Screening of Lactobionic Acid Producing Microorganisms Hiromi

469

(J. Appl. Glycosci., Vol. 49, No. 4, p. 469-477 (2002))

Screening of Lactobionic Acid Producing Microorganisms

Hiromi Murakami,* Jyunko Kawano,' Hajime Yoshizumi,' Hirofumi Nakano and Sumio Kitahata

Osaka Municipal Technical Research Institute (1-6-50, Morinomiya, Joto-ku, Osaka 536-8553, Japan) 1Faculty of Agriculture, Kinki University (3327-204, Nakamachi, Nara 631-8505, Japan)

Lactobionic acid (LA) is derived from lactose and expected to be a versatile material for growing bifidobacterium and forming mineral salts with high solubility in water for supplements. We aimed to develop microbial or enzymatic production systems of LA. To this aim, we screened lactose-oxidizing microorganisms, and obtained a strain of Burkholderia cepacia. The lactose- oxidizing activity existed in the membrane fraction of disrupted cell preparation of the strain. Only oxygen was necessary for lactose-oxidizing activity as a proton acceptor. A crude cell-free enzyme preparation was prepared, and its oxidizing ability and other properties on several saccharides were examined. The cell-free preparation oxidized D-glucose, D-mannose, D-galactose, D-xylose, L- arabinose and D-ribose. It also reacted with lactose, cellobiose, maltose, maltotriose, maltotetaose and maltopentaose. The strain accumulated LA in the culture supernatant with no loss of lactose. The strain is advantageous to production of LA by both fermentation and enzymatic reaction.

Lactose (Lac), one of the most common saccha- pergillus niger,6,7)Phanerochaete chrysosporium8) rides in dairy products, can be obtained easily and Penicillium chrysogenum .9) These strains and from cheese whey and casein whey, the large pool enzymes will not be used for LA production be- of unutilized resources. We screened microorgan cause they are not able to oxidize lactose. In con- isms to convert Lac to lactobionic acid (LA), ƒÀ- trast to these microorganisms, some kinds of

1,4-D-galactosyl-D-gluconate, to use whey effec- plants, marine red algae, Chondrus crispus,10) tively. LA can be used as a bifidus factor,1) a min- Iridophycus flaccidum11) and oranges such as Cit- eral absorption promoter2 and a preservative for rus sinensis var Valencia,12) possess hexose oxi-

isolated organs for transplantation.3-5) In spite of dase (EC 1.1.3.5) activities to oxidize several

such usefulness, an industrial method for produc- mono- and oligosaccharides. These organisms and

tion of LA has not been established yet. We iso- their enzymes are not suitable for LA production lated Burkholderia cepacia No. 216 with a Lac- because cultivations are not easy and their reactivi-

oxidizing activity. The cell-free enzyme prepara- ties on Lac are weak. Glucooligosaccharide oxi-

tion did not require any particular proton acceptor dase from Acremonium strictum 13) catalyzes oxida- other than oxygen and was estimated to be a kind tion of maltooligosaccharides well but the reactiv-

of glucose oxidase (EC 1.1.3.4). ity on Lac is not so high. Lactose dehydrogenase

Glucose oxidases have been reported from As- of Pseudomonas graveolence 14) catalyzes oxidation of Lac, maltose and cellobiose, producing their

* Corresponding autbor (murakami@ omtri.city.osaka.jp). aldobionic-ƒÂ-lactone in the presence of an appro- Abbreviations: Gal, D-galactose; Glc, D-glucose; TOC, priate hydrogen acceptor. The maltose dehydroge- total organic carbon; Yxic, growth yield for the de nase from Corynebacterium sp.15' can oxidize mal- crease of TOC; H2O2, hydrogen peroxide; Lac, lactose; tose, and the galactose dehydrogenase from rat LA, lactobionic acid; Ypic, product yields for the decrease of TOC; SDS, sodium dodecyl sulfate; TLC, liver was reported to react with maltose and cello- thin layer chromatography. biose. But these reactions do not seem to be suit- 470 J. Appl. Glycosci., Vol. 49, No. 4 (2002)

able for practical use because they require specific Enzyme assay. The Lac oxidizing activity was

hydrogen acceptors. measured by monitoring consumption of oxygen This paper describes screening and isolation of a using an oxygen electrode (YSI 5331 oxygen

LA-producing microorganism, B, cepacia, and par- probe, Yellow Springs, Ohio). 1.4 mL of 100 mM tial purification and characterization of its lactose- Lac in 50 mM acetate buffer (pH 5.5) was preincu-

oxidizing activity. We examined capability of the bated at 30•Ž. The reaction was initiated by the strain to oxidize several saccharides and evaluated addition of 200ƒÊL of cell-free enzyme prepara- efficiencies of aldonic acids-production by the tion, and the initial velocity of oxygen consump-

strain. tion was measured.

One unit of Lac-oxidizing activity was defined

MATERIALS AND METHODS as the amount of enzyme which consumed 1 p mol of 02 per min at 30•Ž.

Materials. Lac, D-Glc, maltose, sucrose and •@•@ Catalase activity was measured by monitoring other reagents were purchased from Nacalai the decrease of hydrogen peroxide (H202) at 25•Ž

Tesque Inc. (Kyoto). Polypepton and yeast extract with absorbance at 240 nm.18) A hundred microlit- were products of Nissui Pharmaceuticals (Tokyo). ters of enzyme solution was added to 2.9 mL of a-1,6-Galactobiose16) was a gift from H. Hashimoto 0.06% H2O2 in 50 mM phosphate buffer (pH 7.0) of Faculty of Agriculture, Shinshu University. Glu- in quartz cuvette (1 cm light path). The time re- coamylase from Rhizopus niveus16) was a product quired to decrease the absorbance at 240 nm from of Seikagaku Kogyo Co. (Tokyo). a-Glucosidase 0.450 to 0.400 was measured. This decrease corre- preparation, transglucosidase Amano, was pur- sponded to the decomposition of 3.45 p mol of chased from Amano (Nagoya). Peroxidase from hydrogen peroxide in 3 mL of reaction mixture . horseradish was purchased from Toyobo (Osaka) . One unit of catalase activity was defined as the

Screening of Lac-oxidizing microorganisms. amount of enzyme which decomposed 1 p mol of

About 0.1 g of soil was suspended in 5 mL of H2O2 per min at 25•Ž. sterilized water. The solution was diluted to a hun- Analytical methods. H2O2 was determined by dredth. A hundred p L of the supernatant solution peroxidase chromogen method described below. was spread on agar plates containing 0.1 % Lac , Five hundred microlitters of a sample solution was 0.2% NH4NO3, 0.05% NaCI, 0.1% K2HPO4, 0.1% incubated with 5ƒÊL of 1% 4-aminoantipyrin, 25ƒÊ KH2PO4 and 0.05% MgSO4.7H2O. After being in- L of 5% phenol, 50 ƒÊL of 1.0 U/mL peroxidase cubated at 28•Ž for several days, colonies grown solution, and 420 p L of 10 mM phosphate buffer on the plates were isolated and inoculated into 2 (pH 7.0) at 30•Ž for 15 min. The increase of ab- mL of liquid culture medium supplemented with sorbance was measured at 500 nm for 3 min. Re- 1 % Lac and 1 % polypepton to the above minimum ducing sugar was determined by Somogyi and medium, which was cultured at 28°C for 3 days on Nelson's method.19,20) Total sugar was measured by a reciprocal shaker. The culture liquor was incu- the phenol-sulfuric acid method.21) Protein concen- bated with 1 % Lac and 0.1% SDS in 50 mM phos- tration was measured by the Bradford method.22) phate buffer (pH 7.0) at 40•Ž for 4 h. The reaction Thin layer chromatography. TLC of the reac- mixtures were analyzed by TLC. tion products was carried out to check reaction Cultures. A bacterial strain No. 216 was culti- products by Kieselgel 60 plates (Merck), using vated in 500-mL shaking flasks containing 100 mL ethyl acetate-acetic acid-water (3:1:1 , v/v) as a of medium to obtain Lac-oxidizing activity at 28•Ž solvent. Carbohydrates were detected by heating for 48 h on a reciprocating shaker. The medium the plate at 110-120•Ž after spraying sulfuric acid- was composed of 1 % Lac, 1 % polypepton, 0.1% methanol (50%, wt/wt). yeast extract, 0.05% NaCI, 0.2% NH4NO3, 0.1% High performance liquid chromatography. K2HPO4, 0.1% KH2PO4 and 0.05% MgSOe 7H2O High performance liquid chromatography was car- and pH was adjusted to 7.0. ried out under the following conditions: column, Microbial Production of Lactobionic Acid 471

Asahipak NH2P-50 (Shodex Co., Ltd.); solvent, as a shift reference.

CH3CN/40 mM citrate buffer (60/40, v/v); flow Oxygen demand for the oxidation of Lac. rate, 1.0 mL/mL; temperature, 40•Ž; detection, RI The cell-free enzyme extraction was dialyzed

detector (Shimazu Co., Ltd.). against 10 mM phosphate buffer (pH 7.0) to re-

Preparation of the crude enzyme. The cell- move cytoplasmic low molecular weight sub-

free enzyme was prepared to observe oxidation of stances. The dialyzed enzyme solution (0.21 U/

saccharides without consumption of oxygen and mL, 200 ,CL) was incubated with 14.6 mM Lac in

degradation of substrates and products by cells. 50 mM acetate buffer (pH 5.5) at 30°C with and The cells of B. cepacia No. 216 (wet weight 100 without bubbling of nitrogen gas. The test tubes

g) were suspended in 130 mL of 10 mM phosphate were sealed and the gas phase was replaced with buffer (pH 7.0) and disrupted by passage through a N2 gas while the control tube continued to have air

french pressure cell at 1500 kgf/cm2. After soni- in it. Both reaction mixtures were analyzed by cated for 5 min to reduce the viscosity of the sam- TLC after 0, 1, 2 and 3h.

ple solution, it was centrifuged by 15,000•~g for Time courses of oxidation of v-Glc, maltose, 30 min to remove cell debris. The supernatant was sucrose and Lac by cultivation. The strain was

used as a crude enzyme preparation, and its total cultivated with 300 mL each of four kinds of liq-

and specific activities were 84.0 U and 0.0305 U/ uid medium containing 1 % each of a carbon mg protein. The crude enzyme was ultracentri- source (D-glucose, maltose, sucrose, or Lac), 1.%

fuged by 100,000•~g for 60 min. The precipi- polypepton, 0.1% yeast extract, 0.05% NaCI, 0.2% tated membrane fraction was washed with 50 mM NH4N03, 0.1% K2HP04, 0.1% KH2P04 and 0.05%

acetate buffer (pH 5.5) and ultracentrifuged again. MgS04.7H20. The concentrations of saccharides The precipitate was resuspended in 55 mL of the and aldonic acids were measured by HPLC. The

same buffer, and was used as a membrane-bound absorbance at 660 nm and the amount of total or-

enzyme preparation. Supernatant solution had no ganic carbon (TOC, mgC/mL) of the supernatants activity. The total and specific activities were 40.0 were monitored. After 72 h cultivation, the dry U and 0.184 U/mg. The enzyme was purified 6.0- weights of cells were measured.

folds. This crude enzyme was used as cell-free TOG measurement. TOC of culture superna

preparation with oxidizing activity. The enzyme tants were measured by combustion oxidation- preparation had both oxidase and catalase activi- infrared type TOC analysis method using TOC- ties. 5000 (Shimazu Co., Ltd.) Potassium phthalate was Preparation of a reaction product from Lac. used as TOC reference. The measurement proce-

The crude enzyme (0.5 U) preparation was incu- dures were the same as described in JIS K0102-22. bated with 10 mL of 100 mM lactose in 10 mM Time course of Lac oxidation by the enzyme phosphate buffer (pH 7.0) at 40•Ž for 48 h. The preparation. The enzyme (0.21 U/mL, 5.0 mL) reaction mixture was applied to an active-carbon was incubated with 10 mM Lac in 10 mM phos- column (2•~20 cm) equilibrated with deionized phate buffer (pH 7.0) at 40•Ž, and after 0.5, 1, 2, water. The passed and washed solutions were con- 3, 4 and 5 h, the reaction mixtures were analyzed centrated to 3 mL and applied to Bio-Gel P-2 col- photometrically. The amount of reducing sugar, to umn (3•~100 cm), which was eluted with 8% tal sugar, and hydrogen peroxide were measured. ethanol. The eluted carbohydrates were detected by Effects of pH and temperature on activity and phenol-sulfuric acid method. The product fractions stability of Lac oxidizing activity. The cell-free were collected and freeze-dried. 1 extract was incubated with 100 mM Lac in various

H and 13C-NMR measurements. A sample pHs between pH 2.5 and 11.0, and the initial reac was dissolved in D20, and 1H- and 13C-NMR spec- tion rates were measured under the standard assay tra were recorded at 300 MHz and 75 MHz, re- conditions. The cell-free preparation was stored at

spectively, with a JEOL AL-300 spectrometer. So- 4•Ž for 20 h in various pHs and the residual ac dium 3-(trimethylsilyl)-propanesulfonate was used tivities were assayed under the standard assay con- 472 J. Appl. Glycosci., Vol. 49, No. 4 (2002)

ditions. The enzyme reaction was carried out at pH 5.5

and various temperatures between 10•Ž and 75•Ž.

After 10 min incubation in 10 mM phosphate

buffer (pH 7.0), the Lac-oxidizing activities were measured under the standard conditions.

Oxidation of several saccharides by the enzyme preparation. The enzyme preparation (0.21-0.5 U/mL, 200 1CL) was incubated with 100 mM of

each substrate in 50 mM acetate buffer (pH 5.5) and the initial velocity of oxidation reaction was

measured under the standard assay conditions.

Fig. 1. Time course of the cultivation of B. cepacia No.•@

RESULTS 216.

Pre-cultivation of B. cepacia No. 216 was inoculated to

Screening of Lac-oxidizing microorganisms. 100 mL of liquid medium for the enzyme production in 500-mL of shaking flask, and cultured at 28•Ž for 48 h. We examined the several hundred colonies The activity, pH, absorbance at 660 nm as a indicator of

grown on selection medium plates, and selected a microbial growth, and Lac and LA of the culture superna- couple of strains judging from both the amounts of tant were measured after 4, 8, 24 and 48 h. The washed LA (size of spots on TLC) in the reaction mixtures cells from 5 mL of each culture was suspended to 1 mL

and growth rates of the cells. We obtained a bacte- of 10 mM phosphate buffer (pH 7.0) and disrupted by sonication. Cell debris was removed by centrifugation, rial strain No. 216, which oxidized Lac to accumu- and their supernatants were assayed. -•¢-, activity; --- late LA in the culture liquor. It was a rod-shaped •›--- , growth; ---•¢---, pH; -•›-, Lac (mg/mL); -•œ-, and Gram negative bacterium and was identified as LA (mg/mL). B. cepacia by National Collections of Industrial 3J and Marine Bacteria Japan Co., Ltd., in Shizuoka, H4 ,H5' = 3.3 Hz, H4'), 3.85 (dd, 2H, 3JH5,H6 = 3.3

Japan. Hz, 2Jgeminal =16.1 Hz, H6), 3.82 (dd, 2H, 3JH5' , H6'

= 3 .5 Hz, 2Jgeminal =16.1 Hz, H6'), 3.74 (dd, H, 3J Preparation of a product from Lac and estima HS,H4 - 2.9 Hz, 3JH5,H6 = 3.3 Hz, H5), 3.68 (dd, H, 3J tion of its structure. H3', H2' = 6.4 Hz, 3JH3, H4' = 3.3 Hz, H3'), 3.64 (dd,

A reaction product from Lac was prepared by H, 3JH5', H4' = 3.3 Hz, 3JH5' , H6' = 3.5 Hz, H5), 3.56

the procedure shown in MATERIALS AND METHOD. (dd, H, 3JH2', HI' = 7.7 Hz, 3JH2' ,H3' = 7.5 Hz, H2') ; 13C About 300 mg of product was obtained from 1 -NMR (D20) o 181 .1 (C 1), 106.0 (C1'ƒÀ), 83.9

mmol (342 mg) of Lac. The freeze-dried sample (C2), 78.0 (C3'), 76.5 (C4), 75.2 (CS'), 74.2 (C4'),

(100 mg) was dissolved in 1 mL of D20 and ana 73.8 (C2'), 71.3 (CS), 64.6 (C6), 63.7 (C6'). lyzed by 1H- and 13C-NMR spectroscopies. The structure of the product was confirmed to be 4-0 - Time course of the cultivation.

ƒÀ-D-galactosyl D-gluconate , lactobionic acid (LA), The growth (absorbance at 660 nm), pH, con on the basis of the following data. The assign- centrations of Lac and LA, and Lacoxidizing ac ments of all signals were described below. The tivity of the cells were monitored (Fig. 1). The cell carbons and protons of gluconic residue corre- growth reached stationary phase after 30 h cultiva- spond to C1-C6, H1-H6 and those of galactosyl tion. The pH gradually increased to 7.8 under this residue correspond to C 1' -C6', Hi' -H6'. 1H- 1% Lac condition. The lactose-oxidizing activity NMR (D20) c4.56(d, H, 3JH,-,H2' =7.7 Hz, H1'), reached maximum after 30 h and did not decrease 4.21 (d, H, 3JH4,H3- 3JH4,H5 - 2.8 Hz, H4), 4.13 (dd, for the following 24 h. The Lac decreased and

H, 3JH3,H2=4.8 Hz, 3JH3,H4=2.8 Hz, H3), 3.99 (d, H, completely disappeared after 48 h, while LA in- 3J H2,H3= 4.8 Hz, H2), 3.91 (dd, H, 3JH4', H3' = 3.5 Hz, creased and finally reached 10 mg/mL. Microbial Production of Lactobionic Acid 473

Fig. 2. Time courses of oxidation of D-glucose, maltose, sucrose and Lac by cultivation.

Pre-cultivation of B. cepacia No. 216 was inoculated to 300 mL of four kinds of liquid medium containing l % each of a carbon source (D-glucose, maltose, sucrose, or Lac), 1% polypepton, 0.1% yeast extract, 0.05% NaCI, 0.2% NH4

NO3, 0.1% K2HPO4, and 0.05% MgSO4.7H2O, and cultured at 28•Ž for 72 h. The concentration of saccharides and al donic acid were measured by HPLC. Glc, D-glucose; Suc, sucrose; Mal, Maltose; MA, maltobionic acid; Lac, lactose;

LA, lactobionic acid.

Oxygen demand for the oxidation of Lac. was supplemented) was 16.2, 19.1, 17.3, 20.7 or We did the following experiment to confirm 16.4%, respectively. The Yx/cvalue of Lac medium whether the enzyme would need an appropriate was not lower than those of other carbon sources. hydrogen acceptor other than oxygen or not. The The product yields for the decrease of TOG (Ypic) dialyzed crude enzyme was incubated with Lac (D-Glc, maltose, sucrose, Lac, or no carbon source with and without bubbling of nitrogen gas. Both of was supplemented) were 0, 59.8, 0, 243.0 and 0%, the reaction mixtures were analyzed by TLC. respectively. These values were not calculated When the reaction system was replaced with N2, from molar ratio of substrates and products, and no oxidation of Lac occurred, while a considerable were not proportional to their conversion effi- amount of LA was produced in the control reac ciency, but useful as indicators of the efficiency. tion mixture. The enzyme needed oxygen as a pro- The results showed that Lac was the best substrate ton acceptor, and was thought to be an oxidase. for aldonic acid production. Figure 2 shows the time courses of saccharides Capability and efficiency of oxidizing several and aldonic acids of cultural supernatants. After 22 saccharides of the strain. h cultivation, D-glucose, maltose, and sucrose com- To examine the capability of the strain to oxi pletely disappeared. When maltose was used as a dize several saccharides, the strain was cultured carbon source, a small amount of maltobionic acid using D-Glc, maltose, sucrose and Lac. The was observed after 8 h. It had reached maximum amounts of substrates and products (Fig. 2), concentration (4 mg/mL) at 22 h, decreased rap growth (Fig. 3), and TOG (Fig. 4) were measured. idly and finally disappeared. In the case of Lac, We calculated the growth yield for the decrease of the equivalent molarity of LA was produced. The TOG (YX/c)(%, w/w) and the product yield for the prolonged cultivation did not cause a decrease of decrease of TOG (Ypic)(%, w/w) from these re- LA. sults, and evaluated the efficiency of aldonic acids- Figure 3 shows growth curves and dry cell production. The YX/cvalue of each cultivation (D- weights of each cultivation. Every culture reached Glc, maltose, sucrose, Lac, or no carbon source a stationary phase after 24 h. Dry cell weights of 474 J. Appl. Glycosci., Vol. 49, No. 4 (2002)

Fig. 4. Time courses of TOC of culture supernatants.

The cultivations were carried out in the same way as in Fig. 2 and the TOC (mgC/mL) of the cultural superna-

tants were measured. ---•¢---, D-glucose; -•£-, maltose; -• - , sucrose; -•œ-, lactose; -•›-, no carbon Fig. 3. Growth curves of cultivations and dry cell weights. source.

The cultivations were carried out in the same way as in

Fig. 2 and the absorbances at 660 nm were measured. initial amount and the reducing sugar completely After 72 h cultivation, the cells were centrifuged and vanished. LA has no reducing power and its sensi- washed with 10 mM phosphate buffer (pH 7.0) and dried tivity in phenol-sulfuric acid detection is half of at 105•Ž for 3 h at atmospheric pressure. ---•¢---, D- that of Lac. A trace amount of hydrogen peroxide glucose; -•£-, maltose; -• -, sucrose; -•œ-, lactose; -•›-, no carbon source. was detected, and it gradually increased during the

reaction. The ratio of H2O2 to LA was about

72 h cultivation with D-glucose, maltose, sucrose 1/150-1/300 (H202/LA, molar ratio). or Lac as carbon sources were 0.302, 0.336, 0.340 and 0.178 g, respectively. The dry cell weight un- Effects of pH and temperature on activity and der the same conditions without a carbon source stability of the oxidase. was 0.146 g. The mean value when Lac or no car- The Lac oxidizing activity was the most active at bon source was added was 0.162 g, which was al- pH 5.5 and stable between pH 6.0 and 9.0. The most the half of the average (0.326 g) as the other optimum temperature was 50•Ž, and stable below three were used as carbon sources. 35•Ž. Figure 4 shows the time courses of TOC of culture supernatants. The decrease rates of TOC at•@•@ Oxidation of several saccharides by the enzyme the first 24 h were 0.260, 0.246, 0.272 and 0.121 preparation.•@•@ mg/mL/h, respectively, when D-Glc, maltose, su- The reactivity of the cell-free enzyme prepara- crose or Lac was used as a carbon source, while tion was examined on various mono- and oligosac- the decrease rates of TOC under the same condi- charides. The relative rates, compared to the value tions without a carbon source was 0.122 mg/mL/ obtained when Lac was used as a substrate, are h. This result shows that the decrease rates of shown in Table 1. The most favorable substrate for

TOC up to 24 h cultivations for which Lac or no the enzyme was D-Glc. It oxidized 2- and 4-epimer carbon source was added were about the half of of D-Glc well. The enzyme also reacted with oli- the rates when D-Glc, maltose, or sucrose was gosaccharides such as Lac, cellobiose, maltose, used as a carbon source. maltotriose, maltotetraose and maltopentaose.

Time course of Lac oxidation by the oxidase. Capability of the enzyme to distinguish ano- The time course of the oxidation of Lac was ob- meric type of v-Gk. served by measuring total sugar, reducing sugar, The reactivities of the cell-free extract on w- and hydrogen peroxide in the reaction mixture. Af- and ƒÀ-D-GIc were examined. When the enzyme ter 5 h, the total sugar was reduced to half of the preparation was incubated with maltopentaose, Microbial Production of Lactobionic Acid 475

Table 1. Oxidizing ability of cell free-extract of B. cepacia No. 216 on various saccharides.

Fig. 5. Reactivities of the lactose oxidizing activity on ƒ¿- and ƒÀ-D-glucose.

The enzyme preparation (0.015 U, 100 JCL) was incu-

bated with 50 mi i maltopentaose at 30°C for 3 min, fol- lowed by the addition of glucoamylase or ƒ¿-glucosidase

solution (20,aL each). The consumption of oxygen was

monitored by oxygen electrode. The glucoamylase and a-

glucosidase were previously dialyzed against deionized water, and their activities on maltopentaose (amount of re-

leasing ƒ¿- and ƒÀ-D-glucose) were made similar to each other. Arrows at around 1 min indicate the addition of en-

zyme preparation.

toplasm. This result suggests that oxygen works as a hydrogen acceptor in the reaction and that the enzyme is classified as an oxidase. Table 1 shows that D-Glc was the most efficient maltopentaonic ƒÂ-lactone was produced and oxy- substrate for the enzyme, which suggests that the gen concentration in the reaction mixture de- enzyme is a glucose oxidase. The oxidase from B. creased linearly. The following addition of gluco- cepacia was characterized by a broad substrate amylase to the reaction mixture accelerated the specificity in contrast to rigid substrate specificity consumption rate of oxygen but the addition of ƒ¿- of the ordinary glucose oxidases. The oxidase glucosidase did not change the consumption rate acted on not only aldohexoses, but also aldopento- of oxygen (Fig. 5). It is well known that gluco- ses and oligosaccharides such as D-xylose, L- amylase and ƒ¿-glucosidase hydrolyze maltooligo- arabinose, D-ribose, Lac, cellobiose and maltooli- saccharides and produce ƒÀ- and a-anomer of D- gosaccharides, but not oxidize ketoses and nonre- Glc, respectively. In addition the enzyme oxidized ducing sugars. Well-known glucose oxidases are D-Glc faster than maltopentaose (Table 1). There- highly specific to /3-D-Glc, and their reaction rates fore it was presumed that the enzyme oxidized ƒÀ- of a-D-Glc oxidation are negligible. Figure 5 anomer of D-Glc, but not a-anomer. shows that the oxidase, like other common glucose oxidases, effectively catalyzed the oxidation of ƒÀ-

DISCUSSIONS D-Glc, but could not react on a-D-Glc. Reported

organisms and enzymes, such as glucose oxidases, We obtained a bacterial strain B. cepacia No. hexose oxidases, glucooligosaccharide oxidase, and

216 that had a Lac-oxidizing activity in its mem- lactose dehydrogenase are not suitable for produc- brane fraction. The Lac oxidation occurred only in tion of LA because their reactivities on Lac are the presence of oxygen and was not affected by weak and cultivations are not easy. In contrast to the absence of other hydrogen acceptors from cy- these organisms and enzymes, B. cepacia No. 216 476 J. Appl. Glycosci., Vol. 49, No. 4 (2002) is advantageous to effective production of LA. The the rat. Transplantation, 53, 1206-1210 (1992). 4) J.A. Wahlberg, R. Love, L. Landegaard, J.H. Southard strain has a high reactivity on Lac, and does not and F.O. Belzer: 72-Hour preservation of the canine digest either Lac nor LA. pancreas. Transplantation, 43, 5-8 (1987). When D-Glc, maltose and sucrose were used as 5) Wisconsin Alumni Research Foundation: Preservative carbon sources for cultivation, the saccharides and solution for organs. USA, JAPAN Kokai Tokkyo Koho, their oxidized products did not remain in the cul- 188001, 1991-08-16. ture. When Lac was given as a carbon source, the 6) J.H. Pazur: Glucose oxidase from Aspergillus niger. Methods Enzymol., 9, 82-87 (1966). strain was not able to digest LA but accumulated 7) B.E.P. Swoboda and V. Massey: Purification and prop- it in culture (Fig. 2). The time courses of the culti- rties of the glucose oxidase from Aspergillus niger. J. vation showed that Lac and LA were not assimi- Biol. Chem., 240, 2209-2215 (1965). lated by the strain. The results suggested that Lac 8) R.L. Kelley and C.A. Reddy: Purification and charac- was metabolized only by catabolic procedure and terization of glucose oxidase from ligninolytic culture of Phanerochaete chrysosporium. J. Bacteriol., 166, was converted to LA, whereas other nutrients such 269-274 (1986). as polypepton, yeast extracts, D-Glc, maltose and 9) T. Nakamatsu, T. Akamatsu, R. Miyajima and I. Shio: sucrose were metabolized by both anabolic and Microbial production of glucose oxidase. Agric. Biol. catabolic procedure to be converted to cell compo- Chem., 39, 1803-1811 (1975). nents, energy and carbon dioxide. 10) J.D. Sullivan and M. Ikawa: Purification and characterization of hexose oxidase from the red alge Chon- The culture liquor was composed of a solid drus crispus. Biochim. Biophys. Acta, 309, 11-22 fraction (cells) and a liquid fraction (culture super- (1973). natant). TOC lost from the liquid fraction was ex- 11) R.C. Bean and W.Z. Hassid: Carbohydrate oxidase pelled as carbon dioxide to gas phase or trans- from a red alga, Iridophycus flaccidum. J. Biol. formed to solid material as cell components. The Chem., 218, 425-436 (1956). 12) R.C. Bean, G.G. Porter and B.M. Steinberg: Carbohy- growth yield for the decrease of TOC (Y /c) is that drate metabolism of Citrus fruits. J. Biol. Chem., 236, whatever amount of TOC was lost from liquid was 1235-1240 (1961). utilized as cell components. The result also sug- 13) S. Lin, T. Yang, T. Inukai, M. Yamasaki and Y. Tsai: gested that it was not necessary to supplement the Purification and characterization of a novel glucooli- Lac medium with other carbon sources for rapid gosaccharide oxidase from Acremonium strictum 11. Biochim. Biophys. Acta, 1118, 41-47 (1991). growth of cells. All these results suggest that the 14) Y. Nisizuka and 0. Hayaishi: Enzymatic formation of combination of the strain as an oxidase producer lactobionic acid from lactose. J. Biol. Chem., 237, and Lac as a substrate is an excellent system of al- 2721-2728 (1962). donic acid production. 15) Y. Kobayashi and K. Horikoshi: Purification and properties of NAD + -dependent maltose dehydroge- The author appreciates a generous gift of a-1,6- nase produced by alkalophilic Corynebacterium sp. No. 93-1. Biochim. Biophys. 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20) M. Somogyi: Notes on sugar determination. J. Biol. ラルと水溶性の高い塩を作るなど,さまざまな用途が Chem., 195, 19-23 (1952). 期待される糖質素材である.われわれは,微生物ある 21) M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers いは酵素を用いたラクトビオン酸生産法を確立するた and F. Smith: Colorimetric method for determination of sugars and related substances. Anal. Chem., 28, め,ラクトースの酸化活性を持つ微生物を検索した. 350-356 (1956). その結果,Burkholderia cepaciaの一菌株を得た.ラ 22) M. Bradford: A rapid and sensitive method for the クトースの酸化活性は,菌体内に存在し,菌体を破砕 quantitation of microgram quantities of protein utiliz- ing the principle of protein-dye binding. Anal. Bio- 後,酸素の存在下および非存在下でラクトースに作用 chem., 72, 248-254 (1976). させたところ,酸素の存在下でのみラクトビオン酸の生成が認められた.無細胞抽出液を調製し,さまざま (Received February 26, 2002; Accepted May 27, 2002) な糖質に対する酸化活性を検討した.本粗酵素標品は D-グルコース,D-マンノース,D-ガラクトース,D-キラクトビオン酸生産微生物の検索シロース,L-アラビノース,D-リボース,ラクトー村上洋,河野純子1,吉栖肇1, ス,セロビオース,マルトース,マルトトリオース, 中野博文,北畑寿美雄マルトテトラオース,マルトペンタオースに作用し大阪市立工業研究所た.本菌株は培養上清中に2%のラクトビオン酸をラ (536-8553大阪市城東区森之宮1-6-50) 1近畿大学農学部(63-8505奈良市中町3327-204) クトースの損失なく蓄積した.したがって,本菌株は発酵あるいは菌体反応によるラクトビオン酸生産にラクトビオン酸はラクトースから合成されるアルド適用可能であると考えられる. ビオン酸で,ビフィズス菌選択増殖活性を持ち,ミネ