Screening of Lactobionic Acid Producing Microorganisms ...

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 Nakanoand 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 grow-ing 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-

rides in dairy products, can be obtained easily

from cheese whey and casein whey, the large pool

of unutilized resources. We screened microorgan

isms to convert Lac to lactobionic acid (LA), ƒÀ-

1,4-D-galactosyl-D-gluconate, to use whey effec-

tively. LA can be used as a bifidus factor,1) a min-

eral absorption promoter2 and a preservative for

isolated organs for transplantation.3-5) In spite of

such usefulness, an industrial method for produc-

tion of LA has not been established yet. We iso-

lated Burkholderia cepacia No. 216 with a Lac-

oxidizing activity. The cell-free enzyme prepara-

tion did not require any particular proton acceptor

other than oxygen and was estimated to be a kind

of glucose oxidase (EC 1.1.3.4).

Glucose oxidases have been reported from As-

pergillus niger,6,7)Phanerochaete chrysosporium8)

and Penicillium chrysogenum .9) These strains and

enzymes will not be used for LA production be-

cause they are not able to oxidize lactose. In con-

trast to these microorganisms, some kinds of

plants, marine red algae, Chondrus crispus,10)

Iridophycus flaccidum11) and oranges such as Cit-

rus sinensis var Valencia,12) possess hexose oxi-

dase (EC 1.1.3.5) activities to oxidize several

mono- and oligosaccharides. These organisms and

their enzymes are not suitable for LA production

because cultivations are not easy and their reactivi-

ties on Lac are weak. Glucooligosaccharide oxi-

dase from Acremonium strictum 13) catalyzes oxida-

tion of maltooligosaccharides well but the reactiv-

ity on Lac is not so high. Lactose dehydrogenase

of Pseudomonas graveolence 14) catalyzes oxidation

of Lac, maltose and cellobiose, producing their

aldobionic-ƒÂ-lactone in the presence of an appro-

priate hydrogen acceptor. The maltose dehydroge-

nase from Corynebacterium sp.15' can oxidize mal-

tose, and the galactose dehydrogenase from rat

liver was reported to react with maltose and cello-

biose. But these reactions do not seem to be suit-

* Corresponding autbor (murakami@ omtri.city.osaka.jp). Abbreviations: Gal, D-galactose; Glc, D-glucose; TOC, total organic carbon; Yxic, growth yield for the decrease of TOC; H2O2, hydrogen peroxide; Lac, lactose; LA, lactobionic acid; Ypic, product yields for the de-crease of TOC; SDS, sodium dodecyl sulfate; TLC, thin layer chromatography.

470 J. Appl. Glycosci., Vol. 49, No. 4 (2002)

able for practical use because they require specific

hydrogen acceptors.

This paper describes screening and isolation of a

LA-producing microorganism, B, cepacia, and par-

tial purification and characterization of its lactose-

oxidizing activity. We examined capability of the

strain to oxidize several saccharides and evaluated

efficiencies of aldonic acids-production by the

strain.

MATERIALS AND METHODS

Materials. Lac, D-Glc, maltose, sucrose and

other reagents were purchased from Nacalai

Tesque Inc. (Kyoto). Polypepton and yeast extract

were products of Nissui Pharmaceuticals (Tokyo).

a-1,6-Galactobiose16) was a gift from H. Hashimoto

of Faculty of Agriculture, Shinshu University. Glu-

coamylase from Rhizopus niveus16) was a product

of Seikagaku Kogyo Co. (Tokyo). a-Glucosidase

preparation, transglucosidase Amano, was pur-

chased from Amano (Nagoya). Peroxidase from

horseradish was purchased from Toyobo (Osaka) .

Screening of Lac-oxidizing microorganisms.

About 0.1 g of soil was suspended in 5 mL of

sterilized water. The solution was diluted to a hun-

dredth. A hundred p L of the supernatant solution

was spread on agar plates containing 0.1 % Lac ,

0.2% NH4NO3, 0.05% NaCI, 0.1% K2HPO4, 0.1%

KH2PO4 and 0.05% MgSO4.7H2O. After being in-

cubated at 28•Ž for several days, colonies grown

on the plates were isolated and inoculated into 2

mL of liquid culture medium supplemented with

1 % Lac and 1 % polypepton to the above minimum

medium, which was cultured at 28°C for 3 days on

a reciprocal shaker. The culture liquor was incu-

bated with 1 % Lac and 0.1% SDS in 50 mM phos-

phate buffer (pH 7.0) at 40•Ž for 4 h. The reaction

mixtures were analyzed by TLC.

Cultures. A bacterial strain No. 216 was culti-

vated in 500-mL shaking flasks containing 100 mL

of medium to obtain Lac-oxidizing activity at 28•Ž

for 48 h on a reciprocating shaker. The medium

was composed of 1 % Lac, 1 % polypepton, 0.1%

yeast extract, 0.05% NaCI, 0.2% NH4NO3, 0.1%

K2HPO4, 0.1% KH2PO4 and 0.05% MgSOe 7H2O

and pH was adjusted to 7.0.

Enzyme assay. The Lac oxidizing activity was

measured by monitoring consumption of oxygen

using an oxygen electrode (YSI 5331 oxygen

probe, Yellow Springs, Ohio). 1.4 mL of 100 mM

Lac in 50 mM acetate buffer (pH 5.5) was preincu-

bated at 30•Ž. The reaction was initiated by the

addition of 200ƒÊL of cell-free enzyme prepara-

tion, and the initial velocity of oxygen consump-

tion was measured.

One unit of Lac-oxidizing activity was defined

as the amount of enzyme which consumed 1 p mol

of 02 per min at 30•Ž.

•@•@ Catalase activity was measured by monitoring

the decrease of hydrogen peroxide (H202) at 25•Ž

with absorbance at 240 nm.18) A hundred microlit-

ters of enzyme solution was added to 2.9 mL of

0.06% H2O2 in 50 mM phosphate buffer (pH 7.0)

in quartz cuvette (1 cm light path). The time re-

quired to decrease the absorbance at 240 nm from

0.450 to 0.400 was measured. This decrease corre-

sponded to the decomposition of 3.45 p mol of

hydrogen peroxide in 3 mL of reaction mixture .

One unit of catalase activity was defined as the

amount of enzyme which decomposed 1 p mol of

H2O2 per min at 25•Ž.

Analytical methods. H2O2 was determined by

peroxidase chromogen method described below.

Five hundred microlitters of a sample solution was

incubated with 5ƒÊL of 1% 4-aminoantipyrin, 25ƒÊ

L of 5% phenol, 50 ƒÊL of 1.0 U/mL peroxidase

solution, and 420 p L of 10 mM phosphate buffer

(pH 7.0) at 30•Ž for 15 min. The increase of ab-

sorbance was measured at 500 nm for 3 min. Re-

ducing sugar was determined by Somogyi and

Nelson's method.19,20) Total sugar was measured by

the phenol-sulfuric acid method.21) Protein concen-

tration was measured by the Bradford method.22)

Thin layer chromatography. TLC of the reac-

tion products was carried out to check reaction

products by Kieselgel 60 plates (Merck), using

ethyl acetate-acetic acid-water (3:1:1 , v/v) as a

solvent. Carbohydrates were detected by heating

the plate at 110-120•Ž after spraying sulfuric acid-

methanol (50%, wt/wt).

High performance liquid chromatography.

High performance liquid chromatography was car-

ried out under the following conditions: column,

471Microbial Production of Lactobionic Acid

Asahipak NH2P-50 (Shodex Co., Ltd.); solvent,

CH3CN/40 mM citrate buffer (60/40, v/v); flow

rate, 1.0 mL/mL; temperature, 40•Ž; detection, RI

detector (Shimazu Co., Ltd.).

Preparation of the crude enzyme. The cell-

free enzyme was prepared to observe oxidation of

saccharides without consumption of oxygen and

degradation of substrates and products by cells.

The cells of B. cepacia No. 216 (wet weight 100

g) were suspended in 130 mL of 10 mM phosphate

buffer (pH 7.0) and disrupted by passage through a

french pressure cell at 1500 kgf/cm2. After soni-

cated for 5 min to reduce the viscosity of the sam-

ple solution, it was centrifuged by 15,000•~g for

30 min to remove cell debris. The supernatant was

used as a crude enzyme preparation, and its total

and specific activities were 84.0 U and 0.0305 U/

mg protein. The crude enzyme was ultracentri-

fuged by 100,000•~g for 60 min. The precipi-

tated membrane fraction was washed with 50 mM

acetate buffer (pH 5.5) and ultracentrifuged again.

The precipitate was resuspended in 55 mL of the

same buffer, and was used as a membrane-bound

enzyme preparation. Supernatant solution had no

activity. The total and specific activities were 40.0

U and 0.184 U/mg. The enzyme was purified 6.0-

folds. This crude enzyme was used as cell-free

preparation with oxidizing activity. The enzyme

preparation had both oxidase and catalase activi-

ties.

Preparation of a reaction product from Lac.

The crude enzyme (0.5 U) preparation was incu-

bated with 10 mL of 100 mM lactose in 10 mM

phosphate buffer (pH 7.0) at 40•Ž for 48 h. The

reaction mixture was applied to an active-carbon

column (2•~20 cm) equilibrated with deionized

water. The passed and washed solutions were con-

centrated to 3 mL and applied to Bio-Gel P-2 col-

umn (3•~100 cm), which was eluted with 8%

ethanol. The eluted carbohydrates were detected by

phenol-sulfuric acid method. The product fractions

were collected and freeze-dried. 1

H and 13C-NMR measurements. A sample

was dissolved in D20, and 1H- and 13C-NMR spec-

tra were recorded at 300 MHz and 75 MHz, re-

spectively, with a JEOL AL-300 spectrometer. So-

dium 3-(trimethylsilyl)-propanesulfonate was used

as a shift reference.

Oxygen demand for the oxidation of Lac.

The cell-free enzyme extraction was dialyzed

against 10 mM phosphate buffer (pH 7.0) to re-

move cytoplasmic low molecular weight sub-

stances. The dialyzed enzyme solution (0.21 U/

mL, 200 ,CL) was incubated with 14.6 mM Lac in

50 mM acetate buffer (pH 5.5) at 30°C with and

without bubbling of nitrogen gas. The test tubes

were sealed and the gas phase was replaced with

N2 gas while the control tube continued to have air

in it. Both reaction mixtures were analyzed by

TLC after 0, 1, 2 and 3h.

Time courses of oxidation of v-Glc, maltose,

sucrose and Lac by cultivation. The strain was

cultivated with 300 mL each of four kinds of liq-

uid medium containing 1 % each of a carbon

source (D-glucose, maltose, sucrose, or Lac), 1.%

polypepton, 0.1% yeast extract, 0.05% NaCI, 0.2%

NH4N03, 0.1% K2HP04, 0.1% KH2P04 and 0.05%

MgS04.7H20. The concentrations of saccharides

and aldonic acids were measured by HPLC. The

absorbance at 660 nm and the amount of total or-

ganic carbon (TOC, mgC/mL) of the supernatants

were monitored. After 72 h cultivation, the dry

weights of cells were measured.

TOG measurement. TOC of culture superna

tants were measured by combustion oxidation-

infrared type TOC analysis method using TOC-

5000 (Shimazu Co., Ltd.) Potassium phthalate was

used as TOC reference. The measurement proce-

dures were the same as described in JIS K0102-22.

Time course of Lac oxidation by the enzyme

preparation. The enzyme (0.21 U/mL, 5.0 mL)

was incubated with 10 mM Lac in 10 mM phos-

phate buffer (pH 7.0) at 40•Ž, and after 0.5, 1, 2,

3, 4 and 5 h, the reaction mixtures were analyzed

photometrically. The amount of reducing sugar, to

tal sugar, and hydrogen peroxide were measured.

Effects of pH and temperature on activity and

stability of Lac oxidizing activity. The cell-free

extract was incubated with 100 mM Lac in various

pHs between pH 2.5 and 11.0, and the initial reac

tion rates were measured under the standard assay

conditions. The cell-free preparation was stored at

4•Ž for 20 h in various pHs and the residual ac

tivities were assayed under the standard assay con-


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.

RESULTS

Screening of Lac-oxidizing microorganisms.

We examined the several hundred colonies

grown on selection medium plates, and selected a

couple of strains judging from both the amounts of

LA (size of spots on TLC) in the reaction mixtures

and growth rates of the cells. We obtained a bacte-

rial strain No. 216, which oxidized Lac to accumu-

late LA in the culture liquor. It was a rod-shaped

and Gram negative bacterium and was identified as

B. cepacia by National Collections of Industrial

and Marine Bacteria Japan Co., Ltd., in Shizuoka,

Japan.

Preparation of a product from Lac and estima

tion of its structure.

A reaction product from Lac was prepared by

the procedure shown in MATERIALS AND METHOD.

About 300 mg of product was obtained from 1

mmol (342 mg) of Lac. The freeze-dried sample

(100 mg) was dissolved in 1 mL of D20 and ana

lyzed by 1H- and 13C-NMR spectroscopies. The

structure of the product was confirmed to be 4-0 -

ƒÀ-D-galactosyl D-gluconate , lactobionic acid (LA),

on the basis of the following data. The assign-

ments of all signals were described below. The

carbons and protons of gluconic residue corre-

spond to C1-C6, H1-H6 and those of galactosyl

residue correspond to C 1' -C6', Hi' -H6'. 1H-

NMR (D20) c4.56(d, H, 3JH,-,H2' =7.7 Hz, H1'),

4.21 (d, H, 3JH4,H3 - 3JH4,H5 - 2.8 Hz, H4), 4.13 (dd,

H, 3JH3,H2=4.8 Hz, 3JH3,H4=2.8 Hz, H3), 3.99 (d, H, 3J

H2,H3 = 4.8 Hz, H2), 3.91 (dd, H, 3JH4', H3' = 3.5 Hz,

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

100 mL of liquid medium for the enzyme production in

500-mL of shaking flask, and cultured at 28•Ž for 48 h.

The activity, pH, absorbance at 660 nm as a indicator of

microbial growth, and Lac and LA of the culture superna-

tant were measured after 4, 8, 24 and 48 h. The washed

cells from 5 mL of each culture was suspended to 1 mL

of 10 mM phosphate buffer (pH 7.0) and disrupted by

sonication. Cell debris was removed by centrifugation,

and their supernatants were assayed. -•¢-, activity; ---

•› ---, growth; ---•¢---, pH; -•›-, Lac (mg/mL); -•œ-,

LA (mg/mL).

3JH4 ,H5' = 3.3 Hz, H4'), 3.85 (dd, 2H, 3JH5,H6 = 3.3

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

= 3 .5 Hz, 2Jgeminal =16.1 Hz, H6'), 3.74 (dd, H,

3JHS,H4 - 2.9 Hz, 3JH5,H6 = 3.3 Hz, H5), 3.68 (dd, H,

3JH3', H2' = 6.4 Hz, 3JH3, H4' = 3.3 Hz, H3'), 3.64 (dd,

H, 3JH5', H4' = 3.3 Hz, 3JH5' , H6' = 3.5 Hz, H5), 3.56

(dd, H, 3JH2', HI' = 7.7 Hz, 3JH2' ,H3' = 7.5 Hz, H2') ;

13C -NMR (D20) o 181 .1 (C 1), 106.0 (C1'ƒÀ), 83.9

(C2), 78.0 (C3'), 76.5 (C4), 75.2 (CS'), 74.2 (C4'),

73.8 (C2'), 71.3 (CS), 64.6 (C6), 63.7 (C6').

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

216.

Time course of the cultivation. The growth (absorbance at 660 nm), pH, con

centrations of Lac and LA, and Lacoxidizing activity of the cells were monitored (Fig. 1). The cell

growth reached stationary phase after 30 h cultiva-tion. The pH gradually increased to 7.8 under this 1% Lac condition. The lactose-oxidizing activity reached maximum after 30 h and did not decrease for the following 24 h. The Lac decreased and completely disappeared after 48 h, while LA in-creased and finally reached 10 mg/mL.


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. We did the following experiment to confirm

whether the enzyme would need an appropriate hydrogen acceptor other than oxygen or not. The dialyzed crude enzyme was incubated with Lac with and without bubbling of nitrogen gas. Both of the reaction mixtures were analyzed by TLC. When the reaction system was replaced with N2, no oxidation of Lac occurred, while a considerable amount of LA was produced in the control reaction mixture. The enzyme needed oxygen as a pro-ton acceptor, and was thought to be an oxidase.

Capability and efficiency of oxidizing several saccharides of the strain.

To examine the capability of the strain to oxidize several saccharides, the strain was cultured using D-Glc, maltose, sucrose and Lac. The amounts of substrates and products (Fig. 2),

growth (Fig. 3), and TOG (Fig. 4) were measured. We calculated the growth yield for the decrease of TOG (YX/c) (%, w/w) and the product yield for the decrease of TOG (Ypic) (%, w/w) from these re-sults, and evaluated the efficiency of aldonic acids-

production. The YX/c value of each cultivation (D-Glc, maltose, sucrose, Lac, or no carbon source

was supplemented) was 16.2, 19.1, 17.3, 20.7 or

16.4%, respectively. The Yx/c value of Lac medium was not lower than those of other carbon sources.

The product yields for the decrease of TOG (Ypic)

(D-Glc, maltose, sucrose, Lac, or no carbon source was supplemented) were 0, 59.8, 0, 243.0 and 0%, respectively. These values were not calculated

from molar ratio of substrates and products, and

were not proportional to their conversion effi-ciency, but useful as indicators of the efficiency.

The results showed that Lac was the best substrate

for aldonic acid production.

Figure 2 shows the time courses of saccharides and aldonic acids of cultural supernatants. After 22

h cultivation, D-glucose, maltose, and sucrose com-

pletely disappeared. When maltose was used as a carbon source, a small amount of maltobionic acid

was observed after 8 h. It had reached maximum

concentration (4 mg/mL) at 22 h, decreased rap

idly and finally disappeared. In the case of Lac, the equivalent molarity of LA was produced. The

prolonged cultivation did not cause a decrease of LA.

Figure 3 shows growth curves and dry cell weights of each cultivation. Every culture reached

a stationary phase after 24 h. Dry cell weights of


Fig. 3. Growth curves of cultivations and dry cell weights.

The cultivations were carried out in the same way as in

Fig. 2 and the absorbances at 660 nm were measured.

After 72 h cultivation, the cells were centrifuged and

washed with 10 mM phosphate buffer (pH 7.0) and dried

at 105•Ž for 3 h at atmospheric pressure. ---•¢---, D-

glucose; -•£-, maltose; -• -, sucrose; -•œ-, lac-

tose; -•›-, no carbon source.

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

source.

72 h cultivation with D-glucose, maltose, sucrose or Lac as carbon sources were 0.302, 0.336, 0.340 and 0.178 g, respectively. The dry cell weight un-der the same conditions without a carbon source was 0.146 g. The mean value when Lac or no car-bon source was added was 0.162 g, which was al-most the half of the average (0.326 g) as the other three were used as carbon sources. Figure 4 shows the time courses of TOC of cul-

ture supernatants. The decrease rates of TOC at the first 24 h were 0.260, 0.246, 0.272 and 0.121 mg/mL/h, respectively, when D-Glc, maltose, su-crose or Lac was used as a carbon source, while the decrease rates of TOC under the same condi-tions without a carbon source was 0.122 mg/mL/ h. This result shows that the decrease rates of TOC up to 24 h cultivations for which Lac or no carbon source was added were about the half of the rates when D-Glc, maltose, or sucrose was used as a carbon source.

Time course of Lac oxidation by the oxidase. The time course of the oxidation of Lac was ob-

served by measuring total sugar, reducing sugar, and hydrogen peroxide in the reaction mixture. Af-ter 5 h, the total sugar was reduced to half of the

initial amount and the reducing sugar completely

vanished. LA has no reducing power and its sensi-

tivity in phenol-sulfuric acid detection is half of

that of Lac. A trace amount of hydrogen peroxide

was detected, and it gradually increased during the

reaction. The ratio of H2O2 to LA was about

1/150-1/300 (H202/LA, molar ratio).

Effects of pH and temperature on activity and

stability of the oxidase.

The Lac oxidizing activity was the most active at

pH 5.5 and stable between pH 6.0 and 9.0. The

optimum temperature was 50•Ž, and stable below

35•Ž.

•@•@ Oxidation of several saccharides by the enzyme

preparation.•@•@

The reactivity of the cell-free enzyme prepara-

tion was examined on various mono- and oligosac-

charides. The relative rates, compared to the value

obtained when Lac was used as a substrate, are

shown in Table 1. The most favorable substrate for

the enzyme was D-Glc. It oxidized 2- and 4-epimer

of D-Glc well. The enzyme also reacted with oli-

gosaccharides such as Lac, cellobiose, maltose,

maltotriose, maltotetraose and maltopentaose.

Capability of the enzyme to distinguish ano-

meric type of v-Gk.

The reactivities of the cell-free extract on w-

and ƒÀ-D-GIc were examined. When the enzyme

preparation was incubated with maltopentaose,


Table 1. Oxidizing ability of cell free-extract of B.

cepacia No. 216 on various saccharides.

maltopentaonic ƒÂ-lactone was produced and oxy-

gen concentration in the reaction mixture de-

creased linearly. The following addition of gluco-

amylase to the reaction mixture accelerated the

consumption rate of oxygen but the addition of ƒ¿-

glucosidase did not change the consumption rate

of oxygen (Fig. 5). It is well known that gluco-

amylase and ƒ¿-glucosidase hydrolyze maltooligo-

saccharides and produce ƒÀ- and a-anomer of D-

Glc, respectively. In addition the enzyme oxidized

D-Glc faster than maltopentaose (Table 1). There-

fore it was presumed that the enzyme oxidized ƒÀ-

anomer of D-Glc, but not a-anomer.

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.

DISCUSSIONS

We obtained a bacterial strain B. cepacia No.

216 that had a Lac-oxidizing activity in its mem-

brane fraction. The Lac oxidation occurred only in

the presence of oxygen and was not affected by

the absence of other hydrogen acceptors from cy-

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

substrate for the enzyme, which suggests that the

enzyme is a glucose oxidase. The oxidase from B.

cepacia was characterized by a broad substrate

specificity in contrast to rigid substrate specificity

of the ordinary glucose oxidases. The oxidase

acted on not only aldohexoses, but also aldopento-

ses and oligosaccharides such as D-xylose, L-

arabinose, D-ribose, Lac, cellobiose and maltooli-

gosaccharides, but not oxidize ketoses and nonre-ducing sugars. Well-known glucose oxidases are

highly specific to /3-D-Glc, and their reaction rates

of a-D-Glc oxidation are negligible. Figure 5

shows that the oxidase, like other common glucose

oxidases, effectively catalyzed the oxidation of ƒÀ-

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

organisms and enzymes, such as glucose oxidases,

hexose oxidases, glucooligosaccharide oxidase, and

lactose dehydrogenase are not suitable for produc-

tion of LA because their reactivities on Lac are

weak and cultivations are not easy. In contrast to

these organisms and enzymes, B. cepacia No. 216


is advantageous to effective production of LA. The

strain has a high reactivity on Lac, and does not

digest either Lac nor LA. When D-Glc, maltose and sucrose were used as

carbon sources for cultivation, the saccharides and

their oxidized products did not remain in the cul-

ture. When Lac was given as a carbon source, the

strain was not able to digest LA but accumulated it in culture (Fig. 2). The time courses of the culti-

vation showed that Lac and LA were not assimi-

lated by the strain. The results suggested that Lac

was metabolized only by catabolic procedure and

was converted to LA, whereas other nutrients such as polypepton, yeast extracts, D-Glc, maltose and

sucrose were metabolized by both anabolic and

catabolic procedure to be converted to cell compo-

nents, energy and carbon dioxide. The culture liquor was composed of a solid

fraction (cells) and a liquid fraction (culture super-

natant). TOC lost from the liquid fraction was ex-

pelled as carbon dioxide to gas phase or trans-formed to solid material as cell components. The

growth yield for the decrease of TOC (Y /c) is that whatever amount of TOC was lost from liquid was

utilized as cell components. The result also sug-

gested that it was not necessary to supplement the Lac medium with other carbon sources for rapid

growth of cells. All these results suggest that the combination of the strain as an oxidase producer and Lac as a substrate is an excellent system of al-

donic acid production.

The author appreciates a generous gift of a-1,6-galactobiose from Dr. H. Hashimoto of Faculty of Agricul-ture, Shinshu University.

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(Received February 26, 2002; Accepted May 27, 2002)

ラクトビオン酸生産微生物の検索

村上洋,河野純子1,吉栖肇1,

中野博文,北畑寿美雄

大阪市立工業研究所

(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)

　ラクトビオン酸はラクトースから合成されるアルド

ビオン酸で,ビフィズス菌選択増殖活性を持ち,ミネ

ラルと水溶性の高い塩を作るなど,さまざまな用途が

期待される糖質素材である.われわれは,微生物ある

いは酵素を用いたラクトビオン酸生産法を確立するた

め,ラクトースの酸化活性を持つ微生物を検索した.

その結果,Burkholderia cepaciaの一菌株を得た.ラ

クトースの酸化活性は,菌体内に存在し,菌体を破砕

後,酸素の存在下および非存在下でラクトースに作用

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

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

シロース,L-アラビノース,D-リボース,ラクトー

ス,セロビオース,マルトース,マルトトリオース,

マルトテトラオース,マルトペンタオースに作用し

た.本菌株は培養上清中に2%のラクトビオン酸をラ

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

適用可能であると考えられる.

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