium chloride and 0.005% BSA was added to a mixed solution of 1% soluble starch (500pl) and OSM acetate buffer, pH 6.0 containing 7mM CaC12 (lOOpl), the mixture was incubated at 45°C for 15min, and then the reaction was stopped by the addition of 0.1N hydrochloric acid (700~1). The reaction mixture (150~1) was mixed with 0.2% iodine -2% iodine iodide solution (200~1) and water was added to a volume of 5ml. After standing for 15min at room temperature, the absorbance at 580nm was measured using a spectrophotometer (Shimadzu UV-210A). One unit (CU) of the enzyme activity was defined as the amount of enzyme which increased the absorbance 0.1 per h.

2.3 Determination of Km, Vmax and Ki Kinetic parameters were determined from the data ob-

tained from at least three experiments by the Lineweaver-Burk method and are expressed as mean values. The inhibitor con- stant (Ki) was determined by the same method in the presence of 5mM of each inhibitor.

2.4 Assay of glucoamylase activity One unit of the glucoamylase was defined as the amount of

enzyme which produced lpmole of glucose per min at pH 5.0 and 40°C from soluble starch (1Yo). The resulting glucose was determined by Somogvi’s method [31] using Nelson’s reagent [32]. Total carbohydrate was measured by the phenol-sulfuric acid method [34].

2.5 Digestion of raw starch Starch (250mg dry weight) was suspended in 50mM acetate

buffer (5ml), pH 6.0 (for isoamylase only) or pH 5.0 (for com-

Table I. Purification of Fluvobucterium isoamylase.

bined action with glucoamylase), containing 2mM CaC12 and appropriate amount(s) of enzyme(s) and incubated at 40°C. The reaction mixture was occasionally (at intervals of approx- imately 15min) shaken by hand. The mixture (0.5ml) was taken out at proper time intervals, and the reaction was stopped by the addition of 1N HCl (0.5ml). After dilution of the mixture to 5ml with water, it was centrifuged to remove undi- gested starch. The solubilized starch was measured by the phenol-sulfuric acid method [34].

2.6 lsoelectric focusing Isoelectric focusing was performed at 7OC using a pre-cast

polyacrylamide-gel plate, Servalyt Precotes (Serva, Heidel- berg) pH 3-10, according to the manufacturer’s instruction. The enzyme solution ($1) was loaded and pre-focused at 5.5W for 30min, the focused at 5.5W for 90min. The protein was detected by Comassie brilliant blue staining. A PI calibra- tion kit 3-10 (Pharmacia, Uppsala) was used as a standard.

2.7 SDS-electrophoresis SDS polyacrylamide gel electrophoresis was carried out on

a 12.5% gel by the method of Laemmli and Favre [35] using low-molecular weight kit (LMW kitE, Pharmacia, Uppsala).

2.8 Determination of protein

or by absorbance at 280nm. Protein was determined by the method of Lowry et al. [36]

3 Results and Discussion

3.1 Molecular structure of the enzyme The enzyme was purified to a homogeneous protein by

SDS-PAGE (Fig. 1) through the starch absorption and gel- permeation chromatography on Toyopearl HW55S (Table 1). Richard et al. [9] used glycogen bound ConA-sepharose for Flavobacterium enzyme and Kato et al. [37] used amylose for Pseudomonas enzyme. However, starch-granule adsorption was found to be the most convenient. Recently, Fang et al. [38] also used starch for affinity binding of the enzyme.

The molecular weight was suggested to be 78000 by SDS- PAGE (Fig. 2), which was smaller than that (121000) of other Flavobacterium sp. [lo] and that (80800) of Pseudomonas de- duced from cDNA [ll], but was larger than that (65000) of al- kalophilic strain of Bacillus sp. [7]. The N-terminal amino acid sequence of the enzyme was determined as Ala-Ile-Pro-Asn- Lys-Leu-Gly-Ala-Ala-Tyr-Asp-Ala-Thr-Lys- by Edman degra- dation. The PI of the enzyme was found to be 8.70 (Fig. 3), which differed greatly from that (4.4) of Pseudomonas enzyme 1331.

Step Volume Protein Activity

Total Specific Recovery (ml) ( n d

KJ*) (U*/mg) (010)

Crude 3760 940 337300 360 Starch adsorption 40 13.5 172000 12700

TOYOPEARL HW-55s 1.2 1.28 97800 76400

100 51 29

* 1U = 0.1 ASsO/h

296 Starch/Starke 48 (1996) Nr. 718, S . 295-300

Fig. 1. Sodium dodecyl sulfate gel electrophoresis of Fluvobucterium odorutum KU isoamylase.

\ 78000)

\ 6

11 1 0 0.2 0.4 0.6 0.8 10 1.2

Mobility Fig. 2. Estimation of molecular weight of Fluvobucterium odorutum KU isoamylase by sodium dodecyl sulfate gel electrophoresis. Standards: 1, phosphorylase a (MW 97400); 2, bovine serum albumin (MW 66000); 3, ovalbumin (MW 45000); 4, glyceraldehyde 3-phos- phate dehydrogenase (MW 36000); 5, tripsinogen (MW 24000); 6, myoglobin (MW 172000).

3.2 Optimum conditions for stability and activity of the enzyme and the effect of various materials

The enzyme was fully stable up to 45OC and showed 90% activity at 5OoC at pH 6.0 for lOmin in the presence of Ca2+, but it showed only 77% activity at 45OC and pH 6.0 and was completely inactivated in the presence of EDTA (1 mM). The enzyme was fully stable in the ranges of pH 5.0-7.0 and 5.5- 6.3 at 45OC for lOmin, in the presence and absence of CaC12 (ImM), respectively. It was greatly inhibited by Cu2+ and Hg2+, and partially by Zn2+, p-chloromercuribenzoate, and sodium dodecylsulfate (Table 2). A sulfhydryl group appears to be in- volved in the active site. These responses were not the same as those reported by Sato and Park [lo], because their enzyme

9.0 1 \

::I 8.0

1 2 3 4 5

Distance from cathode (cm) Fig. 3. Isoelectric focusing of Fluvobacterium odorutum KU isoamylase.

Table 2. Effects of Metal Ions and Organic Reagents on the Activity.

Ion and reagent ( h M ) Relative activity (Yo)

Control Cd2+ co2+ cu2+ Fe2+ Fe'+

Hg2+ (O.1mM) Hg2+ Mg2+ Mn2+ Ni2+ Pb2+ Zn2+

p-Chloromercuribenzoate Phenylmethanesulfonyl fluoride

Sodium dodecyl sulfate N-Bromosuccinimide

100 100 105

4 100 100 100

3 100 100 100 100 63 88

104 87

101

was not inhibited greatly by Cu2+ (72O/o), was slightly inhibited Mn2+ and was slightly activated by NiS04 and BaC12 but not by CaC12.

3.3 Effects of temperature and pH on the activity The enzyme exhibited maximum activity at 45OC and 5OoC

in the absence and presence of CaCI2 (1mM) at pH 6, respectively (Fig. 4). The optimum pHs for the activity were at 6.0 and between 4.5-6.0 in the absence and presence of 1mM CaC12, respectively, as shown in Fig. 5 . Below 4OoC, the activity was not influenced by the absence or presence of CaC12. Above 45OC, the activity gradually decreased and almost no activity was found at 55OC in the absence of CaC12, while activity was maintained up to 6OoC in the presence of CaC12. These proper-

Temperature (C) Fig. 4. Temperature optima for Fluvobacterium odorutum KU isoamylase in the presence (0) and absence (0) of calcium chloride ( h M ) .

Starch1Starke 48 (1996) Nr. 718, S . 295-300 2 97

4 5 6 7 8 9

PH Fig. 5. pH-optima for activities of Flavobacterium isoamylase in the presence (0) and absence (0) of CaCI2 (1mM).

ties of the enzyme suggest that the enzyme is useful for the industrial production of glucose, maltose and other linear mal- tooligosaccharides by cooperation with glucoamylase, P-amylase, and other enzymes.

3.4 Action and kinetic properties The enzyme hydrolyzed all the branch linkages of amy-

lopectin (Fig. 6 ) and glycogen (data not shown) but not (1+4)- a-linkages, and the final products were completely hydrolyzed with P-amylase. The kinetic properties of the Fla- vobacterium and the commercial Pseudomonas isoamylases were comparatively characterized (Table 3). The Flavo- bacterium enzyme had similar Km and ko values for amylopectin and glycogen, while the Pseudomonas enzyme showed higher (3340%) ko values for glycogen than amylopectin. Both enzymes had higher Km values for /?-limit dextrins of amylopectin and glycogen than for the mother polysaccharides. Flavobacterium enzyme showed slightly lower and higher ko values for P-limit dextrin of potato amylopectin and rabbit liver glycogen than those of their mother polysac-

0 1 2 3 4 5 6 Time (hr)

Fig. 6. Time-courses of hydrolysis of waxy rice starch with Fla- vobacterium isoamylase. Conditions: Same as standard assay conditions except substrate concentration 2.5mg/ml. Key: 0 and 0, debranching with 0.3 and 0.03RU/mg, respectively; 0 and ., DP by 0.3 and 0.03RU/mg, respectively.

charides, respectively, while Pseudomonas enzyme had higher ko values for /?-limit dextrins than for their respective mother polysaccharides. The catalytic efficiency, reflected by kolKm, indicated that glycogen was a better substrate than amylopectin. Both enzymes were scarcely active for pullulan. These properties were similar to those reported by early workers [9, 1.51.

To clarify the substrate specificity of isoamylase, the Km and ko values for branched cyclodextrins with varied side- chain lengths were measured (Table 4). The ko value was ex- tremely low for G2-CDs and much higher for cyclodextrins with longer side chains up to maltotetraose for both enzymes. The Flavobacterium enzyme showed an increased ko value with increasing side-chain length but the Pseudomonas enzyme showed the highest activity for G3 side chain in the series of a-cyclodextrin. Both enzymes gave the highest ko for G3-P-CL among the substrates tested. It was surprising that

Table 3. Comparison of Kinetic Parameters Between Flavobacterium and Pseudomonas isoamylases on Branched a-Glucans.

Substrate Flavo bacterium Pseudomonas

Km k0 ko/Km x lo-' Km k0 kO/Km x (mM*) ( S - 7 (mM- 'd ) (mM) ( m M - W )

Amylopectin Rice 0.0.5 580 11.6 0.04 600 15.0

Potato 0.05 5.50 11.0 0.03 530 17.7 Potato /?-limit dextrin 0.17 480 2.8 0.06 690 11.5

Oyster 0.04 540 13.5 0.03 740 24.7 Rabbit liver 0.03 550 18.3 0.02 800 40.0

Rabbit liver /?-limit dextrin 0.07 580 8.3 0.04 1100 27.5

Glycogen

Pullulan 7.29 0.76 1 10-4 3.15 1.23 4 10-4

* mM of (1+6)-a-linkage.

Table 4. Comparison of Kinetic Parameters Between Flavobacterium and Pseudomonas isoamylases on Branched Cyclodextrins. ~~ ~~ -

Substrate Flavobacterium Pseudomonas

Km k0 kO/Km Km k0 ko/Km (mM) ( s - 9 (mM) 6 - I )

G2-a-CD 3.1 0.43 0.14 32.6 2.10 0.06 G,-a-CD 14.6 106 7.2 21.0 94.3 4.5 G4-a-CD 6.3 113 18.0 11.4 63.8 5.6 G5-a-CD 11.3 124 11.0 11.8 87.4 7.4 GZ-P-CD 9.1 3.1 0.34 2.3 2.36 1.0 Gj-P-CD 9.6 413 43.1 4.5 269 59.9 GZ-y-CD 6.3 13.3 2.1 2.7 4.67 1.7

298 Starch/Stirke 48 (1996) Nr. 7/8, S . 295-300

the Flavobacterium and Pseudomonas enzymes showed the lowest and the highest Km values for G*-a-CD, respectively. From the comparisons of Km values of three cyclodextrins with G2 side chain, it was suggested that the Flavobacterium enzyme had increasing affinities in the order of B-CD, y-CD and a-CD, while the order for Pseudomonas enzyme was a-CD, )I-CD, and p-CD. Accordingly, the ring size of CD also affected the action of both enzymes differently. This suggests that the enzyme activity is influenced by slight conforma- tional variations of amylopectin or glycogen in solution. The Km values were in the order of G+Gs>G4>G2 for the Fla- vobacterium enzyme and of G2>G3>G5>G4 for the Pseudo- monas enzyme in the a-CD series. The best substrate for both enzymes was found to be G3-p-CD among these branched CDs.

The CDs and their 6-0-a-glucosylated CDs competitively inhibited the hydrolysis of waxy maize starch by Fla- vobacterium enzyme (Fig. 7). The CDs with increased ring size gave the smaller inhibitor constants of the Flavobacterium and Pseudomonas isoamylases (Table 5), indicating greater affinities with increasing ring size. This was out of accordance with Km for G2-a-CD of the Flavobacterium enzyme. The addition of a glucosyl group at C-6 of the a- and P-CDs reduced the inhibitor constants of the Flavobacterium enzyme but not that for y-CDs. This may indicate that the active site of the Fla- vobacterium enzyme recognizes the T-shape structure of the substrate. A slight increase of Ki by glucosylation of y-CD for the Flavobacterium enzyme may be due to the resulting defor- mation of the ring structure caused by the addition of a glucosyl residue because the ring structure of y-CD is fairly flex- ible. However, the increase of inhibition due to the binding of a glucosyl side-chain was not observed with the Pseudomonas enzyme.

3.5 Raw-starch digesting activity The Flavobacterium isoamylase showed a very weak activity

for raw starch digestion, similarly to the Pseudomonas enzyme [39]. It hydrolyzed much more waxy corn starch but hardly hydrolyzed potato starch (Fig. 8). Although the isoamylase had the weak raw-starch-digesting activity, relatively small amounts of the enzyme stimulated the raw-starch-digesting activity of Rhizopus delemar glucoamylase GIII, which had a starch-binding site and the strong activity for raw starches [40] (Fig. 9). This cooperative action with the glucoamylase was to be reported synergistic in the case of Pseudomonas isoamylase [41] and was observed also in other forms of glucoamylases of Rhizopus delemar GI and GI1 [40] and that of Aspergil- lus K-27 [22] irrespective of whether or not it had a starch- binding site (Fig. 10). The raw starch digestion by the cooperative action with glucoamylase was observed on a wide variety of starches a3 shown in Fig. 11. The waxyvarieties of rice and maize were the most susceptible, suggesting that the amylose molecule may make a resistant structure in the starch granule, although amylose itself is a poorer substrate for glucoamylase than amylopectin because of having fewer non-reducing residues. Rice starch was the most susceptible among the normal starches tested, and this is at least partly due to being the smallest granule. The raw-starch digesting action suggests that isoamylase is useful for the industrial production of ethanol and other materials by raw starch-digesting process.

Time (hr) Fig. 8. Hydrolysis of raw waxy corn (0) and potato (A) starches with Flavobacterium KU isoamylase. Conditions: Substrate 5%, isoamylase 0.06RU/mg.

,.,' -6 -4 -2 0 2 4 6 8 10

I/S (ml/mg)

Fig. 7. Double-reciprocal plots of inhibition of Flavobacferium odoraturn isoamylase by a-, p-, and y-CDs (5mM). Conditions: Standard assay conditions but using waxy corn starch as substrate at indicated concentration.

Table 5. Inhibitor Constants (Ki) of Cyclodextrins and 6-0-a-Gluco- syl Cyclodextrins for Isoamylases.

Ki (mM) I ' l o 20 50 100

lsoamylase (RU/g starch)

Fig. 9. Synergistic action of Flavobacferium KU isoamylase and Rhizo- pus delemar glucoamylase GI11 for raw waxy corn starch. The starch (5%) was hydrolyzed at pH 5.0 and 4OoC for 8h with glucoamylase 0.06U/mg and the indicated amounts of isoamylase. a) = (A-(B+C)/B+C)x 100, where A is the hydrolysis with cooper- ated action with isoarnylase and glucoamylase, B and C are the hydrolysis with a sole action of isoamylase and glucoamylase, respectively.

Flavobacterium Pseudomonas ~

a-CD 12.9 GI-a-CD 5.3

8-CD 4.3 GI-P-CD 2.9

7-CD 1.7 GT-Y-CD 2.0

3.6 5.0 1.4 1.5 0.6 0.7

299 StarchlSt8rke 48 (1996) Nr. 718, S. 295-300

lWt n n I l l $, 40 I

20

' 3 8 3 8 3 8 3 8 ( h ) Rdakwlu Rdekmuv Rolekmnar &K-27

GI GI 01

Fig. 10. Synergistic actions of Flavobacterium KU isoamylase and some glucoamylases for raw waxy corn starch. Substrate (5%) was hydrolyzed with glucoamylase (0.06U/mg) and isoamylase (0.02RU/ mg). Key: ., hydrolysis with glucoamylase; 0, hydrolysis with isoamylase; 0, synergistic effect.

Urlaub, H.. and G. Wober: FEBS Lett. 57 (1975), 1-4. Richard. M. E., D. J. Manners, and J . R. Stark: Carbohydr. Res. 76

Sato, H. H., and E K. Park: Starch/Starke 32 (1980), 132-136. Amemura, A. , R. Chakraborty, M . Fujita, 7: Noumi, and M. Futai: J. Biol. Chem. 263 (1988), 9271-9275. Chen, J. H., 2. K Chen, i? E Chow, C. J. Chen, S. 7: Tan, and W: H. Hsu: Biochim. Biophys. Acta 1087 (1990), 309-315. Rani, M. R. S., K. Shibanuma, and S. Hizukuri: Carbohydr. Res.

Hizukuri, S., and Y. Maehara: Carbohydr. Res. 206 (1990), 145- 159. Yokobayashi, K., H. Akai, 7: Sugimoto, M. Hirao, K. Sugimoto, and I: Harada: Biochim. Biophys. Acta 293 (1973), 197-202. Takahashi, K., J. Abe, 7: Kozuma, M. Yoshida, N. Nakamura, and S. Hizukuri: Enzyme Mikrobiol. Technol. (1991), in press. Takeda, E, S. Hizukuri. and B. 0. Juliano: Carbohydr. Res. 148

Abe, J.. S. Hizukuri, K. Koizumi, E Kubota, and 7: Utamura: Carbo- hydr. Res. 176 (1988), 87-95. Hizukuri, S., J. Abe. K. Koizumi, E Okada, I: Kubota, S. Sakai, and I: Mandai: Carbohydr. Res. 185 (1989), 191-198.

(1979), 203-213.

227 (1992). 183-194.

(1986), 299-308.

n s

$, 40

20

Y .l 60

I

38 38 38 38 38 3 8 38 3 8 3 8 ( h ) waxy y x y Rice sweet -water Nagaim0 COm We potato chestnut Potato

Fig. 11. Hydrolysis of various raw starches by the cooperative action of Flavobacterium KU isoamylase and Rhizopus delemar GIII. Condi- tions and keys, see Fig. 10.

4 Conclusion

Flavobacterium odoratum KU isoamylase has several mer- its for industrial production of glucose, maltose and oligosaccharides from starch by its stability, optimal temperature and pH for activity, and high stimulation o f raw-starch digestion by glucoamylase.

Bibliography Maruo, B., and 7: Kobayashi: Nature 167 (1951), 606-607. Harada. T., K . Yokobayashi, and A . Misaki: Appl. Microbiol. 16

Tognoni. A. , P. Carrera. G. Galli. G. Lucchese. B. Camerini, G. Grandi: J. Gen. Microbiol. 135 (1989), 37-45. Gunja-Smith, 2.. J. J. Marshall, E. E. Smith, and W J . Whelan: FEBS Lett. 12 (1970), 96-100. Jeanningros, R., N. Creuzet-Sigar, C. Frixon, and J . Cattaneo: Bio- chim. Biophys. Acta 438 (1976), 186-199. Spencer-Martins, I . : Appl. Environ. Microbiol. 44 (1982), 1253- 1257. Ara, K., K. Saeki, and S. Ito: J. Gen. Microbiol. 139 (1993), 781- 786.

(1968), 1439-1444.

[20] Hizukuri, S., S. Kono. J . Abe, K. Koizumi, and T Tanimoto: Bio-

[21] Koizumi, K., 7: Utamura, I: Kuroyanagi, S. Hizukuri, and J . Abe: J.

[22] Abe. J . , K. Nakashima, H. Nagano, S. Hizukuri, and K. Obata: Car-

[23] Takedo. E, and S. Hizukuri: Biochim. Biophys. Acta 185 (1969),

[24] Takedo. E, and S. Hizukuri: Carbohydr. Res. 148 (1986), 299-308. I251 Takedo. E, N. Tokunaga, C. Takeda. and S. Hizukuri: Starch/Star-

[26] Suzuhi. A., M. Kaneyama, K. Shibanuma, E Takeda. J. Abe, and S.

[27] Hizuhilri. S., Y. Takeda, TShitaozono, J. Abe, A . Otakara, C. Take-

[281 Suzuki. A. . M. Kanayama, I: Takeda, and S. Hizukuri: Denpun

[291 Hizukuri. S.. E Takeda, M. Yasuda, and A . Suzuki: Carbohydr.

[301 Hizukuri. S., K. Shirasaka, and B. 0. Juliano: Starch/Starke 35

[ j l ] Somogvi, M.: J. Biol. Chem. 195 (1952), 19-23. [32] Nelson, N.: J. Biol. Chem. 153 (1944), 375-380. [331 Yokobayashi, K., A. Misaki, and 7: Harada: Biochim. Biophys.

[34] Dubois, M., K. A. Gilles, J. K. Hamilton, P. A . Robers, and F? Smith:

1351 Laemmli, U. K., and M. Favre: J. Mol. Biol. 80 (1973), 575-599. [361 Lowry. 0. H.. N. J . Rosebrough. A . L. Farr, and R. J. Randall: J.

[371 Kato, K., Y. Konishi, A . Amemura, and 7: Harada: Agric. Biol.

[381 Fang, 2, L. Lin, and W: Hsu:Enzyme Microb.Technol.16 (1994),

[391 Lynn, A., and J. R. Stark: Carbohydr. Res. 227 (1992), 379-383. [401 Abe, J. , H . Nagano, and S. Hizukuri: J. Appl. Biochem. 7 (1985),

[41] Ueda, S.: Denpun Kagaku 21 (1974), 210-221.

technol. Appl. Biochem. 11 (1989), 60-73.

Chromatol. 360 (1986), 397-406.

bohydr. Res. 175 (1988e), 85-92.

469-47 1.

ke 38 (1986), 345-350.

Hizuhuri: Cereal Chem. 6 9 (1992), 309-315.

da, and A . Suzuki: Starch/Starke 40 (1988), 165-171.

Kagaku 33 (1986), 191-198.

Res. 9 4 (1981), 205-213.

(1983). 348-350.

Acta 212 (1970), 458-469.

Anal. Chem. 28 (1956), 350-356.

Biol. Chem. 1 9 3 (1951), 265-295.

Chem. 41 (1977), 2077-2080.

247-252.

235-247.

Addresses of authors: Professor Dr. Susumu Hizukuri, Mr. Toshiteru Kozuma, Mr. Hironori Yoshida, Associate Professor Dr. Jun-ichi Abe, Faculty of Agriculture, Kagoshima University, Korimoto-1, Kagoshi- rpa 890 (Japan). Mr. Kosei Takahashi, Dr. Mikio Yamamoto, and Dr. Nobuyuki Nakamura, Research Institute, Nihon Shokuhin Kako Co. Ltd., Fuji, Shizuoka 417 (Japan).

(Received: January 29, 1996).

300 StarchlStarke 48 (1996) Nr. 7/8, S. 295-300

properties of flavobacterium odoratum ku isoamylase

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