Efficient synthesis of glyceroyl β-lactoside and its derivatives through a condensation reaction by cellulase

Biochimica et Biophysica Acta 1620 (2003) 252 – 258 www.bba-direct.com

Efficient synthesis of glyceroyl h-lactoside and its derivatives through a condensation reaction by cellulase Nozomu Yasutake a, Kazuhide Totani b, Yoichiro Harada b, Shinobu Haraguchi b, Takeomi Murata b, Taichi Usui b,* a

Science of Biological Resource, The United Graduate School of Agricultural Science, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan b Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Ohya 836, Shizuoka 422-8529, Japan Received 30 September 2002; received in revised form 3 December 2002; accepted 3 December 2002

Abstract Condensation reaction between lactose and glycerol was effectively catalyzed by utilizing a commercially available cellulase preparation from Trichoderma reesei. The enzyme induced the formation of 1-O-h-lactosyl-(R,S)-glycerol (1) and 2-O-h-lactosyl glycerol (2) in a molar ratio of 7:3 and in a 20% yield based on lactose added. The enzyme also induced the condensation of lactose with 1,3-propanediol to produce O-h-lactosyl propanediol (3) in a yield of 15%. When various alkanols (n: 2 – 8) and allyl alcohol were used in the condensation reaction, the corresponding alkyl and allyl h-lactoside were obtained in the yields of 0.9 – 3.8% of the desired compounds. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Condensation; Synthesis; Lactoside; Trichoderma reesei; Cellulase; Glycerol; Lactose

1. Introduction There has been growing interest in the use of glycosidases for the synthesis of glycoside and oligosaccharide, because they usually not only hydrolyze glycosidic bonds but also catalyze the new formation of glycosides [1 –6]. Glycosidasecatalyzed synthesis has generally been achieved through two types of reactions, condensation and transglycosylation. We have recently found that endo-h-glycosidase, a cellulase from Trichoderma reesei that shows transglycosylation activity, transfers the entire lactose and N-acetyllactosamine from donor substrates, p-nitrophenyl h-lactoside (Lach-pNP) and p-nitrophenyl h-N-acetyllactosaminide (LacNAch-pNP), to hydroxyl groups of acceptor substrates such as alkanols or sugars [2]. However, the use of Lach-pNP and LacNAchpNP is not always suitable for practical synthesis because of the restricted availability of expensive substrates. If such a

Abbreviations: Lach-pNP, p-nitrophenyl h-lactoside; LacNAch-pNP, p-nitrophenyl h-N-acetyllactosaminide; Lach-ethyl, ethyl h-lactoside; Lach-octyl, octyl h-lactoside; TPS, sodium 3-(tri-methylsilyl)-propionate; CMC, sodium carboxymethyl cellulose; Lac, lactose; Glc, D-glucose; Gal, D-galactose * Corresponding author. Fax: +81-54-238-4873. E-mail address: [email protected] (T. Usui).

lactosyl glycoside formation could be mediated through condensation, it would be a very attractive method for the synthesis of lactosyl glycoside. The use of cheap lactose as a substrate has great potential for mass production of useful glycosides. There has been no report of condensation of a lactose unit through an enzymatic process. Our interest was directed toward the establishment of a method of efficient synthesis for obtaining lactosyl h-glycerol as a precursor of neoglycolipid in sufficient amount. Such synthetic substances could be utilized as starting materials for the synthesis of neoglycolipid [7] and new kinds of detergents [8] and as acceptors for glycosidase and glycosyltransferase [9]. In this paper, we describe an efficient method for synthesizing glyceroyl h-lactoside and its derivatives through a condensation reaction by utilizing a commercially available enzyme preparation from T. reesei producing various types of h-1,4glucanases known as cellulases [10 – 13].

2. Materials and methods 2.1. Materials Cellulase (crude enzyme powder) from T. reesei C1 was obtained from Kyowa Hakko Co., Ltd. Lach-pNP, ethyl h-

0304-4165/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0304-4165(03)00004-7

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lactoside (Lach-ethyl), and octyl h-lactoside (Lach-octyl) were prepared by our previously described methods [2]. Butyl h-lactoside (Lach-butyl) was purchased from SigmaAldrich Co. Silicagel (Wakogel C-300) was purchased from Wako and YMC-PolyamineII was from YMC Japan Ltd. DEAE-Sepharose Fast Flow, a MonoP HR 5/20 column with Poly buffer, and PhastGel (Gradient 8-25) for PhastSystem were purchased from Amersham Pharmacia Biotech. All other reagents were of the highest quality commercially available and were used without further purification. 2.2. Analytical methods FAB-mass analysis was carried out in the positive ion mode using a JEOL JMS DX-303HF mass spectrometer coupled to a JEOL DA-800 mass data system. The accelerating voltage of 10 kV and mass resolution of 1000 were used. A sample of 1 Al in H2O or CH3OH was loaded with 1 Al of glycerol as a matrix. 1H- and 13C-NMR spectra of each sample in D2O or CD3OD were recorded on a JEOL JNMLA 500 or JEOL JNM-EX 270 spectrometer at 30 jC. Chemical shifts were expressed in d relative to sodium 3(tri-methylsilyl)-propionate (TPS) as an external standard. 2.3. Analysis of condensation reaction Condensation reactions between lactose and various alkanols using the crude enzyme were performed and analyzed by HPLC and TLC methods as follows. A reaction mixture consisting of 0.9 M of lactose, 27% (v/v) of each alkanol, 167 mg/ml (2 units of Lach-pNP hydrolytic activity) of crude enzyme powder and 50 mM of sodium acetate buffer (pH 5.5) in a fatal volume of 150 Al was incubated at 40 jC for 24 h with vigorous stirring. An aliquot (10 Al) of each assay mixture was diluted with distilled water and heated at 100 jC for 5 min to stop the reaction, and then the assay mixture was analyzed by HPLC (column, Mightysil Si 60, f 0.46  25 cm; solvent, CH3CN/H2O 4/1; flow rate, 1.0 ml/min; detection, RI). The retention times of products by condensation reactions were compared with those of authentic samples of Lach-ethyl, lactose (Lac), D-galactose (Gal), and D-glucose (Glc). Each assay mixture was also analyzed on a TLC plate (Merk Kieselgel 60/F254) with solvents (CHCl3/CH3OH/H2O 60:35:8 or 50:45:10) by the Orcinol – sulfuric acid method [15]. The positions of detected spots were compared with those of authentic samples. 2.4. Preparation of condensation products Glyceroyl h-lactoside [1-O-h-lactosyl-(R,S)-glycerol (1) and 2-O-h-lactosyl glycerol (2)] was synthesized as follows. A mixture containing 0.2 M of lactose, 5.0 M of glycerol, 100 mg/ml of crude enzyme powder (8.4 units of Lach-pNP hydrolytic activity), and 12.5% ethyl acetate in 1 ml of 50

253

mM sodium acetate buffer (pH 5.0) was incubated at 40 jC for 48 h. The reaction was terminated by heating at 100 jC for 5 min, and the supernatant obtained from centrifugation (17,000 rpm, 10 min) was loaded onto a charcoal-Celite column (f 8  40 cm) equilibrated with distilled water. After washing the column with 5 l of distilled water, adsorbed portion was eluted with a linear gradient of ethanol concentration from 0% to 20% in a total volume of 10 l. The neutral sugar contents of eluted fractions were measured at 485 nm by the phenol – sulfuric acid method [16]. As shown in Fig. 2a, the chromatogram showed two peaks (F-1, tubes 50 – 96; F-2, tubes 155– 190). F-2 was concentrated and lyophilized (1.76 g). F-1 mainly contained lactose used as a substrate. F-2 was dissolved in 2.7 ml of distilled water, and aliquots (50 – 100 Al) of the solution were applied to a column of YMC-Polyamine II by HPLC. F-II was separated into two fractions, F-II-1 and F-II-2, as shown in Fig. 2b, and each fraction was concentrated and lyophilized to afford compound 2 (9.6 mg) from F-II-1 and compound 1 (22.4 mg) from F-II-2. Compound 1, m/z 417 (M + H)+ and 439 (M + Na)+. 1HNMR data (in D2O): d 4.48 (d, J 7.6 Hz, H-1), 4.42 (d, J 7.6 Hz, H-1V). Compound 2, m/z 417 (M + H) + and 439 (M + Na)+. 1H-NMR data (in D2O): d 4.64 (d, J 8.1 Hz, H-1), 4.46 (d, J 7.6 Hz, H-1V). 13C-NMR data in D2O are summarized in Table 1. For the synthesis of Lach-ethyl, a reaction mixture containing 0.8 M of lactose, 3.3 M of ethanol, and 150 mg/ml of crude enzyme powder (25 units of Lach-pNP hydrolytic activity) in 2 ml of 50 mM sodium acetate buffer (pH 5.5) was incubated at 37 jC for 72 h. The reaction was terminated by heating at 100 jC for 5 min. The supernatant obtained from the centrifugation (17,000 rpm, 10 min) was evaporated to dryness, dissolved into 5 ml of distilled water, and charged onto a charcoal (Wako HPLC Grade) column (f 2.5  25 cm). The column was developed with a linear gradient of 0 – 20% ethanol at a flow rate of 1 ml/min and a fraction size of 10 ml/tube. The neutral sugar content of each fraction was measured in a similar manner. A fraction from tubes 20– 50 was concentrated and dissolved in 5 ml of CHCl3/CH3OH/H2O 60:35:8 and then charged onto a Silicagel (Wakogel C-300) column (f 2  40 cm). The column was developed with the same solvent at a flow rate of 3.0 ml/min and fraction size of 6 ml/tube. A fraction from tubes 13– 20 was concentrated and lyophilized (23 mg) in a yield of 3.8% based on lactose added. The physical and NMR data were identical to those of the authentic sample reported previously [2]. In a similar manner, O-h-lactosyl propanediol (3), propyl h-lactoside (4), isopropyl h-lactoside (5), butyl h-lactoside (6), and allyl h-lactoside (7) were prepared as lyophilized powders of 102, 13.8, 5.3, 5.8, and 17.1 mg, respectively. Compound 3, m/z 401 (M + H)+ and 461 (M + Na + K)+. 1HNMR data (in D2O): d 4.50 (d, J 8.1 Hz, H-1), 4.46 (d, J 7.8 Hz, H-1V), 1.88 (dd, H-h). Compound 4, m/z 385 (M + H)+ and 407 (M + Na)+. 1H-NMR data (in D2O): d 4.48 (d, J 7.9

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Table 1 13 C-NMR chemical shifts of condensation products (1 – 7) C-1

C-2

C-4

C-5

C-6

77.1

81.3

77.6

62.9

75.4 65.2

71.4

78.2

63.9

2-O-b-lactosyl glycerol (2) Glc 104.6 75.8 Gal 105.8 73.8 Glycerol 64.2 83.8

77.1 75.4 63.7

81.2 71.4

77.6 78.2

62.9 63.9

O-b-lactosyl propanediol (3) Glc 105.0 75.7 Gal 105.8 73.9 Propyl 70.2 34.3

77.3 75.4 61.4

81.3 71.4

77.7 78.2

63.0 63.9

Propyl b-lactoside (4) Glc 104.8 Gal 105.8 Propyl 75.2

75.8 73.9 25.1

77.3 75.2 12.5

81.3 71.5

77.7 78.2

63.0 63.9

Isopropyl b-lactoside (5) Glc 103.1 Gal 105.8 Isopropyl 76.1

75.8 73.9 25.2

77.4 75.4 23.8

81.4 71.4

77.6 78.2

63.0 63.9

Butyl b-lactoside (6) Glc 104.8 Gal 105.9 Butyl 73.3

75.7 73.8 33.8

77.4 75.4 21.3

81.3 71.4 15.9

77.7 78.2

63.0 63.9

Allyl b-lactoside (7) Glc 103.9 Gal 105.8 Allyl 73.6

75.7 73.9 136.2

77.3 75.4 121.7

81.3 71.4

77.6 78.2

63.0 63.9

1-O-b-lactosyl-(R,S)-glycerol (1) Glc 105.4 75.7 105.1 Gal 105.8 73.8 Glycerol 73.6a 73.2a 73.5a

C-3

performed by a slightly modified procedure as follows. F-2 was concentrated to 10 ml with a membrane filter for MW 10,000 (Biomax PBGC Ultrafiltration Disc, Millipore Corp.) and applied to a MonoP column (f 0.5  20 cm) equilibrated with Poly buffer (pH 5.0). The column was washed with 60 ml of the same buffer at a flow rate of 0.5 ml/min and eluted with a linear gradient from pH 5.0 to 3.0 in a total volume of 30 ml. A fraction from tubes 40– 48 showing both hydrolytic activities for Lach-pNP and sodium carboxymethyl cellulose (CMC) was concentrated to 2 ml with the same membrane filter. The purified enzyme was checked by SDS- and IEF-PAGE (PhastSystem, Amersham Pharmacia Biotech). The hydrolytic activities toward Lach-pNP, CMC, and Avicel were measured by our previously reported methods [2]. 2.6. Improvement in productivity

Chemical shifts are shown in parts per million downfield from internal TPS. a Assignment may be interchanged.

Hz, H-1), 4.44 (d, J 7.7 Hz, H-1V), 3.30 (dd, H-2), 1.64 (m, H-h), 0.91 (t, H-g). Compound 5, m/z 385 (M + H)+ and 407 (M + Na)+. 1H-NMR data (in D2O): d 4.58 (d, J 7.8 Hz, H1), 4.46 (d, J 7.6 Hz, H-1V), 3.28 (dd, H-2), 4.12 (m, H-a), 1.23 (t, H-h), 1.23 (t, H-hV). Compound 6, m/z 399 (M + H)+ and 421 (M + Na)+. 1H-NMR data (in D2O): d 4.49 (d, J 8.4 Hz, H-1), 4.46 (d, J 8.4 Hz, H-1V), 3.31 (dd, H-2), 1.62 (m, H-h), 1.38 (t, H-g), 0.92 (t, H-y). Compound 7, m/z 383 (M + H)+ and 405 (M + Na)+.1H-NMR data (in D2O): d 4.54 (d, J 7.8 Hz, H-1), 4.46 (d, J 7.8 Hz, H-1V), 3.35 (dd, H-2), 4.24 (d, H-a), 5.98 (m, H-h), 5.34 (d, H-yg). 13C NMR data in D2O are summarized in Table 1. 2.5. Purification of enzyme Hydrolytic activities for Lach-pNP were fractionated into three fractions, F-1, F-2, and F-3, by a chromatographic procedure on a DEAE-Sepharose Fast Flow column (Fig. 4) as previously described [2]. Further purification of F-2 was

Changes in the amounts of glyceroyl h-lactoside and Lach-ethyl with time were examined as follows. A reaction mixture consisting of 0.9 M of lactose, 27% (v/v) of each alkanol, 10 mU/l (Lach-pNP hydrolytic activity) of F-2, F-3 or crude enzyme powder, and 50 mM of sodium acetate buffer (pH 5.5) was incubated at 40 jC in a total volume of 1 ml at each time. An aliquot (10 Al) of the assay mixture was diluted with distilled water and heated at 100 jC for 5 min to stop the reaction, and the assay mixture was analyzed by HPLC as described above.

3. Results and discussion A commercially available crude cellulase preparation was directly used for the synthesis of lactosyl glycosides through condensation reactions of lactose with various alkanols without further purification. 3.1. Synthesis of glyceroyl b-lactoside and its analog A condensation reaction between lactose and glycerol was performed using a crude enzyme preparation from T. reesei. The molar ratio of lactose and glycerol was 1:25, and the total substrate concentration was 53% (w/v). The reaction was analyzed by HPLC, and several peaks were presumed to be condensation products formed during the incubation (Fig. 1b). The presumed products were observed in an analytical yield of 25% based on lactose added. In this case, the addition of ethyl acetate at 12.5% concentration to the reaction system resulted in a little high yield (about 10%) of 1 and 2. In general, the efficiency is known to be improved in the presence of a minimal amount of water and an excess of substrates. The reaction mixture was fractionated to F-I and F-II by a charcoal-Celite column (Fig. 2a). F-II was further separated into F-II-1 and F-II-2 on a column of YMC-PolyamineII (Fig. 2b). The generation of compounds F-II-1 and F-II-2 reached maximal level at 36–

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Fig. 1. HPLC analysis of the cellulase-catalyzed product of condensation reaction between glycerol and lactose. For HPLC analysis, diluted samples were analyzed by RI (column, Mightysil Si 60, f 0.46  25 cm; solvent, CH3CN/H2O 4:1; flow rate, 1 ml/min). (a) 0 h, (b) 24 h. See Materials and methods.

48 h with a total yield of about 20% based on lactose added. By structural analysis, F-II-1 and F-II-2 were identified as 2O-h-lactosyl glycerol (2) and 1-O-h-lactosyl-(R,S)-glycerol (1), respectively. The molar ratio of 1 and 2 isolated was about 7:3. Positive ion mode FAB-MS spectra of F-II-1 and F-II-2 showed a molecular ion at m/z 417 (M + H)+. This indicates that both products have a sequence of hexose –

Fig. 2. Chromatographic separation of products formed by cellulasecatalyzed condensation between glycerol and lactose. (a) Chromatography of carbohydrate was carried out on a column (f 8  40 cm) of charcoalCelite at room temperature. (b) Further chromatography was performed with an YMC-PolyamineII column (f 2.15  30 cm).

hexose –glycerol. In 13C-NMR chemical shift data of F-II-1 (Table 1), four signals, at 62.9, 63.7, 63.9, and 64.2 ppm, indicated that neither of the primary hydroxyl groups of the glycerol residue was not affected. A large downfield shift (9 ppm) in C-2 of the glycerol residue at 83.8 ppm, compared with that of free glycerol, also indicated that the linkage occurred at the secondary hydroxyl group of the glycerol residue. The results of these analyses revealed that F-II-1 is 2-O-h-lactosyl glycerol (2, Lac-2-Gly). In 13C-NMR data of F-II-2, three signals due to a primary hydroxyl group appeared at 62.9, 63.9, and 65.2 ppm. No signal was observed in the region around 84 ppm, indicating that a linkage occurred at the primary hydroxyl group of glycerol. C-1 of the Glc residue collapsed to two signals, at 105.1 and 105.4 ppm, and C-1 of the glycerol residue also collapsed to two signals, at 73.5 and 73.6 ppm. These collapses of the signals indicated that C-2 of the glycerol residue became asymmetric by the formation of a glycosidic linkage

Fig. 3. Enzymatic lactosylation through a condensation reaction between lactose and glycerol.

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Fig. 4. Anion-exchange chromatography on a DEAE-Sepharose Fast Flow column of crude cellulase after ammonium sulfate precipitation. The enzyme solution prepared from crude enzyme powder (500 mg, 42 units) was applied onto DEAE-Sepharose Fast Flow column (f 2.6  30 cm). Flow rate: 2.5 ml/min. Hydrolytic activities toward the following substrates were assayed as described in Materials and methods. —, 280 nm; -.-, Lach-pNP; -5-, Galh-pNP; -o-, Glch-pNP.

between C-1 of glycerol and C-1 of Glc and those two diastereometric glycosides were formed. Based on these data, F-II-2 was concluded to be 1-O-h-lactosyl-(R,S)-glycerol [1, Lac-1(R,S)-Gly]. The scheme of enzymatic lactosylation through a condensation reaction is shown in Fig. 3. When 1,3-propanediol instead of glycerol was used as a substrate, the enzyme also catalyzed similar condensation to produce mono O-h-lactosyl propanediol (3) in a yield of 17%. 3.2. Synthesis of alkyl b-lactosides Condensation reactions of lactose with various alkanols were carried out. With ethanol, 1-propanol, 1-butanol, and 1-hexanol, TLC analysis of the reaction mixture showed a new spot corresponding to the condensation product in each case (data not shown). Few spots were observed when tertiary alcohol and a long-chain alkanol such as 1-

octanol were used as acceptors. In the synthesis of Lacbethyl, the molar ratio of lactose and ethanol was 1:2 and the total substrate concentration was 40% (w/v). Lacbethyl was easily separated by chromatography on a charcoal-Celite column in a yield of 3.8%. The yield was much lower than that obtained by condensation with glycerol. Other condensation products, propyl b-lactoside (4), isopropyl h-lactoside (5), and butyl h-lactoside (6), were similarly produced and separated by charcoal-Celite chromatography in yields of 2.3%, 0.9%, and 1.0%, respectively. The yields tended to decrease with increase in the length of the alkyl chain of alkanols from C2 to C4. A similar tendency was also observed in the lactosyl transfer reaction from Lach-pNP to various alkanols using the same enzyme [2]. The enzyme also catalyzed the condensation of lactose with allyl alcohol to produce allyl hlactoside (7) in a yield of 2.7%. Compound 7 was separated by one-step chromatography on a charcoal-Celite

Fig. 5. Time course of the cellulase-mediated condensation product between lactose and glycerol (a), and lactose and ethanol (b). The productivity of glyceroyl h-lactoside and ethyl h-lactoside was examined on a 1-ml scale as described in Materials and methods, and samples were analyzed by HPLC during incubation. -.-, F-2 fraction; -o-, F-3 fraction; -E-, crude enzyme powder.

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column. Because the allyl group is a derivative for reactions with electrophiles, 7 is useful as a starting substance for glycopolymer synthesis [14]. 3.3. Improvement in productivity In the condensation reaction utilizing glycosidase, a large excess of glycosidase is required for increasing the productivity of the condensation product [17,18]. However, the efficiency of the reaction is occasionally reduced by trace amounts of activities of other coexisting glycosidases. In our study, there was a need to eliminate such enzyme activities to prevent the degradation of lactose and condensation products. On a column of DEAE-Sepharose Fast Flow (Fig. 4), Lach-pNP hydrolytic activities were divided into three fractions (F-1, F-2, and F-3), which were completely devoid of h-D-galactosidase activity. F-2 and F-3 were used for the enzyme synthesis without further purification. Fig. 5a shows a profile of the condensation reactions by HPLC analysis with lactose and glycerol utilizing F-2, F-3, and the crude enzyme preparation, respectively. With F-2, the total productivity of 1 and 2 was about 2.5-fold higher than that obtained from a reaction utilizing the crude enzyme containing h-D-galactosidase. The formation of 1 and 2 reached a maximum at 40 h with an overall yield of 40% based on lactose added. Once the formation had reached its maximum, the amounts varied little during the subsequent reaction. In contrast to F-2, F-3 did not show any condensation activity. This was also the case for the condensation reaction with lactose and ethanol (Fig. 5b). To confirm that the coexisting h-D-galactosidase in the crude enzyme prevented the efficiency of condensation, condensation reactions with lactose and ethanol using the crude enzyme and F-2 preparations were analyzed on TLC. With the crude enzyme, a spot corresponding to authentic ethyl h-glucoside other than Lach-ethyl was clearly detected as a by-product, but it was not detected with F-2 (data not shown). This indicates that the direct use of the crude enzyme preparation resulted in degradation of lactose and condensation products once formed by coexisting h-D-galactosidase. In the case of condensation of lactose with 1-octanol using F-2, formation of the desired Lach-octyl was confirmed, but the yield was less than 1%. 3.4. Purification of an enzyme possessing condensation activity Most of condensation activity was concentrated in F-2 (Table 2). F-2 was further purified on a MonoP column to reveal whether both the condensation and Lach-pNP hydrolytic activities were catalyzed by the same enzyme molecule. The purified enzyme gave an apparent single protein band on SDS- and IEF-PAGE (PhastSystem, Amersham Pharmacia Biotech). The molecular weight and isoelectric point of the enzyme were estimated to be 55 kDa and 4.5, respectively, and no subunit structure was observed. The

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Table 2 Purification of crude enzyme preparation from T. reesei

(NH4)2SO4 precipitation DEAE-Sepharose Fast Flow F-2 F-3 F-2 fraction, MonoP F-3 fraction, MonoP

Protein (mg)

Lach-pNP specific activity (U/mg)

Yield (%)

765

0.22

100

20.1 126 0.6 7.8

0.82 0.55 2.56 0.40

9.6 40.6 0.9 2.0

Enzyme activity was described as Lach-pNP hydrolytic activity.

purified enzyme not only hydrolyzed Lach-pNP with disaccharide units but also possessed activity to condense lactose and various alkanols. The enzyme also hydrolyzed CMC with a specific activity of 18.5 units/mg but showed only slight activity toward Avicel. These results show that a single cellulolytic enzyme catalyzes the condensation activity [19,20]. In recent studies, transglycosylation activities of cellulases have been examined. In those studies, donor substrates such as cellobiose, lactosyl fluoride, cellulose, xyloglucan, and Lach-pNP, which we reported previously, were used [2,21 –23]. The results of the present study showed that the present enzyme preparation directly condensed a lactose unit to – OH in various alkanols. It is a useful tool for onepot synthesis of h-lactosyl glycerol and monolactosyl glycerol as a precursor of neoglycolipid. This is the first report of condensation using inexpensive lactose as a substrate and the first report to show new characteristics of the wellknown cellulase.

Acknowledgements This work was supported by a grants-in-aid (no. 11660105) from the Ministry of Education, Science, Sports, and Culture of Japan; a research grant from the Ministry of Agriculture, Forestry, and Fisheries of Japan; and a research grant from Showa Sangyo Co., Ltd.

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