J. Gen. Appl. Microbiol., 38, 483-490 (1992)

ISOLATION AND CHARACTERIZATION OF - AND TREHALULOSE- PRODUCING BACTERIA FROM THAILAND SOIL

KEN'ICHIRO TSUYUKI,* TOSHIAKI SUGITANI, YUKIE MIYATA, TADASHI EBASHI, AND YOSHIKAZU NAKAJIMA

Research and Development Department, Mitsui Sugar Co., Ltd., Kawasaki 210, Japan

(Received July 2, 1992)

The bacterial screening to get the strains that produce isomaltulose (palatinose, 6-0-a-D-glucopyranosyl-D-) and trehalulose (1-0-a- D-glucopyranosyl-D-fructose) from , were carried out in Thailand. Many samples such as soil, fruits, etc., were obtained mainly from the northeastern area of Thailand. And several strains named MX-2, -4, -10, -11, and -13 were isolated from these samples. These strains, which were facultative anaerobic, gram-negative and rod-shaped bacteria, belonged to the genus Klebsiella : K. planticola (MX-2, -4, -10, and -11), and K. terrigena (MX-13) The strain MX-10 produced isomaltulose and treha- lulose from sucrose, and the yields of isomaltulose and trehalulose were 65 and 30% as against sucrose, respectively, when 98% of sucrose was transformed. It is interesting that the trehalulose yields were higher in the reactions by the strain MX-10 than those by Protaminobacter rubrum (9% of trehalulose and 86% of isomaltulose). The results obtained by using immobilized cells of MX-10 were described.

Since the isolation of Protaminobacter rubrum CBS 574.77 which produces isomaltulose from sucrose (18), the production of isomaltulose (palatinose, 6- 0-a-D-glucopyranosyl-D-fructose) has been well studied (8, 9,17). Isomaltulose, a structural of sucrose, is a non-cariogenic sugar (5,12-14), and is about half as sweet as sucrose. Recently it has been produced in industrial scale with development of immobilization method (8, 9,17), and has been used in various confectionery products as sugar substitutes in Japan. It was well known that sucrose was converted to isomaltulose and trehalulose by a-glucosyltransferase (3,

* Address reprint requests to: Ken'ichiro Tsuyuki , Research and Development Department, Mitsui Sugar Co., Ltd., 2-1 Mizue-cho, Kawasaki-ku, Kawasaki 210, Japan.

483 484 TSUYUKI, SUGITANI, MIYATA, EBASHI, and NAKAJIMA VOL. 38

8, 9,17), which was produced by Serratia plymuthica (2), Erwinia rhapontici (1), Pseudomonas mesoacidophila (7), and P. rubrum. P. rubrum's a-glucosyltransferase transformed mainly sucrose to isomaltulose (85.7%), and transformed to trehalu- lose (8.7%, 1-0-a-D-glucopyranosyl-D-fructose) and a little amount of and fructose as by-products (8, 9,17) . Trehalulose, which has a good taste, and is non-crystalline, and non-cariogenic (6), and naturally occurs in , was ob- tained as a by-product (10). Because water solubility of trehalulose is higher than that of isomaltulose, trehalulose is suitable in foods, for example jam and jelly. Then we paid attention to not only isomaltulose but also by-product trehalulose. The bacterial screening was conducted in the area of northeastern Thailand, and we could find the strains that produce more trehalulose than P. rubrum. In this paper, taxonomical characterizations of these strains are described. And the production of trehalulose that was examined with immobilization method, was reported together.

MATERIALS AND METHODS

Media and cultivation. Isomaltulose agar medium was used for the screening of bacteria, and contained 1.0% isomaltulose, 0.05% yeast extract, 0.2% Na2HPO4.12H2O, 0.3% NaCI, 0.1% KH2PO4, 0.02% KCI, 0.05% MgSO4.7H2O, 0.1% NH4PO4, 0.01% cycloheximide, and 2.0% agar (pH 7.2), and the culture was done at 37°C for 24-48 h. Sucrose medium, which contained 10% sucrose, 1.0% peptone, 0.5% yeast extract, 0.3% meat extract, 0.2% Na2HPO4 12H20, and 0.3% NaCI (pH 7.0), was used for growth of the strains and productions of trehalulose and isomaltulose from sucrose at 37°C with standing or reciprocal shaking culture. In the case of shaking culture, a shaking flask containing 100 ml of sucrose medium was used for the cultivation for 24 h. In another case, the test tubes containing 10 ml of sucrose medium were used. Sucrose agar slant was prepared by adding 2.0% agar to sucrose medium. And the cultivation medium for jar fermentor, which was added 10% affination syrup and 0.03% anti-foaming agent (Nissan Disfoam CC- 118, Nippon Oil & Fats Co., Ltd.) in sucrose medium instead of sucrose, was used for the cultivation at 30°C for 16.5 h with the aeration rate of 1/4 vvm by using a jar fermentor (MODEL MD-300; B. E. Marubishi Co., Ltd., Tokyo). Isolation of bacteria. Soil and fruit samples were collected in the area of northeastern Thailand. These samples were suspended in the sterilized water and spread over the palatinose agar plates. These plates were cultured at 37°C for 24- 48 h. A part of each single colony obtained was transferred into the sucrose medium in a tese tube, and cultured at 37°C for 24h as standing culture. The culture was used as a sample of thin-layer chromatography (TLC) to select the strain. Another part of the colony was transferred to a sucrose agar slant for the stock culture. Thin-layer chromatography. Zero point five microliter of the cultures pre- pared at Bx. 2 were spotted on TLC plates (TLC aluminum sheets silica gel 60, 1992 Isolation of Trehalulose-Producing Bacteria 485

Merck art. 5553) and developed. TLC was carried out using a solvent system ethylacetate-acetic acid-water (3 : 3 :1 by volume). The spots were visualized with the diphenylamine-aniline-phosphoric acid reagent (11) at 105°C. Measurement of a glucosyltransferase activity. One milliliter of the culture and 4m1 of 25% (w/v) sucrose in calcium acetate buffer (pH 5.6) were mixed well and incubated at 20°C for 60 min. The quantity of reducing sugars in the reacted solutions was determined by Shaffer Somogyi method (16). a-Glucosyltransferase activity was calculated at the quantity of isomaltulose. High performance liquid chromatography (HPLC). An Erma ERC-8711 liquid chromatography pump was used. The conditions used were as follows: column, ERC-NH-1171; solvent, 80% (v/v) acetonitrile-water; temperature, room temperature; flow rate, 1.0 ml/min; detector, ERMA RI detector ERC-7521. The sample solution was filtrated through a membrane filter (pore size, 0.20 tim). Taxonomical identification method. Nutrient agar medium (Difco, Labora- tories, Detroit, Mich.) was used for preculture and growth test at 10°C. Gram reaction was tested by KOH method (4), and oxidase test was carried out with cytochrome-oxidase reagent (Nissui Pharmaceutical Co., Ltd., Tokyo). 0- Nitrophenyl-j3-D-galactopyranoside test (ONPG test), arginine dihydrolase test, lysine decarboxylase test, ornithine decarboxylase test, citrate utilization test, hydrogen sulfide test (H2S), crease test, tryptophan deaminase, indole test, Voges- Proskauer test, and gelatin liquefaction test were carried out by using the API-20E kit (API SYSTEM S. A.). Acid formation tests from glucose, mannitol, inositol, sorbitol, rhamnose, saccharose, melibiose, amygdalin, and arabinose were carried out by using the API-20E kit. Utilization tests of melezitose were carried out by using Barsikow's medium with 10o of melezitose (melezitose 10 g, peptone 10 g, NaCI 5 g, BTB 0.08 g, and list. water 1,000 ml, pH 7.0) (15). Immobilization of the MX-1O cells. The strain MX-10 was cultured with the jar fermentor containing affination medium (3.01) for 16.5 h. Cultured cells were concentrated with a centrifuge (KOKUSAN Co., Ltd., Model H-251) at 15,000>< g for 30 min at 5°C, and then washed with water. The concentrated slurry (1 volume) was mixed well with an aqueous solution of 4% sodium alginate (1 volume), and then the mixture was dropped into an aqueous solution of 0.25 M calcium chloride to gelatinize the mixture into granules. After 1 h aging, this granule gel was washed with water, and mixed well with the same volume of 2% polyethylenimine (pH 5.6, PEI) for 5 min. Then, gel beads were separated from PEI and were mixed with 0.5°/6 glutaraldehyde (GA) solution at 5°C, and agitated for 20 min. After that, the immobilized cell was separated from GA solution and washed well with water, and the immobilized enzyme was obtained. This im- mobilized enzyme was examined for a-glucosyltransferase activity. 486 TSUYUKI, SUGITANI, MIYATA, EBASHI, and NAKAJIMA VOL. 38

Table 1. List of strains and source of isolation.

Table 2. Glucosyltransferase ability of isolates.

RESULTS

Isolation of bacteria Five strains which produced isomaltulose and trehalulose from sucrose were obtained from the samples shown in Table 1. Trehalulose productions of these strains were more than that of P, rubrum (Table 2). In this investigation, one unit (U) of this a-glucosyltransferase was defined as the amount of the enzyme that could convert one micromole of sucrose to the other sugars in 1 min at initial stage of reaction in 20% (w/v) sucrose solution (pH 5.6) at 20°C.

Sugar conversion of the isolates These isolates converted sucrose to isomaltulose, trehalulose, and a little amount of glucose and fructose. Enzyme activity, sugar composition of reacted solution, and the ratios of the amount of isomaltulose to that of trehalulose are shown in Table 2.

Identification of the isolates The five strains, which produced isomaltulose and trehalulose from sucrose, were gram-negative, facultatively anaerobic, non-motile and rod-shaped bacteria. All five strains were positive for the ONPG test and Voges-Proskauer test. These strains produced urease and lysine decarboxylase, but did not produce oxidase, 1992 Isolation of Trehalulose-Producing Bacteria 487

Table 3. Taxonomic features of the isolates.

arginine dihydrolase, ornithine decarboxylase, and tryptophan deaminase. They did not hydrolyze gelatin and did not produce H2S, but utilized citrate. Except for strain MX-10, they could not produce indole. All five strains could grow at 10°C and produced acids from carbohydrates such as D-glucose, L-arabinose, rhamnose, sucrose, melibiose, inositol, mannitol, sorbitol and amygdalin. But, these strains, except for strain MX-13, could not produce acid from melezitose. These results are shown in Table 3.

Characterization of the MX 10 immobilized enzyme On the cultivation of MX-10, the time course of growth, pH, and the enzyme activity are shown in Fig. 1. The enzyme activity peaked at 8 h of cultivation. The immobilized enzyme of MX-10 was prepared from the culture that had 16.5 h cultivation. Absorbance of the culture was 27.2 at 600 nm, and enzyme activity was 488 TSUYUKI, SUGITANI, MIYATA, EBASHI, and NAKAJIMA VOL. 38

Fig. 1. Time course of growth, pH, and the enzyme activity by the isolate MX-10. The strain MX-10 was cultured with the jar fermentor containing affination medium (temp., 30°C; aeration: 1/4 vvm; Kd, 4). •, the enzyme activity (U/ml); A, pH; •, cell growth (ODD).

9.7 U/ml. The immobilized enzyme showed 40.7 U/ml as enzyme activity, and converted sucrose into 65.4% of isomaltulose and 29.7% of trehalulose.

DISCUSSION

Five isolates (MX-2, -4, -10, -11, and -13), which were from the soil samples of Thailand, produced isomaltulose and trehalulose from sucrose. And these five isolates were non-motile, gram-negative, facultatively aerobic, and rod-shaped bacteria. They fermented inositol and indicated positive reaction on the Voges- Proskauer test, but did not produce oxidase and ornithine decarboxylase. There- fore, according to Bergey's Manual of Systematic Bacteriology, Vol. 1, these five isolates would be identified as the genus Klebsiella; four isolates (MX-2, -4, -10, and -11) would be identified as K, planticola, because they could grow at 10°C, but could not utilize melezitose; the isolate MX-13 would be identified as K. terrigena, because it could grow at 10°C, and could utilize melezitose. These five strains, particularly strains MX-l0, -11, and -13, produced trehalu- lose with higher yield from sucrose. MX-13 made trehalulose with the highest ratio but its enzyme activity was lower than the other strains, and MX-11 had a problem of bad sedimentation when we prepared the immobilized enzyme. Then, MX-10 was selected to prepare the immobilized enzyme. On the jar fermentor culture of MX-10 with affination medium, its enzyme 1992 Isolation of Trehalulose-Producing Bacteria 489 activity peaked at 10 h (10.0 U/ml); after 16.5 h, it became 9.7 U/ml. Immobilized enzyme, which was prepared with the culture, had 40.7 U/g of a-glucosyltransferase activity and produced 65.4% of isomaltulose and 29.7% of trehalulose from sucrose, when 98% of sucrose was transformed. As compared with the im- mobilized enzyme of Protaminobacter, it was shown that about 3 times more trehalulose was produced with the obtained MX-10 enzyme (8, 9,17). As we have shown, trehalulose could be produced commercially by MX-10 immobilized enzyme.

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