Oligosaccharides in Food and Agriculture - American Chemical Society

Chapter 9

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 9, 2015 | http://pubs.acs.org Publication Date: April 30, 2003 | doi: 10.1021/bk-2003-0849.ch009

Kojioligosaccharides: Application of Kojibiose Phosphorylase on the Formation of Various Kojioligosaccharides Tetsuya Nakada, Tomayaki Nishimoto, Hiroto Chaen, and Shigeharu Fukuda Amase Institute, Hayashibara Biochemical Laboratories Inc., 7-7 Amase-Minami Machi, Okayanna, Japan

Various kojioligosaccharides such as kojibiose, kojitriose, kojitetraose, 2-O-α-kojibiosyl-β-D-fructofuranoside, 1-O-αkojibiosyl-α-D-glucopyranoside, and 4-O-α-kojibiosyl-D -glucose were enzymatically synthesized using kojibiose phosphorylase (KPase) from a thermophilic anaerobe, Thermoanaerobacter brockii. Combination of KPase with other phosphorylases, such as maltose phosphorylase or trehalose phosphorylase, has potential applications in production of novel saccharides from inexpensive sugars. Structures and functions of kojioligosaccharides are also presented.

104

© 2003 American Chemical Society

In Oligosaccharides in Food and Agriculture; Eggleston, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


105 Kojibiose (2-0-a-D-glucopyranosyl α-D-glucopyranose) is a disaccharide that occurs in koji extract (1), beer, honey (2), and starch hydrolyzate. The best known method for preparation of kojibiose is the isolation from a partial acetolyzate of dextran from Leuconostoc mecenteroides NRRL B-1299 (3)> although the method is obviously a tedious one. Although many researchers have attempted to synthesize kojibiose using glycosidases such as a-glucosidase (4), glucoamylase (5) or sucrose phosphorylase (6), there are some problems such as the formation of by-products and a low efficiency for production. On the other hand, kojioligosaccharides of DP3 or higher, such as kojitriose, kojitetraose and kojipentaose, are rare in nature. Therefore, these have been prepared by chemical synthesis (7-8). We isolated a novel enzyme, kojibiose phosphorylase (KPase) from a thermophilic anaerobe, Thermoanaerobacter brockii ATCC 35047 (9). This enzyme catalyzes the reversible phosphorolysis of kojibiose. In the presence of suitable acceptors such as mono- or oligo-saccharides, KPase also catalyzes a1,2-glucosyl transfer from β-D-glucose-l-phosphate (β-GlP) to the acceptors (10), and produces various kojioligosaccharides (11-12). This paper describes the properties of KPase, the enzymatic synthesis of various kojioligosaccharides, and on some properties and functions of these sugars.

Properties of Thermoanaerobacter Kpase

Properties of Thermoanaerobacter KPase We have previously reported on the purification and characterization of KPase (10). Properties and the deduced amino acid sequence of the enzyme (13) are shown in Table I and Figure 1, respectively. The apparent molecular mass of KPase was estimated to be 500 kDa by gel filtration. On the other hand, SDSPAGE of the purified KPase gave a single protein band with an apparent molecular mass of 80 kDa. These results suggest that the enzyme consists of six identical subunits. The optimum pH was 5.5 for both the phosphorolytic and the synthetic reactions. Maximum activity was observed at 65°C. The enzyme was stable from pH 5.5 to 9.7, and at temperatures up to 65°C. A KPase gene has been cloned from Thermonanaerobacter brockii ATCC 35047 and sequenced to obtain the amino acid sequence of KPase. KPase is composed of 755 amino acid residues, and its molecular mass is calculated to be about 90 kDa. The putative three catalytic amino acid residues, aspartate-362, lysine-614 and glutamate-642 were deduced by site-directed mutational analysis (data not shown).


106 Table I. Enzymatic properties of KPase from Thermoanaerobacter brockii. Molecular mass (Da) SDS-PAGE Gel filtration Isoelecric point Optimum pH Optimum temperature (°C) pH Stability Thermal stability (°C) a

b


0

d

a

83,000 500,000 4.3 5.5 65 5.5-9.7 below 65 b

* Both phosphorolytic and synthetic reaction; ' 30-min reaction;

c>

d>

after 20-hr incubation at 4°C; after 60-min incubation.

MVKHMFLEDV NNLISDDKWL IFONEYNTEV NPRYETLFTL TNGYMGVRGT 50 FEEGSEGERS GNFIAGIFDK SDAOVREIVN AONWLRIKLY VEGEELSLDK 100 CQLIEFKRIL DMKKGILFRS MLIKDSKDRI TRIEGYRFIS RSDLHRSAIK 150 LFVTPVNYSG WGIESIIDG TVLNSADSPK HRVKHLKVAD NSSLNKSGVY 200 LETATIDDDI RIATGSAVRL YHYEDKEKNN IAKFKRFLPL GEMSIEYFEF 250 DGTENKTWI DKFIITYTSR DVKKGLLKST VEKELFAFAG EGIDKELQRH 300 IEVYEELWSV ADINIEGDEE ADKALRFNIF HLMSSVNEND PMVSIAAKAL 350 HGEGYKGHVF V^TEIFMLPF FIYVHPKAAK TLLMYRYNML DAARKNAALN 400 GYKGAQYPWE SADTGEEETP KWGFDYMGNP VRIWTGDLEH HITADIAFAV 450 WEYFRATEDI EFMLNYGAEV IFETARFWVS RCEYVKELDR YEINNVIGPD 500 EFHEHVDNNA YTDYLAKWNI KKGLELINML KEKYPEHYHA ISNKKCLTNE 550 EMEKWKEVEE KIYIPYDKDK KLIEQFEGYF DKKDYVIDKF DENNMPIWPE 600 GVP ITKLGDT QL3@QADWM LMLLLGEEFD EETKRINYEY T@KRTMHKSS 650 LGPSMYAIMG LKVGDHKNAY QSFMRSANVD LVDNQGNTKE GLHAASAGGT 700 WQVWFGFGG MEIDKEGALN INSWLPEKWD KLSYKVFWKG NLIEVIVTKQ 750 EVTVKKLKGK GNIKVKVKGK ELTIE 775 Figure L Amino acid sequence of KPase from Thermoanaerobacter brockii The underlined amino acid sequences are those found from protease digestion products of KPase protein. The open-boxed amino acids are the putative three catalytic residues deducedfromsite directed mutational analysis.

Substrate and Acceptor Specificities of KPase KPase was specifically active on kojibiose and inactive on other disaccharides such as sophorose, trehalose, neotrehalose, nigerose, laminaribiose,



107 maltose, cellobiose, isomaltose, gentiobiose, sucrose and lactose (data not shown). Acceptor specificity was examined using β-GlP sodium salt as a glucosyl donor and various mono-, di-, and oligosaccharides as acceptors. As shown in Table II, D-glucose, L-sorbose, methyl-oc-D-glucopyranoside, and methyl-P-D-glucopyranoside were effective acceptors among various mono saccharides. D- and L-Xylose also acted as acceptors, but their transfer ratio was less than 25%. Furthermore, maltose, trehalose, nigerose, isomaltose, maltotriose, and others were good oligosaccharide acceptors. These results suggest that diand oligosaccharides having a D-glucosyl residue at their non-reducing ends were good acceptors in any case.

Table II. Acceptor Specificity of KPase. Acceptor D-Glucose D-Xylose L-Xylose D-Galactose D-fructose D-Mannose D-Arabinose D-Fucose L-Fucose L-Sorbose D-Ribose L-Rhamnose Methyl-ct-D-glucoside Methy-P-D-glucoside 2-Deoxy-D-glucose ΛΓ-Acetyl-D-glucosamine D-Glucosamine Sorbitol

Product

+++ + + +++ +++ +++ -

Acceptor α,α-Trehalose Neotrehalose Kojibiose Nigerose Maltose Isomaltose Laminaribiose Cellobiose Gentiobiose Maltitol Sucrose Palatinose Maltulose Turanose Lactose Melibiose Lactulose Maltotriose Maltotetraose Maltopentaose a

8

Product

+++ +++ +++ +++ +++ +++ +++ ++ ++ +++ ++ -H-+

+++ +++ +++ +++ +++

The transfer ratio to acceptor was detected by GLC. +++, 50% 2)-0-a-D-glucopyranosyl-(l—>2)-p-D-ihictofuranoside (2-0-a-kojibiosyl-P-D-fructoiuranoside). The structural formulas of kojioligosaccharides that were synthesized are shown in Figure 5.

Properties and Functions of Kojioligosaccharides

Reducing Power and Maillard Reaction of Kojibiose, Kojitriose and Kojitetraose Compared with glucose, the reducing powers of kojioligosaccharides was measured by Nelson-Somogyi method (18). Although kojioligosaccharides are reducing sugars, having an aldehyde group at C - l position of the end glucose, the reducing powers of kojibiose, kojitriose and kojitetraose were much less than 1% of that of glucose (data not shown). As shown in Figure 6, their low reducing power weakens the Maillard reaction of kojioligosaccharides with amino acids. Further, kojioligosaccharides


In Oligosaccharides in Food and Agriculture; Eggleston, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003. t

Figure 5. Several kojioligosaccharides synthesized using KPase. a, kojibiose; b, kojitriose; c, kojitetraose; d, 2-0-a-kojibiosyl-fi-D-fructofuranoside; e, l-O-'a- ' kojibiosyl-a-D-glucopyranoside; f 4-O-a-kojibiosyl-D-glucopyranose.


Κ)


113

Figure 6. Maillard reaction of kojioligosaccharides. Equal volume of 5% sugar solution and 1% glycine solution were mixed and incubated at pH8.0, 100°Cfor 90 min. K2, kojibiose; K3, kojitriose; K4, kojitetraose; G2, maltose; G3, maltotriose; G4, maltotetraose

Figure 7. Acid-formation by a cariogenic bacterium, Streptococcus mutons OMZ-176. Equal volume of 50 % of S. mutans cell suspension and 1% sugar solutions were mixed and incubated at 37°C for 90 min. Changes of pH levels were measured at various intervals. •, sucrose; 0, maltose; o, kojibiose; ; kojitriose; Δ, kojitetraose.


114 have a mild amount of sweetness. These features kojioligosaccharides attractive materials in the food industry.

should

make


Acid Formation by Streptococcus /nutans Acid-formation by Streptococcus mutonsfromkojioligosaccharides was examined (Figure 7). It is well known that Streptococcus mutans causes dental caries. When sucrose is ingested, the bacterium liberates glucosyltransferase to synthesize both adhesive and non-adhesive glucans from sucrose. Adhesive glucan is the major cause of dental plaque because the bacterium lives in it, and converts sugars into organic acids, which degrade the enamel of teeth. Sucrose and maltose decreased pH level, while kojioligosaccharides did not. These results may suggest that kojioligosaccharides are not utilized and converted into adhesive glucan and acids by the bacterium. Kojioligosaccharides do not inhibit the formation of adhesive glucan from sucrose (data not shown). Sugars can be classified into three types: cariogenic, non-cariogenic, and cariostatic types. According to this classification, kojioligosaccharides are of non-cariogenic type.

In vitro Digestibility of Kojioligosaccharides In order to investigate digestibility of kojioligosaccharides, in vitro digestion tests were carried out using human saliva, artificial gastric juice, porcine pancreatic amylase and rat intestinal enzyme according to the method of Okada et al. (19). Maltose was used as a control. No hydrolysis of maltose, kojibiose, kojitriose, kojitetraose, 4-0-ockojibiosyl-D-glucose, or 2-0-a-kojibiosyl-P-D-fructoiuranoside was observed using salivary, artificial gastric juice or pancreatic amylases. Maltose and kojibiose were hydrolyzed by small intestinal enzymes, while kojitriose, kojitetraose, 2-\ L. reuteri JCM1112 L. salivarius JCM1231 Eubacterium E. limosum JCM6421 E. aerofaciens ATCC25986 Bacteroides B. distasonis JCM5825 B. vulgatus JCM5826 B. ovatus JCM5824 Clostridium C. butyricum JCM1391 C. perfringens JCM3816 C. ramosum JCM1298 C. paraputrificum JCM1293 Streptococcus SfaecalislAM\0065 Peptostreptococcus P. prevotii ATCC9321 P. productus ATCC27340 Escherichia E. coli JFO3301

Gl

K2

K3

+++ ++ +++ +++ +++

+++ + +

+++

++ +++ ++ +++ •f ++• •f ++-

K4

-

+++ +++

+++

+

-

-H ++• f +++++ ++ ++ +-H-

+++ +++ +++ ^_

+++ +-H-

++

+++

+++

+++

Utilization test was carried out according to the method of Mitsuoka et al. (20), using glucose as control. Each bacterium was inoculated into a culture medium, containing either of the sugars, and cultivated at 37°C for 4 days. Utilization of sugars were estimated by measuring pH levels of resulting culture broth. +++, < pH 4.9; ++, pH 5.0 pH 5.4; +, pH 5.5 - pH 5.9; -, pH 6.0

Oligosaccharides in Food and Agriculture - American Chemical Society

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