Screening of Lactobionic Acid Producing Microorganisms Hiromi
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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 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- 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 de- crease 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.