Agric. Biol. Chem., 48 (9), 2265-2269, 1984 2265

Structure of Water-insoluble Glucan Synthesized by /?-Transglycosylase of Trichoderma longibrachiatum Toshio Tanaka and Susumu Oi Department of Biology, Faculty of Science, Osaka City University, Osaka 558, Japan Received February 8, 1984

A water-insoluble glucan was synthesized by the /?-transglycosylase of Trichoderma longibra- chiatum in a reasonable yield from 1%cellopentaose as the substrate after 48 hr incubation. The glucan could be completely solubilized by to give , cellobiose and cellotriose, although the reaction time was about 9 times longer than that required for the complete solubilization of the higher cellodextrin (DP 14) synthesized by the jff-transglucosylase of Sclerotinia libertiana. The glucan was separated into two main fractions according to the solubility in NaOH solution, the major soluble in 4 N-NaOHand the minor insoluble in it. The structure of the major fraction was investigated by methylation analysis, and it was clearly shown to be a linear jff-1,4- glucan having an average degree of polymerization of 19. These results indicated that the f$- transglycosylase of Trichoderma longibrachiatum had a strict specificity of forming /?- l ,4-glucosidic linkages and also had a capacity to elongate the linkage to a higher extent.

Typical disproportionating enzymes (a- enzyme could transfer cellobiosyl and cello- transglycosylase) such as amylomaltase^ of E. triosyl moieties as well as glucosyl moieties coli and D-enzyme2'3) are known to synthesize from cellopentaose to the acceptor molecule amylose from lower maltooligosaccharides as such as cellopentaose upwards with the ef- the substrate by transferring glucosyl, mal- ficiency of almost 100%.6) As a result of the tosyl, and maltotriosyl moieties and so on. On transfer action it could synthesize a water- the other hand, the jS-transglucosylase of insoluble glucan using cellopentaose as a start- Sclerotinia libertiana was shown to synthesize ing substrate. Therefore, the transfer action of a higher cellodextrin of DP 14 using cello- the enzyme was attributed to a dispropor- tetraose as the substrate by transferring a tionating reaction on /M ,4-glucosidic linkages. glucosyl moiety.4>5) Since the fact indicated In the present study, weinvestigted the chemi- a capacity of the enzyme to elongate /M,4- cal structure of a high molecular weight com- glucosidic linkages specifically to a higher ex- ponent (a water-insoluble glucan) synthesized tent, the transfer action of the enzyme was by the ^-transglycosylase of Trichoderma regarded as a kind of disproportionating re- longibrachiatum, and compared both speci- action on lower cellooligosaccharides. As de- ficity and capacity of /M,4-glucosidic linkage scribed above, a disproportionating enzyme elongation by the enzyme with that of the /?- action should result in the production of a high transglucosylase of Sclerotinia libertiana. molecular weight component, which has the same glucosidic linkage as its substrate, with accompanying production of lower oligosac- MATERIALS AND METHODS charides. Enzymes. jS-Transglycosylase: The enzyme (277.1 Recently, a /J-transglycosylase was purified units/mg protein) was purified from a wheat bran Koji to a homogeneousstate by several chromato- culture of Trichoderma longibrachiatum as previously re- graphic procedures from a wheat bran Koji ported.^ The specific activity was defined as the amount of culture of Trichoderma longibrachiatum.6) The enzyme which produced a water-insoluble glucan showing 2266 T. Tanaka and S. Oi turbidity (optical density) of 1.0 at 660nm per ml of the reaction mixture in a 24-hr reaction per mgof the enzyme protein. Cellulase: A commercial cellulase "Cellulase Onozuka R-10" was purchased from the Kinki Yakult Mfg. Co., and was used to analyze the chemical structures of water-insoluble glucans. The enzyme activity was as- sayed as described below. A mixture containing 0.8 ml of 0.125% carboxymethylcellulose (CMC) in 0.02 m acetate buffer, pH 6.0, and 0.2ml of the enzyme solution was incubated for 120min at 40°C, and the reducing sugars produced were determined by the Nelson-Somogyi meth- od.7) Cellulase activity was referred to as 1 unit when reducing sugar equivalent to 1 /rniol of D-glucose was Fig. 1. Photograph of the Water-insoluble Glucan produced in 1 min under the given conditions. Synthesized in the Mixture Containing Cellopentaose and jS-Transglycosylase. Substrates. Cellopentaose was prepared according to the method described in our previous paper.6) Carboxy- The reaction mixture (T) containing 3.6 ml of ^-transgly- methylcellulose sodium salt (CMC) was purchased cosylase (3.5 units/ml) in 0.02 mphosphate buffer, pH 6.0, and 3.6ml of 2%cellopentaose was incubated for 48 hr at from Wako Pure Chemical Industries Ltd. The higher cellodextrin (DP 14) was prepared by the enzyme reac- 30°C after adding a few drops of toluene. (E), incubated tion of /?-transglucosylase of Sclerotinia libertiana using without the substrate; (S), incubated without the enzyme. cellotetraose as the substrate as described in our pre- transglycosylase (3.5 units/ml) in 0.02 m phos- Methylation analysis. Sample (5 mg) was methylated by phate buffer, pH 6.0 and 3.6ml of2% cellop- the method of Hakomori.8) In the methylation repeated entaose was incubated for 48 hr after adding a three times, the reaction mixture was extracted with few drops of toluene to protect from microbial chloroform and washed several times with distilled water. infection. The enzyme apparently synthesized After removing chloroform in vacuo, the residue finally a water-insoluble glucan under the conditions obtained was methanolyzed in a sealed tube for 6hr at described above, as shown in Fig. 1. The 110°C with 1.0ml of 2.2n methanolic hydrogen chloride. Methanolysis products were further refluxed in 1.0 ml of precipitate was centrifuged and washed several 2.0n H2SO4for 4hr after removing HC1by repeated times with distilled water. The recovery yield evaporation with methanol. The hydrolysate was neutral- of the precipitate, which was further washed ized by the addition of BaCO3. After filtration, the filtrate with acetone and dried overnight at 60°C, was evaporated to dryness under reduced pressure, and the syrup which remained was reduced and acetylated accord- was about 20%of the cellopentaose initially ing to the method previously described.9) Authentic added. The precipitate was designated as a samples of 2,3,4,6-tetra-O-methyl-D-glucitol acetate and water-insoluble glucan and employed in the 2,3,6-tri-O-methyl-D-glucitol acetate were also prepared study of the chemical structure. from cellobiose as described above. Enzymic hydrolysis of the water-insoluble Chromatography. GLC was performed using a glucan Shimadzu GC-6ATFgas chromatograph equipped with a column of ECNSS-Mon Gaschrom Q (0.3 x200cm) at The susceptibility of the glucan to cellulase 180°C for alditol acetates. TLCwas performed using silica action was examined. A reaction mixture con- gel 60 (Merck) and the following mixture: 1-butanol, taining 0.1 ml of cellulase (0.12 units/ml) in pyridine, water (6 : 4 : 3) as the solvent for 4hr, and sugars 0.02m acetate buffer, pH 6.0 and 0.2ml of the were detected by a quick dip of the plate into ethanol containing 10%of sulfuric acid, followed by heating for glucan suspension (20 mg/ml) in distilled water lOminat 120°C.was incubated at 37°C. At several times, 5^1 portions were withdrawn and analyzed by RESULTS TLC. As shown in Fig. 2, the glucan remaining at the origin of the chromatogram was hy- Synthesis of a water-insoluble glucan drolyzed by the cellulase action to give glu- The reaction mixture containing 3.6 ml of/?- cose, cellobiose, and cellotriose. After 9hr of Structure of Water-insoluble Glucan 2267 incubation the glucan was completely solu- bilized, and the spot at the origin of the chro- matogramdisappeared. It was apparent that the glucan was mainly composed of /M,4- glucosidic linkages. However, the higher cello- (DP 14), which was synthesized by /?- transglucosylase of Sclerotinia libertiana, was completely solubilized within 1 hr of incu- bation under the same conditions. The water- insoluble glucan seemed to have bigger mol- ecules than the higher cellodextrin (DP 14). The course of hydrolysis of the water- insoluble glucan by cellulase was also exam- ined. The reaction mixture (1 ml) containing the cellulase (0.06 units/ml) in 0.5ml of 0.02m acetate buffer, pH 6.0, and 0.5ml of the higher Fig. 2. Thin-layer Chromatogram of cellodextrin (DP 14) suspension (4mg/ml) or Produced by Cellulase Action on Water-insoluble Glucan. 0.5ml of the water-insoluble glucan suspen- Markers: C, cellooligosaccharides (glucose~cello- sion (4 mg/ml) in distilled water was incubated pentaose) from the top to the bottom of the plate. See at 37°C. At several times, 0.1 ml portions were the text for details. withdrawn and reducing sugars were deter- mined. As shown in Fig. 3, the higher cellodex- 50 trin (DP 14) was rapidly hydrolyzed by the cellulase, and the hydrolysis reached a plateau na s * value (40%) after about 1 hr incubation. On IIffl) the other hand, the water-insoluble glucan was >, y To>25 hydrolyzed by the cellulase at a lower rate than T>, '� that of the higher cellodextrin (DP 14). Also, the rate of hydrolysis significantly decreased 0 H 1 2 after 1 hr of incubation, suggesting the pres- I n c u b a t i o n t i m e , h r ence of different molecular weight compo- nents in the susceptibility to the cellulase ac- Fig. 3. Course of Hydrolysis of a Water-insoluble tion. Glucan by Cellulase. #, higher cellodextrin; O, water-insoluble glucan. See the text for details. Solubility of the water-insoluble glucan in NaOHsolution It was probable that the /?-transglycosylase 660nmusing cells of 1-cm pass length. As of Trichoderma longibrachiatum synthesized a shown in Fig. 4, the higher cellodextrin (DP glucan of higher molecular weight than the 14) completely dissolved in 1.0n NaOH, while higher cellodextrin (DP 14). Therefore, the the water-insoluble glucan suspension still re- glucan was examined for solubility in NaOH mained turbid in 4n NaOH. The water- solution to estimate the molecular size. Twomg insoluble glucan (58.7mg) was then dissolved of the water-insoluble glucan or the higher in 3ml of4n NaOH, and the insoluble moiety cellodextrin (DP 14) was suspended in 0.2ml wasseparated by centrifugation. The precip- of distilled water, and mixed with NaOH itate was dried overnight at 60°C after wash- solution (0.1 ml) of various concentrations. ing twice with 4n NaOHand several times After the mixture was kept for 5min at room with distilled water. The dried preparation temperature, the turbidity wasdetermined at (2.5mg) was obtained by the procedures de- 2268 T. Tanaka and S. Oi

r . . . , i-i i - moiety composing the main chain of the glu- can as well as 2,3,4,6-tetra-O-methyl-D- o \\ id \\ glucose. The fact also madeit clear that the 4n c£ \\ NaOH-soluble glucan was a linear /M,4- O \\ -1<0" \\ glucan. B \\ The degree of polymerization of the glucan could be estimated by determining the ratio of the total glucose residues to the non-reducing 0 0.5 1 2 A glucose residues. The ratio of 2,3,4,6-tetra-O- Cone, of NaOH, N methyl-D-glucose to 2,3,6-tri-(9-methyl-D- Fig. 4. Solubility of Water-insoluble Glucan in NaOH. glucose was calculated to be 17.7 from the The higher cellodextrin (2mg) or the water-insoluble glucan (2mg) suspension in 0.2ml of distilled water was peak area on the chromatogram. Accordingly, mixed with NaOHsolution (0.1 ml) of various concen- the average degree of polymerization was esti- trations. The turbidity of each mixture was determined at mated to be 18.7. 660nm using cells of 1-cm path length. #, higher cellodextrin; O, water-insoluble glucan. DISCUSSION

In a disproportionation reaction of malto- and cellooligosaccharide, the specific elon- gation of a particular glucosidic linkage has been demonstrated on the basis of the chemi- cal structure of a higher molecular weight glucan synthesized by the reaction.2'4) The hy-

0 10 20 30 drolysates of an iodine staining glucan, which Retention time,min was produced by D-enzyme from maltotet- Fig. 5. Gas-liquid Chromatogram of Alditol Acetates raose, with salivary a-amylase were shownto Obtained from 4n NaOH-soluble Fraction of Water- consist of only glucose, maltose, and malto- insoluble Glucan. triose.2) Also, the chemical structure of a water-insoluble glucan (higher cellodextrin), 1 : 2,3,4,6-tetra-O-methyl-D-glucose; 2: 2,3,6-tri-O-methyl- the product of jS-transglucosylase of Sclero- D-glucose. Conditions: the column (0.3 x200cm) was packed with 3% ECNSS-Mon Gaschrom Q; 180°C; tinia libertiana, clearly indicated the speci- carrier gas, N2 (90ml/min). ficity of /M,4-glucosidic linkage elongation by the enzyme.4) scribed above. After removing the 4n NaOH- /?-Transglycosylase of Trichoderma longi- insoluble precipitate, the 4n NaOH-soluble brachiatum apparently synthesized a water- fraction was precipitated again by neutralizing insoluble glucan using 1 %cellopentaose as the the supernatant with 1 n HC1. The precipitate substrate (see Fig. 1). The glucan was frac- (50.3mg) was also isolated as a dried sample tionated according to the solubility in NaOH, after washing several times with distilled and the 4n NaOH-soluble glucan was shown water. to be a linear /M,4-glucan having an average degree of polymerization of 19. As shownin Methylation analysis of the 4n NaOH-soluble Fig. 3, enzymatic hydrolysis of the water- glucan insoluble glucan also indicated the presence of Figure 5 shows the gas-liquid chromato- two kinds of glucans different in the rate of gram of glucitol apetates obtained from the hydrolysis by cellulase. As was expected from methylated sample of the 4n NaOH-soluble the rate of hydrolysis of the higher cellodex- glucan. Methyl 2,3,6-tri-O-methyl-D-glucose trin (DP 14), the 4n NaOH-soluble glucan was detected as the alditol acetate of glucose should rapidly hydrolyzed by cellulase. In the Structure of Water-insoluble Glucan 2269 present study, the structure of the 4n NaOH- lose synthase, as shown in synthesis insoluble glucan was not precisely investi- by Acetobacter xylinum. gated. The glucan could not be methylated by The jS-transglycosylase seems to have an the Hakomori method using DMSO as the ability to synthesize a high molecular weight solvent, and the amount obtained as a dried linear /M,4-glucan (cellulose) by itself, al- preparation was too small to be analyzed by though further analyses are needed to confirm any other method. The water-insoluble glucan the fact. It is reasonable that few of the lower itself was completely solubilized by cellulase /?-l,4-glucan molecules (4n NaOH-soluble to give glucose, cellobiose, and cellotriose. glucan) once accumulated in the reaction mix- Probably the 4n NaOH-insoluble glucan was ture specifically accept glycosyl moieties, prob- a linear /M ,4-glucan of high molecular weight, ably by a single chain mechanism,» because and was rather resistant to the cellulase action the ratio of the 4n NaOH-insoluble glucan to because of its size. the total water-insoluble glucan was so low Several glucosyltransferases have been (about 5%) with respect to the recovery yield. knownto synthesize cellulose using nucleotide Whenthe ratio is calculated on a molelcular sugars such as GDPglucose (EC 2.4.1.29) and basis, the value is much lower than that ob- UDPglucose (EC 2.4.1.12) as a glucosyl do- tained above. Therefore, the jS-transglyco- nor.10~14) Glaser reported that a cell-free sylase should not be regarded as a cellulose preparation of Acetobacter xylinum synthe- synthase, but rather be regarded as a specific sized cellulose using a mixed cellodextrin frac- disproportionate enzymewith respect to the tion as a glucosyl acceptor, where the short capacity and specificity of /?-l,4-glucosidic chain cellodextrin was suggested to be provid- linkage elongation. ed by a transglucosidation mediated by other enzymes of the bacterium.13) The mechanism REFERENCES of cellulose synthesis has been mainly investi- gated for higher plants and bacteria but not 1) J. Monod and A. Torriani, Ann. Inst. Pasteur, 78, 65 for fungi. Except for the fungi having chitin (1950). or chitosan as a main polysaccharide compo- 2) S. Peat, W. J. Whelan and W. R. Rees, Nature, 172, nent of the hyphal wall* relatively few fungi 158 (1953). 3) S. Peat, W. J. WhelanandW. R. Rees, /. Chem. Soc, have cellulosic walls; Phytophthora has those 44 (1956). the presence of which has been established 4) T. Tanaka, R. Yamamoto and S. Oi, Agric. Biol. through X-ray analysis and enzymatic hydrol- Chem., 47, 2731 (1983). ysis tests with cellulase.15'16* However, the 5) T. Tanaka, R. Yamamoto, S. Oi and D. J. Nevins, polysaccharide composition of Trichoderma, Carbohydr. Res., 106, 131 (1982). a typical cellulolytic fungus, has not been 6) T. Tanaka and S. Oi, /. Biochem., 95, 847 (1984). 7) N. Nelson, /. Biol. Chem., 153, 375 (1944). well understood, while recent analysis reveal- 8) S. Hakomori, /. Biochem., 55, 205 (1964). ed the presence of cellobiose in the acetoly- 9) T. Tanaka, S. Oi and T. Yamamoto, /. Biochem., 87, sates of the hyphal wall of Trichoderma longi- 297 (1980). brachiatum, suggesting the presence of cellu- 10) J. C. Chambers and A. D. Elbein, Arch. Biochem. lose in the fraction (T. Tanaka and S. Oi, un- Biophys., 138, 620 (1970). published data). 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